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• Whole genome and transcriptome analysis of a sporadic and recurring parathyroid carcinoma

  Parathyroid carcinoma is an extremely rare endocrine malignancy with an estimated incidence of less than 1 per million population. Excessive secretion of parathyroid hormone, extremely high serum calcium level and the deleterious effects of hypercalcemia are the clinical manifestations of the disease. Up to 60% of patients develop multiple disease recurrences and although long-term survival is possible with palliative surgery, permanent remission is rarely achieved. Molecular drivers of sporadic parathyroid carcinoma have remained largely unknown. Previous studies, mostly based on familial cases of the disease, suggested a potential role for the tumor suppressor MEN1 and proto-oncogene RET in benign parathyroid tumorigenesis while the tumor suppressor HRPT2 and proto-oncogene CCND1 may also act as drivers in parathyroid cancer. Here, we report the complete genomic analysis of a sporadic and recurring parathyroid carcinoma. Mutational landscapes of the primary and recurrent tumor specimens were analyzed using high throughput sequencing technologies. Such molecular profiling allowed for identification of somatic mutations never previously identified in this malignancy. These included single nucleotide point mutations in well characterized cancer genes such as mTOR, MLL2, CDKN2C and PIK3CA. Comparison of acquired mutations in patient-matched primary and recurrent tumors revealed loss of PIK3CA activating mutation during the evolution of the tumor from the primary to the recurrence. Structural variations leading to gene fusions and regions of copy loss and gain were identified at a single-base resolution. Loss of short arm of chromosome 1 along with somatic mis-sense and truncating mutations in CDKN2C and THRAP3, respectively, provide new evidence for the potential role of these genes as tumor suppressors in parathyroid cancer. The key somatic mutations indentified in this study can serve as novel diagnostic markers as well as therapeutic targets.

  Study EGAS00001000484

• Integrated Genomic, Epigenetic, and Expression Analyses of Ovarian Cancer Cell Lines

  Epithelial ovarian carcinoma of all subtypes has one of the worst outcomes of any human cancer. To improve our understanding of underlying biology and therapeutic possibilities in this disease, we performed whole genome sequencing, methylation, and expression analyses of a comprehensive set of 47 ovarian cancer cell lines representing different histologic tumor subtypes. Given the challenges of genomic analyses in tumors without matched normal samples, we developed novel approaches for detection of somatic alterations and used these to identify sequence, copy number, and rearrangement changes and integrated these with genome-wide epigenetic and expression alterations. Ovarian cancer cell lines had molecular changes in genes involved in cell cycle, PI3K, RAS, BRCA, chromatin regulating, and p53 pathways, and displayed global mutation and methylation changes similar to primary ovarian carcinoma. We identified alterations not previously implicated in ovarian cancer including amplification or overexpression of ASXL1 and H3F3B, deletion or underexpression of CDC73 and members of the TGF beta receptor pathway, including TGFBR2, SMAD3 and SMAD4, in-frame rearrangements of YAP1-MAML2 and IKZF2-ERBB4, and fusions involving MET, NF1, FBXW7 and CCND1. Integrating cell line alterations with dose-response data using three targeted therapies revealed novel molecular dependencies, including increased sensitivity of tumors with PIK3CA and PPP2R1A alterations to PI3K inhibitor GNE-493, those with MYC amplifications to PARP inhibitor BMN673, and SMAD4 alterations to MEK inhibitor MEK162. Genome-wide analyses revealed intrachromosomal rearrangements as an improved measure of sensitivity to PARP inhibition compared to the homologous recombination defect (HRD) score. This study provides a comprehensive resource of molecular information for ovarian cancer cell line models and a pharmacogenomic platform for developing rational cancer therapeutic strategies.

  Study EGAS00001002998

• Evolutionary dynamics of residual disease in human glioblastoma

  Glioblastoma is the most common and aggressive adult brain malignancy against which conventional surgery and chemoradiation provide limited benefit. Even when a good treatment response is obtained, recurrence inevitably occurs either locally (c.80%) or distally (c.20%), driven by cancer clones that are often genomically distinct from those in the primary tumour. Glioblastoma cells display a characteristic infiltrative phenotype, invading the surrounding tissue and often spreading across the whole brain. We hypothesised that cancer cells responsible for relapse reside in two compartments of residual disease that are left behind after treatment: the infiltrated normal brain parenchyma, and the sub-ventricular zone (SVZ). In this model, residual disease subclones diverge early during glioblastoma evolution, may remain dormant in the normal parenchyma and are responsible for recurrence. To test our hypothesis, we performed whole-exome sequencing of 69 multi- region samples collected using fluorescence-guided resection from 11 patients, including the infiltrating tumour margin (M) and the SVZ for each patient, as well as matched blood. We used a phylogenomic approach to dissect the spatio-temporal evolution of each tumour and unveil the relation between residual disease and the main tumour mass. We also analysed two patients with paired primary-recurrence samples with matched residual disease. Our results suggest that the infiltrative phenotype is an early evolutionary trait of glioblastoma, giving rise to the tumour mass detected at surgery. After treatment, the same infiltrative clone may seed the growth of a recurrent tumour, thus representing the ‘missing link’ between the primary tumour and recurrent disease. These results are consistent with recognised clinical phenotypic behaviour and suggest that more specific therapeutic targeting of cells in the infiltrated brain parenchyma may improve patient’s outcome.

  Study EGAS00001003043

• Sequencing of paediatric High Grade Gliomas and DIPG

  Paediatric high grade glioma (pHGG) and diffuse intrinsic pontine glioma (DIPG) are a phenotypically diverse collection of tumours which molecular profiling studies have refined into age- and location-based subgroups driven by unique genetic and epigenetic alterations. Despite this, the rarity of these tumours means individual studies are underpowered to investigate subgroup-specific changes and to identify low frequency events recurrently targeting specific pathways. We have collated genomic data from 143 unpublished cases up to 30 years of age along with those from 18 previously published datasets in an integrated analysis comprising 1067 unique patients across all anatomical compartments of the CNS, with histone mutation status available for 893 cases (n=67 H3.3G34R/V, n=316 H3.3K27M, n=68 H3.1/H3.2K27M). Distinct clinicopathological and molecular subgroups are defined by these histone mutations, with several novel co-segregating mutations identified through these aggregated analyses, including loss of FBXW7 in H3.3G34R/V, TOP3A rearrangements in H3.3K27M, and BCOR mutations in H3.1K27M. Histone wild-type subgroups can be further refined by the presence of key oncogenic events (PDGFRA or EGFR amplification/mutations) or methylation profiles which molecularly more closely resemble pleomorphic xanthoastrocytoma or other low grade gliomas. The burden of genomic aberrations decreases with age, highlighting the infant population as biologically and clinically distinct. Across the whole cohort, we identify novel previously unrecognised pathway dysregulation in small subsets of tumours (e.g. splicing, WNT, immune response) which make up the umbrella classification of paediatric diffusely infiltrating gliomas. The integrated dataset helps to further define the molecular diversity of the disease, opening up novel avenues for biological study and providing a basis for functionally defined future treatment stratification.

  Study EGAS00001002314

• UK10K NEURO IMGSAC

  In the UK10K project we propose a series of complementary genetic approaches to find new low frequency/rare variants contributing to disease phenotypes. These will be based on obtaining the genome wide sequence of 4000 samples from the TwinsUK and ALSPAC cohorts (at 6x sequence coverage), and the exome sequence (protein coding regions and related conserved sequence) of 6000 samples selected for extreme phenotypes. Our studies will focus primarily on cardiovascular-related quantitative traits, obesity and related metabolic traits, neurodevelopmental disorders and a limited number of extreme clinical phenotypes that will provide proof-of-concept for future familial trait sequencing. We will analyse directly quantitative traits in the cohorts and the selected traits in the extreme samples, and also use imputation down to 0.1% allele frequency to extend the analyses to further sample sets with genome wide genotype data. In each case we will investigate indels and larger structural variants as well as SNPs, and use statistical methods that combine rare variants in a locus or pathway as well as single-variant approaches.The IMGSAC cohort is an international collection of families containing children ascertained for ASDs (autism spectrum disorders). The affected individuals are have been phenotyped, including using the ADI-R and ADOS instruments. Individuals with a past or current medical disorder of probable etiological significance or TSC have been excluded. Where possible, karyotyping has been performed on one affected individual per family to exclude Fragile X syndrome. Many of the samples have been genotyped, using the Affymetrix 10k and Illumina 1M platforms. All samples to be included in the current study are of UK origin.For further information on this cohort please contact Jeremy Parr (jeremy.parr@newcastle.ac.uk).

  Study EGAS00001000120

• Combined MEK and BCL-2/XL inhibition is effective in high-grade serous ovarian cancer patient-derived xenograft models and BIM levels are predictive of responsiveness

  Most patients with late stage high-grade serous ovarian cancer (HGSOC) initially respond to chemotherapy but inevitably relapse and develop resistance, highlighting the need for novel therapies to improve patient outcomes. The MEK/ERK pathway is activated in a large subset of HGSOC, thus making it an attractive therapeutic target. Here, we systematically evaluated the extent of MEK/ERK pathway activation and efficacy of pathway inhibition in a large panel of well-annotated HGSOC patient-derived xenograft (PDX) models. The vast majority of models were nonresponsive to the MEK inhibitor cobimetinib (GDC-0973) despite effective pathway inhibition. Proteomic analyses of adaptive responses to GDC-0973 revealed that GDC-0973 upregulated the pro-apoptotic protein BIM, thus priming the cells for apoptosis regulated by BCL2-family proteins. Indeed, combination of both MEK inhibitor and dual BCL-2/XL inhibitor (ABT-263) significantly reduced cell number, increased cell death and displayed synergy in vitro in most models. In vivo, the GDC-0973 and ABT-263 combination was well tolerated and resulted in greater tumor growth inhibition than single agents. Detailed proteomic and correlation analyses identified two subsets of responsive models – those with high BIM at baseline that was increased with MEK inhibition and those with low basal Bim and high pERK levels. Models with low BIM and low pERK were non-responsive. Our findings demonstrate that combined MEK and BCL-2/XL inhibition has therapeutic activity in HGSOC models and provide a mechanistic rationale for clinical evaluation of this drug combination as well as the assessment of the extent to which BIM and/or pERK levels predict drug combination effectiveness in chemoresistant HGSOC.

  Study EGAS00001003427

• Ischemic stroke in a Swedish case-control study.

  Background. Genetic risk scores (GRS), summing up the total effect of several single nucleotide polymorphisms (SNPs) in genes associated with either coronary risk or cardiovascular risk factors, have been tested for association with ischemic stroke with conflicting results. Recently an association was found between a GRS, based on 29 SNPs discovered by genome-wide association studies (GWAS) and hypertension. The aim of our study was to investigate the possible association of the same GRS with ischemic stroke on top of other “traditional risk factors”, also testing its potential improvement in indices of discrimination and reclassification, in a Swedish case-control study. Methods and results. Twenty-nine SNPs were genotyped in 3,677 stroke cases and 2,415 controls included in the Lund Stroke Register (LSR), the Malmö Diet and Cancer (MDC) study and the Sahlgrenska Academy Study on Ischemic Stroke (SAHLSIS). The analysis was conducted in the entire material, and separately for the three samples. After adjustment for hypertension, diabetes mellitus and smoking habits, the GRS was associated with ischemic stroke in the entire material [OR (95% CI) 1.086 (1.029-1.147) per SD increase in the GRS p=0.003] with similar trends in all three samples; LSR [1.050 (0.967-1.140)]; p=0.25], MDC [1.168 (1.060-1.288); p=0.002] and SAHLSIS [1.124 (0.997-1.267); P=0.055]. Measures of discrimination and reclassification improved marginally using the GRS. Conclusions. A blood pressure GRS is independently associated with ischemic stroke risk in three Swedish case-control studies, however, the effect size is low and adds marginally to prediction of stroke on top of traditional risk factors including hypertension.

  Study EGAS00001000936

• Post_Mortem_Tissue_COVID19_RNA

  Single cell analysis of post mortem tissue samples from SARS-CoV2-infected patients We aim to directly identify the human cell types infected by SARS-CoV-2 and measure the cellular response to COVID19 infection across 20 different tissues from infected patient autopsies. We have examined the expression pattern of viral entry receptors across healthy human tissues to predict several candidate target cell types across the airway and heart. In addition, high prevalence of cardiac failure and abnormal renal function in COVID19 patients implicates heart and kidney involvement, but the pathogenesis of organ specific damage - whether via a direct cytopathic mechanism or an indirect inflammatory response - remains unknown . Currently, we lack confirmation of target cell types and cellular processes in infected tissues as autopsies are discouraged in most countries due to health and safety risks. Our collaborators Drs Michael Osborn and Brian Hanley (Imperial College) have outlined guidelines to perform post-mortem in COVID19 patients (Hanley et al., 2020) and have established a programme of autopsies for research to be performed in a high-risk facility at Westminster Public Mortuary. Here, we propose to identify infected cell types and aberrant molecular pathologies in this precious tissue resource using single cell and spatial genomics. We will prioritise three organ systems: the human airway, heart and the kidney. We will directly examine the cellular identities of SARS-CoV-2 infected cell types and identify the cellular responses to infection across these organs. This fundamental knowledge will help guide future treatment choices for COVID19. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/

  Study EGAS00001004442

• ctDNA monitoring using patient-specific sequencing and integration of variant reads - Lung cohort

  Circulating tumor-derived DNA (ctDNA) can be used to monitor cancer dynamics noninvasively. Detection of ctDNA can be challenging in patients with low-volume or residual disease, where plasma contains very few tumor-derived DNA fragments. We show that sensitivity for ctDNA detection in plasma can be improved by analyzing hundreds to thousands of mutations that are first identified by tumor genotyping. We describe the INtegration of VAriant Reads (INVAR) pipeline, which combines custom error-suppression methods and signal-enrichment approaches based on biological features of ctDNA. With this approach, the detection limit in each sample can be estimated independently based on the number of informative reads sequenced across multiple patient-specific loci. We applied INVAR to custom hybrid-capture sequencing data from 176 plasma samples from 105 patients with melanoma, lung, renal, glioma, and breast cancer across both early and advanced disease. By integrating signal across a median of >105 informative reads, ctDNA was routinely quantified to 1 mutant molecule per 100,000, and in some cases with high tumor mutation burden and/or plasma input material, to individual parts per million. This resulted in median Area Under the Curve (AUC) values of 0.98 in advanced cancers, and 0.80 in early stage and challenging settings for ctDNA detection. We generalized this method to whole-exome and whole-genome sequencing, showing that the INVAR may be applied without requiring personalized sequencing panels, so long as a tumor mutation list is available. As tumor sequencing becomes increasingly performed, such methods for personalized cancer monitoring may enhance the sensitivity of cancer liquid biopsies.

  Study EGAS00001004447

• Induced Pluripotent Cells Derived from Differentiated Rod Photoreceptors Undergo Efficient Retinogenesis in Three-Dimensional Cultures

  "Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are used to study lineage-specific differentiation in culture. We developed a standardized quantitative protocol called STEM-RET to compare eye field specification, optic cup formation, and retinal differentiation across stem cell populations. We reprogrammed individual rod photoreceptors into iPSCs by inducible expression of Oct3/4, Klf4, Sox2, and Myc. We also developed ESC lines from rods by somatic cell nuclear transfer. Using the STEM-RET protocol, we compared the abilities of ESCs, fibroblast-derived iPSCs (f-iPSCs), rod photoreceptor-derived iPSCs (r-iPSCs), and rod photoreceptorderived ESCs to form retinae using STEM-RET. r-iPSCs were the most efficient at producing differentiated retina. Retinae derived from f-iPSCs lacked an inner nuclear layer (INL) and had a concomitant reduction in amacrine cells and other INL cell populations. The INL-specific LIM homeobox gene, Lhx9, was hypermethylated in fiPSCs and concomitantly downregulated in retinae derived from those stem cells. ChIP-seq analysis for H3K36me3, H3K4me1, H3K4me3, H3K9/14Ac, H3K27me3, H3K9me3, CTCF and 5 hydroxymethyl cytosine showed that the major difference between the r-iPSCs and the f-iPSCs were in the genes marked by the CTCF insulator protein. Importantly, CTCF binding enrichment at rod photoreceptor specific genes was at least 10-fold higher in r-iPSCs relative to f-iPSCs. Taken together, our data suggest that both inhibitory and permissive epigenetic marks important for retinal development can be retained in iPSCs and such marks can be exploited to select the most favorable stem cell population to study retinal development or produce photoreceptor precursors for cell transplantation."

  Study EGAS00001001288

• DSRCT RNA genomic sequencing

  Purpose: To further understand the molecular pathogenesis of desmoplastic small round cell tumor (DSRCT), a rare but often fatal malignancy occurring primarily in young males, we used next-generation RNA sequencing to investigate the gene expression profiles intrinsic to this disease.Experimental Design: RNA from DSRCT tumor samples were obtained from the Cooperative Human Tissue Network biorepository. RNA sequencing was completed using the Illumina HiSeq 2000 system. Presence of the EWSR1-WT1 fusion was verified using both RNA-sequencing and PCR. Validation studies, including WT1 ChIP-seq, RNA sequencing, and EWS-WT1 knockdown experiments, were performed on an established DSRCT cell line, JN-DSRCT-1. Protein expression of clinically relevant genes determined by RNA sequencing was confirmed using immunohistochemistry. A panel of immune signature genes was also evaluated to identify possible immune therapeutic targets. Results: We analyzed the RNA from 14 tumor samples, of which two were confirmed negative for the diagnostic EWSR1-WT1 translocation. Principal component analysis of the sequencing data indicated that fusion-negative and fusion-positive tumors are transcriptionally distinct entities. Overlapping the significant ChIP-seq peaks with genes found to be highly expressed in the RNA sequencing data revealed that IGF2 and FGFR4 were both highly expressed, and targets of the EWS-WT1 fusion gene. This finding has potential clinical relevance for targeted therapeutic intervention. In addition, we identified the immune checkpoints CD200 and CD276 as potentially targetable genes whose expression is independent of the EWS-WT1 fusion gene in vitro.Conclusions: Despite the transcriptional complexity of this disease, we were able to identify IGF2, FGFR4, CD200, and CD276 as potential therapeutic targets for patients suffering from DSRCT.

  Study EGAS00001002770

• Genetic diversity and continuity of the population of the UAE

  With high consanguinity rates on the Arabian Peninsula, it would not have been unexpected if the population of the UAE was shown to be relatively homogenous. However, this study of 1,000 UAE nationals, provided a contrasting perspective, one of a relatively heterogeneous population. Located at the apex of Europe, Asia and Africa, the observed diversity could be explained by a plethora of migration patterns since the first Out-of-Africa movement. A strategy to explore the extent of genetic variation of the population of the UAE is presented. The first step involved a comprehensive population stratification study that was instructive for subsequent Whole Genome Sequencing (WGS) of suitable representatives (which is described elsewhere). When this UAE data was compared to previous smaller studies from the region, the findings were consistent with a population that is a diverse and admixed group of people. However, rather than sharp and distinctive clusters, a continuous genotype distribution was observed. The analysis showed a continuous gradient of ancestral populations, suggesting that admixture on the south eastern tip of Arabian Peninsula occurred gradually. When visualized using a unique technique that combined admixture ratios and Principal Component Analysis (PCA), unappreciated diversity was revealed while mitigating projection bias of conventional PCA. The analytical strategy used here highlights the complementary nature of data from genotype array and WGS for anthropological studies. Specifically, genotype array data was instructive to select representative subjects for WGS. Furthermore, from the 2.3 million allele frequencies obtained from genotype arrays, we identified 46,481 loci with allele frequencies that were significantly different with respect to other world populations. This comparison of allele frequencies facilitates variant prioritization in common diseases. In addition, these loci bear great potential as biomarkers in anthropological and forensic studies.

  Study EGAS00001004362

• Matching of actionable mutations with therapies in cancer patients: comparison of three commercial decision support platforms

  Purpose: In oncology, precision medicine centers on tailoring the most appropriate therapy to a patient according to their tumor molecular profile. We addressed the question whether there is a consensus in the classification of actionability of somatic variants and their alignment to treatment recommendations. To this end, we evaluated three commercial clinical decision support tools to assess their variant classification and drug matching strategies.Methods: In this two centers study 48 patients with metastatic breast (n=12), colorectal (n=17) or non-small cell lung cancer (n=19) were recruited based on circulating tumor DNA levels in peripheral blood. In individual patients’ plasma samples, we established somatic copy number alterations and somatic mutations across a 77 cancer-associated gene panel and exposed the obtained molecular profiles to the decision support packages NAVIFY Mutation Profiler (NMP; Roche), Qiagen Clinical Insight (QCI) Interpret (Qiagen) and CureMatch Bionov (CureMatch).Results: In the plasma samples, altogether 492 somatic alterations were assessed. Each decision support platform varied in their format of data input, mode of variant classification and strategies for identifying druggable targets and clinical trials, which resulted in discrepancies in tier classification as well as designations of actionability. The frequency of concordant actionable events for tier I-A or tier I-B classifications, i.e. those with the strongest clinical evidence, was only 4.3%, 9.5% and 28.4% when comparing NMP with QCI, NMP with CureMatch, and CureMatch with QCI, respectively. As a consequence, the obtained treatment recommendations differed drastically, and alignment of treatment recommendations was limited to established predictive markers.Conclusions: The lack of standards for the extraction of treatment recommendations from comprehensive tumor molecular profiling data challenge the promising concepts of precision oncology.

  Study EGAS00001004383

• Post_Mortem_Tissue_COVID19_spatial

  Single cell analysis of post mortem tissue samples from SARS-CoV2-infected patients We aim to directly identify the human cell types infected by SARS-CoV-2 and measure the cellular response to COVID19 infection across 20 different tissues from infected patient autopsies. We have examined the expression pattern of viral entry receptors across healthy human tissues to predict several candidate target cell types across the airway and heart. In addition, high prevalence of cardiac failure and abnormal renal function in COVID19 patients implicates heart and kidney involvement, but the pathogenesis of organ specific damage - whether via a direct cytopathic mechanism or an indirect inflammatory response - remains unknown . Currently, we lack confirmation of target cell types and cellular processes in infected tissues as autopsies are discouraged in most countries due to health and safety risks. Our collaborators Drs Michael Osborn and Brian Hanley (Imperial College) have outlined guidelines to perform post-mortem in COVID19 patients (Hanley et al., 2020) and have established a programme of autopsies for research to be performed in a high-risk facility at Westminster Public Mortuary. Here, we propose to identify infected cell types and aberrant molecular pathologies in this precious tissue resource using single cell and spatial genomics. We will prioritise three organ systems: the human airway, heart and the kidney. We will directly examine the cellular identities of SARS-CoV-2 infected cell types and identify the cellular responses to infection across these organs. This fundamental knowledge will help guide future treatment choices for COVID19. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/

  Study EGAS00001004441

• Whole genome sequencing of a breast cancer cohort with known functional homologous recombination status

  Optimal patient selection for PARP inhibitors and double strand break (DSB) inducing chemotherapy is of great clinical importance. In principle, the use of these therapies can be extended beyond germline BRCA1/2 mutated cancers. Although several methods for detection of BRCA-like tumors have been developed, a golden standard is lacking. We have developed the RECAP test, a functional HR assay exploiting the formation of RAD51 foci in proliferating cells after ex vivo irradiation of fresh primary breast cancer (BrC) tissue. The RECAP test seems robust and has an advantage over genetic tests, due to its functional character for exploring the HR phenotype. Another type of HRD test is the BRCAness classifier, which is based on specific genomic patterns derived from copy number data of BRCA1/2 mutated BrCs that also occur in sporadic cancers. The BRCAness classifier is a robust test that is easily applicable in the clinic and has also been validated to predict in vivo response to high dose chemotherapy in clinical trials. More recently, a new HRD test based on genomic signatures which can be extracted from whole genome sequencing has been published, HRDetect. Studies comparing these different HRD tests within the same cohort of patients do not yet exist. Here, we have the unique opportunity to compare three HRD tests within the same cohort. We aim to compare three different HRD tests (RECAP, BRCAness classifier and HRDetect) within the same cohort of primary BrCs (n=41). As a secondary aim, we will analyze additional genetic alterations in HRD tumors (both BRCA-deficient and BRCA-proficient HRD tumors).

  Study EGAS00001005572

• The clinical utility of genomics in childhood cancer extends beyond targetable mutations

  Survival of children with relapsed cancer remains unacceptably low. Cancer genomic profiling may identify new therapeutic options, enhance diagnostic accuracy, and improve understanding of tumors’ origins and continued evolution. We deeply sequenced 864 cancer-associated genes and the complete genomes and transcriptomes for 300 pediatric and adolescent/young adult (AYA) patients with poor prognosis or rare tumors. Using integrative somatic-germline analyses, we assessed the clinical utility of cancer genomics in this population. We analyzed tumor mutational signatures and burden to interrogate the relevance of germline variants in DNA repair genes and the impact of therapeutic exposures on tumor evolution. We also evaluated the frequency of changes to tumor drivers and therapeutic targets at relapse in those with serial samples. Integrative analysis yielded clinically actionable variants in a majority of patients. Improved diagnostic accuracy led to modified management in a subset. Therapeutically targetable variants were of unanticipated timing and type, with many of these derived from the germline. Enrichment in corroborating mutational signature 3 (‘BRCAness’) in patients with germline homologous recombination defects suggests they are drivers of pediatric cancers and potential targets for PARP inhibition. We show that mutation burden, which may in part be driven by prior therapeutic exposures, was significantly elevated in a minority of patients. Over one-third of patients with sequential samples analyzed showed a change in therapeutically-targetable drivers. Comprehensive somatic-germline cancer genomic profiling is useful at multiple points in the care trajectory for pediatric/AYA patients, supporting its integration into early clinical management. Re-biopsy at the time of relapse should be considered, and specific attention to mutational burden is indicated. Defective DNA repair genes in the germline may represent important targetable drivers.

  Study EGAS00001006034

• ctDNA monitoring using patient-specific sequencing and integration of variant reads - Breast cohort

  Circulating tumor-derived DNA (ctDNA) can be used to monitor cancer dynamics noninvasively. Detection of ctDNA can be challenging in patients with low-volume or residual disease, where plasma contains very few tumor-derived DNA fragments. We show that sensitivity for ctDNA detection in plasma can be improved by analyzing hundreds to thousands of mutations that are first identified by tumor genotyping. We describe the INtegration of VAriant Reads (INVAR) pipeline, which combines custom error-suppression methods and signal-enrichment approaches based on biological features of ctDNA. With this approach, the detection limit in each sample can be estimated independently based on the number of informative reads sequenced across multiple patient-specific loci. We applied INVAR to custom hybrid-capture sequencing data from 176 plasma samples from 105 patients with melanoma, lung, renal, glioma, and breast cancer across both early and advanced disease. By integrating signal across a median of >105 informative reads, ctDNA was routinely quantified to 1 mutant molecule per 100,000, and in some cases with high tumor mutation burden and/or plasma input material, to individual parts per million. This resulted in median Area Under the Curve (AUC) values of 0.98 in advanced cancers, and 0.80 in early stage and challenging settings for ctDNA detection. We generalized this method to whole-exome and whole-genome sequencing, showing that the INVAR may be applied without requiring personalized sequencing panels, so long as a tumor mutation list is available. As tumor sequencing becomes increasingly performed, such methods for personalized cancer monitoring may enhance the sensitivity of cancer liquid biopsies.

  Study EGAS00001004446

• Integrative analysis of exome-seq, RNA-seq, ATAC-seq (bulk and single-cell), and Hi-C data generated from 3-D spatially mapped samples acquired during surgical resection from 10 patients diagnosed with IDH-WT glioblastoma

  Glioblastoma (GBM), the most common primary brain cancer in adults, remains incurable with no targeted therapies approved despite decades of investigation into its molecular landscape. Treatment failure is attributed to intratumoral heterogeneity in the GBM genome and epigenome, which foster tumor evolution and selection of resistant clones. However, tumor evolution and intratumoral heterogeneity remain poorly understood on the level of the whole tumor as most studies are based on single samples per patient and lack spatial context. Here, we have used 3-D neuronavigation during surgical resection for 10 primary IDH-WT GBM patients to collect 102 samples representing maximal tumor diversity, each mapped by 3-D spatial coordinates. We have applied a strategic set of genomic and epigenomic assays spanning multiple levels of resolution to discover, orthogonally validate, and functionally assess drivers of tumor evolution and intratumoral heterogeneity. These include extrachromosomal DNA amplifications, chromothripsis events, inversions, and translocations that disrupt both the GBM genome and epigenome while generating fusion transcripts and opportunities for therapeutic intervention. We define epigenomic programs that contribute to GBM evolution and intratumoral heterogeneity, revealing their 3-D spatial patterning within whole tumors and their cell type(s) of origin in single-cell data from the same tumor samples. Notably, we distinguish neuronal, glial, and immune programs aberrantly active in tumor cells from their counterparts in normal cells and discover NEUROD1, JUN/FOS, and NF1 transcription factors as key drivers of GBM evolution and growth. Collectively, these data provide unprecedented insight into GBM evolution and intratumoral heterogeneity from single-cell to whole-tumor resolution, redefining current understanding and providing a rich resource of targets for therapeutic investigation.

  Study EGAS00001006785

• 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

• cfDNA dataset from the urine supernatant of ovarian cancer patients and healthy controls

  Background Ovarian cancer is the deadliest gynecological malignancy worldwide, due to frequent diagnosis at advanced stage. Simple and non-invasive methods for earlier and accurate detection are urgently needed. Here, the presence of tumor-derived DNA in home-collected urine, cervicovaginal self-samples and clinician-taken cervical scrapes of ovarian cancer patients was explored by DNA methylation and copy number analysis. Methods A total of 428 samples (urine, cervicovaginal self-samples, and clinician-taken cervical scrapes) of 110 healthy controls and 54 patients with benign (n=25) or malignant (n=29) ovarian masses were analyzed. Different urine fractions were examined (full void urine, urine supernatant, and urine sediment). All samples were tested for 12 methylation markers by quantitative methylation-specific PCR. Shallow whole-genome sequencing was performed to detect copy number aberrations and verify the presence of tumor-derived DNA. Results Full void urine was most discriminatory between healthy controls and ovarian cancer patients (C2CD4D, p=0.008; CDO1, p=0.022; MAL, p=0.008), followed by cervical scrapes (C2CD4D, p=0.001; CDO1, p=0.004). A significant difference between benign and malignant ovarian masses was only observed for GHSR in the urine sediment (p=0.024). Methylation levels in cervicovaginal self-samples did not discriminate between healthy controls, benign and malignant ovarian masses. Copy number changes were identified in 17% of urine supernatant samples and smaller fragment sizes were seen in urine supernatant samples with a high tumor fraction. Conclusions This study demonstrates the presence of ovarian cancer-derived DNA in home-collected urine. Additional studies are warranted to further explore the clinical applicability of this approach.

  Study EGAS00001007238

• Center for Common Disease Genomics [CCDG] - Cardiovascular: Genetics of Coronary Heart Disease - Characterizaton of Coronary Prone Pedigrees

  The primary cohort includes samples from 499 men and 154 women who survived a myocardial infarction, percutaneous transluminal angioplasty, or coronary artery bypass grafting (CABG) by age 55 years for men or age 65 years for women. Patients were seen at least six months after their acute event. Samples from another 488 men and 541 women that showed a nearly identical age, gender, and measured blood pressure distribution were collected as population-based controls. These controls tended to live in urban Salt Lake City, where the vast majority of the population is Caucasian. Blood samples were collected in the morning after 12h to 16h of fasting and prepared according to guidelines of the Lipid Research Clinic's program Manual of Laboratory Operations. All samples have serum, DNA and/or white blood cells, and a host of measurements (e.g. standard clinical chemistries, LDL and HDL cholesterol, triglycerides, glucose, HbA1C, insulin, blood pressures, anthropometric data, information on medical conditions and drug use, etc.). Additional details about the cohort or sample preparation were published previously. Here we present in total 1,475 patients that were successfully sequenced. This number includes 228 women, 419 men, and 7 of unreported gender survivors as well as 339 women and 482 men that served as population-based controls.

  Study phs001901

• Transcriptome sequencing of gingivo-buccal oral squamous cell carcinoma for integrative analysis: alterations in expression of genes attributable to methylation changes

  Gingivo-buccal oral squamous cell carcinoma (OSCC-GB) is the most common cancer among men in India and is associated with high mortality. Although OSCC-GB is known to be quite different from tongue cancer in its genomic presentation and its clinical behavior, it is treated identically as tongue cancer. Predictive markers of prognosis and therapy that are specific to OSCC-GB are, therefore, required. To achieve this, we have carried out transcriptome (n=72) profiling of paired tumor-normal tissues collected from OSCC-GB patients from India. RNA sequencing was performed on tumor and paired adjacent normal samples from 72 OSCC-GB patients. From each patient, total RNA was extracted from tumor and normal tissue samples using AllPrep DNA/RNA Mini Kit (QIAGEN). The quality and concentration of isolated total RNA were checked using Agilent 2100 Bioanalyzer and NanoDrop 2000 (Thermo Fisher Scientific). The OD260/OD280 ratio was ≥ 2 and RNA Integrity Number (RIN) was ≥ 7.0 for all sequenced samples. Ribosomal RNA (rRNA) was removed from the RNA samples, using Ribo-Zero Magnetic Kit (Epicentre). Sequencing libraries were prepared from rRNA depleted samples using TrueSeq RNA Sample Preparation Kit (Illumina). Each triplex cDNA library pool was sequenced as 100bp paired-end on HiSeq-2000 or HiSeq-2500 (Illumina). Protocols suggested by the manufacturers were used for all assays.

  Study EGAS00001003893

• Patient-derived tumor organoids for personalized medicine in a rare case of hepatocellular carcinoma with neuroendocrine differentiation.

  We report the case of a 74-year-old man with a very rare subtype of hepatocellular carcinoma with neuroendocrine differentiation (HCC-NED). The patient presented with two independent tumors, a gastrointestinal stromal tumor in the stomach and a hepatocellular carcinoma in the liver. Both tumors were surgically removed in curative intent. Histopathological work-up of the liver tumor revealed poorly differentiated hepatocellular carcinoma (Edmondson-Steiner grade IV) with diffuse expression of neuroendocrine markers synaptophysin (SYP) and chromogranin (CHGA). Three months after resection, multifocal recurrence of the HCC with neuroendocrine differentiation (HCC-NED) was observed. In the meantime, tumor organoids have been generated from the resected HCC-NED and extensively characterized. Sensitivity to a number of drugs approved for the treatment of HCC or neuroendocrine carcinomas was tested in vitro. Based on their in vitro efficacy, etoposide and carboplatin were used as first line palliative combination treatment. Because genomic analysis revealed a NTRK1-mutation (kinase domain) and tumor organoids were sensitive to entrectinib, a pan-TRK inhibitor, the patient received entrectinib as second line therapy. After only two weeks, treatment had to be discontinued due to deterioration of the patient’s general condition. In conclusion, we demonstrate for the first time that preclinical drug testing using organoids is feasible in selected HCC cases.

  Study EGAS00001005887

• Machine Learning Signal Enrichment for Ultrasensitive Plasma Tumor Burden Monitoring

  In solid tumor oncology, circulating tumor DNA (ctDNA) is poised to transform care through accurate assessment of minimal residual disease (MRD) and therapeutic response monitoring. To overcome the sparsity of ctDNA fragments in low tumor fraction (TF) settings and increase MRD sensitivity, we previously leveraged genome-wide mutational integration through plasma whole genome sequencing (WGS). We now introduce MRD-EDGE, a composite machine learning-guided WGS ctDNA single nucleotide variant (SNV) and copy number variant (CNV) detection platform designed to increase signal enrichment. MRD-EDGESNV uses deep learning and a ctDNA-specific feature space to increase SNV signal-to-noise enrichment in WGS by 300X compared to previous WGS error suppression. MRD-EDGECNV also reduces the degree of aneuploidy needed for ultrasensitive CNV detection through WGS from 1 Gb to 200 Mb, thereby expanding its applicability to a wider range of solid tumors. We harness the improved performance to identify MRD following surgery in multiple cancer types, track changes in tumor burden in response to neoadjuvant immunotherapy in non-small cell lung cancer (NSCLC) and demonstrate ctDNA shedding in precancerous colorectal adenomas. Finally, the radical signal-to-noise enrichment in MRD-EDGESNV enables de novo mutation calling in melanoma and small cell lung cancer (SCLC) without matched tumor, yielding clinically informative TF monitoring for patients on immune checkpoint inhibition (ICI).

  Study EGAS00001007451

• Machine learning guided signal enrichment for ultrasensitive plasma tumor burden monitoring

  In solid tumor oncology, circulating tumor DNA (ctDNA) is poised to transform care through accurate assessment of minimal residual disease (MRD) and therapeutic response monitoring. To overcome the sparsity of ctDNA fragments in low tumor fraction (TF) settings and increase MRD sensitivity, we previously leveraged genome-wide mutational integration through plasma whole genome sequencing (WGS). We now introduce MRD-EDGE, a composite machine learning-guided WGS ctDNA single nucleotide variant (SNV) and copy number variant (CNV) detection platform designed to increase signal enrichment. MRD-EDGESNV uses deep learning and a ctDNA-specific feature space to increase SNV signal-to-noise enrichment in WGS by 300X compared to previous WGS error suppression. MRD-EDGECNV also reduces the degree of aneuploidy needed for ultrasensitive CNV detection through WGS from 1 Gb to 200 Mb, thereby expanding its applicability to a wider range of solid tumors. We harness the improved performance to identify MRD following surgery in multiple cancer types, track changes in tumor burden in response to neoadjuvant immunotherapy in non-small cell lung cancer (NSCLC) and demonstrate ctDNA shedding in precancerous colorectal adenomas. Finally, the radical signal-to-noise enrichment in MRD-EDGESNV enables de novo mutation calling in melanoma and small cell lung cancer (SCLC) without matched tumor, yielding clinically informative TF monitoring for patients on immune checkpoint inhibition (ICI).

  Study EGAS00001007545

• Genetic_variation_in_Kuusamo

  The aim of this study is to characterize as completely as possible the genetic variation and structure of Kuusamo and surrounding regions. We will perform low coverage, whole genome sequencing in a relatively limited number (approximately 200) of individuals from the FINRISK study both of whose parents are born in Kuusamo, Posio or Taivalkoski. Sequencing will be performed at the Wellcome Trust Sanger Institute (WTSI). We have previously shown using simulations and genome wide SNP data that the genetic characteristics agree with known genealogical data, and that approximately 200 individuals suffice to capture the majority of the genetic variation in this population.We have also developed methods for long-range haplotyping and long-range imputation, which are more accurate than traditional methods. This will allow us to identify a majority of the founder haplotypes in this population. The information from the haplotypes identified in this study can be used in other studies to impute from genome-wide SNP data the full genome sequence of samples ascertained for specific phenotypes.

  Study EGAS00001000020

• Offspring Sex Impacts DNA Methylation and Gene Expression in Placentae from Women with Diabetes during Pregnancy

  Exposure to diabetes in utero is known to increase the offspring's likelihood of developing metabolic disease in adulthood, but the mechanisms involved are unknown. It has been proposed that early exposure to hyperglycemia and elevated insulin levels may lead to malprogramming of the fetus leading to the subsequent development of diabetes and obesity. Epigenetic modifications of the genome including DNA methylation, provide a plausible mechanism that allows for permanent propagation of gene activity states from one generation of cells to the next. The placenta, a fetal tissue easily accessible for study, is a complex organ that is essential in regulating fetal growth. The changes in placental nutrient transport associated with diabetes during pregnancy (DDP) have significant effects on the developing fetus, indicating that the placenta plays a critical role in fetal programming. The aim of our study was to investigate whether exposure to DDP alters genome-wide DNA methylation in the placenta obtained from term pregnancies resulting in differentially methylated loci of metabolically relevant genes and downstream changes in RNA and protein expression.

  Study phs001535

• University of North Carolina at Chapel Hill (UNC) Hepatocellular Carcinoma Study by Exome Sequencing (HCCSES)

  Genetic alterations in specific driver genes lead to disruption of cellular pathways and are critical events in the instigation and progression of hepatocellular carcinoma. As a prerequisite for individualized cancer treatment, we sought to characterize the landscape of recurrent somatic mutations in hepatocellular carcinoma. We performed whole exome sequencing on 87 hepatocellular carcinomas and matched normal adjacent tissues to an average coverage of 59x. The overall mutation rate was roughly 2 mutations per Mb, with a median of 45 non-synonymous mutations that altered the amino acid sequence (range 2 to 381). We found recurrent mutations in several genes with high transcript levels: TP53 (18%), CTNNB1 (10%), KEAP1 (8%), C16orf62 (8%), MLL4 (7%) and RAC2 (5%). Significantly affected gene families include the nucleotide-binding domain and leucine rich repeat containing family, calcium channel subunits, and histone methyltransferases. In particular, the MLL family methyltransferases for histone H3 lysine 4 were mutated in 20% of tumors. Conclusion: The NFE2L2-KEAP1 and MLL pathways are recurrently mutated in multiple cohorts of hepatocellular carcinoma.

  Study phs000627

• Role of the Cervico-Vaginal Microbiome in Spontaneous Preterm Birth

  Preterm birth is the leading cause of neonatal morbidity and mortality. A failure to predict and understand the causes of preterm birth have limited effective interventions and therapeutics. From a cohort of 2,000 pregnant women, we performed a nested case control study on 107 well-phenotyped cases of spontaneous preterm birth (sPTB) and 432 women delivering at term. Modern and innovative Bayesian modeling of vaginal microbiota identified features of these communities associated with PTB. Seven bacterial taxa were shown to have relative abundances significantly associated with an increased risk of sPTB, with a stronger effect in African American women. However, higher vaginal levels of β-defensins significantly decreased the risk of sPTB associated with the vaginal microbiota in an ethnicity-dependent manner. These findings hold promise for the development of novel diagnostics that could more accurately identify women at risk for sPTB early in pregnancy and offer new therapeutic strategies that would include immune modulators and microbiome-based therapeutics to reduce this significant health burden.

  Study phs001739

• Genome-Wide Discovery of Novel Colon Cancer Predisposing Mutations

  Cancer risk shows clear heritability, but the inherited genetic factors remain largely unknown. This project will seek to identify new genes in which mutations confer hereditary risk for early onset colorectal cancer, with important implications for both biological understanding and clinical prediction and prevention. Identifying cancer predisposing mutations can provide new biological insights and significantly impact important clinical decision making regarding treatment, surveillance and preventive approaches. Hereditary colon cancer patients commonly present to Cancer Genetics clinics, but the majority of these patients do not have identifiable mutations in known candidate genes. We have performed whole exome sequencing to discover novel colorectal cancer susceptibility genes. The patients have been recruited from the Memorial Sloan Kettering Cancer Center (PIs Kenneth Offit, Nathan Ellis, and Zsofia Stadler), and Case Western University and University Hospitals of Cleveland (PIs Joseph Willis and Sanford Markowitz). All exome sequencing was performed at the Broad Institute of Harvard and MIT; samples sequence capture was performed using Agilent SureSelect Human All Exon Kit v2 and sequencing was performed on an Illumina HiSeq 2000 or 2500.

  Study phs000824

• Next Generation Mendelian Genetics: Congenital Hyperinsulinism

  The NHGRI Next Generation Mendelian Genetics project uses exome resequencing to identify variants in unsolved Mendelian diseases. Congenital Hyperinsulinism (CHI) is the most common cause of hypoglycemia in the newborn and it is due to mutations in 8 different genes (ABCC8, KCNJ11, GLUD1, GCK, HADH, SLC16A1, HNF4A and UCP2). It is a heterogeneous disease with variable onset (birth to adulthood) and a persistent, intermittent, or transient course with possible later conversion to non-autoimmune diabetes. Although mutations in the two subunits of the KATP channel (ABCC8 and KCNJ11) account for 50% of the cases, the other half is still genetically unexplained. CHI can be inherited in a dominant or recessive and can also present as a 'de novo' mutation. We became part of this study when we submitted 8 DNA samples for exome sequencing, from patients with CHI of Caucasian ancestry, which had no mutations identified in ABCC8 or KCNJ1, with the goal to identify new mutations in known genes or new mutations in new genes or genetic variants.

  Study phs000539

• CSER: Incorporating Genomics into the Clinical Care of Diverse NYC Children (NYCKidSeq)

  The NYCKidSeq program will significantly advance the implementation of genomic medicine, particularly for children, young adults and their families in Harlem and the Bronx. The study will assess the clinical utility of genomic medicine in three broad areas of pediatric disorders, while engaging a range of providers and community advisors to overcome the well-documented barriers to inclusion of underserved and underrepresented populations in genomic research. The study will also include testing, analyzing, and implementing a novel communication tool, Genomic Understanding, Information and Awareness (GUÍA), to facilitate the return of genomic test results. The use of GUÍA will enhance the understanding of these genomic testing results by families, patients, and care providers at all levels of expertise, in two health systems. Healthcare system leadership will be engaged to provide insights into their readiness for genomic implementation. Overall, the NYCKidSeq program will inform the genomics and clinical communities about how to implement genomic medicine in a diverse population in a clinically useful, technologically savvy, culturally sensitive, and ethically sound manner.

  Study phs002337

• BrainSpan Atlas of the Human Brain

  The NIH-funded BrainSpan (www.brainspan.org) and PsychENCODE (www.psychencode.org). Consortia sought to generate and analyze multi-dimensional genomics data from the developing and adult human brain in healthy and disease states. One of the main goals has been to perform large-scale and integrated analysis of the genome, transcriptome, and epigenome of the human brain to broaden our understanding of human neurodevelopment. This dataset consists of sixteen regions, including eleven neocortical areas, of human donors of both sexes and various ethnic groups. In the first stage of this project, we provide the genome-wide exon-level transcriptome data generated using the Affymetrix GeneChip Human Exon 1.0 ST Arrays, and the genome-wide genotyping data for 2.5 million markers using the Illumina Human Omni 2.5-Quad Bead Chips. In the second stage of this project, we provide whole-genome sequencing data, transcriptome data by mRNA-Seq, small RNA data by smRNA-seq, DNA cytosine methylation by Infinium HumanMethylation450 BeadChip, and epigenomic/epigenetic data by ChIP-Seq for H3K4me3, H3K27me3, H3K27ac and CTCF.

  Study phs000755

• 99 Cases of Small Cell Lung Cancer Study

  Small cell lung cancer (SCLC) is an aggressive disease with poor survival. A few sequencing studies performed on limited number of samples have revealed potential disease-driving genes in SCLC, however, much still remains unknown, particularly in the Asian patient population. Here we conducted whole exome sequencing (WES) and transcriptomic sequencing of primary tumors from 99 Chinese SCLC patients. Dysregulation of tumor suppressor genes TP53 and RB1 was observed in 82% and 62% of SCLC patients, respectively, and more than half of the SCLC patients (62%) harbored TP53 and RB1 mutation and/or copy number loss. Additionally, Serine/Arginine Splicing Factor 1 (SRSF1) DNA copy number gain and mRNA over-expression was strongly associated with poor survival using both discovery and validation patient cohorts. Functional studies in vitro and in vivo demonstrate that SRSF1 is important for tumorigenicity of SCLC and may play a key role in DNA repair and chemo-sensitivity. These results strongly support SRSF1 as a prognostic biomarker in SCLC and provide a rationale for personalized therapy in SCLC. The manuscript has been accepted by Plos Genetics.

  Study phs001083

• CHDWB Rare Regulatory Alleles and Gene Expression Study

  In order to evaluate whether rare regulatory variants in the vicinity of promoters are likely to impact gene expression, we conducted a novel burden test for rare variants at the extreme of expression. After targeted sequencing of 2kb promoter regions of 472 genes in 410 healthy adults, burden tests were performed by calculating the summed rare allele counts in ranked expression level bins using Illumina whole blood microarray gene expression data. The results clearly show an enrichment of rare variants at both extremes of gene expression. The rare regulatory variant effects were also partitioned into subsets of genes based on their regulatory functions, positions relative to transcription start sites, disease relatedness, with some intriguing biases. The enrichment of rare regulatory variants in extremely expressed genes was replicated in an independent sample of 75 individuals with RNASeq and whole genome sequence information. Participants were from the Emory-Georgia Tech Center for Health Discovery and Well Being (CHDWB) longitudinal cohort study, but only baseline gene expression data was used.

  Study phs001021

• Adoptive Cell Therapy of Autologous T cell Receptor-Engineered T Cells Targeting the p53 Neoantigens in Human Solid Tumors

  Adoptive T cell therapy (ACT) using T cell receptor (TCR)-engineered T cells can mediate regression of advanced human cancers. How TCR-transduced T cells change post-ACT in vivo is currently not well understood. We screened 78 patients for TP53 mutations by whole exome sequencing and whole transcriptome sequencing. Of the 78 patient samples, 49 patient samples were previously reported (phs001003) and 2 additional patients were reported in phs002735. In this accession we report the sequencing data of remaining 23 patients and include 6 additional samples from previously published patients. Additionally, we treated a patient with chemo-refractory breast cancer that expressed mutant p53 R175H with autologous T cells transduced with an allogeneic TCR targeting p53 R175H with human leukocyte antigen (HLA) restriction of A*02. The patient had a partial response for 6 months with 55% tumor regression. The T cells from the infusion product and the peripheral blood T cells at 6 weeks post-ACT were analyzed using 10X single cell transcriptome analysis.

  Study phs002928

• The Cardiopulmonary Effects of Particulate Exposure

  The study was initially designed as a longitudinal cohort study of cardiovascular effects of particulate exposure. Participants were boilermaker construction workers who were exposed to particulate matter (PM) component (e.g. a focus on metal constituents) while performing welding, grinding or cutting work in the welding plants. Participants were followed up approximately half a year for a non-work day and a work day from 1999 to 2012, and blood, urine, blood pressure, as well as real-time PM2.5 samples were collected from each participant. In this specific study, we conducted epigenome-wide DNA methylation profiling through Center for Inherited Disease Research (CIDR) services using Illumina Infinium HumanMethylation450, which will give us more compete epigenome-wide coverage. Genome-wide DNA methylation was carried out on paired samples pre- and post-work shift at a welding apprentice school. With transcriptomic and metabolomic data already generated from the same panel study, we will be able to conduct integrative analysis to gain insights into mechanisms of gene-metal interactions and maximize the probability of finding genes functionally relevant to PM-induced adverse cardiovascular outcomes.

  Study phs000968

• Transcriptional Profiling of Macrophages In Situ in Metastatic Melanoma Reveals Localization-Dependent Phenotypes and Function

  Therapeutic interventions modulating immune function at the tumor site could improve outcomes in cancer. Here we analyzed metastatic melanoma, a tumor type highly responsive to T-cell based therapies, and found that the tumor-infiltrating T cells are localized closely to CD14+ monocytes/macrophages rather than to melanoma cells. Using novel immunofluorescence-guided laser capture micro-dissection, we analyzed transcriptomes of CD3+ T cells, CD14+ monocytes/macrophages and melanoma cells in non-dissociated tissue. CD14+ cells localized in tumors beds displayed specific transcriptional signatures distinct from CD14+ cells that were localized in tumor stroma.The 8 distinct metastatic melanoma samples are from the Cooperative Human Tissue Network (CHTN). 64 RNA-seq files are available from 16 intraCD14+, 16 stromaCD14+, 16 cancer cells, 8 intraCD3+ and 8 stromaCD3+ cells. 2 LCM harvests were completed for the CD14+ and cancer cells on two sections of the samples. One harvest was done for the CD3+ samples.The processed data (TPM/raw counts tables) are available on GEO under GSE180124. 

  Study phs002564

• Study of Tumor Recurrence Related to the Expression of the PAX3-FOXO1 Oncogenic Transcription Factor in Fusion-Positive Rhabdomyosarcoma

  Alveolar rhabdomyosarcoma (ARMS) is a highly aggressive pediatric soft tissue cancer characterized by a recurrent 2;13 chromosome translocation. This translocation generates a PAX3-FOXO1 (P3F) fusion gene, which encodes a chimeric transcription factor with oncogenic activity. Using a human myoblast system that models targeted therapy directed against P3F, we demonstrated that the expression of the fibroblast growth factor 8 (FGF8), a direct transcriptional target of P3F, is necessary and sufficient for PAX3-FOXO1-independent tumor recurrence in addition to be required for the tumorigenic activity of primary tumors expressing P3F. In addition, we showed that FGF8 is expressed in human rhabdomyosarcoma cell lines that express P3F. We also demonstrated that FGF8 contributes to their proliferation and transformation in vitro, and is sufficient to maintain their oncogenicity after the loss of P3F expression. We suggest that targeting the expression of the PAX3-FOXO1 transcription factor can generate resistance and recommend that additional anticipated therapeutic approaches, such as targeting FGF8 pathway, may avoid this resistance mechanism.

  Study phs002344

• Genes-Environments and Admixture in Latino Asthmatics (GALA II) Study

  A case-control pharmacogenetic study of bronchodilator drug response among racially admixed Latino children with asthma. Each participant had two spirometry measurements using the KoKo PFT System. With the first spirometry test, participant was administered with 4 puffs of HFA Albuterol. The second albuterol measurement was based on age, for participants under 16 years of age, additional 2 puffs were administered and for those over 16 years of age, additional 4 puffs were administered. The overall goal is to identify genetic factors which are predictive of drug response in children with asthma. The principal tools include a questionnaire and biological specimen collection. Participants are 8-21 years old at time of recruitment. Children with asthma have physician-diagnosed asthma, symptoms and medications. The GALAII Study is utilized in the following dbGaP substudies. To view genotypes, analysis, other molecular data, and derived variables collected in these substudies, please click on the following substudies below or in the "Substudies" box located on the right hand side of this top-level study page phs001180 GALAII Study. phs001274phs001274 GALAII GWAS

  Study phs001180

• National Heart, Lung, and Blood Institute (NHLBI) Data Coordinating Center, Microbiome of the Lung and Respiratory Track, Lung HIV Microbiome Project (LHMP)

  Humans interact with countless microorganisms in multiple environments. The Lung HIV Microbiome Project (LHMP) characterized the microbiome of the lung and respiratory tract. This effort provided initial data to develop further hypotheses addressing differences between HIV-infected and HIV-uninfected individuals. The project involved multiple studies, either single center or multiple-center studies. The research design of each study varies with the objective of that study. The Lung HIV Microbiome Project (LHMP) worked collaboratively to ensure the clinical and scientific integrity and success of this project. The LHMP characterized the microbiome of the lung alone or in combination with the oropharyngeal cavities in HIV-infected individuals and HIV-uninfected controls using molecular techniques to identify bacteria. Knowledge of the lung microbiome and that of other components of the respiratory tract in healthy and diseased states may lead to the identification of predictors of disease progression and therapeutic targets for translation into better preventive and treatment strategies. Study datasets and biospecimens are available for request from https://biolincc.nhlbi.nih.gov/studies/lhmp/.

  Study phs000769

• Single-Cell Analysis of Somatic Mutations in Human Bronchial Epithelial Cells in Relation to Aging and Smoking

  While lung cancer risk among smokers is dependent on smoking dose, it remains unknown if this increased risk reflects an increased rate of somatic mutation accumulation in normal lung cells. Here we applied single-cell whole genome sequencing of proximal bronchial basal cells from 33 subjects, aged between 11 and 86 years, with smoking histories varying from never smoking to 116 pack years. We found an increase in the frequency of single-nucleotide variants and small insertions and deletions with chronological age in never-smokers, with mutation frequencies significantly elevated among smokers. However, when plotted against smoking pack-years, mutations were found to follow the linear increase in cancer risk only until about 23 pack years, after which no further increase in mutation frequency was observed, pointing towards individual selection for mutation avoidance. Known lung cancer-defined global mutation signatures tracked with both age and smoking. Within study power limits, no significant enrichment for somatic mutations in lung cancer driver genes was observed.

  Study phs002758

• HiDEF-seq Single-Molecule Sequencing of Single-Strand Mismatches and Damage

  Our study sought to resolve, with single-molecule resolution, the mismatches and damage events that precede DNA mutations. Using a novel single-molecule, long-read sequencing method (HiDEF-seq) we detected base substitutions when present in either one or both DNA strands. We compared the fidelity for dsDNA mutations to that of the same samples profiled by NanoSeq duplex sequencing. We also detected cytosine deamination, a common type of DNA damage, with single-molecule resolution. We profiled 134 samples from diverse tissues, including from individuals with cancer predisposition syndromes. These samples revealed single-strand mismatch and damage signatures. Since double-strand DNA mutations are only the endpoint of the mutation process, our approach enables new studies of how mutations arise in a variety of contexts, especially in cancer and aging.The data available via dbGaP includes raw HiDEF-seq sequencing files (Pacific Biosciences subreads in BAM format), raw NanoSeq sequencing files (Illumina FASTQ format), and raw germline sequencing files (Illumina FASTQ format).The HiDEF-seq main publication contains a supplementary table with a list of accession IDs for each sample's files.

  Study phs003604

• Gabriella Miller Kids First Pediatric Research Project in Cornelia de Lange Syndrome, Related Diagnosis and Structural Birth Defects

  This study involves a cohort of 400 individuals and family members with Cornelia de Lange Syndrome (CdLS) and CdLS-like phenotypes for which a molecular etiology has not yet been established. CdLS is a developmental disorder characterized by development delays, cognitive impairment, short stature, hearing loss, specific facial features, and structural birth defects such as differences of the limbs, heart, kidneys, and GI tract. Our approach to tease out genetic contributions to birth defects has been to identify the underlying causes of syndromic birth defects which are often Mendelian in nature and therefore lend themselves more readily to genetic causal identification. Once identified, these genetic causes of syndromic forms of birth defects can be leveraged to understand the genetic contributions to isolated birth defects seen in constellation in syndromes such as CdLS. This work will lead to the identification of genes critical in human embryonic development, provide novel insights into transcriptional regulation and help to identify genetic causes and candidate genes for isolated birth defects seen in constellation in similar diagnoses.

  Study phs002174

• Transcriptomics sequencing analysis of pre-invasive lung adenocarcinoma in never-smokers

  The prevalence of lung adenocarcinoma (LUAD) has increased sharply in East Asia. Early diagnosis leads to better survival rates, but this requires an improved understanding of the molecular changes during early tumorigenesis, particularly in non-smokers. We performed whole exome-sequencing and RNA-sequencing for samples from 94 East Asian patients with precancerous lesions (25 with atypical adenomatous hyperplasia [AAH]; 69 with adenocarcinoma in situ [AIS]) and 73 patients with early invasive lesions (minimally invasive adenocarcinoma [MIA]). Cellular analysis revealed that the activities of endothelial and stromal cells could categorize tumors into molecular subtypes within pathology-defined lesion types. These subtypes were linked with the advanced radiology-defined lesion type and coordinated with immune cell infiltration throughout the progression of LUAD. Characterization of these lesion types identified positively selected mutation patterns, and suggested that angiogenesis of the late-stage AIS type potentially contributes to tissue invasion of the MIA type. Our findings offer a novel resource that may help to improve early diagnosis and patient prognosis, and also suggest possible approaches for early disease interception.

  Study EGAS50000000439

• Genomic Alterations in Normal Breast Tissues Preceding Breast Cancer Diagnosis (GANPBC)

  This study was designed to examine the genomic alterations in histologically normal breast tissues of women who subsequently developed breast cancer and compare it to breast tissues from women who did not. Breast core needle biopsy tissues from 160 healthy women at the Susan G. Komen Tissue Bank were requested for this study and sequenced using whole exome sequencing. The samples included tissues from 79 participants who, during the annual follow-up with Susan G. Komen self-reported a subsequent diagnosis of invasive or non-invasive breast cancer after tissue submission; these cases were classified as pre-diagnosis normal tissues. The remaining 81 samples were from women who had no cancer diagnosis at their last follow-up and were classified as healthy control samples. Among the pre-diagnosis samples, the average time from tissue donation to breast cancer diagnosis was 4.7 years (range: 0-11). For the healthy controls, the average time from tissue donation to the last follow up was 6.1 years (range: 0-14).

  Study phs003822

• IL-27 promotes tumour control by enhancing cytotoxic T cell function

  Cytotoxic CD8+ T lymphocytes (CTL) are essential for anti-tumour immunity but are frequently dysfunctional in established tumours. Here, we show that expression of a CTL signature is strongly associated with IL-27 expression in diverse human and murine tumours. In mice, IL-27 acts directly on tumour-specific CTL to promote their persistence and effector function in the tumor microenvironment. Moreover, treatment with inducible IL-27 overexpression or a half-life-extended IL-27 protein in vivo is well tolerated, induces regression of large established tumours, drives an enhanced cytotoxic program in anti-tumour CTL, and synergizes with PD-L1 blockade. In cancer patients treated with anti-PD-1 or anti-PD-L1 therapy, high expression of IL-27 is associated with improved clinical response. Finally, IL-27 increases cytotoxicity and limits terminal differentiation of human CD8+ T cells during chronic antigen exposure. Our data demonstrate that endogenous IL-27 is essential for anti-tumour immunity and that IL-27 receptor agonism can safely improve anti-tumor T cell responses alone or in combination with PD-L1 blockade.

  Study EGAS50000000694

• Acute Respiratory Distress Network (ARDSNet) Study 02 Late Steroid Rescue Study (LaSRS) (ARDSNet-LaSRS-BioLINCC)

  Data Access NOTE: Please refer to the “Authorized Access” section below for information about how access to the data from this accession differs from many other dbGaP accessions. Biospecimens: Access to Biospecimens is through the NHLBI Biologic Specimen and Data Repository Information Coordinating Center (BioLINCC). Biospecimens from ARDSNet-LaSRS include BAL Slides, Bronchial Lavage, and Plasma. Please note that use of biospecimens in genetic research is subject to a tiered consent.Objectives: Since previous reports suggested that corticosteroids may improve survival, this project was developed as a multicenter, randomized controlled trial of corticosteroids in patients with persistent ARDS.Background: Persistent acute respiratory distress syndrome (ARDS) is characterized by excessive fibroproliferation, ongoing inflammation, prolonged mechanical ventilation, and a substantial risk of death.Participants: There were 180 participants.Conclusions: The results do not support the routine use of methylprednisolone for persistent ARDS despite the improvement in cardiopulmonary physiology. In addition, starting methylprednisolone therapy more than two weeks after the onset of ARDS may increase the risk of death. (NEJM April 20, 2006; Vol 354, No. 16, pp 1671-84).

  Study phs003769

• Whole exome sequencing of pre-invasive lung adenocarcinoma in non-smokers

  The prevalence of lung adenocarcinoma (LUAD) has increased sharply in East Asia. Early diagnosis leads to better survival rates, but this requires an improved understanding of the molecular changes during early tumorigenesis, particularly in non-smokers. We performed whole exome-sequencing and RNA-sequencing for samples from 94 East Asian patients with precancerous lesions (25 with atypical adenomatous hyperplasia [AAH]; 69 with adenocarcinoma in situ [AIS]) and 73 patients with early invasive lesions (minimally invasive adenocarcinoma [MIA]). Cellular analysis revealed that the activities of endothelial and stromal cells could categorize tumors into molecular subtypes within pathology-defined lesion types. These subtypes were linked with the advanced radiology-defined lesion type and coordinated with immune cell infiltration throughout the progression of LUAD. Characterization of these lesion types identified positively selected mutation patterns, and suggested that angiogenesis of the late-stage AIS type potentially contributes to tissue invasion of the MIA type. Our findings offer a novel resource that may help to improve early diagnosis and patient prognosis, and also suggest possible approaches for early disease interception.

  Study EGAS50000000438

• Anti-myeloperoxidase IgM B cells in anti-neutrophil cytoplasmic antibody-associated vasculitis

  Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a prototypic autoimmune disease, with a subset of AAV patients manifesting anti-myeloperoxidase (MPO) IgG. Patients with AAV respond positively to B cell-targeting and complement-targeting therapies, but disease flares are not uncommon. Here, by comparing samples from healthy individuals and MPO+ AVV patients, we show that B cell autoreactivity against MPO in the circulation of patients is dominated by CD27+IgM+ B cells whereas MPO-specific IgG+ cells are infrequent. Additionally, while naive anti-MPO-IgM B cells are present in both patients and controls and produce anti-MPO IgM upon stimulation, anti-MPO-IgM memory B cells and serum anti-MPO IgM are features of patients. Our results thus hint that defective elimination of B cell reactivity to MPO in the human repertoire, the presence of activated IgM+ anti-MPO B cells in disease, and a dominant role for anti-MPO IgM in complement activation, may all contribute to MPO+ AAV etiology and thereby serve as potential target for therapy.

  Study EGAS50000000753

• Genomic Analysis of Relapsed/Refractory DLBCL

  Molecular phenotyping techniques have established a landscape of genomic variants in diagnostic Diffuse Large B Cell Lymphoma (DLBCL), however these have not yet been applied in large scale studies of relapsed/refractory DLBCL resulting in incomplete characterization of mechanisms driving tumor progression and treatment resistance. Here, we performed an integrated multiomic analysis on relapsed/refractory DLBCL samples. Tumors from 87 relapsed/refractory DLBCL patients were included in this study and were available for data deposition [Tumor whole exome sequencing (WES): n=75, Germline WES: n=73, RNA sequencing (RNA-Seq): n=72]. Tumor DNA was extracted from formalin-fixed, paraffin embedded (FFPE) tissue sections, germline DNA was extracted from blood, and WES of all samples was performed using the Agilent SureSelect XT All Exon v6 kit and sequencing was carried out on an Illumina NovaSeq, 100 x 2 paired end reads. Tumor RNA was extracted from FFPE tissue sections and RNA-Seq was performed using the Illumina TruSeq RNA Exome Kit (Illumina) for library preparation, sequencing platform HiSeq 4000, 100 x 2 paired end reads.

  Study phs003868

• LEF1 knockdown effects on human T cell transcriptome and chromatin accessibility profiles.

  LEF1 (Lymphoid Enhancer-Binding Factor 1) is a key transcription factor in T cell development and function, and its dysregulation has been implicated in various immune-related disorders. This study aimed to comprehensively characterize the transcriptional and epigenetic changes induced by LEF1 knockdown in human T cells. CD3+ T cells isolated from three healthy donors were transfected with either LEF1-specific siRNA or control siRNA, followed by activation with CD3/CD28 Dynabeads and culture in IL-2-containing media. Cells were harvested at 24, 48 and 72 hours post-transfection for multi-omics analysis including bulk RNA-seq, bulk ATAC-seq, single-cell CITE-seq (For CITE-seq, 48 and 72 hours only). This dataset provides insights into LEF1-dependent gene regulatory networks and chromatin accessibility patterns in activated human T cells, contributing to our understanding of T cell biology and potential therapeutic targets. This dataset is part of a comprehensive study investigating functional roles of risk polymorphisms in autoimmune diseases, particularly focusing on the rs58107865 variant at the LEF1 locus.

  Study JGAS000818

• Single Cell RNA-Sequencing of BCG Naive and Recurrent Non-Muscle Invasive Bladder Cancer Reveals a CD6/ALCAM-Mediated Immune-Suppressive Pathway

  Non-muscle invasive bladder cancer (NMIBC) represents 70–80% patients with newly diagnosed bladder cancer, and Bacillus Calmette-Guerin (BCG) remains a cornerstone treatment for intermediate- and high-risk NMIBC to prevent disease recurrence and progression. However, a significant number of patients experience recurrence after their first course of BCG, posing significant challenges in the management of the disease. We conducted single cell RNA sequencing (scRNA-Seq) on freshly collected NMIBC samples, distinguishing between those naive to BCG treatment and those that recurred post-BCG treatment. We observed a clear activation of inflammatory pathways across cell types during recurrence, but these were not associated with canonical immune checkpoint or T cell exhaustion phenotypes. Analysis of cell-to-cell communication revealed enhanced interactions between T cells and urothelial cells in BCG-recurrence, predominantly modulated by CD6 and activated leukocyte cell adhesion molecule (ALCAM). Furthermore, we found CD6hi T cells to be immunosuppressed and enriched in recurrent samples, suggesting a potential role for CD6 as an immune evasion signal in NMIBC.

  Study phs003742

• Single-Cell Multiomics of the Immune Microenvironment in T-Cell Acute Lymphoblastic Leukemia

  We present single-cell transcriptome and surface protein expression (CITE-seq) data from four healthy donors and fifteen pediatric T cell acute lymphoblastic leukemia (T-ALL) patients at diagnosis and at different treatment timepoints (days 1 – 6 and at time of remission). Mononuclear cells from peripheral blood (PB; longitudinal) or bone marrow (BM; diagnosis only) were isolated and CD45high immune cells and CD45dimCD7+ malignant cells were sorted and subsequently pooled to enrich for immune cells. For comparison,CD45high immune cells from four healthy donors were also sorted. Single-cell sequencing of the transcriptome and surface protein expression (204 antibodies) was performed using the 10x Genomics platform.Multiomics analyses resulted in the identification of a T-ALL subgroup characterized by a remodeled immune microenvironment at initial diagnosis. This immune landscape is dominated by the presence of non-malignant CD4-CD8-TCRab T cells that interact with CXCL16 expressing non-classical monocytes. Leukemia cell intrinsic transcriptional rewiring in these patients is associated with activation of Rap1 signaling.

  Study phs004269

• Prevalence and Clinical Characteristics of hearing loss caused by MYH14 mutation

  Mutations in MYH14 are reported to cause autosomal dominant nonsyndromic hereditary hearing loss (ADNSHL), and 35 variants have been reported to cause hearing loss in various ethnic groups, but the information on prevalence as well as clinical features are fragmented. In this study, genetic screening for MYH14 variants was carried out using a large series of Japanese hearing loss patients to reveal the information. Massively parallel DNA sequencing of 68 target candidate genes was utilized in 8074 (including 1336 ADNSHL) unrelated Japanese hearing loss patients (probands) to identify genomic variations responsible for hearing loss. We identified 20 patients with 14 mutations from 15 pedigrees. The prevalence was proved to be 0.19% (15/8074) among hearing loss patients, and 1.12% (15/1336) among ADNSHL patients. The 12 mutations out of 14 mutations identified were novel. The patients typically showed late onset, slowly progressive modelate flat-type hearing loss. The clinical information revealed by the present study would contribute to further diagnosis and management of MYH14-associated hearing loss.

  Study JGAS000323

• A multicenter study of susceptibility genes to type 1 diabetes

  The first genome-wide association study of fulminant type 1 diabetes mellitus was performed in Japanese individuals. As previously reported using a candidate gene approach, a strong association was observed with multiple SNPs in the HLA region, and the strongest association was seen for rs9268853 in the class II DR region (P=1.56 x 10-23, odds ratio [OR] 3.18). In addition, rs11170445 in CSAD/lnc-ITGB7-1 on chromosome 12q13.13 showed an association at a genome-wide significance level (P=7.58 x 10-9, OR 1.96). Fine mapping of the region revealed that rs3782151 in CSAD/lnc-ITGB7-1 showed the lowest P value (P=4.60 x 10-9, OR 1.97 [95% CI 1.57-2.48]). The risk allele of rs3782151 is a cis-expression quantitative trait locus (eQTL) for ITGB7 that significantly increases the expression of this gene. CSAD/lnc-ITGB7-1 was strongly associated with the susceptibility to fulminant, but not classical autoimmune, type 1 diabetes, implicating this locus in the distinct phenotype of fulminant type 1 diabetes.

  Study JGAS000144

• Identification and Characterization of Cancer Mutations in Japanese LungAdenocarcinoma without Sequencing of Normal Tissue Counterparts

  We analyzed whole-exome sequencing data from 97 Japanese lung adenocarcinomapatients and identified several putative cancer-related genes and pathways. Particularly, we observed that cancer-related mutation patterns were significantly different between different ethnic groups. As previously reported, mutations in the EGFR gene were characteristic to Japanese, while those in the KRAS gene were more frequent in Caucasians. Furthermore, during the course of this analysis, we found that cancer-specific somatic mutations can be detected without sequencing normal tissue counterparts. 64% of the germline variants could be excluded using a total of 217 external Japanese exome datasets. We also show that a similar approach may beused for other three ethnic groups, although the discriminative power depends on the ethnic group. We demonstrate that the ATM gene and the PAPPA2 gene could be identified as cancer prognosis related genes. By bypassing the sequencing of normaltissue counterparts, this approach provides a useful means of not only reducing the time and cost of sequencing but also analyzing archive samples, for which normal tissue counterparts are not available.

  Study JGAS000001

• Immuno-genomic Profiling of Biopsy Specimens Predicts Neoadjuvant Chemotherapy Response in Esophageal Squamous Cell Carcinoma

  Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers and is primarily treated with platinum-based neoadjuvant chemotherapy (NAC). Some ESCCs respond well to NAC. However, biomarkers to predict NAC sensitivity and their response mechanism in ESCC remain unclear. We introduce a model by machine learning and validated its potential to predict the NAC response in ESCC. To create this model, we performed whole genome sequencing and RNA-seq analysis on totally 141 ESCC biopsy specimens before NAC treatment and analyzed their immuno-genomic features and association with NAC response. Neutrophils infiltration could play an important role in ESCC response to NAC. We also demonstrated that specific copy number alterations and copy number signatures in the ESCC genome were significantly associated with NAC response. The interactions between the tumor genome and immune features of ESCC are likely to be a new indicator of therapeutic capability and a new therapeutic target for ESCC, and machine learning prediction for NAC response is useful.

  Study JGAS000535

• Spatial predictors of response to immunotherapy in microsatellite stable metastatic colorectal cancer

  Microsatellite stable (MSS) colorectal cancers (CRC) are largely unresponsive to immune checkpoint inhibition (ICI), prompting investigation into strategies to enhance sensitivity. The MAYA trial, which utilized temozolomide (TMZ) in MGMT-silenced MSS mCRC, hypothesized that TMZ-induced hypermutation could sensitize tumors to ICI. This phase II trial met its primary endpoint, demonstrating durable clinical responses with TMZ combined with ipilimumab and nivolumab. To elucidate factors influencing response heterogeneity, we conducted multi-omic spatial profiling of samples from patients who participated in the MAYA trial, including baseline and on-treatment tissue and blood specimens. While increased neoantigen load following TMZ exposure did not consistently predict for deep responses, spatial profiling revealed key determinants. Lymphocyte proportions, particularly CD8+KI67+ cells, within stromal and tumor compartments, along with macrophage composition (CD68+CD163+ cells) at the tumor-stromal interface, were predictive of response. Treatment pressures dynamically altered the tumor microenvironment composition and activated peripheral immune cells. This study is the first to identify spatial predictors of response to this promising novel treatment approach for MSS CRC.

  Study EGAS50000001567

• DERMATLAS__Hidradenoma_papilliferum_WES

  Virtually no other tumour type is associated with so many different forms as skin cancer. Histologically, tumours of the skin may arise from epithelium, including epidermis, hair follicle, sebaceous or sweat gland, melanocytes, dermal-associated mesenchymal structures or tissue resident immune cells, making for a diversity of clinical presentations. Importantly, many skin tumour types have an extremely poor prognosis. Many skin tumour subtypes have never undergone molecular profiling, or if they have, targeted sequencing has been used and the number of cases analyzed has been so limited that deriving firm conclusions about the profile of driver genes, DNA mutational signatures and germline alleles has not been possible For 70 key skin tumour subtypes defined by the World Health Organization, that have not undergone extensive genetic analysis previoulsy, we propose to perform whole exome and transcriptome sequencing, from a range of body sites, to build a genomic atlas of dermatological tumours, including detailed maps of SNVs, copy number alterations, genome-wide methylation and expression profiles. The cases in this cohort are hidradenoma papilliferums.

  Study EGAS00001005714

• DERMATLAS__Porocarcinoma_WES

  Virtually no other tumour type is associated with so many different forms as skin cancer. Histologically, tumours of the skin may arise from epithelium, including epidermis, hair follicle, sebaceous or sweat gland, melanocytes, dermal-associated mesenchymal structures or tissue resident immune cells, making for a diversity of clinical presentations. Importantly, many skin tumour types have an extremely poor prognosis. Many skin tumour subtypes have never undergone molecular profiling, or if they have, targeted sequencing has been used and the number of cases analyzed has been so limited that deriving firm conclusions about the profile of driver genes, DNA mutational signatures and germline alleles has not been possible For 70 key skin tumour subtypes defined by the World Health Organization, that have not undergone extensive genetic analysis previoulsy, we propose to perform whole exome and transcriptome sequencing, from a range of body sites, to build a genomic atlas of dermatological tumours, including detailed maps of SNVs, copy number alterations, genome-wide methylation and expression profiles. The cases in this cohort are porocarcinomas.

  Study EGAS00001005720

• HipSci - Human Induced Pluripotent Stem Cells Initiative

  Human induced pluripotent stem (iPS) cells can be generated from adult tissues and differentiated into a range of specific cell types (Yamanaka and Blau, 2010). They therefore provide unprecedented opportunities for investigating disease mechanisms and understanding the phenotypic consequences of genetic variation within the normal population. HipSci brings together diverse constitutents in genomics, proteomics, cell biology and clinical genetics to create a UK national iPS cell resource and use it to carry out cellular genetic studies. Between 2013 and 2016 we aim to generate iPS cells from over 500 healthy individuals and 500 individuals with genetic disease. We will then use these cells to discover how genomic variation impacts on cellular phenotype and identify new disease mechanisms. Strong links with NHS investigators will ensure that studies on the disease-associated cell lines will be linked to extensive clinical information. Further key features of the project are an open access model of data sharing; engagement of the wider clinical genetics community in selecting patient samples; and provision of dedicated laboratory space for collaborative cell phenotyping and differentiation.

  Study EGAS00001001465

• CD36 defines CML cells less sensitive to imatinib

  Tyrosine kinase inhibitors (TKIs) are highly effective for treatment of chronic myeloid leukemia (CML), but very few patients are cured. Major drawbacks with TKIs are their low efficacy in eradicating the leukemic stem cells responsible for disease maintenance and relapse upon TKI cessation. Here, we performed RNA-sequencing of flow-sorted primitive (CD34+CD38low) and progenitor (CD34+CD38+) chronic phase CML cells and identified transcriptional upregulation of 32 cell surface molecules relative to corresponding normal bone marrow cells. Focusing on novel markers with increased expression on primitive CML cells, we confirmed upregulation of the scavenger receptor CD36 and the leptin receptor (LEPR) by flow cytometry. We also delineate a subpopulation of primitive CML cells expressing CD36 that is less sensitive to imatinib treatment. Using CD36 targeting antibodies, we show that the CD36 positive cells can be targeted and killed by antibody dependent cellular cytotoxicity (ADCC). In summary, CD36 defines a subpopulation of primitive CML cells with decreased imatinib sensitivity that can be effectively targeted by ADCC using an anti-CD36 antibody.

  Study EGAS00001002421

• Ovarian cancer organoid biobank

  Ovarian cancer (OC) is a heterogeneous disease usually diagnosed at a late stage. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of OC are limited and hard to establish. We present a novel protocol that enables efficient derivation and long-term expansion of OC organoids. Utilizing this protocol, we have established 56 organoid lines from 32 patients, representing the spectrum of ovarian neoplasms, including non-malignant borderline tumors, as well as mucinous, clear-cell, endometrioid, low- and high-grade serous carcinomas. OC organoids recapitulate histological and genomic features of the pertinent lesion from which they were derived, illustrating intra- and inter-patient heterogeneity, and can be genetically modified. We show that OC organoids can be used for drug screening assays and capture different tumor subtype responses to the gold standard platinum-based chemotherapy, including acquisition of chemoresistance in recurrent disease. Finally, OC organoids can be xenografted, enabling in vivo drug sensitivity assays. Taken together, this demonstrates their potential application for research and personalized medicine.

  Study EGAS00001003073

• Uveal melanoma patient with germline MBD4 nonsense mutation

  There is currently no effective treatment for metastasised uveal melanoma (UM). Recently, it was reported that a UM patient was responsive to checkpoint inhibitor (CI) treatment, due to a high tumour mutation burden correlated with a germline loss-of-function MBD4 mutation. Here, we report on another UM patient who carried an MBD4 germline nonsense variant (p.Leu563Ter) and her tumour showed a 5-fold higher than average mutation burden. We confirmed the association between germline loss-of-function variant in MBD4 and CI response. The patient experienced stable disease (10 months) and survived two years with metastatic disease, which is twice as long as median survival. Additionally, the frequency of MBD4 loss-of-function variants in reported UM cohorts was >20 times higher than in an aggregated population genome database (P < 5x10-5), implying a potential role as UM predisposition gene. These findings provide a strong basis for the inclusion of MBD4 in screening of potential UM-prone families as well as stratification of immunotherapy.

  Study EGAS00001003362

• RNA-seq study of a Princess Margaret Cancer Centre human acute myeloid leukemia patient cohort

  The PMCC AML RNAseq dataset consists of 81 AML patient samples (clinical data in Supplemental Table 11 of manuscript), processed in two batches. These patient samples are able to engraft in the NSG (NOD.Cg PrkdcscidIl2rgtm1Wjl /SzJ) mouse model. Five patients (90543, 598, 90240, 110484, 100500) were included in both batches. Viaably frozen material from the Leukemia Tissue Bank at Princess Margaret Cancer Centre/ University Health Network were thawed by dropwise addition of X-VIVO + 50% fetal calf serum supplemented with DNase (100μg/mL final concentration, Roche). RNA was extracted from bulk peripheral blood mononuclear cells (PBMC) using the RNeasy Micro Kit (Qiagen Inc.). A paired-end 76 base-pair flow-cell lane Illumina High seq 2000 yielded an average of 240 million sequence reads aligning to genome per sample at the Genome Sciences Centre, BC Cancer Agency for cohort 1. Cohort 2 was subjected to 125 bp, paired-end RNA-sequencing on the Illumina HiSeq 2500 with an average of 50 million reads/sample at the Centre for Applied Genomics, Sick Kids Hospital.

  Study EGAS00001004792

• Pilot_experiment_on_functional_genomics_in_osteoarthritis

  The aim of this work is to apply an integrated systems approach to understand the biological underpinnings of large joint (hip and knee) osteoarthritis which culminates in the need for total joint replacement (TJR). In this pilot we will assess the feasibility of the approach in the relevant tissue. We will obtain diseased and non-diseased tissue (cartilage and endochondral bone) following TJR, coupled with a blood sample, from 12 patients. We will generate genotype data on the HumanCoreExome array and will characterise the 12 pairs of diseased and non-diseased tissue samples in terms of methylation (Illumina 450k array), transcription (RNASeq) and expression (quantitative proteomics). The pilot will help assess the feasibility of isolating sufficient levels of starting material for the different approaches, and will instigate the development of analytical approaches to synthesising the resulting data. If successful, it is envisaged that we will proceed with expanding the work to 500 patients in the first year, with a target total sample size of 3,000 in the longer term. The key collaborators in this work are Mark Wilkinson (Sheffield) and Jyoti Choudhary

  Study EGAS00001001212

• FetalQuant-SD: Accurate quantification of fetal DNA fraction by shallow-depth sequencing of maternal plasma DNA

  Noninvasive prenatal testing using massively parallel sequencing of maternal plasma DNA has been rapidly adopted in clinical use worldwide. Fetal DNA fraction in a maternal plasma sample is an important parameter for accurate interpretations of these tests. However, there is a lack of methods involving low-sequencing depth and yet would allow a robust and accurate determination of fetal DNA fraction in maternal plasma for all pregnancies. In this study, we have developed a new method to accurately quantify the fetal DNA fraction by analysing the maternal genotypes and sequencing data of maternal plasma DNA. Fetal DNA fraction was calculated based on the proportion of non-maternal alleles at single-nucleotide polymorphisms where the mother is homozygous. This new approach achieves a median deviation of 0.6% between predicted fetal DNA fraction and the actual fetal DNA fraction using as low as 0.03-fold sequencing coverage of the human genome. We believe that this method will further enhance the clinical interpretations of noninvasive prenatal testing using genome-wide random sequencing.

  Study EGAS00001001611

• Field_effect_of_healthy_and_diseased_livers

  Recent work in the Campbell group has revealed somatic mutations present in normal, non-cancerous human skin. A subset of the mutations conferred selective advantages to the host cells, leading to clonal expansions and raising the risk for future cancer development. Capturing such somatic mutations in normal tissue is important to advance our understanding about carcinogenesis and could provide prospective medical insights. In this project, our goal is to detect somatic mutations in normal (pre-cancerous) liver tissue. Using Laser Microdissection technology, we will dissect individual liver lobules from patient samples and submit these to sequencing. For each patient sample, we aim to sequence multiple lobules to characterise the mutagenic burden. Samples will be taken from patients with different liver disease aetiologies, including alcoholism and obesity, with a view on distinguishing the prevalent mutation types occurring in each disease context. We will perform targeted sequencing, initially using the WTSI cancer panel. Later we aim to use a novel bait set that captures both cancer genes as well as genes relevant to the non-cancerous samples (ie. genes implicated in hereditary disorders, immune sequences).

  Study EGAS00001002382

• Genome-wide mutational consequences of nucleotide excision repair-deficiency through XPC deletion in a human adult stem cell culture

  Nucleotide excision repair (NER) is one of the main DNA repair pathways that protect cells against genomic damage. Deficiency in this pathway can contribute to the development of cancer and accelerate aging. NER-deficiency is an important determinant for cancer treatment outcome, as NER-deficient tumors are selectively sensitive to cisplatin treatment. While NER-deficiency has been linked to mutational Signature 5, not all NER-deficient tumors are characterized by a high Signature 5 contribution, illustrating the importance to further characterize the mutational consequences of NER-deficiency. Here, we analyzed the mutational profile of a human adult stem cell (organoid) culture that is deficient in NER through XPC deletion by CRISPR-Cas9 gene-editing, and subsequent whole-genome sequencing analysis of a clonally derived organoid. We found that XPC deletion results in an increase in base substitution load and specifically induces Signature 8 mutations, a mutational signature with previously unknown etiology. Presence of Signature 8 may, therefore, serve as a novel biomarker for NER-deficient tumors and could improve personalized treatment strategies.

  Study EGAS00001002681

• Using human induced pluripotent stem cells (iPSC) and iPSC-derived neurons to explore cellular phenotypes associated with schizophrenia.

  Schizophrenia and major mental illness are common devastating conditions. Whilst partially effective treatments are available, none are disease-modifying. Notwithstanding important insights provided by animal models there is a need for novel models of mental illness to develop more effective therapies. A study of sporadic schizophrenia-derived human induced pluripotent stem cells (hiPS) has demonstrated a neuronal phenotype representing a powerful proof of concept of in vitro disease modeling, even for diseases with complex causation such as schizophrenia. These techniques have been used here to examine the behaviour of neural stem cells and neurons derived from fibroblasts from subjects carrying NDE1 copy number variations (CNVs) associated with increased risk of major mental illness. Using hiPS from individuals with and without disease-associated CNVs we have investigated how disease risk is conferred at a cellular and molecular level through detailed comparative studies of neural progenitor proliferation, neuronal morphology, physiology, cytoskeletal organization, synaptic function, glutamate (NMDA) receptor expression and also RNA sequencing and proteomic studies of these patient-derived cells.

  Study EGAS00001002895

• Pheno-Seq, linking 3D phenotypes of clonal tumor spheroids to gene expression

  3D-culture systems have advanced cancer modeling by reflecting physiological characteristics of in-vivo tissues, but our understanding of functional intratumor heterogeneity including visual phenotypes and underlying gene expression is still limited. Transcriptional heterogeneity can be dissected by single-cell RNA-sequencing, but these technologies suffer from low RNA-input and fail to directly correlate gene expression with contextual cellular phenotypes. Here we present pheno-seq for integrated high-throughput imaging and transcriptomic profiling of clonal tumor spheroids derived from 3D models of breast and colorectal cancer. Specifically, we identify characteristic expression signatures that are associated with heterogeneous invasive and proliferative behavior including a rare cell subtype. Furthermore, we identify functionally relevant transcriptional regulators missed by single-cell RNA-seq, link visual phenotypes defined by heterogenous expression to inhibitor response and infer single-cell regulatory states by deconvolution. We anticipate that directly linking molecular features with patho-phenotypes of cancer cells will improve the understanding of intratumor heterogeneity and consequently prove to be useful for translational research.

  Study EGAS00001002999

• Dissecting intratumor heterogeneity of nodal B cell lymphoma on the transcriptional, genetic, and drug response level

  Tumor heterogeneity encompasses both the malignant cells and their microenvironment. While heterogeneity between individual patients is well-known to affect the efficacy of anti-cancer, most personalized treatment approaches do not account for intratumor heterogeneity. We addressed this issue by studying the heterogeneity of lymph node-derived B cell non-Hodgkin lymphoma (B-NHL) by single cell RNA-sequencing (scRNA-seq) and transcriptome-informed flow cytometry. We identified transcriptionally distinct malignant subclones and compared their drug response and genomic profiles. Malignant subclones of the same patient responded strikingly different to anti-cancer drugs ex vivo, which recapitulated subclone-specific drug sensitivity during in vivo treatment. Tumor infiltrating T cells represented the majority of non-malignant cells, whose gene expression signatures were similar across all donors, whereas the frequencies of T cell subsets varied significantly between the donors. Our data provide new insights into the heterogeneity of B-NHL and highlight the relevance of intratumor heterogeneity for personalized cancer therapies.

  Study EGAS00001004335

• Pilot_experiment_on_functional_genomics_in_osteoarthritis

  The aim of this work is to apply an integrated systems approach to understand the biological underpinnings of large joint (hip and knee) osteoarthritis which culminates in the need for total joint replacement (TJR). In this pilot we will assess the feasibility of the approach in the relevant tissue. We will obtain diseased and non-diseased tissue (cartilage and endochondral bone) following TJR, coupled with a blood sample, from 12 patients. We will generate genotype data on the HumanCoreExome array and will characterise the 12 pairs of diseased and non-diseased tissue samples in terms of methylation (Illumina 450k array), transcription (RNASeq) and expression (quantitative proteomics). The pilot will help assess the feasibility of isolating sufficient levels of starting material for the different approaches, and will instigate the development of analytical approaches to synthesising the resulting data. If successful, it is envisaged that we will proceed with expanding the work to 500 patients in the first year, with a target total sample size of 3,000 in the longer term. The key collaborators in this work are Mark Wilkinson (Sheffield) and Jyoti Choudhary

  Study EGAS00001001213

• Integrative genomic analysis identifies multiple subtypes and therapeutic targets in acute erythroid leukemia

  Acute erythroid leukemia (AEL) is a high risk leukemia of poorly understood genetic basis, and controversy regarding diagnosis in the spectrum of myelodysplasia and myeloid leukemia. We compared genomic and clinical features of 159 childhood and adult AEL cases to non-AEL myeloid disorders, and identified patterns of mutation that describe 5 age-related subgroups with distinct transcriptional profiles: adult, TP53-mutated; NPM1-mutated; KMT2A-mutated/rearranged; adult, DDX41-mutated; and pediatric, NUP98-rearranged. Mutational spectra were age-dependent and significantly associated with outcome, with NPM1 mutations and high HOXB9 expression being associated with favorable prognosis, and TP53, FLT3 and RB1 alterations associated with poor survival. Targetable signaling mutations were present in 45% of cases, and include recurrent mutations of ALK and NTRK1, the latter of which drive erythroid leukemogenesis sensitive to TRK inhibition. This genomic landscape of AEL provides the framework for accurate diagnosis and risk stratification of this disease, and the rationale for testing targeted therapies in this form of leukemia that has hitherto and dismal outcome.

  Study EGAS00001002537

• Genes associated with pancreas development and function maintain open chromatin in iPSCs generated from human pancreatic beta cells

  Current in-vitro islet differentiation protocols suffer from heterogeneity and low efficiency. Induced-pluripotent stem cells (iPSCs) derived from pancreatic beta-cells (BiPSCs) preferentially differentiate towards endocrine pancreas-like cells versus those from fibroblasts (FiPSCs). We interrogated genome-wide open chromatin in BiPSCs and FiPSCs via ATAC-seq, and identified ~8.3k significant, differential open chromatin sites (DOCS) between the two iPSC subtypes (FDR<0.05). DOCS where chromatin was more accessible in BiPSCs (Bi-DOCS) were significantly enriched for known regulators of endodermal development, including bivalent and weak enhancers, and FOXA2 binding sites (FDR<0.05). Bi-DOCS were associated with genes related to pancreas development and beta-cell function, including transcription factors mutated in monogenic diabetes (PDX1, NKX2-2, HNF1A; FDR<0.05). Moreover, Bi-DOCS correlated with enhanced gene expression in BiPSC-derived definitive endoderm and pancreatic progenitor cells. Bi-DOCS therefore highlight genes and pathways governing islet-lineage commitment, which can be exploited for differentiation protocol optimisation, diabetes disease modelling, and therapeutic purposes.

  Study EGAS00001002591

• Genomic and functional fidelity of small cell lung cancer patient-derived xenografts

  Small cell lung cancer (SCLC) patient-derived xenografts (PDXs) can be generated from biopsies or circulating tumor cells (CTCs), though scarcity of tissue and low efficiency of tumor growth have previously limited these approaches. Applying an established clinical-translational pipeline for tissue collection and an automated microfluidic platform for CTC-enrichment, we generated 17 biopsy-derived PDXs and 17 CTC-derived PDXs in a two-year timeframe, at efficiencies of 89% and 38%, respectively. Whole exome sequencing showed that somatic alterations are stably maintained between patient tumors and PDXs. Early-passage PDXs maintain the genomic and transcriptional profiles of the founder PDX. In vivo treatment with etoposide and cisplatin (EP) in 30 PDX models demonstrated greater sensitivity in PDXs from EP naïve patients, and resistance to EP corresponded to increased expression of a MYC gene signature. Finally, serial CTC-derived PDXs generated from an individual patient at multiple time points accurately recapitulated the evolving drug sensitivities of that patient’s disease. Collectively, this work highlights the translational potential of this strategy.

  Study EGAS00001002853

• Somatic_mutation_profiling_of_intestinal_crypts_from_IBD

  In this project we want to investigate the role that somatic mutations in intestinal stem cells play in IBD. We will use laser capture microdissection to ascertain epithelial cells from an individual crypts/vilus. We will extract DNA from these and use a new low-input material library prep methods to make accurate libraries for sequencing. In this pilot phase of the project we will perform 15X whole-genome sequencing of a small number of crypts biopsied from four patients. Biopsies have been taken from inflammed and noninflammed patches to compare mutation rates and mutational signatures. We will also test if crypts from inflammed patches of a single patient are clonal (either within a given inflammed patch or across inflammed patches). We will quantify the rate of cancer driver mutations in inflammed and non-inflammed crypts. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/

  Study EGAS00001002896

• DERMATLAS__Hidradenoma_papilliferum_RNAseq

  Virtually no other tumour type is associated with so many different forms as skin cancer. Histologically, tumours of the skin may arise from epithelium, including epidermis, hair follicle, sebaceous or sweat gland, melanocytes, dermal-associated mesenchymal structures or tissue resident immune cells, making for a diversity of clinical presentations. Importantly, many skin tumour types have an extremely poor prognosis. Many skin tumour subtypes have never undergone molecular profiling, or if they have, targeted sequencing has been used and the number of cases analyzed has been so limited that deriving firm conclusions about the profile of driver genes, DNA mutational signatures and germline alleles has not been possible For 70 key skin tumour subtypes defined by the World Health Organization, that have not undergone extensive genetic analysis previoulsy, we propose to perform whole exome and transcriptome sequencing, from a range of body sites, to build a genomic atlas of dermatological tumours, including detailed maps of SNVs, copy number alterations, genome-wide methylation and expression profiles. The cases in this cohort are hidradenoma papilliferums.

  Study EGAS00001005715

• DERMATLAS__Porocarcinoma_RNAseq

  Virtually no other tumour type is associated with so many different forms as skin cancer. Histologically, tumours of the skin may arise from epithelium, including epidermis, hair follicle, sebaceous or sweat gland, melanocytes, dermal-associated mesenchymal structures or tissue resident immune cells, making for a diversity of clinical presentations. Importantly, many skin tumour types have an extremely poor prognosis. Many skin tumour subtypes have never undergone molecular profiling, or if they have, targeted sequencing has been used and the number of cases analyzed has been so limited that deriving firm conclusions about the profile of driver genes, DNA mutational signatures and germline alleles has not been possible For 70 key skin tumour subtypes defined by the World Health Organization, that have not undergone extensive genetic analysis previoulsy, we propose to perform whole exome and transcriptome sequencing, from a range of body sites, to build a genomic atlas of dermatological tumours, including detailed maps of SNVs, copy number alterations, genome-wide methylation and expression profiles. The cases in this cohort are porocarcinomas.

  Study EGAS00001005721

• DERMATLAS__Poroma_RNAseq

  Virtually no other tumour type is associated with so many different forms as skin cancer. Histologically, tumours of the skin may arise from epithelium, including epidermis, hair follicle, sebaceous or sweat gland, melanocytes, dermal-associated mesenchymal structures or tissue resident immune cells, making for a diversity of clinical presentations. Importantly, many skin tumour types have an extremely poor prognosis. Many skin tumour subtypes have never undergone molecular profiling, or if they have, targeted sequencing has been used and the number of cases analyzed has been so limited that deriving firm conclusions about the profile of driver genes, DNA mutational signatures and germline alleles has not been possible For 70 key skin tumour subtypes defined by the World Health Organization, that have not undergone extensive genetic analysis previoulsy, we propose to perform whole exome and transcriptome sequencing, from a range of body sites, to build a genomic atlas of dermatological tumours, including detailed maps of SNVs, copy number alterations, genome-wide methylation and expression profiles. The cases in this cohort are poromas.

  Study EGAS00001005759

• Single-nuclei gene-expression analysis of pheochromocytoma and paraganglioma links tumor subtypes with tumor microenvironment

  Pheochromocytomas and paragangliomas (PCPG) are rare neuroendocrine tumors associated with autonomic nerves. We used single nuclei-RNA-seq (snRNA-seq) for analysis of 30 PCPG representing 13 known driver genes, plus two normal adrenal medullas to dissect cell composition, refine PCPG subtypes and compare PCPG and normal tissue expression. Incorporating bulk-tissue and snRNA-seq data we identified seven PCPG gene-expression subtypes with genotype and cell type associations. The tumor microenvironment reflected HIF pathway activation and neovascularization by detection of abundant tip-like endothelial cells and pericytes concordant with VEGFA expression in neoplastic cells with mutations in VHL, genes encoding SDH subunits and MAML3-fusions. Macrophages were abundant in PCPG with distinct tumor-associated features. Schwann-cell-like cells were also enriched in some subtypes. The transcriptional profile of neoplastic PCPG cells resembled normal chromaffin cells but early chromaffin and neuroblast cell markers were expressed in some subtypes including GPR139, a putative therapeutic target for a subset of pseudohypoxic PCPG.

  Study EGAS00001005861

• Serum proteomics of aortic diseases

  Aortic diseases are a rare but potentially life-threatening condition. We present a serum proteomic study for a spectrum of aortic diseases including thoracic aortic aneurysms (n = 11), chronic dissections (n = 9), acute aortic dissections (n = 11), and surgically treated dissections (n = 19) as well as healthy controls (n = 10) and patients of coronary heart disease (n = 10) to represent non-aortic cardiovascular disease. In total, we identified and quantified 425 proteins across all 70 samples. The different aortic diseases represented distinguishable proteome profiles. We identified protein clusters that positively or negatively correlate with disease severity, including increase of cytosolic tissue leakage proteins and decrease of components of the coagulation and complement system. Further, we identified a serum proteome fingerprint of acute aortic dissections, consisting, among others, of enriched inflammatory markers such as C-reactive protein and members of the S100 protein family. The study underlines the applicability of serum proteomics for the investigation of aortic diseases and highlights the possibility to establish disease-specific prognostic markers.

  Study EGAS00001006201

• Salivary Gland Cancer TSO500 dataset

  Salivary gland cancer (SGC) is a rare cancer for which systemic treatment options are limited. Therefore, it is important to characterize its genetic landscape in search for actionable aberrations. This research aimed to identify these actionable aberrations by NGS-based analysis of DNA (single and multiple nucleotide variants, copy number variants, microsatellite instability and tumor mutational burden) in a large cohort of SGC patients. DNA was extracted from archival tissue of 119 patients with various SGC subtypes and sequenced using a targeted NGS panel encompassing 523 cancer related genes (TruSight Oncology 500, TSO500). The 119 patients included 46 AdCC patients, 43 SDC patients, 15 MEC patients, 9 AciCC patients and 6 patients with other subtypes (1 secretory carcinoma, 1 polymorphic adenocarcinoma (PAC), 1 adenocarcinoma NOS, 1 myoepithelial carcinoma, 1 epithelial/myoepithelial carcinoma and 1 mixed PAC/myoepithelial carcinoma). Median exon coverage ranged from 80-904 (median 308) unique reads and the median percentage of exon coverage with at least 100 unique reads was median 96.6% in these 119 samples.

  Study EGAS00001006232

• Genetic and epigenetic variation at regulatory regions contribute to cancer evolution under endocrine treatment

  Comprehensive profiling of hormone-dependent breast cancer (HDBC) has identified hundreds of protein-coding alterations contributing to cancer initiation, but only a handful have been linked to endocrine therapy resistance, potentially contributing to 40% of relapses. If other mechanisms underlie the evolution of HDBC under adjuvant therapy is currently unknown. In this work, we employ integrative functional genomics to dissect the contribution of cis-regulatory elements (CREs) to cancer evolution by focusing on 12 megabases of non-coding DNA, including clonal enhancers, gene promoters, and boundaries of topologically associating domains. Massive parallel perturbation in vitro reveals context-dependent roles for many of these CREs, with a specific impact on dormancy entrance and endocrine therapy resistance9. Profiling of CRE somatic alterations in a unique, longitudinal cohort of patients treated with endocrine therapies identifies non-coding changes involved in therapy resistance. Overall, our data uncover actionable transient transcriptional programs critical for dormant persister cells and unveil new regulatory nodes driving evolutionary trajectories towards disease progression.

  Study EGAS00001006340

• DERMATLAS__Tubular_adenoma

  IVirtually no other tumour type is associated with so many different forms as skin cancer. Histologically, tumours of the skin may arise from epithelium, including epidermis, hair follicle, sebaceous or sweat gland, melanocytes, dermal-associated mesenchymal structures or tissue resident immune cells, making for a diversity of clinical presentations. Importantly, many skin tumour types have an extremely poor prognosis. Many skin tumour subtypes have never undergone molecular profiling, or if they have, targeted sequencing has been used and the number of cases analyzed has been so limited that deriving firm conclusions about the profile of driver genes, DNA mutational signatures and germline alleles has not been possible For 70 key skin tumour subtypes defined by the World Health Organization, that have not undergone extensive genetic analysis previoulsy, we propose to perform whole exome and transcriptome sequencing, from a range of body sites, to build a genomic atlas of dermatological tumours, including detailed maps of SNVs, copy number alterations, genome-wide methylation and expression profiles. The cases in this cohort are tubular adenomas.

  Study EGAS00001006686

• The miR-185/PAK6 Axis Predicts Therapy Response and Regulates Survival of Drug-Resistant Leukemic Stem Cells in Chronic Myeloid Leukemia

  Overcoming drug resistance and targeting cancer stem cells remain challenges for curative cancer treatment. To investigate the role of miRNAs in regulating drug resistance and leukemic stem cell (LSCs) fate, we performed global transcriptome profiling in treatment-naïve chronic myeloid leukemia (CML) stem/progenitor cells and identified that miR-185 levels anticipate their response to ABL tyrosine kinase inhibitors (TKIs). miR-185 functions as a tumor suppressor; its restored expression impaired survival of drug-resistant cells, sensitized them to TKIs in vitro, and markedly eliminated long-term repopulating LSCs and infiltrating blast cells, conferring a survival advantage in pre-clinical xenotransplantation models. Integrative analysis with mRNA profiles uncovered PAK6 as a crucial target of miR-185 and pharmacological inhibition of PAK6 perturbed the RAS/MAPK pathway and mitochondrial activity, sensitizing therapy-resistant cells to TKIs. Thus, miR-185 presents as a potential predictive biomarker, and dual targeting of miR-185-mediated PAK6 activity and BCR-ABL may provide a valuable strategy for overcoming drug resistance in patients.

  Study EGAS00001004153

• DERMATLAS__Tubular_adenoma_RNAseq

  IVirtually no other tumour type is associated with so many different forms as skin cancer. Histologically, tumours of the skin may arise from epithelium, including epidermis, hair follicle, sebaceous or sweat gland, melanocytes, dermal-associated mesenchymal structures or tissue resident immune cells, making for a diversity of clinical presentations. Importantly, many skin tumour types have an extremely poor prognosis. Many skin tumour subtypes have never undergone molecular profiling, or if they have, targeted sequencing has been used and the number of cases analyzed has been so limited that deriving firm conclusions about the profile of driver genes, DNA mutational signatures and germline alleles has not been possible For 70 key skin tumour subtypes defined by the World Health Organization, that have not undergone extensive genetic analysis previoulsy, we propose to perform whole exome and transcriptome sequencing, from a range of body sites, to build a genomic atlas of dermatological tumours, including detailed maps of SNVs, copy number alterations, genome-wide methylation and expression profiles. The cases in this cohort are tubular adenomas.

  Study EGAS00001006685

• Youth-GEMs, the project using data to define the mental health trajectories of young people

  Mental health trajectories are shaped by genetic, cognitive, social, and environmental factors that are particularly influential during human development. How exactly the interplay between these factors is impacting mental health, however, is yet unknown. The project “Gene Environment interactions in Mental health trajectories of Youth (Youth-GEMs)”, funded by the European Commission under the Horizon Europe programme, aims to help reducing mental suffering among European youth. How do Youth-GEMs contribute to bettering mental health in young people? Youth-GEMs aims to identify specific neurological, (epi)genetic and environmental drivers on mental health trajectories of young people. To do so, the project is working on a public knowledge base on functional genomics of the developing human brain, between birth and 24 years old. Reliable predictive models for trajectories of onset and course of mental illness in young people of the general population in Europe will be developed. Additionally, a secure and FAIR data infrastructure in mental health is being produced. Engaging and empowering young people Youth-GEMs is also working on the engagement of young people in the scientific field of Psychiatry. For that, the project is developing initiatives such as Science Café, intended to create a learning community where ideas and stories are shared. The 10th Science Café will take place today, with the participation of Lauren Fromont from the EGA. How do the EGA contribute to Youth-GEMs? The contributions require careful data management and harmonisation: we are dealing with an extraordinary amount of data types, sources, and time points (since the studies are longitudinal). To analyse them together and to determine which factors and interactions are truly key to account for mental health trajectories, the variables need to be transformed in a common “language”, i.e., harmonised under a common data model. Further, to guarantee FAIRness of data storage and sharing for future projects that will further advance our knowledge in the mental health field, we are ensuring that data are processed securely but can also be discovered easily. Find out more information about the project in the Youth-GEMs website.

  Blog youth-gems-mental-health

• Introducing Federated EGA Affiliates: a newly defined tier in the Federated EGA Network

  We're happy to announce that we are now accepting Affiliates in the Federated EGA Network! Federated EGA is a global network of repositories enabling secure discovery and access to sensitive human data. Currently, Federated EGA consists of Central EGA (CEGA), which serves the global community, and nine national Nodes that serve respective jurisdictions. Did you know that the Federated EGA (FEGA) has always envisioned a third tier within the network? This brings us to FEGA Affiliates, recently defined in the newly approved "Federated EGA Structure and Organisation" document. This new tier broadens the range of institutions or organisations that can become part of FEGA. Thus begins a new era for the network. What is a FEGA Affiliate? A FEGA Affiliate archives and serves controlled-access data within its own governance scope, while still participating in federated discovery through the EGA catalogue. Affiliates can be individual organisations or institutions, healthcare genomics initiatives, consortia of institutions, as well as project-specific consortia (national or international). In practical terms, a FEGA Affiliate: Hosts and safeguards the data it archives. Submits non-personal metadata to CEGA so studies are discoverable and can receive EGA accessions. Manages permissions and data access workflows for its datasets. Provides data access and/or distribution, along with support, to authorised users. Key takeaways about FEGA Affiliates FEGA is now organised into three tiers: CEGA, FEGA Nodes, and FEGA Affiliates. Affiliates archive data from their own organisation and/or selected partner institutions (for example, an international consortium). Affiliates make studies discoverable through the EGA catalogue by submitting non-personal metadata for accessioning. Access decisions and conditions are managed locally by the Affiliate (e.g. distribution or access in a trusted research environment). Are you considering whether your institution or project might be a good fit as a FEGA Affiliate? Would you like to learn more about this tier? We have published a blog post that addresses potential common questions, providing answers on this topic. You can read it here.

  Blog fega-affiliates

• Genome Diversity in Africa Project: Uganda (2021-02-17)

  Globally, human populations show structured genetic diversity as a result of geographical dispersion, selection and drift. Understanding this genetic variation can provide insights into the evolutionary processes that shape both human adaptation and variation in disease. Populations from SSA have the highest levels of genetic diversity. This characteristic, in addition to historical genetic admixture, can lead to complexities in the design of studies assessing the genetic determinants of disease and human variation. However, such studies of African populations are also likely to provide new opportunities to discover novel disease susceptibility loci and variants and refine gene-disease association signals. A systematic assessment of genetic diversity within SSA would facilitate genomic epidemiological studies in the region. The Genome Diversity in Africa Project (GDAP) aims to produce a comprehensive catalogue of human genetic variation in SSA, including single nucleotide polymorphisms (SNPs), structural variants, and haplotypes. This resource will make a substantial contribution to understanding patterns of genetic diversity within and among populations in SSA, as well as providing a global resource to help design, implement and interpret genomic studies in SSA populations and studies comprising globally diverse populations, complementing existing genomic resources. Specifically, we plan to carry out high depth whole genome sequencing of up to 2000 individuals across Africa (25 individuals from each ethnolinguistic group). Our scientific objectives are to: 1) develop a resource that provides a comprehensive catalogue of genetic variation in populations from SSA accessible to the global scientific community; 2) characterise population genetic diversity, structure, gene flow and admixture across SSA; 3) develop a cost-efficient, next-generation genotype array for diverse populations across SSA; and 4) facilitate whole genome-sequencing association studies of complex traits and diseases by developing a reference panel for imputation and resource for enhancing fine-mapping disease susceptibility loci. These scientific objectives will be supported by cross-cutting operational activities, including network and management of the consortium, research ethics, and research capacity building in statistical genetics and bioinformatics . This dataset contains all the data available for this study on 2021-02-17.

  Dataset EGAD00001006976

• DERMATLAS__Leiomyosarcoma_WES

  irtually no other tumour type is associated with so many different forms as skin cancer. Histologically, tumours of the skin may arise from epithelium, including epidermis, hair follicle, sebaceous or sweat gland, melanocytes, dermal-associated mesenchymal structures or tissue resident immune cells, making for a diversity of clinical presentations. Importantly, many skin tumour types have an extremely poor prognosis. Many skin tumour subtypes have never undergone molecular profiling, or if they have, targeted sequencing has been used and the number of cases analyzed has been so limited that deriving firm conclusions about the profile of driver genes, DNA mutational signatures and germline alleles has not been possible For 70 key skin tumour subtypes defined by the World Health Organization, that have not undergone extensive genetic analysis previoulsy, we propose to perform whole exome and transcriptome sequencing, from a range of body sites, to build a genomic atlas of dermatological tumours, including detailed maps of SNVs, copy number alterations, genome-wide methylation and expression profiles. The cases in this cohort are leiomyosarcomas.

  Study EGAS00001007628

• DERMATLAS__Poroma_WES

  Virtually no other tumour type is associated with so many different forms as skin cancer. Histologically, tumours of the skin may arise from epithelium, including epidermis, hair follicle, sebaceous or sweat gland, melanocytes, dermal-associated mesenchymal structures or tissue resident immune cells, making for a diversity of clinical presentations. Importantly, many skin tumour types have an extremely poor prognosis. Many skin tumour subtypes have never undergone molecular profiling, or if they have, targeted sequencing has been used and the number of cases analyzed has been so limited that deriving firm conclusions about the profile of driver genes, DNA mutational signatures and germline alleles has not been possible For 70 key skin tumour subtypes defined by the World Health Organization, that have not undergone extensive genetic analysis previoulsy, we propose to perform whole exome and transcriptome sequencing, from a range of body sites, to build a genomic atlas of dermatological tumours, including detailed maps of SNVs, copy number alterations, genome-wide methylation and expression profiles. The cases in this cohort are poromas.

  Study EGAS00001007705

• DERMATLAS__SG_basal_cell_adenoma_and_adenocarcinoma_WES

  Virtually no other tumour type is associated with so many different forms as skin cancer. Histologically, tumours of the skin may arise from epithelium, including epidermis, hair follicle, sebaceous or sweat gland, melanocytes, dermal-associated mesenchymal structures or tissue resident immune cells, making for a diversity of clinical presentations. Importantly, many skin tumour types have an extremely poor prognosis. Many skin tumour subtypes have never undergone molecular profiling, or if they have, targeted sequencing has been used and the number of cases analyzed has been so limited that deriving firm conclusions about the profile of driver genes, DNA mutational signatures and germline alleles has not been possible For 70 key skin tumour subtypes defined by the World Health Organization, that have not undergone extensive genetic analysis previoulsy, we propose to perform whole exome and transcriptome sequencing, from a range of body sites, to build a genomic atlas of dermatological tumours, including detailed maps of SNVs, copy number alterations, genome-wide methylation and expression profiles. The cases in this cohort are SG basal cell adenomas and adenocarcinomas.

  Study EGAS00001007745

• DERMATLAS__SG_basal_cell_adenoma_and_adenocarcinoma_RNAseq

  Virtually no other tumour type is associated with so many different forms as skin cancer. Histologically, tumours of the skin may arise from epithelium, including epidermis, hair follicle, sebaceous or sweat gland, melanocytes, dermal-associated mesenchymal structures or tissue resident immune cells, making for a diversity of clinical presentations. Importantly, many skin tumour types have an extremely poor prognosis. Many skin tumour subtypes have never undergone molecular profiling, or if they have, targeted sequencing has been used and the number of cases analyzed has been so limited that deriving firm conclusions about the profile of driver genes, DNA mutational signatures and germline alleles has not been possible For 70 key skin tumour subtypes defined by the World Health Organization, that have not undergone extensive genetic analysis previoulsy, we propose to perform whole exome and transcriptome sequencing, from a range of body sites, to build a genomic atlas of dermatological tumours, including detailed maps of SNVs, copy number alterations, genome-wide methylation and expression profiles. The cases in this cohort are SG basal cell adenomas and adenocarcinomas.

  Study EGAS00001007746

• RNA sequencing data of 257 samples from 106 patients with HR+/HER2- breast cancer treated with AC plus paclitaxel or letrozole plus ribociclib (SOLTI-1402 CORALLEEN trial)

  Early-stage Luminal B breast cancer is frequent and is a major cause of breast cancer death due to its poor prognosis. Our proposal aims to study the biology behind the sensitivity and resistance of Luminal B breast cancer to chemotherapy (CHT) or a non-CHT regimen composed of hormone therapy in combination with ribociclib, a CDK4/6 inhibitor. To accomplish this, we first completed the SOLTI-1402 CORALLEEN phase II trial, a study where 106 patients with early-stage Luminal B breast cancer were randomized to standard neoadjuvant CHT for 6 months, or neoadjuvant letrozole and ribociclib for 6 months. After treatment, patients underwent surgery. The primary results of the study, which showed that the response rate to letrozole+ribociclib was similar to CHT, was reported (Prat et al; Lancet Oncol). Tumor biopsies were available at baseline, week 3 and surgery. A total of 257 samples were analyzed using the Illumina TruSeq Stranded Total RNA w/Ribo Zero Gold with MiSeq in TGL (Sequencer NovaSeq S4/PE/100x).

  Study EGAS00001007060

• Sequential Antigen-loss and Branching Evolution in Lymphoma after Anti-CD19 and Anti-CD20 Targeted T Cell Engaging Immunotherapy

  CD19 CAR T cells and CD20 targeting T cell engaging bispecific antibodies have been approved in B-cell Non-Hodgkin lymphoma lately, heralding a new clinical setting where patients are treated with both approaches, sequentially. The aim of our study was to investigate the selective pressure of CD19 and CD20 directed therapy on the clonal architecture in lymphoma. Using a broad analytical pipeline, we identified truncating mutations in the gene encoding CD20 conferring antigen loss in 80% of patients relapsing from CD20 bispecs. Pronounced T cell exhaustion was identified in cases with progressive disease and retained CD20 expression. We also confirmed CD19 loss after CAR T cell therapy and report the case of sequential CD19 and CD20 loss. We observed branching evolution with re-emergence of CD20 positive subclones at later time points and spatial heterogeneity for CD20 expression in response to targeted therapy. Our results highlight immunotherapy as an evolutionary bottleneck selecting for antigen-loss variants but also complex evolutionary pathways underlying disease progression from these novel therapies.

  Study EGAS00001007561

• DERMATLAS__Leiomyoma_WES

  Virtually no other tumour type is associated with so many different forms as skin cancer. Histologically, tumours of the skin may arise from epithelium, including epidermis, hair follicle, sebaceous or sweat gland, melanocytes, dermal-associated mesenchymal structures or tissue resident immune cells, making for a diversity of clinical presentations. Importantly, many skin tumour types have an extremely poor prognosis. Many skin tumour subtypes have never undergone molecular profiling, or if they have, targeted sequencing has been used and the number of cases analyzed has been so limited that deriving firm conclusions about the profile of driver genes, DNA mutational signatures and germline alleles has not been possible For 70 key skin tumour subtypes defined by the World Health Organization, that have not undergone extensive genetic analysis previoulsy, we propose to perform whole exome and transcriptome sequencing, from a range of body sites, to build a genomic atlas of dermatological tumours, including detailed maps of SNVs, copy number alterations, genome-wide methylation and expression profiles. The cases in this cohort are leiomyomas.

  Study EGAS00001007629

• DERMATLAS__Leiomyoma_RNAseq

  Virtually no other tumour type is associated with so many different forms as skin cancer. Histologically, tumours of the skin may arise from epithelium, including epidermis, hair follicle, sebaceous or sweat gland, melanocytes, dermal-associated mesenchymal structures or tissue resident immune cells, making for a diversity of clinical presentations. Importantly, many skin tumour types have an extremely poor prognosis. Many skin tumour subtypes have never undergone molecular profiling, or if they have, targeted sequencing has been used and the number of cases analyzed has been so limited that deriving firm conclusions about the profile of driver genes, DNA mutational signatures and germline alleles has not been possible For 70 key skin tumour subtypes defined by the World Health Organization, that have not undergone extensive genetic analysis previoulsy, we propose to perform whole exome and transcriptome sequencing, from a range of body sites, to build a genomic atlas of dermatological tumours, including detailed maps of SNVs, copy number alterations, genome-wide methylation and expression profiles. The cases in this cohort are leiomyomas.

  Study EGAS00001007630

• DERMATLAS__Leiomyosarcoma_RNAseq

  Virtually no other tumour type is associated with so many different forms as skin cancer. Histologically, tumours of the skin may arise from epithelium, including epidermis, hair follicle, sebaceous or sweat gland, melanocytes, dermal-associated mesenchymal structures or tissue resident immune cells, making for a diversity of clinical presentations. Importantly, many skin tumour types have an extremely poor prognosis. Many skin tumour subtypes have never undergone molecular profiling, or if they have, targeted sequencing has been used and the number of cases analyzed has been so limited that deriving firm conclusions about the profile of driver genes, DNA mutational signatures and germline alleles has not been possible For 70 key skin tumour subtypes defined by the World Health Organization, that have not undergone extensive genetic analysis previoulsy, we propose to perform whole exome and transcriptome sequencing, from a range of body sites, to build a genomic atlas of dermatological tumours, including detailed maps of SNVs, copy number alterations, genome-wide methylation and expression profiles. The cases in this cohort are leiomyosarcomas.

  Study EGAS00001007631