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• Gene expression-based prediction of pazopanib efficacy in sarcoma (HIPO, H021)

  The multi-receptor tyrosine kinase (RTK) inhibitor pazopanib is approved for the treatment of advanced non-adipocytic soft-tissue sarcoma and has also shown activity in other sarcoma subtypes. However, its clinical efficacy is highly variable, and no reliable predictors exist to date to select patients who are most likely to benefit from this drug. We analyzed the molecular profiles and clinical outcomes of pazopanib-treated sarcoma patients enrolled in a prospective observational study by the German Cancer Consortium, DKTK MASTER, that employs whole-genome/exome sequencing (WGS/WES) and transcriptome sequencing to inform the care of adults with advanced cancer across histologies who are younger than 51 and patients with rare tumors, including rare subtypes of more common entities, regardless of age.​ Among a total of 109 patients with available WGS/WES data, there was no correlation between clinical parameters, specific genetic alterations, or mutational signatures and clinical outcome. In contrast, analysis of a subcohort of 62 patients who underwent molecular analysis before pazopanib treatment and had transcriptome sequencing data available showed that mRNA levels of NTRK3 (hazard ratio [HR]=0.53, p=0.021), IGF1R (HR=1.82, p=0.027), and KDR (HR=0.50, p=0.011) were independently associated with progression-free survival (PFS). Based on the expression of these RTK genes, i.e., the features NTRK3-high, IGF1R-low, and KDR-high, we developed a pazopanib efficacy predictor (PEP) that stratified patients into three groups with significantly different PFS (p<0.0001). Application of the PEP to an independent cohort of pazopanib-treated sarcoma patients from DKTK MASTER (n=43) confirmed its potential to separate patient groups with significantly different PFS (p=0.02), whereas no such association was observed in sarcoma patients from DKTK MASTER (n=77) or The Cancer Genome Atlas sarcoma cohort (n=256) who were not treated with pazopanib. A score based on the combined expression of NTRK3, IGF1R, and KDR allows identification of sarcoma patients with good, intermediate, and poor outcome following pazopanib therapy and warrants investigation as a predictive tool in prospective studies to optimize the use of this drug in the clinic.

  Study EGAS00001005836

• TK-EPN862 - Patient-dervied xenograft model of Posterior Fossa A Ependymoma - RNAseq

  Ependymomas (EPN) are the third most common malignant brain cancer in children. Treatment strategies for pediatric EPN have remained unchanged over recent decades, with 10-year survival rates stagnating at just 67% for children aged 0-14 years. Moreover, patients who survive treatment for the most common subtype of pediatric EPN (posterior fossa A (PFA)), often suffer long-term neurological side effects as a result of therapy. It is evident that there is a need for safer, more effective treatments for pediatric EPN patients. There are ten distinct subgroups of EPN, each with their own molecular and prognostic features. To identify and facilitate the testing of new treatments for EPN, in vivo laboratory models representative of the diverse molecular subtypes are required. Here, we describe the establishment of a patient-derived orthotopic xenograft (PDOX) model of PFA EPN, derived from a metastatic cranial lesion. Patient and PDOX tumors were analyzed using immunohistochemistry, DNA methylation profiling, whole genome sequencing (WGS) and RNA sequencing. Both patient and PDOX tumors classified as PFA EPN by methylation profiling, and shared similar histological features consistent with this molecular subgroup. RNA sequencing revealed that gene expression patterns were maintained across the primary and metastatic tumors, as well as the PDOX. Copy number profiling revealed gains of chromosomes 7, 8 and 19, and loss of chromosomes 2q and 6q in the PDOX and matched patient tumor. No clinically significant single nucleotide variants were identified, consistent with the low mutation rates observed in PFA EPN. Overexpression of EZHIP RNA and protein, a common feature of PFA EPN, was also observed. Despite the aggressive nature of the tumor in the patient, this PDOX was unable to be maintained past two passages in vivo. Others who have successfully developed PDOX models report some of the lowest success rates for EPN compared to other pediatric brain cancer types attempted, with loss of tumorigenicity not uncommon, highlighting the challenge in propagating these tumors in the laboratory. Here, we discuss our collective experiences with PFA EPN PDOX model generation and propose potential approaches to improve future success in establishing preclinical EPN models

  Study EGAS00001006844

• ACT-Discover: identifying karyotype heterogeneity in pancreatic cancer evolution using ctDNA

  Background Liquid biopsies, and the dynamic tracking of somatic mutations within circulating tumour DNA (ctDNA) can provide insight into the dynamics of cancer evolution and the intra-tumour heterogeneity that fuels treatment resistance. However, identifying and tracking dynamic changes in somatic copy-number alterations (SCNAs), which have been associated with poor outcome and metastasis, using ctDNA is challenging. Pancreatic adenocarcinoma is a disease which has been considered to harbour early punctuated events in its evolution, leading to an early fitness peak, with minimal further subclonal evolution. Methods To interrogate the role of SCNAs in pancreatic adenocarcinoma cancer evolution, we applied whole-exome sequencing of 55 longitudinal cell-free DNA (cfDNA) samples taken from 24 patients (including 8 from whom a patient-derived xenograft (PDX) was derived) with metastatic disease prospectively recruited into a clinical trial. We developed a method, Aneuploidy in Circulating Tumour DNA (ACT-Discover), that leverages haplotype phasing of paired tumour biopsies or PDXs to identify SCNAs in cfDNA with greater sensitivity. Results SCNAs were observed within 28 of 47 evaluable cfDNA samples. Of these events, 30% could only be identified by harnessing the haplotype-aware approach leveraged in ACT-Discover. The exceptional purity of PDX tumours enabled near-complete phasing of genomic regions in allelic imbalance, highlighting an important auxiliary function of PDXs. Finally, although the classical model of pancreatic cancer evolution emphasises the importance of early, homogenous somatic events as a key requirement for cancer development, ACT-Discover identified substantial heterogeneity of SCNAs, including parallel focal and arm-level events, affecting different parental alleles within individual tumours. Indeed, ongoing acquisition of SCNAs was identified within tumours throughout the disease course, including within an untreated metastatic tumour. Conclusions This work demonstrates the power of haplotype phasing to study genomic variation in cfDNA samples and reveals undiscovered intra-tumour heterogeneity with important scientific and clinical implications. Implementation of ACT-Discover could lead to important insights from existing cohorts, or underpin future prospective studies seeking to characterise the landscape of tumour evolution through liquid biopsy.

  Study EGAS00001007077

• Mitochondrial DNA mosaicism in human somatic cells

  Genomic alterations accumulate in the somatic cells throughout an individual’s lifetime. Recent sequencing studies have documented widespread mutations in the nuclear genome and the frequent clonal competition of normal cells carrying mutations. However, the landscape of mitochondrial DNA (mtDNA) heteroplasmy in normal human tissues is poorly understood. This study investigated the whole genome sequences (WGSs) of 2,096 clones established from non-neoplastic healthy single cells obtained from 31 donors. In addition, we analyzed 31 WGSs of neoplastic cells, including 12 clones established from adenomatous polyps from one individual with MUTYH-associated polyposis and 19 matched colorectal carcinomas from individuals who donated normal colorectal clones.

  Dataset EGAD50000000373

• PRADO trial - Personalized response-directed surgery and adjuvant therapy after neoadjuvant ipilimumab plus nivolumab in stage III melanoma

  The dataset includes WES sequencing data on PRE- treatment biopsies of lymph node metastasis (n=79) The technology used for sequencing is llumina HiSeq. The PRADO trial tested a personalized response-directed treatment approach based on the pathologic response after neoadjuvant ipilimumab plus nivolumab in stage III melanoma patients. In patients achieving a major pathologic response, in their index lymph node (the largest lymph node metastasis at baseline), therapeutic lymph node dissection (TLND) and adjuvant therapy were omitted. Patients with partial response underwent TLND only, whereas patients with pathologic non-response underwent TLND and adjuvant systemic therapy ± synchronous radiotherapy.

  Dataset EGAD50000000393

• Raw scRNA-seq data from B-lineage cells in the blood of celiac disease patients

  Raw scRNA-seq data from 355 IgA+ peripheral blood B-lineage cells of two untreated celiac disease patients. The data was generated with the Smart-seq2 protocol and sequenced on a NextSeq500 instrument (Illumina) with 75 bp paired-end reads in high-output mode. The dataset contains R1 and R2 reads for each single cell (fastq.gz files) for cells passing quality control based on number of detected genes, reads, mitochondrial genes, reads mapping to the reference transcriptome and a productively rearranged immunoglobulin heavy chain IgA isotype reconstructed by the computational tool BraCeR. Metadata for the cells is provided in a csv file.

  Dataset EGAD50000000340

• Single cell RNAseq and TCRseq data from tumor and blood samples from 4 patients with muscle invasive bladder cancer

  For this dataset we performed single cell RNAseq paired with single cell TCR-seq on tumor and blood samples from 4 patients. This dataset contains 4 tumor samples as well as 4 blood samples. Each sample is made up of 2 sets of paired fastq files. The first pair contains reads corresponding to RNA transcripts (_Transcripts in file name), while the second pair contain reads corresponding to TCRs (_VDJ in file name). Sequenced on the Illumina NovaSeq6000 platform in a paired-end run using an SP flow cell (v1.5, 300 cycles).

  Dataset EGAD50000001381

• Genotyping microarray data of molecular tumorboard patients in the context of the HRD-manuscript published in IJC

  Intensity files (.idat) derived from the Infinium CytoSNP-850K BeadChip microarray (Illumina) were used to analzye a possible Homologous-Recombination-Repair Deficiency (Molecular Rationale for a PARP-Inhibitor therapy) of n= 39 tumor patients (each n= 39 .idat files of the green and red chanel respectively) presented in the Molecular Tumorboard at the University Hospital Erlangen. Using these files a Genomic Instability Score was bioinformatically deducted (R-based scripts, e.g. ASCAT, scarHRD) based upon the presence of three parameteres "Loss of Heterozygosity, LOH", "Large Scale Transitions, LST" and "Telomeric Allelic Imbalances, TAIs". The associated manuscript is published in International Journal of Cancer, IJC.

  Dataset EGAD00010002736

• SCANDARE HNSCC DNA targeted panel sequencing

  SCANDARE (NCT03017573) is a multicentric biobanking study, enrolling adult patients with newly diagnosed head and neck squamous cell carcinoma (HNSCC), triple negative breast cancer (TNBC), ovarian and cervical cancer. Tumor tissue and blood samples are collected at several time points during patient's journey, including at diagnosis, post-neoadjuvant chemotherapy in case of neoadjuvant treatment, at surgery, at recurrence and at disease progression following treatment initiated at recurrence. Since its launch in 2017 at Institut Curie, SCANDARE has enabled the longitudinal collection and preservation of samples for in-depth analyses. Here, we describe molecular alterations in 232 FFPE samples at baseline from 83 HNSCC patients using DNA targeted panel sequencing, with FASTQ files deposited in the dataset.

  Dataset EGAD50000001654

• Polymorphisms in the Mitochondrial Genome associated with Bullous Pemphigoid in Germans

  This study is the first to interrogate the whole mtDNA in BP patients and controls and to implicate multiple novel mtDNA variants in disease susceptibility. Whole mtDNA of German BP patients (n=180) and age- and sex-matched healthy controls (n=188) were sequenced using next generation sequencing (NGS) technology, followed by the replication study using Sanger sequencing of an additional independent BP (n=89) and control cohort (n=104). While the BP and control groups showed comparable mitochondrial haplogroup distributions, the haplogroup T exhibited a tendency of higher frequency in BP patients suffering from neurodegenerative diseases (ND) compared to BP patients without ND (p= 0.1448, Fisher’s exact test)

  Dataset EGAD00001005379

• Mutational analysis of an oligoprogressive sarcomatoid hepatocellular carcinoma treated with an immune checkpoint inhibitor.

  The dataset is composed of three sequenced tumor samples: (I) Meta-bone-557 (bone metastasis obtained from occipital lesion resection during treatment with Liposomal doxorubicin); (II) Meta-CNS-888 (brain metastasis obtained from surgical resection during treatment with Nivolumab); (III) Primary-liver-463 (primary hepatic tumor obtained from surgical resection during treatment with Nivolumab). Genomic DNA from tumor samples was extracted using GeneRead DNA FFPE kit (Qiagen), containing Uracyl-D Glycosylase, according to the manufacturer’s instructions. Whole-exome libraries were prepared using SureSelect XT Clinical Research Exome Target Enrichment kit (Agilent Technologies # 5190-7338). Sequences (150bp paired-end) were generated on a NextSeq 500 sequencing platform (Illumina).

  Dataset EGAD00001006982

• Chracterising cellur pathways underlying CD3/CD28 activation of human CD4+ cells

  We devised an approach to disentangle the TCR and CD28 pathways upon stimulation in naive and memory primary human CD4+ T cells (Tcons) in response to defined stimulatory signals. Sorted Tcons were activated using a titration of anti-CD3 and anti-CD28 in combination as well as individually. As a control we cultured cells in the same conditions but without the stimuli. In total, we defined seven conditions from four individuals for sequencing. 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 EGAD00001006610

• Chracterising cellur pathways underlying CD3/CD28 activation of human CD4+ cells

  We devised an approach to disentangle the TCR and CD28 pathways upon stimulation in naive and memory primary human CD4+ T cells (Tcons) in response to defined stimulatory signals. Sorted Tcons were activated using a titration of anti-CD3 and anti-CD28 in combination as well as individually. As a control we cultured cells in the same conditions but without the stimuli. In total, we defined seven conditions from four individuals for sequencing. 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 EGAD00001006612

• Targeted sequencing of healthy individuals, aged individuals and AML patients

  Targeted sequencing using BD Rhapsody with 462 mRNA and 97 antibodies of healthy young and aged adult as well as AML bone marrow mononuclear cells from iliac crest aspirations. Please note raw and integrated gene expression data, cell type annotation, metadata and dimensionality reduction are available as Seurat v3 objects through figshare. Access link is https://figshare.com/s/0fda29b169c719223ee3.

  Dataset EGAD00001008188

• Induction Failure in Childhood and Young Adult T-cell Acute Lymphoblastic Leukemia

  We carried out WGS and RNAseq on a cohort of 48 children and young adults with induction failure in T-cell Acute Lymphoblastic Leukemia (T-ALL) to identify genomic drivers of treatment resistance. The study includes WGS for 33 tumour/normal pairs and 15 tumour-only samples. In addition, there is RNAseq data for 37 cases.

  Dataset EGAD00001009058

• RNA-Seq data of hepatoma (Huh7 derived) and Ph5CH cells and primary hepatocytes

  The innate immune response of cells of hepatic origin (Huh7, Huh7.5, PH5CH and primary human hepatocytes (PHH), 66 samples) was analyzed by transcriptome analysis (RNAseq) upon supernatant delivery or transfection of synthetic dsRNA (poly(I:C)). Expression of TLR3 and RIG-I was reconstituted by lentiviral transduction in Huh7 and Huh7.5 cells. The sequencing is single RNA-Seq on an Illumina HiSeq 4000 with the Illumina TruSeq stranded mRNA kit.

  Dataset EGAD00001007083

• Genetic architecture of autism spectrum disorder in India

  The dataset consists of whole exome sequencing data (fastq format) of 100 non-syndromic autism spectrum disorder patients from India. Whole exome sequencing data is generated using Agilent SureSelect v6 capture kit and Illumina HiSeq sequencing platform. Paired end fastq files are available.

  Dataset EGAD00001008621

• Genomic signatures define three subtypes of EGFR-mutant stage II-III non-small-cell lung cancer with distinct adjuvant therapy outcomes

  This dataset contains targeted sequencing data of 204 surgical samples from resected NSCLC. Genomic profiling identifies five predictive biomarkers, which is then integrated into the Multiple-gene INdex to Evaluate the Relative benefit of Various Adjuvant therapies (MINERVA) score. The MINERVA score categorizes patients into three subgroups with relative disease-free survival and overall survival benefits from either adjuvant gefitinib or chemotherapy. This study demonstrates that predictive genomic signatures could potentially stratify resected EGFR-mutant NSCLC patients and provide precise guidance towards future personalized adjuvant therapy.

  Dataset EGAD00001008157

• Targeted and exome sequencing data from 24 prostate cancer patients with somatic hypermutation

  This dataset contains all available targeted and exome sequencing paired fastq files from our study, "Identification of hypermutation and defective mismatch repair in ctDNA from metastatic prostate cancer". Patient identifiers are denoted by the first three characters of the sample aliases (e.g. "P01"), and additional information is appended to reflect the panel used (targeted 73 gene panel: "PC", or whole-exome panel: "WXS"), and whether the sample represents cell-free DNA ("cfdna") or paired white-blood cell control ("wbc"). Several patients have multiple serial collections available, and these are denoted by the characters "C1, C2, C3," etc. All samples were sequenced using Illumina technology.

  Dataset EGAD00001005474

• scRNAseq of aplastic anemia and healthy immune cells co-cultured with autologous HSPCs

  This dataset contains 8 batches of bone marrow or peripheral blood cells of patients with aplastic anemia and bone marrow cells of healthy controls. The samples from different experimental conditions have been multiplexed with Totalseq-C antibodies (feature barcoding). To demultiplex the samples, see instructions in 10.5281/zenodo.2590196 and https://github.com/janihuuh/aa_manu. Each sample is analysed with Chromium V(D)J and 5' Gene Expression Platform v2 (10X Genomics). The raw data includes fastq files for Gene expression, feature barcodes and V(D)J Expression. The processed data have been deposited in the ArrayExpress database at EMBL-EBI (www.ebi.ac.uk/arrayexpress).

  Dataset EGAD00001012120

• LLDeep DAG2+: scRNA-seq in PBMCs

  Single cell transcriptomics of PBMCs of 47 donors from the Lifelines Deep cohort (general population, Northern part of the Netherlands). Cells of five or six different donors were pooled together in one sample pool, resulting in eight different sample pools. In total, 28.855 cells were captured and their transcriptomes were sequenced to an average depth of 74k. Genotype data was available for each donor, which allowed us to use the Demuxlet method that uses variable SNPs between the pooled individuals to determine which cell belongs to which individual. Since genotype information is lacking of 2 individuals, the transcriptome of only 45 individuals could be retrieved.

  Dataset EGAD00001003459

• RNASeq of bone marrow endothelial cells upon regeneration, (fetal) niche formation, and steady-state.

  Comprehensive transcriptional characterization of bone marrow endothelial cells by RNA sequencing was performed to determine the molecular properties/signatures of endothelium during bone marrow recovery and niche formation. Regenerative bone marrow endothelium was FACS-isolated from bone marrow aspirates of Acute Myeloid Leukemia patients 17 days after receiving chemotherapy (n=3). Niche-forming endothelial cells were FACS-isolated from fetal bones (gestational age 15-20 weeks) (n=3). Healthy adult bone marrow endothelial cells (n=7) were used as steady-state controls. cDNA was prepared using the SMARTer procedure (SMARTer Ultra Low RNA Kit, Clonetech). The provided file type is FASTQ.

  Dataset EGAD00001003904

• A Study to Evaluate Denosumab in Young Patients With Primary Breast Cancer (D-Beyond)

  This is a prospective, single arm phase IIa trial in which patients with early breast cancer will receive pre-operatively two doses of denosumab 120mg subcutaneously one week apart (maximum 12 days) followed by surgery. Tumor, normal breast tissue and blood samples will be collected at baseline and at surgery. Post-operative treatment will be at the discretion of the investigator.Primary objective: to determine if a short course of RANKL inhibition with denosumab can induce a decrease in tumor proliferation rates as determined by Ki67 immunohistochemistry (IHC) in newly diagnosed, early stage breast cancer in pre-menopausal women.

  Dataset EGAD00001004391

• Tam-Seq of HGSOC samples for fixative comparison study

  Targeted amplicon sequencing of samples as part of the study "Methanol-based fixation is superior to buffered formalin for next-generation sequencing of DNA from clinical cancer samples. The amplicon panel consists of 48 amplicons in TP53, PTEN, EGFR, PIK3CA, KRAS and BRAF genes as described previously [Forshew, STM 2012]. All libraries were pooled and quantify using DNA 1000 kit on Agilent 2100 Bioanalyzer and KAPA SYBR FAST ABI Prism qPCR Kit (KAPA Biosystems) on 7900HT Fast Real-Time PCR System (Applied Biosystems) according to the supplier's recommendations. Reads were aligned using bwa-mem v0.7.12-r1039 to the 1000 genomes version of human genome build GRCh37, retaining duplicate reads.

  Dataset EGAD00001001937

• Regulatory Genomics of Human Embryonic Development

  We used epigenetic profiling to map active enhancers in the developing human limb and cortex as described in two published studies: Cotney J, Leng J, Yin J, Reilly SK et al. The evolution of lineage-specific regulatory activities in the human embryonic limb. Cell 2013;154(1):185-96. Reilly SK, Yin J, Ayoub AE, Emera D et al. Evolutionary changes in promoter and enhancer activity during human corticogenesis. Science 2015;347(6226):1155-9. We also used ChIP-seq to map binding sites for the chromatin modifier CHD8 in the developing human brain, as described in one published study: Cotney J, Muhle RA, Sanders SJ, Liu L et al. The autism-associated chromatin modifier CHD8 regulates other autism risk genes during human neurodevelopment. Nat Commun 2015;6:6404. We are also depositing primary human sequence reads related to processed datasets in the Gene Expression Omnibus under the following accession numbers: GSE42413 (Cotney et al. 2013); GSE63649 (Reilly et al. 2015); GSE57369 (Cotney et al. 2015).

  Study phs001226

• Novel Strategies to Eliminate Resistance to B-cell Receptor Inhibitor Therapy in Lymphoid Malignancies

  Ibrutinib and acalabrutinib are BTK inhibitors (BTKi) used as frontline therapies for chronic lymphocytic leukemia (CLL). Clonal evolution during ibrutinib treatment has previously been studied, but limited study of clonal evolution in patients receiving acalabrutinib has been done. Our group analyzed 216 blood samples from 38 CLL patients at multiple time points during their BTKi treatment. Samples were prepared by standard protocols and sequenced using 200x Illumina sequencing. Using the longitudinal data, we reconstructed the clonal evolution of each patient using somatic mutations found within the B-cell populations. Three patterns of clonal evolution were identified: 1) new clone emergence, 2) clonal selection, and 3) clonal replacement. As previously seen in ibrutinib treatment, the evolution of subclones containing CLL-driver mutations was associated with worse clinical outcomes for patients treated with acalabrutinib (Log-rank p=0.04). Understanding clonal evolution during treatment with acalabrutinib can help determine and modify the clinical course of patients with CLL.

  Study phs003042

• Characterizing Individual Cells Obtained from Bone Marrow Biopsies of MPN Patients

  We profiled the transcriptomes of individual cells obtained from bone marrow aspirates of 7 treatment-naïve newly diagnosed JAK2-mutant myeloproliferative neoplasm (MPN) patients (3 polycythemia vera and 4 essential thrombocythemia patients), and 2 healthy controls. The submitted data corresponds to sequencing reads mapped to the human reference genome GhRC38 build using CellRanger for a total of 9 samples (one per patient). In addition, to reconstruct the lineage histories of single cells in MPN patients, we performed whole-genome sequencing of single-cell-derived colonies of hematopoietic stem and progenitor cells from 2 essential thrombocythemia (ET) patients: 22 JAK2-mutant colonies and 20 WT colonies for patient ET 1; and 13 JAK2-mutant colonies and 21 WT colonies for patient ET 2. The submitted data consists of sequencing reads aligned to the human reference genome GhRC38 build using bwa-mem. Each file corresponds to the whole-genome sequencing data for one colony.

  Study phs002308

• iRHOM2 Deficiency Causes Environmentally Directed Immunodysregulatory Disease

  We have conducted a study of two kindreds each with a unique mutation in the gene RHBDF2 that encodes the protein iRHOM2. Whole exome (kindred 1) and whole genome sequencing (kindred 2) have uncovered the first human loss-of-function mutations of RHBDF2 in 4 individuals. The affected patients are all children, 8 years old or younger, and were brought to our attention initially due to their suffering recurrent infections. Sequencing of their family members revealed parents with heterozygous RHBDF2 mutations, and heterozygous and wild type siblings – all of which were unaffected by this disease. A comprehensive investigation of this gene mutation through in vivo and in vitro experiments revealed that a loss of the protein iRHOM2 impairs the activity of the protease ADAM17 and subsequently compromises immune responses. This condition leads to the afflicted patients becoming vulnerable to different infections based on their unique environment and presenting with diverse clinical phenotypes. 

  Study phs002478

• Somatic Mutations and Cell Lineage in the Human Brain

  Genetic mutations causing human disease are conventionally thought to be inherited from one's parents and present in all somatic (body) cells. Increasingly however, somatic mutations are implicated in neurological diseases. Somatic mutations that arise during the cell divisions of prenatal brain development are inherited in clonal fashion and can cause neurodevelopmental diseases, even when present at low levels of mosaicism. In this study we use whole genome, RNA, and ATAC sequencing of single cells and bulk tissue to identify somatic mutations in control, and some disease, brains to: 1) identify and catalogue the mutations which shape the somatic neuronal genome; 2) perform a cell lineage analysis of the human brain using clonal somatic mutations in cortical neurons; 3) determine patterns of somatic mutations at different ages and in aging related disease phenotypes; and 4) relate cell lineage patterns to cell phenotype in the human brain through cell type-specific analyses.

  Study phs001485

• Rare Mendelian Disease in Old Order Amish and Mennonite Patients

  The Clinic for Special Children (CSC) has integrated biochemical and molecular methods into a rural pediatric practice serving Old Order Amish and Mennonite (Plain) children. Among the Plain people, we have used single nucleotide polymorphism (SNP) microarrays to genetically map recessive disorders to large autozygous haplotype blocks (mean = 4.4 Mb) that contain many genes (mean = 79). For some, uninformative mapping or large gene lists preclude disease-gene identification by Sanger sequencing. Seven such conditions were selected for exome sequencing at the Broad Institute; all had been previously mapped at the CSC using low density SNP microarrays coupled with autozygosity and linkage analyses. Using between 1 and 5 patient samples per disorder, we identified sequence variants in the known disease-causing genes SLC6A3 and FLVCR1, and present evidence to strongly support the pathogenicity of variants identified in TUBGCP6, BRAT1, SNIP1, CRADD, and HARS. Our results reveal the power of coupling new genotyping technologies to population-specific genetic knowledge and robust clinical data.

  Study phs000623

• L1-Seq and Genome-Wide SNP Genotyping in a Multiethnic Asian Population

  Insertions of the human-specific subfamily of LINE-1 (L1) retrotransposon are highly polymorphic across individuals and can critically influence the human transcriptome. We hypothesized that L1 insertions could represent genetic variants determining important human phenotypic traits, and performed an integrated analysis of L1 elements and single nucleotide polymorphisms (SNPs) in several human populations. We found that a large fraction of L1s were in high linkage disequilibrium (LD) with their surrounding genomic regions and that they were well-tagged by SNPs. However, L1 variants were only partially captured by SNPs on standard SNP arrays, so that their potential phenotypic impact would be frequently missed by SNP array-based genome-wide association studies. We next identified potential phenotypic effects of L1s by looking for signatures of natural selection linked to L1 insertions; significant extended haplotype homozygosity (EHH) was detected around several L1 insertions. This suggests that some of these L1 insertions may have been the target of recent positive selection.

  Study phs000732

• ALLELE Consortium Glioblastoma Project

  Glioblastoma patient samples were sequenced for this project. The purposes of this project are: (1) to establish an umbrella protocol as a mechanism for identifying patients (in the USA and international sites) who agree to the collection, storage, and broad usage of their specimens and ongoing collection, and review of their medical information for the purposes of minimal-risk research and use of such data in clinical trials on which the patient may enroll; (2) to develop operations that facilitate investigator access to the data and/or tissue to conduct research, while minimizing risks to participants and promoting responsible conduct; and (3) to provide the infrastructure for the sharing of de-identified, coded samples, clinical data, and genomic data between participating investigators and institutions. Eligible patients include patients with known or suspected nervous system tumors or other neurologic diseases. Whole exome sequencing from the stated project will be released under this submission.

  Study phs003000

• BarcUVa-Seq (Biology of Colorectal Cancer Risk Enhancers)

  This study was designed to generate a cohort of normal colon mucosal biopsies from the "average risk" population. Its primary aims included the development of an RNA-seq dataset of normal colon mucosal biopsies across three colon regions (ascending, transverse and descending). Further, we aimed to provide plausible prioritization to genetic components of colorectal cancer risk by employing methods such as expression quantitative trait loci analysis. In doing so, we provide a novel reference dataset for the study of colon cancer risk in the healthy colon epithelium. RNA-seq was performed in batches on an Illumina HiSeq 2500 (batch 1-7) or NovaSeq 6000 (batch 8) on a total of 463 samples. Matched genotyping data was generated on the Illumina OncoArray BeadChip. Phasing was carried out using Minimac 3 and imputation was performed using The Haplotype Reference Consortium panel on the Michigan Imputation Server.Genotype and RNA-seq data will be made available in dbGaP.

  Study phs003338

• Type I Interferon Exacerbates Mycobacterium Tuberculosis Induced Human Macrophage Death

  In this study, we tested whether type I interferons (IFN-I), namely IFN-alpha and IFN-beta, may promote Mycobacterium tuberculosis (Mtb) pathogenesis by exacerbating cell death of human monocyte-derived macrophages (MDM) infected with Mtb. We differentiated the MDM under two different published methods (see Document Method.pdf for Table 1 of Methods). To determine if MDM infected with Mtb upregulates IFN-I transcripts, signaling factors, or IFN-I stimulated genes (ISGs), the MDM were infected with Mtb or treated with an activator of stimulator of IFN genes (STING), diamidobenzimidazole (di-ABZI). STING activation acted as a positive control for IFN-I induction and ISG upregulation. Indeed, di-ABZI treated MDM differentiated under both methods upregulated ISGs 24 hpi. At 24 hour post treatment, MDM differentiated under one method, but not under another method, upregulated ISGs, such as ISG15, IFITM3, and CXCL10, upon Mtb infection. MDM differentiated under both methods upregulated ISGs upon di-ABZI treatment.

  Study phs003607

• Ultra-sensitive ctDNA monitoring required for predicting response and resistance to immunotherapy in advanced melanoma

  Circulating tumor DNA (ctDNA) monitoring in peripheral blood has shown promise for disease surveillance and prognostication of ICI response. Yet even in the metastatic context, ctDNA signals can be missed, suggesting the need for increased assay sensitivity. We used a tumor-informed ctDNA assay capable of detection down to 1 part-per-million (PPM) ctDNA to profile 188 plasma samples from 23 melanoma patients receiving ICI over several years. Residual disease was detected in ctDNA over a broad range, from 130,000 PPM down to 2.3 PPM, with approximately a quarter of detections below 50 PPM. Early on-treatment increases in ctDNA level, and both ctDNA-based molecular clearance (mCR) and molecular progression (mPD) were all prognostic of both OS and PFS. Additionally, mCR consistently preceded radiographic imaging. Our results show the importance and utility of an ultra-sensitive ctDNA assay in advanced melanoma treated by ICI with potential implications for other cancers.

  Study EGAS50000000550

• Genome sequencing identifies splice-disrupting variants in childhood heart disease

  We used genome sequencing (GS) to identify cardiac specific splice disrupting variants that were subsequently validated in a subset of participants. In turn, we developed a heart-specific model for canonical and non-canonical splice variants, which was applied to patients with congenital heart disease (CHD). These included patients with two of the most common forms of cyanotic CHD i.e. tetralogy of Fallot (TOF) and dextro-transposition of the great arteries (TGA). In addition to identifying canonical splice-disrupting variants in known CHD-related genes in 1% cases, this approach identified putatively damaging non-canonical splice-disrupting variants in 11% of isolated CHD, with deeply intronic variants representing 53% of non-canonical splice-disrupting variants in CHD genes. GS was critical for the identification of variants that would not be captured by routine clinical genetic tests including exome sequencing, while cardiac RNA-Seq allowed for high specificity in the interpretation of splice-disrupting effects in the heart.

  Study EGAS50000000586

• Aberrant RNA-splicing (neojunctions) offers a new source for targets, and our neoantigen discovery platform (SNIPP) characterizes a novel class of clonally-expressed splicing-derived neoantigens that elicit a CD8+ T-cell-mediated tumor killing response.

  RESULTS: Our pipeline identified 789 public neojunctions, with 32 neojunctions concurrently identified in transcriptomic and proteomic glioma data and predicted to be presented by HLA-A*02:01 with high confidence. IVS and subsequent 10x VDJ scRNA-seq identified TCR clonotypes reactive against neojunctions in RPL22 (n=7) and GNAS (n=1), the latter being highly intratumorally-conserved (detected in > 90% of spatially-mapped biopsies across 17/56 patients (26.78%)). TCR-transduced T-cells demonstrated recognition and immunogenic activation against endogenously processed and presented neoantigens in multiple GBM PDX cell lines. Furthermore, IDH1-mutant oligodendroglioma samples demonstrated significantly elevated expression of neojunctions over IDH1-mutant astrocytoma and IDH1wt subtypes. Differential gene expression (DESeq2) identified decreased expression of splicing factors due to oligodendroglioma-specific co-deletion of Chromosomes 1p/19q. siRNA knockdown of these splicing factors (e.g. SF3A3, SNRPD2) in IDH1wt glioma cells resulted in significantly increased expression of corresponding neojunctions.

  Study EGAS00001007986

• High Resolution Maps of the HeLa 3D Genome Using Hi-C

  We use in situ Hi-C to probe the three-dimensional architecture of genomes, constructing haploid and diploid maps of nine cell types (including HeLa). The densest, in human lymphoblastoid cells, contains 4.9 billion contacts, achieving 1-kilobase resolution. We find that genomes are partitioned into contact domains (median length, 185kb), which are associated with distinct patterns of histone marks and segregate into six subcompartments. We identify ~10,000 loops. These loops frequently link promoters and enhancers, correlate with gene activation, and show conservation across cell types and species. Loop anchors typically occur at domain boundaries and bind CTCF. CTCF sites at loop anchors occur predominantly (>90%) in a convergent orientation, with the asymmetric motifs 'facing' one another. The inactive X-chromosome splits into two massive domains and contains large loops anchored at CTCF-binding repeats. The data for the other (non-HeLa) cell types has been uploaded to GEO.

  Study phs001010

• WFS1 mutation screening in a large series of Japanese hearing loss patients: Massively parallel DNA sequencing-based analysis

  A heterozygous mutation in the Wolfram syndrome type 1 gene (WFS1) causes autosomal dominant nonsyndromic hereditary hearing loss, DFNA6/14/38, or Wolfram-like syndrome. To date, more than 40 different mutations have been reported to be responsible for DFNA6/14/38. In the present study, WFS1 variants were screened in a large series of Japanese hearing loss (HL) patients to clarify the prevalence and clinical characteristics of DFNA6/14/38 and Wolfram-like syndrome. Massively parallel DNA sequencing of 68 target genes was performed in 2,549 unrelated Japanese HL patients to identify genomic variations responsible for HL. The detailed clinical features in patients with WFS1 variants were collected from medical charts and analyzed. We successfully identified 13 WFS1 variants in 19 probands: eight of the 13 variants were previously reported mutations, including three mutations (p.A684V, p.K836N, and p.E864K) known to cause Wolfram-like syndrome, and five were novel mutations.

  Study JGAS000123

• Measles oncolytic virus as an immunotherapy for recurrent/refractory pediatric medulloblastoma and atypical teratoid rhabdoid tumor

  Pediatric recurrent medulloblastoma (MB) and atypical teratoid rhabdoid tumor (ATRT) are largely incurable and warrant novel therapies. We investigated a) the safety of a measles virus variant, MV-NIS, in a pediatric phase 1 study and b) the mechanisms of MV-NIS and potential benefit of combination with immune checkpoint inhibition (ICI). Pediatric patients with recurrent MB or ATRT were treated with intratumoral injections for local recurrence or via lumbar puncture for disseminated recurrence. We evaluated local immune responses to MV-NIS with and without ICI via single-cell and bulk RNA sequencing in an intracranial, immunocompetent, syngeneic murine model. MV-NIS given intratumorally or via repeat intrathecal dosing was safe. MV-NIS prolonged survival in murine models but did not demonstrate additive benefit with ICI. No changes in tumor-infiltrating immune-cell composition or activation were observed in response to MV-NIS treatment; however, MV-NIS induced local expression of neutralizing antibodies, complement cascade, and phagocytosis-related genes.

  Study EGAS50000000811

• Adjuvant nivolumab in resected esophageal or gastroesophageal junction cancer: exploratory biomarker analyses from CheckMate 577

  Adjuvant nivolumab demonstrated a significant improvement in disease-free survival (DFS; primary endpoint) versus placebo in patients with resected esophageal or gastroesophageal junction cancer and residual pathologic disease following neoadjuvant chemoradiotherapy (CRT) in the phase III CheckMate 577 study, leading to global approval and widespread adoption. Exploratory biomarker analyses were performed using whole-exome sequencing, RNA sequencing, and immunohistochemistry. Hazard ratios for DFS favored nivolumab versus placebo in patients with higher inflammation and proliferation gene expression signature scores; lower M2 macrophage, endothelial, and stromal GES scores; and higher densities of CD3+/CD8- T cells and natural killer cells. Assessments of pre-CRT and post-CRT tumor tissue found post-CRT increases in programmed death ligand 1 combined positive score in 51% of patients, which appeared to be associated with greater DFS benefit. Through these analyses, we identified patient subpopulations that appear to derive improved DFS benefit from adjuvant nivolumab in this setting.

  Study EGAS50000001663

• MGA-NUTM1 fusion in high grade spindle cell sarcoma

  NUT carcinoma (NC) with mesenchymal differentiation have rarely been described in the literature. In this report, we describe the characterization of two cases of high grade spindle cell sarcoma harboring a novel MGA-NUTM1 fusion. Whole genome sequencing identified the presence of complex rearrangements resulting in a MGA-NUTM1 fusion gene in the absence of other significant somatic mutations. The fusion protein was predicted to retain nearly the entire protein sequence of both MGA (exons 1-22) and NUTM1 (exons 3-8). In contrast to typical cases of NC, these cases were successfully treated with aggressive local control measures (surgery and radiation) and both patients remain alive without disease. These cases describe a new subtype of NUTM1-rearranged tumors warranting expansion of diagnostic testing to evaluate for the presence of MGA-NUTM1 or alternative NUTM1 gene fusions in the diagnostic workup of high grade spindle cell sarcomas or small round blue cell tumors of ambiguous lineage.

  Study EGAS00001003341

• Dynamics of neoantigen landscape during immunotherapy

  Immune checkpoint inhibitors have shown significant therapeutic responses against tumors containing increased mutation-associated neoantigen load. We have examined the evolving landscape of tumor neoantigens during the emergence of acquired resistance in non-small cell lung cancer patients after initial response to immune checkpoint blockade with anti-PD1 or anti-PD-1/anti-CTLA4 antibodies. Analyses of matched pretreatment and resistant tumors identified genomic changes resulting in loss of 7 to 18 putative mutation-associated neoantigens in resistant clones. Peptides generated from the eliminated neoantigens elicited clonal T cell expansion in autologous T cell cultures, suggesting that they generated functional immune responses. Neoantigen loss occurred through elimination of tumor subclones or through deletion of chromosomal regions containing truncal alterations and were associated with changes in T cell receptor clonality. These analyses provide insights into the dynamics of mutational landscapes during immune checkpoint blockade and have implications for development of immune therapies that target tumor neoantigens.

  Study EGAS00001002704

• Autozygosity_pilot_Born_in_Bradford

  The offspring of first cousin marriages have ~6% of their genome autozygous, i.e. homozygous identical by descent, or even more if there was further consanguinity in their ancestry. In the UK there are large populations with very high first cousin marriage rates of 20-50%. Sequencing the exomes of a sample of these individuals has the potential both to support genetic health programmes in these populations, and to provide genetic research information about rare loss of function mutations. This pilot study based on existing cohort samples from the Born In Bradford study will identify homozygous individuals for almost all variants down to an allele frequency around 1%, plus individuals carrying hundreds of new homozygous rare loss-of-function variants, and will support development of community relations and ethics for a wider study currently being designed. The data deposited in the EGA consist of low coverage whole exome sequencing on these samples.

  Study EGAS00001000462

• Whole exome sequencing of long-term, never relapse exceptional responders of trastuzumab-treated HER2+ metastatic breast cancer

  Trastuzumab has significantly improved the overall survival of patients with HER2+ metastatic breast cancer (MBC). However, outcomes can vary, with patients progressing within 1-year of treatment or exceptional cases of complete response to trastuzumab for ≥ 10 years. Identification of the underlying genomic aberrations of “exceptional responders (ExRs)” compared to “rapid non-responders (NR)” increases our understanding of the mechanisms involved in MBC progression and identification of biomarkers of trastuzumab response and resistance. Whole exome sequencing was performed on six ExRs compared to five NR. The overall fraction of genome copy number alteration (CNA) burden was higher in NR patients (P=0.07), while more significantly pronounced in copy number gains (P=0.03) in NR compared to ExRs. Delineation of the distribution of CNA burden across the genome identified a greater degree of CNA burden in NR within chr8 (P=0.02) and in chr17 (P=0.06) and conferred a statistically significant benefit in overall survival.

  Study EGAS00001004486

• Integrated genomic characterization of IDH1 mutant Glioma malignant progression

  Gliomas represent approximately 30% of all central nervous system tumors, and 80% of malignant brain tumors. To understand the molecular mechanisms underlying the malignant progression of low-grade Isocitrate Dehydrogenase 1 (IDH1) mutant gliomas, we studied paired tumor samples from 41 patients, comparing higher-grade, progressed samples to their lower-grade counterparts. Integrated genomic analyses, including whole-exome sequencing, copy number, gene expression and DNA methylation profiling, demonstrated non-linear clonal expansion of the original tumors and identified oncogenic pathways driving progression. These include activation of the MYC and RTK-RAS-PI3K pathways, up-regulation of the FOXM1- and E2F2-mediated cell cycle transition genes, as well as epigenetic silencing of developmental transcriptional factors bound by Polycomb repressive complex 2 in human embryonic stem cells. Our results not only provide mechanistic insight into the genetic and epigenetic mechanisms driving glioma progression but also identify inhibition of the bromodomain and extra-terminal (BET) family as a potential therapeutic approach.

  Study EGAS00001001588

• Resident memory CD8 T cells persist for years in human small intestine

  Resident memory CD8 T cells (Trm) have been shown to provide effective protective responses in the small intestine (SI) in mice. A better understanding of the generation and persistence of SI CD8 Trm cells in humans may have implications for intestinal immune-mediated diseases and vaccine development. Analyzing normal and transplanted human SI we demonstrated that the majority of SI CD8 T cells were bona fide CD8 Trm cells that survived for over 1 year in the graft. Intraepithelial and lamina propria CD8 Trm cells showed a high clonal overlap and a repertoire dominated by expanded clones, conserved both spatially in the intestine and over time. Functionally, lamina propria CD8 Trm cells were potent cytokine-producers, exhibiting a polyfunctional (IFN-γ+ IL-2+ TNF-α+) profile, and efficiently expressed cytotoxic mediators after stimulation. These results suggest that SI CD8 Trm cells could be relevant targets for future oral vaccines and therapeutic strategies for gut disorders.

  Study EGAS00001003676

• Familial adult myoclonic epilepsy in Sri Lankan and Indian families

  Familial adult myoclonic epilepsy 1 (FAME1), first recognised in Japanese families, was recently shown to be caused by a TTTCA repeat insertion in intron 4 of SAMD12 on chromosome 8. We performed whole genome sequencing on two families with FAME, one of Sri Lankan origin and the other of Indian origin, and identified a TTTCA repeat insertion in SAMD12 in both families. Haplotype analysis revealed that both families shared the same core ancestral haplotype reported in Japanese and Chinese families with FAME1. Mutation dating, based on the length of shared haplotypes, estimated the age of the ancestral haplotype to be approximately 670 generations, or 17,000 years old. Our data extend the geographic range of this repeat expansion to Southern Asia and potentially implicate an even broader regional distribution given the age of the variant. This finding suggests patients of Asian ancestry with suspected FAME should be screened for the SAMD12 TTTCA expansion.

  Study EGAS00001004012

• Immune profiling reveals enrichment of distinct immune signatures in high-risk oral potentially malignant disorders

  Oral epithelial dysplasia (OED) associates with an increased risk of oral squamous cell carcinoma (OSCC). Changes in the immune landscape of OED that accompany transformation remain understudied. We evaluated gene co-expression in benign lesions (fibroepithelial polyps; FEP), high-risk OED (mod-sev OED) and early stage OSCC. We demonstrate that transcripts indicating immune responses are enriched in high-risk OED. In particular, immune signatures representing high lymphocyte infiltration and cytotoxic responses are prominent, indicating active immunosurveillance. Expression of immune checkpoint molecules was enriched in high-risk OED and further increased in early stage OSCC. Seventy percent of high-risk OED displayed a T cell-inflamed phenotype histology suggesting that they might be responsive to anti-PD-1 immunoprevention. Other immunoprevention strategies are required for the 30% of high-risk OED are not immune-enriched. Overall, our data point the way for new approaches to non-surgical management of OED.

  Study EGAS00001005520

• Dissecting features of epigenetic variants underlying cardiometabolic risk using full-resolution epigenome profiling in regulatory elements

  Sparse profiling of CpG methylation in blood by microarrays have identified epigenetic links to common diseases. We apply methylC-capture sequencing (MCC-Seq) in a clinical population of ~200 adipose tissue and matched blood samples (Ntotal ~400), providing high-resolution methylation profiling (>1.3M CpGs) at regulatory elements. We link methylation to cardiometabolic risk through associations to circulating plasma lipid levels and identify lipid-associated CpGs with unique localization patterns in regulatory elements. We show distinct features of tissue-specific versus tissue-independent lipid-linked regulatory regions by contrasting with parallel assessments in ~800 independent adipose tissue and blood samples from the general population. We follow-up on adipose-specific regulatory regions under (1) genetic and (2) epigenetic (environmental) regulation via integrational studies. Overall, the comprehensive sequencing of regulatory element methylomes reveals a rich landscape of functional variants linked genetically as well as epigenetically to plasma lipid traits.

  Study EGAS00001003415