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• 300BCG study ATAC-seq data: human population variation of trained immunity

  ATAC-seq dataset for the paper: Title Multi-omics analysis of human population variation in immune function and in vivo response to BCG vaccination Abstract Immune responses are tightly regulated, yet highly variable between individuals. To investigate human population variation of trained immunity, we immunized healthy individuals with Bacillus Calmette-Guérin (BCG). This live attenuated vaccine induces not only an adaptive immune response against tu-berculosis, but also triggers innate immune activation and memory. We established personal immune profiles and chromatin accessibility maps over a time course of BCG vaccination in 323 individuals. This large resource uncovered genetic and epigenetic predictors of baseline immunity and BCG vaccine response. We found that BCG vaccination enhances the innate immune response only in individuals with dormant immune states at baseline, suggesting that exogeneous induction of trained immunity is not a universal booster of innate immunity, but specifically elevates weak innate immune responses. This study advances our understanding of BCG’s heterologous immune-stimulatory effects and trained immunity in humans. Moreover, our results highlight the value of epigenetic cell states as an “endo-phenotype” that connects immune function with genotype and the environment.

  Dataset EGAD50000000123

• Genomic Characterization of Patient-Derived Xenograft Models to Improve Targeted Therapy for HER2+ Breast Cancer Brain Metastases Treatments

  We have developed orthotopic patient-derived xenograft models of HER2 positive breast cancer metastasized into the brain of patients to test novel therapeutic strategies. In this study, we identified a novel combinatorial therapeutic strategy that has resulted in a durable remission and markedly increased overall survival in majority of patient-derived xenograft (PDX) models tested. We performed whole exome sequencing analysis of these PDX tumors and their matched blood and patient samples to investigate drug sensitive and resistance mechanisms. Our sequencing data revealed an interesting association of genotyping and phenotyping with tumors responses to drug treatment.

  Study phs001063

• A whole-genome sequencing study for evolutionary history of Tibetans and their genetic adaptations to high altitude

  Due to a unique adaptation to high altitude, the Tibetan Plateau population has been the subject of much research interest. In this study, we conducted whole genome sequencing of 27 Tibetan individuals. Through our analysis, we inferred a detailed history of demography and revealed the natural selection of Tibetan population. We provided evidence of genetic separation between the two subpopulations of Han and Tibetans as early as 44 to 58 thousand years ago, replicated previously reported high altitude adaptation genes, including EPAS1 and EGLN1, and reported three new candidate genes, including PTGIS, VDR, and KCTD12.

  Study phs001338

• Distinct immune cell infiltration patterns in pancreatic ductal adenocarcinoma (PDAC) exhibit divergent immune cell selection and immunosuppressive mechanisms

  Pancreatic ductal adenocarcinoma has a dismal prognosis. A comprehensive analysis of single-cell multi-omic data from matched tumour-infiltrated CD45+ cells and peripheral blood in 12 patients, and two published datasets, reveals a complex immune infiltrate. Patients have either a myeloid-enriched or adaptive-enriched tumour microenvironment. Lymphocyte enrichment is intrinsically linked with highly distinct B and T cell clonal selection, diversification, and differentiation. Using TCR data, we see the largest clonal expansions in CD8 effector memory, senescent cells, and highly activated regulatory T cells which are induced within the tumour from naïve cells. We identify pathways that potentially lead to a suppressive microenvironement, including targets of immunotherapy and checkpoints including TIGIT/PVR and SIRPA/CD47. PDAC patient tumors from the APACT clinical trial revealed that patients with myeloid enrichment had the shorter overall survival compared to those adaptive cell enrichment. We identify multiple therapeutic targets that should form the basis for rational design including boosting of B cell responses, targeting immunosuppressive macrophages, and specific Treg depletion approaches. The dataset is scRNA-seq data from PDAC patients.

  Dac EGAC50000000319

• M116 scRNA-seq

  scRNA-seq was performed using Chromium Next GEM Single Cell 3’ Kit v3.1 (10X Genomics) according to the manufacturer’s instructions. Briefly, M116's PBMCs were thawed and quantified in order to calculate the number of cells to be loaded. Then, barcoded Single Cell 3ʹ Gel Beads, a master mix containing PBMCs, and Partitioning Oil were combined onto the Chromium Next GEM Chip G to generate Gel Beads-In-Emulsion (GEMs), where polyadenylated mRNAs were reverse transcribed. This way, all generated cDNAs from the same cell shared a common 10X Barcode. Following the reverse transcription, GEMs were broken and cDNAs were amplified and cleaned up with SPRIselect beads (Beckman Coulter®). Next, a portion of these cDNAs were enzymatically fragmented and subjected to adaptor ligation before using them as a PCR template for the incorporation of i5/i7 indexes. Finally, libraries were purified with SPRIselect beads, quantified and quality checked by using the 2200 TapeStation (Agilent®), and subjected to paired-end 150-bp sequencing (Novaseq systems, Illumina®) following standard practices at external facilities (Novogene®). Files types included in this dataset are raw Fastq files.

  Dataset EGAD50000001289

• High Sensitivity ctDNA Analysis Using a Novel Panel and NOIR-SS Technology for Monitoring Advanced Urothelial Carcinoma

  This dataset contains sequencing data generated by targeted amplicon analysis using the NOIR-SS method in 15 patients with advanced urothelial carcinoma. Formalin-fixed paraffin-embedded (FFPE) tumor tissues (15 samples) and plasma cfDNA (50 samples) were analyzed. Tumor tissues were subjected to targeted panel sequencing to identify mutations in TP53, FGFR3, TERT, and KRAS/HRAS. Plasma cfDNA was analyzed using a tumor-informed approach focusing on the mutations identified in matched tumor tissues. Sequencing was performed on the Ion PGM or Ion S5 XL platform. Raw sequencing data are provided in FASTQ format under controlled access.

  Study JGAS000836

• Research for candidate genes of splenic epidermoid cyst

  Splenic epidermoid cyst is a very rare benign tumor-like lesion filled with fluid or semi-fluid material. It occurs mostly in 2nd or 3rd decades of life with some female dominance. Most cases are asymptomatic and the clinical symptoms are dependent on the size of the cyst. Exome re-sequencing is an efficient strategy to selectively sequence the protein-coding regions of the genome, and has increasingly been used to identify novel genetic variants underlying Mendelian diseases. In this study, exome re-sequencing of 8 Japanese patients with splenic epidermoid cyst were conducted.

  Study JGAS000008

• Integrative understanding of human immune system by functional genomics and development of intervention strategies for the prevention of autoimmune diseases

  To elucidate the regulation of B cell recptor repertoire in B cell subsets and its contribution to autoimmune diseases, B cell subsets from 36 Systemic Lupus Erythematosus(SLE), 90 Systemic Sclerosis(SSc), 85 Myositis(IIM), 21 Mixed Connective Tissue Disease(MCTD), 18 Sjogren's syndrome(SjS), 27 Rheumatoid Arthritis(RA), 23 Behcet's disease(BD), 18 Adult Onset Still���s Disease(AOSD), 24 ANCA-associated Vasculitis(AAV), 16 Takayasu���s Arteritis(TAK) and 137 healthy controls(HC) were collected (Naive_B, USM_B, SM_B, DN_B, Plasmablast. RNA-seq and BCR-mapping was performed with each B cell subset samples.

  Study JGAS000485

• cfDNA analysis reveals relation of POLR1D amplification to bevacizumab resistance in colorectal cancer patients

  Bevacizumab is an approved anti-angiogenic drug for patients with metastasized colorectal cancer (mCRC) targeting VEGF. The survival benefit of anti-VEGF therapy in mCRC patients is limited to a few months and acquired resistance mechanisms are greatly unknown. Using plasma DNA, we studied the evolution of tumor genomes in a cohort of patients with mCRC (n=150) and observed a recurrent focal amplification (8.7% of cases) on chromosome 13q12.2. Analysis of TCGA data (n=619) suggested an association with later stages, which we confirmed by longitudinal plasma analyses. We defined the minimally amplified region and studied the mechanistic consequences of copy number gain of the involved genes. The amplification of one gene, POLR1D, impacted cell proliferation, resulting in upregulation of VEGFA, an important regulator of angiogenesis which has been implicated in the resistance to bevacizumab. In several patients, we observed the emergence of this 13q12.2 amplicon under bevacizumab treatment, which was invariably associated with evolution of therapy resistance. Hence, we describe a novel resistance mechanism against a widely applied treatment in mCRC patients which will impact clinical management .

  Dataset EGAD00001005761

• Genetic landscape of SMM

  Identifying high risk smoldering myeloma patients and progression mechanism is a prerequisite to implement effective inception strategies and curve myeloma related morbidity and mortality. We hypothesize that genomics may help identify determinants of progression that may help predict outcome and offer effective chemo preventive targets. Eighty-two patients underwent a custom targeted panel sequencing with an additional capture for the translocation loci. These results were compared to 223 newly diagnosed patients (EGAS00001003223 and EGAD00001004117) and 17 MGUS and 10 early myeloma patients. DNA was obtained from either CD138+ cells from the bone marrow of multiple myeloma patients (tumor) or from stem cell harvests or peripheral blood cells from the same patient (control). 100 ng of DNA was fragmented, end-repaired, and adapters ligated using the HyperPlus kit (KAPA Biosystems). After PCR amplification the libraries were hybridized with probes against either a targeted panel consisting of 140 genes and chromosomal regions (Nimblegen) using SeqCap reagents (Nimblegen). Hybridized libraries underwent further amplification before being sequenced on a NextSeq500 (Illumina) using 75 bp paired end reads. Overall, these data highlight the importance of dysregulation of the MAPK pathway in the progression to MM.

  Dataset EGAD00001005285

• The Precision Medicine in Liver Cancer across an Asia-pacific NETwork (PLANET) - Intratumoural immune heterogeneity

  The clinical relevance of immune landscape intratumoural heterogeneity (immune-ITH) and its role in tumour evolution remain largely unexplored. Here, we uncover significant spatial and phenotypic immune–ITH from multiple tumour sectors and decipher its relationship with tumour evolution and disease progression in hepatocellular carcinomas (HCC). Immune–ITH is associated with tumour transcriptomic-ITH, mutational burden, and distinct immune microenvironments. Tumours with low immune–ITH experience higher immunoselective pressure and escape via loss of heterozygosity in human leukocyte antigens and immunoediting. Instead, the tumours with high immune-ITH evolve to a more immunosuppressive/exhausted microenvironment. This gradient of immune pressure along with immune-ITH represents a hallmark of tumour evolution, which is closely linked to the transcriptome-immune networks that contributes to disease progression and immune inactivation. Remarkably, high immune-ITH and its transcriptomic signature are predictive for worse clinical outcome in HCC patients. This in-depth investigation of ITH provides evidence on tumour-immune co-evolution along HCC progression.

  Dataset EGAD00001006570

• Hypertension delays viral clearance and exacerbates airway hyperinflammation in patients with COVID-19

  In Coronavirus Disease 2019 (COVID-19), hypertension and cardiovascular diseases are major risk factors for critical disease progression. However, the underlying reasons and the effect of the main anti-hypertensive therapies—angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs)—remain unclear. Combining clinical data (n = 144) and single-cell sequencing data of airway samples (n = 48) with in vitro experiments, we observed a distinct inflammatory predisposition of immune cells in patients with hypertension that correlated with critical disease progression. ACEI treatment associated with dampened COVID-19-related hyperinflammation and with increased cell intrinsic anti-viral responses, whereas ARB treatment related to enhanced epithelial–immune cell interactions. Macrophages and neutrophils of patients with hypertension, in particular under ARB treatment, exhibited higher expression of the pro-inflammatory cytokines CCL3 and CCL4 and the chemokine receptor CCR1. Although the limited size of our cohort does not allow us to establish clinical efficacy, our data suggest that the clinical benefits of ACEI treatment in patients with COVID-19 who have hypertension warrant further investigation.

  Dataset EGAD00001006828

• Assessment of epigenetic variation in human iPS cells-Medip

  n order to progress human induced pluripotent stem cells (hiPSCs) towards the clinic, several outstanding questions must be addressed. It is possible to reprogram different somatic cell types into hiPSCs and from studies in the mouse, it appears that an epigenetic memory of the starting cell type is carried over to hiPSCs. However a comprehensive comparative study of the characteristics of these hiPSCs has been missing from the literature. Importantly studies which aimed to address these aspects of hiPSCs have used cells from different patients. In order to avoid this important confounding variable and to keep the genetic background constant, tissue samples were procured from the patients and reprogrammed to iPS cells. The methylation status of these iPS cells will be compared. Protocol: Primary cell cultures were generated and reprogrammed to iPS cells. DNA was extracted and immunoprecipitated using anti-methyl cytosine and anti-hydroxymethyl cytosine antibodies. 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/

  Dataset EGAD00001000827

• The mutational landscape of human somatic and germline cells

  During the course of a lifetime normal human cells accumulate mutations. Here, using multiple samples from the same individuals we compared the mutational landscape in 29 anatomical structures from soma and the germline. Two ubiquitous mutational signatures, SBS1 and SBS5/40, accounted for the majority of acquired mutations in most cell types but their absolute and relative contributions varied substantially. SBS18, potentially reflecting oxidative damage, and several additional signatures attributed to exogenous and endogenous exposures contributed mutations to subsets of cell types. The mutation rate was lowest in spermatogonia, the stem cell from which sperm are generated and from which most genetic variation in the human population is thought to originate. This was due to low rates of ubiquitous mutation processes and may be partially attributable to a low cell division rate of basal spermatogonia. The results provide important insights into how mutational processes affect the soma and germline.

  Dataset EGAD00001006641

• Tobacco exposure and somatic mutations in normal human bronchial epithelium

  That tobacco smoking causes lung cancer is well-established, but we lack quantitative understanding of its effects on genomes of normal bronchial epithelium. We sequenced whole genomes of 632 colonies derived from single bronchial epithelial cells across 16 subjects. Tobacco smoking is the major influence on mutation burden, adding 1000-10,000+ mutations/cell, massively increasing both between-subject and within-subject variance, and generating several distinct signatures of substitutions and indels. A population of cells in subjects with smoking history had mutation burdens equivalent to that expected for never-smokers: these cells lacked tobacco-specific mutational signatures, were four-fold more frequent in ex-smokers than current smokers, and had significantly longer telomeres than their more mutated counterparts. Driver mutations increased in frequency with age, affecting 4-14% of cells in middle-aged never-smokers. In current smokers, ≥25% of cells carried driver mutations and 0-6% cells had 2 or even 3 drivers. Thus, tobacco smoking increases mutation burden, cell-to-cell heterogeneity and driver mutations, but quitting promotes replenishment of bronchial epithelium from mitotically quiescent cells that have avoided tobacco mutagenesis.

  Dataset EGAD00001005193

• CBD-RAW-SC-GEX: 10X Single-Cell Gene Expression

  Raw Illumina sequencing data and CellRanger BAM output files. For further information regarding this dataset, please contact Stephen Sansom at contact@combat.ox.ac.uk.

  Dataset EGAD00001008007

• BLUEPRINT: Epigenetic programming during monocyte to macrophage differentiation and trained innate immunity

  Monocyte differentiation into macrophages represents a cornerstone process for host defense. Concomitantly, immunological imprinting of either tolerance or trained immunity determines the functional fate of macrophages and susceptibility to secondary infections. Transcriptomes (RNA-Seq) and epigenomes (ChIP-Seq H3K4me1,H3K4me3,H3K27ac) in four primary cell types: monocytes, in vitro differentiated naive, tolerized and trained macrophages were characterized. Inflammatory and metabolic pathways were modulated in macrophages, including decreased inflammasome activation, and pathways functionally implicated in trained immunity were identified. Strikingly, B-glucan training elicits an exclusive epigenetic signature, revealing a complex network of enhancers and promoters. Analysis of transcription factor motifs in DNase I hypersensitive sites at cell-type specific epigenetic loci unveiled differentiation and treatment specific repertoires. Altogether, this study provides a resource to understand the epigenetic changes that underlie innate immunity in humans.

  Dataset EGAD00001001011

• Multiomic characterization of clonotypic B cells

  DNA was purified using AMPure XP magnetic beads (Beckman Coulter [BC] Life Sciences, Indianapolis, IN) and its quality was evaluated in an Agilent 4200 Tapestation using the Genomic DNA ScreenTape system (Agilent, Santa Clara, CA). DNA was quantified using the Qubit System (Invitrogen). Extracted DNA from AL patients was fragmented to 200bp by a Covaris S220 ultrasonicator (Covaris, Woburn, MA) and used to generate and capture libraries following the manufacturer’s indications from Twist Bioscience’s Human Core Exome Enrichment Kit (Twist Bioscience). Extracted DNA from MM patients and HA was fragmented to an average size of 225 bp using a Covaris S220 ultrasonicator (Covaris, Woburn, MA) and subjected to DNA library construction using Chromium Genome Reagent Kit V2 for Exome Assays (10X Genomics). Target enrichment was performed with SureSelectXT Human All Exon V6 Capture Library (Agilent) or SureSelectXT2 Human All Exon V5+UTRs (Agilent), and sequence targets were captured and amplified in accordance with the manufacturer’s protocol. Final libraries were combined with standard Illumina sequencing primers and paired-end sequenced (150bp) on a HiSeq X platform (Illumina, CA) or in a NovaSeq 6000 (Illumina).

  Dataset EGAD50000002162

• Spatial and temporal intra-tumoural heterogeneity in advanced High-Grade Serous Ovarian Cancer: implications for surgical and clinical outcomes

  Limited evidence exists on the extent and impact of spatial and temporal heterogeneity of high grade serous ovarian cancer (HGSOC) on tumour evolution and patients surgical and clinical outcome. We investigated this through systematic mapping of multi-site tumours at initial presentation and matched relapse from 49 chemo-naïve HGSOC patients with high tumour load, operated upfront. Our data provides a unique insight into the tumour evolution and metastatic pathways of HGSOC across time and space, how this complexity relates to surgical and clinical outcome and its consequences on clinical decision-making.

  Study EGAS00001007164

• Personalized Onco-Genomic Project for pediatric and adolescent patients in British Columbia

  This is an extension of the BC Cancer Agency's Personalized Onco-Genomics (POG) project to children and adolescents treated at BC Children's Hospital. The project provides access to whole genome and transcriptome sequencing, and integrated bioinformatics analyses, of relapsed, refractory or poor prognosis cancers with an aim to identify actionable variants that can inform clinical decision making.

  Study EGAS00001006967

• T-cell reconstitution after reduced dose ATLG induction in kidney transplant patients

  We analyzed the T-cell receptor (TCR) repertoires from ten kidney transplant recipients. Five out of the ten kidney transplant recipients received ATLG while the other five recipients received basiliximab as induction therapy. TCR repertoires of CD4+ and CD8+ positive T-cells were assessed prior to transplantation and within the first month after transplantation as well as at three- and 12-months post-transplant. In addition, the pre-formed alloreactive TCR repertoire for each kidney transplant recipient was identified using mixed lymphocyte reaction and donor reactive T-cells were subjected to TCR beta sequencing. This dataset comprises a total of 106 samples. NGS TCR beta libraries of all samples were sequenced on an Illumina NextSeq 500 and raw sequencing data (in the form of fastq files) as well assembled clonotypes and their counts (in the form of clonotype tables) are provided.

  Dataset EGAD00001008478

• WES and RNA-seq of triple-negative breast cancers from the MyBrCa cohort

  The dataset contains a genomics characterization of 35 triple-negative Asian breast tumours from the Malaysian Breast Cancer cohort. This includes whole-exome sequencing of tumour tissue at 80X, whole-exome sequencing of matched normal (blood) tissue at 40X, and RNA-seq of tumour tissue at 40X coverage (>15 million reads). Whole-exome libraries were prepared using the Nextera Rapid Capture Exome Kit; exome capture was performed in pools of 3 and subjected to paired end 75 sequencing on a NovaSeq 6000 platform. RNA libraries were prepared using the TruSeq Stranded Total RNA HT kit with Ribo-Zero Gold as per manufacturer’s instructions and also subjected to paired end 75 sequencing on a NovaSeq 6000 platform. Uploaded bam files have been mapped to the hs37d5 human genome and processed using the standard GATK pipelines. Paired clinical, demographic, genotyping, and overall survival data for these patients are available from the associated publications or by request.

  Dataset EGAD00001009311

• Somatic mutation and clonal evolution normal breast tissue WGS

  Cancer is a genetic disease caused by an accumulation of mutations, however many of these mutations have been identified in pathologically normal tissue. We aim to use laser-capture microscopy (LCM) to sample individual clones from breast tissue to identify whether cancer-associated mutations appear in this normal tissue, assess the mutational burden present, and identify the mutational processes causing these mutations. We will sample from a wide age range of individuals (<20 to >70 years old) to determine whether these processes differ in pre- and post-menopausal women. We will also be comparing the tissue from healthy individuals (samples from breast reduction surgery) to those at elevated risk of breast cancer (mastectomy from BRCA1/2 patients) and those who have breast cancer (adjacent normal, distal normal, and tumour tissue from mastectomy). This will allow us to determine how these processes are different between these groups of individuals, and gain insight into the earliest stages of tumour development. . This dataset contains all the data available for this study on 2023-03-09.

  Dataset EGAD00001010123

• Alloreactive T-cell receptor (TCR) repertoire in kidney transplantation

  We analyzed the T-cell receptor (TCR) repertoires from twelve kidney transplant recipients. Six out of the twelve kidney transplant recipients experienced a cellular rejection after kidney transplantation. TCR repertoires of CD4+ and CD8+ positive T-cells were assessed prior to transplantation and after transplantation at time of allograft biopsy using RNA based T-cell receptor beta next generation sequencing (NGS). In addition, the pre-formed alloreactive TCR repertoire for each kidney transplant recipient was identified using mixed lymphocyte reaction and donor reactive T-cells were subjected to TCR beta sequencing. In two out of the six patients with cellular rejection the TCR repertoire of graft infiltrating T-cells was additionally captured. This dataset comprises a total of 98 samples. NGS TCR beta libraries of all samples were sequenced on an Illumina NextSeq 500 and raw sequencing data (in the form of fastq files) as well assembled clonotypes and their counts (in the form of clonotype tables) are provided.

  Dataset EGAD00001007695

• Dataset for other_cancer-RNA

  Rare cancer sequencing data of 95 runs in tumor/control pairs, which were uploaded to umbrella studies. The sequencing was always paired

  Dataset EGAD00001008847

• Somatic mutant selection is altered by prior NOTCH1 mutation in ageing esophageal epithelium

  Authors: Charlotte King1, Emilie Abbie1, Joanna C. Fowler1, Irina Abnizova1, Roshan K. Sood1, Swee Hoe Ong1, Michael W. J. Hall1,2, Faye Lynch-Williams3, Benjamin A. Hall4, Philip H. Jones1,2,5 Abstract: In cancer evolution, genome alterations often occur in a specific order, implying selection depends on the prior clonal genotype 1-3. It is unknown if similar constraints operate in normal epithelia. Here, we mapped mutations in normal mid-esophagus of aged UK subjects. Mutant NOTCH1 clones colonized most of the epithelium by age 60 and above 70 tissue was saturated with mutants under strong competitive selection. Mutant TP53 was more strongly selected in donors over 60 years of age. Samples predominantly mutant for NOTCH1 showed increased selection of NOTCH2 mutants and weaker selection of mutant TP53 compared with samples that were mostly NOTCH1 wild type. In mouse esophagus lacking Notch1 we observed strong selection of mutant Notch2 and other genes not selected in wild type esophagus. In normal ageing esophagus, the first driver mutation may change the trajectory of subsequent somatic evolution by altering mutant selection.

  Dataset EGAD00001015259

• Organoid Derivation Project: WGS (2023-06-22)

  Sequencing of tissue samples and their derived organoids from oesophageal, pancreatic and colorectal cancer patients. . This dataset contains all the data available for this study on 2023-06-22.

  Dataset EGAD00001011090

• Neuroblastoma patient Total RNA Seq data

  Tumor Total RNA Seq data of primary neuroblastomas. This is an update of the „Berlin Neuroblastoma Dataset” (EGAS00001004022). This data was used for the analysis of circular RNA expression and regulation in neuroblastoma.

  Dataset EGAD00001008124

• Myeloid cell programming in patients with non-medullary thyroid carcinoma

  This dataset contains single-cell RNA sequencing data from patients with thyroid cancer (n=7), multinodal Goiter (n=3) and healthy individuals (n=5). Mononuclear cells were taken from both the peripheral blood and the bone marrow compartments. We used a pooled single-cell design where multiple individuals were pooled in a single sample for sequencing (NextSeq 500-V2) and later demultiplexed using their genotype data. Associated metadata contains information on the phenotypes per individual, the pooling design and the linkage between the supplied files and sequenced pools. Due to limitations from EGA in uploading single-cell data, the raw fastq files were processed as follows: (i) I1/I2/R1/R2 fast files were concatenated over the different lanes. (ii) Concatenated I1 and I2 files were interleaved, as were the concatenated R1 and R2 files to generate two fastq files per pool containing all the information. To interleave the fastq files, the BBmap tool bbmap/reformat.sh was used, which can also be used to de-interleave the files.

  Dataset EGAD00001008108

• Transcriptome analysis for intrahepatic cholangiocarcinoma

  Intrahepatic cholangiocarcinomas (iCCs) are characterized by their rarity, difficulty in diagnosis, and overall poor prognosis. We performed comprehensive transcriptomic characterization of treatment-naive iCC. Whole transcriptome analyses identified two prognostic subtypes, concordant with previous reports.The findings could assist in patient stratification with iCCs and in developing rational therapeutic strategies.

  Dataset EGAD00001008544

• Somatic mutant selection is altered by prior NOTCH1 mutation in ageing esophageal epithelium

  Authors: Charlotte King1, Emilie Abbie1, Joanna C. Fowler1, Irina Abnizova1, Roshan K. Sood1, Swee Hoe Ong1, Michael W. J. Hall1,2, Faye Lynch-Williams3, Benjamin A. Hall4, Philip H. Jones1,2,5 Abstract: In cancer evolution, genome alterations often occur in a specific order, implying selection depends on the prior clonal genotype 1-3. It is unknown if similar constraints operate in normal epithelia. Here, we mapped mutations in normal mid-esophagus of aged UK subjects. Mutant NOTCH1 clones colonized most of the epithelium by age 60 and above 70 tissue was saturated with mutants under strong competitive selection. Mutant TP53 was more strongly selected in donors over 60 years of age. Samples predominantly mutant for NOTCH1 showed increased selection of NOTCH2 mutants and weaker selection of mutant TP53 compared with samples that were mostly NOTCH1 wild type. In mouse esophagus lacking Notch1 we observed strong selection of mutant Notch2 and other genes not selected in wild type esophagus. In normal ageing esophagus, the first driver mutation may change the trajectory of subsequent somatic evolution by altering mutant selection.

  Dataset EGAD00001015260

• Somatic mutant selection is altered by prior NOTCH1 mutation in ageing esophageal epithelium

  Authors: Charlotte King1, Emilie Abbie1, Joanna C. Fowler1, Irina Abnizova1, Roshan K. Sood1, Swee Hoe Ong1, Michael W. J. Hall1,2, Faye Lynch-Williams3, Benjamin A. Hall4, Philip H. Jones1,2,5 Abstract: In cancer evolution, genome alterations often occur in a specific order, implying selection depends on the prior clonal genotype 1-3. It is unknown if similar constraints operate in normal epithelia. Here, we mapped mutations in normal mid-esophagus of aged UK subjects. Mutant NOTCH1 clones colonized most of the epithelium by age 60 and above 70 tissue was saturated with mutants under strong competitive selection. Mutant TP53 was more strongly selected in donors over 60 years of age. Samples predominantly mutant for NOTCH1 showed increased selection of NOTCH2 mutants and weaker selection of mutant TP53 compared with samples that were mostly NOTCH1 wild type. In mouse esophagus lacking Notch1 we observed strong selection of mutant Notch2 and other genes not selected in wild type esophagus. In normal ageing esophagus, the first driver mutation may change the trajectory of subsequent somatic evolution by altering mutant selection.

  Dataset EGAD00001015257

• Whole blood RNA-sequencing of covid-19 patients and healthy controls

  This dataset contains whole blood transcriptome data generated from 93 patients with COVID-19 across a range of severities and 23 healthy controls. All patients were PCR positive for SARS-CoV-2 and disease severity ranged from asymptomatic to severe disease requiring ventilation. Individuals without symptoms, or with mild symptoms, were recruited from routine screening of healthcare workers, while COVID-19 patients were recruited at or soon after admission to Addenbrooke’s or Royal Papworth hospitals. Blood samples were taken at recruitment and then again four weeks later. Further details of the cohort and the generation of the RNA-Sequencing data can be obtained from Bergamaschi, L. et al. Longitudinal analysis reveals that delayed bystander CD8+ T cell activation and early immune pathology distinguish severe COVID-19 from mild disease. Immunity 54, 1257-1275 e8 (2021).

  Dataset EGAD00001007776

• Somatic mutant selection is altered by prior NOTCH1 mutation in ageing esophageal epithelium

  Authors: Charlotte King1, Emilie Abbie1, Joanna C. Fowler1, Irina Abnizova1, Roshan K. Sood1, Swee Hoe Ong1, Michael W. J. Hall1,2, Faye Lynch-Williams3, Benjamin A. Hall4, Philip H. Jones1,2,5 Abstract: In cancer evolution, genome alterations often occur in a specific order, implying selection depends on the prior clonal genotype 1-3. It is unknown if similar constraints operate in normal epithelia. Here, we mapped mutations in normal mid-esophagus of aged UK subjects. Mutant NOTCH1 clones colonized most of the epithelium by age 60 and above 70 tissue was saturated with mutants under strong competitive selection. Mutant TP53 was more strongly selected in donors over 60 years of age. Samples predominantly mutant for NOTCH1 showed increased selection of NOTCH2 mutants and weaker selection of mutant TP53 compared with samples that were mostly NOTCH1 wild type. In mouse esophagus lacking Notch1 we observed strong selection of mutant Notch2 and other genes not selected in wild type esophagus. In normal ageing esophagus, the first driver mutation may change the trajectory of subsequent somatic evolution by altering mutant selection.

  Dataset EGAD00001015256

• Somatic mutant selection is altered by prior NOTCH1 mutation in ageing esophageal epithelium

  Authors: Charlotte King1, Emilie Abbie1, Joanna C. Fowler1, Irina Abnizova1, Roshan K. Sood1, Swee Hoe Ong1, Michael W. J. Hall1,2, Faye Lynch-Williams3, Benjamin A. Hall4, Philip H. Jones1,2,5 Abstract: In cancer evolution, genome alterations often occur in a specific order, implying selection depends on the prior clonal genotype 1-3. It is unknown if similar constraints operate in normal epithelia. Here, we mapped mutations in normal mid-esophagus of aged UK subjects. Mutant NOTCH1 clones colonized most of the epithelium by age 60 and above 70 tissue was saturated with mutants under strong competitive selection. Mutant TP53 was more strongly selected in donors over 60 years of age. Samples predominantly mutant for NOTCH1 showed increased selection of NOTCH2 mutants and weaker selection of mutant TP53 compared with samples that were mostly NOTCH1 wild type. In mouse esophagus lacking Notch1 we observed strong selection of mutant Notch2 and other genes not selected in wild type esophagus. In normal ageing esophagus, the first driver mutation may change the trajectory of subsequent somatic evolution by altering mutant selection.

  Dataset EGAD00001015258

• Somatic mutation and clonal evolution normal breast tissue TGS (2020-01-15)

  Cancer is a genetic disease caused by an accumulation of mutations, however many of these mutations have been identified in pathologically normal tissue. We aim to use laser-capture microscopy (LCM) to sample individual clones from breast tissue to identify whether cancer-associated mutations appear in this normal tissue, assess the mutational burden present, and identify the mutational processes causing these mutations. We will sample from a wide age range of individuals (<20 to >70 years old) to determine whether these processes differ in pre- and post-menopausal women. We will also be comparing the tissue from healthy individuals (samples from breast reduction surgery) to those at elevated risk of breast cancer (mastectomy from BRCA1/2 patients) and those who have breast cancer (adjacent normal, distal normal, and tumour tissue from mastectomy). This will allow us to determine how these processes are different between these groups of individuals, and gain insight into the earliest stages of tumour development. . This dataset contains all the data available for this study on 2020-01-15.

  Dataset EGAD00001005787

• Cryopreservation of human cancers conserves tumour heterogeneity for single-cell multi-omics analysis

  Single-cell RNA-Sequencing of three primary breast cancers, two primary prostate cancers, and a metastatic melanoma sample from Wu et al. (2021) Genome Medicine study. Each tumour was sequenced across different cryopreservation conditions including Fresh Tissue (FT), cryopreserved single-cell suspensions (CCS), cryopreserved solid tissue fragments (CT) and a cryopreserved after overnight cold storage (CO). Data was generated using the Chromium controller (10X Genomics) and sequenced on the NextSeq platform.

  Dataset EGAD00001007030

• Public Access Defibrillation Community Trial (PAD)(PAD-BioLINCC)

  Data Access NOTE: Please refer to the “Authorized Access” section below for information about how access to the data from this accession differs from many other dbGaP accessions. Objectives The Public Access Defibrillation (PAD) Community Trial sought to evaluate broad implementation of Public Access Defibrillators (PAD) in urban community units. Survival to hospital discharge of participants with out-of-hospital cardiac arrest was the main outcome measure. Survival was compared in community units (e.g., apartment or office buildings, gated communities, sports venues, senior centers, shopping malls) served by non-medical responders trained in CPR and use of automated external defibrillators (AEDs), to units receiving the traditional optimum community standard of care (i.e., rescuers trained to recognize a cardiac emergency, call 911, and initiate CPR). Background Sudden out-of-hospital cardiac arrest (OOH-CA) remains a significant cause of death, in spite of recent declines in overall mortality from cardiovascular disease. Existing methods of emergency resuscitation are inadequate due to time delays inherent in transporting trained responders with defibrillation capabilities to the side of the OOH-CA victim. Existing Emergency Medical Services (EMS) systems typically combine paramedic Emergency Medical Technician (EMT) services with some level of community involvement, such as bystander cardiopulmonary resuscitation (CPR) training. Some communities include automated external defibrillators (AEDs) at isolated sites or in mobile police or fire vehicles. Such an approach typically varies in effectiveness, with an incremental improvement in effectiveness seen in communities that organize and integrate services with the existing EMS system. However, optimal improvement in survival from sudden OOH-CA may require a program that utilizes volunteer non-medical responders (who may not have a traditional duty to respond to an emergency) trained to use AEDs. Participants The PAD trial was a prospective, randomized community based trial. More than 19,000 volunteer responders from 993 community units in 24 North American regions participated. The two study arms had similar unit and volunteer characteristics. Participants with treated out-of-hospital cardiac arrest in the two groups were similar in age (mean: 69.8 years), proportion of men (67 perecnt), rate of cardiac arrest in a public location (70 percent), and rate of witnessed cardiac arrest (72 percent). Conclusions Community units with volunteers trained in CPR and AEDs had significantly more participants surviving to hospital discharge than units with volunteers trained to use CPR only. There were 30 survivors among 128 definite cardiac arrests in the CPR+AED units and 15 survivors among 107 definite cardiac arrests in the CPR only units (p = 0.03). Serious adverse effects were rarely reported. No volunteers received inadvertent shocks, and no participants were shocked unnecessarily. AED maintenance problems were infrequent. A few participating volunteers reported severe stress related to responding to emergency situations. Although residential complexes represented 16% of the units and 29% of the treatable cardiac arrests, only 5% of the survivors were from residential complexes. Such information should be helpful for individual facilities that are considering implementing PAD programs. (NEJM 2004; 351:637-46).

  Study phs003858

• Acute Respiratory Distress Network (ARDSNet) Studies 10 and 12 Statins for Acutely Injured Lungs from Sepsis (SAILS) (ARDSNet-SAILS-BioLINCC)

  Data Access NOTE: Please refer to the “Authorized Access” section below for information about how access to the data from this accession differs from many other dbGaP accessions. Biospecimens Access to Biospecimens is through the NHLBI Biologic Specimen and Data Repository Information Coordinating Center (BioLINCC). Biospecimens from ARDSNet-SAILS include Plasma, DNA, and Urine. Please note that use of biospecimens in genetic research is subject to a tiered consent. Objectives The SAILS trial was intended to assess the efficacy and safety of oral rosuvastatin in participants with sepsis-induced Acute Lung Injury (ALI) and test the hypothesis that rosuvastatin therapy would improve the clinical outcomes of critically ill participants with sepsis-associated acute respiratory distress syndrome (ARDS). Background In ARDS, inflammation in the lungs and other organs can cause life-threatening organ failure. Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) can modulate inflammatory responses. Previous observational studies suggested that statins improved clinical outcomes in participants with sepsis. Participants There were 745 participants. Design Participants were randomly assigned in permuted blocks to receive either enteral rosuvastatin or placebo. A 40 mg loading dose of the study drug was administered within four hours after randomization. Subsequently, maintenance doses of 20 mg were administered daily until the third day after discharge from the intensive care unit, study day 28, hospital discharge, or death, whichever came first. The primary outcome measure was mortality before hospital discharge home or until study day 60 if the participant was still in a health care facility. Secondary outcome measures included the number of ventilator-free days to day 28, organ-failure-free days to day 14, and ICU-free days to day 28. Conclusions The study was stopped because of futility after 745 of an estimated 1000 participants had been enrolled. There was no significant difference between study groups in 60 day in-hospital mortality or in mean ventilator-free days. The rosuvastatin group had fewer days free of hepatic or renal failure. Thus, rosuvastatin therapy did not improve clinical outcomes in participants with sepsis-associated ARDS and may have contributed to hepatic and renal organ dysfunction.

  Study phs003736

• Whole Genome Sequencing and Variant Calling for Autism Families

  We extracted DNA from whole blood or lymphoblast-derived cell lines and assessed the DNA quality with PicoGreenTM and gel electrophoresis. Whole genome sequencing was performed (Illumina HiSeq2000 and Illumina HiSeq X). WGS reads were mapped to the human reference genome assembly hg19 (GRCh37) using Burrows-Wheeler Aligner v.0.7.12 (TCAG) or Isaac v.2.0.13 (Macrogen). For each genome, we performed local realignment and quality recalibration and detected SNVs and small indels using GATK Haplotype Caller v.3.4.6 without genotype refinement. We detected CNVs using ERDS (estimation by read depth with single nucleotide variants) and CNVnator. We detected structural variants using Manta v.0.29.6. When available by the variant caller (i.e. GATK and Manta), trio-based joint variant calling was conducted for each family.

  Dataset EGAD00001008452

• The patterns and dynamics of genomic instability in metastatic pancreatic cancer

  Pancreatic cancer is an aggressive malignancy with a five-year mortality of 97–98%, usually due to widespread metastatic disease. Previous studies indicate that this disease has a complex genomic landscape, with frequent copy number changes and point mutations, but genomic rearrangements have not been characterized in detail. Despite the clinical importance of metastasis, there remain fundamental questions about the clonal structures of metastatic tumours, including phylogenetic relationships among metastases, the scale of ongoing parallel evolution in metastatic and primary sites, and how the tumour disseminates. Here we harness advances in DNA sequencing to annotate genomic rearrangements in 13 patients with pancreatic cancer and explore clonal relationships among metastases. We find that pancreatic cancer acquires rearrangements indicative of telomere dysfunction and abnormal cell-cycle control, namely dysregulated G1-to-S-phase transition with intact G2–M checkpoint. These initiate amplification of cancer genes and occur predominantly in early cancer development rather than the later stages of the disease. Genomic instability frequently persists after cancer dissemination, resulting in ongoing, parallel and even convergent evolution among different metastases. We find evidence that there is genetic heterogeneity among metastasis-initiating cells, that seeding metastasis may require driver mutations beyond those required for primary tumours, and that phylogenetic trees across metastases show organ-specific branches. These data attest to the richness of genetic variation in cancer, brought about by the tandem forces of genomic instability and evolutionary selection.

  Study EGAS00000000064

• Characterizing the Role of the Immune Microenvironment in Multiple Myeloma Progression at a Single Cell Level

  The plasma cell disease multiple myeloma (MM) is highly dependent on its bone marrow microenvironment (BMME). Early alterations within the bone marrow microenvironment that contribute to the progression of multiple myeloma (MM) from its precursor stages could hold the key to identifying novel therapeutic approaches, yet the intrinsic variability in cellular populations between patients together with differences in sample processing and analysis methods have made it difficult to identify consistent changes between datasets. Here, we used single-cell RNA sequencing of BM cells from precursor stages, monoclonal gammopathy of unknown significance (MGUS), smoldering MM (SMM) and newly diagnosed MM and analyzed our data in combination with a previously published dataset that used a similar patient population and sample processing. Despite vast interpatient heterogeneity, some alterations were seen consistently in both datasets. We identified changes in immune cell populations as disease progressed that were characterized by a substantial decrease in memory and naïve CD4 T cells and an increase in CD8+ effector T cells and T-regulatory cells. These alterations were further accompanied by an enrichment of non-clonal memory B cells and an increase of CD14 and CD16 monocytes in MM compared to its precursor stages. These results provide crucial information of the immune changes associated with the progression to clinical MM and can help to develop immune based strategies for patient stratification and early therapeutic intervention. 

  Study phs002756

• Multimodal Immune Profiling to Determine Mechanisms of COVID-19 Clinical Trajectory in Uganda

  We conducted a prospective cohort study of adults (RESERVE-U-C19) admitted to Entebbe Regional Referral Hospital (ERRH), a national COVID-19 referral center, across three viral variant-driven phases of the pandemic in Uganda. In this cohort, which reflects the entire spectrum of SARS-CoV-2 infection, we integrated clinical data with serum proteomics (N=306) and whole-blood transcriptomics (N=100) to determine a range of host responses associated with COVID-19 severity, SARS-CoV-2/HIV co-infection, and circulating viral variants. Beyond the prognostic importance of myeloid cell-driven immune activation and lymphopenia, we show that multifaceted impairment of host protein synthesis and extensive redox imbalance define core metabolic signatures of severe COVID-19, with central roles for IL-7, IL-15, and lymphotoxin-α in COVID-19 respiratory failure. While prognostic signatures were generally consistent in SARS-CoV-2/HIV-coinfection, type I interferon responses uniquely scaled with COVID-19 severity in persons living with HIV. Throughout the pandemic, COVID-19 severity peaked during phases dominated by A.23/A.23.1 and Delta B.1.617.2/AY variants, with Delta phase COVID-19 distinguished by exaggerated pro-inflammatory myeloid cell and inflammasome activation, NK and CD8+ T-cell depletion, impaired host protein synthesis, and upregulation of viral cell entry and trafficking pathways. Data from this study available through dbGaP include raw RNA-Seq data from whole-blood of adult participants (N=100).

  Study phs003246

• NIMH (National Institute of Mental Health) De Novo Mutation Identification in Taiwanese Schizophrenia Trios

  The substantial reproductive impact of schizophrenia, for which affected individuals have fewer than half as many offspring as unaffected individuals do, implies that mutations of largest effect will frequently be de novo mutations. Ascertaining exome sequence variation in father-mother-offspring trios allows such mutations to be identified and distinguished from the far-larger amount of rare variation that is inherited by each individual. The pursuit of this approach in a large, well-powered cohort of trios can also provide lessons that inform the development of such gene discovery strategies more generally in human genetics. Schizophrenia trios from the Taiwanese population are being collected by Dr. Ming Tsuang (PI, UC San Diego, California) and investigators in Taiwan (PI, Dr. Hai Gwo Hwu; both funded by NIMH grant 1R01MH085560; Expanding Rapid Ascertainment Networks of Schizophrenia Families in Taiwan). A total of 3800 trios are anticipated to be collected by May 2013. This represents a highly homogenous national sample from the same ancestral population. DNA samples will be obtained from the NIMH Repository, Rutgers University Cell and DNA Repository (described below) and stored at the Broad Institute. Genetic and data analyses will be performed at the Broad Institute. We propose to sequence the whole exome of trios by hybrid capture and Illumina next generation sequencing and perform targeted genotyping and validation of variants (SNPs, indels and CNVs) using several molecular methods, to include emulsion-based PCR and Sanger sequencing.

  Study phs001196

• Genome and Transcriptome Assembly Reveals SVA-Mediated Aberrant Splicing in X-Linked Dystonia Parkinsonism

  X-linked Dystonia-Parkinsonism (XDP, DYT3) is a long-standing quandary in human disease genetics. XDP is predominantly observed on Panay island in the Philippines. This study is one of the first of its kind to interrogate an unsolved Mendelian disorder by integrating genome and transcriptome assembly methods using Illumina, 10X Genomics, Pacific Biosciences, and Agilent genome targeting technologies. These data provide strong evidence for a pathogenic link between a noncoding SVA retrotransposon and XDP. We demonstrate that this Mendelian disorder is associated with a sine-VNTR-Alu (SVA) retrotransposon that inserted into the TAF1 gene and is shared by all XDP probands, yet never observed in controls from worldwide populations. Transcriptome assembly in iPSC-derived neural stem cells (NSCs) and neurons revealed that this SVA caused aberrant splicing and significant intron retention, which was negatively correlated with TAF1 expression. Remarkably, CRISPR/Cas9 excision of the SVA rescued the aberrant transcriptional signature and normalized expression of TAF1 in patient-derived NSCs. We have also interrogated iPSC-derived microglia to investigate the contribution of glial components to the cellular and molecular deficit, effect of TNF treatment on XDP transcriptional signatures. To further understand what XDP may have in common with other hereditary dystonias (such as DYT6), we are further comparing these signatures to ones associated with dystonia-specific variants in proteins that, like TAF1, are also involved in regulating transcription, i.e. THAP1. 

  Study phs001525

• National Institute on Alcohol Abuse and Alcoholism (NIAAA) Postmortem Prefrontal Cortex eQTL and mQTL Study

  Mapping expression and methylation quantitative trait loci (eQTLs and mQTLs) using genome-wide genotype, transcriptome, and DNA methylome data can provide an unbiased approach to identify novel functional variants. One critical issue of the eQTL/mQTL analysis is that the role of genetic variants in regulating gene expression (or DNA methylation) can be tissue- or cell-specific. To unravel causal variants from disease-associated loci, it would be necessary to map eQTLs and mQTLs in both patients and controls. Those eQTLs and mQTLs in patients are more likely to be causal variants for the disease. Identification of disease- and tissue-specific eQTLs and mQTLs will help disentangle the role of the specific tissue in disease development as well. In the present study, we mapped eQTLs and mQTLs in postmortem prefrontal cortex of patients with alcoholism or alcohol use disorder (AUD) and matched control subjects by integrative analyses of genome-wide SNP genotype, mRNA transcriptome, and DNA methylome data. As AUD is comorbid with schizophrenia (SCZ), bipolar disorder (BIP), major depressive disorder (MDD), and attention deficit/hyperactivity disorder (ADHD), we further examined the co-localization of PFC eQTLs/mQTLs and GWAS-identified SNPs (mainly noncoding SNPs) that were associated with AUD as well as comorbid SCZ, BIP, MDD, and ADHD. Our study demonstrated the enrichment of PFC eQTLs/mQTLs in the above psychiatric disorder-associated genetic variants identified by genome-wide association studies (GWAS).

  Study phs001981

• Gabriella Miller Kids First Pediatric Research Program in Craniofacial Microsomia

  The Gabriella Miller Kids First Pediatric Research Program (Kids First) is a trans-NIH effort initiated in response to the 2014 Gabriella Miller Kids First Research Act and supported by the NIH Common Fund. This program focuses on gene discovery in pediatric cancers and structural birth defects and the development of the Gabriella Miller Kids First Pediatric Data Resource (Kids First Data Resource). All of the WGS and phenotypic data from this study are accessible through dbGaP and kidsfirstdrc.org, where other Kids First datasets can also be accessed. Craniofacial microsomia (CFM), also termed hemifacial microsomia or oculo-auricular-vertebral spectrum, is the third most common congenital craniofacial condition. CFM comprises a variable phenotype and the most common features include malformations of the ear (i.e. microtia) and lower jaw (i.e. mandibular hypoplasia) on one or both sides. Microtia in the absence of other anomalies is believed to represent the mildest form of CFM. The cause of CFM is unknown for most affected individuals. We have established a cohort through previous studies and collected DNA to identify the genetic contributions to CFM, which could facilitate diagnosis, tailored treatments and guide prevention strategies. Successful completion of this proposal will advance knowledge in the genetic architecture of susceptibility to CFM and will provide insight about the biological mechanisms underlying craniofacial development. The results from this study have the potential to further research on the etiology of other craniofacial conditions, and the pathogenesis of typical and atypical craniofacial development.

  Study phs002130

• Genomic Analysis of Pediatric Low Grade Gliomas

  Pediatric low-grade gliomas (PLGGs) are among the most common solid tumors in children but, apart from mutations or duplications in the BRAF kinase in specific subclasses, few genetic driver events are known. Diffuse PLGGs compose a set of uncommon subtypes that exhibit invasive growth and are therefore especially challenging clinically. These tumors are particularly poorly understood. We performed high-resolution copy-number analysis of 44 diffuse PLGGs to identify recurrent alterations. Diffuse PLGGs exhibited fewer such alterations than adult low-grade gliomas, but we identified several significantly recurrent events. The most significant event, 8q13.1 gains, was observed in 28% of diffuse astrocytoma WHO grade II (DA2) and resulted in partial duplication of the transcription factor MYBL1 with truncation of its C-terminal negative-regulatory domain. A similar recurrent deletion-truncation breakpoint was identified in two angiocentric gliomas in the related gene MYB on 6q23.3. Whole genome sequencing of a MYBL1-rearranged diffuse astrocytoma grade II demonstrated MYBL1 tandem duplication and few other events. Two truncated MYBL1 transcripts identified in this tumor induced anchorage-independent growth when expressed in 3T3 cells and tumor formation in nude mice. Truncated transcripts were also expressed in two additional tumors with MYBL1 partial duplication. Our results define clinically relevant molecular subclasses of diffuse PLGGs and highlight a potential role for the MYB family in the biology of low-grade gliomas. "Reprinted from www.pnas.org/cgi/doi/10.1073/pnas.1300252110 with permission from PNAS."

  Study phs000614

• A New Pipeline to Predict and Confirm Tumor Neoantigens Predicts Better Response to Checkpoint Blockade

  Mutations that drive oncogenesis in cancer can generate neoantigens that may be recognized by the immune system. Identification of these neoantigens remains challenging due to the complexity of the major histocompatibility complex (MHC) antigen and T-cell receptor interaction. Here we describe the development of a systematic approach to efficiently identify and validate immunogenic neoantigens. Whole exome sequencing of tissue from a melanoma patient was used to identify nonsynonymous mutations, followed by MHC binding prediction and identification of tumor clonal architecture. The top 18 putative class I neoantigens were selected for immunogenicity testing via a novel in vitro pipeline in HLA-A201 healthy donor blood. Naive CD8 T cells from donors were stimulated with allogeneic dendritic cells pulsed with peptide pools and then with individual peptides. The presence of antigen-specific T cells was determined via functional assays. We identified one putative neoantigen that expanded T cells specific to the mutant form of the peptide and validated this pipeline in a subset of patients with bladder tumors treated with durvalumab (N = 5). Within this cohort, the top predicted neoantigens from all patients were immunogenic in vitro. Finally, we looked at overall survival in the whole durvalumab-treated bladder cohort (N = 37) by stratifying patients by tertile measure of tumor mutation burden (TMB) or neoantigen load. Patients with higher neoantigen and TMB load tended to show better overall survival.

  Study phs002269

• Tumor Mutation Burden, Expressed Neoantigen and Immune Microenvironment in Diffuse Gliomas

  A consistent correlation between tumor mutation burden (TMB) and tumor immune microenvironment has not been observed in gliomas as in other cancers. Driver germline and somatic mutations, TMB, neoantigen, and immune cell signatures were analyzed using whole exome sequencing (WES) and transcriptome sequencing of tumor and WES of matched germline DNA in a cohort of 66 glioma samples (44 IDH-mutant and 22 IDH-wildtype). Fourteen samples revealed a hypermutator phenotype (HMP). Eight pathogenic (P) or likely pathogenic (LP) germline variants were detected in 9 (19%) patients. Six of these 8 genes were DNA damage repair genes. P/LP germline variants were found in 22% of IDH-mutant gliomas and 12.5%of IDH-wildtype gliomas (p= 0.7). TMB was correlated with expressed neoantigen but showed an inverse correlation with immune score (R =−0.46, p= 0.03) in IDH-wildtype tumors and no correlation in IDH-mutant tumors. Antigen Processing and Presentation (APP) score correlates with immune score. The APP is higher in non-hypermutator phenotype (NHMP) samples versus HMP samples in the IDH-wildtype gliomas, but higher in HMP samples versus NHMP samples in the IDH-mutant gliomas. Conclusion: TMB was inversely correlated with the immune score in the IDH-wildtype gliomas, and showed no correlation in the IDH-mutant tumors. The APP was correlated with the immune score, and may be further investigated as a biomarker for response to immunotherapy in gliomas. Studies of germline variants in a large glioma cohort are warranted.

  Study phs002653

• Extent and Significance of Bacterial DNA Integrations in the Human Cancer Genome

  The integration of exogenous DNA into the human genome can cause somatic mutations associated with oncogenesis. For example, the insertion of HPV DNA into human chromosomes is the single most important event leading to tumorigenesis in cervical cancer. It is also now preventable with vaccines against HPV. In contrast to viral DNA integrations, the instances and repercussions of bacterial DNA integration into the somatic human genome are less clear. Yet bacterial DNA integrations (BDI) found to be associated with tumorigenesis could also be prevented using therapeutics like vaccines that limit exposure to the bacteria. Previous analysis of publicly available sequencing data from the Cancer Genome Atlas (TCGA) showed BDIs from Pseudomonas spp. rRNA into the 5'-UTR of four proto-oncogenes as well as in Ig (the immunoglobulin gene) of gastric cancer samples. The original samples from TCGA were not available to validate these findings. Therefore, a different cohort of gastric cancer patients was identified in order to establish the presence BDIs. Whole exome sequencing was completed on seven tumor samples and six adjacent gastric samples, as well as one intestinal metaplasia sample and one non-atrophic gastritis sample. Four of those samples were also investigated with transcriptomics and whole genome sequencing. Given that 15-20% of cancers worldwide are linked to infections, more work is needed to understand the role of infectious agents in oncogenesis and the mechanisms underlying that role.

  Study phs001936

• Medulloblastoma exome sequence analysis

  Medulloblastomas are the most common malignant brain tumors in children. Identifying and understanding the genetic events that drive these tumors is critical for the development of more effective diagnostic, prognostic and therapeutic strategies. Recently, our group and others described distinct molecular subtypes of medulloblastoma based on transcriptional and copy number profiles. Here, we utilized whole exome hybrid capture and Illumina sequencing to identify somatic mutations across the coding regions of 92 primary medulloblastoma/normal pairs. Overall, medulloblastomas exhibit low mutation rates consistent with other pediatric tumors, with a median of 0.35 non-silent mutations per megabase. We identified twelve genes mutated at statistically significant frequencies, including previously known mutated genes in medulloblastoma such as CTNNB1, PTCH1, MLL2, SMARCA4 and TP53. Recurrent somatic mutations were identified in an RNA helicase gene, DDX3X, often concurrent with CTNNB1 mutations, and in the nuclear co-repressor (N-CoR) complex genes GPS2, BCOR, and LDB1, to our knowledge novel findings in medulloblastoma and all cancer. We show that mutant DDX3X potentiates transactivation of a TCF promoter and enhances cell viability in combination with mutant but not wild type beta-catenin. Together, our study reveals the alteration of Wnt, Hedgehog, histone methyltransferase and now N-CoR pathways across medulloblastomas and nominates the RNA helicase DDX3X as a component of pathogenic beta-catenin signaling in medulloblastoma. "Reprinted from 'MEDULLOBLASTOMA EXOME SEQUENCING UNCOVERS SUBTYPE-SPECIFIC SOMATIC MUTATION', with permission from Nature"

  Study phs000504

• Molecular Characterization of Hemimegalencephaly

  Somatic mutations, in which a fraction of the cells in the body have a deleterious mutation, are well recognized in cancer but only recently appreciated in neurological disease. Given a somatic mutation in a population of progenitor cells, all daughter cells inherit the mutation and are able to express the resultant phenotype as a function of the differentiation program. We recently identified the first de novo somatic mutations in the developing brain in the condition hemimegalencephaly (HME), a catastrophic focal epilepsy condition associated with a malformation of cerebral cortical development (MCD). HME is one of the most severe MCD syndromes, characterized by massive hamartomatous overgrowth of either of the two cerebral hemispheres. Cerebral hemispherectomy is a frequent treatment for refractory epilepsy, allowing the sampling of diseased tissue. By comparing DNA from a diseased brain with DNA from blood/saliva, we identified de novo somatic mutations in PIK3CA, AKT3 or MTOR, part of the mTOR pathway. Mutations were present in 8-40% of sequenced alleles in various brain regions sampled during surgery and in some codons known to activate proteins.• Population informationPatient samples were collected from multiple centers. The population is Caucasian and Hispanic• Molecular technologies employedWhole-exome sequencing• Principal findings of the studyWe were the first to describe mTOR-related and non-mTOR mutations causing hemimegalencephaly, and we described two-hit mutational models to explain the genetic pathogenesis of hemimegalencephaly

  Study phs002156

• Single Cell Genotypic and Phenotypic Analysis of Measurable Residual Disease in Acute Myeloid Leukemia

  Acute myeloid leukemia (AML) is a group of blood cancers with high mortality. Even though complete remission is often achieved, majority of the AML patients still relapse. Measurable residual disease (MRD), defined as the population of cancer cells which persists following chemotherapy, is responsible for AML relapse. Understanding the biology enabling MRD clones to resist therapy is necessary to guide the development of curative treatments more effectively. Discriminating residual leukemic clones, preleukemic clones, and normal precursors remains a challenge with current MRD tools. We developed a single cell MRD (scMRD) assay by combining flow cytometric enrichment of the targeted precursor/blast population with integrated single cell DNA sequencing (scDNA-seq) and immunophenotyping (Tapestri, Missionbio). The platform utilizes a custom amplicon panel covering 109 amplicons across 31 genes frequently mutated in AML and a panel of 42 oligo-conjugated antibodies against surface makers. We applied our scMRD assay to sequence 30 MRD samples (multiplexed into 6) from 29 AML patients to study the clonal architecture and protein correlation. In addition, 2 MRD samples were sequenced with scDNA+protein sequencing technology without enrichment or multiplexing. Five patients with AML at diagnosis were also sequenced with scDNA+protein sequencing technology without enrichment or multiplexing. In order to establish the sensitivity of our scMRD assay, we spiked AML cells into normal bone marrow cells. We sequenced 12 such spiked samples after multiplexing.

  Study phs003233

• MILK-Omics: Systems Biology of Human Milk and Its Links to Maternal and Infant Health

  The primary objective of the project is to generate a systems-level view of human milk in the context of healthy mothers and their term infants. The study uses biospecimens and clinical data from the Mothers and Infants LinKed for Healthy Growth Study (MILk Study), a prospective observational cohort of approximately 500 exclusively breastfeeding women and their infants who are followed to 6 months of age. The project focuses on mature milk samples collected at 1 month postpartum and infant gut microbiome samples collected at 1 and 6 months of age. Specific Aim 1 is to identify maternal genetic and clinical factors that shape human milk gene expression. Using bulk and single-cell RNA sequencing of the milk cell pellet and genotypes from maternal DNA, we will identify novel genetic determinants of the milk transcriptome and assess potential modification of these genetic associations by gestational weight gain, diet, and other clinical factors. Specific Aim 2 is to describe key features of the normative human milk biosystem and their interactions with one another. Specifically, machine learning techniques will be used to characterize interaction networks and correlational structures among the following features of human milk: transcriptomics, microbiomes, oligosaccharides, metabolomics, lipidomics, and milk macronutrient composition. Specific Aim 3 is to establish how the milk biosystem is related to variation in infant gut microbiomes and health. dbGaP will house the maternal genotype and milk transcriptomics data.The infant microbiome sequence data are associated with BioProject PRJNA1019702.

  Study phs003408

• Neoadjuvant immune checkpoint blockade in women with mismatch repair deficient endometrial cancer

  Neoadjuvant immune checkpoint blockade (ICB) has shown unprecedented activity in mismatch repair deficient (MMRd) colorectal cancers, but its effectiveness in MMRd endometrial cancer (EC) remains unknown. In this investigator-driven, phase I, feasibility study (NCT04262089), 10 women with MMRd EC of any grade, planned for primary surgery, received two cycles of neoadjuvant pembrolizumab (200 mg IV) every three weeks. A pathologic response (primary objective) was observed in 5/10 patients, with 2 patients showing a major pathologic response. No patient achieved a complete pathologic response. A partial radiologic response (secondary objective) was observed in 3/10 patients, 5/10 patients had stable disease and 2/10 patients were non-evaluable on Magnetic Resonance Imaging (MRI). All patients completed treatment without severe toxicity (exploratory objective). At median duration of follow-up of 22.5 months, two non-responders experienced disease recurrence. In-depth analysis of the loco-regional and systemic immune response (predefined exploratory objective) showed that monoclonal T cell expansion significantly correlated with treatment response. Tumour-draining lymph nodes displayed clonal overlap with intra-tumoural T cell expansion. All pre-specified endpoints, efficacy in terms of pathologic response as primary endpoint, radiologic response as secondary outcome and safety and tolerability as exploratory endpoint, were reached. Neoadjuvant ICB with pembrolizumab proved safe and induced pathologic, radiologic, and immunologic responses in MMRd EC, warranting further exploration of extended neoadjuvant treatment.

  Study EGAS50000000483

• Diagnostic Genomic Analysis is Prognostic in AYA ALL Patients Treated on a MRD-Stratified Paediatric Protocol

  Background: Over the last decade, next generation sequencing has enabled classification of multiple new recurrent genomic drivers of acute lymphoblastic leukemia (ALL). We provide a comprehensive genomic survey of adolescent and young adult (AYA) patients treated on the Australasian Leukaemia and Lymphoma Group (ALLG) ALL06 study. ALL06 clinical trial described the efficacy and tolerability of AYA ALL patients aged 15-39 years treated on a single minimal residual disease (MRD)-stratified platform. In this study we have taken the ALL06 cohort and retrospectively performed comprehensive genomic analyses assessing response of AYA patients with various genomic subtypes treated with a paediatric approach. Methods: Genomic risk classification of B- and T-ALL patients (n=57) enrolled to the study was based on mRNA-Sequencing, MLPA, immunophenotyping and cytogenetics. Results: Using this approach, 36/40 (90%) B- and 13/17 (76.5%) T-ALL patients were classified according to genomic risk. We have linked outcome data with each patient’s genomic risk and found a strong correlation between adverse genomic risk and MRD at the end of consolidation, translating to inferior overall and relapse free survival. Patients with adverse risk genomics who achieved negative MRD status had improved responses compared to those with persistent MRD. Patients with standard risk genomics had excellent responses regardless of their MRD status. Conclusion: Our data argue strongly for incorporation of a genomic risk classification into future ALL treatment paradigms at the time of diagnosis.

  Study EGAS50000000752

• Ultra-sensitive tumor-informed ctDNA monitoring of treatment response in advanced esophagogastric cancer patients

  To explore whether ultra-sensitive circulating tumor DNA (ctDNA) profiling enables earlier prediction of treatment response and detection of disease progression, we applied NeXT Personal, an ultra-sensitive bespoke tumor-informed liquid biopsy platform, to profile tumor samples from the KeyLargo study, a phase II trial in which metastatic esophagogastric cancer (mEGC) patients received capecitabine, oxaliplatin, and pembrolizumab. A total of 24 patients were evaluated, and all were ctDNA-positive at baseline. ctDNA levels varied from 406,067 down to 1.5 parts per million (PPM) with a median limit of detection of 2.03 PPM. ctDNA dynamics were highly correlated with changes in tumor size (ρ = 0.59, p = 7.3×10-9). Lack of early molecular response (50% or greater decrease in ctDNA levels at first available time point after 30 days, C2D1 or C4D1) was associated with worse overall survival (OS) (HR 6.6, 95% CI 1.8-24.1, p = 0.005) and progression-free survival (PFS) (HR 15.4, 95% CI 2.7-87.0, p = 0.002). Lack of molecular clearance of ctDNA was associated with worse OS (HR 6.9, 95% CI 1.5-30.8, p = 0.012) and PFS (HR 19.2, 95% CI 2.4-152.8, p = 0.005). Molecular progression (ctDNA increase) preceded imaging-derived progression by a median lead time of 65 days. These results suggest that ultra-sensitive liquid biopsy approaches could improve treatment decision-making for mEGC patients receiving chemotherapy and immunotherapy.

  Study EGAS50000001224

• Longitudinal Multi-Omic Immune Resource Reveals Dynamic Responses in Healthy Human Aging

  The generation and maintenance of protective immunity is a dynamic interplay between host and environment that is impacted by age. Understanding fundamental changes in the healthy immune system that occur over a lifespan is critical in developing interventions for age-related susceptibility to infections and diseases. Here, we use multi-omic profiling (scRNA-seq, proteomics, flow cytometry) to examine human peripheral immunity in over 300 healthy adults, with 96 young and older adults followed over two years with yearly vaccination. The resulting resource includes scRNA-seq datasets of >16 million PBMCs, interrogating 71 immune cell subsets from our new Immune Health Atlas. This study allows unique insights into the composition and transcriptional state of immune cells at homeostasis, with vaccine perturbation, under Cytomegalovirus (CMV) infection and across age. We find that T cells specifically accumulate age-related transcriptional changes more than other immune cells, independent from inflammation and chronic perturbation. Moreover, impaired memory B cell responses to vaccination are linked to a Th2-like state shift in older adults' memory CD4 T cells, revealing possible mechanisms of immune dysregulation during healthy human aging. This extensive resource is provided with a suite of exploration tools at https://apps.allenimmunology.org/aifi/insights/dynamics-imm-health-age/ to enhance data accessibility and further the understanding of immune health across age. Please note that the Sound Life Cohort raw data are spread across three dbGap submissions: phs003841.v1 (this study), phs003944.v1, and phs003400.v1.

  Study phs003841

• Delineating pediatric brain tumor progression using single-nuclei sequencing

  Pediatric brain cancers are the most common solid tumors in children with varying survival rates depending on molecular features of cancer cells and associated microenvironment. Several studies have used single-cell RNA sequencing to define transcriptional programs active in cancer cells and highlighted supportive roles of non-cancerous cells embedded in these tumors. Although application of single-cell RNA sequencing technology has expanded our understanding of intratumoral heterogeneity and underlying transcriptional mechanisms in brain tumors at diagnosis, mechanisms of disease remains elusive. Therefore, the purpose of this proposal is to characterize cellular and transcriptional states of malignant and non-malignant compartments at primary diagnosis and at relapse. In this study, we performed single-nuclei sequencing of 42 patient samples taken at various disease stages (initial diagnosis, progressive disease, and relapsed disease) from 20 patients with low-grade glioma or Atypical Teratoid Rhabdoid Tumor (ATRT). Bulk whole genome and RNA-seq data for these samples are available from the Children’s Brain Tumor Network and the Gabriella Miller Kids First Data Resource Center. Findings from our study will help define the mechanistic shifts of tumor and non-cancerous cells over course of disease progression. Collectively with the single-cell Pediatric Cancer Atlas funded, this project lays the foundation of an important data set focused on lethal relapsed cancers that can continue to be expanded to identify cancer-intrinsic and extrinsic microenvironmental factors supporting recurrence of pediatric brain tumors.

  Study EGAS50000001288

• Space Associated Stem Cell Hallmarks of Aging in Astronauts

  This study examined the effects of the stress of space, specifically low earth orbit, on hematopoietic stem and progenitor cells (HSPCs) that maintain lifelong hematopoiesis and immune responses. Enriched CD34+ HSPCs were derived from 7 astronauts before, during and after 10 and 21-day ISS missions on the International Space Station. The samples were used as input to whole transcriptome sequencing by bulk RNA-Seq performed at The Scripps Research Institute Next Generation Sequencing Core on Illumina NextSeq 2000 sequencer and single cell RNA-Seq performed at The Salk Institute for Biological Studies on Illumina NovaSeq X Plus sequencer. Subsequent expression, RNA editing, and retrotransposon analyses were performed to assess HSPC and immune subpopulation survival dynamics. Overall, these analyses revealed space-associated stem cell hallmarks of aging (SASHA), including spaceflight dependent alterations in HSPC survival and self-renewal, ADAR1, telomere maintenance, mobilization and cell cycle gene expression, combined with space-induced clonal hematopoietic mutations, APOBEC3C activation and retrotransposon deregulation to warrant countermeasure development to enable long-duration spaceflight. Post-spaceflight analysis revealed increased C-to-T and clonal hematopoiesis-associated mutations, repetitive element and base deaminase deregulation, decreased telomere maintenance gene expression, and decreased self-renewal capacity compared to ground controls. These results demonstrate the space-associated aging effects in HSPCs after month-long exposure to LEO, providing insights into utilizing the LEO environment to model space-accelerated aging and other age-related malignancies.

  Study phs004267

• Genomic landscapes of endometrioid and mucinous ovarian cancers and morphologically similar tumor types

  While endometrioid and mucinous ovarian cancers represent nearly a quarter of ovarian cancers, their molecular characteristics and pathologic origins are poorly understood. To characterize the genomic and epigenomic alterations of these ovarian cancer subtypes and evaluate links to morphologically similar tumors from other sites, we performed sequence, copy number, mutation signature, rearrangement, and methylation analyses from tumor samples and matched normal tissues of 133 patients. These analyses included patients with ovarian endometrioid (n=44), ovarian mucinous (n=43), uterine endometrial (n=15), and gastrointestinal mucinous cancers (n=31). In addition to identifying genes previously known to be involved in these tumors, we identified alterations in RAD51C, NOTCH4, SMARCA1/4 and JAK1 in ovarian endometrioid, ESR1 in uterine endometrioid, and SMARCA4 in ovarian mucinous cancers. Whole genome sequencing revealed rearrangements involving PTEN, NF1, and NF2 in ovarian endometrioid tumors, and NF1 and MED1 in ovarian mucinous cancers. The number of alterations and affected genes in ovarian and uterine endometrioid cancers were similar as were genome-wide methylation profiles, suggesting that these tumors may have a similar tissue of origin. By contrast, methylation patterns in ovarian mucinous cancers were more similar to other gastrointestinal mucinous cancers, including mucinous cancers of the stomach, colon, and pancreas. These analyses provide insights into the genomic landscapes and origins of mucinous and endometrioid ovarian carcinomas, providing new avenues for early clinical intervention and management of patients with these cancers.

  Study EGAS50000000523

• CRISPR screening in human trophoblast stem cells reveals both shared and distinct aspects of human and mouse placental development

  The placenta serves as the interface between the mother and fetus, facilitating the exchange of gases and nutrients between their separate blood circulation systems. Trophoblasts in the placenta play a central role in this process. Our current understanding of mammalian trophoblast development relies largely on mouse models. However, given the diversification of mammalian placentas, findings from the mouse placenta cannot be readily extrapolated to other mammalian species, including humans. To fill this knowledge gap, we performed CRISPR knockout (KO) screening in human trophoblast stem cells (hTSCs). We targeted genes essential for mouse placental development and identified more than 100 genes as critical regulators in both human hTSCs and mouse placentas. Among them, we further characterized in detail two transcription factors, DLX3 and GCM1, and revealed their essential roles in hTSC differentiation. Moreover, a gene function-based comparison between human and mouse trophoblast subtypes suggests that their relationship may differ significantly from previous assumptions based on tissue localization or cellular function. Notably, our data reveal that hTSCs may not be analogous to mouse TSCs or the extraembryonic ectoderm (ExE) in which in vivo TSCs reside. Instead, hTSCs may be analogous to progenitor cells in the mouse ectoplacental cone and chorion. This finding is consistent with the absence of ExE-like structures during human placental development. Our data not only deepen our understanding of human trophoblast development but also facilitate cross-species comparison of mammalian placentas.

  Study JGAS000659

• Tumor-reactive heterotypic CD8 T cell clusters from clinical samples

  Emerging evidence suggests a correlation between CD8+ T cell-tumor cell proximity and anti-tumor immune response. However, it remains unknown whether these cells exist as functional clusters that can be isolated from clinical samples. By conventional and imaging flow cytometry, we show here that from 21/21 human melanoma metastases, we could isolate heterotypic clusters, comprising CD8+ T cells interacting with one or more tumor cells and/or antigen-presenting cells (APCs). Single-cell RNA-sequencing revealed that T cells from clusters were enriched for gene signatures associated with tumor reactivity and exhaustion. Clustered T cells exhibited increased TCR clonality indicative of expansion, while TCR-matched T cells showed more exhaustion and co-modulation when conjugated to APCs than to tumor cells. T cells that were expanded from clusters ex vivo exerted on average 9-fold increased killing activity towards autologous melanomas, which was accompanied by enhanced cytokine production. Upon adoptive cell transfer into mice, T cells from clusters showed improved patient-derived melanoma control, which was associated with increased T cell infiltration and activation. Together, these results demonstrate that tumor-reactive CD8+ T cells are enriched in functional clusters with tumor cells and/or APCs and that they can be isolated and expanded from clinical samples. Typically excluded by single cell gating in flow cytometry, these distinct heterotypic T cell clusters serve as a valuable source to decipher functional tumor-immune cell interactions, while they may also be therapeutically explored.

  Study EGAS50000000785

• Evolutionary origin and methylation status of human intronic CpG islands that are not present in mouse

  Imprinting of the human RB1 gene is due to the presence of a differentially methylated CpG island (CGI) in intron 2, which is part of a retrogene derived from the PPP1R26 gene on chromosome 9. The murine Rb1 gene does not have this retrogene and is not imprinted. We have investigated whether the RB1/Rb1 locus is unique with respect to these differences. For this we have compared the CGIs from human and mouse by in silico analyses and found out that there are at least 2033 human intronic CGIs that are not present in the mouse. Among these CGIs, 104 show sequences similarities elsewhere in the human genome, which suggests that they arose by retrotransposition. We could narrow down the time points when most of these CGIs appeared during evolution. Their methylation status was analysed in two monocyte methylome datasets from whole genome bisulfite sequencing and in 18 published methylomes. Five CGIs appear to be differentially methylated, which suggests that they are subject of genomic imprinting. They are located in introns of the following genes: RB1, ASRGL1, PARP11, PDXDC1 and MYO1D. Four of these CGIs are parts of retrogenes. Our study supports the notion that imprinting builds on host defence mechanisms by which the genome protects itself against retrotransposed and foreign DNA and that the epigenetic fate of the retrotransposed DNA depends on selective forces at the integration site.

  Study EGAS00001000719

• Genetic and epigenetic characterization of adenoid cystic carcinoma

  Translocation events are frequent in cancer and may create chimeric fusions or regulatory rearrangements that drive oncogene overexpression. Although regulatory rearrangements are increasingly recognized in hematopoietic and even solid tumors, the underlying mechanisms remain obscure. Here we identify super-enhancer translocations that drive overexpression of the oncogenic transcription factor MYB as a unifying theme in adenoid cystic carcinoma (ACC). Whole genome sequencing data and chromatin state maps for 13 primary tumors and xenografts reveal distinct chromosomal rearrangements that juxtapose super-enhancer clusters to the MYB locus. Chromosome conformation capture confirms that the translocated enhancers physically interact with the MYB promoter. Remarkably, the MYB product itself binds to the translocated enhancers, thus creating a positive feedback loop that sustains its own expression. MYB also binds a large number of active enhancers that drive opposing regulatory programs in alternate cell lineages in ACC. MYB cooperates with the transcription factor TP63 in the myoepithelial component of the tumors, but promotes a Notch program in the luminal epithelial component. Bromodomain inhibitors, which disrupt enhancer function, slow tumor growth in ACC primagraft models in vivo, but are ineffective against high grade tumors that harbor activating mutations in the Notch pathway. Thus, our study identifies super-enhancer translocations as a unifying feature of ACC, and provides insight into the mechanism by which sustained MYB overexpression drives alternate cell fates in this disease.Processed ChIP-seq data is available on GEO under accession number GSE76465.

  Study EGAS00001001457

• Tissue-specific mutation accumulation in human adult stem cells during life

  Gradual accumulation of mutations in human adult stem cells during life is associated with various age-related diseases, including cancer. The number of stem cell divisions throughout life is believed to be a major determinant for mutation accumulation and could explain the extreme variation of cancer incidence across different organs. Yet, mutation patterns and rates of healthy adult stem cells remain unknown. Here, we determined genome-wide mutation patterns in primary adult stem cells of the small intestine, colon and liver of human donors with ages ranging from 3 to 87 years. We find that the number of mutations increases linearly with age up to several thousand mutations per cell at 87 years of age, while mutation spectra remain constant throughout life. Small intestine and colon stem cells have a 2-fold higher mutation rate per year compared with liver stem cells. These differences could be exclusively attributed to the mutagenic action of spontaneous deamination of cytosine residues and may reflect the high stem cell division rate in these tissues. The genomic distribution of somatic mutations is non-random and predominantly associated with DNA replication dynamics in the small intestine and colon, and with transcription in the liver. These results indicate that a stable balance between various mutagenic and DNA repair processes is maintained throughout life and that the activity of these processes in adult stem cells varies between tissues.

  Study EGAS00001001682

• Genomic Alterations in Gingivo-buccal Cancer: ICGC-India Project_YR01

  As a part of the ICGC, India has undertaken genomic studies on gingivobuccal cancer of the oral cavity, which is the most prevalent form of cancer among men in India. There are various known environmental (life-style) correlates of this cancer, the most important of which are tobacco chewing and HPV infection. Paired DNA samples – isolated from the tumor tissue and from the blood of fifty patients – have been analyzed to catalog germline and somatic mutations. Association between each observed genomic alteration and exposure to environmental risk factors is being explored.Detailed clinical characterization of the patients, collection of data on demographic and environmental exposures, and isolation of DNA samples from blood and tumor tissues collected from each patient are being done at the Advanced Centre for Research, Treatment and Education on Cancer, Mumbai. Exome capture and deep resequencing are being performed at the National Institute of Biomedical Genomics, Kalyani. We have analyzed the exomes of paired blood and tumor DNA samples using two orthogonal platforms – exome capture by Roche-Nimblegen SeqCap Ez kit followed by sequencing on Roche-454 GS FLX, and exome capture by Illumina TrueSeq Exome Enrichment kit followed by sequencing on Illumina HiSeq-2000. Each exome is being sequenced at a mean depth of 30x. In addition, we have used Illumina Omni Quad SNP-chips to generate genotype data at 1.14 million genomic markers. The average concordance between SNP-chip and sequence data is 99.8%.

  Study EGAS00001000249

• Efficacy of JAK/STAT pathway inhibition in murine xenograft models of early T-cell precursor (ETP) acute lymphoblastic leukemia

  Early T-cell precursor (ETP) acute lymphoblastic leukemia (ALL) is a recently described subtype of T-ALL characterized by a unique immunophenotype and genomic profile as well as a high rate of induction failure. Frequent mutations in cytokine receptor and JAK/STAT signaling pathways led us to hypothesize that ETP-ALL is dependent on JAK/STAT signaling. Here we demonstrate aberrant activation of the JAK/STAT pathway in ETP-ALL blasts relative to non-ETP T-ALL. Moreover, ETP-ALL showed hyperactivation of STAT5 in response to IL7, an effect that was abrogated by the JAK1/2 inhibitor ruxolitinib. In vivo, ruxolitinib displayed activity in 6/6 patient-derived murine xenograft models of ETP-ALL, with profound single-agent efficacy in 5 models. Ruxolitinib treatment decreased peripheral blast counts relative to pre-treatment levels and compared to control (P<0.01) in 5/6 ETP-ALL xenografts, with marked reduction in mean splenic blast counts (P<0.01) in 6/6 samples. Surprisingly, both JAK/STAT pathway activation and ruxolitinib efficacy were independent of the presence of JAK/STAT pathway mutations, raising the possibility that the therapeutic potential of ruxolitinib in ETP-ALL extends beyond those cases with JAK mutations. These findings establish the preclinical in vivo efficacy of ruxolitinib in ETP-ALL, a biologically distinct subtype for which novel therapies are needed.

  Study EGAS00001001146

• Assessing mitochondrial bioenergetics in coronary artery disease: A translational left ventricular tissue study in humans (The AMBITION study).

  Severe or recurrent myocardial ischemia can lead to long-term left ventricular (LV) dysfunction and heart failure in patients with coronary artery disease (CAD). In this study, we assessed for chronic metabolomic and transcriptomic adaptations within LV myocardium of patients with evidence of preoperative inducible ischemia undergoing coronary artery bypass grafting (CABG). During surgery, paired transmural LV biopsies were acquired on the beating heart from a region of inducible ischemia (determined by stress perfusion cardiovascular magnetic resonance), and a remote LV segment. From 33 patients, 63 LV biopsies were acquired on the beating heart during CABG, comparing to analysis of LV samples from 11 donor hearts. The global myocardial ATP/ADP ratio was reduced in CAD patients as compared to donor LV tissue with increased expression of oxidative phosphorylation (OXPHOS) genes encoding the electron transport chain complexes across multiple cell types. Paired analyses of biopsies obtained during CABG from LV segments with or without inducible ischemia revealed no significant difference in the ATP/ADP ratio, broader metabolic profile or expression of ventricular cardiomyocyte genes implicated in OXPHOS. Finally, differential metabolite analysis suggested dysregulation of several intermediates in patients with impaired LVEF; this notably included the tricyclic acid cycle metabolite, succinate. Overall, our results suggest that viable human myocardium in patients with stable CAD has global alterations in bioenergetic and transcriptional profile without large regional differences between areas with or without inducible ischemia.

  Study EGAS00001007351

• Genetic variability in exon 1 of the glucocorticoid receptor gene NR3C1 is associated with postoperative complications

  Patients having major surgery experience inflammation post-operatively, which may contribute to post-operative morbidity. Endogenous glucocorticoids (GCs) are an essential part of the stress response, but this response varies between individuals, which may in turn affect clinical outcome in general, and post-operative inflammation, specifically. Exon 1 of the NR3C1 gene, encoding the glucocorticoid receptor, contains an established region of differential regulation. DNA methylation patterns in this region have been found to differ between individuals. We investigated the methylation status and genotype in the Cytosine-phosphate-Guanine (CpG) island in exon 1 of NR3C1 in 24 patients who underwent major abdominal surgery and correlated it with post-operative complications. DNA was extracted from peripheral blood leukocytes and underwent targeted bisulfite sequencing of the CpG island. Complications were graded according to the Clavien-Dindo classification and 14 out of 24 had post-operative complications. Multifactorial and partial least square methods were used to analyse the data. A homogenous demethylated pattern was observed in all patients and no single CpG methylation was associated with post-operative complications. Four single nucleotide polymorphisms (SNPs) were significantly correlated with higher Clavien-Dindo scores. Genetic variability in the chr5:143,402,505-143,405,805 region of exon 1 of the glucocorticoid receptor gene NR3C1, but not DNA methylation, was associated with more severe post-operative complications in patients having major abdominal surgery. This indicates that the response to GCs may be of clinical importance for inflammatory conditions.

  Study EGAS00001005737

• Whole exome sequencing of an invasive diffuse intrinsic pontine glioma sampled from different sites

  The lateral ventricle subventricular zone (SVZ) is a frequent site of high-grade glioma (HGG) spread and tumor invasion of the SVZ is a predictor of a worse clinical prognosis. A range of HGG types invade the stem cell niche of the SVZ, including both adult glioblastoma and pediatric high-grade gliomas such as diffuse intrinsic pontine glioma (DIPG). The cellular and molecular mechanisms mediating this frequent invasion of the SVZ are poorly understood. Here we demonstrate that neural precursor cell (NPC):glioma cell communication underpins the propensity of glioma to colonize the SVZ. SVZ NPCs secrete chemoattractant signals toward which glioma cells home. Biochemical and proteomic analyses of factors secreted by SVZ NPCs revealed a discrete list of candidate proteins. Necessity and sufficiency testing implicated the neurite outgrowth-promoting factor pleiotrophin, along with three required binding partners (secreted protein acidic and rich in cysteine (SPARC), SPARC-like protein 1, and heat shock protein 90B), as key mediators of this chemoattractant effect. Pleiotrophin protein expression is strongly enriched in the SVZ, and knockdown of pleiotrophin expression starkly reduced glioma invasion of the SVZ in the adult murine brain. Pleiotrophin, in complex with the three binding partners, activated the Rho/ROCK pathway in DIPG cells, and inhibition of ROCK resulted in decreased DIPG invasion toward SVZ neural precursor cell-secreted factors. These findings demonstrate a pathogenic role for neural precursor cell – glioma interactions and potential therapeutic targets to limit glioma invasion.

  Study EGAS00001002326

• Identification of a Fumarate-Hydratase Deficient-like RCC Subtype with Convergent Pathway Alterations

  The development of molecularly targeted therapies builds upon the identification of tumor clades with shared molecular abnormalities. Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a familial syndrome resulting from germline mutation of the two-hit tumor suppressor gene, Fumarate Hydratase (FH). HLRCC renal tumors present often in young adults, exhibit unique histological features and are particularly aggressive. HLRCC is uncommon and poorly understood, and whether there are sporadic renal tumors with similar characteristics, is unclear. Notably, FH can also be inactivated somatically in RCC, and should be included among FH-deficient (FHD) kidney tumors. We identified a tumor clade without FH mutations but with similar morphological and molecular characteristics as well as aggressive clinical behavior, which we name FHDL (FHD-like). We show that FHD/FHDL tumors represent a unique subtype of CIMP (CpG Island Methylator Phenotype) with the highest levels of overall genome methylation and CDKN2A silencing. FHD/FHDL tumors are characterized by convergent activation of the Hippo pathway as determined by frequent and mutually exclusive mutations in NF2 and PTPN14. AKR1B10, an NRF2 target, was upregulated in FHD/FHDL tumors as well as GATA3, which suggests a shared origin in the renal medulla and is consistent with topological studies. There is loss of 5-hydroxymethylcytosine in both FHD and FHDL tumors. These data define a group of particularly aggressive renal cancers with shared molecular features and potential targets for therapeutic intervention.

  Study EGAS00001002646

• Pseudodiastrophic dysplasia expands the known phenotypic spectrum of defects in proteoglycan biosynthesis

  Pseudodiastrophic dysplasia (PDD) is a severe skeletal dysplasia associated with prenatal manifestation and early lethality. Clinically, PDD is classified as a ‘dysplasia with multiple joint dislocations’ however, the molecular aetiology of the disorder is currently unknown. In order to identify the genetic defects underlying PDD, we performed whole exome sequencing (WES) on 3 patients from 2 unrelated families, clinically diagnosed with PDD. WES resulted in the identification of bi-allelic variants in the established skeletal dysplasia genes, B3GAT3 (Family 1) and CANT1 (Family 2). Mutations in these genes have previously been reported to cause ‘multiple joint dislocations, short stature, and craniofacial dysmorphism with or without congenital heart defects’ (B3GAT3) and Desbuquois dysplasia 1 (CANT1); disorders in the same nosological group as PDD. Follow-up of the B3GAT3 variants demonstrated significantly reduced B3GAT3/GlcAT-I expression. Downstream in vitro functional analysis revealed abolished biosynthesis of glycosaminoglycan side chains on proteoglycans. Functional evaluation of the CANT1 variant showed impaired nucleotidase activity, which results in inhibition of glycosaminoglycan synthesis through accumulation of uridine diphosphate. For the families described in this study, the PDD phenotype was caused by mutations in the known skeletal dysplasia genes B3GAT3 and CANT1, demonstrating the advantage of genomic analyses in delineating the molecular diagnosis of skeletal dysplasias. This finding expands the phenotypic spectrum of B3GAT3- and CANT1- related skeletal dysplasias to include PDD, and highlights the significant phenotypic overlap of conditions within the proteoglycan biosynthesis pathway.

  Study EGAS00001004145

• A no-stop mutation in MAGEB4 is a possible cause of rare X-linked azoospermia and oligozoospermia in a consanguineous Turkish family

  Purpose: The purpose of this study was to identify mutations that cause non-syndromic male infertility using whole exome sequencing of family cases.Methods: We recruited a consanguineous Turkish family comprising nine siblings with male triplets; two of the triplets were infertile as well as one younger infertile brother. Whole exome sequencing (WES) performed on two azoospermic brothers, identified a mutation in the MAGEB4 gene which was confirmed via Sanger sequencing and then screened for on control groups and unrelated infertile subjects. The effect of the mutation on mRNA and protein levels were tested after in vitro cell transfection. Structural features of MAGEB4 were predicted throughout the conserved MAGE domain.Results: The novel single base substitution (c.1041A>T) in the X-linked MAGEB4 gene was identified as a no-stop mutation. The mutation is predicted to add 24 amino acids to the C-terminus of MAGEB4. Our functional studies were unable to detect any effect either on mRNA stability, intracellular localization of the protein or the ability to homo / heterodimerize with other MAGE proteins. We thus hypothesize that these additional amino acids may affect the proper protein interactions with MAGEB4 partners. Conclusion: The whole exome analysis of a consanguineous Turkish family revealed MAGEB4 as a possible new X-linked cause of inherited male infertility. This study provides the first clue to the physiological function of a MAGE protein.

  Study EGAS00001002272

• Germline variants collaborate with somatic mutations and initiate and/or drive disease in primary myelodysplastic syndrome (MDS) and therapy-related myeloid neoplasms (t-MN)

  Therapy-related myeloid neoplasms (T-MN) are poorly characterized secondary hematological malignancies following chemotherapy/radiotherapy exposure. We compared the clinical and mutational characteristics of T-MN (n=129) and primary myelodysplastic syndrome (P-MDS, n=108) patients. Although the somatic mutation frequency was similar between T-MN and P-MDS patients (93% in both groups), the pattern was distinct. TP53 mutations were more frequent in T-MN (29.5% versus 7%), while spliceosomal complex mutations were more common in P-MDS (56.5% versus 25.6%).In contrast to P-MDS, the ring sideroblasts (RS) phenotype was not associated with better survival in T-MN, most probably due to genetic association with TP53 mutations. SF3B1 was mutated in 96% of P-MDS with ≥15% RS, but in only 32% T-MN. TP53 mutations were detected in 92% T-MN with ≥15% RS and SF3B1 wild-type cases. Interestingly, T-MN and P-MDS patients with “Very low” or “Low” Revised International Prognostic Scoring System (IPSS-R) showed similar biological and clinical characteristics. In a Cox-regression analysis, TP53 mutation was a poor prognostic factor in T-MN, independent of IPSS-R cytogenetic, disease-modifying therapy and NRAS mutation.Our data have direct implications for T-MN management and provide evidence that, in addition to conventional disease parameters, mutational analysis should be incorporated in T-MN risk stratification.

  Study EGAS00001003547

• Whole Tumor spatial heterogeneity in metastatic melanoma

  Heterogeneous inter-tumoral responses, sustained periods of apparent clinical benefit despite lack of objective response to various therapies, and even spontaneous remission are well known within a subpopulation of advanced melanoma patients. The molecular and cellular dynamics facilitating long-term survival remain poorly defined, particularly in the current era ofexposure to multiple potentially active therapies. We studied an exceptional case of long-term survival in a patient with non-responding metastatic melanoma in order to characterize the clonal and microenvironmental factors.We performed immunogenomic analysis of 3 metachronous tumors, and of 67 intratumor sub-regions of one metastatic lesion, usingwhole exome sequencing, RNA-sequencing, immunohistochemistry, and T cell receptor sequencing. Longitudinal analysesidentified mutations in severalknownresistance genes affecting distinctmetastases. Intra-tumoral immune signatures revealed spatially-distinct pockets of immune activation and suppression throughout the metastasis. A specific T cell VBeta CDR3 rearrangement was identified as both dominant and recurrent not only across multiple spatial points within a single tumor mass, but also across metachronous tumors spanning the patient’s disease course. Immunophenotyping of this T cell population with single-cell sequencingsuggestedrepeatedT-cell priming events leading to the persistence of both activated and exhausted T cells bearing this same TCR-Beta at any given time.Our findings highlight genomic and immune heterogeneityin metastatic disease, extending to the intra-tumoral leveland provide a rationale for integrative spatiotemporal analysis of metastatic lesions in the context of immune checkpoint blockade.

  Study EGAS00001003292

• Exploiting evolutionary steering in cancer therapy

  Drug resistance, mediated by intra-tumour heterogeneity and clonal evolution, is arguably the biggest problem in cancer therapy today. However, evolving resistance to one drug may come at the cost of increased sensitivity to another due to so-called evolutionary trade-offs. This weakness can be exploited in the clinic using an approach called ‘evolutionary herding’ that aims at controlling the tumour cell population to delay or prevent resistance. However, model systems able to recapitulate cancer evolution experimentally are lacking and current in vitro techniques based on small populations, re-plating and escalating dose, are unsuitable to develop evolutionary herding strategies. We present a novel methodology for evolutionary herding in vitro and ex vivo using patient-derived organoids. Our approach is based on a combination of single-cell barcoding, very large populations of 108-109 cells grown without re-plating, realistic high drug doses, time-course monitoring of cancer clones, and mathematical modelling of tumour evolutionary dynamics. We demonstrate evolutionary herding in non-small cell lung cancer in vitro and in patient-derived colorectal cancer organoids (PDO). We show that herding causes controlled evolutionary bottlenecks that lead to collateral sensitivity. Through genomic analysis, we were also able to determine the mechanisms that drive such sensitivity. Our approach allows modelling evolutionary trade-offs experimentally to test patient-specific evolutionary herding strategies that can be translated into the clinic to control treatment resistance.

  Study EGAS00001003200

• Targets of MEK inhibition in DIPG

  The survival of children with diffuse intrinsic pontine glioma (DIPG) remains dismal, with new treatments desperately needed. In the era of precision medicine, targeted therapies represent an exciting treatment opportunity, yet resistance can rapidly emerge, playing an important role in treatment failure. In a prospective biopsy-stratified clinical trial, we combined detailed molecular profiling (methylation BeadArray, exome, RNAseq, phospho-proteomics) linked to drug screening in newly-established patient-derived models of DIPG in vitro and in vivo. We identified a high degree of in vitro sensitivity to the MEK inhibitor trametinib in samples which harboured genetic alterations targeting the MAPK pathway, including the non-canonical BRAF_G469V mutation, and those affecting PIK3R1 and NF1. However, treatment of PDX models and of a patient with trametinib at relapse failed to elicit a significant response. We generated trametinib-resistant clones in the BRAF_G469V model through continuous drug exposure, and identified acquired mutations in MEK1/2 (MEK1_K57N, MEK1_I141S and MEK2_I115N) with sustained pathway up-regulation. These cells showed the hallmarks of mesenchymal transition, and expression signatures overlapping with inherently trametinib-insensitive primary patient-derived cells that predicted an observed sensitivity to dasatinib. Combinations of trametinib with dasatinib and the downstream ERK inhibitor ulixertinib showed highly synergistic effects in vitro. These data highlight the MAPK pathway as a therapeutic target in DIPG, and show the importance of parallel resistance modelling and rational combinatorial treatments likely to be required for meaningful clinical translation.

  Study EGAS00001004495

• RRBS sequencing of 7 tumour regions and a normal sample from a single TRACERx patient.

  Background: Epigenetic heterogeneity within a tumour can play an important role in tumour evolution and the emergence of resistance to treatment. It is increasingly recognised that the study of DNA methylation (DNAm) patterns along the genome - so-called `epialleles' - offers greater insight into epigenetic dynamics than conventional analyses which examine DNAm marks individually. Results: We have developed a Bayesian model to infer which epialleles are present in multiple regions of the same tumour. We apply our method to reduced representation bisulfite sequencing (RRBS) data from multiple regions of one lung cancer tumour and a matched normal sample. The model borrows information from all tumour regions to leverage greater statistical power. The total number of epialleles, the epiallele DNAm patterns, and a noise hyperparameter are all automatically inferred from the data. Uncertainty as to which epiallele an observed sequencing read originated from is explicitly incorporated by marginalising over the appropriate posterior densities. The degree to which tumour samples are contaminated with normal tissue can be estimated and corrected for. By tracing the distribution of epialleles throughout the tumour we can infer the phylogenetic history of the tumour, identify epialleles that differ between normal and cancer tissue, and define a measure of global epigenetic disorder. Conclusions: Detection and comparison of epialleles within multiple tumour regions enables phylogenetic analyses, identification of differentially expressed epialleles, and provides a measure of epigenetic heterogeneity.

  Study EGAS00001002484

• Renal_habitat_WXS

  This project aims at correlating genetic diversity (as well as transcriptomic, metabolic and immune diversity) observed in multiple targeted tumour biopsies and circulating tumour DNA with heterogeneity on multiparametric MRI. To this end, 20 patients with RCC (15 patients with localised/locally advanced disease and 5 with mRCC) will undergo multiparametric MRI including metabolic assessment by 13C-MRSI as well as measurements of tumour perfusion, oxygenation and water diffusion. Based on unique combinations of these imaging parameters, tumour habitats will be defined through cluster mapping. Intra-operative biopsy and immediate freezing of tumour cores from different imaging habitats will be performed. Multiregional ex-vivo tumour biopsies will be spatially registered to the MR data by using 3D printed tumour moulds generated from high-resolution volumetric sequences. These moulds allow slicing tumour specimens in identical planes to the MR acquisition and increase the accuracy of tissue sampling. The biopsies will then undergo molecular profiling such as metabolomic and RNA sequencing (including mRNA immune profiling), as well as DNA sequencing to assess driver and clonal mutations in different tumour habitats and guide design of appropriate cfDNA probes. The targeted cfDNA capture panel will be employed to determine the ability of cfDNA to capture the full genetic diversity observed in the primary tumour and to relate cfDNA to various imaging habitats in both renal and pancreatic cancer. Data integration will be undertaken between imaging habitats and molecular profiles for each region.

  Study EGAS00001003703

• Single_cell_characterization_of_T_cell_lymphoma_

  Lymphomas are types of blood cancer derived from lymphocytes, and can be classified into two main categories, Hodgkin's lymphomas (HL) and non-Hodgkin lymphomas (NHL). T-cell lymphoma is a type of NHL that develops from T lymphocytes and accounts for 10-15% of total NHL cases. As a rare disease, T-cell lymphoma is functionally, pathologically, and clinically complex and heterogeneous. The 2016 revision of the WHO classification has listed 29 subtypes of mature T/NK-cell neoplasms, among which over 25 are T lymphocyte in origin. These subtypes of T-cell lymphomas have different biological behaviours and clinical prognosis. Clinically, T-cell lymphoma ranges from indolent (slow-growing) to aggressive (fast-growing and spreading) disease. According to the guideline booklet provided by the Lymphoma Research Foundation (https://lymphoma.org/aboutlymphoma/nhl/), indolent T-cell lymphoma subtypes include adult T-cell leukaemia/lymphoma (ATLL), cutaneous T-cell lymphoma (CTCL), and mycosis fungoides (MF), while the aggressive subtypes include anaplastic large cell lymphoma (ALCL), angioimmunoblastic T-cell lymphoma (AITL), peripheral T-cell lymphoma (PTCL), and T lymphoblastic lymphoma (TLBL). PTCL and ALCL represent the most common subtypes of aggressive T-cell lymphoma whereas TLBL is a rare and aggressive subtype mostly diagnosed in children. In this study, we will use single cell RNA sequencing and single cell multiomic methods three aggressive T-cell lymphoma subtypes (ALCL, PTCL and TLBL) and try to gain a deeper insight into the genotypic and phenotypic features of these T-cell lymphomas.

  Study EGAS00001005750

• Genomic stratification and liquid biopsy in a rare adrenocortical carcinoma (ACC) case, with dual lung metastases

  Adrenocortical carcinoma is a rare malignancy with a poor prognosis and few treatment options. Molecular characterization of this cancer remains limited. We present a case of an adrenocortical carcinoma (ACC) in a 37-yr-old female, with dual lung metastases identified 1 yr following commencement of adjuvant mitotane therapy. As standard therapeutic regimens are often unsuccessful in ACC, we undertook a comprehensive genomic study into this case to identify treatment options and monitor disease progress. We performed targeted and whole-genome sequencing of germline, primary tumor, and both metastatic tumors from this patient and monitored recurrence over 2 yr using liquid biopsy for ctDNA and steroid hormone measurements. Sequencing revealed the primary and metastatic tumors were hyperhaploid, with extensive loss of heterozygosity but few structural rearrangements. Loss-of-function mutations were identified in MSH2, TP53, RB1, and PTEN, resulting in tumors with mismatch repair signatures and microsatellite instability. At the cellular level, tumors were populated by mitochondria-rich oncocytes. Longitudinal ctDNA mutation and hormone profiles were unable to detect micrometastatic disease, consistent with clinical indicators of disease remission. The molecular signatures in our ACC case suggested immunotherapy in the event of disease progression; however, the patient remains free of cancer. The extensive molecular analysis presented here could be applied to other rare and/or poorly stratified cancers, to identify novel or repurpose existing therapeutic options, thereby broadly improving diagnoses, treatments, and prognoses.

  Study EGAS00001003497

• Prostate cancer ancestral genomic disparity

  Prostate cancer is characterised by significant global disparity; mortality rates in SubSaharan Africa are double to quadruple those in Eurasia. Hypothesising unknown interplay between genetic and non-genetic factors, tumour genome profiling envisages contributing mutational processes. Through whole-genome sequencing of treatment-naïve prostate cancer from 183 ethnically/globally distinct patients (African versus European), we generate the largest cancer genomics resource for Sub-Saharan Africa. Identifying ~2 million somatic variants, Africans carried the greatest burden. We describe a new molecular taxonomy using all mutational types and ethno39 geographic identifiers, including Asian. Defined as Global Mutational Subtypes (GMS) A–D, although Africans presented within all subtypes, we found GMS-B to be ‘African-specific’ and GMS-D ‘African-predominant’, including Admixed and European Africans. Conversely, Europeans from Australia, Africa and Brazil predominated within ‘mutationally-quiet’ and ethnically/globally ‘universal’ GMS-A, while European Australians shared a higher mutational burden with Africans in GMS45 C. GMS predicts clinical outcomes; reconstructing cancer timelines suggests four evolutionary trajectories with different mutation rates (GMS-A, low 0.968/year versus D, highest 1.315/year). Our data suggest both common genetic factors across extant populations and regional environmental factors contributing to carcinogenesis, analogous to gene-environment interaction defined here as a different effect of an environmental surrounding in persons with different ancestries or vice versa. We anticipate GMS acting as a proxy to intrinsic and extrinsic mutational processes in cancers, promoting global inclusion in landmark studies.

  Study EGAS00001006425

• Whole transcriptome profiling of liquid biopsies from tumour xenografted mouse models: validation cohort

  While cell-free DNA (cfDNA) in liquid biopsies is widely being used and investigated, free circulating RNA (extracellular RNA, exRNA) has the potential to improve therapy response monitoring and cancer detection due to its dynamic nature. A fundamental open question hampering the initiation of large-scale liquid biopsy collections for tumour exRNA analysis is that it remains unclear in which blood subcompartment tumour-derived exRNAs primarily reside. We set out to develop a host-xenograft deconvolution framework, exRNAxeno, with cDNA mapping strategies to either a combined human-mouse reference genome or both species genomes in parallel, that can be applied to exRNA sequencing data from liquid biopsies of human xenograft mouse models, enabling to distinguish (human) tumoural RNA from (murine) host RNA, and as such to specifically analyse tumour-derived exRNA. Subsequently, the preferred exRNAxeno combination pipeline was applied to total exRNA sequencing data from blood platelets and three plasma fractions from a breast cancer patient-derived and neuroblastoma cell line-derived xenograft mouse model. We show that tumoural exRNA concentrations are not determined by plasma platelet levels, while host exRNA concentrations increase with platelet content. Furthermore, a large variability in exRNA levels and gene content across individual mice is observed. In general, the tumoural gene detectability in plasma is correlated with the RNA expression levels in the tumour tissue or cancer cell line. Our results unravel new aspects of tumour-derived exRNA biology in xenograft mouse models.

  Study EGAS00001006582

• Atypical B cells and impaired SARS-CoV-2 neutralisation following heterologous vaccination in the elderly

  Age is a major risk factor for hospitalization and death after SARS-CoV-2 infection, even in vaccinees. Suboptimal responses to a primary vaccination course have been reported in the elderly, but there is little information regarding the impact of age on responses to booster third doses. Here we show that individuals 70 or older who received a primary two dose schedule with AZD1222 and booster third dose with mRNA vaccine achieved significantly lower neutralizing antibody responses against SARS-CoV-2 spike pseudotyped virus compared to those below 70 at one month post booster. Neither the concentration of serum binding anti spike IgG antibody, nor the frequency of spike- specific B cells showed differences by age grouping. Impaired neutralization potency and breadth post-third dose in the elderly was associated with enrichment of circulating “atypical” spike-specific B cells expressing CD11c and FCRL5. Presence of these atypical B cells was confirmed by scRNA seq. However, when considering individuals who received three doses of mRNA vaccine, we did not observe statistically significant differences in neutralization or enrichment in atypical B cells by age grouping following dose 3. Together, our data reveal impaired neutralising antibody responses in the elderly after heterologous vaccination with AdV followed by mRNA booster dose, but not in homologous three dose mRNA vaccination highlighting that different vaccine formats differentially instruct the memory B cell response.

  Study EGAS00001007385

• The long term effects of chemotherapy on normal blood - WGS dataset

  In developed countries, ~10% of individuals are exposed to systemic chemotherapy for cancer and other diseases. Many chemotherapeutic agents act by increasing DNA damage in cancer cells, hence triggering cell death. However, there is limited understanding of the extent and consequences of collateral DNA damage to normal tissues. To investigate the impact of chemotherapy on mutation burdens and cell population structure of a normal tissue we sequenced blood cell genomes from 23 individuals, aged 3-80 years, treated with a range of chemotherapy regimens. Substantial additional mutation loads with characteristic mutational signatures were imposed by some chemotherapeutic agents, but there were differences in burden between different classes of agent, different agents of the same class and different blood cell types. Chemotherapy also induced premature changes in the cell population structure of normal blood, similar to those of normal ageing. The results constitute an initial survey of the long-term biological consequences of cytotoxic agents to which a substantial fraction of the population is exposed during the course of their disease management, raising mechanistic questions and highlighting opportunities for mitigation of adverse effects.

  Dataset EGAD00001015339

• NIHR BioResource Common Disease Patients 2016

  NIHR BioResource Common Disease Patients 2016. The dataset includes 13489 samples from blood donors, they were not screened for any particular disease, and therefore they are representative of the general population. Genomic data includes 845487 snps collected using the UK BioBank V1 Affymetrix array. Phenotypic data includes gender, age, ethnicity and disease. According to our internal quality check there are 81 duplicates in this dataset.

  Dataset EGAD00010002059

• Linked Read Sequencing of Gastric Cancer Metastases for Complex SV Resolution

  In this study, linked read sequencing was performed on two ovarian metastases and matched normal tissue, from a patient with primary diffuse gastric cancer. Linked read sequencing is a DNA preparation technology whereby high molecular weight molecules of DNA are uniquely barcoded prior to fragmentation and sequencing, thus retaining information about genomic contiguity. This study performed an extended analysis of linked read sequencing data to resolve the complex structures of structural variants in the cancer genomes. Complex structural rearrangements were identified in the genomic region surrounding the known oncogene FGFR2, and the association between FGFR2 and gastric cancer metastasis was demonstrated in an organoid model.

  Study phs001362

• RNA sequencing data from glioblastoma primary cell lines treated with indisulam data access committee

  Four glioblastoma primary cell lines (BT592/pt1, BT1007/pt2, BT1009/pt3; BT1012/pt4) were derived from glioblastoma Cavitron Ultrasonic Aspirator (CUSA) material and cultured as neurospheres (GB-NS) in DMEM/F12+Glutamax medium containing B27 supplement (Thermofisher, Waltham, MA, USA) and the mitogenic factors epidermal growth factor (EGF) and fibroblast growth factor b (bFGF) (Peprotech, Rocky Hill, NJ, USA). GB-NS were confirmed mycoplasma-free by PCR Test. Cells were seeded in triplicate in 6-well plates, treated with 5 μM indisulam (Sigma Aldrich, Merk), or vehicle control, and collected after 6 and 16 hours for RNA extraction. Total RNA was extracted using TRIzol Reagent (Life Technologies, Thermo Fisher, cat n. 15596026), RNeasy Mini Kit, and RNase-Free DNase Set (Qiagen, cat n. 74104 and 79254). Sample QC, RNA library preparations, and sequencing reactions were conducted at GENEWIZ (Germany GmbH). The concentration of RNA was quantified using a Qubit Fluorometer (Life Technologies, Carlsbad, CA) and RNA integrity was assayed using a TapeStation. Sequencing libraries were prepared with RNA with PolyA selection and ERCC spike-in with standard ERCC kit (ERCC RNA Spike-In Mix,Thermo Fisher 4456740). Final libraries (Illumina, RNA with PolyA selection) were sequenced on a NovaSeq platform, producing 2×150 base paired-end reads.

  Dac EGAC50000000407

• Development, maturation and maintenance of human prostate inferred from somatic mutations

  Stem cells within prostate epithelium frequently undergo malignant transformation, but there is limited information on their clonal dynamics and mutation burden in healthy human prostates. We sequenced whole genomes from 409 microdissections of prostate epithelium across 8 donors, using phylogenetic reconstruction with spatial mapping in a 59-year-old man’s prostate to provide high-resolution reconstruction of tissue dynamics across the lifespan. Somatic mutation burden increases linearly with age, at ~16 mutations/year/clone, and is higher in peripheral than peri-urethral regions. The 24-30 independent glandular subunits are established as rudimentary ductal structures during fetal development by 5-10 embryonic cells each. Puberty induces formation of further side branches and terminal acini by local stem cells disseminated through the rudimentary ducts during development. During adult tissue maintenance, clonal expansions are small, with limited geographic scope and minimal migration. Driver mutations are rare in normal ageing prostate epithelium, but the one canonical driver we did observe generated a sizable intraepithelial clonal expansion. By resolving unbiased, continuously occurring lineage-marking mutations, we define stem cell dynamics through embryogenesis, puberty and ageing, with relevance for prostate cancer.

  Dataset EGAD00001006591

• BAM files of targeted next-generation DNA sequencing data of 13 chordoid gliomas of the third ventricle

  BAM files of targeted next-generation DNA sequencing data of 13 chordoid gliomas of the third ventricle (2 paired tumor-normal samples and 11 tumor-only samples). Genomic DNA was extracted from formalin-fixed, paraffin-embedded blocks of tumor tissue from 13 patients with chordoid glioma of the third ventricle using the QIAamp DNA FFPE Tissue Kit (Qiagen). Genomic DNA was also extracted from leukocytes in a peripheral blood sample from one of the patients and a non-neoplastic gastric biopsy specimen from one of the patients. Capture-based next-generation DNA sequencing was performed at the University of California, San Francisco Clinical Cancer Genomics Laboratory, using an assay that targets all coding exons of approximately 500 cancer-related genes, select introns of 47 genes, and TERT promoter with a total sequencing footprint of 2.8 Mb (UCSF500 Cancer Panel). Sequencing libraries were prepared from genomic DNA, and target enrichment was performed by hybrid capture using a custom oligonucleotide library (Nimblegen SeqCap EZ Choice). Captured libraries were sequenced as paired-end 100 bp reads on an Illumina HiSeq 2500 instrument. Duplicate sequencing reads were removed computationally to allow for accurate allele frequency determination and copy number calling.

  Dataset EGAD00001003818

• Evolution and clinical impact of genetic epistasis within EGFR-mutant lung cancers: multi-timepoint exome sequencing of a single patient's disease

  This dataset is a time-series of EGFR-mutant NSCLC clinical specimens from an individual patient profiled using tumor-based whole exome sequencing and the data is in BAM format. DNA was extracted from FFPE for primary tumor and frozen tumor tissue samples and matched non-tumor tissue using the Qiagen Allprep DNA/RNA Mini Kit.  The library preparation protocol was based on the Agilent SureSelect Library Prep and Capture System. DNA was resuspended in a low TE buffer and sheared (Duty Cycle 5%; Intensity 175; Cycles/Burst: 200; Time: 300s, Corvaris S2 Utrasonicator).  Bar-coded exome libraries were prepared using the Agilent Sure Select V5 library kit per manfucaturer’s specifications. The libraries were run on the HiSeq2500. Raw paired end reads (100bp) in FastQ format generated by the Illumina pipeline were aligned to the full hg19 genomic assembly obtained from USCS, gencode 14, using bwa version 0.7.12. Picard tools version 1.117 was used to sort, remove duplicate reads and generate QC statistics. Tumor DNA was sequenced to median depth of 303X (range 114.39-383.41) and the matched germline DNA to average depth of 231.65.

  Dataset EGAD00001003769

• SS18-SSX-mediated hijacking of BAF complexes drives synovial sarcoma

  Synovial sarcoma (SS) is defined by a recurrent t(x;18) chromosomal translocation, which produces the hallmark SS18-SSX oncogenic fusion. Incorporation of SS18-SSX into BAF complexes renders BAF complexes aberrant in two distinct manners: the addition of 78aa of SSX onto SS18, and concomitant loss of BAF47 assembly. However, the importance and functional contributions of each of these perturbations on BAF complex targeting and gene expression regulation remain unclear. Here we use an integrative set of genomic approaches in human cancer cell lines and primary tumor samples to define the mechanistic consequences of the SS18-SSX fusion oncoprotein. We find that SS18-SSX hijacks BAF complexes to broad polycomb domains to activate bivalent genes, driving a unique gene expression program distinct from other loss-of-function BAF complex malignancies. Importantly, restoration of BAF47 rescues enhancer activation but is dispensable for proliferative arrest in cell lines. These results demonstrate that gain-of-function SS18-SSX-mediated BAF complex targeting and gene activation is the driving event in SS, and present a mechanism by which distinct functions of BAF complexes can be co-opted to drive oncogenesis.

  Dataset EGAD00001004135

• CyTOF of 27 DLBCLs

  Diffuse large B-cell lymphoma (DLBCL) is the most common histologic subtype of non-Hodgkin lymphoma and is notorious for its clinical heterogeneity. Patient outcomes can be predicted by cell-of-origin (COO) classification, demonstrating that the underlying transcriptional signature of malignant B-cells informs biological behavior in the context of standard combination chemotherapy regimens. In the current study, we used mass cytometry (CyTOF) to examine tumor phenotypes at the protein level with single cell resolution in a collection of 27 diagnostic DLBCL biopsy specimens from treatment naïve patients. We found that malignant B-cells from each patient occupied unique regions in 37-dimensional phenotypic space with no apparent clustering of samples into discrete subtypes. Interestingly, variable MHC class II expression was found to be the greatest contributor to phenotypic diversity. Within individual tumors, a subset of cases showed multiple phenotypic subpopulations, and in one case we were able to demonstrate direct correspondence between protein-level phenotypic subsets and DNA mutation-defined subclones. In summary, CyTOF analysis can resolve both inter- and intra-tumoral heterogeneity among primary samples, and reveals that each case of DLBCL is unique and may be comprised of multiple, genetically distinct subclones.

  Dataset EGAD00001005419

• P647 Targeted resequencing project

  In collaboration with Dr Robert Semple we have identified a family harbouring an autosomal dominant variant, which leads to severe insulin resistance (SIR), short stature and facial dysmorphism. This family is unique within the SIR cohort in having normal lipid profiles, preserved adiponectin and normal INSR expression and phosphorylation. DNA is available for 7 affected and 7 unaffected family members across 3 generations. All 14 samples have been genotyped using microsatellites and the Affymetrix 6.0 SNP chip. Linkage analysis identified an 18.8Mb haplotype on chromosome 19 as a possible location of the causative variant. However, Exome sequencing of 3 affected and 1 unaffected family members has not identified the causative variant suggesting the possibility of an intronic or intergenic variant in this region or elsewhere in the genome. We propose to conduct whole genome sequencing of 5 members of the pedigree at a depth of 20X. The chosen samples are two sets of parents plus one member of an unaffected branch of the pedigree who shares the risk haplotype on chromosome 19. Sequencing of the two sets of parents will be used along with the genome-wide SNP data to impute 4 affected children giving an effect sample size of 6 affected individuals.

  Dataset EGAD00001000383

• Timing the landmark events in the evolution of clear cell renal cell cancer

  Clear cell renal cancer is characterized by near-universal loss of the short arm of chromosome 3 (3p). This event arises through unknown mechanisms, but critically results in the loss of several tumor suppressor genes. We analyzed whole genomes from 95 biopsies across 33 patients with clear cell renal cancer (ccRCC) recruited into the Renal TRACERx study. We find novel hotspots of point mutations in the 5'-UTR of TERT, targeting a MYC-MAX repressor, that result in telomere lengthening. The most common structural abnormality generates simultaneous 3p loss and 5q gain (36% patients), typically through chromothripsis. Using molecular clocks, we estimate this occurs in childhood or adolescence, generally preceding emergence of the most recent common ancestor by years to decades. Similar genomic changes recent common ancestor by years to decades. Similar genomic changes are seen in inherited kidney cancers. Modeling differences in age-incidence between inherited and sporadic cancers suggests that the number of cells with 3p loss capable of initiating sporadic tumors is no more than a few hundred. Targeting essential genes in deleted regions of chromosome 3p could represent a potential preventative strategy for renal cancer.

  Dataset EGAD00001003445

• CRISPR/Cas9-mediated genome editing of Schistosoma mansoni acetylcholinesterase

  Here we report successful gene knock-in (KI) in the eggs of Schistosoma mansoni by combining CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs). We targeted the acetylcholinesterase (AChE) gene of S. mansoni using two synthetic guide RNAs (gRNAs), X5 and X7, respectively. Liver eggs of S. mansoni were exposed to CRISPR-vector containing X5 or X7 by electroporation. Simultaneously, eggs were transfected with a ssODN donor encoding a stop codon in all six frames. Next generation sequencing analysis revealed that CRISPR/Cas9-mediated editing in S. mansoni eggs resulted in Homology-Directed Repair (HDR) when template DNA ssODN provided. Furthermore, soluble egg antigen (SEA) from AChE-modified eggs exhibited markedly reduced AChE activity compared with controls, indicative that programmed Cas9 cleavage mutated the AChE gene. Following injection of modified schistosome eggs into the tail veins of mice, a significant decrease in granuloma size in the lungs of these animals. Notably, an enhanced Th2 response induced by eggs in lung, and splenocytes small intestine-draining mesenteric lymph node cells was also generated in mice injected with X5-KI eggs in different methods. These findings further demonstrate the power and utility of CRISPR/Cas9-based genome editing for undertaking functional genomics studies in schistosomes.

  Dataset EGAD00001006573

• Combination Therapies for Personalised Cancer Medicine in 11-18

  This study involves mutagenizing 11-18 with ENU to identify those mutations which engender resistance to targeted treatment. In the last decade we have begun to move towards the use of targeted therapeutics in the clinic, often resulting in dramatic response rates in cancer patients. However, clinical experience has demonstrated time and time again that such responses are invariably followed by the development of drug resistance. Identification of the underlying mechanisms may enable us to reverse such resistance. Traditional methods of modelling this resistance in vitro can lead to a plethora of potential resistance mechanisms that can be difficult to decipher.

  Study EGAS00001002579

• Combination Therapies for Personalised Cancer Medicine in drug resistant EGFR mutant lung cancer

  This study involves mutagenizing a range of different cell lines with ENU to identify those mutations which engender resistance to targeted treatment. In the last decade we have begun to move towards the use of targeted therapeutics in the clinic, often resulting in dramatic response rates in cancer patients. However, clinical experience has demonstrated time and time again that such responses are invariably followed by the development of drug resistance. Identification of the underlying mechanisms may enable us to reverse such resistance. Traditional methods of modelling this resistance in vitro can lead to a plethora of potential resistance mechanisms that can be difficult to decipher.

  Study EGAS00001002509