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• Somatic Mutation in Normal Bladder Study

  Adult human somatic tissues are landscapes of mutant clones, but little is known about how these landscapes are shaped by known cancer risk factors. The goal of this study was to determine how the main bladder cancer risk factors (sex and smoking) change the mutational landscape of the normal urothelium. We collected normal urothelium from 45 donors at autopsy by brushing ~2cm² of the surface epithelium in the upper part (dome) and lower part (trigone) of the bladder (79 samples total). To identify mutations with high sensitivity we employed ultradeep (~5000x) DNA error-correcting duplex-sequencing on a panel of 15 protein coding genes and the TERT promoter, previously described to be under positive selection in normal urothelium or relevant to bladder cancer. We identified thousands of clonal driver mutations, which demonstrate pervasive positive selection in the normal urothelium. Males showed a significantly higher number of driver truncating mutations in RBM10, CDKN1A and ARID1A, despite similar rates of non-coding mutations, indicating stronger positive selection and clonal expansion in the male urothelium. We also discovered clonal expansions driven by activating TERT promoter mutations in the normal bladder, which were strongly associated with age and smoking. These results demonstrate that sex and smoking shape the clonal landscape of the normal bladder and indicate that the high mutational content of normal tissues might be leveraged to study the effect of mutations in vivo (i.e. natural saturation mutagenesis). We will deposit in dbGaP de-identified relevant clinical information from the 45 donors and the fastq files derived from duplex sequencing of the 79 samples in the study. We will also deposit fastq files derived from duplex sequencing of 3 control cord blood samples.

  Study phs004105

• Tracking the evolution of Therapy-Related Myeloid Neoplasms using chemotherapy signatures

  Patients treated with cytotoxic therapies, including autologous stem cell transplantation, are at risk for developing therapy-related myeloid neoplasms (tMN). Pre-leukemic clones (i.e., clonal hematopoiesis; CH) are detectable years before the development of these aggressive malignancies, though the genomic events leading to transformation and expansion are not well-defined. Here, leveraging distinctive chemotherapy-associated mutational signatures from whole-genome sequencing data and targeted sequencing of pre-chemotherapy samples, we reconstruct the evolutionary life-history of 39 therapy-related myeloid malignancies. A dichotomy is revealed, in which neoplasms with evidence of chemotherapy-induced mutagenesis from platinum and melphalan are hypermutated and enriched for complex structural variants (i.e., chromothripsis) while neoplasms with non-mutagenic chemotherapy exposures are genomically similar to de novo acute myeloid leukemia. Using chemotherapy-associated mutational signatures as temporal barcodes linked to a discrete clinical exposure in each patient’s life, we estimate that several complex events and genomic drivers are acquired after chemotherapy is administered. For patients with prior multiple myeloma who were treated with high-dose melphalan and autologous stem cell transplantation, we demonstrate that tMN can develop from either a reinfused CH clone that escapes chemotherapy exposure and is selected following reinfusion, or from TP53-mutant CH that survives direct myeloablative conditioning and acquires melphalan-induced DNA-damage. Overall, we reveal a novel mode of tMN progression that is not reliant on direct mutagenesis or even exposure to chemotherapy. Conversely, for tMN that evolve under the influence of chemotherapy-induced mutagenesis, distinct chemotherapies not only select pre-existing CH, but also promote the acquisition of recurrent genomic drivers.

  Study EGAS00001006903

• Whole genome sequencing of tumor samples from advanced pre-treated NSCLC patients recruited in a phase I/II single-arm trial utilising CVA21, an oncolytic coxsackie virus, in combination with pembrolizumab.

  Acquired or de novo resistance to immune checkpoint inhibitors occurs in the majority of advanced non-small cell lung cancers (NSCLC). There is an unmet need to improve outcomes for patients with this condition. Oncolytic viruses represent an attractive treatment approach due to their dual activity in inducing tumor cell lysis directly and potentially augmenting anti-tumor immunity. Here, we present the safety, efficacy and translational findings from a phase I/II single-arm trial utilising CVA21, an oncolytic coxsackie virus, in combination with pembrolizumab in patients with advanced pre-treated NSCLC. We performed paired biopsies pre- and post-treatment in 10 patients who received intravenous CVA21 and pembrolizumab, 8 of whom had prior treatment with immune checkpoint inhibitor (ICI) therapy. Whole genome sequencing and spatial proteomics were performed to comprehensively characterise the response to CVA21. Combination CVA21/pembrolizumab (anti PD-1) therapy was well tolerated with no serious treatment-related adverse events. Partial responses were seen in two patients with prior acquired anti-PD-1 resistance and disease stabilisation in 6 patients, giving a clinical benefit rate of 80%. High baseline tumor mutational burden and PD-L1 expression were observed in patients with better response to treatment. Interestingly, an increase in antigen presentation and CD8+ T cell infiltration was observed on treatment compared with baseline in patients with better progression-free survival. This study demonstrates the potential of CVA21 to modulate the immunogenicity of tumor cells and remodelling the tumor microenvironment, providing novel insights for patient selection for trials involving novel immunotherapeutic approaches.

  Study EGAS00001008258

• Ultra-Fast Patient-Derived Xenografts Identify Functional and Spatial Tumour Heterogeneities that Drive Therapeutic Resistance

  Treatment of patients with advanced cancers increasingly relies on agents targeting specific molecular or cellular aberrations. These expensive therapies often fail because of pre-existing drug resistance. Intra-tumoural molecular and micro-environmental heterogeneity resistance can create resistance in one or more subclones of a tumour, leading to spatio-functional heterogeneity. We created a rapid approach for quantifying tumour functional phenotypes based on tumour-implantation into the chorioallantoic membrane of chick embryos. We apply this technique to 43 separate biopsies from 6 patients with renal cell carcinoma (RCC) with 36 replicates per biopsy, achieving a 93.6% engraftment rate (1449/1548). Challenging these models with sunitinib, an anti-angiogenic therapy frequently used in RCC allowed us to quantify spatio-functional heterogeneity in individual patients. Prediction of de novo drug resistance in advanced cancer patients would facilitate precision medicine by tailoring systemic therapies and improve clinical trial design outcome measures. We describe a patient-derived xenograft platform where fresh patient tumour samples are implanted into the chorioallantoic membrane (CAM) of chick embryos at high engraftment efficiencies, permitting large scale “tumour avatar” studies to guide the selection of drugs and to predict clinical drug resistance outcomes in renal cell carcinoma (RCC) patients. Applying this “tumour avatar” model in a co-clinical trial on RCC patients, we observe intra-tumoural functional heterogeneity with sunitinib treatment, revealing the presence of drug-resistant clones prior to the initiation of therapy. Multi-regional exome Matched DNA sequencing of the primary tumour and metastases revealed DNA mutational signatures associated with sunitinib resistance at baseline and after treatment. These These studies demonstrate a rapid and affordable method of findings confirm the existence of genetic and surveying functional tumour heterogeneities heterogeneity.

  Study EGAS00001002627

• Resolving the Full Spectrum of Human Genome Variation using Linked-Reads

  Large-scale population based analyses coupled with advances in technology have demonstrated that the human genome is more diverse than originally thought. Standard short-read approaches, used primarily due to accuracy, throughput and costs, fail to give a complete picture of a genome. They struggle to identify large, balanced structural events, cannot access repetitive regions of the genome and fail to resolve the human genome into its two haplotypes. Here we describe an approach that retains long range information while harnessing the power of short reads. Starting from only 1ng of DNA, we produce barcoded short read libraries. The use of novel informatic approaches allows for the barcoded short reads to be associated with the long molecules of origin producing a novel datatype known as 'Linked-Reads'. This approach allows for simultaneous detection of small and large variants from a single Linked-Read library. We have previously demonstrated the utility of whole genome Linked-Reads (lrWGS) for performing diploid, de novo assembly of individual genomes. In this manuscript, we show the utility of reference based analysis using a single Linked-Read library for full spectrum genome analysis. We demonstrate the ability of Linked-Reads to reconstruct megabase scale haplotypes and to recover parts of the genome that are typically inaccessible to short reads, including phenotypically important genes such as STRC, SMN1 and SMN2. We demonstrate the ability of both lrWGS and Linked-Read Whole Exome Sequencing (lrWES) to identify complex structural variations, including balanced events, single exon deletions, and single exon duplications. The data presented here show that Linked-Reads provide a scalable approach for comprehensive genome analysis that is not possible using short reads alone.

  Study EGAS00001003121

• We evaluate the potential for routine WGS using ONT by sequencing the well-characterised reference sample NA12878 and the genome of an individual with ataxia-pancytopenia syndrome accompanied by severe immune dysregulation.

  Whole-genome sequencing (WGS) is becoming widely used in clinical medicine in diagnostic contexts and to inform treatment choice. While current sequencing technologies have been extremely successful, their reliance on short read lengths necessarily involves some limitations: accurately assaying certain genomic regions and classes of variation can be problematic. Recent advances in throughput and cost have made WGS using the Oxford Nanopore Technologies (ONT) MinION long-read single-molecule sequencer a potential solution to these challenges. Here we evaluate the potential for routine WGS using ONT by sequencing the well-characterised reference sample NA12878 and the genome of an individual with ataxia-pancytopenia syndrome accompanied by severe immune dysregulation, to 82× and 30× respectively. For NA12878, we evaluated single-nucleotide variant (SNV) calls based on data from multiple base-calling algorithms. We demonstrate that phasing metrics from a novel, reference panel-free, long-read-based method can improve variant-calling performance from otherwise modest levels, resulting in a false discovery rate of 7.1% and false negative rate of 8.5%; remaining errors are concentrated near homopolymers and in regions of reduced sequencing coverage. In the clinical sample, we are able to identify and directly phase two non-synonymous de novo variants in SAMD9L, (OMIM #159550) inferring that they both lie on a common paternal haplotype by overlap with parental genotypes at nearby common variants. This work demonstrates that methodological innovation can substantially reduce variant-calling error rates in ONT data, and that with on-going improvements in throughput, base-calling and dedicated long-read-based SNV-calling methodology, ONT offers promise as an option for clinical WGS.

  Study EGAS00001003469

• Circulating Tumor DNA in Checkpoint Inhibitor treated Lung Cancer

  BackgroundImmune checkpoint inhibitors (ICI) are increasingly used to treat non-small cell lung cancer (NSCLC) and there is a critical need for novel predictive/prognostic biomarkers. We evaluated whether dynamics of circulating tumor (ctDNA) can identify NSCLC patients who benefit from single agent ICI.MethodsCirculating cell-free DNA (cfDNA) from 177 patients was analyzed prior ICI and at first treatment evaluation using a Commercial 77-gene panel. We performed de novo mutation calling and corrected for variants related clonal hematopoiesis. ctDNA changes were evaluated as predictors of tumor response estimated with PET/diagnostic CT and survival.ResultsMutations were detected in 159 of 174 (91.4%) analyzable patients. Most responders presented decreasing ctDNA levels, while non-responders had mixed molecular responses. Patients with a >50% molecular ctDNA decrease or no detectable ctDNA demonstrated improved PFS (5.9 versus 1.8 months; HR 0.49, 95%CI, 0.35-0.67, P < .001), and OS (18.9 versus 9.9 months; HR 0.41, 95%CI, 0.30-0.57, P < .001) compared to patients not achieving these endpoints. Clearance of mutations (and no detected ctDNA) had a similar effect (PFS HR 0.45, 95%CI, 0.32-0.61, P < .001; OS 0.37,95%CI, 0.27-0.52, P < .001) compared to patients having stable or additional mutations. After adjusting for clinical factors both >50% ctDNA decrease (HR 0.4, 95% CI, 0.3-0.6, P < .001) and clearance of mutations (HR 0.42, 95% CI, 0.3-0.6, P < .001) remained independent predictors for PFS and OS.ConclusionsctDNA testing reveals significant prognostic albeit not predictive information in ICI treated advanced NSCLC. This relevant information can be obtained with a commercial easy-to-implement ctDNA test that will facilitate extensive testing.

  Study EGAS00001004847

• Characterization of DLBCL with a PMBL gene expression signature

  Primary mediastinal large B-cell lymphoma (PMBL) is a type of aggressive B-cell lymphoma that typicallyaffects young adults, characterized by presence of a bulky anterior mediastinal mass. Lymphomas withgene expression features of PMBL have been described in non-mediastinal sites, raising questions abouthow these tumors should be classified.Here, we investigated whether these "non-mediastinal PMBLs" are indeed PMBLs or instead represent adistinct group within DLBCL. From a cohort of 325 de novo DLBCL cases, we identified tumors frompatients without evidence of anterior mediastinal involvement that expressed a PMBL expression signature(nm-PMBLsig-pos, n=16, 5%). The majority of these tumors expressed MAL and CD23 – proteins typicallyobserved in bona fide PMBL (bf-PMBL). Evaluation of clinical features of nm-PMBLsig-pos cases revealedclose associations with DLBCL, and the majority displayed a germinal center B-cell-like cell-of-origin(GCB). In contrast to bf-PMBL, nm-PMBLsig-pos patients presented at an older age, did not show pleuraldisease, and bone/bone marrow involvement was observed in three cases. However, while clinicallydistinct from bf-PMBL, nm-PMBL-sig-pos tumors resembled bf-PMBL at the molecular level with upregulationof immune response, JAK-STAT, and NF-kB signatures. Mutational analysis revealed frequent somatic genemutations in SOCS1, IL4R, ITPKB and STAT6, as well as CD83 and BIRC3, with the latter genes beingsignificantly more frequently affected than in GCB-DLBCL and bf-PMBL.Our data establish nm-PMBLsig-pos lymphomas as a group of DLBCL with distinct phenotypic and geneticfeatures, and potential implications for gene expression- and mutation-based subtyping of aggressive Bcell lymphoma and related targeted therapies.

  Study EGAS00001005057

• Single_cell_measurements_to_characterise_B_cell_repopulation_in_SLE_after_rituximab_therapy__a_pilot_study

  Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterised by autoantibodies against DNA and nuclear proteins. Kidney involvement (lupus nephritis) is one of the most serious manifestations and is the leading determinant of long-term prognosis. Therapies targeting B cells (e.g. rituximab) are used in the treatment of severe lupus, including lupus nephritis. Rituximab is a monoclonal antibody that targets the CD20 surface protein on the surface of B cells. Following rituximab treatment, there is near complete depletion of B cells in peripheral blood. Peripheral blood B cells slowly return over a period of months. The speed of repopulation varies but typically B cells have returned to baseline levels between 9 and 12 months post rituximab therapy. Return of B cells may be associated with increased disease activity, but this is not always the case. For example, some patients remain in clinical remission for several years following one cycle of rituximab, despite B cell repopulation, whilst others relapse as soon as the B cell number reaches the normal range. The reasons for the different response remain unclear. The B cell subpopulations that return following B cell depleting therapy have been previously studied using flow cytometry but have never been characterised using single cell -omic technologies and thus no data on their molecular signatures are available. Previous studies have shown that naïve B cells were the predominant repopulating B-cells, indicating de novo production of B cells. Obtaining an integrated understanding of the molecular programs of these cells at the single cell level will enhance our ability to predict disease outcome, to identify patients with poorer B-cell drug responses, understand disease heterogeneity and design more effective B-cell targeted therapies approach.

  Study EGAS00001006798

• OpACIN-neo – A Multicenter Phase 2 Study to identify the Optimal neo-Adjuvant Combination scheme of Ipilimumab and Nivolumab – Whole exome sequencing

  The outcome of patients with macroscopic stage III melanoma is poor. Neoadjuvant treatment with ipilimumab plus nivolumab at the standard dosing schedule induced pathological responses in a high proportion of patients in two small independent early-phase trials. However, toxicity of the standard ipilimumab plus nivolumab dosing schedule was high, preventing its broader clinical use. The aim of the OpACIN-neo study (NCT02977052) to identify a dosing schedule of ipilimumab plus nivolumab that is less toxic but equally effective. 86 patients with macroscopic stage III melanoma were randomised to one of three dosing schedules: arm A, two cycles of ipilimumab 3 mg/kg plus nivolumab 1 mg/kg once every 3 weeks; arm B, two cycles of ipilimumab 1 mg/kg plus nivolumab 3 mg/kg once every 3 weeks; or arm C, two cycles of ipilimumab 3 mg/kg once every 3 weeks directly followed by two cycles of nivolumab 3mg/kg once every 2 weeks. Within the first 12 weeks, grade 3–4 immune-related adverse events were observed in 12 (40%) of 30 patients in group A, six (20%) of 30 in group B, and 13 (50%) of 26 in group C. Pathological responses occurred in 24 (80%) patients in group A, 23 (77%) in group B, and 17 (65%) in group C. The 2-year estimated relapse-free survival was 84% for all patients, 97% for patients achieving a pathologic response and 36% for nonresponders (p<0.001). High tumor mutational burden (TMB) and high interferon-gamma-related gene expression signature score (IFN-γ score) were associated with pathologic response and low risk of relapse; pathologic response rate (pRR) was 100% in patients with high IFN-γ score/high TMB, patients with high IFN-γ score/low TMB or low IFN-γ score/high TMB had pRRs of 91% and 88%; while patients with low IFN-γ score/low TMB had a pRR of only 39%. These data demonstrate long-term benefit in patients with a pathologic response and show the predictive potential of TMB and IFN-γ score.

  Study EGAS00001004832

• OpACIN-neo – A Multicenter Phase 2 Study to identify the Optimal neo-Adjuvant Combination scheme of Ipilimumab and Nivolumab – RNA sequencing

  The outcome of patients with macroscopic stage III melanoma is poor. Neoadjuvant treatment with ipilimumab plus nivolumab at the standard dosing schedule induced pathological responses in a high proportion of patients in two small independent early-phase trials. However, toxicity of the standard ipilimumab plus nivolumab dosing schedule was high, preventing its broader clinical use. The aim of the OpACIN-neo study (NCT02977052) to identify a dosing schedule of ipilimumab plus nivolumab that is less toxic but equally effective. 86 patients with macroscopic stage III melanoma were randomised to one of three dosing schedules: arm A, two cycles of ipilimumab 3 mg/kg plus nivolumab 1 mg/kg once every 3 weeks; arm B, two cycles of ipilimumab 1 mg/kg plus nivolumab 3 mg/kg once every 3 weeks; or arm C, two cycles of ipilimumab 3 mg/kg once every 3 weeks directly followed by two cycles of nivolumab 3mg/kg once every 2 weeks. Within the first 12 weeks, grade 3–4 immune-related adverse events were observed in 12 (40%) of 30 patients in group A, six (20%) of 30 in group B, and 13 (50%) of 26 in group C. Pathological responses occurred in 24 (80%) patients in group A, 23 (77%) in group B, and 17 (65%) in group C. The 2-year estimated relapse-free survival was 84% for all patients, 97% for patients achieving a pathologic response and 36% for nonresponders (p<0.001). High tumor mutational burden (TMB) and high interferon-gamma-related gene expression signature score (IFN-γ score) were associated with pathologic response and low risk of relapse; pathologic response rate (pRR) was 100% in patients with high IFN-γ score/high TMB, patients with high IFN-γ score/low TMB or low IFN-γ score/high TMB had pRRs of 91% and 88%; while patients with low IFN-γ score/low TMB had a pRR of only 39%. These data demonstrate long-term benefit in patients with a pathologic response and show the predictive potential of TMB and IFN-γ score.

  Study EGAS00001004833

• Nurminen et al ("GP2Men") Study Primary and Metastatic Prostate Cancer Whole Genome Sequence Data

  We used novel processing techniques to obtain whole genome data together with 3D anatomic and histomorphologic analysis in two men (GP5 and GP12) with high risk PrCa undergoing radical prostatectomy. A total of 22 whole genome-sequenced sites (16 primary cancer foci and 6 lymph node metastatic) were analyzed using evolutionary reconstruction tools and spatio-evolutionary models. Probability models were used to trace spatial and chronological origins of the primary tumor and metastases, chart their genetic drivers, and distinguish metastatic and non-metastatic subclones.

  Study EGAS50000000005

• SUM-seq data for spontaneously differentiated iPSCs to EBs

  Clonal CRISPRi/a hiPS cell lines were plated to induce spontaneous differentiation in KSR medium (KSR medium: knockout DMEM/F12 (Gibco, 12660012), 2 mM GlutaMAX, 1× NEAAs, 20% knockout serum replacement (Gibco, 10828010) and 0.1 mM β-mercaptoethanol (Merck-Millipore)). non-TC plates and dishes were used. At the day of plating, KSR medium was supplemented with Y-27632 (10 µM). The next day, medium was replaced with fresh KSR medium. Medium changes were performed every other day. EBs were transferred to 15 ml conical tubes and allowed to sediment at room temperature for 5 min. The supernatant was replaced with fresh KSR medium and cells were transferred onto non-TC treated plates and maintained at 37 °C, 5% CO2. On day 12 of differentiation, the KSR medium was replaced with Essential 6 medium (Gibco, A1516401). EBs were collected and subjected to SUM-seq library preparation as described in Lobato-Moreno et al. 2025 Nature Methods.

  Dataset EGAD50000001589

• Single-cell RNA sequencing of control and Notch inhibitor treated HCC PDX model

  We generated single-cell RNA-seq data for anti-Notch2 (n=3), anti-JAG1 (n=1) and anti-gD (n=4) treated LIV78 tumors. NCR nude mice bearing similarly sized LIV78 tumors (volumes between 300-900mm3) were injected intravenously with 30mg/kg body weight of either anti-Notch2, anti-Jag1 or anti-gD control antibody 72hrs prior to tumor harvest and tumor cell isolation. Following the depletion of CD45+ and Ter119+ cells, the samples were processed for single-cell RNA-seq (scRNAseq) as described previously (Long et al., 2019) using the Chromium Single Cell 3’ Library and Gel bead kit v2, following the manufacturer’s manual. cDNAs and libraries were prepared following the manufacturer’s manual (10X Genomics). Libraries were profiled by Bioanalyzer High Sensitivity DNA kit (Agilent Technologies) and quantified using Kapa Library Quantification Kit (Kapa Biosystems, Wilmington, MA). Each library was sequenced in one lane of HiSeq 2500 (Illumina) following the manufacturer’s sequencing specification (10X Genomics) to produce the resulting FASTQ files.

  Dataset EGAD50000000738

• Mechanisms of patient response to Dabrafenib in Melanoma

  Samples will be from the BRF113683 (BREAK-3) study which is a Phase III Randomized, Open-label Study Comparing GSK2118436 to Dacarbazine (DTIC) in Previously Untreated Subjects With BRAF Mutation Positive Advanced (Stage III) or Metastatic (Stage IV) Melanoma (n=250 enrolled)*NGS [Agilent capture (Sanger V2 panel): 360 genes and 20 gene fusions; Illumina HiSEQ Sequencing]*CNV: [via NGS or Affy SNP 6.0 or Illumina Omni (TBD)]Bioinformatics: Analysis will be performed using core Sanger informatics pipelines similar to those previously described (Papaemmanuil E et al. (2013) Blood. 22:3616 -3627). Briefly, copy number analysis will be performed using the ASCAT algorithm, and base substitutions, small insertions and deletions using the CAVEMAN and Pindel algorithms, respectively. Statistical approaches including generalized linear models will be used to predict clinical variables such as maximum clinical response and duration of response using genetic data. Sanger and EBI to conduct analysis; Raw data and correlation with clinical endpoints to be analyzed by both EBI/Sanger and GSK (unique pipeline analyses to increase call confidence)

  Dataset EGAD00001001375

• Tagged-amplicon deep sequencing

  This dataset contains a collection of tumour samples from high grade serous ovarian carcinoma patients with recurrent disease collected near the point of diagnosis as well as tumour samples collected after patient relapse upon or some time after study entry. Whole blood samples were also collected from patients at study entry for the purpose of germline variant detection. The majority of diagnosis samples were preserved in neutral buffered formalin whereas the majority of post-relapse samples were preserved in universal molecular fixative (UMFIX, Sakura Finetek USA, Inc). DNA was extracted and the tagged-amplicon deep sequencing assay was applied (TAm-Seq, Forshew et al. 2002, Sci Transl Med) with the aid of fluidigm access array technology. Targeted loci were sequenced at very high depths (typically >100X) for the detection of both somatic and germline variants. Sequenced reads were aligned to the GRCh37 reference genome (release hs37d5).

  Dataset EGAD00001011058

• Targeted Myeloid DNA-Panelsequencing, DKFZ

  Targeted myeloid DNA-Panelsequencing from purified bone marrow blasts of 104 treatment naive AML patients from the discovery cohort. For more details, we refere to Jayavelu, Wolf, Buettner et al. Libraries were prepared from 40 ng DNA using the QIASeq Human Myeloid Neoplasms Panel (Qiagen) according to the manufacturer’s protocol. Samples were tagged with the QIAseq 96-Unique Dual Index Set A for Illumina platforms (Qiagen) to yield unique combinations of i5 and i7 barcodes for each sample. Sample fragment size distribution and concentration was estimated using the Agilent High Sensitivity DNA kit on a 2100 Bioanalyzer (Agilent). Samples were pooled in an equimolar fashion, denatured, and diluted to 1.5 pM according to Illumina’s recommendations. The diluted library was sequenced on a NextSeq 500 benchtop sequencer (Illumina) using NextSeq High Output cartridges. Demultiplexing was performed using the BaseSpace cloud platform (Illumina).

  Dataset EGAD00001008501

• The effect of freezing delay of cell-type specific transcriptome responses in human brain via snRNA-seq

  In other analysis in the current manuscript, we find a similar gene signature (to dissociation based artifacts in mouse and human tissue) is present in post-mortem microglia and astrocytes, across all snRNA-seq datasets analyzed, although it is highly variable between subjects. ​ Using acutely-resected neurosurgical tissue, we performed single-nucleus RNA-seq and reveal that a similar signature can be detected in microglia following prolonged exposure to room temperature. Tissue handling and methods details, as well as sequencing and analysis details) can be found in the methods section of related manuscript (Marsh et al., 2022). Together, these results suggest that the presence of this signature in post-mortem brain samples may be the result of a combination of acute pre-mortem (agonal state, cause of death, comorbidities, etc.) and post-mortem (post-mortem interval (PMI), storage time, RNA quality, etc.) variables and may not represent normally present cell state.

  Dataset EGAD00001008541

• RNA-Seq of Whole Blood from Patients with Intracranial Aneurysms

  Rupture of intracranial aneurysms (IAs) causes intracranial hemorrhaging that leads to high rates of neurological deficits and death. Although rupture rates are low, clinicians must decide whether to treat or monitor these potentially dangerous lesions. In the current clinical practice, the most common metric to measure risk of rupture is aneurysm size. However, clinical data show that small aneurysms can also rupture. As a result, alternative clinical stratification scores have been proposed, including the PHASES (Population, Hypertension, Age, Size, Earlier subarachnoid hemorrhage, and Site) score based on patient demographics and IA characteristics to stratify ruptured and unruptured IAs, and the Rupture Resemblance Score (RRS) that stratifies ruptured and unruptured IAs based on hemodynamic and morphological properties. However, all metrics require imaging on digital subtraction angiography (DSA), which is invasive, expensive, requires the use of X-rays, and is associated with transient or permanent neurological and non-neurological complications. We hypothesize that individuals with dangerous IAs have detectable gene expression differences in their blood that could be used as biomarkers to determine rupture risk. We propose to use whole blood transcriptomes to develop a "one-stop" diagnostic test that can detect the presence of IAs and determine the risk of rupture based on circulating RNA expression biomarkers using our prototype AneuScreenTM platform. We aim to develop and validate biomarkers to predict risk, as calculated by the currently-used metric of aneurysm size, the clinical PHASES score, and the RRS. Here, we collected blood samples from consented individuals who were receiving cerebral imaging at the Gates Vascular Institute (Buffalo, NY) for intracranial aneurysm. RNA extracted from blood samples (n=43) was subjected to RNA-sequencing and added to our existing database (n=44). When combined with our previous data, we had transcriptome data from 68 aneurysms (after additional sample filtering). This dataset was stratified into low- and high-risk according to IA size, PHASES score, and RRS score. Differentially expressed genes were identified and used to construct predictive models. Fastq files and basic demographic information for the 43 newly-sequenced intracranial aneurysm samples on this project will be available through dbGaP.

  Study phs003072

• Longitudinal Studies of Patients with Familial Platelet Disorder with Associated Myeloid Malignancy (FPDMM)

  FPDMM (OMIM #601399) is a rare autosomal dominant disease characterized by defective megakaryocytic development, thrombocytopenia, platelet functional defects, and a life-long risk of developing hematological malignancies. FPDMM is caused by germline mutations in the RUNX1 gene, which encodes a master regulator of hematopoiesis. The overall lifetime risk of hematological malignancies in FPDMM patients is 35 - 45%. It is hypothesized that mutations in other genes cooperate with the germline RUNX1 mutations for leukemia development. However, it is not clear which genes need to be mutated for leukemia to develop and which biomarkers or clinical tests can be used to predict which patients will progress to leukemia. Likewise, there are no treatments and/or preventative measures that can reduce or stop disease progression except for stem cell transplantation, which is not available for all patients and may be associated with significant complications. To address these questions, we launched a longitudinal natural history study of patients associated with RUNX1 germline mutations at the NIH Clinical Center in early 2019 (Trial Registration ID: NCT03854318). The goals of this study are to: (1) perform comprehensive longitudinal phenotyping of FPDMM patients to document the full spectrum of both hematopoietic and non-hematopoietic manifestations of the disease; and (2) perform extensive genomic analyses of FPDMM patient samples to identify cooperating genomic variants, which may confer growth advantage that leads to clonal expansion of affected hematopoietic cells that may serve as the precursor to malignancies. The data available through dbGaP include exome sequencing and RNA-seq data from these patients. Diverse germline mutation types were identified in the RUNX1 gene, including splice site mutations and large deletions, in addition to nucleotide substitutions and small indels. Somatic mutations, especially in genes associated with clonal hematopoiesis of indeterminate potential (CHIP), were found in a substantial proportion of FPDMM patients who were leukemia-free. The findings suggest that the genomes in these patients are prone to somatic mutations and monitoring the dynamic changes of these somatic mutations prospectively may provide a way to predict progression to malignancies.

  Study phs003075

• NHLBI GO-ESP: Heart Cohorts Exome Sequencing Project (Ischemic Stroke Genetic Study, ISGS)

  The NHLBI "Grand Opportunity" Exome Sequencing Project (GO-ESP), a signature project of the NHLBI Recovery Act investment, was designed to identify genetic variants in coding regions (exons) of the human genome (the "exome") that are associated with heart, lung and blood diseases. These and related diseases that are of high impact to public health and individuals from diverse racial and ethnic groups will be studied. These data may help researchers understand the causes of disease, contributing to better ways to prevent, diagnose, and treat diseases, as well as determine whether to tailor prevention and treatments to specific populations. This could lead to more effective treatments and reduce the likelihood of side effects. GO-ESP is comprised of five collaborative components: 3 cohort consortia - HeartGO, LungGO, and WHISP - and 2 sequencing centers - BroadGO and SeattleGO. The Ischemic Stroke Genetics Study (ISGS) is a study of newly onset cases (~600) with ischemic stroke (a stroke due to sudden interruption of blood flow to a part of the brain) compared with sex- and age-matched non-stroke participants. The study was conducted to determine the genes and their variants that contribute to an individual's risk of developing an ischemic stroke. The coordination of the recruitment and flow of the samples occurred at the Mayo Clinic, Jacksonville, FL, under the direction of James F. Meschia, MD. The University of Virginia (Stephen S. Rich, PhD) served as the analytic site for the genetic data. All GWAS data on ISGS participants have been deposited into dbGaP. As part of the NHLBI Exome Sequencing Project, DNA from a subset of ISGS participants will undergo exome sequencing. For the NHLBI ESP, a subset of 92 individuals with lacunar (small vessel) or atherosclerotic (large vessel) TOAST subtypes were selected from among all ISGS participants, excluding those individuals with TOAST subtypes of stroke of other etiology or of stroke with undetermined etiology. All 92 samples pass initial quality control metrics and 89 samples completed exome sequencing. A total of 75 participants with appropriate consent and variant calls had their genetic and phenotypic data deposited into dbGaP.

  Study phs000546

• Longitudinal Studies of Brain Structure and Function in MPS Disorders

  Mucopolysaccharidosis (MPS) types I, II, IV, VI and VII, are inherited diseases that result from the bodies inability to break down large sugar molecules that are by-products of metabolism. MPS affects most organs of the body and causes abnormalities in the liver, spleen, bones, and brain. We are studying the central nervous system (primarily the brain) so we can better understand the nature of the problems individuals with MPS I, II, IV, VI and VII have so we can find ways of better treating these problems. We would like to find out about the changes in the brain of individuals with MPS I, II, IV, VI and VII using data from MRI and neuropsychological tests. This is a longitudinal study so patients who enroll will need to be seen 3 times over 5 years. The longitudinal nature of the study allows us to make conclusions about how changes in some structures of the brain and changes in cognitive ability are related. The research objectives are: To identify abnormalities of brain (and central nervous system) structure and function in patients with MPS I, II, IV, VI and VII, regardless of how they are being treated or have been treated in the past; and to track disease progression over time. To examine the degree to which independent variables (e.g., age at first treatment, severity of disease, types of medical abnormalities, mutation, medical events, and sensory abnormalities) have an impact on both functional and structural outcomes as well as on quality-of-life. To identify, through longitudinal study, those measures that most accurately reflect the current disease state. This is a longitudinal study of 75-100 individuals with either MPS I, II, IV, VI and VII. Those participating in the study will be evaluated 3 times over 5 years. The primary site for this study is the University of Minnesota but there are an additional 6 centers in the United States and Canada that are also participating and will provide data for analysis. You will need to be able to travel to Minnesota or one of the participating centers in order to be a part of this study.

  Study phs001328

• Drug development for pediatric cancers: the importance of patient' s genomic data re-use

  Cancer in children is uncommon and the overall prognosis for most pediatric cancers is good. However, while the combined survival rates have improved over the last decades, certain childhood malignancies, such as high-grade gliomas or metastatic sarcomas, still remain incurable in most patients. New strategies for targeting those devastating diseases are imperative, and patient genomic data can become a key asset in the process [1]. The Pediatric Cancer Genome Project’s datasets At EGA, we store over 500 datasets with sequencing data from pediatric cancer patients. A remarkable case is the datasets belonging to the Pediatric Cancer Genome Project (PCGP) from St. Jude Children’s Research Hospital–Washington University (Table 1 - EGAS list). PCGP is an ambitious effort to identify the mutations that drive childhood cancer and find new cures. Those datasets include 600 patients with complete tumor and normal genomes from 15 different tumor types. PCGP datasets have been an important resource for various studies that pooled genomic data from different public and in-house datasets to perform extensive genomic characterizations and publish comprehensive pan-cancer pediatric studies [2],[3]. Other interesting pediatric cancer datasets are from the Pediatric Brain Tumor Consortium, Sickkids or ICGC PedBrain project. How data reuse impacts drug development for pediatric cancers Recently, in October 2024, Nature Communications published a work led by the University of Michigan where authors used data from EGA (PCGP) check the table below as part of their strategy to pursue the identification of new tumor vulnerabilities susceptible to becoming novel therapeutic targets in diffuse midline glioma. Diffuse midline gliomas (DMG) are treatment-resistant and uniformly fatal pediatric brain tumors. The prognosis of this brainstem tumor is dismal with a median overall survival of 9–12 months from diagnosis. In this study, the authors first developed a lab model of the disease and reanalyzed data from the EGA to identify genes potentially linked to tumor severity. This approach revealed involvement of specific metabolic pathways, suggesting new possibilities for therapeutic intervention. In mice, the study shows that the use of statins can improve survival. For a brief overview of the underlying science, diffuse midline gliomas present intratumor heterogeneity with subpopulations of less-differentiated oligodendrocyte precursors and more differentiated astrocytes. Authors established in vitro models to recapitulate both phenotypes and identify metabolic programs in both subpopulations. To determine the clinical relevance, the authors re-used gene expression data from 76 DMG patients identifying a gene signature predicting decreased overall survival. After extensive metabolic characterization of subpopulations, authors defined strategies to target specific metabolic vulnerabilities. Pre-clinical experiments in mice using OXPHOS inhibitors and statins showed a reduction in tumor burden and increased overall survival [4]. In this scenario, the re-use of high-quality patient’s sequencing data appeared as an advantageous strategy to support the clinical relevance of in-vitro and pre-clinical findings. On the other hand, in rare conditions such as childhood cancer, the availability of public datasets and the possibility of pooling data from different sources can be the only way to obtain impactful results that lead to improvements for patients. References Davidoff, A. M. Pediatric Oncology. Seminars in pediatric surgery 19, 225–233 (2010). Gröbner, S. N. et al. The landscape of genomic alterations across childhood cancers. Nature 555, 321–327 (2018). Venu Thatikonda et al. Comprehensive analysis of mutational signatures reveals distinct patterns and molecular processes across 27 pediatric cancers. Nature Cancer 4, 276–289 (2023). Mbah, N. E. et al. Therapeutic targeting of differentiation-state dependent metabolic vulnerabilities in diffuse midline glioma. Nature Communications 15, (2024). Datasets and Studies ID Title Access Policy Year EGAD00001000134 Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma McGill-DKFZ Pediatric Brain Tumour Consortium 2016 EGAS0000100192 Somatic Histone H3 Mutations in Diffuse Intrinsic Pontine Gliomas and Non-Brainstem Paediatric Glioblastomas St. Jude Children's Research Hospital - Washington University Pediatric Cancer Genome Project 2017 EGAS00001000575 Whole genome sequencing and whole exome sequencing of DIPG tumors and matched normal tissue The hospital for sick children ("SickKids") 2016 EGAD00001000792 Exome sequencing reads of paediatric glioblastoma McGill-DKFZ Pediatric Brain Tumour Consortium 2016 EGAD00001002006 Whole genome sequencing of paediatric glioblastoma in the ICGC PedBrain project ICGC PedBrain project 2016

  Blog drug-development-for-pediatric-cancers

• Comprehensive cancer predisposition gene testing in an adult multiple primary tumor series shows a broad range of deleterious variants and atypical tumor phenotypes

  Multiple primary tumors (MPT) affect a substantial proportion of cancer survivors and may result from various causes including inherited predisposition. Currently, germline genetic testing of MPT cases for cancer predisposition gene (CPG) variants is mostly targeted by tumor type. We ascertained pre-assessed MPT cases from genetics centers (defined as ≥2 primaries by age 60 years or ≥3 by 70) and performed whole genome sequencing (WGS) on 460 individuals from 440 families. Despite previous negative genetic assessment/molecular investigations, pathogenic variants in moderate and high-risk CPGs were detected in 67/440 (15.2%) of probands. WGS detected variants that would not be (or were not) detected by targeted resequencing strategies including structural variants at low frequency (6/440 (1.4%) of probands). In most individuals with a germline variant assessed as pathogenic or likely pathogenic (P/LP), at least one of their tumor types was characteristic of variants in the relevant CPG. However, in 29 probands (42.2% of those with a P/LP variant) the tumor phenotype appeared discordant. The frequency of individuals with truncating or splice site CPG variants and at least one discordant tumor type was significantly higher than a control population (χ2=43.642 P=<0.0001). 2/67 (3%) of probands with P/LP variants had evidence of multiple inherited neoplasia allele syndrome (MINAS) with deleterious variants in two CPGs. Summing together variant detection rates from a similarly ascertained previous MPT case series, the present results suggest that first-line comprehensive CPG analysis in a clinical genetics referral-based MPT cohort would detect a deleterious variant in about a third of cases.

  Dataset EGAD00001004088

• PREDICT-HD Huntington Disease Study

  The purpose of this project is to make clinical measurements from the PREDICT-HD consortium available through the dbGaP mechanism. The phenotype data will first be converted into a community open standard and subsequently exported to dbGaP for archival and open access distribution of the results of the studies. This will permit members of the scientific community to utilize a permanent resource for investigating the interactions of phenotypes upon an international cohort of early Huntington Disease. In version 2 cut of the data we provided HD CAG repeat lengths for both alleles as well as enrollment age of all participants. We have also generated unique identifiers prospectively compatible with the larger initiative GWAS in Huntington's Disease project (also on DbGaP). As such, the version 1 cut of the data was mainly proof of concept and should be deprecated. Going forward, all updates will add-on to version 2 cut of the data. In version 3 cut of the data, we provided baseline or the first usable MRI T1-weighted imaging analysis subcortical and cortical segmentations and cortical parcellations based on a customized Freesurfer 5.2 pipeline developed at The University of Iowa. The customizations to the standard pipeline were mainly to improve bias field correction and image normalization such that segmentation of gray, white, internal csf, dura and surface CSF are optimized for the Freesurfer pipeline. There are 1111 subjects with results in this data release. In version 4 cut of the data, we provided all longitudinal clinical measurements for all subjects (total of 1476) assessmented or enrolled through the end of 2013. Additionally, we are providing measurements on 39 baseline FDG PET images spatially normalized by SPM5 into MNI space, relative regional metabolic values computed in 120 volumes of interest (VOI) defined in the Automated Anatomical Labeling (AAL) Atlas (Tzourio-Mazoyer et al. 2002), and global metabolic values calculated by SPM standard mean voxel value (within per image fullmean/8 mask). This project is a funded ancillary study of PREDICT-HD. In version 5 cut of the data, we provided the first of many forthcoming results from ancilliary studies of PREDICT-HD. In this data cut, we provide individual subject results derived from structural MRI data. The earliest MRI session for each subject was used. The results summarized represent source based morphometry loading coefficients for 23 components (see: "Patterns of Co-Occurring Gray Matter Concentration Loss across the Huntington Disease Prodrome", Ciarochi et al., 2016, Front Neurol. 2016; 7: 147, Published online 2016 Sep 21. doi: 10.3389/fneur.2016.00147]. In this version 6 cut of the data, we provide a full set of derived data, more than 10,000 raw MRI images, and ancillary study data sets. For sample information please link to: PREDICT-HD Biospecimen Resources

  Study phs000222