3883 results for "RNA AND sequencing OR transcriptome"

in 26.39 milliseconds.

• Using Genomics to Reduce Breast Cancer Disparities in the African Diaspora

  Differences in breast cancer incidence and mortality rates between North American Caucasian and African American women are well-described and transcend socioeconomic issues. Black women are diagnosed with breast cancer at a younger median age; have more clinically aggressive disease and stage-for-stage; and have higher mortality rates than age-matched Caucasian women. Black women in West Africa, the origin of the slave trade in the US in the 19th century and thus the founder population for most African Americans, have even higher rates of early-onset, poor-prognosis breast cancer than African American women. Racial difference in the distribution of intrinsic molecular subtypes has been well characterized in the US and throughout the African Diaspora as well. Despite the large efforts on characterizing racial/ethnic differences, however, the reasons women of African ancestry are disproportionately affected by breast cancer incidence and mortality remain poorly understood - largely due to paucity of data on inherent genomic differences that contribute to the disparities in incidence and progression of breast cancer across populations. West Africa Breast Cancer Study (WABCS) is an initiative that aims to comprehensively understand the genetic architecture of breast cancer in West Africans, the founder population of a large proportion of black women in the United States. The objective of the study was to provide a better understanding of the molecular genetic factors that influence prognosis in Nigerian breast cancer patients, and determine which of these alterations may be amenable to available therapy. To that end, we examined the molecular features of breast cancers of indigenous African women using a combination of whole-genome, whole-exome, and transcriptome sequencing (WGS, WES, and RNA-seq) on 194 tumors from Nigerian patients. The goal of this project was to obtain answers to two related research questions using an unscreened population without genetic admixture in Nigeria: 1) why are women of African ancestry more likely to develop aggressive young onset breast cancer? 2) What are the associated genomic and non-genomic risk factors? We hypothesize that the genomic determinants of breast cancer molecular subtypes in women of African ancestry are also molecular drivers of tumor progression and represent targets for interventions to improve clinical outcomes and close the mortality gap. By identifying causal links between genetic variants that promote aggressive tumor progression in Nigerian women in comparison to women from different population found in TCGA and ICGC, the present dataset will have significant public health impact on millions of women in the African Diaspora. The potential to identify novel pathways for interventions to reduce the increasing mortality gap between women of African and European ancestry is huge

  Study phs001687

• Oropharynx cancers sequencing study (RNA-seq from FFPE)

  This is a study of transcriptomics in HPV-associated oropharynx tumors. The goals of this study were to characterize HPV+ oropharynx tumor subtypes and to examine HPV integration events and their downstream effects. HPV-host fusion transcripts and recurrent HPV integration genes were identified. Various alternative isoforms and splicing events of HPV genes were also studied. Multiple potential biomarkers of prognosis were associated with clinical outcomes.

  Study EGAS50000000893

• scRNA-seq and snRNA-seq of trophoblast stem cells (TSCs) differentiation into extravillous trophoblast organoids (EVTs)

  This dataset is part of a study that aims to compare in vivo human trophoblast differentiation into EVTs to different in vitro trophoblast organoids using single-cell and single-nuclei RNA sequencing. This specific dataset includes scRNA-seq and snRNA-seq data from trophoblast stem cells (TSCs). Trophoblast stem cell (TSC) lines BTS5 and BTS11 derived by Okae and colleagues were grown as described previously (Okae et al. 2018) together with EVT differentiation media. This study shows that the main regulatory programs mediating EVT invasion in vivo are preserved in in vitro models of EVT differentiation from primary trophoblast organoids and trophoblast stem cells. Data for primary trophoblast organoids is available under E-MTAB-12650.

  Dataset EGAD00001010017

• Single-cell Transcriptomic and TCR Repertoire Profiling of DENV-specific CD8+ T Cells Across Dengue Disease Severities

  This dataset contains single-cell RNA sequencing (Smart-seq2) and paired TCR repertoire data of sorted dengue virus (DENV)-specific CD8⁺ T cells from individuals with differing clinical outcomes: asymptomatic dengue (AD), dengue fever (DF), and dengue hemorrhagic fever (DHF). Samples were obtained from HLA-A11⁺ and HLA-A24⁺ individuals during acute and convalescent phases, with antigen-specific CD8⁺ T cells isolated using HLA tetramers loaded with immunodominant DENV epitopes. The study identifies phenotypically distinct T cell subsets associated with disease severity, TCR avidity, and antigen reactivity to current or prior DENV serotypes. The dataset supports the understanding of CD8⁺ T cell heterogeneity, TCR features, and potential mechanisms of CD8⁺ T cell-mediated protection versus immunopathogenesis in dengue infection.

  Dataset EGAD00001015637

• Exploring the Microbiome-Gut-Brain Axis with Respect to Psychoneurological Symptoms for Children with Solid Tumors

  The human body hosts tens of trillions of microbes, a number 10 times greater than the number of human body cells. The collection of these human-associated microbes and their genomes is called the “human microbiome”. More than 90% of these microbes live in the gastrointestinal (GI) tract, representing 500 species on average. With the development of high-throughput sequencing technology and bioinformatics tools (e.g., 16S ribosomal RNA or 16S rRNA sequencing and shotgun metagenomics analysis), recent progress has been made to elaborate the critical role that the gut microbiome plays in human health and disease. A significant disruption (dysbiosis) of the composition and function of the gut microbiome is associated with carcinogenesis, chemotherapeutic metabolism, and treatment-related symptom toxicities. The current state of science on the gut microbiome in cancer has only recently started in animal models and adults, and has not been well investigated in children diagnosed and treated for cancer, nor with respect to associations with cancer treatment-related toxicities. This proposed study will explore the hypothesis that dysbiosis of the gut microbiome is associated with cancer treatment-related GI and psychoneurological symptoms. We will collect both stool samples (for the gut microbiome) and patient-reported outcomes across chemotherapy.

  Study phs002960

• Patient-derived pediatric brain tumor organoids faithfully recapitulate primary tumors

  Extensive molecular analysis by many groups has revealed a heterogeneous landscape of embryonal brain tumors, including medulloblastoma (MB) and atypical teratoid/rhabdoid tumor (ATRT). MB, the most common malignant pediatric brain tumor, is comprised of four molecularly distinct subgroups: Wingless (WNT), Sonic Hedgehog (SHH), Group 3 (G3), and Group 4 (G4). Among these, MB-SHH harboring MYCN amplification and TP53 mutation and MB-G3 with MYC overexpression or amplification constitute the most aggressive tumors, have the poorest clinical outcomes, and lack effective therapeutic options for recurrent disease. Here we describe the establishment of tumor organoid (TO) models from patient-derived orthotopic xenografts (PDOX) of MYCN-amplified and TP53-mutant MB-SHH, MYC-amplified MB-G3 and two ATRT which all grew in mice when reimplanted into the cortices of naïve recipient animals (TOX). Methylation, bulk and single-cell RNA sequencing, and whole genome sequencing demonstrated that TOs and TOXs faithfully recapitulated the molecular landscape and heterogeneity of PDOXs and primary patient tumors compared to established cell lines. The development of TOs will facilitate in vitro high-throughput drug or CRISPR screens without the need for use tumor-bearing mice. This will also accelerate the identification and validation of new vulnerabilities and in vivo preclinical testing toward clinical trials.

  Study EGAS00001008305

• Ewing and Ewing-like-sarcoma tumoroid biobank reveals distinct drug sensitivities: translocation makes the difference

  Ewing sarcoma (ES) and Ewing-like sarcomas (ELS) are highly aggressive tumors found in soft tissues and bone of mostly children and young adults. Despite being different in many aspects, including genetics, possible cell-of-origin, and pathology, patients with any of these entities all receive the same therapeutic regimen. Currently, there is a lack of preclinical cell models that can predict drug response of these entities in vitro. Here, we describe a pediatric ES and ELS tumor organoid (tumoroid) biobank containing tumoroids with different translocations, including EWSR1::FLI1, EWSR1::ERG, CIC::DUX4, and BCOR-rearrangements. Using histology, whole genome sequencing and RNA sequencing, we demonstrate that these tumoroids retain key properties of their matching patient tumors. In addition, we traced tumor subclones in the tumoroids across patient-matched longitudinal samples, which showed that cellular heterogeneity was maintained. We subsequently performed drug screening on the tumoroid models, which unveiled entity-specific drug sensitivity to various cytotoxic compounds and targeted compounds, including MCL-1 inhibitors for CIC::DUX4 sarcomas. Taken together, this newly established ES and ELS patient-derived tumoroid biobank generates a unique source of material for future basic cancer research and drug-screening.

  Study EGAS00001007941

• Multi-omics integration reveals only minor long-term molecular and functional sequelae in immune cells of individuals recovered from COVID-19

  The majority of COVID-19 patients experience mild to moderate disease course and recover within a few weeks. An increasing number of studies characterized the long-term changes in the specific anti-SARS-CoV-2 immune responses, but how COVID-19 shapes the innate and heterologous adaptive immune system after recovery is less well known. To comprehensively investigate the post-SARS-CoV-2 infection sequelae on the immune system, we performed a multi-omics study by integrating single-cell RNA-sequencing, single-cell ATAC-sequencing, genome-wide DNA methylation profiling, and functional validation experiments in convalescent COVID-19 and healthy individuals. We showed that immune responses generally recover without major sequelae after COVID-19. However, subtle differences persist at the transcriptomic level in monocytes, with downregulation of the interferon pathway, while DNA methylation also displays moderate changes in convalescent COVID-19 individuals. However, these differences did not affect the cytokine production capacity of PBMCs upon different bacterial, viral, and fungal stimuli, although baseline release of IL-1RA and IFN?? was higher in convalescent individuals. In conclusion, despite minor differences in epigenetic and transcriptional programs, the immune system of convalescent COVID-19 patients largely recovers to the homeostatic level of healthy individuals.

  Study EGAS00001005529

• Genomic Analysis of Fibrolamellar Hepatocellular Carcinoma

  Fibrolamellar hepatocellular carcinoma (FL-HCC) is a very rare and distinct subtype of hepatocellular carcinoma that occurs in the previously healthy livers of adolescents and young adults. The low incidence rate has kept FL-HCC an understudied cancer, thus the list of FL-HCC markers is limited and the genetic alterations driving its growth have remained unknown. In order to gain a better understanding of the molecular mechanisms underlying FL-HCC initiation and progression, we performed an in-depth genomic analyses of one tumor followed by immunohistochemistry validation on seven other tumors. We showed the expression of neuroendocrine markers in FL-HCC. In addition, DNA/RNA sequencing revealed that common cancer pathways are not visibly altered in FL-HCC but identified two novel structural variants, both resulting in fusion transcripts. Through in vitro studies, we demonstrated the oncogenic properties of these fusion transcripts. These experiments further highlight the tumorigenic role of gene fusions in the etiology of pediatric solid tumors and identify both candidate biomarkers and possible therapeutic targets for this lethal pediatric disease.

  Study phs000828

• Tissue and Fluid Analysis in Ocular Inflammatory Disease

  Granulomas are found in multiple infectious and non-infectious human diseases, yet the pathogenic drivers of granulomatous disease are still poorly understood. To enrich for pathologic inflammatory cells, we leveraged the intrinsic immunologic isolation of the eye by examining granulomatous uveitis, a form of ocular inflammation. We performed single-cell RNA-sequencing to obtain an unbiased gene expression survey of ocular immune cells in the anterior chamber of four live human donors with granulomatous uveitis. We paired this with antigen-receptor sequence analysis to identify clonally expanded lymphocytes. Each individual had robust clonal expansion arising from a single T or B cell lineage, suggesting distinct pathogenic processes in each patient. This variability in clonal expansion was mirrored by individualized combinations of TH1, TH1/TH17, and Tregs in ocular CD4 T cells. In contrast, ocular CD8 T cells were more transcriptionally similar across donors. Finally, clonally expanded, activated B cells displayed evidence of plasmablast differentiation and class-switching within the ocular microenvironment. Collectively, our study identified conserved and individualized features of granulomatous inflammation, illuminating parallel pathophysiologic mechanisms.

  Study phs002449

• Epigenetic Landscape of Human Parathyroids

  The goal of this study is to produce a comprehensive map of the chromatin landscape of human parathyroids. Data from 20 subjects with parathyroid adenoma causing primary hyperparathyroidism were used. For the sequencing platform, Illumina HiSeq 2000 with V3 high-output kits were used. For the data processing pipeline, reads were aligned to hg19 using Bowtie or BWA. For RNA-seq data, transcript levels were estimated per gene to RPKM using Cufflink. For ChIP-seq and ATAC-seq data, enrichment peaks were estimated compared to inputs or background using MACS2 after merging reads from replicates. For DNase-seq, significant peaks compared to background distribution were computed using Hotspot. For Hi-C data, significant interactions were detected using Juicer HiCCUPS and 5 kb resolution for the merged data from replicates. Chromatin states for parathyroids were computed using ChromHMM based on ChIP-seq profiles in parathyroids of H3K4me3, H3K4me1, H3K27ac, H3K36me3, H3K27me3 and H3K9me3 and the emission matrix from 18 states pre-trained in the Roadmap Epigenomics. Gene expression, binding positions of transcription factor, open chromatin regions, 3D-chromatin interaction, and chromatin states are submitted.

  Study phs003302

• Proteogenomic Analysis of CALGB40601 (ALLIANCE) a Neoadjuvant Phase III HER2-Positive Breast Cancer Trial

  80 frozen core needle biopsies from 54 HER2+ breast cancer patients from the CALGB 40601 trial (NCT00770809) were processed for proteogenomic analysis to identify genes, proteins, and phosphosites associated with pathological complete response to neodjuvant anti-HER2 therapies. The final set included 22 patients treated with trastuzumab plus lapatinib in combination with pacletaxel (THL arm), 24 patients treated with trastuzumab in combination with pacletaxel (TH arm), and 8 patients treated lapatinib in combination with pacletaxel (TL arm). Principal findings of this study included identifying negative cases misdiagnosed as HER2+ that did not respond to targeted therapy and observing higher levels of two biomarker candidates in non-pCR tumors. Mass spec-based proteomics and phosphoproteomics data generated from 75 biopsies (all 54 patients) will be made available via the Proteomic Data Commons. Whole exome- (67 biopsies from 49 patients) and RNA-sequencing (52 biopsies from 39 patients) data for the subset profiled with proteogenomics will be made available through dbGaP, but genomic and transcriptomic profiling data for the whole cohort has previously been deposited (phs001570).

  Study phs003576

• Systemic Inflammation and Lymphocyte Activation Precede Rheumatoid Arthritis

  The Allen Institute for Immunology-University of California, San Diego-University of Colorado Transition to Rheumatoid Arthritis (ALTRA) project is a prospective longitudinal cohort study to understand the pre-clinical development of rheumatoid arthritis (RA). We performed cross-sectional and longitudinal multi-omics analyses of peripheral immunity in the anti-citrullinated protein autoantibody-positive (ACPA+) at-risk for RA period. We provide FASTQ files from (1) single-cell RNA sequencing (scRNA-seq) of ACPA+ at-risk (n=45), ACPA+ early RA (n=11), and site-matched ACPA- controls (n=38); (2) scRNA-seq of longitudinal samples from ACPA+ at-risk who developed clinically active RA during our study (n=16 participants, n=75 samples) and ACPA- controls (n=29 subjects, n=89 samples); (3) trimodal TEA-seq of a subset of ACPA+ at-risk (n=7) and ACPA- controls (n=5). Using these data, we characterize pathogenesis prior to clinical RA diagnosis and suggest the at-risk state exhibits substantial immune alterations that could potentially be targeted for early intervention to delay or prevent autoimmunity.

  Study phs003944

• Response​ to Tagraxofusp in Blastic Plasmacytoid Dendritic Cell Neoplasm

  Tagraxofusp is a CD123-targeted therapy comprised of a recombinant human interleukin-3 (IL-3) fused to a truncated diphtheria toxin (DT) payload. It is the first approved treatment specifically for patients with Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN). In order to identify biomarkers of response, bone marrow samples from 12 BPDCN patients who were treated with tagraxofusp in the pivotal phase II trial (NCT02113982) were profiled longitudinally using gene panel and/or single-cell RNA sequencing. Durable responders had either wildtype or missense TET2 mutations while transient and non-responders had at least one truncating TET2 mutation. Those who had progression at any one point during the treatment course (transient responders) and non-responders had enrichment of the chemokines CCL3, CCL4, and CCL5. Residual tumor cells following tagraxofusp treatment were identified in all patients, whose resistance may be due to their lower levels of TXNRD1 possibly preventing the potentiation of tagraxofusp. Thus, response to tagraxofusp may be related to culmination of intrinsic TET2 mutations, levels of TXNRD1, and extrinsic effects of chemokines within bone marrow.

  Study phs003895

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

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

  Study JGAS000535

• Genomic Analyses Identify Recurrent MEF2D Fusions in Acute Lymphoblastic Leukemia

  Chromosomal rearrangements are initiating events in acute lymphoblastic leukemia (ALL). Here, using RNA-sequencing (RNAseq) of 560 ALL cases, we identify rearrangements between MEF2D (myocyte enhancer factor 2D) and five genes (BCL9, CSF1R, DAZAP1, HNRNPUL1 and SS18) in 22 B progenitor ALL (B-ALL) cases with a distinct gene expression profile, the most common of which is MEF2D-BCL9. Examination of an extended cohort of 1164 B-ALL cases identified 30 cases with MEF2D rearrangements which include an additional fusion partner, FOXJ2, thus MEF2D-rearranged cases comprise 5.3% of cases lacking recurring alterations. MEF2D-rearranged ALL is characterized by a distinct immunophenotype, DNA copy number alterations at the rearrangement sites, older diagnosis age, and poor outcome. The rearrangements result in enhanced MEF2D transcriptional activity, lymphoid transformation, activation of HDAC9 expression and sensitive to histone deacetylase inhibitors treatment. Thus, MEF2D-rearranged ALL represents a distinct form of high-risk leukemia, for which new therapeutic approaches should be considered.

  Study EGAS00001001952

• Gene expression adaptation of metastases to their host tissue

  The adaptation of metastatic cells to their host tissue critically determines the pathogenicity of a cancer and therefore patient survival. Yet, it remains elusive to what extent the host environment drives gene expression programs in metastatic cells. Here we identify adaptive mechanisms that enable metastases to establish themselves in a novel tissue context. We performed single-cell RNA-sequencing on malignant and benign tissue samples from untreated donors with colorectal adenocarcinoma and liver metastasis to deduce tissue adaptive expression patterns. A novel computational approach identified genes and pathways that consistently adapted to the host tissue at the transition from the primary tumor to the paired metastasis across donors. This analysis revealed that the majority of expression changes in the metastasis reflect an expression signature reminiscent of benign liver epithelial cells. Cellular processes adapting to the liver environment include basic cellular functions such as energy metabolism, as well as tissue-specific pathways such as the regulation of lipid metabolism by PPAR-α. These adaptations potentially increase the pathogenicity of the metastatic cells and may provide new therapeutic strategies.

  Study EGAS50000000817

• Extensive three-dimensional intratumor proteomic heterogeneity revealed by multiregion sampling in high-grade serous ovarian tumor specimens

  Enriched tumor epithelium, tumor-associated stroma, and whole tissue were collected by laser microdissection from thin sections across spatially separated levels of ten high-grade serous ovarian carcinomas (HGSOCs) and analyzed by mass spectrometry, reverse phase protein arrays, and RNA sequencing. Unsupervised analyses of protein abundance data revealed independent clustering of an enriched stroma and enriched tumor epithelium, with whole tumor tissue clustering driven by overall tumor “purity.” Comparing these data to previously defined prognostic HGSOC molecular subtypes revealed protein and transcript expression from tumor epithelium correlated with the differentiated subtype, whereas stromal proteins (and transcripts) correlated with the mesenchymal subtype. Protein and transcript abundance in the tumor epithelium and stroma exhibited decreased correlation in samples collected just hundreds of microns apart. These data reveal substantial tumor microenvironment protein heterogeneity that directly bears on prognostic signatures, biomarker discovery, and cancer pathophysiology and underscore the need to enrich cellular subpopulations for expression profiling. (Companion proteomic data is available in the ProteomeXchange Consortium, PRIDE: PXD026570).

  Study EGAS00001005786

• CD36 defines CML cells less sensitive to imatinib

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

  Study EGAS00001002421

• Immunogenomics of colorectal cancer response to immune checkpoint blockade

  Molecular and cellular determinants of response to immune checkpoint inhibitors are mostly unknown. Here we use whole exome, RNA and T cell receptor sequencing, imaging mass cytometry, immunohistochemistry and PD1-PDL1 interaction detection to profile 543 regions from 16 colorectal cancers (CRCs) subsequently treated with Pembrolizumab or Nivolumab. Independent of treatment response, CRCs express low levels of PD1 and PDL1 proteins and form less PD1-PDL1 complex than other cancers. Non hypermutated CRCs have no benefit from treatment, show WNT activation and low T cell infiltrates. Among hypermutated CRCs, those with durable benefit have clonal immunogenic alterations, clonally expanded T cells and potential for immune escape through dysfunctional antigen presentation. Responsive tumours are enriched in PD1-expressing cytotoxic and proliferating CD8 T cells and PDL1-expressing antigen presenting macrophages, which form high-density clusters of interacting cells. Our study shows that immune checkpoint inhibitors are most effective in highly infiltrated CRCs where they may release the interactions between macrophages and CD8 T cells thus promoting their priming and expansion in intra-tumour niches.

  Study EGAS00001004438

• Mesenchymal inflammation drives genotoxic stress in hematopoietic stem cells and predicts disease evolution in human pre-leukemia

  Mesenchymal niche cells may drive tissue failure and malignant transformation in the hematopoietic system but the molecular mechanisms and their relevance to human disease remain poorly defined. We demonstrated that perturbation of mesenchymal cells in a mouse model of the preleukemic disorder Shwachman-Diamond syndrome induces mitochondrial dysfunction, oxidative stress and activation of DNA damage responses in hematopoietic stem and progenitor cells. In this study we demonstrate, through massive parallel RNA sequencing of highly purified mesenchymal cells in a range of human preleukemic syndromes, TP53-S100A8/9-TLR4 inflammatory signaling as a common driving mechanism of genotoxic stress, which could be attenuated by TLR4 blockade. S100A8/9 expression in mesenchymal cells predicted outcome in myelodysplastic syndromes, the principal human preleukemic condition, independent of known prognostic variables. Collectively, findings reveal a concept of mesenchymal niche-induced genotoxic stress in heterotypic stem and progenitor cells through inflammatory signaling as an actionable determinant of disease outcome in human preleukemia. The data further provide novel conceptual and mechanistic insights into the intimate link between inflammation and cancer.

  Study EGAS00001001926

• Engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus

  Short hairpin RNA (shRNA) expression strategies that allow safe and persistent target mRNA knockdown are key to the success of many in vitro or in vivo RNAi applications. Here, we propose a novel solution which is expression of a promoterless miRNA-adapted shRNA (shmiRNA) from an engineered genomic miRNA locus. For proof-of-concept, we genetically “vaccinated” liver cells against a human pathogen, by using TALEns or CRISPR to integrate an anti-hepatitis C virus (HCV) shmiRNA into the liver-specific miR-122/hcr gene. Reporter assays and qRT-PCR confirmed anti-HCV shmiRNA expression as well as miR-122 integrity and functionality. Specificity and safety of shmiRNA integration were validated via PCR, cDNA and miRNA profiling, and whole genome sequencing. A subgenomic HCV replicon and a full-length reporter virus, but not a Dengue virus control, were significantly impaired in the modified cells. Our original combination of DNA engineering and RNAi expression technologies should benefit numerous applications, from basic miRNA research, to human cell and gene therapy.

  Study EGAS00001001252

• Gene fusion and transcriptomic landscapes of sarcomas

  Sarcoma represents a highly heterogeneous group of tumors. We report here the first unbiased and systematic search for gene fusions and analysis of transcriptomic profiles in 100 monomorphic sarcomas cases using RNA-sequencing profiling. Fusion genes were detected in two thirds of samples. Similarly to fusion genes such as PAX3/7-FOXO1, EWSR1/FUS-ETS, SS18-SSX, BRD3/4-NUTM1 or EWSR1/FUS/TAF15-NR4A3 characterizing well-defined entities, we show that all sarcomas displaying CIC fusions- whatever their fusion partner (being DUX4, NUTM1 or FOXO4) - form a transcriptionally homogeneous group of tumors. Likewise, tumors with either a BCOR fusion (with CCNB3, MAML3 or ZC3H7B partners) or with a BCOR internal duplication form a single biological entity. We also found that EML4-ALK fusions also characterize a homogeneous infantile fibrosarcoma subgroup. In contrast, fusions like VGLL2-NCOA2/CITED2, TMP3/TRP-NTRK1 or ETV6-NTRK3 are observed in more heterogeneous tumors. Finally, we also describe a new group of bone sarcomas characterized by EWSR1- or FUS-TFCP2 fusions.

  Study EGAS00001002189

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

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

  Study EGAS00001002895

• MINCR is a MYC-induced lncRNA able to modulate MYC’s transcriptional network in Burkitt lymphoma cells

  Despite the established role of the transcription factor MYC in cancer, little is known about the involvement of lncRNAs in mediating MYC’s function. Here we have intersected RNA-sequencing data from MYC-inducible cell lines, from a cohort of 91 mature B-cell lymphomas and from sorted germinal-center B-cells. By this approach, we identified 13 lncRNAs differentially expressed in IG-MYC-positive Burkitt lymphoma and regulated by MYC in the model cell lines. Among them we focused on a lncRNA that we named MINCR, showing a strong correlation with MYC expression in MYC-positive lymphomas and in pancreatic ductal adenocarcinomas. RNAi experiments showed that MINCR controls cellular viability by influencing the expression of MYC-regulated cell cycle genes. Finally we provide evidences that down-regulation of AURKA, AURKB and CTD1 may explain the reduction in cellular proliferation observed upon MINCR knock-down. We therefore suggest that MINCR acts as a modulator of MYC transcriptional control of cell cycle genes.

  Study EGAS00001001199

• HCA_Female_Reporductive_Adult_WSSS_RNA

  Cell atlas of the human placenta The Human Cell Atlas initiative is an ambitious global research effort which aims to describe every cell in the human body across all developmental stages to generate a reference map to accelerate progress in science and medicine particularly relating to human development and disease. The initiative is led by investigators based at the Wellcome Sanger Institute and the Broad Institute of MIT and Harvard and funding support from the Wellcome and NIH. This specific project will assess human development. We aim to interrogate the cellular composition and molecular regulators underpinning development and maturation through single cell RNA-sequencing/spatial transcriptomics and computational algorithms to predict cellular developmental trajectories and cell-cell interactions. We will subsequent validate these findings in situ using allied imaging technologies e.g. immunohistochemistry and RNAscope and in vitro culture of human intestinal organoids (HIOs). This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/

  Study EGAS00001004210

• HCA_Placenta_Adult_Vento_RNA

  The Human Cell Atlas initiative is an ambitious global research effort which aims to describe every cell in the human body across all developmental stages to generate a reference map to accelerate progress in science and medicine particularly relating to human development and disease. The initiative is led by investigators based at the Wellcome Sanger Institute and the Broad Institute of MIT and Harvard and funding support from the Wellcome and NIH. This specific project will assess human development. We aim to interrogate the cellular composition and molecular regulators underpinning development and maturation through single cell RNA-sequencing/spatial transcriptomics and computational algorithms to predict cellular developmental trajectories and cell-cell interactions. We will subsequent validate these findings in situ using allied imaging technologies e.g. immunohistochemistry and RNAscope and in vitro culture of human intestinal organoids (HIOs). This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/

  Study EGAS00001004069

• Proteogenomics reveals two distinct biological pilocytic astrocytoma subgroups

  Pilocytic astrocytoma (PA) is the most common pediatric brain tumor and driven by aberrant MAPK signaling, typically mediated by BRAF alterations. While five-year overall survival rates exceed 95%, tumor recurrence constitutes a major clinical challenge in incompletely resected tumors despite chemotherapeutic or radiation based therapies. Therefore, we used proteogenomics to discern the biological heterogeneity of PA to improve classification of this tumor entity and identify novel therapeutic targets. Our proteogenomics approach integrates RNA sequencing and LC/MS-based proteomic profiling data from a cohort of 58 confirmed, primary PA samples. An integrative genomics approach was conducted to discern the biological heterogeneity of PA and to identify aberrant pathway activation in these biological subgroups. In summary, Pilocytic astrocytomas segregate into two groups where younger patients are significantly associated with Group 1. Importantly, we validate the two distinct biological subgroups in two non-overlapping cohorts. The biological heterogeneity seen here may improve biological classification and reveal novel therapeutic targets specifically useful for non-resectable tumors with high risk of recurrent or progressive disease.

  Study EGAS00001006402

• Upper cortical layer-driven network impairment in schizophrenia

  Schizophrenia is one of the most wide-spread and complex mental disorders. To characterize the impact of schizophrenia, we performed single nucleus RNA sequencing (snRNA-seq) of >220,000 neurons from the dorsolateral prefrontal cortex of patients with schizophrenia and matched controls. Additionally, >115,000 neurons were analyzed topographically by immunohistochemistry. Compositional analysis of snRNA-seq data revealed a reduction in abundance of GABAergic neurons and a concomitant increase in principal neurons, most pronounced for upper cortical layer subtypes, which was substantiated by histological analysis. Many neuronal subtypes showed extensive transcriptomic changes, the most dramatic - in upper layer GABAergic neurons, including downregulation in energy metabolism and upregulation in neurotransmission. Transcription factor network analysis demonstrated a developmental origin of transcriptomic changes. Finally, Visium spatial transcriptomics further corroborated upper layer neuron vulnerability in schizophrenia. Overall, our results point towards general network impairment within upper cortical layers as a core substrate associated with schizophrenia symptomatology. Study is available on bioRxiv (https://doi.org/10.1101/2020.11.17.386458) and in upcoming publication.

  Study EGAS00001006495

• Endocrine therapy reprogramming of breast cancer facilitates metastatic escape via upregulation of P-Rex1/Rac1 signalling

  The estrogen receptor (ER) drives growth in ~70% of breast cancers, and endocrine therapies prevent recurrence. However, 30% of patients recur, with many fatal cases occurring late (after >5 years), presenting a challenge due to their often non-proliferative nature and limited options. To study this, we generated slow-growing resistant cells that form small primary tumours but readily metastasize. Single-cell RNA sequencing (scRNAseq) revealed that endocrine therapy reprograms these cells, notably upregulating the Rac1 signalling component P-Rex1. We found in clinical cohorts that P-Rex1 in high in ER+ breast cancer. Intravital imaging demonstrated that Rac1 signalling is active in ER+ cells following endocrine therapy. Targeting the Rac1 pathway with small molecule inhibitors (NSC23766, R-ketorolac) reduced survival and motility in resistant cells, inhibited in vivo Rac1 activity, and reduced tumour burden when combined with tamoxifen in a drug-refractory patient-derived xenograft. This work identifies the P-Rex1/Rac1 axis, as a potential therapeutic target for late recurring ER+ breast cancer.

  Study EGAS00001008353

• “Castration-persistence” is a distinct state of tolerance to androgen receptor targeting therapies in prostate cancer

  Androgen receptor (AR) signalling is important in prostate cancer progression, and therapies that specifically target this pathway are the mainstay of treatment for advanced disease. Treatment however is non-curative, and resistance develops inevitably with time. Although the mechanisms that drive progressive ‘castration resistant’ disease are reasonably well characterized, how tumours survive acute pathway inhibition is unclear. We performed a neo-adjuvant study of a novel combination of AR targeting therapies, and noted that the objective response to treatment was highly variable. To determine what was driving tumour persistence in poorly responding patients, we comprehensively characterised pre- and post-treatment samples were using both whole genome and RNA-sequencing. We find that ‘castration-persistence’ is a distinct state from ‘castration-resistance’, and is mediated by global transcriptional reprogramming leading to transitional EMT state, which is shared with benign luminal epithelial cells. This appears to be AP-1 and KLF driven, and represents and integration of multiple signalling pathways, particularly IGF2, offering a number of tractable strategies to improve clinical response to AR targeting therapies.

  Study EGAS00001003172

• Ongoing Replication Stress Tolerance and Clonal T Cell Responses Distinguish Liver and Lung Recurrence and Patient Outcomes in Pancreatic Ductal Adenocarcinoma

  Patients with metastatic pancreatic ductal adenocarcinoma (PDAC) survive longer when disease spreads to the lung but not to the liver. We generated overlapping, multi-omic datasets to identify molecular and cellular features that distinguish patients whose disease develops liver metastasis (liver cohort) from those whose disease progression results in lung metastasis without liver metastases (lung cohort). Lung cohort patients generally survived longer than liver cohort patients, independent of tumor subtype. We developed a pORG gene signature that distinguishes primary tumors in the liver and lung cohorts. We identified ongoing replication stress (RS) response pathways in high pORG/liver cohort tumors, while low pORG/lung cohort tumors had greater densities of lymphocytes and shared T cell clonal responses. Our study demonstrates that liver-avid PDAC is associated with tolerance to ongoing RS, limited tumor immunity, and less favorable outcomes; whereas low RS, lung-avid/liver-averse tumors are associated with active tumor immunity that may account for favorable outcomes. As expected, we found high frequencies of KRAS, TP53, CDKN2A, and SMAD4 gene alterations in our tumor samples. High pORG activity (GSVA scores) in our primary tumors appear to be positively correlated with alteration frequencies in both TP53 and CDKN2A. We did not see a correlation with alterations in KRAS as almost all the tumor samples have KRAS alterations. From a de-identified dataset of 1,873 patients diagnosed with and/or treated for PDAC at our institution between 2004 and 2020, we identified 422 patients for which we had specimens with sequencing data (N=374) and/or specific evidence of disease metastasis site(s) from the OHSU cancer registry or disease-relevant computed tomography (CT) scans to allow cohort classification. Note that our study includes RNA-Seq, DNA-Seq panel, and TCR-Seq (T-cell Receptor Sequencing) data, but that this dbGaP submission just includes the patients/samples with RNA-Seq and/or DNA-Seq panel data. Therefore, this submission includes 290 samples from 278 patients. TCR sequence data is available on the Adaptive Biotechnologies platform. Clinical course timepoints, patient demographics, stage, grade, nodal involvement, resection margins, and angiolymphatic invasion were provided as deidentified data by the OHSU cancer registry with quality control data verification in a subset by pathologists (BB and TM). We reviewed all available computed tomography (CT) scans for all patients with primary tumor resection dates recorded by the cancer registrar, with tumor samples analyzed by RNA-seq, DNA-seq, or TCR-seq, and/or with additional information indicating metastatic spread (e.g., metastatic samples received for related studies). We abstracted the site of all lesions proven to be metastatic by biopsy and/or that clearly increased in size during progression or decreased in size during treatment as long as a radiologist described the lesion as “likely”, “suspicious for”, “concerning for”, or “favor” metastasis. Clinical imaging was reviewed by a radiologist (AG) to validate patient assignments to the liver, lung, and neither liver nor lung (other recurrence site) cohorts. Time to recurrence was calculated from the earliest of either the recurrence date provided by the OHSU cancer registry, or the date of earliest lesion abstracted from CT reports. The subject attributes reported in this submission were up to date at the time of our data freeze, which occurred July 2021.

  Study phs003597

• Whole exome sequencing and RNA sequencing of cervical cancer

  We designed this study to identify molecular markers involved in the development and prognosis of cervical adenocarcinoma. In this study, we would like to elucidate the molecular biological characteristics of cervical adenocarcinoma in relation to the carcinogenesis of other cervical cancers (squamous cell carcinoma), vaginal and vulvar cancers. In addition, some squamous cell carcinomas of the cervix develop at a young age and have a poor prognosis. Furthermore, small cell carcinoma of the cervix also progresses rapidly and has a poor prognosis, making the establishment of diagnostic and therapeutic methods an urgent task.

  Study JGAS000586

• Whole exome sequencing and RNA sequencing of cervical cancer

  We designed this study to identify molecular markers involved in the development and prognosis of cervical adenocarcinoma. In this study, we would like to elucidate the molecular biological characteristics of cervical adenocarcinoma in relation to the carcinogenesis of other cervical cancers (squamous cell carcinoma), vaginal and vulvar cancers. In addition, some squamous cell carcinomas of the cervix develop at a young age and have a poor prognosis. Furthermore, small cell carcinoma of the cervix also progresses rapidly and has a poor prognosis, making the establishment of diagnostic and therapeutic methods an urgent task.

  Study JGAS000582

• Whole exome sequencing (WES) of EBV- primary central nervous system lymphoma (PCNSL)

  This dataset contains whole exome sequencing (WES) of 29 HIV- EBV- primary central nervous system lymphoma (PCNSL) tumors and 5 activated B-cell-like PCNSL (ABC-PCNSL) and activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) cell lines (TK, HKBML, OCI-Ly3, HBL-1, TMD-8). Cases with secondary involvement of the CNS were excluded. DNA was extracted with AllPrep DNA/RNA FFPE kit and libraries were prepared using the Agilent SureSelect Human All Exon v6 + UTR kit. Paired end reads were aligned to GRCh37 using bwa mem v.0.7.17, and reads were sorted and duplicated were marked in the final BAM files. Access to this data is controlled. There are a number of steps that a researcher must take to obtain access to this data, including execution of a Data Access Agreement between the institutions. The process is overseen by the Technology Development Office; please contact our general email address TDOadmin@phsa.ca. Please only click the "request data" button on the EGA website after a Data Access Agreement is fully executed.

  Dataset EGAD50000000451

• RNA-seq analysis of transcriptome variation with human ESC subclones

  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/ .

  Dataset EGAD00001004266

• Haplotype-specific assembly of shattered chromosomes in oesophageal adenocarciomas

  Genomic and epigenomic sequencing of 5 oesphageal adenocarciomas with evidence of chromothripsis. Genomic sequencing includes: Pacbio circular consensus sequencing, Pacbio continuous long read sequencing, 10X linked read and Illumia HiSeq X Ten sequencing. Epigenomic sequencing includes: Hi-C chromosome capture, ATAC-seq, ChIP seq (for H3K27ac, H3K4me3, H3K27me3 and CTCF) and long read RNA sequencing. All data types have the bam files which have not undergone haplotype resolution (demarcated as unresolved) and some data types also have haplotype resolved reads (demarcated as resolved).

  Dataset EGAD00001010871

• RNA sequencing of undifferentiated sarcomas

  Undifferentiated sarcomas (USARCs) of adults are diverse, rare, and aggressive soft tissue cancers. Recent sequencing efforts have confirmed that USARCs exhibit one of the highest burdens of structural aberrations across human cancer. Here, we sought to unravel the molecular basis of the structural complexity in USARCs by integrating DNA sequencing, ploidy analysis, gene expression, and methylation profiling. We identified whole genome duplication as a prevalent and pernicious force in USARC tumorigenesis. Using mathematical deconvolution strategies to unravel the complex copy-number profiles and mutational timing models we infer distinct evolutionary pathways of these rare cancers. In addition, 15% of tumors exhibited raised mutational burdens that correlated with gene expression signatures of immune infiltration, and good prognosis.

  Study EGAS00001003291

• Breast Cancer FRT RNA seq

  We propose to definitively characterise the somatic genetics of ER+ve, HER2-ve breast cancer through generation of comprehensive catalogues of somatic mutations in breast cancer cases by high coverage genome sequencing coupled with integrated transcriptomic and methylation analyses.

  Dataset EGAD00001000338

• RNA sequencing data of pediatric TCF3::PBX1 acute lymphoblastic leukemia

  TCF3::PBX1 leukemia is a rare subtype of B-cell acute lymphoblastic leukemia (B-ALL), occurring in approximately 3–5% of pediatric cases. It results from a chromosomal translocation t(1;19)(q23;p13), generating the TCF3::PBX1 fusion gene, which disrupts normal transcriptional regulation and contributes to leukemogenesis. Although historically associated with poor prognosis, contemporary risk stratification and intensified therapy have significantly improved outcomes, and both 5-years DFS and OS are nowadays ≥ 0.96.

  Study EGAS50000001797

• The Transcriptional Landscape of SHH Medulloblastoma

  To further understand the biology of Sonic hedgehog medulloblastoma and its molecular subtypes, we studied 250 human Shh-MB using strand-specific RNA sequencing. We identified novel alterations within the cAMP dependent pathway and found that 18% of tumors have genetic events that directly target the abundance and/or stability of MYCN. We also discovered an extensive network of fusions in focally amplified regions, and several loss-of-function fusions in tumor suppressor genes PTCH, SUFU and NCOR1. Molecular convergence on a core of specific genes by nucleotide variants, copy number aberrations, and gene fusions highlights key roles of specific pathways in the pathogenesis of Sonic hedgehog medulloblastoma.

  Dataset EGAD00001006305

• Genomic characterisation of SDH deficient renal cell carcinoma - WGS

  SDH deficient renal cell carcinomas are a rare and recently defined subtype of kidney cancer, often associated with an inherited mutation in one of the SDH gene subunits. This dataset sought to understand the genomic events that underpin tumour formation, from putative cell of origin, characterisation of the tumour microenvironment, to the genomic evolution of these rare tumours. We performed whole genome and RNA sequencing of 4 patients with SDH deficient renal cell carcinomas, including one patient who had an additional paraganglioma. An addition patient in this cohort had the initial diagnosis revised to a clear cell renal cell carcinoma.

  Dataset EGAD00001008469

• B cell receptor silencing reveals origin and dependencies of high-grade B cell lymphomas with MYC and BCL2 rearrangements

  High-grade B cell lymphomas with MYC and BCL2 rearrangements (HGBCL-DH-BCL2) represent highly aggressive Germinal center (GC) derived B cell malignancies with dismal prognosis and limited therapeutic options. In the present study, we investigated the expression of BCR complex components, including immunoglobulin (IG) heavy (H) and light (L) chains, and CD79A and CD79B signaling subunits in HGBCL-DH-BCL2, scoring 65% of cases with undetectable IGH immunoreactivity (IGHUND). Transcriptomics (bulk RNA-seq) and genetic (whole exome/genome sequencing) analyses indicated that IGHUND and IGH+ HGBCL-DH-BCL2 lymphomas represent distinct biological entities. Specifically, IGHUND HGBCL-DH-BCL2 exhibit a GC DZ-like molecular program, and select subset-specific gene mutations pointing to distinct evolutionary trajectories respect to the IGH+ counterparts. IG profiling revealed that IGHUND HGBCL-DH-BCL2 preserved potentially productive, hypermutated IGH variable (V) gene rearrangements, indicating a selective advantage conferred to the malignant B cells by residual IGH molecules. Accordingly, disruption of the productively rearranged IGHG1 gene in BCRnull HGBCL-DH-BCL2 cell line model confirmed the dependency on IGH expression for optimal fitness. Using transcriptomics and in situ RNA-scope analysis, we reported that IGHUND HGBCL-DH-BCL2 originated from IG-class switched GC B cells, while IGH-expressing tumors were selected for continuous IGM expression. Moreover, while IGH+ HGBCL-DH-BCL2 favor IGM/IG-Kappa expression, IGHUND counterparts complete IG-Lambda light chain rearrangements. Notably, BCR silencing, caused by accelerated IGH turnover and reduced IGH expression, precedes HGBCL-DH-BCL2 onset, inducing RAG1/2-dependent IG light chain editing and eventually facilitating t(8;22)/IGL::MYC translocations. Finally, IGH silencing observed in HGBCL-DH-BCL2 cell models, which leads to intracellular retention/silencing of CD79B, results in reduced sensitivity to the CD79B-targeting antibody-drug conjugate Polatuzumab-vedotin. Collectively, HGBCL-DH-BCL2 originates from isotype-switched t(14;18)+ GC B cells that undergo IG light chain editing fuelling intra-clonal diversification, BCR silencing, and the acquisition of t(8;22) translocation, while preserving IGH dependency.

  Study EGAS50000001047

• Identifying transcriptional programs underlying anti-EGFR small molecule response and resistance with TraCe-seq

  Genetic and non-genetic heterogeneity within cancer cell populations represents a major challenge to anti-cancer therapies. We currently lack robust methods to determine how pre-existing and adaptive features affect cellular responses to therapies. Here, by conducting clonal fitness mapping and transcriptional characterization using expressed barcodes and single-cell RNA-sequencing, we have developed TraCe-seq, a method that captures at clonal resolution the origin, fate, and differential early adaptive transcriptional programs of cells in a complex population in response to distinct treatments. We used TraCe-seq to benchmark how next-generation dual EGFR inhibitors-degraders compare to standard EGFR kinase inhibitors in EGFR-mutant lung cancer cells. To QC the TraCe-seq strategy, single-cell RNA-seq libraries were generated from a variety of human cancer cell lines transduced with the TraCe-seq library to validate the TraCe-seq strategy. Specifically, 5 different cell lines (PC9, MCF-10A, MDA-MB-231, NCI-H358, and NCI-H1373) were each transduced with a unique TraCe-seq barcode. The transduced cells were selected with puromycin only, dissociated to single cell suspensions, and then mixed together. The complex mixture of the 5 cell lines was profiled by 10X scRNA-seq. Furthermore, transduced NCI-H1373 cells were sorted by FACS to enrich for the top 50% of eGFP positive cells, and sorted cells were cultured briefly and used to construct scRNA-seq libraries and profiled by 10x scRNA-seq. To carry out the full TraCe-seq experiment, ~600 PC9 cells carrying unique TraCe-seq barcodes were expanded over 12 doublings to establish the barcoded population. A subset of the barcoded PC9 population was used to generate scRNA-seq libraries and profiled by 10x scRNA-seq prior to treatment to establish a baseline transcription profile for each barcoded clone. The rest of the cells were then treated for four days with 1 µM erlotinib, 1 µM GNE-069, or 1 µM GNE-104 respectively. scRNA-seq libraries were then generated form the treated cells and profiled by 10x scRNA-seq.

  Study EGAS00001005405

• Mapping the epigenomic landscape of human monocytes following innate immune activation reveals context-specific mechanisms driving endotoxin tolerance

  Background Monocytes are key mediators of innate immunity to infection, undergoing profound and dynamic changes in epigenetic state and immune function which are broadly protective but may be dysregulated in disease. Here, we aimed to advance understanding of epigenetic regulation following innate immune activation, acutely and in endotoxin tolerant states. Methods We exposed human primary monocytes from healthy donors (n=6) to interferon-γ or differing combinations of endotoxin (lipopolysaccharide), including acute response (2hr) and two models of endotoxin tolerance: repeated stimulations (6+6hr) and prolonged exposure to endotoxin (24hr). Another subset of monocytes was left untreated (naïve). We identified context-specific regulatory elements based on epigenetic signatures for chromatin accessibility (ATAC-seq) and regulatory non-coding RNAs from total RNA sequencing. Results We present an atlas of differential gene expression for endotoxin and interferon response, identifying widespread context specific changes. Across assayed states, only 24-29% of genes showing differential exon usage are also differential at the gene level. Overall, 19.9% (6884 of 34616) of repeatedly observed ATAC peaks were differential in at least one condition, the majority upregulated on stimulation and located in distal regions (64.1% vs 45.9% of non-differential peaks) within which sequences were less conserved than non-differential peaks. We identified enhancer-derived RNA signatures specific to different monocyte states that correlated with chromatin accessibility changes. The endotoxin tolerance models showed distinct chromatin accessibility and transcriptomic signatures, with integrated analysis identifying genes and pathways involved in the inflammatory response, detoxification, metabolism and wound healing. We leveraged eQTL mapping for the same monocyte activation states to link potential enhancers with specific genes, identifying 1,946 unique differential ATAC peaks with 1,340 expression associated genes. We further use this to inform understanding of reported GWAS, for example involving FCHO1 and coronary artery disease. Conclusion This study reports context-specific regulatory elements based on transcriptomic profiling and epigenetic signatures for enhancer-derived RNAs and chromatin accessibility in immune tolerant monocyte states, and demonstrates the informativeness of linking such elements and eQTL to inform future mechanistic studies aimed at defining therapeutic targets of immunosuppression and diseases.

  Study EGAS00001007362

• Genomic Profiling of Peripheral T-cell Lymphoma

  Peripheral T-cell lymphomas (PTCLs) are malignant and highly aggressive hematologic tumors arising from mature post-thymic T cells. The diagnosis of PTCL includes diverse lymphoma subgroups, altogether accounting for about 15% of all non-Hodgkin lymphomas. Despite recent advances in uncovering the mutational landscape of PTCLs, the prognoses of most PTCLs remain poor. To overcome the limits of exome sequencing and identify new genomic abnormalities in PTCL, we performed RNAseq and whole genome sequencing. From our analyses, we identified a recurrent in-frame deletion (VAV1 Δ778-786) generated by a focal deletion-driven alternative splicing mechanism as well as novel VAV1 gene fusions (VAV1-THAP4, VAV1-MYO1F, and VAV1-S100A7). These genetic lesions result in increased activation of VAV1 catalytic-dependent (MAPK, JNK) and non-catalytic-dependent (nuclear factor of activated T cells, NFAT) VAV1 effector pathways. These results support an oncogenic role for VAV1 signaling in the pathogenesis of PTCL (Abate et al., 2017). Recently, we have also identified a novel chimeric oncogene, FYN-TRAF3IP2, in the RNA-seq and whole genome sequencing analyses. FYN-TRAF3IP2 leads to aberrant NF-κβ activity downstream of T-cell receptor signaling and induces NF-κβ-driven T-cell transformation when expressed in hematopoietic progenitors in mice. This novel gene fusion supports the oncogenic driver role of NF-κβ signaling in the pathogenesis of PTCL (Kim et al., in review).

  Study phs001962

• Whole Tumor spatial heterogeneity in metastatic melanoma

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

  Study EGAS00001003292

• Exploratory novel biomarker and resistance mechanism of milademetan, an MDM2 inhibitor, in MDM2 amplified intimal sarcoma from an open-label phase 1b/2 trial ���NCCH1806/MK004���

  Amplified Murine double minute 2 (MDM2) is found in > 70% of intimal sarcoma, known as one of the ultra-rare sarcomas. Milademetan (DS3032, RAIN-32) is a novel, specific, small-molecule MDM2 inhibitor that disrupts MDM2 and the tumor suppressor protein p53 interactions in tumor cells. We conducted a phase 1b/2 trial (Trial registration No: JMA-IIA00402) in patients with amplified MDM2 wild-type TP53 intimal sarcoma as a sub-study under the nationwide large registry for rare cancers in Japan (MASTERKEY Project). Eleven patients were enrolled, and ten were included in the efficacy analysis. Two (20%) patients had durable responses for > 15 months. Milademetan provided clinical benefits in patients with amplified MDM2 intimal sarcoma. Predictive biomarkers other than amplified MDM2 and acquired resistance mechanisms for milademetan are unknown. Whole-exome and RNA sequencing analyses of pre-treatment tissue samples were conducted to identify determinants of response. Genomic alterations were analyzed for 10 patients, and gene expression was analyzed for 9 patients using their pre-treatment tissue samples. Targeted sequencing of cell-free DNA (cfDNA) samples (liquid biopsy) was also conducted sequentially at three points [before treatment with milademetan (baseline), at Cycle 2 Day1, and at the time of disease progression] to identify determinants of response and resistance. From whole-exome and RNA sequencing analyses of pre-treatment tissue samples, we could not find any molecular pathways associated with the anti-tumor activity of milademetan. Focusing on 8 genes (CDK4, CDKN2A, CDKN2B, EGFR, ERBB3, MDM2, PDGFRA, TP53) known to be frequently affected in intimal sarcoma and 10 genes (AKT1, ATM, BBC3, CDKN1A, CDKN1C, CHEK2, MDM4, PMAIP1, PPM1D, TWIST1) reported to be associated with MDM2 inhibitor responses; we found that anti-tumor activity correlated with amplified TWIST1 (p-value = 0.028) and negatively with CDKN2A loss (p-value = 0.071). Eight of the 10 patients had their cfDNA collected sequentially at baseline, at Cycle 2 Day1 and disease progression; however, one did not consent to the exploratory analysis study using cfDNA, and one had cfDNA collected at baseline but not at disease progression due to ongoing treatment. Of the eight patients, TP53 mutations in cfDNA were detected in one and five patients at baseline and disease progression, respectively. The cfDNA allele frequency of TP53 mutations increased with disease progression. CDKN2A loss and amplified TWIST1 could be associated with the anti-tumor activity of milademetan in patients with MDM2 amplified intimal sarcoma. Acquired TP53 mutations were detected in sequential liquid biopsies as loss-of-function mutations, and these TP53 mutations might compromise the anti-tumor activity.

  Study JGAS000619

• Differential Presence of Exons in Cell-Free DNA Reveals Different Patterns in Colorectal Cancer Between Metastatic and Non-Metastatic Patients

  Tumor genotyping during disease follow-up is gaining an increasing interest for monitoring and clinical management in colorectal cancer (CRC). Growing evidences suggest that single biopsies are inefficient for molecular profiling, due to clonal evolution and intratumoral heterogeneity of primary tumors and/or metastasis. Circulating free DNA (cfDNA) has emerged as an alternative source of genetic material that seems to be representative of the continuously changing tumor molecular features. Thus, the so-called 'liquid biopsy' also offers the additional advantages of accessibility and non-invasiveness for the patient. Novel strategies based on next-generation sequencing (NGS) of cfDNA are being currently developed, with most studies focusing on targeted deep sequencing panels of potential clinically actionable genes. Here we describe an alternative approach, consisting of exome-sequencing in cfDNA at a relatively shallower depth to provide a more general overview of the circulating genome. In this study, two groups of colorectal cancer patients, with either disseminated (M) or localized disease (N), were compared using whole-exome sequencing and a RNA-seq bioinformatics analysis pipeline. Thus, a set of 379 exons present at different levels in cfDNA from both groups were identified, giving rise to a completely new concept in this field, termed as 'Differential Presence of Exons'. Differentially present exons (DPEs) were subsequently used for M and N patients clustering and classification, allowing for the design of a predictive algorithm with encouraging preliminary results in a small subset of patients that could not be initially classified attending to the selection criteria (U group, unassignable).

  Study EGAS00001002687

• Dataset for soft_tissue_tumor-RNA

  Rare cancer sequencing data of 243 runs in tumor/control pairs, which were uploaded to umbrella studies. The sequencing was always paired

  Dataset EGAD00001008859