999 results for "1993+article+mouse+line+professor"

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• DNA methylation at HBV integrants and flanking host genomes

  Integration of DNA viruses into the human genome plays an important role in various types of tumors, including hepatitis B virus (HBV)-related hepatocellular carcinoma. However, the molecular details and clinical impact of HBV integration on either the human or HBV epigenomes are unknown. Here, we show that methylation of the integrated HBV DNA is related to the methylation status of the flanking human genome. We developed a next-generation sequencing-based method for structural methylation analysis of integrated viral genomes (denoted G-NaVI). This method is a novel approach that enables enrichment of viral fragments for sequencing using unique baits based on the sequence of the HBV genome. We detected integrated HBV sequences in the genome of the PLC/PRF/5 cell line and found variable levels of methylation within the integrated HBV genomes. Allele-specific methylation analysis revealed that the HBV genome often became significantly methylated when integrated into highly methylated host sites. After integration into unmethylated human genome regions such as promoters, however, the HBV DNA remains unmethylated and may eventually play an important role in tumorigenesis. The observed dynamic changes in DNA methylation of the host and viral genomes may functionally affect the biological behavior of HBV. These findings may impact public health given that millions of people worldwide are carriers of HBV. We also believe our assay will be a powerful tool to increase our understanding of the various types of DNA virus-associated tumorigenesis.

  Study JGAS000015

• Open_Targets_020_Epigenomes_of_Cell_Lines

  There is currently a drive to establish cell based assay systems of greater human biological and disease relevance through the use of well characterised transformed cell lines, primary cells and complex cellular models (e.g. co-culture, 3D models). However, although the field is gaining valuable experience in running more non-standard & complex cell assays for target validation and compound pharmacology studies, there is the lack of a systematic approach to determine if this expansion in cell assay models is reflected in increased human biological and disease relevance. The increasing wealth of publically available transcriptomic, and epigenome (ENCODE and Epigenome Roadmap) data represents an ideal reference mechanism for determining the relationship between cell types used for target & compound studies to primary human cells and tissues from both healthy volunteers & patients. The CTTV020 epigenomes of cell line project aims to generate epigenetic and transcriptomic profiles of cell lines and compare these with existing and newly generated reference data sets from human tissue and cell types. The aim is to identify assay systems which will provide greater confidence in translating target biology and compound pharmacology to patients. Multiple cell types commonly used within research have been grouped according to biology. Examples include erythroid, lung epithelial, hepatocyte cell types and immortalised models of monocyte / macrophage biology. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute please see http://www.sanger.ac.uk/datasharing/

  Study EGAS00001003138

• Bioinformatic Methods and Bridging of Assay Results for Reliable Tumor Mutational Burden Assessment in Non-Small Cell Lung Cancer

  Tumor mutational burden (TMB) has emerged as a predictive biomarker of response to immune checkpoint inhibitors. Standardization of TMB measurement is essential for implementing diagnostic tools to guide treatment. Here we evaluate bioinformatic TMB analysis by whole exome sequencing (WES) in formalin-fixed, paraffin-embedded samples. In CheckMate 026, TMB was retrospectively assessed in 312 patients with non-small cell lung cancer (58% of the intent-to-treat population) who received first-line nivolumab treatment or chemotherapy. We examined the sensitivity of TMB assessment to bioinformatic filtering methods and assessed concordance between TMB data derived by WES and the FoundationOne CDx™ assay. TMB scores comprising synonymous, indel, frameshift, and nonsense mutations (all mutations) were 3.1-fold higher than data including missense mutations only, but values were highly correlated (Spearman’s r = 0.99). Scores from CheckMate 026 samples including missense mutations only were similar to those generated from data in The Cancer Genome Atlas, but those including all mutations were generally higher. Using databases for germline subtraction (instead of matched controls) showed a trend for race-dependent increases in TMB scores. Parameter variation can therefore impact TMB calculations, highlighting the need for standardization. Encouragingly, WES and FoundationOne CDx outputs were highly correlated (Spearman’s r = 0.90) and differences could be accounted for by empirical calibration, suggesting that reliable TMB assessment across assays, platforms and centers is achievable.

  Study EGAS00001003661

• Platelet RNAseq data for SLFN14 K219N patients

  Pathogenic missense variants in SLFN14, which encodes an RNA endoribonuclease protein that regulates rRNA degradation, are known to cause inherited thrombocytopenia with impaired platelet aggregation and ATP secretion. Despite rather mild laboratory defects, these patients display an obvious bleeding phenotype. The function of SLFN14 in megakaryocyte (MK) and platelet biology is unknown. This study aims to characterize the platelet transcriptome in patients with a SLFN14 K219N variant and model the disease in the immortalized megakaryocyte cell line imMKCL. Total platelet RNA was sequenced for two patients and 19 healthy controls. Differential gene expression analysis yielded a total of 2999 and 2888 significantly (|log2FC|>1, FDR<0.05) up- and downregulated genes, respectively. Remarkably, these downregulated genes were not enriched for any biological pathway while upregulated genes were enriched for pathways involved in (mitochondrial) translation and transcription with a significant upregulation of 134 ribosomal protein genes (RPG). Heterozygous and homozygous SLFN14 K219N imMKCL showed a defect in MK differentiation that aggravated during cell passaging of undifferentiated cells and in proplatelet formation. SLFN14 defective platelets and MK showed signs of rRNA degradation while this was absent in undifferentiated imMKCL cells. Upregulation of RPG through increased mTOR signaling in SLFN14 K219N MK seems to be a compensatory response for rRNA degradation. Indeed, mTOR inhibition with rapamycin resulted in further enhanced rRNA degradation in SLFN14 K219N MK. Taken together, our study indicates dysregulation of mTOR-regulated ribosomal biogenesis as the disease mechanism for SLFN14-related thrombocytopenia.

  Study EGAS00001006339

• Whole genome study of Hurthle cell thyroid carcinoma

  Oncocytic thyroid carcinoma, also known as Hürthle cell thyroid carcinoma, accounts for only a small percentage of all thyroid cancers. However, this malignancy often presents at an advanced stage and poses unique challenges to patients and clinicians. Surgical resection of the tumor accompanied in some cases by radioactive iodine treatment, radiation and chemotherapy are the established modes of therapy. Knowledge of the perturbed oncogenic pathways can provide better understanding of the mechanism of disease and thus opportunities for more effective clinical management. Initially, two oncocytic thyroid carcinomas and their matched normal tissues were profiled using whole genome sequencing. Subsequently, 72 oncocytic thyroid carcinomas, one cell line and 5 Hürthle cell adenomas were examined by targeted sequencing for the presence of mutations in multiple endocrine neoplasia I (MEN1) gene. We report the identification of MEN1 loss-of-function mutations in approximately 7% of patients diagnosed with oncocytic thyroid carcinoma. Whole genome sequence data also revealed large regions of copy number variation encompassing nearly the entire genomes of these tumours. Menin, a ubiquitously expressed nuclear protein, is a well-characterized tumor suppressor whose loss is the cause of multiple endocrine neoplasia type 1 syndrome. Menin is involved in several major cellular pathways such as regulation of transcription, control of cell cycle, apoptosis and DNA damage repair pathways. Mutations of this gene in a subset of Hürthle cell tumors point to a potential role for this protein and its associated pathways in thyroid tumorigenesis.

  Study EGAS00001000940

• Dissociation of solid tumour tissues with cold active protease for single-cell RNA-seq minimizes conserved collagenase-associated stress responses

  Background Single-cell RNA sequencing (scRNAseq) is a powerful tool for studying complex biological systems, such as tumour heterogeneity and tissue microenvironments. However, the sources of technical and biological variation in primary solid tumour tissues and patient-derived mouse xenografts for scRNAseq, are not well understood. Here, we used low temperature (6°C) protease and collagenase (37°C) to identify the transcriptional signatures associated with tissue dissociation across a diverse scRNAseq dataset comprising 128,481 cells from patient cancer tissues, patient-derived breast cancer xenografts and cancer cell lines.Results We observe substantial variation in standard quality control (QC) metrics of cell viability across conditions and tissues. From FACS sorted populations gated for cell viability, we identify a sub-population of dead cells that would pass standard data filtering practices, and quantify the extent to which their transcriptomes differ from live cells. We identify a further subpopulation of transcriptomically “dying” cells that exhibit up-regulation of MHC class I transcripts, in contrast with live and fully dead cells. From the contrast between tissue protease dissociation at 37°C or 6°C, we observe that collagenase digestion results in a stress response. We derive a core gene set of 512 heat shock and stress response genes, including FOS and JUN, induced by collagenase (37°C), which are minimized by dissociation with a cold active protease (6°C). While induction of these genes was highly conserved across all cell types, cell type-specific responses to collagenase digestion were observed in patient tissues. We observe that the yield of cancer and non-cancer cell types varies between tissues and dissociation methods.Conclusions The method and conditions of tumour dissociation influence cell yield and transcriptome state and are both tissue and cell type dependent. Interpretation of stress pathway expression differences in cancer single cell studies, including components of surface immune recognition such as MHC class I, may be especially confounded. We define a core set of 512 genes that can assist with identification of such effects in dissociated scRNA-seq experiments.

  Study EGAS00001003753

• plasma DNA LINE-1 targeted bisulfite sequencing: a new non-invasive multi-cancer detection marker

  The detection of circulating tumor DNA, which allows non-invasive tumor molecular profiling and disease follow-up, promises optimal and individualized management of patients with cancer. However, detecting small fractions of tumor DNA released when the tumor burden is reduced remains a challenge. We developed a PCR-based targeted bisulfite method coupled to deep sequencing to detect methylation patterns of L1PA elements,which we named DIAMOND (for Detection of Long Interspersed Nuclear Element Altered Methylation ON plasma DNA). We used sodium bisulfite chemical conversion to achieve base-pair resolution analysis and designed a multiplexed PCR based on 8 amplicons covering L1PAs .

  Study EGAS50000000446

• Partner independent fusion gene detection by multiplexed CRISPR Cas9 enrichment and long read Nanopore sequencing (FUDGE)

  Targeted long-read nanopore sequencing. Abstract: Fusion genes are hallmarks of various cancer types and important determinants for diagnosis, prognosis and treatment. Fusion gene partner choice and breakpoint-position promiscuity restricts diagnostic detection, even for known and recurrent configurations. To accurately and impartially identify fusions, we developed FUDGE: FUsion Detection from Gene Enrichment. FUDGE couples target-selected and strand-specific CRISPR/Cas9 activity for fusion gene driver enrichment - without prior knowledge of fusion partner or breakpoint-location – to long-read Nanopore sequencing with the bioinformatics pipeline NanoFG. FUDGE has flexible target-loci choices and enables multiplexed enrichment for simultaneous analysis of several genes in multiple samples in one sequencing run. We observe on-average 665 fold breakpoint-site enrichment and identify nucleotide resolution fusion breakpoints - within two days. The assay identifies cancer cell line and tumor sample fusions irrespective of partner gene or breakpoint-position. FUDGE is a rapid and versatile fusion detection assay, providing unparalleled opportunity for diagnostic pan-cancer fusion detection.

  Dataset EGAD00001006111

• Angelman, Rett, Prader-Willi Syndromes Consortium (ARP) Angelman Syndrome Natural History Protocol

  This study is a longitudinal multidisciplinary investigation on the natural history, morbidity and mortality of Angelman Syndrome (AS). We will collect detailed longitudinal data on a cohort of AS individuals to gain a better understanding of the disease progression, and to follow the natural history of the clinical features of this cohort including assessment of quality of life and longevity. The participants to be recruited for the study will include 1) patients who have a documented molecular diagnosis of AS and 2) patients with a clear clinical diagnosis of Angelman Syndrome as determined by the Principal Investigator (PI) and the Co-investigators in this study but who do not have a known molecular defect. One of the goals of the natural history study will be to characterize the phenotypic differences between patients with Class I deletions and those with Class II deletions, particularly with respect to the issue of autism. A blood sample may be collected on the participants in order to create a DNA repository, and in some cases, to establish cell lines if further material is required for molecular studies. Alternatively, DNA may be obtained by buccal mucosa swabs/brushing. In those AS patients with known deletions involving the 15q11-q13 regions, a blood sample will be collected to perform comparative genome hybridization (CGH) microarray studies to characterize the extent of the deletion. No drugs or treatments will be administered through this protocol. In rare instances, a skin biopsy to establish a fibroblast cell line may be requested (using separate consent).

  Study phs000576

• Molecular Evolution of Cancer

  Recently developed methods that utilize partitioning of long genomic DNA fragments, and barcoding of shorter fragments derived from them, have succeeded in retaining long-range information in short sequencing reads. These so-called read cloud approaches represent a powerful, accurate, and cost-effective alternative to single-molecule long-read sequencing. We developed software, GROC-SVs, that takes advantage of read clouds for structural variant detection and assembly. We apply the method to two 10x Genomics data sets, one chromothriptic sarcoma with several spatially separated samples, and one breast cancer cell line, all Illumina-sequenced to high coverage. Comparison to short-fragment data from the same samples, and validation by mate-pair data from a subset of the sarcoma samples, demonstrate substantial improvement in specificity of breakpoint detection compared to short-fragment sequencing, at comparable sensitivity, and vice versa. The embedded long-range information also facilitates sequence assembly of a large fraction of the breakpoints; importantly, consecutive breakpoints that are closer than the average length of the input DNA molecules can be assembled together and their order and arrangement reconstructed, with some events exhibiting remarkable complexity. These features facilitated an analysis of the structural evolution of the sarcoma. In the chromothripsis, rearrangements occurred before copy number amplifications, and using the phylogenetic tree built from point mutation data, we show that single nucleotide variants and structural variants are not correlated. We predict significant future advances in structural variant science using 10x data analyzed with GROC-SVs and other read cloud-specific methods.

  Study phs001255

• Transcriptome profiling of megakaryocytes and platelets: application to GP9- and IKZF5-related thrombocytopenia

  Platelets are anucleate cells produced in the bone marrow and derived from large progenitor cells called megakaryocytes (MKs). Platelets receive RNA transcripts from their progenitorial MKs during thrombopoiesis. However, the correspondence between platelet and MK transcriptomes is poorly understood, particularly in the context of germline mutations that cause platelet formation defects or thrombocytopenia. We have studied the effects of two such mutations on MK and platelet transcriptomes. We generated immortalized MK cell line (imMKCL)-based models of Bernard-Soulier syndrome and IKZF5-related thrombocytopenia. MKs derived from imMKCLs with either a homozygous deletion of GP9 (GP9-/-) or a heterozygous Y121F variant in IKZF5 (IKZF5WT/Y121F) exhibited reduced proplatelet formation (reductions of 96% and 57%, respectively). Platelets from patients with either GP9-/- or IKZF5WT/Y121F genotypes had broad transcriptomic dysregulation, suggesting that (pro)platelet formation defects due to mutations in glycoprotein receptor and transcription factor genes such as GP9 and IKZF5 affect the trafficking of transcripts from MKs into platelets. RNA-seq data from MKs at four stages of differentiation revealed widespread but distinct changes in expression over time between the GP9-/- and the IKZF5WT/Y121F genotypes. Dysregulated genes in GP9-/- MKs were enriched for RNA metabolism and actin/tubulin folding pathways while those in IKZF5WT/Y121F MKs were enriched for cell cycle pathways. Most of these genes were also dysregulated in the platelets of patients with the corresponding diseases. Our results suggest that patients with inherited forms of thrombocytopenia present with a transcript trafficking defect during platelet formation.

  Study EGAS50000001276

• Organoid cultures of early-onset colorectal cancers reveal distinct and rare genetic profiles

  Sporadic early-onset colorectal cancer (EOCRC) has bad prognosis, yet is poorly represented by cell line models. We examine the key mutational and transcriptomic alterations in an organoid biobank enriched in EOCRCs. We established paired cancer (n=32) and normal organoids (n=18) from 20 patients enriched in microsatellite-stable EOCRC. Exome and transcriptome analysis was performed. We observed a striking diversity of molecular phenotypes, including PTPRK-RSPO3 fusions. Transcriptionally, RSPO fusion organoids resembled normal colon organoids and were distinct from APC mutant organoids, with high BMP2 and low PTK7 expression. Single cell transcriptome analysis confirmed the similarity between RSPO fusion organoids and normal organoids, with a propensity for maturation on Wnt withdrawal, whereas the APC mutant organoids were locked in progenitor stages. CRISPR/Cas9 engineered mutation of APC in normal human colon organoids led to upregulation of PTK7 protein and suppression of BMP2, but less so with an engineered RNF43 mutation. The frequent co-occurrence of RSPO fusions with SMAD4 or BMPR1A mutation was confirmed in TCGA database searches. RNF43 mutation was found in organoid from a leukaemia survivor with a novel mutational signature; and organoids with POLE proofreading mutation displayed ultramutation. The cancer organoid genomes were stable over long culture periods, while normal human colon organoids tended to be subject to clonal dominance over time. These organoid models enriched in EOCRCs with linked genomic data fill a gap in existing CRC models and reveal distinct genetic profiles and novel pathway cooperativity.

  Study EGAS00001004063

• Pediatric Papillary Thyroid Carcinoma Whole Exome Sequencing

  BACKGROUND: Pediatric papillary thyroid carcinoma (PPTC) is distinct from adult-onset disease. Despite higher rates of regional and distal metastases and more frequent recurrence, there is, paradoxically, a highly favourable prognosis. Historically, only 40-50% of childhood PTC are driven by genomic variants common to adult PTC, while the oncogenic drivers in the remainder are unknown. METHODS: Fifty-two PPTCs were genotyped by candidate gene testing, followed, in a subset (n=32), by whole exome (WES) and transcriptome sequencing (RNASeq) to identify oncogenic variants in driver-unknown tumors and to explore gene expression patterns. FINDINGS: Thirty-one tumors (60%) carried variants identified by candidate gene testing, specifically: BRAFV600E(22%), NRASQ61R(2%), RET-CCDC6(19%), RET-NCOA4(15%) and PAX8-PPARG. Among the 21 remaining tumors with negative first-line genotyping, oncogenic variants were identified in 19 (90%). These were enriched with oncogenic fusions, with 11 non-recurrent fusion transcripts identified, including two that had not been previously described: STRN-RET and TG-PBF. Most fusions were associated with 3’ receptor tyrosine kinase (RTK) moieties: RET, MET, ALK and NTRK3. Gene expression analysis defined three distinct clusters, which demonstrated differing expression of genes involved in thyroid differentiation and MAPK signaling. INTERPRETATION: Exome and transcriptome-wide sequencing identify genomic drivers of PPTC in 98% of tumors, whereas conventional genotyping leaves many tumors genetically unexplained. PPTC is driven, in the majority of cases, by oncogenic fusion transcripts involving one of a limited number of RTKs. These observations have broad implications for diagnostic testing and for therapeutic options in children with advanced disease.

  Study EGAS00001005187

• Biallelic tumor suppressor loss and DNA repair defects in de novo small cell prostate cancer

  Small cell prostatic carcinoma (SCPC) is an aggressive pathology that is managed similar to small cell lung cancer. SCPC can evolve from prostatic adenocarcinoma in response to androgen deprivation therapy but in rare cases is present at initial cancer diagnosis. The molecular etiology of de novo SCPC is incompletely understood due to the scarcity of tumor tissue and the brief life expectancy of patients. Through a retrospective search of our regional oncology pharmacy database, we identified 18 patients diagnosed with de novo SCPC between 2004 and 2017. Ten patients had pure SCPC pathology and the remainder had some admixed adenocarcinoma foci, but all were treated with first line platinum-based chemotherapy. Median overall survival was 28 months. We performed targeted DNA sequencing, whole exome sequencing and mRNA profiling on formalin-fixed paraffin embedded archival tumor tissue. We observed frequent biallelic deletion and/or mutation of tumor suppressors TP53, RB1 and PTEN, similar to treatment related SCPC. Indeed, at the RNA level pure de novo SCPC closely resembled treatment-related SCPC. However, five patients had biallelic loss of DNA repair genes including BRCA1, BRCA2, ATM and MSH2/6: potentially underlying the high genomic instability of this rare disease variant. Two patients with pure de novo SCPC harbored ETS gene rearrangements to androgen-driven promoters, consistent with evolution of de novo SCPC from an androgen-driven ancestor. Overall, our results reveal a highly aggressive molecular landscape underlies this unusual pathologic variant, and propose opportunities for targeted therapy strategies in a disease with few treatment options.

  Study EGAS00001003007

• Open_Targets_020_Epigenomes_of_Cell_Lines

  There is currently a drive to establish cell based assay systems of greater human biological and disease relevance through the use of well characterised transformed cell lines, primary cells and complex cellular models (e.g. co-culture, 3D models). However, although the field is gaining valuable experience in running more non-standard & complex cell assays for target validation and compound pharmacology studies, there is the lack of a systematic approach to determine if this expansion in cell assay models is reflected in increased human biological and disease relevance. The increasing wealth of publically available transcriptomic, and epigenome (ENCODE and Epigenome Roadmap) data represents an ideal reference mechanism for determining the relationship between cell types used for target & compound studies to primary human cells and tissues from both healthy volunteers & patients. The CTTV020 epigenomes of cell line project aims to generate epigenetic and transcriptomic profiles of cell lines and compare these with existing and newly generated reference data sets from human tissue and cell types. The aim is to identify assay systems which will provide greater confidence in translating target biology and compound pharmacology to patients. Multiple cell types commonly used within research have been grouped according to biology. Examples include erythroid, lung epithelial, hepatocyte cell types and immortalised models of monocyte / macrophage biology. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/

  Study EGAS00001003136

• Study on the consequences of prenatal famine exposure on DNA methylation.

  The potential of the epigenome to undergo environmentally-induced changes during early development is central to its postulated role in human disease. Here, we report a genome-scale view of DNA methylation differences after early gestational malnutrition that was caused by the Dutch Hunger Winter, a severe famine at the end of World War II. We adopted an annotation guided analysis of reduced-representation bisulfite sequencing data generated in 24 middle-aged individuals who had been exposed to famine in utero and 24 unexposed sibling controls. We observed differential DNA methylation primarily at genomic regions with regulatory potential, including enhancers active during early development. In-depth annotation of 181 prenatally-induced differentially methylated regions (P-DMRs) showed that they were predominantly located in gene bodies and co-occurred with histone marks denoting an active state. In line with the early gestational timing of the exposure, the P-DMRs mapped to genes that were enriched for differential expression patterns during blastocyst development and early organogenesis. Validation and further explorative analyses of 6 P-DMRs mapping to SMAD7, CDH23, INSR, RFTN1, CPT1A and KLF13 highlighted the critical role of gestational timing, indicated that differential methylation extended along pathways involved in growth, development and metabolism, and suggested associations with birth weight and serum LDL cholesterol in adulthood and confirmed an enhancer function for the INSR and CPT1A P-DMRs. Our results point toward a link between prenatal malnutrition and epigenetic modulation of growth and lipid metabolism that may underlie the adverse metabolic phenotype of exposed individuals in later life.

  Study EGAS00001000668

• Sporadic and endemic Burkitt lymphoma have frequent FOXO1 mutations but distinct hotspots in the AKT recognition motif

  FOXO1 has an oncogenic role in adult germinal center derived lymphomas, in which mutations, predominately within the AKT recognition motif, cause nuclear retention of FOXO1 resulting in increased cell proliferation. To determine the prevalence and distribution of FOXO1 mutations in pediatric Burkitt lymphoma (BL), we sequenced a large number of sporadic and endemic BL patient samples. We report a high frequency of FOXO1 mutations in both sporadic and endemic BL at diagnosis, occurring in 23/78 (29%) and 48/89 (54%) samples respectively, as well as 8/16 (50%) cases at relapse. Mutations of T24 were the most common in sporadic BL but were rare in endemic cases, in which mutations of residue S22, also within the AKT recognition motif, were the most frequent. FOXO1 mutations were almost always present in the major tumor cell clone but were not associated with outcome. Analysis of other recurrent mutations reported in BL revealed that FOXO1 mutations were associated with mutations of DDX3X and ARID1A, but not MYC, TCF3/ID3 or members of the phosphatidylinositol 3’ OH kinase (PI3K) signaling pathway. We further show common nuclear retention of the FOXO1 protein, irrespective of mutation status, suggesting alternative unknown mechanisms for maintaining FOXO1 transcriptional activity in BL. CRISPR/Cas9 knockout of FOXO1 in an endemic cell line produced a significant decrease in cell proliferation, supporting an oncogenic role for FOXO1 in endemic BL. Thus, FOXO1 is frequently mutated in both sporadic and endemic BL and may offer a potential therapeutic target for pediatric BL patients worldwide.

  Study EGAS00001003719

• MYO5B mutations in Pheochromocytoma/Paraganglioma promote cancer progression

  Identification of additional cancer-associated genes and secondary mutations driving the metastatic progression in pheochromocytoma and paraganglioma (PPGL) is important. We recently reported novel recurrent nonsynonymous mutations in the MYO5B gene in metastatic PPGL. Here, we explored the functional impact of these MYO5B mutations, and analyzed MYO5B expression in primary PPGL tumor cases in relation to mutation status.Immunohistochemistry and mRNA expression analysis in 30 primary PPGL tumors revealed an increased MYO5B expression in metastatic compared to non-metastatic tumor cases. In addition, subcellular localization of MYO5B protein was altered from cytoplasmatic to membranous in some metastatic tumors, and the strongest and most abnormal expression pattern was observed in a PGL case harboring a MYO5B:p.G1611S mutation. Mutation analysis of additional PPGL tumors revealed additional, and hence recurrent, mutations in the paralog MYO5A. Three missense mutations in MYO5B were functionally studied using site directed mutagenesis and stable transfection into human neuroblastoma cells (SK-N-AS) and embryonic kidney cells (HEK293 cell line). Functional analysis of the mutants verified a significant increased proliferation rate in all MYO5B mutated clones (p.L587P, p.G1611S, and p.R1641C). The two somatically derived mutations p.L587P and p.G1611S were also found to increase the migration rate, and all three mutants affected intracellular transport by increased transferrin uptake. Downstream expression analysis of MYO5B mutants demonstrated a transcriptional activation of genes involved in glucose metabolism, proliferation, homeostasis, and migration. Our study validates the functional role of novel MYO5B mutations in proliferation and migration, and suggest the MYO5-pathway to be involved the malignant progression of PPGL tumors.

  Study EGAS00001003991

• Open_Targets_020_Epigenomes_of_Cell_Lines

  There is currently a drive to establish cell based assay systems of greater human biological and disease relevance through the use of well characterised transformed cell lines, primary cells and complex cellular models (e.g. co-culture, 3D models). However, although the field is gaining valuable experience in running more non-standard & complex cell assays for target validation and compound pharmacology studies, there is the lack of a systematic approach to determine if this expansion in cell assay models is reflected in increased human biological and disease relevance. The increasing wealth of publically available transcriptomic, and epigenome (ENCODE and Epigenome Roadmap) data represents an ideal reference mechanism for determining the relationship between cell types used for target & compound studies to primary human cells and tissues from both healthy volunteers & patients. The CTTV020 epigenomes of cell line project aims to generate epigenetic and transcriptomic profiles of cell lines and compare these with existing and newly generated reference data sets from human tissue and cell types. The aim is to identify assay systems which will provide greater confidence in translating target biology and compound pharmacology to patients. Multiple cell types commonly used within research have been grouped according to biology. Examples include erythroid, lung epithelial, hepatocyte cell types and immortalised models of monocyte / macrophage biology. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/

  Study EGAS00001003137

• Open_Targets_020_Epigenomes_of_Cell_Lines

  There is currently a drive to establish cell based assay systems of greater human biological and disease relevance through the use of well characterised transformed cell lines, primary cells and complex cellular models (e.g. co-culture, 3D models). However, although the field is gaining valuable experience in running more non-standard & complex cell assays for target validation and compound pharmacology studies, there is the lack of a systematic approach to determine if this expansion in cell assay models is reflected in increased human biological and disease relevance. The increasing wealth of publically available transcriptomic, and epigenome (ENCODE and Epigenome Roadmap) data represents an ideal reference mechanism for determining the relationship between cell types used for target & compound studies to primary human cells and tissues from both healthy volunteers & patients. The CTTV020 epigenomes of cell line project aims to generate epigenetic and transcriptomic profiles of cell lines and compare these with existing and newly generated reference data sets from human tissue and cell types. The aim is to identify assay systems which will provide greater confidence in translating target biology and compound pharmacology to patients. Multiple cell types commonly used within research have been grouped according to biology. Examples include erythroid, lung epithelial, hepatocyte cell types and immortalised models of monocyte / macrophage biology. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/

  Study EGAS00001003139

• The University of Hong Kong Intestinal Metaplasia Organoids Study

  Background: Gastric intestinal metaplasia (IM) is a pre-cancerous stage spanning a morphological spectrum that is poorly represented by human cell line models. Objective: We aim to establish and characterize human IM cell models to better understand IM progression along the cancer spectrum. Design: A large human gastric IM organoid (IMO) cohort (n=28), their clonal derivatives and normal gastric organoids (n=42) for comparison were established. Comprehensive multi-omics profiling and functional characterization were performed. Results: Single-cell transcriptomes revealed IMO cells spanning a spectrum from hybrid gastric/intestinal to advanced intestinal differentiation. Their lineage trajectories connected different cycling and quiescent stem and progenitors, highlighting differences in gastric to IM transition and the potential origin of IM from STMN1 cycling isthmus stem cells. Hybrid IMOs showed impaired differentiation potential, high lineage plasticity beyond gastric or intestinal fates, and reactivation of a fetal gene program. Cell populations in gastric IM and cancer tissues were highly similar to those derived from IMOs and exhibited a fetal signature. Genomically, IMOs showed elevated mutation burden, frequent chromosome 20 gain, and epigenetic de-regulation of many intestinal and gastric genes. Functionally, IMOs were FGF10 independent and showed downregulated FGFR2. Several IMOs exhibited a cell-matrix adhesion independent (CMi) subpopulation that displayed chromosome 20 gain but lacked key cancer driver mutations, potentially representing the earliest neoplastic precursor of IM-induced gastric cancer. Conclusions: Overall, our IM organoid biobank captured the heterogeneous nature of IM, revealing mechanistic insights on IM pathogenesis and progression, offering an ideal platform for studying early gastric neoplastic transformation and chemoprevention.

  Study EGAS00001007899

• Duplexseq of the interstrand crosslinks_WGS

  One of the most dangerous forms of DNA damage are interstrand crosslinks (ICLs), which covalently crosslink the two strands of the DNA double helix. The repair of these lesions is crucial for cellular survival due to their ability to block transcription and DNA replication. Initially, the major pathway that has been described in ICL repair involves a network of 22 genes that are mutated in a severe human genetic disease known as Fanconi Anemia (FA). Using synthetic lethality screens in the near-haploid human HAP1 cell line, we recently identified two potentially novel regulators of ICL repair, C1orf112 and THAP12. Loss of C1orf112 and THAP12 causes hypersensitivity to ICL-inducing DNA damaging agents, such as Mitomycin C (MMC). Additionally, C1orf112-depleted cells show elevated levels of micronuclei and accumulation of DNA damage in S-phase. To better understand how C1orf112 and THAP12 mediate the repair of ICLs, we want to perform mutational signature analysis, using the BotSeq method. Therefore, WT, C1orf112 and THAP12 knockout cells were cultured in vehicle or MMC treated conditions for 10 days and the genomic DNA was isolated. FANCA and FANCD2 knockout cells are taken along as controls in this experimental setting. . This dataset contains all the data available for this study on 2023-04-20.

  Dataset EGAD00001010298

• Characterization of mutational signatures in human cancer cell lines reveals episodic APOBEC mutagenesis

  Multiple signatures of somatic mutations have been identified in human cancer genomes. To investigate whether mutational signatures continue to be generated, and if so their temporal patterns of activity, subsets of cell lines were cultured in vitro for extended periods and subjected to single cell cloning and whole genome or exome sequencing or directly to single cell whole genome sequencing. As expected, signatures of past exogenous exposures, such as tobacco smoke and ultraviolet light, were not generated in vitro. In contrast, signatures of normal and defective DNA repair and replication continued to be generated at essentially constant mutation rates. Signatures of APOBEC cytidine deaminase DNA-editing activity exhibited a distinctive pattern with substantial fluctuations in mutation rate over time and episodic bursts of mutations. The initiating factors for these bursts are unclear although retrotransposon mobilisation may play a role. This cell line set now constitutes a comprehensive resource of live experimental models of mutational processes of both known and unknown aetiologies potentially retaining the patterns of activity and regulatory influences operative in human cells in vivo.

  Dataset EGAD00001004201

• Successful Clinical Response in Pneumonia Therapy (SCRIPT)

  The goal of this study is to iteratively identify, validate and refine biomarkers to predict clinical failure, and identify novel targets for therapy, beyond traditional antibiotics, for patients with severe pneumonia requiring intubation, including severe community-acquired pneumonia (CAP), hospital-acquired pneumonia, and ventilator-associated pneumonia (HAP/VAP). We hypothesize that factors within the alveolar microenvironment in many patients with pneumonia prevent the active process of pneumonia resolution.The major emphasis will be on HAP. Mechanically ventilated CAP patients are a very high-risk group for subsequent VAP, and therefore CAP samples will be included to determine baseline bacteria, microbiome, and host defense patterns for those that do or do not develop HAP/VAP. Hospital-acquired pneumonia is the leading cause of death from nosocomial infections. Even in patients receiving pathogen-appropriate therapy, reported clinical failure rates are increasing, approaching 50% in recent clinical trials. Current approaches to improve outcomes in patients with pneumonia almost exclusively focus on the development of antibiotics to address the problem of resistance. However, the prevalence of antibiotic-resistant pathogens is insufficient to explain the high rates of clinical failure. Instead, we hypothesize that factors within the alveolar microenvironment in many patients with pneumonia prevent the active process of pneumonia resolution. These factors include pathogen-specific adaptations to the injured lung that modulate the innate and adaptive immune response, as well as changes to the alveolar microbiome, driven by selective pressure within our modern intensive care units. Understanding this complex adaptive system requires the integration of clinical information with unbiased measures of the host, pathogen, and microbiome changes within the alveolar microenvironment. These measures must be repeated over time to identify factors that distinguish successful from failed therapy for pneumonia sufficiently early during the clinical course to allow for effective interventions. To address this challenge, we have assembled a talented group of investigators in the Successful Clinical Response In Pneumonia Therapy (SCRIPT) Systems Biology Center. We will leverage our routine clinical practice of safe alveolar sampling in mechanically ventilated patients with pneumonia with repeated non-bronchoscopic bronchoalveolar lavage (NBBAL) sampling over the course of pneumonia. From this fluid, we will combine flow cytometry with multi-omic technologies (cell population-specific transcriptomics, epigenomics, shotgun microbiome sequencing, and pathogen-specific sequencing). Our systems scientists will integrate these genomic data with robust clinical -omics with the goal of identifying biomarkers that can be prospectively tested in patients and causally evaluated in humanized mouse models.The addition of a pooled sample artificially increases the number of automatically computed subjects by one.

  Study phs002300

• Development of cell lines and mouse models of bone and soft tissue sarcoma to establish novel treatment

  Malignant giant-cell tumors are extremely rare bone sarcomas that transform from conventional giant-cell tumors during long periods of treatment. We have applied the culture methods and succeeded in establishing patient-derived organoids (PDO) of rare sarcomas. This study aimed to investigate the genomic characteristics of our established novel organoids from human malignant giant-cell tumors. The developed tumors with xenograft were genomically analyzed to compare their characteristics with those of the original tumors. Genetic changes over time throughout treatment were analyzed, and the genomic status of the established organoid was confirmed. Next generation sequencing revealed that the established organoids lacked the H3-3A G34W mutation. The xenografted organoids of the malignant giant-cell tumor had phenotypes genetically similar to those of the original tumor. The established organoids were confirmed to be derived from human malignant giant-cell tumors. In summary, the present study demonstrated a novel organoid model of a malignant giant-cell tumor that was genetically confirmed to be a malignant transformed tumor. Our organoid model could be used to elucidate the molecular pathogenesis of a malignant giant-cell tumor and develop novel treatment modalities.

  Study JGAS000618