14305 results for ""Pennsylvania+German"+ACS+study+'Socius'+open+access"

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• Pharmacogenomic analysis of patient-derived tumor cells in gynecologic cancers

  Gynecologic malignancy is one of the leading causes of mortality in female adults worldwide. Comprehensive genomic analysis has revealed a list of molecular aberrations that are essential to tumorigenesis, progression, and metastasis of gynecologic tumors. However, targeting such alterations has frequently led to treatment failures due to underlying genomic complexity and activation of various tumor cell survival pathway molecules. A compilation of molecular characterization of tumors with pharmacological drug response is the next step towards clinical application of patient-tailored treatment regimens. Toward this goal, we have established a library of 139 gynecologic tumors including cervical, endometrial, and epithelial ovarian cancers (EOCs) and uterine sarcomas that were genomically and/or pharmacologically annotated and explored dynamic pharmacogenomic associations against 37 molecularly targeted drugs. We discovered lineage-specific drug sensitivities based on subcategorization of gynecologic tumors and identified TP53 mutation as a molecular determinant that elicit therapeutic response to poly (ADP-Ribose) polymerase (PARP) inhibitor. We further identified transcriptome expression of inhibitor of DNA biding 2 (ID2) as a potential predictive biomarker for treatment response to olaparib. Together, our results demonstrate potential utility of rapid drug screening combined with genomic profiling for precision treatment of gynecologic cancers.

  Study EGAS00001003556

• Treatment-mediated selection of lethal prostate cancer clones defined by copy number architectures

  Despite initial responses to hormone treatment, metastatic prostate cancer invariably evolves to a lethal state. To characterize the intra-patient relationships of metastases that evade treatment, we performed genome- wide copy number profiling and bespoke approaches targeting the androgen receptor (AR) on 142 metastatic regions from 10 organs harvested post-mortem from nine men who died from prostate cancer. We identified diverse and patient-unique alterations clustering around the AR in metastases from every patient with evidence of independent acquisition of related genomic changes within an individual and, in some patients, the co-existence of AR-neutral clones. Using the genomic boundaries of pan-autosome copy number change, we confirmed a common clone of origin across metastases and diagnostic biopsies; and identified in individual patients, clusters of metastases occupied by dominant clones with diverged autosomal copy number alterations. Autosome-defined clusters were characterized by cluster-specific AR gene architectures that in two index cases were topologically more congruent than by chance (p-values 0.03, 3.07x10-8). Integration with anatomical site suggested patterns of spread and points of genomic divergence. Copy number boundaries identified treatment-selected clones with putatively distinct lethal trajectories.

  Study EGAS00001006598

• DNA methylation landscape of prostate cancer

  Metastases from primary prostate cancers to rare locations, such as the brain, are becoming more common due to longer life expectancy thanks to improved treatments. Epigenetic dysregulation is a feature of primary prostate cancer, and distinct DNA methylation profiles are associated with the mutually exclusive SPOP mutant or TMPRSS2-ERG fusion genetic backgrounds. Using a cohort of 42 patients with prostate cancer brain metastases (PCBM), with matched primary samples available for 17 patients, we carried out DNA methylation analysis using Illumina EPIC array. We examined the epigenetic distinction between primary PCa and PCBM, the association between epigenetic alterations and mutational background, and particular epigenetic alterations that may be associated with PCBM in particular. Multiregion sampling of PCBM revealed epigenetic heterogeneity in metastases. We found aberrant methylation in PCBM is associated with mutational background, PRC2 complex activity and upregulation of EZH2, an effect that is particularly pronounced in SPOP mutant PCBM. While we detected a hypermethylator phenotype in PCBM, we also observed hypomethylation occurring at the promoters of genes involved in neuroactive ligand-receptor interactions or the calcium signalling pathway, such as GABRB3 and GRM5, a change not observed in non-brain prostate cancer metastases.

  Study EGAS00001006670

• Pre-neoplastic liver colonization by 11p15.5 altered mosaic cells in young children with hepatoblastoma

  In children, liver tumors are a heterogeneous group representing 1-2% of all cancers with the most common diagnosis being hepatoblastoma (HB). While HBs are predominantly sporadic, around 15% of cases develop as part of predisposition syndromes such as familial adenomatous polyposis (APC mutation) or Beckwith-Wiedemann (BWS; 11p15.5 locus altered). Here, we identified a mosaic genetic alteration of 11p15.5 locus in the liver in 17% of HB patients without a clinical diagnosis of BWS syndrome. These mosaic 11p15.5 alterations were never found in children with other types of liver tumor (hepatocellular carcinoma or adenoma, fibrolamellar carcinoma). Bulk transcriptomic analysis revealed the abundance of extracellular matrix, angiogenesis, and progenitor gene signatures in mosaic livers. In situ hybridization assay RNAscope allowed for the detection of 11p15.5 altered hepatocytes mosaicism in liver FFPE sections of HB patients presenting IGF2 overexpression and H19 downregulation together with an alteration of the zonal architecture of the liver. Spatial transcriptomics analysis revealed an overexpression of the IGF2 pathway, lipogenesis, and platelet activation in 11p15.5 altered hepatocytes. These data provide insights for 11p15.5 mosaicism detection as well as functional consequences of these alterations during the early steps of carcinogenesis.

  Study EGAS00001006692

• Targeted therapy of advanced parathyroid carcinoma guided by genomic and transcriptomic profiling (hipo_021)

  Parathyroid carcinoma (PC) is an orphan malignancy with a high risk of recurrence after surgery. Tumor-directed systemic treatments for PC are not established. We used whole-genome and RNA sequencing in four patients with advanced PC to identify molecular alterations that could guide clinical management. In two cases, the genomic and transcriptomic profiles provided targets for experimental therapies that resulted in biochemical response and prolonged disease stabilization: (i) immune checkpoint inhibition with pembrolizumab based on high tumor mutational burden and a single-base substitution signature associated with APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) overactivation; (ii) multi-receptor tyrosine kinase inhibition with lenvatinib due to overexpression of FGFR1 and RET and, later in the course of disease, PARP inhibition with olaparib prompted by signs of defective homologous recombination DNA repair. In addition, our data provided new insights into the molecular landscape of PC with respect to the genome-wide footprints of specific mutational processes and pathogenic germline alterations. These data underscore the potential of comprehensive molecular analyses to improve care for patients with ultra-rare cancers based on insight into disease biology.

  Study EGAS00001006747

• HLA-DR is absent in primitive macrophages through epigenetic silencing of master regulator CIITA

  The earliest macrophages are generated during embryonic development from erythro-myeloid progenitors (EMPs) via primitive haematopoiesis. This process is poorly understood in humans and in the mouse is thought to be spatially restricted to the yolk sac. Human fetal placental macrophages, Hofbauer cells (HBC), arise during the primitive haematopoietic wave at ~18 days post conception. Here we identify a population of placental erythro-myeloid progenitors (PEMPs) in the early human placenta that give rise to HBC. PEMP are fetal CD34+CD43+ progenitors found exclusively at early gestational timepoints. Transcriptomic analyses reveal that PEMP have conserved features of primitive yolk sac EMPs, including the lack of HLF expression. Using in vitro single-cell culture experiments we show that PEMP generate HBC-like cells that completely lack HLA-DR expression  and demonstrate that this is a conserved feature of all macrophages generated by primitive haematopoiesis in humans. Finally, we demonstrate that the class II transactivator, CTIIA, the master regulator HLA-DR expression, is epigenetically silenced during primitive haematopoiesis, leading to HLA-DRneg macrophages. These findings demonstrate that HBC are derived locally from PEMP and establish the human placenta as an additional site of primitive haematopoiesis.

  Study EGAS00001006981

• RNAseq

  Familial Mediterranean Fever (FMF) is a prototypical periodic fever syndrome caused by genetic variation in MEFV. While it is known that especially IL-1β responses are dysregulated in FMF, its innate immune landscape has not been comprehensively described. Therefore, we extensively characterized the function of monocytes and neutrophils in patients with FMF in between disease attacks, as well as their circulating inflammatory proteome. We found that monocyte IL-1 and IL-6 production was enhanced following a range of stimulations, in concordance with alterations in the plasma inflammatory proteome. In contrast, neutrophil function in FMF was normal. Additionally, ATAC-seq and RNA-seq analyses revealed important epigenetic and transcriptional reprogramming arguing for monocyte function dysregulation. Interestingly, chromatin-accessibility was down-regulated in genomic regions related to cellular responses. This argues that the primary immune dysregulation in monocytes due to MEFV mutations leads to a chronic inflammatory profile that is subsequently associated with counterregulatory epigenetic and transcriptional changes reminiscent of tolerance (rather than trained immunity) induction. These data increase our understanding of the innate immune changes in FMF, that can be used in the design of novel approaches to manage chronic inflammation of these patients.

  Study EGAS00001007165

• Functional single-cell characterization of immune aplastic anemia shows convergence of NK and NK-like CD8+ T cells with disease-associated TCR signature

  Immune aplastic anemia (AA) is a life-threatening bone marrow (BM) failure disorder driven by an autoimmune T cell attack against the hematopoietic stem and progenitor cells (HSPCs). However, the autoantigen targets and the role of other immune cells are unknown. Here, by analyzing an international cohort of 156 AA patients with scRNA+TCRαβ-seq, TCRβ-seq, flow cytometry, and plasma cytokine profiling with comparisons to healthy controls and patients with other hematological disorders, we identify NK cells and CD8+ TEMRA cells expressing NK receptors with AA-associated TCRβ-seq motifs as the most dysregulated immune cell populations in AA BM. Functional co-culture experiments with scRNA-TCRαβ-seq readout utilizing primary HSPCs and immune cells provide evidence that NK cellscannot kill HSPCs alone, but sensitize them to CD8+ T cell-mediated killing. Furthermore, HSPCs induce strong activation of T cell clones with CD8+ TEMRA NK-like phenotype and AA-associated TCR motifs. Our results reveal the convergent evolution of innate and adaptive immune cells in AA, where NK cells support CD8+ T cell-mediated autoimmunity.

  Study EGAS00001007602

• Single-cell profiling of co-cultures of GSCCs and macrophages

  Glioblastoma (GBM) remains the most malignant primary brain tumor, with a median survival rarely exceeding 2 years. Tumor heterogeneity and an immunosuppressive microenvironment are key factors contributing to the poor response rates of current therapeutic approaches. GBM-associated macrophages (GAMs) often exhibit immunosuppressive features that promote tumor progression. However, their dynamic interactions with GBM tumor cells remain poorly understood. Here, we used patient-derived GBM stem cell cultures and combined single-cell RNA sequencing of GAM-GBM co-cultures and real-time in vivo monitoring of GAM-GBM interactions in orthotopic zebrafish xenograft models to provide insight into the cellular, molecular, and spatial heterogeneity. Our analyses revealed substantial heterogeneity across GBM patients in GBM-induced GAM polarization and the ability to attract and activate GAMs – features that correlated with patient survival. Differential gene expression analysis, immunohistochemistry on original tumor samples, and knock-out experiments in zebrafish subsequently identified LGALS1 as a primary regulator of immunosuppression. Overall, our work highlights that GAM-GBM interactions can be studied in a clinically relevant way using co-cultures and avatar models, while offering new opportunities to identify promising immune-modulating targets.

  Study EGAS00001007482

• Massively parallel functional dissection of schizophrenia associated non-coding genetic variants

  Schizophrenia (SCZ) is a severe mental disorder affecting 1% of the world population. SCZ is characterized by an underlying genetic architecture that is highly polygenic. Genome wide association studies have identified thousands of genetic variants that are statistically linked to the disease. However, the translation of these associations into insights on the pathomechanisms has been challenging because the causal genetic variants, their molecular function, and their target genes remain largely unknown. To address these questions, we combined induced pluripotent stem cell technology with a massively parallel variant annotation pipeline (MVAP) to functionally characterize 35,000 SCZ associated non-coding genetic variants. This approach identified a set of 620 (1.7%) single nucleotide polymorphisms as functional on the molecular level in a highly cell type and condition specific fashion. Subsequent multi-modal integration of epigenomic data combined with CRISPR screening in human neurons enabled us to systematically translate SCZ variant associations into target genes, biological processes, and ultimately alterations of neuronal physiology. These results provide a new high-resolution map of functional variant-gene combinations and offer comprehensive biological insights into the developmental context and stimulus dependent molecular processes modulated by SCZ genetic variation beyond statistical association.

  Study EGAS00001007542

• Functional single-cell characterization of immune aplastic anemia shows convergence of NK and NK-like CD8+ T cells with disease-associated TCR signature

  Immune aplastic anemia (AA) is a life-threatening bone marrow (BM) failure disorder driven by an autoimmune T cell attack against the hematopoietic stem and progenitor cells (HSPCs). However, the autoantigen targets and the role of other immune cells are unknown. Here, by analyzing an international cohort of 156 AA patients with scRNA+TCRαβ-seq, TCRβ-seq, flow cytometry, and plasma cytokine profiling with comparisons to healthy controls and patients with other hematological disorders, we identify NK cells and CD8+ TEMRA cells expressing NK receptors with AA-associated TCRβ-seq motifs as the most dysregulated immune cell populations in AA BM. Functional co-culture experiments with scRNA-TCRαβ-seq readout utilizing primary HSPCs and immune cells provide evidence that NK cellscannot kill HSPCs alone, but sensitize them to CD8+ T cell-mediated killing. Furthermore, HSPCs induce strong activation of T cell clones with CD8+ TEMRA NK-like phenotype and AA-associated TCR motifs. Our results reveal the convergent evolution of innate and adaptive immune cells in AA, where NK cells support CD8+ T cell-mediated autoimmunity.

  Study EGAS00001007604

• Immune and clinicopathological features predict HER2-positive breast cancer prognosis in the neoadjuvant NeoALTTO and CALGB 40601 trials

  The identification of prognostic drivers in patients receiving neoadjuvant therapy is crucial for treatment optimization in HER2-positive breast cancer, with the immune microenvironment being a key factor. Here, we investigated the complexity of B and T cell receptor (BCR and TCR) repertoires, reflecting antigen-specific immune response, in the context of two phase III trials, namely NeoALTTO and CALGB 40601, evaluating neoadjuvant paclitaxel with trastuzumab and/or lapatinib in women affected by HER2-positive breast cancer. BCR/TCR features were heterogeneous, and BCR measures describing clonal expansion demonstrated independent prognostic value. A model developed in NeoALTTO and validated in CALGB 40601 predicted event-free survival (EFS) by integrating hormone receptor and clinical nodal status, treatment response (breast pCR)breast pathological complete response (pCR) as well as tumor-infiltrating lymphocytes and BCR evenness, measuring the equality of BCR clonal distribution. The HER2-EveNT score derived from the model allows the identification of patients with 5-year EFS > 90%, and, in those not achieving pCR, of a subgroup of immune-enriched tumors with an excellent outcome despite residual disease.

  Study EGAS00001007563

• Discovery of immunotherapy targets for pediatric solid and brain tumors by exon-level expression

  Immunotherapy with chimeric antigen receptor T cells for pediatric solid and brain tumors is constrained by available targetable antigens. Cancer-specific exons present a promising reservoir of targets; however, these have not been explored and validated systematically in a pan-cancer fashion. To identify cancer specific exon targets, we analyze 1,532 RNA-seq datasets from 16 types of pediatric solid and brain tumors for comparison with normal tissues using a newly developed workflow. We find 2,933 exons in 157 genes encoding proteins of the surfaceome or matrisome with high cancer specificity either at the gene (n=148) or the alternatively spliced isoform (n=9) level. Expression of selected alternatively spliced targets, including the EDB domain of fibronectin 1, and gene targets, such as COL11A1, are validated in pediatric patient derived xenograft tumors. We generate T cells expressing chimeric antigen receptors specific for the EDB domain or COL11A1 and demonstrate that these have antitumor activity. The full target list, explorable via an interactive web portal (https://cseminer.stjude.org/), provides a rich resource for developing immunotherapy of pediatric solid and brain tumors using gene or AS targets with high expression specificity in cancer.

  Study EGAS00001007766

• Genome‐wide postnatal changes in immunity following fetal inflammatory response

  The fetal inflammatory response (FIR) increases the risk of perinatal brain injury, particularly in extremely low gestational age newborns (ELGANs, < 28 weeks of gestation). One of the mechanisms contributing to such a risk is a postnatal intermittent or sustained systemic inflammation (ISSI) following FIR. The link between prenatal and postnatal systemic inflammation is supported by the presence of well‐established inflammatory biomarkers in the umbilical cord and peripheral blood. However, the extent of molecular changes contributing to this association is unknown. Using RNA sequencing and mass spectrometry proteomics, we profiled the transcriptome and proteome of archived neonatal dried blood spot (DBS) specimens from 21 ELGANs. Comparing FIR‐affected and unaffected ELGANs, we identified 782 gene and 27 protein expression changes of 50% magnitude or more, and an experiment‐wide significance level below 5% false discovery rate. These expression changes confirm the robust postnatal activation of the innate immune system in FIR‐affected ELGANs and reveal for the first time an impairment of their adaptive immunity. In turn, the altered pathways provide clues about the molecular mechanisms triggering ISSI after FIR, and the onset of perinatal brain injury.

  Study EGAS00001003635

• Pan-neuroblastoma analysis reveals age- and signature-associated driver alterations

  Neuroblastoma is a pediatric malignancy with heterogeneous clinical outcomes depending on age and other factors. To better understand neuroblastoma pathogenesis, here we analyze whole-genome, whole-exome and/or transcriptome sequencing data from 702 neuroblastoma samples, half of which are new data. Over 95% of samples harbor at least one recurrent driver alteration and most aberrations, including MYCN, ATRX, and TERT alterations, differ in frequency by age. MYCN alterations occur at median 2.3 years of age, TERT at 3.8 years, and ATRX at 5.6 years, suggesting age-specific susceptibility to genetic alterations. Mutational signature analysis shows that reactive oxygen-species (ROS)-induced mutations are the most common cause of single nucleotide variant (SNV) drivers in neuroblastoma, including most ALK and Ras pathway SNVs. ROS-induced mutagenesis is both an early event, as it causes truncal variants, and continuous as it also induces relapse-specific mutations. ROS-induced mutations are more abundant in neuroblastomas with MYCN amplification, 17q gains, and increased expression of mitochondrial ribosome genes. We observe recurrent FGFR1 N546K or internal tandem duplication variants in 6 patients and ALK N-terminal structural alterations in 5 samples, identifying additional patients amenable to precision therapy.

  Study EGAS00001003931

• SINGLE-CELL RNA SEQUENCING Single-cell RNA sequencing was performed on 13 ‘mild-moderate’ and 10 ‘critical’ COVID19 PBMC samples

  Summary Epidemiological and clinical reports have indicated that SARS-CoV-2 virulence hinges upon the triggering of an aberrant host immune response, more so than on direct virus-induced cellular damage. To elucidate the immunopathology underlying COVID-19 severity, we performed cytokine and multiplex immune profiling in mild-moderate and critically ill COVID-19 patients. Hypercytokinemia in COVID-19 differed from the IFN-γ-driven cytokine storm in macrophage activation syndrome, and was more pronounced in critical versus mild-moderate COVID-19. Systems modelling of cytokine levels paired with deep-immune profiling showed that classical monocytes drive this hyper-inflammatory phenotype and that a reduction in T-lymphocytes correlates with disease severity, with CD8+ cells being disproportionately affected. Expression of antigen presenting machinery was also reduced in critical disease. Furthermore, we found that neutrophils contributed to disease severity and local tissue damage by amplification of hypercytokinemia and the formation of neutrophil extracellular traps. Together these findings suggest a myeloid-driven immunopathology, in which hyperactivated neutrophils and an ineffective adaptive immune system act as mediators of COVID-19 disease severity.

  Study EGAS00001005039

• RNA-sequencing of tumors from 45 patients with recurrent or metastatic gastric cancer treated with immune checkpoint inhibitors

  Genomic profiling can provide prognostic and predictive information to guide clinical care. Biomarkers that reliably predict patient response to chemotherapy and immune checkpoint inhibition in gastric cancer are lacking. In this work, we used our machine learning algorithm NTriPath to identify a gastric-cancer specific 32-gene signature. Using unsupervised clustering on expression levels of these 32 genes in tumors from 567 patients, we identified four molecular subtypes that were prognostic for survival. We then built a support vector machine with linear kernel and the binary classifier to generate a risk score that is prognostic for 5-year overall survival and validated the risk score using three independent datasets. We also found that the molecular subtypes predicted response to adjuvant 5-fluorouracil and platinum after gastrectomy and immune checkpoint inhibitors in patients with metastatic or recurrent disease. The 32-gene signature is a promising prognostic and predictive biomarker to guide the clinical care of gastric cancer patients and should be validated in a prospective manner. This EGA archive includes both St.Mary hospital cohort samples (n=28) and Yonsei immunotherapy patient samples (n=17).

  Study EGAS00001005588

• BIOKEY: A single-cell catalogue of the dynamic changes underlying Checkpoint Immunotherapy response in Early Breast Cancer

  Checkpoint immunotherapy combined with neoadjuvant chemotherapy improves complete pathologic response in a subset of breast cancer patients. Here, we applied single-cell profiling to tumor biopsies collected before and during anti-PD1 therapy. One-third of tumors exhibited proliferative T-cells expanding along CD8+ or CD4+ lineages, which were either characterized by increased cytotoxicity and exhaustion or improved T-helper function, respectively. Lineage tracing in non-expanding tumors revealed at which point in the lineage T-cells were impaired, while gene expression modeling along these lineages revealed novel genes and underlying transcription factors involved in T-cell expansion. Interestingly, different dendritic and myeloid cell phenotypes could either stimulate or inhibit expanding T-cells, while cell-to-cell communication revealed an integrated immune context highly predictive of T-cell expansion, consisting of immune-stimulatory/-inhibitory interactions between cancer and various immune cell types. Our data yield unprecedented insights into the dynamic changes underlying checkpoint immunotherapy response in breast cancer.

  Study EGAS00001004809

• The Vaginal Microbiome: Disease, Genetics and the Environment

  The vagina is an interactive interface with the environment, and as such is covered by a protective epithelial surface. This surface is colonized by a biofilm consisting of bacteria and other microorganisms, which through a variety of mechanisms serve to protect the host from invasion by pathogens. Alterations in the normal vaginal microflora; i.e., dysbioses, particularly those associated with bacterial vaginosis, the most common disorder of the female reproductive tract, contribute to risk of sexually transmitted infections, infection by HIV, and are associated with increased risk of adverse pregnancy outcomes including but not limited to spontaneous preterm birth. Physiologic alterations in the host (e.g., menopause and pregnancy), which are beginning to be investigated as potential selective conditions for change in the "normal" vaginal flora, and their impact on disease susceptibility and transmission, remain to be more definitively elucidated. The effects of chronically abnormal physiologic states (e.g., diabetes mellitus) on normal vaginal flora have not been well described or studied. Finally, an almost unexplored area of inquiry is the genetic contribution, including race/ethnicity, to the establishment and maintenance of a "normal" vaginal flora, under normal and physiologically altered circumstances. Our research will shed light on how the vaginal microbiome contributes to adverse obstetrical outcomes and sexually transmitted infections in diverse populations. This project addresses the following questions: First: Do the genes of the host contribute to the composition of the vaginal microbiome? We hypothesize that a woman's genetic composition significantly affects the ability of certain commensal, parasitic and pathogenic microbes to colonize and/or infect the genital tract. Thus, we will compare and quantify the microbial populations inhabiting the vaginas of monozygotic and dizygotic twins from the Mid Atlantic Twin Registry, and, in the process, address the question of whether there is a relationship between the microbiomes of the vagina, mouth and GI tract. Second: What changes in the vaginal microbiome are associated with common physiological perturbations or non-infectious pathological states of the host?We hypothesize that "altered" physiologic (pregnancy, menopause) and pathologic (chronic disease, hysterectomy) conditions, or environmental "exposures" (exogenous hormones, antibiotics, chronic immunosuppressant, smoking; douching) can predictably alter the vaginal microenvironment. These alterations will lead to changes in microbial populations within the vagina. Changes in the microbial populations may have impacts, positive or more likely negative, on the spontaneous and future well-being of the affected individual. We are characterizing the effects of these "altered" physiologic and pathologic conditions, and environmental exposures, on the composition of the vaginal microbiome. Finally, we are examining the impact of the important physiological changes incurred during pregnancy on the composition of the microbiome of the female reproductive tract. Third: What changes in the vaginal microbiome are associated with relevant infectious diseases and conditions? We are testing the hypothesis that infectious diseases predictably alter the vaginal microbiome, and that these changes have an impact on the disease susceptibility, process, and outcome. A predilection for bacterial vaginosis, vaginitis, HIV infection, or other sexually transmitted diseases, is likely associated with a women's vaginal microbial composition. Thus, we will characterize samples from women with a variety of these infectious conditions to determine the contribution of their microbiomes to the disease process and susceptibility. We are addressing these questions using a combination of high throughput 'nextgen' sequencing technologies. Early studies were performed using the Roche 454 FLX and the first generation Illumina Genome Analyzer II instruments and more recently employing Illumina MiSeq and HiSeq 2500 and 4000 platforms, installed in the Genomics Core of the Nucleic Acids Research Facilities at VCU. Thus, segments of the 16S rRNA genes are amplified from the complex samples taken from various target sites in and around the vagina of each study participant. These segments are sequenced and subjected to taxonomic classification protocols to identify and quantify the bacterial taxa present in each sample. Additionally, total DNA isolated from these samples are subjected to shotgun sequence analysis to empirically reconstruct the metabolic potential of these microbiomes. Finally, specific bacterial clones are completely sequenced and analyzed to associate the unavoidable strain and isolate diversity with the clinical phenotypes presented.

  Study phs000256

• Human Microbiome Project Demonstration Study of Cutaneous Microbiome in Psoriasis

  Psoriasis, a highly prevalent disease of humans of unknown cause, is a chronic inflammatory disorder primarily involving skin, with distinctive clinical characteristics. With the newly developed tools that facilitate microbiome research, it now is possible to assess whether the cutaneous microbiome plays a role in the pathogenesis of this disorder. Preliminary data from our studies suggest that the cutaneous microbiome in psoriasis is complex and possibly different from normal. To deal with this complexity, we propose to examine the cutaneous microbiome in relation to psoriasis with explorations at several taxonomic and informatic levels. Our overall objective is to examine how changes in the normal cutaneous microbiome contribute to the pathogenesis of psoriasis. Since causality is complex and often difficult to prove, and beyond the scope of this RFP, our overall hypothesis is that there are alterations in the cutaneous microbiome in areas of skin affected by psoriasis in comparison with the range observed in clinically unaffected areas, or in healthy persons. We also hypothesize that the characteristics of the microbiome may affect clinical responses to the immunomodulatory agents used to treat psoriasis. An alternative hypothesis is that effective treatment of psoriasis with systemic immunomodulatory agents will not substantially affect the disordered microbial ecosystem. Such observations would provide evidence for the roles of the microbiota in this disorder. Since an important consideration in microbiome research is the optimal level (e.g. phylum, genus, species, strain, gene) at which to examine a scientific question, and we are not yet certain what are the optimal levels for psoriasis, this also will be examined. Our studies of psoriasis should allow development of both approaches and tools that will have general utility for Microbiome research. To test our hypothesis, we propose the following specific aims: 1) To understand the cutaneous microbiome species composition overlaying psoriatic lesions; 2)To investigate differences in metagenome content for psoriatic lesions compared to normal skin; 3) To identify differences in the transcriptional profiles of the microbiome and the host between normal skin and psoriatic lesions using high-throughput sequencing; and 4) To estimate the effects of systemic immunomodulatory therapy for psoriasis on microbiome composition. In total, these studies should help us understand the role of the microbiome in psoriasis pathogenesis. We sought to characterize and compare the cutaneous microbiota of psoriatic lesions (lesion), unaffected contralateral skin from psoriatic patients (normal), and similar skin loci in matched healthy controls (control) in order to discern patterns that govern skin colonization and their relationship to clinical diagnosis. Using high-throughput 16S rRNA sequencing, we assayed the cutaneous bacterial communities of 51 matched triplets and characterized these samples using community data analysis techniques.

  Study phs000251

• Efficacy of two different FGFR-inhibitors in a patient with extrahepatic cholangiocarcinoma harboring an FGFR2 mutation

  Cholangiocarcinomas (CCAs) is a type of cancer with few effective systemic therapies. Elucidation of the molecular landscape of the disease from genomic studies based on next generation sequencing (NGS) has contributed to the introduction of new targeted therapies. One of these treatments consists of a class of small molecules that target members of the FGFR family of receptor tyrosine kinases. These drugs are effective and have been approved for cholangiocarcinomas with fusions or rearrangements of FGFR genes. In contrast, the role of these inhibitors in cholangiocarcinomas with mutations in FGFR genes is less well defined. We report here a patient with a cholangiocarcinoma bearing a FGFR2 p.Ser252Trp mutation. The patient was treated with two different FGFR inhibitors, as the first caused ocular toxicity. She obtained clinical benefit from both. This case illustrates the efficacy of FGFR inhibitors on cholangiocarcinoma with specific point mutations. This is the first case to report the clinical benefit of these drugs in FGFR2 p.Ser252Trp mutation. Clinical benefit can be sustained, as seen in our patient. Our case also shows that FGFR inhibitors-induced adverse effects, such as ocular toxicities, may not recur after re-challenge with an alternative drug of the same class.

  Study EGAS50000000015

• Genome Variation among HIV-Resistant People with Hemophilia

  Rare individuals are highly resistant to infection with human immunodeficiency virus (HIV). Studies of candidate genes resulted in the discovery of a 32bp deletion in the CC-chemokine receptor 5 gene (CCR5Δ32), which rendered this critical co-receptor for primary HIV infection to be non-functional. Pharmacologic and vaccine-induced blockade of CCR5 is being pursued to treat and prevent HIV infection and other conditions. The allele frequency of CCR5Δ32 among persons of European ancestry is approximately 10%. CCR5Δ32/Δ32 homozygotes are almost totally resistant to HIV infection. People with severe hemophilia A require frequent replacement with clotting Factor VIII (FVIII) to control hemorrhage. Prior to the discovery of HIV in 1984 and licensure of recombinant FVIII in the late 1980s, people with severe hemophilia A were treated with plasma-derived FVIII and thus were intensively exposed to HIV. Only 5% of such patients were not infected with HIV. Of these, approximately 1/3 were CCR5Δ32/Δ32 homozygotes. The remaining 2/3 of these people who were highly resistant to HIV remain unexplained. This project seeks to discover genome variations among people who are highly resistant to HIV infection. Such variation is likely to serve as a target for reducing the morbidity and incidence of HIV.

  Study phs000445

• Analysis of the Whole Transcriptomes of Human Testis with Complete Spermatogenesis and of Human Testes with Sertoli Cell-Only Syndrome

  Sertoli cell-only syndrome is severe form of human male infertility in which most seminiferous tubules appear to lack all spermatogenic cells, including spermatogonial stem cells (SSCs). However, a few small tubule segments of some patients have active spermatogenesis and, thus, functional stem cell niches and SSCs. Normally SSCs replicate, migrate and refill adjacent empty niches, but this does not appear to occur in SCO syndrome. We hypothesized that this failure occurs because most niches are dysfunctional. As Sertoli cells are essential to formation of these niches, we used RNAseq to compare the transcriptomes of human testes with qualitatively normal (complete) spermatogenesis (n=4) with the transcriptomes of human testes with SCO syndrome (n=7). We then focused our analysis on the expression of transcripts that bioinformatic analyses identified as Sertoli cell signature transcripts. Results show that Sertoli cells in SCO testes express abnormally low levels of GDNF, FGF8 and BMP4, all of which are important regulators of mouse SSCs and/or progenitor spermatogonia. Sertoli cells in SCO testes express significantly reduced levels of transcripts for proteins that polarize the Sertoli cell plasma membrane and regulate the trafficking of cell adhesion and gap junction proteins in and out of that plasma membrane.

  Study phs001777

• Whole Transcriptome Sequencing of Human Tumor Cells and Hematopoietic Stem and Progenitor Cells During Aging and Bone Marrow Disorders

  Whole transcriptome sequencing analyses were performed on human hematopoietic stem and progenitor cells during aging and bone marrow disorders. For this purpose, normal and diseased bone marrow or peripheral blood-derived mononuclear cells were FACS-purified into hematopoietic stem (CD34+CD38-Lin-) or hematopoietic progenitor (CD34+CD38+Lin-) populations. Primary human samples from healthy individuals were used along with comparative analyses in some pre-malignant and malignant conditions, including myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and plasma cell dyscrasias (e.g., multiple myeloma). Studies were also performed on total mononuclear cell fractions from patients with newly-diagnosed or relapsed multiple myeloma, or plasma cell leukemia. Briefly, RNA was extracted from all samples and quality was assessed using a Bioanalyzer instrument. Only samples with RNA integrity (RIN) values >7 were subjected to library preparation for RNA-sequencing. The SMARTer v4 cDNA amplification kit was used along with library construction by the NEB Ultra DNA kit for Illumina. Paired-end sequencing was performed on Illumina NextSeq instruments (Scripps Next Generation Sequencing Core), with all samples run over two separate flow-cells (to generate additional sequencing reads); >95M reads were generated in total for each sample. The RNA-sequencing data are available as FASTQ files.

  Study phs002291

• Germ Cell and Associated Heme Malignancies Evolve from a Common Shared Precursor

  Germ cell tumors (GCTs) are the most common cancer in men between the ages of 15-40. While most patients are cured, those with disease arising in the mediastinum have distinctly poor outcomes. One in every 17 patients with primary mediastinal non-seminomatous GCTs develop an incurable hematologic malignancy and prior data intriguingly suggests a clonal relationship exists between hematologic malignancies and GCTs in these cases. To date however, the precise clonal relationship between GCTs and the diverse additional somatic malignancies arising in such individuals has not been determined. Here, we traced the clonal evolution and characterized the genetic features of each neoplasm from a cohort of fifteen patients with GCTs and associated hematologic malignancies. We discovered that GCTs and hematologic malignancies developing in such individuals evolved from a common shared precursor, nearly all of which harbored allelically imbalanced TP53 and/or RAS pathway mutations. Hematologic malignancies arising in this setting genetically resembled mediastinal GCTs rather than de novo myeloid neoplasms. Our findings argue that this scenario represents a unique clinical syndrome, distinct from de novo GCTs or hematologic malignancies, initiated by an ancestral precursor which gives rise to the parallel evolution of GCTs and blood cancers in these patients. Reprinted from PMID: 32897884, with permission from JCI.

  Study phs002231

• TNF-induced dynamic regulation of mRNA stabilome in rheumatoid arthritis fibroblast-like synoviocytes

  During rheumatoid arthritis (RA), TNF activates fibroblast-like synoviocytes (FLS) inducing in a temporal order a constellation of genes, which perpetuate synovial inflammation. Although the molecular mechanisms regulating TNF-induced transcription are well characterized, little is known about the impact of mRNA stability on gene expression and the impact of TNF on decay rates of mRNA transcripts in FLS. To address these issues we performed RNA sequencing and genome-wide analysis of the mRNA stabilome in RA FLS. We found that TNF induces a biphasic gene expression program: initially, the inducible transcriptome consists primarily of unstable transcripts but progressively switches and becomes dominated by very stable transcripts. This temporal switch is due to: a) TNF-induced prolonged stabilization of previously unstable transcripts that enables progressive transcript accumulation over days and b) sustained expression and late induction of very stable transcripts. TNF- induced mRNA stabilization in RA FLS occurs during the late phase of TNF response, is MAPK-dependent, and involves several genes with pathogenic potential such as IL6, CXCL1, CXCL3, CXCL8/IL8, CCL2, and PTGS2. These results provide the first insights into genome-wide regulation of mRNA stability in RA FLS and highlight the potential contribution of dynamic regulation of the mRNA stabilome by TNF to chronic synovitis.

  Study phs001371

• Identification and Targeting of Inflammatory Macrophage-Fibroblast Crosstalk in Rheumatoid Arthritis

  Macrophages tailor their function according to the signals found in tissue microenvironments, assuming a wide spectrum of phenotypes. A detailed understanding of macrophage phenotypes in human tissues is limited. Using single-cell RNA sequencing, we defined distinct macrophage subsets in the joints of patients with the autoimmune disease rheumatoid arthritis (RA), which affects ~1% of the population. The subset we refer to as HBEGF+ inflammatory macrophages is enriched in RA tissues and is shaped by resident fibroblasts and the cytokine tumor necrosis factor (TNF). These macrophages promoted fibroblast invasiveness in an epidermal growth factor receptor-dependent manner, indicating that intercellular cross-talk in this inflamed setting reshapes both cell types and contributes to fibroblast-mediated joint destruction. In an ex vivo synovial tissue assay, most medications used to treat RA patients targeted HBEGF+ inflammatory macrophages; however, in some cases, redirecting them into a state is not expected to resolve inflammation. These data highlight how advances in our understanding of chronically inflamed human tissues and the effects of medications therein can be achieved by studies on local macrophage phenotypes and intercellular interactions. Reprinted from Kuo, Ding et al., Science Translational Medicine 2019, PMID: 31068444, with permission from American Association for the Advancement of Science.

  Study phs001340

• Genomic Sequencing of Cervical Cancers

  Cervical cancer is responsible for 10-15% of cancer related deaths in women worldwide. The etiological role of infection with high-risk human papilloma viruses (HPV) in carcinomas of the cervix is well established. In general, the development of cervical carcinomas follows a progression from persistent HPV infection through precancerous lesions to invasive cancer. Previous studies have implicated somatic mutations in PIK3CA, PTEN, TP53, STK11 and KRAS as well as chromosome-arm level copy number alterations in the pathogenesis of cervical carcinomas. Here, we report whole exome sequencing analysis of 118 cervical carcinoma-normal paired samples from patients in Norway and Mexico, as well as transcriptome sequencing of 80 cases and whole genome sequencing of 13 tumor-normal pairs. Novel somatic mutations include recurrent E322K substitutions in the MAPK1 gene encoding the ERK2 kinase and inactivating mutations in the HLA-B gene. In addition, recurrent somatic mutations in FBXW7, EP300, and NFE2L2 are novel in the context of primary cervical carcinomas. Analysis of HPV integration sites revealed recurrent integration into the RAD51B locus as well as co-occurrence of HPV genome integration and copy number gains within several genomic loci. These findings shed new light on the pathogenesis of cervical carcinomas and suggest potential novel therapeutic targets.

  Study phs000600

• Single Cell and Tissue Level Functional Genomics Analysis of Astrocyte-Related Mechanisms in Taupathy

  This data is the first part of a larger single cell and tissue level functional genomics analysis of dementias with tau pathology.  Cellular resolution will clarify the role of glial genes and pathways in neurodegenerative disease. This includes understanding the role of microglia and astrocytes in neurodegenerative disorders characterized by Tau protein pathology. To achieve a refined view of microglial neuroinflammatory signaling in tau-associated neurodegeneration, we performed a functional genomic analysis that integrated time-course gene expression data from mouse models expressing human mutant MAPT (the tau gene) with single nuclear sequencing data obtained from patients with frontotemporal dementia with Tau protein pathology (Pick’s disease). This included microglia-specific gene expression data obtained from single nuclear sequencing of postmortem frozen precentral gyrus samples collected from 16 total individuals, including 8 subjects with clinical diagnosis of behavior variant frontotemporal dementia and neuropathological diagnosis of Pick’s disease (Tau protein pathology) and controls (n=8). Our findings present detailed molecular profiles of disease-associated microglial transitions over the course of tau-associated neurodegeneration. Furthermore, we demonstrate overlap between microglia profiles obtained from precentral gyrus of patients with Pick’s disease with those up-regulated during the neurodegenerative stage in mice expressing human MAPT mutations.

  Study phs002197

• Whole genome sequencing of core-binding factor leukemia

  Pediatric de novo acute myeloid leukemia (AML) is a heterogeneous disease that can be divided into clinically distinct subtypes based on the presence of specific chromosomal abnormalities or gene alterations. One of the best characterized subtypes of AML involves leukemias with alterations of the core-binding factor (CBF)-complex, which comprises the FAB subtypes M2 and M4Eo and associates with a favorable outcome. Patients with the AML M2 subtype harbor a translocation between chromosomes 8 and 21 [t(8;21)] that yields the chimeric fusion gene RUNX1(AML1)-RUNX1T1(ETO), while patients with AML M4Eo express the chimeric fusion gene CBFβ-SMMHC(MYH11) as a result of an inversion/translocation event of chromosome 16 [inv(16)/t(16;16)]. In an effort to define the total complement of genetic changes in CBF-leukemia, we performed paired-end whole genome sequencing (WGS) on diagnostic leukemia blasts and matched germ line samples from 17 pediatric CBF-leukemia patients using the Illumina platform. Somatic alterations, including single nucleotide variations (SNVs) and structural variations (SVs), including insertions, deletions, inversions, and inter- and intra-chromosomal rearrangements, were detected using complementary analysis pipelines (Bambino, CREST and CONSERTING). Recurrent screening of identified mutations will be performed in a cohort of approximately 94 cases of CBF-leukemias.

  Study phs000414

• International Multi-Center ADHD Genetics Project

  The goal of the project is to complete a 600,000 tag SNP genome-wide association scan of 958 parent-child trios from the International Multisite ADHD Genetics (IMAGE) project, in order to assess the association of SNP markers with ADHD, analyze quantitative ADHD phenotypes, complete copy number analyses, assess parent of origin effects and season of birth effects, and test for epistasis among apparently uncorrelated genes. Acquiring DNA Samples All consent forms stipulate that the samples can only be used by researchers who have been approved by the National Institute of Mental Health (NIMH), National Institutes of Health. All consent forms, except those used at the Zürich site (N=141 subjects), explicitly indicate that the samples may be used by researchers from commercial enterprises seeking to benefit financially from the analysis of the samples. The Zürich consent does not prohibit such use. The Zürich consent form also included an "opt out" that allowed the subjects to indicate that they did not want their samples stored at the NIMH repository or used by researchers external to the project. No subjects enrolled in the project opted out. Consent groups and participant set ADHD (ADHD): 2758 (924 trios)

  Study phs000016

• Cross-Species Single-Cell Analysis of Pancreatic Ductal Adenocarcinoma Reveals Antigen-Presenting Cancer-Associated Fibroblasts

  Cancer-associated fibroblasts (CAFs) are major players in the progression and drug resistance of pancreatic ductal adenocarcinoma (PDAC). CAFs constitute a diverse cell population consisting of several recently described subtypes, although the extent of CAF heterogeneity has remained undefined. Here we employ single-cell RNA-sequencing to thoroughly characterize the neoplastic and tumor microenvironment content of human PDAC tumors. Six human PDAC tumor specimens from six patients were collected, and processed for single-cell RNA-sequencing analysis. Adjacent-normal pancreas tissue was also collected from two of the patients. Tumor samples were digested, and fluorescence-activated cell sorting was used to isolate viable cells. For one tumor sample, viable, CD45-negative, CD31-negative, and EpCAM-negative cells were also isolated to enrich for CAFs. The 10X Chromium platform was then used to isolate single cells for RNA-sequencing analysis. This work has demonstrated the differences in immune cell populations between adjacent-normal and tumor tissues, and identified subpopulations of epithelial cells and CAFs present in PDAC tumors. This high-throughput analysis is a resource to better understand the cell populations present in PDAC, and may ultimately aid in the development of more effective therapies for this deadly malignancy.

  Study phs001840

• Integrated Genomic Analysis of Periampullary Tumors at The Human Genome Sequencing Center, Baylor College of Medicine (HGSC-BCM)

  Adenocarcinomas arising in the complex environment of the ampulla of Vater constitute a histopathological heterogenous group, presumably originating from the different epithelial cellular constituents present at the site: pancreas, bile duct, and intestinal duodenum. These tumors have been described in many different ways: intra-ampullary, periampullary, intra-ampullary papillary-tubular neoplasm, ampullary-ductal, periampullary-duodenal, and ampullary-not otherwise specified. These varied classifications reflect the difficulty in classifying these tumors into specific groups. Only the tumors clearly localized in the bile duct or duodenum are identified as distal cholangiocarcinomas (CAC) or duodenal adenocarcinomas (DUOAC). The current classification is based on macroscopic features that may distinguish the epithelium of origin, microscopic features, clinicopathological criteria, histopathology and expression of differential markers. This classification is subjective and prone to inter-observer variability and significantly impacts on treatment selection and therapeutic development. In order to define subtypes of periampullary cancer with clinical relevance, we performed whole exome sequencing and copy number analysis of 160 cancers arising in the periampullary region, 62 of these clearly arising from either the bile duct (n = 44), or the duodenum (n = 18) and 98 periampullary cancers (AMPAC) where the epithelium of origin could not be clearly defined.

  Study phs000895

• Trisomy21: Risk Factors for Chromosome Nondisjunction (T21NDJ)

  The overall goal of the project is to identify genetic risk factors associated with chromosome 21 nondisjunction in the oocyte. The dataset derives from multi-site collection of live birth probands with Down syndrome due to standard trisomy 21 (T21) and their biological parents. The type of nondisjunction error (NDJ)(maternal or paternal) in all cases has been determined to be maternal in origin based on the chromosome 21 variants contributed from parent to proband. The Center of Inherited Disease with support from NICHD has conducted genome-wide genotyping using the Illuminia Human OmniExpress Plus Exome array on approximately 800 women who have been identified through their offspring with DS and have been characterized as having a maternal meiois I (MI) or meiosis II (MII) nondisjunction error. Genotypes from biological fathers of the offspring with DS can be used with the data on mothers to better define the type of nondisjunction error (MI or MII) and to refine the chromosome 21 recombination profile. We provide the type of nondisjunction error, knowing that this will be updated based on the new panel. We do not provide the recombination profile, as this can be best defined using the new comprehensive set of SNPs in the OmniExpress panel.

  Study phs000718

• NIDDK IBD Genetics Consortium Repository Immunochip

  The Immunochip is a custom Illumina Infinium chip comprising 196,524 SNPs and small indels selected primarily based on GWAS analysis of 12 autoimmune and inflammatory diseases. The chip has two purposes: fine mapping of 289 established associations corresponding to 187 distinct loci, and deep replication of suggestive, but not yet proven, associations. Fine-mapping regions were defined as 0.2cM centered on GWAS hit SNPs, and all SNPs and short indels in these regions from the 1000 Genomes Project low coverage pilot CEU samples, as well as variants discovered in resequencing experiments conducted by groups collaborating in the chip design were selected for inclusion. Replication of autoimmune and inflammatory GWAS (including Crohn's disease and ulcerative colitis) contributed the bulk of the remaining SNP lists. Approximately 25,000 SNPs were included as replication of unrelated diseases as part of the WTCCC2 project, which serve as useful null SNPs for these analyses. In total, approximately 240,000 SNPs were selected for inclusion, with an assay design success rate of ~80%. Reprinted from Jostins et al. (2012), with permission from Springer Nature (see https://www.nature.com/reprints/permission-requests.html). Jostins, L., et al. (2012) Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature, 491, 119-24. PMID: 23128233.

  Study phs001721

• Germline hypomorphic CARD11 mutations in severe atopic disease

  We described five patients from four unrelated families who had severe atopic dermatitis and susceptibility to mild skin viral infections. Some patients also exhibited other allergic symptoms such as asthma and food allergy. We performed targeted sequencing for affected patients A-I of family A and B-I of family B, whole exome sequencing for D-I, D-II.1 and healthy mother D-II.3 of family D, and whole genome sequencing for C-I and healthy mother C-II.2 of family C. We identified three different novel damaging missense mutations of CARD11 (GeneID:84433) in patients A-I (L194P), B-I (R975W), and C-I (E57D). In patients D-I and D-II.1, a dup183_196 (p.K196_V197insMKEERDSYNDELVK) mutation was found in the coil-coil domain of the protein. These mutations act as dominant negatives impeding the scaffolding function of the wild type protein, which leads to downstream NF-kB and mTORC1 signaling defects after TCR stimulation. Sanger sequencing later identified the corresponding CARD11 mutations in the affected family members (C-II.1, D-II.2) that are not included in the dbGaP submission. Hypomorphic CARD11 dominant negative mutants are novel causes of severe atopic disease.

  Study phs001369

• Discovery of Non-ETS Gene Fusions in Human Prostate Cancer using Next Generation RNA Sequencing

  Half of prostate cancers harbor gene fusions between TMPRSS2 and members of the ETS transcription factor family. To date little is known about the presence of non-ETS fusion events in prostate cancer. We employed next-generation transcriptome sequencing (RNA-Seq) in order to explore the whole transcriptome of 25 human prostate cancer samples for the presence of chimeric fusion transcripts. We generated more than 1 billion sequence reads and used a novel computational approach (FusionSeq) in order to identify novel gene fusion candidates with high confidence. In total, we discovered and characterized seven new cancer-specific gene fusions, two involving the ETS genes ETV1 and ERG, and five involving non-ETS genes such as CDKN1A (p21), CD9 and IKBKB (IKK-beta), genes known to exhibit key biological roles in cellular homeostasis or assumed to be critical in tumorigenesis of other tumor entities, as well as the oncogene PIGU and the tumor suppressor gene RSRC2. The novel gene fusions are found to be of low frequency but interestingly, the non-ETS fusions were all present in prostate cancer harboring the TMPRSS2-ERG gene fusion. Future work will focus on determining if the ETS rearrangements in prostate cancer are associated or directly predispose to a rearrangement prone phenotype.

  Study phs000310

• Somatic Mutation Profile by Next Generation Sequencing in HER2+ Breast Cancer

  The HER pathway is the driving force behind 30% of human breast cancers. It is important to understand how targeted therapies block different cellular pathways, and mechanisms of escape from this blockage. Therapies directed at HER2 establish a successful treatment paradigm, but de novo and acquired resistance exist. The HER signaling system is a complex network with four receptors and eleven ligands, a phosphorylation signaling cascade, and many transcription factors, all complicated by both positive and negative feedback circuits. Analysis of genomes, exomes and transcriptomes by next generation sequencing is aimed at uncovering the genetic factors responsible for patient responses to HER2-directed therapies. We are sequencing HER2-overexpressing cancers, in order to identify potential somatic changes that may better select patients who will benefit from therapy, to determine new targets that may overcome resistance, and to improve outcomes with known current HER2-targeted therapies. Whole exome capture sequencing will determine somatic mutation profiles in HER2-overexpressing tumors, to comprehensively characterize the somatic alterations, with the goal of identifying those patients most likely to respond, as well as discovering new targets that may overcome resistance to HER2-directed therapy. We augment the whole exome data with RNA-seq data to determine expression levels of somatic mutations we discover.

  Study phs000770

• Targeted MitoExome Sequencing of Mitochondrial OXPHOS Diseases (Massachusetts General Hospital)

  Human disorders of mitochondrial oxidative phosphorylation (OXPHOS) represent a devastating collection of inherited diseases. These disorders impact at least 1:5000 live births, and are characterized by multi-organ system involvement. They are characterized by remarkable locus heterogeneity, with mutations in the mtDNA as well as in over 77 nuclear genes identified to date. It is estimated that additional genes may be mutated in these disorders. To discover the genetic causes of mitochondrial OXPHOS diseases, we performed targeted, deep sequencing of the entire mitochondrial genome (mtDNA) and the coding exons of over 1000 nuclear genes encoding the mitochondrial proteome. We applied this 'MitoExome' sequencing to 124 unrelated patients with a wide range of OXPHOS disease presentations from the Massachusetts General Hospital Mitochondrial Disorders Clinic. The 2.3Mb targeted region was captured by hybrid selection and Illumina sequenced with paired 76bp reads. The total set of 1605 targeted nuclear genes included 1013 genes with strong evidence of mitochondrial localization from the MitoCarta database, 377 genes with weaker evidence of mitochondrial localization from the MitoP2 database and other sources, and 215 genes known to cause other inborn errors of metabolism. Approximately 88% of targeted bases were well-covered (>20X), with mean 200X coverage per targeted base.

  Study phs000339

• Rapid Evolution of a Skin Lightening Allele in Southern African KhoeSan

  The genetic architecture and polygenicity of skin pigmentation is explored in KhoeSan communities, including Nama individuals whose phenotype and genotype data are provided in this dataset. By pairing genotypes and quantitative spectrophotometric skin pigmentation phenotypes, we show that skin pigmentation is highly heritable in KhoeSan, yet known pigmentation loci only explains a small fraction of the phenotypic variance. Using genome-wide association analyses, we identified both canonical and non-canonical skin pigmentation loci. We show that this phenotype is more polygenic and complex than previously characterized. (Martin et al., Cell 2017. PMID: 29195075) Following up on the top associated signal with large effect size in the gene SLC24A5, we demonstrate that the canonical Eurasian nonsynonymous allele was introduced into KhoeSan via a recent migration ~2 kya and was under extremely strong selection. The derived allele was present at high frequency despite controlling for gene flow. With high-throughput sequences in the captured SLC24A5 region, we show that the most common derived haplotype is identical amongst Europeans, eastern African and KhoeSan. Using 4-population demographic simulations with selection, we show that the allele was introduced into the KhoeSan only 2,000 ya via a back-to-Africa migration and then experienced a selective sweep.

  Study phs001753

• Generation of a Cellular Atlas of Human Adipose Tissue

  White adipose tissue (WAT), once regarded as morphologically and functionally bland, is now recognized to be dynamic, plastic, heterogenous, and involved in a wide array of biological processes including energy homeostasis, glucose and lipid handling, blood pressure control, and host defense.  High fat feeding and other metabolic stressors cause dramatic changes in adipose morphology, physiology, and cellular composition, and alterations in adiposity are associated with insulin resistance, dyslipidemia, and Type 2 diabetes (T2D). Here we provide detailed cellular atlases of human and murine subcutaneous and visceral white fat at single-cell resolution across a range of body weight. We identify subpopulations of adipocytes, adipose stem and progenitor cells (ASPCs), vascular, and immune cells and demonstrate commonalities and differences across species and dietary conditions. We link specific cell types to increased risk of metabolic disease, and we provide an initial blueprint for a comprehensive set of interactions between individual cell types in the adipose niche in leanness and obesity. We also utilize bulk RNA sequencing data of enzymatically digested human adipocytes to scale these findings to a larger cohort. These data comprise an extensive resource for the exploration of genes, traits, and cell types in the function of WAT across species, depots, and nutritional conditions. 

  Study phs002766

• Joint-Specific TF Regulation in RA

  The mechanisms responsible for the distribution and severity of joint involvement in rheumatoid arthritis (RA) are not known. To explore whether site-specific fibroblast-like synoviocyte (FLS) biology might be associated with location-specific synovitis and explain the predilection for hand (wrist/metacarpal phalangeal joints) involvement in RA, we generated bulk, whole-genome transcriptomic and chromatin accessibility data from FLS. Samples were matched in age, sex, and were homogeneous in that all had significant joint damage with longstanding disease requiring surgical intervention. Analysis revealed joint-specific patterns of FLS phenotypes, with proliferative, migratory, proinflammatory, and matrix-degrading characteristics observed in resting FLS derived from the hand joints compared with hip or knee. TNF-stimulation amplified these differences, with greater enrichment of proinflammatory and proliferative genes in hand FLS compared with hip and knee FLS. Hand FLS also had the greatest expression of markers associated with an ‘activated' state relative to the ‘resting' state, with the greatest cytokine and MMP expression in TNF-stimulated hand FLS. Predicted differences in proliferation and migration were biologically validated with hand FLS exhibiting greater migration and cell growth than hip or knee FLS. Distinctive joint-specific FLS biology associated with a more aggressive inflammatory response might contribute to the distribution and severity of joint involvement in RA.

  Study phs003633

• Tumor detection by analysis of both symmetric- and hemi-methylation of plasma cell-free DNA

  Aberrant DNA methylation plays a critical role in tumorigenesis. While DNA methylation has been used for cancer detection and classification, DNA hemi-methylation, a novel epigenetic mark has not been analyzed extensively in cancer epigenomes. Here we report a strand-specific (ss) sequencing method (MeDIP-Seq) for plasma cell free (cf) DNA (sscf-MeDIP-Seq), which can analyze both symmetrically methylated DNA regions (DMRs) as well as hemi-methylated regions (DHMRs). Using the sscf-MeDIP-Seq method, we analyzed plasma cfDNA methylomes of 271 samples from subjects with liver cancer and brain cancer and from individuals without cancer (controls). Among them, 215 samples were chosen as the discovery cohort for the identification of DMRs and DHMRs specific in each subject group and for the training of machine learning models of multi-cancer detection (MCD) using DMRs, DHMRs and DMRs+DHMRs as inputs. These models were then used to predict the 56 samples in the validation cohort. We found that models trained with DMRs+DHMRs as inputs in general outperformed models trained with DMRs or DHMRs alone, with AUROC being 0.971, 0.981, and 0.99 in predicting control, liver and brain cancer samples in the validation cohort, respectively, by the DMR+DHMR-trained models.

  Study phs003462

• Single cell RNAseq of stenotic, inflamed and non-inflamed transmural lesions from patients with Crohn's disease

  This dataset contains a gene-cell matrix derived from single-cell RNA sequencing (scRNA-seq) data of ileal tissue from Crohn's disease (CD) patients and colorectal cancer (CRC) patients. It includes: Crohn's Disease Patients: A trio of transmural lesions (stenotic, inflamed, and non-inflamed) from each patient. Colorectal Cancer Patients: Unaffected ileal tissue used as external non-inflamed control. Cell Level Metadata: The dataset includes relevant cell-level metadata such as cell type annotations used in the study. Experimental Details: Platform: 10x Genomics Chromium Single Cell 3' GEX Sequencing: Illumina NovaSeq Processing: Data processed with Cell Ranger software. Resulting count matrices were merged for downstream analysis, including integration and dimensionality reduction. Dataset Composition: Crohn's Disease Patients: 10 patients with 3 samples each (non-inflamed, inflamed, stenotic), totaling 30 samples. Colorectal Cancer Patients: 5 patients with 1 sample each of unaffected tissue, totaling 5 samples. Data Provided: Merged Raw Count Matrix: The final merged raw count matrix used for downstream analysis. Cell Metadata File: Contains details of sample, tissue, and patient for each cell in the count matrix. Barcodes File: Indicate each cell barcode which also encodes the sample, tissue, and patient details for each cell. CD.S_Inf: Stenotic Corhn's disease inflamed samples CD.S_Sten: Stenotic CD patient stenosis sample CD.S_Prox: Stenotic CD Patient - proximal non-inflamed sample CC.C_Prox: CRC Patient proximal unaffected sample eg: A barcode 'CC.C_1_Prox_AAGTCGTAGACCCTTA' indicates CRC Patient unaffected proximal sampe from CRC Patient no.1 and the nucleic acid sequence indicate a unique cell from this sample. Total Samples: Crohn's Disease (CD) Patients: 30 samples Colorectal Cancer (CRC) Patients: 5 samples Patient_no Sample Sample_type 1 CC.C_1 CC.C_1_Prox CC.C_Prox 2 CD.S_1 CD.S_1_Prox CD.S_Prox 3 CD.S_1 CD.S_1_Infl CD.S_Infl 4 CD.S_1 CD.S_1_Sten CD.S_Sten 5 CC.C_2 CC.C_2_Prox CC.C_Prox 6 CD.S_2 CD.S_2_Prox CD.S_Prox 7 CD.S_2 CD.S_2_Infl CD.S_Infl 8 CD.S_2 CD.S_2_Sten CD.S_Sten 9 CC.C_3 CC.C_3_Prox CC.C_Prox 10 CC.C_4 CC.C_4_Prox CC.C_Prox 11 CD.S_3 CD.S_3_Prox CD.S_Prox 12 CD.S_3 CD.S_3_Infl CD.S_Infl 13 CD.S_3 CD.S_3_Sten CD.S_Sten 14 CD.S_4 CD.S_4_Prox CD.S_Prox 15 CD.S_4 CD.S_4_Infl CD.S_Infl 16 CD.S_4 CD.S_4_Sten CD.S_Sten 17 CC.C_5 CC.C_5_Prox CC.C_Prox 18 CD.S_5 CD.S_5_Prox CD.S_Prox 19 CD.S_5 CD.S_5_Infl CD.S_Infl 20 CD.S_5 CD.S_5_Sten CD.S_Sten 21 CD.S_6 CD.S_6_Prox CD.S_Prox 22 CD.S_6 CD.S_6_Infl CD.S_Infl 23 CD.S_6 CD.S_6_Sten CD.S_Sten 24 CD.S_7 CD.S_7_Prox CD.S_Prox 25 CD.S_7 CD.S_7_Infl CD.S_Infl 26 CD.S_7 CD.S_7_Sten CD.S_Sten 27 CD.S_8 CD.S_8_Prox CD.S_Prox 28 CD.S_8 CD.S_8_Infl CD.S_Infl 29 CD.S_8 CD.S_8_Sten CD.S_Sten 30 CD.S_9 CD.S_9_Prox CD.S_Prox 31 CD.S_9 CD.S_9_Infl CD.S_Infl 32 CD.S_9 CD.S_9_Sten CD.S_Sten 33 CD.S_10 CD.S_10_Prox CD.S_Prox 34 CD.S_10 CD.S_10_Infl CD.S_Infl 35 CD.S_10 CD.S_10_Sten CD.S_Sten

  Dataset EGAD50000000559

• Cell type mapping of inflammatory muscle diseases highlights selective myofiber vulnerability in inclusion body myositis

  Inclusion body myositis (IBM) is the most prevalent inflammatory muscle disease in older adults with no effective therapy available. In contrast to other inflammatory myopathies like subacute immune-mediated necrotizing myopathy (IMNM), IBM follows a chronic disease course with both inflammatory and degenerative features of pathology. Moreover, causal factors and molecular drivers of IBM progression are largely unknown. Therefore, we paired single-nucleus RNA sequencing with spatial transcriptomics from patient muscle biopsies to map cell type-specific drivers underlying IBM pathogenesis compared to IMNM muscles and non-inflammatory skeletal muscle samples. In IBM muscles, we observed a selective loss of type 2 myonuclei paralleled by increased levels of cytotoxic T and cDC1 cells. IBM myofibers were either characterized by upregulation of cell stress markers featuring GADD45A and NORAD, or protein degradation markers including RNF7 associated with p62 aggregates. GADD45A upregulation was preferentially seen in type 2A myofibers associated with severe tissue inflammation. We also noted IBM-specific upregulation of ACHE encoding acetylcholinesterase, which can be regulated by NORAD activity and result in functional denervation of myofibers. Our results provide promising insights into possible mechanisms of myofiber degeneration in IBM and suggest a selective type 2 fiber vulnerability linked to genomic stress and denervation pathways.

  Study EGAS50000000310

• IMPRESS: Improved methylation profiling using restriction enzymes and smMIP sequencing, combined with a new biomarker panel, creating a multi-cancer detection assay

  Background Despite the worldwide progress in cancer diagnostics, more sensitive diagnostic biomarkers are needed. The methylome has been extensively investigated in the last decades, but a low-cost, bisulfite-free detection method for multiplex analysis is still lacking. Methods We developed a methylation detection technique called IMPRESS, which combines methylation-sensitive restriction enzymes and single-molecule Molecular Inversion Probes. We used this technique for the development of a multi-cancer detection assay for eight of the most lethal cancer types worldwide. We selected 1791 CpG sites that can distinguish tumor from normal tissue based on DNA methylation. These sites were analysed with IMPRESS in 35 blood, 111 tumor and 114 normal samples. Finally, a classifier model was built. Results We present the successful development of IMPRESS and validated it with ddPCR. The final classifier model discriminating tumor from normal samples was built with 358 CpG target sites and reached a sensitivity of 0.95 and a specificity of 0.91. Moreover, we provide data that highlight IMPRESS’s potential for liquid biopsies. Conclusions We successfully created an innovative DNA methylation detection technique. By combining this method with a new multi-cancer biomarker panel, we developed a sensitive and specific multi-cancer assay, with potential use in liquid biopsies.

  Study EGAS50000000624

• Identifying New Genetic Subtypes in Follicular Lymphoma

  Follicular lymphoma (FL) exhibits considerable variability in biological features and clinical trajectories across patients. To dissect the diversity of FL, we utilized a Bernoulli mixture model to identify genetic subtypes in 713 pre-treatment tumor tissue samples. Our analysis revealed the existence of five subtypes with unique genetic profiles that correlated with clinicopathological characteristics. The clusters were enriched in specific mutations as follows: CS (CREBBP and STAT6), TT (TNFAIP3 and TP53), GM (GNA13 and MEF2B), Q (quiescent, for low mutation burden), and AR (mutations of mTOR pathway-related genes). The subtype Q was enriched for patients with stage I disease and was associated with a lower proliferative history compared to the other subtypes. The AR subtype was unique in its enrichment for IgM-expressing FL cases and was associated with advanced-stage and more than 4 nodal sites. The existence of subtypes was validated in an independent cohort of 418 samples from the GALLIUM trial. Notably, patients assigned to the TT subtype consistently experienced inferior progression-free survival when treated with immunochemotherapy. Collectively, our findings offer insight into core pathways distinctly linked with each FL cluster and are expected to be informative in the era of targeted therapies.

  Study EGAS50000000435

• Melanoma Genome Sequencing Project

  Melanoma is the most prevalent cause of skin cancer morbidity and mortality. In order to characterize the full range of somatic mutations that may drive the growth of melanoma, we are sequencing tumor and normal DNA from a set of roughly 150 melanomas. For the majority of samples (approximately 90% of the cases), mutations in protein coding genes will be assessed in the exonic DNA of tumor-normal pairs using hybrid capture and paired-end DNA sequencing. For a few of DNA samples (approximately 10% of the cases), the entire genomes will be analyzed to assess the possible contribution of complex structural rearrangements contributing to oncogenesis. The sequencing data are supplemented by copy number profiling on the same tumors using high-density SNP arrays. Integration of these approaches will enable the unbiased and comprehensive characterization of both known and novel recurrent DNA alterations that arise in melanoma.Versions 1-4 contain whole exome and bulk RNA sequencing data for patients with melanoma.Version 5 - We compared somatic genomic profiles from matched pre-treatment and post-resistance tumor biopsies in patients with metastatic melanoma who exhibited heterogeneous responses to immune checkpoint inhibitors (ICIs). This version includes whole exome sequencing (WES) data from 14 patients.

  Study phs000452

• Human inflammatory cardiomyopathies following SARS-CoV2 infection and COVID-19 vaccination

  Title: The cellular and molecular responses in human inflammatory cardiomyopathies following SARS-CoV2 infection and COVID-19 vaccination. Abstract: Abstract: Myocarditis is a cardiac disorder with multiple etiologies characterised by inflammatory cell infiltration and can occur after SARS-CoV-2 infection or rarely following mRNA-based COVID-19 vaccination. The underlying cellular and molecular mechanisms driving these pathologies remain poorly understood.Here we performed single-nucleus-RNA-sequencing in left ventricular endomyocardial biopsies from patients with Non-COVID-19 myocarditis, following SARS-CoV-2 infection (Post-COVID-19) and COVID-19 vaccination (Post-Vaccination). We detected specific cytokine expression patterns highlighting a particular role of interferon-γ in Post-COVID-19 and upregulated IL16 and IL18 expression as a Post-Vaccination hallmark. While the myeloid response was similar between groups, CD4+T-cell proportions were higher in Post-Vaccination and cytotoxic CD8+T and NK cells expanded in Post-COVID-19. Endothelial cells showed gene expression changes suggestive of vascular barrier function deficiency in Post-COVID-19 and angiogenesis response to cardiac inflammation in all groups. Together, our results illuminate shared and distinct cellular and molecular architectures of Non-COVID-19, Post-COVID-19 and Post-Vaccination associated myocardial inflammation in human hearts.

  Study EGAS50000000769

• Use of new methodologies to achieve a thorough molecular characterization in pediatric acute leukemia

  In childhood leukemia, a better genetic characterization is needed to define new clinic-biological entities, refine the risk-stratification of patients and help to reduce the toxicity by applying a risk-directed therapy. This project aims to obtain information that will allow us to personalize the management of pediatric patients with leukemia as much as possible, so that we can offer a complete diagnosis, a sensitive follow-up and a more targeted treatment to the real risk of each case. The use of massive genome analysis technologies such as transcriptome sequencing (RNA-Sequencing) will allow a comprehensive biological characterization of pediatric leukemia patients, especially those cases that we have not been able to characterize by conventional diagnostic techniques. We believe that the RNA-Seq technique will allow us to identify new biomarkers that will be useful for: 1) defining new biological entities with their own clinical behavior; 2) refining the stratification of patients into risk groups; 3) finding targets for a better follow-up of MRD and 4) identifying therapeutic targets for the application of targeted therapies to reduce toxicity. PI: Camós-Guijosa, Mireia; Vega-García, Nerea Funding: PI21/00213 Instituto de Salud Carlos III (ISCIII)

  Study EGAS50000000701

• Childhood Cancer Data Initiative (CCDI): Pediatric In Vivo Testing Program –Sarcoma, Kidney, and Liver Cancers

  The goal was to complete whole exome sequencing (WES) and RNA sequencing (RNA-Seq) on patient derived xenograft samples. Cancer in children is rare with approximately 15,700 new cases diagnosed annually in children 21 years or younger in the U.S. Through use of multimodality therapy (surgery, radiation therapy, and aggressive chemotherapy), 70% of patients will be 'cured' of their disease, and 5-year Event-Free Survival (EFS) exceeds 80%. Consequently, the number of patients that can be enrolled in phase I/II clinical trials is small, and most patients will have been extensively treated, hence drug/radiation resistant. Thus, preclinical studies that accurately translate into effective clinical therapy are an essential component of pediatric drug development. Our group has contributed to studies in the Pediatric Preclinical Testing Program (PPTP) and Pediatric Preclinical Testing Consortium (PPTC) that have led to clinical studies through Children's Oncology Group (COG). Of importance, we have developed and characterized over 330 Patient Derived Xenografts (PDX), developed from tumors both at diagnosis and relapse, that can be used to facilitate pediatric drug development as directed by FDA under the Research to Accelerate Cures and Equity for Children Act (RACE for Children Act).

  Study phs003160

• National Cancer Institute (NCI) Early Onset Malignancies Initiative (EOMI): Molecular profiling of Breast, Colon, Kidney, Liver, Multiple Myeloma, and Prostate among Racially and Ethnically Diverse Populations

  The Early Onset Malignancies Initiative (EOMI) is focused on collecting specimens from racially and ethnically diverse population groups with six different types of cancer (breast, prostate, colon, liver, kidney, and multiple myeloma). This initiative is a partnership between the Center for Research Strategy, the Center for Cancer Genomics and the Division of Cancer (DCP). DCP's NCI Community Oncology Research Program (NCORP) is a critical partner. The acquisition of the cancer and matching normal biospecimens in this initiative are from newly diagnosed early onset cancer patients who identify as Non-Hispanic Black, Hispanic, American Indian or Alaska Native, or Non-Hispanic White. The Minority/Underserved NCORPs sites will collect the samples for this project. By carrying out this initiative in the NCORP's national network of investigators, cancer care providers, and research institutions, the EOMI will harness the community-based partnerships to improve participation among these racial/ethnic groups. The NCORP investigators, as participants in NCI's clinical trials network, will annotate the biologic samples with clinical information that will enhance the ability to answer questions about molecular determinants of early disease, treatment response, and/or prognosis. The EOMI will be a step forward in addressing racial/ethnic disparities and in understanding cancer in underserved populations.

  Study phs001952

• Single-Cell Genomic and Transcriptomic Analysis of the Aging Human Brain

  We studied human brain aging using single-cell whole genome sequencing and single-nucleus RNA sequencing of the pre-frontal cortex from 19 donors ranging in age from 0.4 years to 104 years. All donors were free of neuropathology and none received a clinical neurological diagnosis prior to death. snRNA-seq libraries were prepared in batches that always included old and young donors to mitigate batch effects in the preparations. Neurons for single-cell WGS were collected from the same tissue region as the snRNA-seq for each donor but represent independent cells. Multiplexed error-robust fluorescent in-situ hybridization (MERFISH) data was also collected from pre-frontal cortex samples of a subset of the 19 donors. Transcriptomic results identified infant-specific cell clusters, and common-down regulation of cell-essential genes involved in ribosomes, transport, homeostasis and metabolism during aging, across all cell types. scWGS identified two-age associated mutational signatures, as well as gene-length and expression-level dependent somatic mutation rates in neurons. Raw sequencing data in fastq format for single-nucleus RNA-seq and single-cell DNA-seq, as well as cell and gene count matrix, transcript metadata, and cell metadata for MERFISH will be available in dbGaP.

  Study phs003445

• Examination of Engineered LINE-1 Integration Events in HeLa Cells

  Long Interspsesed Element-1 (LINE1 or L1) encodes two proteins (ORF1p and ORF2p) required for its mobilization (i.e. retrotransposition) in the human genome. Human ORF2p, a 150kDa protein, has endonuclease (EN) and reverse transcriptase (RT) activities. L1 EN is classified as apurinic/apyrimidin-like endonuclease (APE) and is responsible for initiating target-site primer reverse transcription (TPRT) during the L1 retrotransposition cycle. During canonical TPRT, the L1 EN activity makes a single-strand endonucleolytic nick at a double-strand DNA target sequence in genomic DNA. The endonucleolytic nick is typically made at an L1 EN degnerate consensus cleavage sequence (5'-TTTT/A-3') exposing a free 3' hydroxy group that can be used as a primer by the ORF2p L1 RT activity to reverse transcribe the associated L1 RNA. Here, we characterized over 21,000 de novo engineered L1 retrotransposition events in HeLa cells to determine if L1 exhibits overt integration preferences in the human genome. Bru-seq of HeLa cells to determine actively transcribed regions of the genome was also performed. This Bru-seq data was used to determine if engineered L1 integration events preferentially integrate into transcribed regions of the genome, and to determine which strand during transcription may present itself as preferential during L1 retrotransposition.

  Study phs001669

• Transcriptome sequencing of human colon organoid after co-cultivation with Bifidobacterium longum

  The large intestine has a dense milieu of indigenous bacteria, generating a complex ecosystem with crosstalk between individual bacteria and host cells. In vitro host cell modeling and bacterial interactions at the anaerobic interphase have elucidated the crosstalk molecular basis. Although classical cell lines derived from patients with colorectal cancer including Caco-2 cells are used, whether they adequately mimic normal colonic epithelial physiology is unclear. To address this, we performed transcriptome profiling of Caco-2 and Monolayer cells derived from healthy Human Colonic Organoid (MHCO) cultured hemi-anaerobically. Coculture with the anaerobic gut bacteria, Bifidobacterium longum subsp. longum differentiated the probiotic effects of test cells from those of physiologically normal intestinal and colorectal cancer cells. We cataloged non- or overlapping gene signatures where gene profiles of Caco-2 cells represented absorptive cells in the small intestinal epithelium, and MHCO cells showed complete colonic epithelium signature, including stem/progenitor, goblet, and enteroendocrine cells colonocytes. Characteristic gene expression changes related to lipid metabolism, inflammation, and cell-cell adhesion were observed in cocultured live Bifidobacterium longum and Caco-2 or MHCO cells. B. longum-stimulated MHCO cells exhibited barrier-enhancing characteristics, as demonstrated in clinical trials. Our data represent a valuable resource for understanding gut microbe and host cell communication.

  Study JGAS000740

• Hematopoietic Tet2 inactivation enhances the response to checkpoint blockade immunotherapy

  Somatic mutations inactivating TET2 are among the most common drivers of clonal hematopoiesis (CH). While TET2 inactivation is associated with monocyte-derived inflammation and improved chimeric antigen-receptor-T cell function, its impact on immunotherapy response is unknown. In murine models, hematopoietic Tet2 mutation enhanced the immune checkpoint blockade (ICB) response via the combined presence of phagocytes, CD4 and CD8 T cells. This effect was lost with myeloid- or T-cell restricted Tet2 inactivation, or in mice with 20% Tet2-mutant hematopoiesis. Mechanistically, in Tet2-mutant tumor-infiltrating leukocytes (TILs), ICB preferentially restricted cell states linked to tumor progression while inducing anti-tumor states. Tet2-mutant monocytes activated costimulatory programs, while Tet2-mutant T cells showed enhanced T cell memory signatures, alongside decreased exhaustion and regulatory phenotypes. This murine data was clinically relevant, since tumors from colorectal cancer and melanoma patients with TET2 driver mutation-CH (TET2-CH) showed enhanced immune infiltration, inflammation, and T cell activation. In melanoma patients treated with ICB, TET2-CH was associated with 6-fold greater odds of clinical benefit. Collectively, we demonstrate that hematopoietic Tet2 inactivation primes leukocytes for anti-tumor states associated with immunotherapy response and provides a potential biomarker for personalized therapy.

  Study EGAS50000001191

• Recurrent intra-tumour heterogeneity is a hallmark of metastatic prostate cancer

  The evolution of cancers from low grade to metastatic disease is a major determinant of cancer mortality. Cancer evolution involves a complex interplay between cell intrinsic genetics and transcriptional alterations and the extrinsic microenvironment. To define the key mechanisms underpinning metastatic development, we focused on the most lethal form of prostate cancer, metastatic castration-resistant prostate cancer, and employed single-cell multi-omics and whole-genome sequencing to deeply profile 34 metastatic lesions from 9 patients obtained from rapid autopsy. We found that intra-tumour heterogeneity is an indicator of key evolutionary processes, characterised by recurrent tumour populations acting as critical functional components of the tumour ecosystem, irrespective of clonal and microenvironmental backgrounds. Unexpectedly, microenvironments across metastatic sites played a limited role while clonal evolution primarily promoted transcriptional noise. Intra-patient functional convergence of tumour ecosystems was observed across metastatic sites, pointing towards system-level selection pressures that drive the heterogeneity landscape of mCRPC. Our findings reveal functional evolutionary convergence of metastatic disease into units of intra-tumour heterogeneity identifying critical determinants of metastatic progression for therapeutic targeting. Note: matched WGS for some tumour single-cell experiments, as well as germline WGS for patients CA0027, CA0034, CA0035, CA0043, and CA0076, can be found in EGAS00001006598.

  Study EGAS50000001312

• Comprehensive gene analysis of colorectal cancer cases

  Colorectal cancer is the third most common morbidity and the fourth leading cause of cancer death in the world.To characterize the mutation profiles of colorectal cancer with liver metastasis,we performed both whole-exosome sequencing and RNA sequencing for 12 cases of colorectal cancer with liver metastasis (group A) and 16 cases of colorectal cancer without liver metastasis (group B). As a result of driver mutation search by Mutsig pipeline, PTEN was identified as a significant mutant gene in group A alone.ADAMTS10,NELL1 and RXFP3 were identified as candidate genes for metastasis by MuTect,SID and VarScan pipeline.Furthermore, in all samples, the copy number of chromosomes 1p,4,7,8,13,15, 17p,18,20 and 22q were significantly abnormal. Chromosomes 1p,4,15,18,17p,22q were deleted and chromosomes 7, 8,13 and 20 were amplified by CNV.Pathway analysed revealed Wnt/Ca2+ and PCP pathway was involved in metastasis of cl cancer.9 kinds of fusion genes were identified in deFuse pipeline. Among them, ADAP1 fusion genes were identified in 2 samples, and ADAP1 fusion genes were further identified in 3 samples by our in-house algorithm. It is suggested that new fusion genes present in such multiple samples may be involved in the development of colorectal cancer.

  Study JGAS000128

• Rapid detection of expanded short tandem repeats in personal genomics using hybrid sequencing.

  Long expansions of short tandem repeats (STRs); i.e., DNA repeats of 2-6 nucleotides, are associated with some genetic diseases. Cost-efficient high throughput sequencing can quickly produce billions of short reads that would be useful for uncovering disease-associated STRs. However, enumerating STRs in short reads remains largely unexplored because of the difficulty in elucidating STRs much longer than 100 bp, the typical length of short reads. We propose ab initio procedures for sensing and locating long STRs promptly by utilizing the frequency distribution of all STRs and paired-end read information. We validated the reproducibility of this method using biological replicates and used it to locate an STR associated with a brain disease (SCA31). Subsequently, we sequenced this STR site in eleven SCA31 samples using SMRT? sequencing (Pacific Biosciences), determined 2.3-3.1 kb sequences at nucleotide resolution, and revealed that (TGGAA)- and (TAAAATAGAA)-repeat expansions determined the instability of the repeat expansions associated with SCA31. Our method could also identify common STRs, (AAAG)- and (AAAAG)-repeat expansions, which are remarkably expanded at four positions in an SCA31 sample. This is the first proposed method for rapidly finding disease-associated long STRs in personal genomes using hybrid sequencing of short and long reads.

  Study JGAS000002

• BHD-associated kidney cancer

  Background Birt-Hogg-Dub? (BHD) syndrome is a hereditary kidney cancer syndrome, which predisposes patients to develop kidney cancer, cutaneous fibrofolliculomas and pulmonary cysts. The responsible gene, FLCN is a tumor suppressor for kidney cancer which plays an important role for energy homeostasis through the regulation of mitochondrial oxidative metabolism. However, how FLCN-deficiency leads to renal tumorigenesis is unclear. Results Whole-exome sequencing analysis of BHD-associated kidney cancer revealed that copy number variations (CNV) of BHD-associated kidney cancer are considerably different from those already reported in sporadic cases. Somatic mutation analysis revealed BHD-associated kidney cancer has quite few common mutations in specific genes; however, chromatin remodeling genes in total were frequently mutated in BHD-associated kidney cancer (17/29 tumors, 59%). Metabolite analysis revealed that glycolysis, pentose phosphate pathway and lipid metabolism were upregulated whereas the rate of beta oxydation and TCA cycle is decreased in BHD-associated kidney cancer compared to normal adjacent kidney. Conclusions BHD-associated kidney cancer displays a unique molecular characteristics which is totally different from that of sporadic kidney cancer, providing the mechanistic insight into tumorigenesis under FLCN deficiency as well as the foundation for the development of novel therapeutics for kidney cancer.

  Study JGAS000115

• Genomic and Genetic Analysis of Brain Tumors and Analysis of Their Clinicopathological Significance

  The treatment outcome of glioma patients remains disappointing, and more studies are needed to improve therapeutic strategies. In particular, it is crucial to understand the mechanisms underlying resistance to treatment and malignant transformation, as well as the intratumoral heterogeneity and interaction of tumor cells and host immunity. Recent advances in omics analysis techniques, such as next-generation sequencing (NGS), have enabled us to obtain omics data more easily and accelerated our ability to integrate this information and precisely characterize cases. In our project, we have already collected more than 100 pairs of human glioma/normal tissue samples, of which some are multiple specimens collected from initial and recurrent tumors, and some include multiple specimens collected from different regions within a single tumor. We used our in-house algorithms, composed of alignment and variant calling programs, to analyze omics data from multiple platforms, such as whole-exome sequencing (WES), RNA sequencing (RNA-seq), and methylation array, with our original cohort of glioma patients. Using several informatics tools, the information about genetic and epigenetic status and expression signatures were analyzed. Those data are expected to provide key information for investigating the mechanisms discussed above. Through this project, we hope to improve strategies for personalized therapy based on comprehensive omics data.

  Study JGAS000004

• The impact of CCR8+ regulatory T cells on cytotoxic T cell function in human lung cancer

  Regulatory T cells (Tregs) suppress the host immune response and maintain immune homeostasis. Tregs also promote cancer progression and are involved in resistance to immune checkpoint inhibitor treatments. Recent studies identified selective CCR8 expression on tumor-infiltrating Tregs; CCR8+ Tregs have been indicated as a possible new target of cancer immunotherapy. Here, we investigated the features of CCR8+ Tregs in lung cancer patients. CCR8+ Tregs were highly activated and infiltration of CCR8+ Tregs in tumors was associated with poor prognosis in lung cancer patients. We also investigated their immune suppressive function, especially the influence on cytotoxic T lymphocyte cell function. The Cancer Genome Analysis revealed that CD8 T cell activities were suppressed in high CCR8-expressing tumors. Additionally, depletion of CCR8+ cells enhanced CD8 T cell function in an ex vivo culture of lung tumor-infiltrating cells. Moreover, CCR8+ Tregs, but not CCR8��� Tregs, induced from human PBMCs markedly suppressed CD8 T cell cytotoxicity. Finally, we demonstrated the therapeutic effect of targeting CCR8 in a murine model of lung cancer. These findings reveal the significance of CCR8+ Tregs for immunosuppression in lung cancer, especially via cytotoxic T lymphocyte cell suppression, and suggest the potential value of CCR8-targeted therapy for cancer treatment.

  Study JGAS000454

• Phylogenetic analysis of paired breast carcinomas identifies genetic events associated with clonal recurrence and invasive progression

  Development of ipsilateral breast carcinoma following diagnosis of breast ductal carcinoma in situ (DCIS) has been assumed to represent recurrence of the primary tumour. However, this may not be the case and it is important to know how often recurrences are new tumours. Ipsilateral primary-recurrence pairs (n=78) were sequenced to test their clonal relatedness. Shared genetic events were identified from whole exome sequencing (n=54 pairs) using haplotype-specific copy number and phylogenetic analysis. The remaining pairs were sequenced by a targeted panel or low-coverage whole genome sequencing. We included 32 non-recurrent DCIS to compare recurrent and non-recurrent disease. We found that 7% of DCIS recurrences were non-clonal by whole exome sequencing, indicative of a new breast carcinoma. Lower resolution methods detected a higher non-clonality rate (29%). Comparing primary DCIS with their recurrences found that evolution of DCIS to invasive disease was associated with increased ploidy and copy number events. TP53 mutations were enriched in DCIS with clonal recurrence compared with non-recurrent DCIS. Our results verify that de novo “recurrent tumours” of independent origin occur in patients who may be at high risk. (https://www.biorxiv.org/content/10.1101/2024.05.19.594731v2.full)

  Study EGAS50000001298

• Emergence of oncofetal plasticity is ubiquitous in early colorectal cancers

  Metastasis formation is classically considered a late-stage event in colorectal cancer (CRC) evolution. Yet, the time and patterning by which metastatic competence is acquired remain poorly understood. Here, we show that metastasis-associated oncofetal cell states already emerge at the earliest stages of CRC, concurrent with invasive front formation. However, while necessary for metastasis, we detect them ubiquitously among early non-metastatic cancers, highlighting additional bottlenecks like immune evasion. To understand how oncofetal cells first emerge, we generated multiregional organoid models that reflect successive tumor progression stages within individual early-stage CRCs. Whole-genome sequencing and growth factor-dependency assays exclude tumor cell-intrinsic acquired traits. In contrast, single-cell spatial atlases of the tumor-microenvironment before and after malignant transformation revealed stereotypic patterning of fibroblast subtypes resembling normal tissue architecture, resulting in distinct regional microenvironments. At the onset of malignant growth into the submucosa, the first cancer-associated fibroblasts (CAFs) to appear strongly resemble submucosal trophocytes and colocalize with oncofetal cell states at invasive fronts. Functionally, fibroblast-organoid co-cultures confirm that these trophocyte-like CAFs induce plastic transitioning to oncofetal states. Thus, interactions between tumor and submucosal fibroblasts directly following malignant transformation dictate the timing and location at which oncofetal plasticity first occurs during CRC progression.

  Study EGAS50000001532

• Assessing gene expression profiling from FFPE Patient Samples: A Comparison of Two Library Preparation Approaches and Recommendations

  Next-Generation Sequencing (NGS) has transformed cancer research and clinical practice, with Whole Exome Sequencing (WES) driving advances in mutational profiling and personalized oncology. Yet, transcriptomic signatures remain essential for understanding disease mechanisms, including therapy resistance pathways. RNA sequencing (RNA-seq), however, faces unique challenges when dealing with low-input or degraded RNA, as often found in archival formalin-fixed paraffin-embedded (FFPE) tissues. Although previous studies have compared library preparation protocols, rapidly evolving technologies call for updated evaluations. Here, we present a direct comparison of two FFPE-compatible stranded RNA-seq library preparation kits: TaKaRa™ SMARTer® Stranded Total RNA-Seq Kit v2 (Kit A) and Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus (Kit B). Both kits generated high-quality RNA-seq data, yet important differences emerged. Notably, Kit A achieved comparable gene expression quantification to Kit B while requiring 20-fold less RNA input, a crucial advantage for limited samples, albeit with increased sequencing depth. We critically discuss these results in relation to RNA availability, technical performance, cost-effectiveness, processing time, and automation potential, offering practical guidance for selecting optimal RNA-seq strategies in clinical and translational research settings

  Study EGAS50000001066

• Microbiomic and immunogenic biomarkers of adjuvant chemotherapy efficacy in stage III colorectal cancer

  The standard treatment for stage III colorectal cancer (CRC) is surgery, followed by adjuvant chemotherapy (AC). However, the efficacy of AC is limited, and approximately 30% of patients experience recurrence. Therefore, easily assessable molecular biomarkers that can predict response to AC are needed. Here, we aimed to identify predictive biomarkers of response to AC using tumor transcriptomic data. Tumor specimens from 253 patients who underwent surgery for stage III CRC between 1997 and 2019 were analyzed using RNA sequencing. Transcriptional subtyping and analyses of gut microbiome, immune cell fractions, and T-cell receptor (TCR) were performed to identify factors associated with recurrence-free survival (RFS) in patients treated with or without AC. Of 253 patients, 118 (46.6%) received AC. Twenty phyla and 799 species of the microbiomes were identified, among which Fusobacteria were identified in 247 patients (97.6%). Multivariable Cox proportional hazards regression analysis of patients who received AC showed that intratumoral Fusobacterium and M1 macrophage abundance and diversity of TCR alpha and delta (TRAD) were independently associated with RFS. Fusobacterium and M1 macrophage abundance, and TRAD diversity were associated with the response to AC, suggesting that these factors could serve as biomarkers for personalized treatment in stage III CRC.

  Study JGAS000875

• Shedding light over COVID-19 susceptibility and severity

  “One of the most remarkable features of SARS-CoV-2 infection is the variation in consequence ranging from asymptomatic to life-threatening” state the authors of the majestic Nature paper released in accelerated pre-view on July 2021. This variation in consequences represent not only an issue of burning medical relevance, but also a scientific and intellectual challenge for scientists around the globe. Researchers are known to be easily triggered by a challenge, and they recently demonstrated to be even better at double their efforts to focus on an urgent societal need. In this work led and coordinated by the COVID-19 Host Genetics Initiative (COVID-19 HGI), researchers gathered data from 49,562 cases and two million controls from 46 studies performed worldwide. The majority of the data has been deposited at the EGA, where 29 datasets have been collected under the study number EGAS00001005304. Data gathered from diverse sources conferred a huge robustness to the results. Genetic data from different ancestry origin were included in the analyses, even though data from patients with European origin was highly predominant (approx. 75%). Separated analysis of the different ancestries gave rise to relevant differences in the results, highlighting how important it is to take this aspect in consideration for future studies. Data from the individual studies was compiled into three categories according to COVID-19 severity for a meta-analysis: (a) critically ill cases (b) moderate or severe cases (c) all cases. They were paired with non-COVID-19-reported controls. All three analysis resulted in a total of 13 genetic loci associated with COVID-19 severity. Out of those, 9 loci associated specifically with critical illness, while 6 (a few overlapped) associated with moderated illness. Some genomic position matched previous findings. Interestingly, working with such an enormous amount of data allowed the depth to discriminate association with disease susceptibility vs severity, an issue of large medical relevance. Several loci mapped into biological relevant genes, like TYK2, PPP1R15A and FOXP4, previously implicated in immune response and/or lung functions. Researchers participating in the COVID-19 Host Genetics Initiative have worked in an unprecedented time-tight collaboration, prioritizing data sharing and collective results to tackle the pandemic affecting the world. The scientific community won’t miss any of the lessons to learn from this, and the EGA is ready to keep working to enable safe data sharing and fruitful collaborations.

  Blog covid-19-susceptibility-and-severity

• UK10K NEURO ABERDEEN

  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. This sample set comprises cases of schizophrenia with additional cognitive measurements, collected in Aberdeen, Scotland.For further information on the Aberdeen cohort please contact David St Clair (d.stclair@abdn.ac.uk).

  Study EGAS00001000109

• Single_Cell_Sequencing_of_Sperm__scSperm_

  The aim of this project is to genotype and sequence single spermatozoa from two men, one in his twenties and the other in his seventies. The resulting data is used to quantify the mutations that have arisen in the gametes of both individuals in order to better understand the effect of aging on mutation rates and modes.Project Outline. In order to quantify mutations, semen from two individuals are sequenced. 48 single sperm cells are isolated from each individual, and their DNA is extracted. The resulting genomes are amplified using PicoPlex, GenomiPhi MDA, Repli-G MDA, and MALBAC. QC step is applied to check the quality of WGA DNA using standard Sequenom plex (26 SNPs). A subset of 32 amplification products which pass the intiall QC, are genotyped using Affymetrix SNP6 chips. 12 of the genotyped amplification products are also sequenced. In addition, one multi-cell sample per individual is sequenced as a reference and for validation purposes.Altogether, 12 single cell sperm genomes and two multi-cell genomes are sequenced, coming to a total of 14 genomes. Of the single cell sperm genomes, 2 are sequenced to 50x coverage, and the other 10 to 25x coverage. Both multi-cell genomes are sequenced to 25x coverage.

  Study EGAS00001000935

• The Genomic Landscape of Core-Binding Factor Acute Myeloid Leukemias

  Pediatric de novo acute myeloid leukemia (AML) is a heterogeneous disease that can be divided into clinically distinct subtypes based on the presence of specific chromosomal abnormalities or gene alterations. One of the best characterized subtypes of AML involves leukemias with alterations of the core-binding factor (CBF)-complex, which comprises the FAB subtypes M2 and M4Eo and associates with a favorable outcome. Patients with the AML M2 subtype harbor a translocation between chromosomes 8 and 21 [t(8;21)] that yields the chimeric fusion gene RUNX1(AML1)-RUNX1T1(ETO), while patients with AML M4Eo express the chimeric fusion gene CBFβ-SMMHC(MYH11) as a result of an inversion/translocation event of chromosome 16 [inv(16)/t(16;16)]. In an effort to define the total complement of genetic changes in CBF-leukemia, we performed paired-end whole genome sequencing (WGS) on diagnostic leukemia blasts and matched germ line samples from 17 pediatric CBF-leukemia patients using the Illumina platform. Somatic alterations, including single nucleotide variations (SNVs) and structural variations (SVs), including insertions, deletions, inversions, and inter- and intra-chromosomal rearrangements, were detected using complementary analysis pipelines (Bambino, CREST and CONSERTING). Recurrent screening of identified mutations will be performed in a cohort of approximately 94 cases of CBF-leukemias.

  Study EGAS00001000349

• ERG ALTERATIONS DEFINE A NOVEL SUBTYPE OF ACUTE LYMPHOBLASTIC LEUKEMIA

  Structural chromosomal alterations are a hallmark of acute lymphoblastic leukemia (ALL) yet many patients lack an identifiable abnormality on conventional cytogenetic analysis. Here, using integrated analysis of genome-wide DNA copy number and gene expression data of 1764 childhood and adult ALL cases, including whole-genome, exome and mRNA-sequencing of 72 cases, we report a novel subtype of B-progenitor ALL (B-ALL) representing 4-13% of B-ALL cases characterized by a distinct gene expression profile, and focal deletions of ERG (ETS related gene) in 56% of cases. These cases are characterized by expression of a ERG transcript that utilizes a novel exon in intron 6 of ERG spliced to the canonical reading frame of ERG exons 7-10, encoding a 27 kDa C-terminal ERG protein that retains the ETS and transactivating domains but lacks the N-terminal pointed and regulatory domains. This protein is a competitive inhibitor of wild-type ERG and is leukemogenic in vivo. ERG-altered ALL cases have a favourable outcome despite the presence of alterations associated with poor prognosis in non-ERG ALL, such as deletions of IKZF1. These findings identify a new subtype of leukemia characterized by a novel mechanism of ETS transcription factor dysregulation in cancer.

  Study EGAS00001000514

• An RCOR1 loss-associated gene expression signature identifies a prognostically significant DLBCL subgroup

  Effective treatment of diffuse large B cell lymphoma (DLBCL) is plagued by heterogeneous responses to standard therapy, and molecular mechanisms underlying unfavorable outcomes in lymphoma patients remain elusive. Here, we profiled 148 genomes with 91 matching transcriptomes in an R-CHOP-treated DLBCL cohort to uncover molecular subgroups linked to treatment failure. Systematic integration of high-resolution genotyping arrays and RNA-seq data revealed novel deletions in RCOR1 to be associated with unfavorable progression free survival (P = 0.001). Integration of expression data from the clinical samples with data from RCOR1 knockdowns in the lymphoma cell lines KM-H2 and Raji yielded an RCOR1 loss-associated gene signature comprising of 233 genes. This signature identified a subgroup of patients with unfavorable overall survival (OS) (P = 0.023). The prognostic significance of the 233-gene signature for OS was reproduced in an independent cohort comprising of 195 R- CHOP treated patients (P = 0.039). Additionally, we discovered that within the IPI low risk group, the gene signature provides additional prognostic value that was independent of the cell-of-origin phenotype. Taken together, we present a novel and reproducible molecular subgroup of DLBCL, impacting risk-stratification of R- CHOP treated DLBCL patients and revealing a new avenue for therapeutic intervention strategies.

  Study EGAS00001001000

• Accurate sample assignment in a multiplexed, ultra-sensitive, high-throughput sequencing assay for minimal residual disease

  High throughput sequencing (HTS) (next generation sequencing) of the rearranged immunoglobulin and T-cell receptor genes promises to be cheaper and more sensitive than current methods for monitoring minimal residual disease (MRD) in patients with acute lymphoblastic leukemia. However, adoption of new approaches by clinical laboratories requires careful evaluation of all potential sources of error and the development of strategies to ensure the highest accuracy. Timely and efficient clinical use of HTS platforms will depend on combining multiple samples (multiplexing) in each sequencing run. Here we examine immunoglobulin heavy chain gene HTS on the Illumina MiSeq platform for MRD (HTS-MRD). We identify errors associated with multiplexing that could potentially impact on the accuracy of MRD analysis. We optimise a strategy combining high purity, sequence-optimised oligonucleotides, dual-indexing and an error-aware demultiplexing approach to minimise errors and maximise sensitivity. We present a probability-based demultiplexing pipeline, Error-Aware Demultiplexer (EAD) - that is suitable for all MiSeq sequencing strategies and accurately assigns samples to the correct identifier without excessive loss of data. Finally using controls quantified by digital PCR, we show that HTS-MRD can accurately detect as few as 1 in 106 copies of specific leukemic MRD.

  Study EGAS00001001303

• Rare SNPs in receptor tyrosine kinases are negative outcome predictors in multiple myeloma

  Multiple myeloma (MM) is a plasma cell disorder that is characterized by a great genetic heterogeneity. Recent next generation sequencing studies revealed an accumulation of tumor-associated mutations in receptor tyrosine kinases (RTKs) which may also contribute to the activation of survival pathways in MM. To investigate the clinical role of RTK-mutations in MM, we deep-sequenced the coding DNA-sequence of EGFR, EPHA2, ERBB3, IGF1R, NTRK1 and NTRK2 which were previously found to be mutated in MM, in 75 uniformly treated MM patients of the "Deutsche Studiengruppe Multiples Myelom". Subsequently, we correlated the detected mutations with common cytogenetic alterations and clinical parameters. We identified 11 novel non-synonymous SNVs or rare patient-specific SNPs, not listed in the SNP databases 1000 genomes and dbSNP, in 10 primary MM cases. The mutations predominantly affected the tyrosine-kinase and ligand-binding domains and no correlation with cytogenetic parameters was found. Interestingly, however, patients with RTK-mutations, specifically those with rare patient-specific SNPs, showed a significant lower overall, event-free and progression-free survival. This indicates that RTK SNVs and rare patient-specific RTK SNPs are of prognostic relevance and suggests that MM patients with RTK-mutations could potentially profit from treatment with RTK-inhibitors.

  Study EGAS00001001665

• Recurrent somatic JAK-STAT mutations within a novel RUNX1-mutated pedigree

  The acquisition of somatic mutations is an emerging field of investigation in familial leukemia. Currently, genetic profiles in familial MDS/AML are considered analogous to sporadic disease, although the patterns of clonal evolution within families are poorly defined. We performed whole exome profiling of tumour samples from a novel RUNX1 mutated family, to determine the stepwise evolution of MDS/AML across 4 young siblings. Three siblings developed monocytic AML/RAEB2 at 5 years of age, with hepatosplenomegaly and somatic mutations upregulating JAK-STAT signalling, the latter are typically detected in <5% of sporadic MDS/AML. Two siblings acquired the canonical JAK2 V617F mutation, while another acquired a unique missense mutation of SH2B3, a negative regulator of JAK2. Notably, 2/3 siblings demonstrated dosage amplification of these mutations due to acquired uniparental disomy of chromosomes 9p and 12q (encompassing JAK2 and SH2B3, respectively). All 4 siblings were heterozygous for the 46/1 JAK2 haplotype associated with predisposition to sporadic V617F myeloproliferative disorders, which likely influenced the acquisition of JAK2 mutations. Our findings provide further evidence that relatives with shared germline mutations may acquire somatic mutations in a non-random manner leading to convergent patterns of disease evolution.

  Study EGAS00001001862

• Whole genome sequencing of tumour and normal paired samples of diffuse intrinsic pontine gliomas

  Diffuse intrinsic pontine glioma (DIPG) have a universally dismal prognosis (median 9-12 months), with neither chemotherapeutic nor targeted agents showing any substantial survival benefit in clinical trials in children with these tumours. DIPG are highly infiltrative malignant glial neoplasms of the ventral pons, which due to their location within the brain, make them unsuitable for surgical resection and consequently have a universally dismal clinical outcome. Recent high-throughput sequencing approaches have revealed a striking prevalence of K27M mutations in the genes encoding the histone variants H3.3 (H3F3A) or H3.1 (HIST1H3B) in the childhood brain tumour DIPG. This K-to-M substitution confers a trans-dominant ablation of global H3K27 trimethylation, which likely profoundly alters gene expression through de-repression of polycomb repressive complex 2 (PRC2) target genes. Despite these advances in our understanding of the distinct biology of these tumours, approaches for specific novel therapeutic interventions are not clear, and little has been reported of the secondary mutations accompanying these changes. We carried out whole genome sequencing on a series of 20 tumour normal pairs of pre-treatment biopsy samples of DIPG, for which the patients underwent a safe stereotactic procedure, and whole exome sequencing on a further six tumour normal pairs obtained at autopsy.

  Study EGAS00001000572

• Pediatric Non-Down Syndrome Acute Megakaryoblastic Leukemia is Characterized by Distinct Genomic Subsets with Varying Outcomes

  Acute Megakaryoblastic Leukemia (AMKL) is a subtype of acute myeloid leukemia (AML) in which cells morphologically resemble abnormal megakaryoblasts.  While extremely rare in adults, AMKL accounts for 4-15% of newly diagnosed childhood AML1-3.  A significant proportion of pediatric AMKL cases occur in children with Down syndrome (DS).  These patients have excellent outcomes and are characterized at the genomic level by a founding GATA1 mutation4-6.  In contrast, AMKL in patients without DS (non-DS-AMKL) is frequently associated with poor outcomes.  Previous efforts have identified chimeric oncogenes in a significant number of cases, including RBM15-MKL1, CBFA2T3-GLIS2, KMT2A gene rearrangements and NUP98-KDM5A7-9.  The etiology of 30-40% of cases, however, remains unknown.  To better understand the genomic landscape of non-DS-AMKL, we performed RNA and exome sequencing on specimens from 99 patients (75 pediatric and 24 adult).  We demonstrate that pediatric non-DS-AMKL is a heterogeneous malignancy that can be divided into seven subgroups with varying outcomes.  These subgroups are characterized by chimeric oncogenes with cooperating mutations in epigenetic and kinase signaling genes.  Overall, these data shed light on the etiology of AMKL and provide useful information for treatment tailoring. 

  Study EGAS00001002183

• Whole-exome and targeted sequencing of pediatric hyperdiploid B-cell precursor acute lymphoblastic leukemia

  High hyperdiploidy defines the largest genetic entity of childhood acute lymphoblastic leukemia (ALL). Despite its relatively low recurrence risk, this subgroup generates a high proportion of relapses. The cause and origin of these relapses remains obscure. We therefore explored the mutational landscape in high hyperdiploid (HD) ALL with whole-exome (n=19) and subsequent targeted deep sequencing of 60 genes in 100 relapsing and 51 non-relapsing cases. We identified multiple clones at diagnosis that were primarily defined by a variety of mutations in receptor tyrosine kinase (RTK)/Ras pathway and chromatin-modifying genes. The relapse clones consisted of reappearing as well as new mutations, and overall contained more mutations. Although RTK/Ras pathway mutations were similarly frequent between diagnosis and relapse, both intergenic and intragenic heterogeneity was essentially lost at relapse. CREBBP mutations, however, increased from initially 18-30% at relapse, then commonly co-occurred with KRAS mutations (P<0.001) and these relapses appeared primarily early (P=0.012). Our results confirm the exceptional susceptibility of HD ALL to RTK/Ras pathway and CREBBP mutations, but, more importantly, suggest that mutant KRAS and CREBBP might cooperate and equip cells with the necessary capacity to evolve into a relapse-generating clone.

  Study EGAS00001001113

• Ethiopia_Genome_Project__high_coverage_

  The Ethiopian area stands among the most ancient ones ever occupied by human populations and their ancestors. Particularly, according to archaeological evidences, it is possible to trace back the presence of Hominids up to at least 3 million years ago. Furthermore, the present day human populations show a great cultural, linguistic and historic diversity which makes them essential candidate to investigate a considerable part of the African variability. Following the typing of 300 Ethiopian samples on Illumina Omni 1M (see Human Variability in Ethiopia project, previously approved by the Genotyping committee) we now have a clearer idea on which populations living in the area include the most of the diversity.This project therefore aims to sequence the whole genome of 300 individuals at low (4-8x) depth belonging to the six most representative populations of the Ethiopian area to produce a unique catalogue of variants peculiar of the North East Africa. Furthermore 6 samples (one from each population) will also be sequenced at high (30x) depth to ensure full coverage of the diversity spectrum.The retrieved variants will be of great help in evaluating the demographic dynamics of those populations as well as shedding light on the migrations out of Africa.

  Study EGAS00001000237

• Whole-Genome sequencing of hepatocellular carcinomas

  The French ICGC project on liver tumors is coordinated by Pr Jessica Zucman-Rossi and funded by Inca (French Institute for Cancer). The aim of the present project is to identify the catalog of somatic and germline mutations in liver tumors using whole genome and whole exome sequencing together with CGH-SNP, methylome and transcriptomic profiling. For this purpose, a series of 500 liver tumors are collected through the French National Liver Collection and these samples will be analyzed using the different omics technologies. The data will be deposited in the ICGC and EGA database to be publically available for the scientific community. Hepatocellular carcinoma (HCC) accounts for more than 90% of liver cancers, and is a major health problem. It is the 3rd cause of cancer-related mortality. Advances in genomic analyses have formed a comprehensive understanding of different underlying pathobiological layers resulting in hepatocarcinogenesis. Thus, the development of next-generation sequencing technologies has made it possible to generate more comprehensive catalogues of somatic alteration events (single nucleotide substitutions, structural variations, and epigenetic changes) in liver cancer genome than ever before. The dataset will include 50 whole genome sequencing tumor/germline pairs, of which 6 are deposited in February 2014.

  Study EGAS00001000706

• UK10K_RARE_NEUROMUSCULAR

  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 neuromuscular disorder samples are part of the “rare disease” group, and will undergo exome sequencing. For further information with regard to this cohort please contact Francesco Muntoni (f.muntoni@ucl.ac.uk).

  Study EGAS00001000101

• UK10K_RARE_FIND

  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 Raymond samples will be part of the “rare disease” group, and will undergo exome sequencing. For further information with regard to this cohort please contact Lucy Raymond (flr24@cam.ac.uk).

  Study EGAS00001000128

• UK10K_RARE_CILIOPATHIES

  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 Ciliopathies samples will be part of the rare disease group, and will undergo exome sequencing. For further information with regard to this cohort please contact Phil Beales (p.beales@ich.ucl.ac.uk).

  Study EGAS00001000126

• UK10K_RARE_SIR

  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 SIR (Severe Insulin Resistance) samples are part of the “rare disease” group, and will undergo exome sequencing. For further information with regard to this cohort please contact Robert Semple (rks16@cam.ac.uk).

  Study EGAS00001000130

• UK10K RARE CHD

  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 CHD (congenital heart disease) samples will be part of the “rare disease” group, and will undergo exome sequencing.For further information with regard to this cohort please contact Shoumo Bhattacharya (shoumo@me.com).

  Study EGAS00001000125

• Genomic Alterations in Gingivo-buccal Cancer: ICGC-India Project_YR02

  As a part of the ICGC, India has undertaken genomic studies on gingivobuccal cancer of the oral cavity, which is the most prevalent form of cancer among men in India. There are various known environmental (life-style) correlates of this cancer, the most important of which are tobacco chewing and HPV infection. Paired DNA samples – isolated from the tumour tissue and from the blood of fifty six patients – have been analyzed to catalog germline and somatic mutations. Association between each observed genomic alteration and exposure to environmental risk factors is being explored.Detailed clinical characterization of the patients, collection of data on demographic and environmental exposures, and isolation of DNA samples from blood and tumour tissues collected from each patient are being done at the Advanced Centre for Research, Treatment and Education on Cancer, Mumbai. Exome capture and deep resequencing are being performed at the National Institute of Biomedical Genomics, Kalyani. We have analyzed the exomes of paired blood and tumour DNA samples using exome capture by Illumina TrueSeq Exome Enrichment kit followed by sequencing on Illumina HiSeq-2000. Each exome is being sequenced at a mean depth of 70x for Tumour and 40x for Blood. In addition, we have used Illumina Omni 2.5 SNP-chips to generate genotype data.

  Study EGAS00001001028

• P647_Targeted_resequencing_project

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

  Study EGAS00001000305

• A somatic reference standard for cancer genome sequencing with COLO829

  Identification of somatic alterations in cancer has become feasible with the massive adoption of next generation sequencing. However, due to variability in sequencing and informatics pipelines, a common somatic reference is needed. We thus performed paired whole genome sequencing of a metastatic melanoma cell line (COLO829) and a matched lymphoblastoid line (COLO829BL) across three institutions (TGen, Illumina, Genome Sciences Centre at the British Columbia Cancer Agency). We performed a meta-analysis of all data, in combination with the originally reported analysis of these cell lines by Pleasance et al (PMID: 20016485), and report a somatic reference standard based on consensus events. DNA extractions, library preparation, sequencing, and analysis were separately performed at each site. Results were compiled with sequence data previously generated for the cell lines to identify true positive variants, collectively representing a somatic reference standard. Overall, common somatic events include point mutations, small insertion/deletions, and genes affected by concurrent copy number changes. We additionally show divergence of somatic mutations in COLO829 associated with differences in cell line lineage. We present this reference to the community as a standard for enabling interpretation and identification of somatic mutations across institutions and analytical pipelines.

  Study EGAS00001001385

• Whole exome sequencing on primary retinoblastoma tissues and matching lymphocyte DNA.

  Retinoblastoma is a rare childhood cancer initiated by RB1 mutation or MYCN amplification, while additional alterations may be required for tumor development. However, the view on single nucleotide variants is very limited. To better understand oncogenesis, we determined the genomic landscape of retinoblastoma. We performed exome sequencing of 71 retinoblastomas and matched blood DNA. Next, we determined the presence of single nucleotide variants, copy number alterations and viruses. Aside from RB1, recurrent gene mutations were very rare. Only a limited fraction of tumors showed BCOR (7/71, 10%) or CREBBP alterations (3/71, 4%). No evidence was found for the presence of viruses. Instead, specific somatic copy number alterations were more common, particularly in patients diagnosed at later age. Recurrent alterations of chromosomal arms often involved less than one copy, also in highly pure tumor samples, suggesting within-tumor heterogeneity. Our results show that retinoblastoma is among the least mutated cancers and signify the extreme sensitivity of the childhood retina for RB1 loss. We hypothesize that retinoblastomas arising later in retinal development benefit more from subclonal secondary alterations and therefore, these alterations are more selected for in these tumors. Targeted therapy based on these subclonal events might be insufficient for complete tumor control.

  Study EGAS00001001690

• Ethiopia_Genome_Project__low_coverage_

  The Ethiopian area stands among the most ancient ones ever occupied by human populations and their ancestors. Particularly, according to archaeological evidences, it is possible to trace back the presence of Hominids up to at least 3 million years ago. Furthermore, the present day human populations show a great cultural, linguistic and historic diversity which makes them essential candidate to investigate a considerable part of the African variability. Following the typing of 300 Ethiopian samples on Illumina Omni 1M (see Human Variability in Ethiopia project, previously approved by the Genotyping committee) we now have a clearer idea on which populations living in the area include the most of the diversity.This project therefore aims to sequence the whole genome of 300 individuals at low (4-8x) depth belonging to the six most representative populations of the Ethiopian area to produce a unique catalogue of variants peculiar of the North East Africa. Furthermore 6 samples (one from each population) will also be sequenced at high (30x) depth to ensure full coverage of the diversity spectrum.The retrieved variants will be of great help in evaluating the demographic dynamics of those populations as well as shedding light on the migrations out of Africa.

  Study EGAS00001000238

• UK10K OBESITY TWINSUK

  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. These TwinsUK samples are a subset of the TwinsUK cohort samples with a BMI>=40 that will be exome sequenced by the Obesity group.

  Study EGAS00001000306

• PSCP_bisulphite analysis in hESCs (2018-08-13)

  Many studies over the past 10 years, culminating in the recent report of the International Stem Cell Initiative (ISCI, 2011) have shown that hPSC acquire genetic and epigenetic changes during their time in culture. Many of the genetic changes are non-random and recurrent, probably because they provide a selective growth advantage to the undifferentiated cells. Some are shared by embryonal carcinoma cells, the malignant counterparts of ES cells. The origins of these growth advantages are poorly understood, but may come from altered cell cycle dynamics, resistance to apoptosis or altered patterns of differentiation. Less is known about the nature and consequences of epigenetic changes, but it is likely that these similarly affect hPSC behaviour; e.g., enhanced expression of DLK1, an imprinted gene, is associated with altered hPSC growth (Enver et al 2005). Inevitably, these genetic and epigenetic changes will impact on our ability to use hPSC for regenerative medicine, either because malignant transformation of the undifferentiated cells or their differentiated derivatives to be used for transplantation compromises safety, or because they impede the function of those differentiated derivatives, or because they affect the efficiency with which the undifferentiated cells can be expanded and differentiated into desired cell types. Focusing initially upon the existing clinical grade hESC lines, later moving to iPSC, we will Consolidate and extend knowledge of the rate, type and functional impact of the genetic variations that occur during hPSC culture. We will use whole genome and exome sequencing as well as SNP arrays, together with clonal analysis and other cytogenetics techniques. Common changes will be compared with those found in the normal human population, at low frequency in the original cell population or observed during iPSC generation in the HIPSCI project currently based at the WTSI. These studies will provide a better understanding of the range of genetic changes that occur in hPSC beyond the CNVs already identified. In conjunction with cancer genome resources and expertise at WTSI, bioinformatic analyses of these hPSC data will allow us to assess potential impact on hPSC behaviour pertinent to applications in regenerative medicine, notably the likelihood that specific changes arising in undifferentiated PSC cultures may be associated with potential malignant transformation of differentiated progeny. 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/ . This dataset contains all the data available for this study on 2018-08-13.

  Dataset EGAD00001004295

• Mutational landscape of normal epithelial cells in Lynch Syndrome patients

  Lynch Syndrome (LS) is an autosomal dominant disease conferring a high risk of colorectal cancer due to germline heterozygous mutations in a DNA mismatch repair (MMR) gene. Although cancers in LS patients show elevated somatic mutation burdens, information on mutation rates in normal tissues and understanding of the trajectory from normal to cancer cell is limited. Here we whole-genome sequenced 152 crypts from normal and neoplastic epithelial tissues from LS patients. In normal tissues the repertoire of mutational processes and mutation rates were similar to those found in wild type individuals. A morphologically normal colonic crypt with an increased mutation burden and mutational signatures consistent with MMR deficiency was identified, which may represent a very early stage of LS pathogenesis. Phylogenetic tress of tumour crypts indicated that the most recent ancestor cell of each tumour was already MMR deficient and had experienced multiple clonal evolution cycles. This study demonstrates the genomic stability of epithelial cells with heterozygous germline MMR gene mutations and highlights important differences in the pathogenesis of LS from other colorectal cancer predisposition syndromes.

  Dataset EGAD00001008092

• Whole Exome Sequencing PPGL

  Whole exome sequencing (WES) libraries were prepared with the SureSelectXT Human All Exon V6+COSMIC target enrichment system (Agilent, 5190-9307) by following kit manufacturer`s instructions. 200 ng of genomic DNA from FFPE tumoral samples and 1 µg from FF or germline samples were used as starting material. SureSelectXT HS (Agilent, G9704A) and SureSelectXT2 (Agilent, G9621A) reagent kits were alternatively used for FFPE derived and good quality samples, respectively. DNAs were fragmented on a Covaris S2 sharing instrument based on initial sample’s integrity. Fragmented DNA samples were processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to Illumina's adapters. Adapter-ligated libraries were PCR amplified with Illumina PE primers and subsequently hybridized to SureSelect Oligo Capture Library Mix. Biotinylated hybrids were captured and the enriched fraction eluted. Libraries were completed by limited-cycle PCR with Illumina TruSeq primers and KAPA HiFi HotStart DNA pol (Roche KK2501). Sample-specific libraries were combined into equimolarly balanced pools and applied to Illumina flow cells for cluster generation. Sequencing was performed on either the Illumina HiSeq2500 or NovaSeq6000 in a paired-end 100bp reads mode to a median target coverage of ~200x and ~100x for tumors and germline, respectively.

  Study EGAS00001006043

• The Spatial Heterogeneity in Multiple Myeloma - from the Subclonal Architecture to the Immune Microenvironment (partly hipo_K08K)

  Multiple myeloma (MM) cells show pronounced heterogeneity not only within a given patient but also between spatially separated tumor loci in the bone marrow. Understanding this spatial heterogeneity is emerging as a critical challenge for the successful treatment of the disease. Yet, our understanding of spatial differences in the subclonal architecture, molecular signatures and interactions with the tumor microenvironment remains very limited. To address this shortcoming, we performed bulk and single-cell multi-region sequencing, including random bone marrow samples from the iliac crest and paired imaging-guided focal lesion specimens from 15 newly diagnosed MM patients. We found a median of 5 subclones per patient and unique subclones in focal lesions. Central features of spatial heterogeneity included a consistent down-regulation of the chemoattractant cytokines CXCL7 and CXCL12 in focal lesion tumor cells as well as a significant depletion of macrophages in the focal lesion microenvironment. In contrast, a site-specific expansion of T-cell clones was not detected in focal lesions. In conclusion, our results demonstrate the relevance of considering spatial heterogeneity with potential implications for immunotherapies and molecular studies analyzing the role of gene signatures and MM-microenvironment interactions in disease progression.

  Study EGAS00001006090

• Pancreatic, Small-intestinal and Pulmonary Neuroendocrine Tumors

  Purpose: The primary origin of neuroendocrine tumor metastases can be difficult to determine by histopathology alone, but is critical for therapeutic decision making. DNA methylation-based profiling is now routinely used in the diagnostic workup of brain tumors. This has been enabled by the availability of cost-efficient array-based platforms. We have extended these efforts to augment histopathological diagnosis in neuroendocrine tumors. Experimental Design and Results: We compiled data of 69 small-intestinal, pulmonary, and pancreatic neuroendocrine tumors. These data were used to build a ridge regression calibrated random forest classification algorithm (NEN-ID) that predicts the origin of tumor samples with high accuracy (> 95%). The model was validated during 3x3 nested cross validation and tested in a local (n=26) and external (n=172) cohort. In addition, we show that our diagnostic approach is robust across a range of possible confounding experimental parameters such as tumor purity and array quality. A software infrastructure and online user interface was built to make the model available to the scientific community. Conclusions: This DNA methylation-based prediction model can be used in the workup for patients with neuroendocrine tumors of unknown primary. To facilitate validation and clinical implementation, we provide a user-friendly, publicly available web-based version of NEN-ID.

  Study EGAS00001004878

• Sclerosing epithelioid fibrosarcoma case report

  Sclerosing epithelioid fibrosarcoma (SEF) is a rare and aggressive soft tissue sarcomathought to originate in fibroblasts of the tissues surrounding tendons, ligaments and muscles. Minimally responsive to conventional cytotoxic chemotherapies, greater than 50% of SEF patients experience relapse and/or metastatic disease. SEF is most commonly discovered in middle-aged and elderly adults, but also rarely in children. A common gene fusion occurring between the EWSR1 and CREB3L1 genes has been observed in 80-90% of SEF cases. We describe here the youngest SEF patient reported to date (a 3-year-old Caucasian male) who presented with numerous bony and lung metastases. Additionally, we perform a comprehensive literature review of all SEF-related articles published since the disease was first characterized. The patient described in this report experienced persistent disease progression despite being heavily treated with multiple surgeries, radiation, numerous chemotherapies and targeted therapeutics. Primary tumor, metastatic and relapse sites were sequenced by whole genome, whole exome, and deep transcriptome next generation sequencing with comparison to a patient-matched normal blood sample. Consistent across all sequencing analyses was the disease-defining EWSR1-CREB3L1 fusion as a single feature consensus. We provide an analysis of our genomic findings and a discussion as to potential therapeutic strategies for SEF.

  Study EGAS00001005214

• Pancreatic tropism of metastatic renal cell carcinoma

  Renal cell carcinoma (RCC) is characterized by a particularly broad metastatic swath, and enigmatically, when the pancreas is a destination, the disease is associated with improved survival. Intrigued by this observation, we sought to characterize the clinical behavior, therapeutic implications, and underlying biology. While pancreatic metastases (PM) are infrequent, we identified 31 patients across two institutional cohorts and show that improved survival is independent of established prognostic variables, that these tumors are exquisitely sensitive to anti-angiogenic agents and resistant to immune checkpoint inhibitors (ICI), and that they are characterized by a distinctive biology. Primary tumors of patients with PM exhibited frequent PBRM1 mutations, 3p loss, and 5q amplification, along with a lower frequency of aggressive features such as BAP1 mutations and loss of 9p, 14q, and 4q. Gene expression analyses revealed constrained evolution with remarkable uniformity, reduced T-effector gene signatures and increased angiogenesis. Similar findings were observed histopathologically. Thus, RCC metastatic to the pancreas is characterized by indolent biology, heightened angiogenesis, and an uninflamed stroma, likely underlying its good prognosis, sensitivity to anti-angiogenic therapies, and refractoriness to ICI. These data suggest that metastatic organotropism may be an indicator of a particular biology with prognostic and treatment implications for patients.

  Study EGAS00001004208

• CancerLocator: Non-Invasive Cancer Diagnosis and Tissue-of-Origin Prediction Using Methylation Profiles of Cell-Free DNA

  Background: The detection and characterization of cell-free DNA in plasma is one of the most promising new areas in cancer diagnosis. Liquid biopsy, unlike traditional tissue biopsy, has the potential to diagnose a variety of different malignancies. Results: Here we propose a probabilistic method, CancerLocator, which exploits the diagnostic potential of cell-free DNA by determining not only the presence but also the location of tumors. CancerLocator simultaneously infers the proportions and the tissue-of-origin of tumor-derived cell-free DNA in a blood sample using genome-wide DNA methylation data. We comprehensively evaluate CancerLocator with simulations and real data, and compare its performance with that of two established multi-class classification methods. We show that the predicted tumor burdens are highly consistent with the true values. In addition, when the proportion of tumor-derived DNAs in the cell-free DNAs is low, the two popular machine learning methods completely fail for cancer diagnosis, while CancerLocator successfully overcomes the challenge. CancerLocator also achieves promising results on patient plasma samples, despite the fact that the DNA methylation data from these samples has very low sequencing coverage. Conclusions: CfDNA methylation may be developed as an important approach for non-invasive early cancer diagnosis.

  Study EGAS00001002211

• Direct Detection of Early-stage Cancers Using Circulating Tumor DNA

  Early detection and intervention are likely to be the most effective means for reducing morbidity and mortality of human cancer. However, development of methods for noninvasive detection of early-stage tumors has remained a challenge. We have developed an approach called targeted error correction sequencing (TEC-Seq) that allows ultrasensitive direct evaluation of sequence changes in circulating cell-free DNA using massively parallel sequencing. We have used this approach to examine 58 cancer-related genes encompassing 81 kb. Analysis of plasma from 44 healthy individuals identified genomic changes related to clonal hematopoiesis in 16% of asymptomatic individuals but no alterations in driver genes related to solid cancers. Evaluation of 200 patients with colorectal, breast, lung, or ovarian cancer detected somatic mutations in the plasma of 71, 59, 59, and 68%, respectively, of patients with stage I or II disease. Analyses of mutations in the circulation revealed high concordance with alterations in the tumors of these patients. In patients with resectable colorectal cancers, higher amounts of preoperative circulating tumor DNA were associated with disease recurrence and decreased overall survival. These analyses provide a broadly applicable approach for noninvasive detection of early-stage tumors that may be useful for screening and management of patients with cancer.

  Study EGAS00001002577