23014 results for "ega"

in 402.50 milliseconds.

• 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

• Expanding the SPTBN1 Spectrum: A Case of Neurodevelopmental Disorder with GI and Musculoskeletal Involvement

  Background: Mutations in the SPTBN1 gene, which encodes the βII-spectrin protein, are associated with a recently described neurodevelopmental syndrome characterized by developmental delay, intellectual disability, autistic traits, seizures, and dysmorphic features. While the phenotypic spectrum continues to evolve, gastrointestinal (GI) involvement and vertical transmission remain rarely reported features. Case Presentation: We describe a 13-year-old male from Georgia with a heterozygous missense mutation in SPTBN1 (c.3799C>G, p.Leu1267Val), inherited from his clinically unaffected mother. The patient presented with cognitive and motor delays, autistic traits, behavioral challenges, feeding difficulties, growth delay, musculoskeletal anomalies (pectus excavatum, pes planus), and persistent GI symptoms including abdominal pain and gallbladder sludge. Despite therapy with ursodeoxycholic acid and nutritional counseling, the gallbladder abnormalities persisted over a 12-month follow-up. Feeding therapy could not be initiated due to access barriers, although parental efforts led to modest dietary improvement. Upon recent visit pes planus was also confirmed. Conclusion: This case contributes to the growing body of literature on SPTBN1-related neurodevelopmental disorders, highlighting the potential for a broader phenotypic spectrum than currently recognized. In addition to the well-documented behavioral, cognitive, and musculoskeletal features, our patient presented with notable gastrointestinal involvement—specifically, gallbladder sludge and feeding disorder—which are not commonly reported in association with SPTBN1 mutations. Although causality cannot be established from a single case, the observed GI manifestations may be indirectly related to underlying neurobehavioral features such as ASD and associated eating disorders. The identification of a vertically transmitted missense variant (c.3799C>G, p.Leu1267Val) adds to the limited but growing evidence of inherited SPTBN1 mutations, expanding our understanding of transmission patterns. Given the novelty of the findings, we emphasize the need for multidisciplinary management, including feeding and behavioral therapy, as well as longitudinal follow-up to assess developmental, nutritional, and hepatobiliary outcomes. Future studies and case series are warranted to determine whether pes planus, pectus excavatum, gastrointestinal symptoms, including biliary tract involvement, represent an underrecognized component of the SPTBN1 phenotypic spectrum.

  Study EGAS50000001213

• German early-onset prostate cancer cohort of the Pan-Prostate Cancer Genome (PPCG) project

  Prostate cancer is the most frequent malignant tumor in males and the second most frequent cause of cancer-related death. Currently, in Germany, more than 60,000 prostate cancers are diagnosed every year. Although most of these patients are treated in a curative attempt, more than 10,000 German men die from prostate cancer annually. Owing to the demographic changes of our society, a further doubling of prostate cancer incidences during the next 20 years is expected. Prostate cancer is generally considered a tumor of elderly men. However, a fraction of prostate cancers are diagnosed at the age of 55 years or less. For several reasons, these “early onset prostate cancers” may represent a key entity for the understanding of prostate cancer biology. First, it is likely that early onset prostate cancers represent a distinct molecular subgroup of prostate cancer (PCa), potentially characterized by relatively small numbers of genetic changes, some of which may be particularly strong driver mutations for PCa development. Second, a fraction of prostate cancers in young individuals could represent classical prostate cancers that are detected at a very early stage and might therefore have accumulated molecular changes/mutations occurring that are most instrumental for prostate cancer early detection. Third, PCa with hereditary backgrounds are likely to accumulate in the age group below 55 years. A comparison with other sample sets (e.g. from other ICGC consortia), a systematic genomic analysis of young men with PCa could therefore lead to the detection of mechanisms for hereditary PCa. Fourth, a better understanding of these tumors is particularly relevant as finding optimal treatment regimens is most critical in young cancer patients. We are analyzing the entire genomic DNA sequences of at least 200 PCas (and matched non-tumorous DNA) of young men (≤ 55y), to at least 30 fold coverage, and integrate single-nucleotide variants and genomic structural variations with differential methylation, mRNA and miRNA expression data.Tissues were collected at the University Medical Center Hamburg-Eppendorf. Sequencing was performed at DKFZ and NCT (Heidelberg), EMBL (Heidelberg), and MPIMG (Berlin). Data management and bioinformatic data analyses is being conducted at DKFZ.

  Study EGAS00001003373

• Ancient Yakuts genomes and metagenomes

  Read data and bam alignments for 124 ancient yakuts

  Dataset EGAD50000001903

• Automated machine-learning approach for next generation profiling of sequence alterations, mutation burden, microsatellite instability, and structural variants in human cancers

  Sequence and structural alterations together with tumor mutation burden (TMB) and microsatellite instability (MSI) have been identified as biomarkers for the determination of response to targeted and immune checkpoint inhibitor therapies. However, widespread clinical adoption of these biomarkers has historically been limited due to barriers such as evidence of clinical utility and reimbursement. We have developed 2.2 Mb targeted NGS system and an automated machine-learning analysis approach (PGDx elio™ tissue complete, ETC) that has been FDA cleared for examination of 500+ cancer-related genes and 68 mononucleotide repeats for identification of sequence and structural alterations, TMB, and MSI in solid cancers in a clinical setting. We designed and trained this approach using sequence data from 4,174 cancers and >124,000 in silico alterations and evaluated the methodology in >2,550 tumor or non-cancerous normal samples. Independent analyses of ETC sequence changes in 440 formalin fixed paraffin embedded (FFPE) tumor or cell line samples using MSK-IMPACT™, FoundationOne®, and ddPCR revealed a positive percent agreement (PPA) >97% with high sensitivity as low as 3% mutant allele fraction. We observed high concordance between panel-wide and whole-exome TMB for 307 pan-cancer FFPE tumors (Pearson r=0.95, p < 0.0001) using samples with ≥20% tumor cellularity. Comparison of the mutation context and repeat-based MSI approach in ETC with a multiplex MSI PCR assay in 223 samples revealed a PPA of 99% and negative predictive agreement (NPA) >99%. We confirmed the accuracy and precision of TMB and MSI measurements across three independent laboratories (CV of <5% and average PPA >99%, respectively). Finally, evaluation of amplifications and translocations against DNA and RNA-based approaches exhibited >98% NPA and PPA of 86% and 82% respectively. These results demonstrate high analytical performance for determination of sequence and structural changes, TMB, and MSI using a targeted NGS panel and provide a scalable and facile approach for evaluating these biomarkers in a clinical laboratory.

  Study EGAS00001005556

• UK10K NEURO IOP COLLIER

  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 Genetics and Psychosis (GAP) set consists of samples from subjects with schizophrenia, ascertained as a new-onset sample. This set is of UK origin, with data on cognition, brain imaging and other endophenotypes. The Maudsley twin series consists of probands ascertained from the Maudsley Twin Register, defined as patients of multiple birth who had suffered psychotic symptoms. This set is of UK origin, with data on cognition, brain imaging and other endophenotypes, with DNA available from an MZ or DZ affected or unaffected co-twin. The Maudsley family study (MFS) consists of over 250 families who have a history of schizophrenia or bipolar disorder Within the Maudsley Family Study, biological markers of psychosis include neuropsychological tests, Evoked Response Potentials Tests (ERPs), MRI scans, dermatoglyphics and eye tracking. Early risk factors for psychosis and clinical symptoms are also investigated. This set is of UK origin, with DNA available from both affected and unaffected relatives in many of the probands. For further information on this study please contact David Collier (david.collier@kcl.ac.uk).

  Study EGAS00001000121

• UK10K NEURO GURLING

  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 consists of DNA from multiply affected schizophrenia families. The families have been diagnosed using the SADS-L clinical instrument which gives diagnoses at the probable level of the research diagnostic criteria (RDC). In addition all diagnoses are available using DSMIIIR criteria. These criteria are widely accepted as being valid and reliable for the diagnosis of schizophrenia. All families have been collected to ensure that they are uni-lineal for transmission of schizophrenia, i.e. they have only one affected parent with schizophrenia, or a relative of only one transmitting or obligate carrier parent with schizophrenia. Families with bi-lineal transmission of schizophrenia (i.e. with both parents being affected) were not sampled for this study. All families have multiple cases of schizophrenia and related disorders. The families have been selected to ensure there are no cases of bipolar disorder within them and that they do not contain bipolar disorder in any relatives on either side of the family.For further information on this cohort please contact Hugh Gurling (h.gurling@ucl.ac.uk).

  Study EGAS00001000225

• Therapeutic Resistance to PI3K-alpha Inhibitors

  We have studied the tumor genomic evolution in a patient with metastatic breast cancer bearing an activating PIK3CA mutation. The patient was treated with the PI3Kα inhibitor BYL719 and achieved a lasting clinical response on BYL719, but eventually progressed to treatment and died shortly thereafter. A rapid autopsy was performed and a total of 14 metastatic lesions were collected for further analysis. In order to identify possible genetic determinants of acquired resistance to PI3Kα inhibition, we took a three-step approach. First, we examined both the primary tumor (before BYL719 treatment) and the new lung metastasis by whole genome sequencing (DNA from the spleen was used as a normal control). Then, we analyzed the primary tumor, lung metastasis, and the peri-aortic lesion that remained stable (responding) at the time of progression to BYL719 therapy by whole exome sequencing. Finally, to confirm and expand our findings, we sequenced the primary tumor and all the metastatic lesions to >500-fold coverage using a custom targeted deep-sequencing assay, MSK-IMPACT. Using this 3-step approach, we have demonstrated that patient’s lesion underwent parallel genetic evolution at multiple metastatic sites with different PTEN genomic alterations leading to a convergent PTEN- null phenotype resistant to PI3Kα inhibition. In order to expand our observations, we analyzed paired samples (pre-treatment and at progression) from six additional patients enrolled in the BYL719 trial. Acquired bi-allelic loss of PTEN was found in one additional patient treated with BYL719 whereas in two patients PIK3CA mutations present in the primary tumor were no longer detected at the time of progression. To functionally characterize our findings, inducible PTEN knockdown in sensitive cells resulted in resistance to BYL719, while simultaneous PI3K-p110β blockade reverted this resistance phenotype, both in cell lines and in PTEN-null xenografts derived from our patient. We conclude that parallel genetic evolution of separate sites with different PTEN genomic alterations leads to a convergent PTEN-null phenotype resistant to PI3Kα inhibition.

  Study EGAS00001000991

• Genomic analysis of patient-derived xenograft models reveals intratumor-heterogeneity in endometrial cancer and can predict tumor growth inhibition with talazoparib

  Background: Endometrial cancer (EC) is a major gynecological cancer with increasing incidence. It comprised of four molecular subtypes with differing etiology, prognoses, and response to chemotherapy. In the future, clinical trials testing new single agents or combination therapies will be targeted to the molecular subtype most likely to respond. Pre-clinical models that faithfully represent the molecular subtypes of EC are urgently needed, we sought to develop and characterize a panel of novel EC patient-derived xenograft (PDX) models. Methods: Here, we report whole exome or whole genome sequencing of 11 PDX models and the matched primary tumor. Analysis of multiple PDX lineages and passages was performed to study tumor heterogeneity across lineages and/or passages. Based on recent reports of frequent defects in the homologous recombination (HR) pathway in EC, we assessed mutational signatures and HR deficiency scores and correlated these with in vivo responses to the PARP inhibitor (PARPi) talazoparib in six PDXs representing the copy number high/p53-mutant and mismatch-repair deficient molecular subtypes of EC. Results: PDX models were successfully generated with similar histomorphology from all four molecular subtypes of EC and uterine carcinosarcomas. We also observed a wide range of inter-tumor and intra-tumor heterogeneity across different PDX lineages, however one arm of most PDX models recapitulated the molecular landscape of the primary tumor without major genomic drift. An in vivo response to talazoparib was detected in four copy number high models. Two models (carcinosarcomas) showed a response consistent with stable disease and two models (one copy number high serous EC and another carcinosarcoma) showed significant tumor growth inhibition, albeit one consistent with progressive disease, however all lacked the HR deficiency genomic signature. Conclusions: EC PDX models represent the four molecular subtypes of disease and can capture intra-tumoral heterogeneity of the original primary tumor. PDXs of the copy number high molecular subtype showed sensitivity to PARPi, however, deeper and more durable responses will likely require combination of PARPi with other agents.

  Study EGAS00001004666

• Histological Transformation and Progression in Follicular Lymphoma: a Clonal Evolution Study

  Background: Follicular lymphoma (FL) is an indolent, yet incurable B-cell malignancy. A subset of patients experience increased mortality rate driven by two distinct clinical end points: histological transformation and early progression after immuno-chemotherapy. The nature of tumor clonal dynamics leading to these clinical endpoints is poorly understood and previously determined genetic alterations do not explain the majority of transformed cases or accurately predict early progressive disease. We contend that detailed knowledge of the expansion patterns of specific cell populations plus their associated mutations would provide insight into therapeutic strategies and disease biology over the timecourse of FL clinical histories. Methods and Findings: Using a combination of whole genome sequencing, targeted deep sequencing and digital droplet PCR on matched diagnostic and relapse specimens, we deciphered the constituent clonal populations in n=15 transformation patients and n=6 progressors, and measured their change in abundance over time. We observed widely divergent patterns of clonal dynamics in transformed cases relative to progressed cases. Transformation specimens were generally composed of clones that were rare or absent in diagnostic specimens, consistent with dramatic clonal expansions that came to dominate the transformation specimen. This pattern was independent of time to transformation and treatment modality. By contrast, progression specimens were composed of clones that were already present in the diagnostic specimens and exhibited only moderate clonal dynamics, even in the presence of immuno-chemotherapy. Analysis of somatic mutations impacting 94 genes was undertaken in a validation cohort consisting of 395 samples from 277 patients in order to decipher disrupted biology in the two clinical endpoints. We found 12 genes were more commonly mutated in transformed samples than in preceding FL, including TP53, B2M, CCND3, GNA13, S1PR2 and P2RY8. Moreover, 10 genes were more commonly mutated in diagnostic specimens of early progressors including TP53, BTG1, MKI67 and XBP1. Conclusions: Our results illuminate contrasting modes of evolution shaping the clinical histories of transformation and progression. They have implication for interpretation of evolutionary dynamics in the context of treatment-induced selective pressures, and indicate that transformation and progression will require different clinical management strategies.

  Study EGAS00001001709

• Foundation Medicine Genomic Data Used to Identify Prognostic Markers and Fusion Genes in Multiple Myeloma

  The purpose of this study is to identify prognostic markers and treatment targets using a clinically certified sequencing panel in multiple myeloma. Mutational burden was associated with maf and proliferation gene expression groups, and a high-mutational burden was associated with a poor prognosis. We identified homozygous deletions that were present in multiple myeloma within key genes, including CDKN2C, RB1, TRAF3, BIRC3 and TP53, and that bi-allelic inactivation was significantly enriched at relapse. Alterations in CDKN2C, TP53, RB1 and the t(4;14) were associated with poor prognosis. Alterations in RB1 were predominantly homozygous deletions and were associated with relapse and a poor prognosis which was independent of other genetic markers, including t(4;14), after multivariate analysis. Bi-allelic inactivation of key tumor suppressor genes in myeloma was enriched at relapse, especially in RB1, CDKN2C and TP53 where they have prognostic significance. In addition, chromosomal rearrangements that result in oncogenic kinase activation are present in many solid and hematological malignancies, but none have been reported in multiple myeloma (MM). Here we detected fusion genes in 1.5% of patients. These fusion genes were in-frame and the majority of them contained kinase domains from either receptor tyrosine kinases (ALK, ROS1, NTRK3, and FGFR1) or cytoplasmic kinases (BRAF, MAP3K14, and MAPK14) which would result in the activation of MEK/ERK, NF-κB or inflammatory signaling pathways. Fusion genes were present in smoldering MM, newly diagnosed MM and relapse patient samples indicating they are not solely late events. Most fusion genes were subclonal in nature, but one EML4-ALK fusion was clonal indicating it is a driver of disease pathogenesis. Samples with fusions of receptor tyrosine kinases were not found in conjunction with clonal Ras/Raf mutations indicating a parallel mechanism of MEK/ERK pathway activation. Fusion genes involving MAP3K14 (NIK), which regulates the NF-κB pathway, were detected as were t(14;17) rearrangements involving NIK in 2% of MM samples. Activation of kinases in myeloma through rearrangements presents an opportunity to use treatments existing in other cancers.

  Study EGAS00001002874

• Scottish High Grade Serous Ovarian Cancer

  Scottish HGSOC samples were collected via local Bioresource facilities at Edinburgh, Glasgow, Dundee and Aberdeen and stored in liquid Nitrogen until required. HGSOC patients were determined from pathology records and were included in the study where there was matched tumour and whole blood samples. On receipt of tumour material the tumour was processed as follows: firstly, the tumour sample was divided into two for DNA and RNA extraction. Slivers of tissue were cut from the front and rear faces of the DNA sample, then fixed in formalin and embedded in paraffin wax. Sections from the front and rear tissues from all samples were examined by H&E staining supplemented by WT1/p53 immunohistochemistry if required. Following pathology review, samples were only included if they met the following criteria: they were confirmed as HGSOC and there was greater than 40% tumour cellularity throughout the tumour, determined using the H&E sections. Somatic DNA was extracted using the Qiagen DNeasy Blood and tissue kit (cat no 69504). The tissue was initially homogenised using a Qiagen Bioruptor, followed by the manufacturers recommended protocol (including RNase digestion step). Germline DNA was extracted from 1-3ml whole blood using the Qiagen FlexiGene kit (cat no 51206) following the manufacturers recommended protocol. The resulting DNA underwent quality control as follows: firstly, A260 and A280nm were measured on a Denovix DS-11 Fx to qualitatively illustrate A260/280nm and A260/230nm ratios as surrogate measures of DNA purity. A260/280 had to be 1.8 or greater and A260/230 had to be 2.0 or greater. Then, DNA was quantified using LifeTechnologies Qubit dsDNA BR kit (cat no Q32850) and we required a minimum of 50ul at 25ng/ul for WGS. Thirdly, DNA was diluted to 25ng/ul and a representative sample was loaded onto a 0.8% TAE gel, ran at 100v for 60mins and then imaged using a BioRad ChemiDoc imaging system to visualise the DNA quality. Only when all 4 quality control requirements were satisfied was the DNA sequenced. The DNA was sequenced at the Glasgow Precision Oncology Laboratories.

  Study EGAS00001004410

• Germline elongator mutations in sonic hedgehog medulloblastoma

  Cancer genomics has illuminated a wide spectrum of genes and core molecular processes contributing to human malignancy. Still, the genetic and molecular basis of many cancers remains only partially explained. Genetic predisposition accounts for 5-10% of cancer diagnoses and genetic events cooperating with known somatic driver events are poorly understood. Analyzing established cancer predisposition genes in medulloblastoma (MB), a malignant childhood brain tumor, we recently identified pathogenic germline variants that account for 5% of all MB patients. Here, by extending our previous analysis to include all protein-coding genes, we discovered and replicated rare germline loss-of-function (LoF) variants across Elongator Complex Protein 1 (ELP1) on 9q31.3 in 15% of pediatric MBSHH cases, thus implicating ELP1 as the most common MB predisposition gene and increasing genetic predisposition to 40% for pediatric MBSHH. Inheritance was verified based on parent-offspring and pedigree analysis, which identified two families with a history of pediatric MB. ELP1-associated MBs were restricted to the molecular SHHa subtype and were characterized by universal biallelic inactivation of ELP1 due to somatic loss of chromosome 9q. The majority of ELP1-associated MBs exhibited co-occurring somatic PTCH1 (9q22.32) alterations, suggesting that ELP1-deficiency predisposes to tumor development in combination with constitutive activation of SHH signaling. ELP1 is an essential subunit of the evolutionary conserved Elongator complex, whose primary function is to enable efficient translational elongation through tRNAs modifications at the wobble (U34) position. Biochemical, transcriptional, and proteomic analyses revealed that ELP1-associated MBSHH are characterized by a destabilized core Elongator complex, loss of Elongator-dependent tRNA modifications, codon-dependent translational reprogramming, and induction of the unfolded protein response (UPR), consistent with deregulation of protein homeostasis due to Elongator-deficiency in model systems. Our findings suggest that genetic predisposition to proteome instability is a previously underappreciated determinant in the pathogenesis of pediatric brain cancer. These results provide strong rationale for further investigating the role of protein homeostasis in other pediatric and adult cancer types and potential opportunities for novel therapeutic interference.

  Study EGAS00001004126

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

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

  Study EGAS00001005836

• Evolution and clinical impact of genetic epistasis within EGFR-mutant lung cancers

  The current understanding of tumorigenesis is largely centered on a monogenic driver oncogene model. This paradigm is incompatible with the prevailing clinical experience in most solid malignancies: monotherapy with a drug directed against an individual oncogenic driver typically results in incomplete clinical responses and eventual tumor progression1-7. By profiling the somatic genetic alterations present in over 2,000 cases of lung cancer, the leading cause of cancer mortality worldwide, we show that combinations of functional genetic alterations, i.e. genetic collectives dominate the landscape of advanced-stage disease. We highlight this polygenic landscape and evolution of advanced-stage non-small cell lung cancer (NSCLC) through the spatial-temporal genomic profiling of 7 distinct tumor biopsy specimens and 6 plasma specimens obtained from an EGFR-mutant NSCLC patient at (1) initial diagnosis of early-stage disease, (2) metastatic progression, (3) sequential treatment and resistance to 2 EGFR inhibitors, (4) death. The comprehensive genomic analysis of this case, coupled with circulating free (cf) tumor DNA profiling of additional advanced-stage EGFR-mutant NSCLC clinical cohorts with associated treatment responses uncovered features of evolutionary selection for multiple concurrent gene alterations: including the presence of EGFR inhibitor-sensitive (EGFRL858R;EGFRexon19del) or inhibitor-resistant (EGFRT790M;EGFRC797S) forms of oncogenic EGFR along with cell cycle gene alterations (e.g. in CDK4/6, CCNE1, RB1) and activating alterations in WNT/β-catenin and PI3K pathway genes, which our data suggest can cooperatively impart non-redundant functions to limit EGFR targeted therapy response and/or promote tumor progression. Moreover, evidence of an unanticipated parallel evolution of both EGFR T790M and two distinct forms of oncogenic PIK3CA was observed. Our study provides a large-scale clinical and genetic dataset of advanced-stage EGFR-mutant NSCLC, a rationale for specific polytherapy strategies such as EGFR and CDK4/6 inhibitor co-treatment to potentially enhance clinical outcomes, and prompts a re-evaluation of the prevailing paradigm of monogenic-based molecular stratification for targeted therapy. Instead, our findings highlight an alternative model of genetic collectives that operate through epistasis to drive lung cancer progression and therapy resistance.

  Study EGAS00001002604

• 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

• Characterization of Cell Free Plasma Methyl-DNA From Xenografted Tumors to Guide the Selection of Diagnostic Markers for Early-Stage Cancers

  Circulating cell-free methyl-DNA (mcfDNA) contains promising cancer markers but its low abundance and possibly diverse origin pose challenges toward the accurate diagnosis of early stage cancers. By whole-genome bisulfite sequencing (WGBS) of cell-free DNA (cfDNA) from about 0.5 mL plasma of mice xenografted with human tumors, we obtained and aligned the reads to the human genome, filtered out the mouse and carrier bacterial sequences, and confirmed the tumor origin of methyl-cfDNA (mctDNA) by methylation-sensitive restriction enzyme digestion prior to species-specific PCR. We estimated that human tumor-specific reads (ctDNA) or mctDNA comprised about 0.29 or 0.01%, respectively of the xenograft mouse cfDNA, and about 0.029 or 0.001% of the cfDNA of human early stage cancer patients. Similar WGBS of early stage (0-II, node- and metastasis-free) breast, lung or colorectal cancer samples identified hundreds of specific DMRs (differentially methylated regions) compared to healthy controls. Their association with tumourigenesis was supported by stage-dependent methylation, tumor suppressor or oncogene clusters, and genes also identified in the xenograft samples. Using 20 three-cancer-common and 17 colorectal cancer-specific DMRs in combination (top 0.0018% of the WGBS methylation clusters) was sufficient to distinguish the stage I colorectal cancers from breast and lung cancers and healthy controls. Our data thus confirmed the tumor origin of mctDNA by sequence specificity, and provide a selection threshold for authentic tumor mctDNA markers toward precise diagnosis of early stage cancers solely by top DMRs in combination. Biological Materials were provided by the Ontario Tumour Bank, which is funded by the Ontario Institute for Cancer Research Reference for citation: Ling Liu, Jinghua Feng, Julian Polimeni, Manli Zhang, Hai Nguyen, Urmi Das, Xu Zhang, Harminder Singh, Xiao-Jian Yao, Etienne Leygue, Sam K.P. Kung, and Xie J. Characterization of cell free plasma methyl-DNA from xenografted tumours to guide the selection of diagnostic markers for early-stage cancers. Frontiers in Oncology, 2021 Feb. 5. DOI: 10.3389/fonc.2021.615821.

  Study EGAS00001006175

• Chromatin profiles classify castration-resistant prostate cancers suggesting therapeutic targets

  Untreated prostate cancers rely on androgen receptor (AR) signaling for growth and survival, forming the basis for the initial efficacy of androgen deprivation therapy (ADT). Yet, the disease can relapse and progress to a lethal stage termed castration-resistant prostate cancer (CRPC). Reactivation of AR signaling represents the most common driver of CRPC growth and next generation AR signaling inhibitors (ARSIs) are now used in combination with ADT as a first line therapy. However, ARSIs can result in selective pressure generating AR-independent tumors. The transition from AR-dependence frequently accompanies a change in phenotype resembling developmental trans-differentiation or ‘lineage plasticity’. Neuroendocrine prostate cancer, which lacks a defined pathologic classification, is the most studied type of lineage plasticity. However, most AR-null tumors do not exhibit neuroendocrine features and are classified as ‘double-negative prostate cancer’, the drivers of which are poorly defined. Lineage plasticity studies in CRPC are limited by the lack of genetically defined patient-derived models that recapitulate the disease spectrum. To address this, we developed a biobank of organoids generated from patient biopsies to study the landscape of metastatic CRPC and allow for functional validation assays. Proteins called transcription factors (TFs) are drivers of tumor lineage plasticity. To identify the key TFs that drive the growth of AR-independent tumors, we integrated epigenetic and transcriptomic data generated from CRPC models. By presenting a map of the chromatin accessibility and transcriptomic landscape of CRPC using a diverse biobank of organoids, PDXs and cell lines that recapitulate the heterogeneity of metastatic prostate cancer, we validate CRPC-AR and CRPC-NE subtypes and reveal two subtypes of AR-negative/low samples as well as their respective masterTFs. Additional analysis revealed a model in which YAP, TAZ, TEAD and AP-1 function together and drive oncogenic growth in CRPC-SCL samples. Overall, we show here how an approach to stratify CRPC patients into four subtypes using their transcriptomic signatures can potentially inform upon appropriate clinical decisions.

  Study EGAS00001006059

• HCA_Thymus_Disease_DiGeorge_Transplant_RNA

  Single cell transcriptomics study of thymic transplant biopsies Allogeneic thymus transplantation is the only curative therapy for complete DiGeorge Syndrome (cDGS), a rare severe primary immunodeficiency characterised by athymia. GOSH is one of only two centres worldwide to offer this treatment. Despite a lack of major histocompatibility complex (MHC)-matching between donor and host, transplanted thymus becomes repopulated by recipient bone marrow derived precursor cells and supports development of functional T-cells. The mechanisms underlying thymopoiesis in this context are poorly understood, but over time we observe reconstitution of T-cell immunity, with the ability to produce host naïve T-cells showing a broad T-cell receptor (TCR) repertoire and to generate MHC-restricted T-cell proliferative responses. Although lifesaving, the achieved immunological reconstitution is typically not complete with circulating T-cell numbers usually remaining below the age related normal ranges. Additionally, we observe persistence of donor-derived T-cells of unknown clinical significance. To gain more insight into the mechanisms by which MHC-mismatched transplanted thymus supports T-cell development with self-tolerance, as well as into the basis of suboptimal T-cell immunity, we now aim to investigate immune reconstitution after thymus transplantation in further detail by using single-cell transcriptomics, applied to thymic transplant biopsies and peripheral blood samples collected during standard post-transplant patient care. By identifying which lineages of host- derived cells repopulate the thymic tissue after transplantation, we will be able to address the role of MHC in positive and negative T-cell selection during T-cell differentiation. We will also be able to clarify the exact ontogeny of the persistent donor T-cells, as well as their possible role. Understanding the mechanisms of action of HLA-mismatched transplanted thymus will contribute to treatment optimisation. Additionally, our research provides a unique opportunity to further investigate key immunological concepts, such as tolerance and autoimmunity, challenging existing paradigms in thymus immunology. 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 EGAS00001004025

• High-resolution testing of ctDNA dynamics predicts survival in metastatic NSCLC

  One of the great challenges of therapeutic oncology is determining who might achieve survival benefit from a particular therapy. Circulating tumor DNA (ctDNA) provides real-time assessments of patient prognosis and response to treatment using a simple blood draw. While ctDNA positivity is established as a poor prognostic factor, studies on longitudinal ctDNA dynamics have been small and non-randomized, with ctDNA assessments done at disparate time points. To address this, we performed high-sensitivity longitudinal ctDNA testing in 466 patients across 5 time points (1,954 samples total) in a randomized phase III study comparing different chemotherapy-immunotherapy combinations. We leverage machine learning to jointly model multiple ctDNA metrics to predict overall survival in a training/testing framework. _ . Treatment initiation correlated with reductions in ctDNA levels, and training of our machine learning model suggests that assessment of ctDNA dynamics at C3D1 (cycle 3 day 1) of chemo-IO treatment may be optimal to predict OS. The model performs well in the hold-back test data, enabling stratification of patients with Stable Disease (SD) into high-risk vs low-intermediate-risk (HR = 3.2 [2.0-5.3], p <0.001; median 7.1 versus 22.3 months respectively); similarly, the model stratifies patients with a Partial Response (PR) (HR =3.3 [1.7-6.4], p <0.001; median 8.8 versus 28.6 months). Importantly, the model validates well in an external cohort of patients in a different treatment setting and assayed with a different ctDNA technology, in which model predictions similarly identified high-risk patients (OS HR=3.73 [1.83-7.60], logrank p=0.00012). Simulations of clinical trial scenarios employing our ctDNA model further suggest that early ctDNA testing outperforms early radiographic imaging for predicting trial outcomes (increasing the rate of ‘True Go’ decisions by 5.2 - 22.8% depending on the drug combination). Overall, we show that measuring ctDNA dynamics during the course of therapy dramatically improves patient risk stratification, and may provide a means to differentiate between competing therapies at an early time point during clinical trials.

  Study EGAS00001006703

• Influence of Genomic Landscape on Cancer Immunotherapy for Newly Diagnosed Ovarian Cancer: Biomarker Analyses from the IMagyn050 Randomized Clinical Trial

  The IMagyn050 trial (NCT03038100), which evaluated the efficacy of Atezo vs placebo (Pla) with carboplatin, paclitaxel and bevacizumab (CPB) in front line ovarian cancer patient (pts), did not meet its co-primary endpoints of PFS in ITT or PD-L1+ (Moore et al. JCO 2021). The aim of this exploratory study was to evaluate whether patients with BRCA1/2-mutated or homologous recombination-deficient (HRD) ovarian cancers benefitted from atezolizumab in the phase III IMagyn050 (NCT03038100) trial. Methods: Patients with newly diagnosed ovarian cancer were randomized to either atezolizumab or placebo with standard chemotherapy and bevacizumab. PD-L1 status of tumor-infiltrating immune cells was determined centrally (VENTANA SP142 assay). Genomic alterations, including deleterious BRCA1/2 alterations, genomic loss of heterozygosity (gLOH), tumor mutation burden (TMB), and microsatellite instability (MSI), were evaluated using the FoundationOne assay. HRD was defined as gLOH ≥16%, regardless of BRCA1/2 mutation status. Potential associations between progression-free survival (PFS) and genomic biomarkers were evaluated using standard correlation analyses and log-rank of Kaplan-Meier estimates. Results Among biomarker-evaluable samples, 22% (234/1050) harbored BRCA1/2 mutations and 46% (446/980) were HRD. Median TMB was low irrespective of BRCA1/2 or HRD. Only 3% (29/1024) had TMB ≥10 mut/Mb and 0.3% (3/1022) were MSI-high. PFS was better in BRCA2-mutated versus BRCA2-non-mutated tumors and in HRD versus proficient tumors. PD-L1 positivity (≥1% expression on immune cells) was associated with HRD but not BRCA1/2 mutations. PFS was not improved by adding atezolizumab in BRCA2-mutated or HRD tumors; there was a trend toward enhanced PFS with atezolizumab in BRCA1-mutated tumors. Conclusion Most ovarian tumors have low TMB despite BRCA1/2 mutations or HRD. Neither BRCA1/2 mutation nor HRD predicted enhanced benefit from atezolizumab. This is the first randomized double-blind trial in ovarian cancer demonstrating that genomic instability triggered by BRCA1/2 mutation or HRD is not associated with improved sensitivity to immune checkpoint inhibitors.

  Study EGAS00001006838

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

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

  Study EGAS00001006844

• METABRIC

  The elucidation of breast cancer subgroups and their molecular drivers requires integrated views of the genome and transcriptome from representative numbers of patients. We present an integrated analysis of copy number and gene expression in a discovery and validation set of 997 and 995 primary breast tumours, respectively, with long-term clinical follow-up. Inherited variants (CNVs, SNPs) and acquired somatic copy number aberrations (CNAs) were associated with expression in 40% of genes, although the landscape was dominated by cis and trans-acting CNAs. By delineating expression outlier genes driven in cis by CNAs, we identified putative cancer genes, including deletions in PPP2R2A, MTAP, and MAP2K4. Unsupervised analysis of paired DNA/RNA profiles revealed novel subgroups with distinct clinical outcomes, which reproduced in the validation cohort. These include a high-risk, ER-positive 11q13/14 cis-acting subgroup and a favourable prognosis subgroup devoid of CNAs. Trans-acting aberration 0152hotspots were found to modulate subgroup-specific gene networks, including a TCR deletion-mediated adaptive immune response in the 0152CNA-devoid sub-group and a Basal-specific chromosome 5 deletion-driven mitotic network. Our results provide a novel molecular stratification of the breast cancer population, derived from the impact of somatic copy number aberrations on the transcriptome.

  Study EGAS00000000083

• APOBEC mutagenesis is a common process in normal human small intestine

  The single base substitution mutational signatures SBS2 and SBS13, likely caused by APOBEC cytosine deaminases, are common in many human cancer types. However, the stimulus activating APOBEC mutagenesis is unknown and understanding of when it occurs in the progression from normal to cancer cell is limited. Here, as part of a wider survey of human tissues, we whole genome sequenced 342 microdissected normal epithelial crypts from the small intestines of 39 individuals. SBS2/13 mutations were present in 17% normal small intestine crypts and were likely due to APOBEC3A activity. Localised clusters of SBS2/13 mutations (kataegis) were also commonly found. APOBEC mutation burdens were variable between individuals and between crypts from the same individual. Crypts with SBS2/13 often had immediate crypt neighbours without SBS2/13, suggesting that the underlying cause of SBS2/13 is cell-intrinsic rather than a widely distributed microenvironmental exposure, or needs to be permitted by cell-intrinsic conditions. APOBEC mutagenesis occurred throughout the human lifespan, including in young children, and was episodic with a small number of episodes occurring during the life history of a single cell. The results indicate that APOBEC mutagenesis is more common in the small intestine epithelium than in many other cell types, and is an episodic process in vivo initiated or permitted by cell intrinsic factors.

  Dataset EGAD00001008764

• Effects of 2DG, Galactose, or Oligomycin on the epigenome remodeling induce by T cells activation.

  T cell activation is known to require a metabolic shift towards glycolysis, triggered by TCR-engagement . To substantiate the role of glycolysis in transcriptional reprogramming during antigen-driven T cell activation, we utilized a well-established ex-vivo model to recapitulate the process of antigen stimulation without the requirement for additional antigen-presenting cells . We initially explored and validated the conditions for both metabolic and epigenetic reprogramming. Antigen-mediated CD4+ T cells activation initiates context-specific gene-expression programs that drive effector functions and cell fates through changes in the epigenetic landscape . To evaluate global changes in histone modifications, we isolated CD4+ T cells from peripheral blood (PB) of healthy control (HC) human donors and either maintained them in resting conditions or stimulated with anti-CD3/CD28 for 24h. Rapid changes in the CD4+ epigenome and transcriptome were observed within 24h as measured by RNA-sequencing (RNA-seq) and H3K27ac chromatin immunoprecipitation followed by DNA-sequencing (ChIP-seq).To explore the requirement for metabolic reprogramming in regulating transcriptional responses after initial TCR engagement, CD4+ T cells from PB of healthy donors were isolated and activated in either the presence or absence of oligomycin (inhibitor of ATP synthase blocking the production of ATP by OXPHOS), 2-deoxyglucose (2DG; competitively inhibiting production of glucose-6-phosphate from glucose), or replacement of glucose with galactose. Galactose is metabolized through glycolysis, utilizing the Leloir pathway which is energy neutral, forcing cells to use all the pyruvate production to sustain the TCA cycle for OXPHOS and ATP synthesis at the expense of lactate production. Employing ChIP-seq and RNA-seq, inhibition of OXPHOS-mediated ATP production by oligomycin had only a limited effect on H3K27-acetylation or activation-induced transcription during the first 24h of activation. In contrast, inhibition of glycolysis by 2DG treatment compromised both H3K27-acetylation and transcriptional changes. However, under conditions of galactose-metabolism where lactate production is abrogated, activated CD4+ T cells were still able to globally remodel H3K27 acetylation and reprogram the transcriptome.

  Study EGAS00001007115

• 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

• Lindsey E. Jones et al. Patient-derived cells from recurrent tumors that model the evolution of IDH-mutant glioma. Neuro-Oncology Advances, 2020. We established robust, versatile, and well-characterized Patient Derived Cells (PDC) of an IDH1-mutant astrocytoma and an IDH1-mutant oligodendroglioma that represents defined evolutionary stages of chemotherapy-induced hypermutation. The PDCs retain tumor subtype defining features over time as well as classic hallmarks of cancer, including anchorage independent growth and cell immortality. The integrated phylogenies composed of PDCs,single-cell-derived PDCs, patient-derived xenografts, and corresponding spatiotemporal tumor tissue samples also provide new insight into the clonality, evolutionary pattern, and immense mutational load of hypermutated IDH mutant gliomas.

  IDH-mutant lower-grade gliomas can undergo malignant progression via temozolomide-driven hypermutation. Patient-derived cells (PDC) that model the genetically distinct hypermutated (HM) tumor subgroup are generally lacking, and few if any human brain tumor cell models are from defined evolutionary time points. Here, we characterize multiple PDC derived from independent surgical specimens of IDH1-mutant recurrences, including an ATRX and TP53-mutant astrocytoma and a 1p/19q co-deleted and TERT promoter-mutant oligodendroglioma. We determined the evolutionary time points represented by each PDC using exome sequencing and phylogenetic reconstruction, comparing the PDC and single cell clones of the PDC (scPDC) to multiple spatiotemporal tumor tissue samples, PDC-derived xenografts (PDX) and patient-matched blood. The tumor samples exhibited TMZ-induced mutagenesis and a branching pattern of evolution. We found clear evidence of two fully independent founder HM clones in the tumor tissue that are faithfully represented by independent PDC. The PDC, scPDC and PDX also shared the mutagenesis signature and represent the mid and later evolutionary time points of their corresponding tumors. The PDC maintained the tumor subtype-defining features over many passages, including heterozygosity of the IDH1 R132H mutation, production of 2-hydroxyglutarate (2-HG), and subtype-specific telomere maintenance mechanisms. The PDC from both tumors exhibited anchorage-independent growth in soft agar. The oligodendroglioma PDC formed infiltrative intracranial tumors with characteristic oligodendroglioma histology, initially with a long period to tumor formation. We conclude that the PDC, scPDC and PDX faithfully model the heterogeneous clonal origins of the corresponding tumor tissue. The multilevel analysis also provides new insight into the intratumoral heterogeneity and vast mutational load of HM glioma. The PDC from multiple evolutionary time points presented in the context of full clinical timelines may be useful to model evolution and intratumoral heterogeneity, important sources of therapeutic failure.

  Study EGAS00001003992

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

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

  Study EGAS00001004832

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

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

  Study EGAS00001004833

• Multi-omic and functional analysis for classification and treatment of sarcomas with FUS-TFCP2 or EWSR1-TFCP2 fusions(H021/INF)

  Many rare cancers are not well understood, and pathogenesis-directed therapies are often lacking, resulting in poor patient outcomes. Leveraging two (inter)national precision oncology trials that enroll large numbers of rare cancers, we investigated the clinical, histopathologic, molecular, and functional characteristics of rhabdomyosarcoma (RMS) with fusions of FUS or EWSR1 to the TFCP2 transcription factor, a recently discovered ultra-rare entity whose classification, pathogenesis, and optimal treatment are unclear. Unusually for fusion-driven sarcomas, most cases had highly rearranged genomes, including chromothripsis, and signs of defective homologous recombination DNA repair. All tumors showed extreme expression of a truncated TERT variant and the ALK receptor tyrosine kinase, which was additionally affected by intragenic deletions and aberrant splicing, resulting in the expression of shortened variants in 58% of cases. Three ALK variants were oncogenic in immortalized cells, and patient-derived tumor cells expressing two variants responded to certain ALK inhibitors. Other recurrent alterations included CDKN2A/MTAP co-deletions and mutations in PAPPA2, encoding an IGFBP5-specific proteinase, in 67% and 25% of cases, respectively. DNA methylation analysis, along with 19 other soft-tissue sarcoma classes, revealed a close relationship with undifferentiated sarcoma but not other RMS subtypes, suggesting that TFCP2-rearranged RMS is a separate entity, possibly arising from a distinct cell of origin. Expression of TFCP2 fusions in immortalized human cells blocked late myogenic differentiation and significantly induced genes that were also highly expressed in patient tumors, including ALK, TERT, and two known regulators of skeletal muscle cells, IGFBP5 and PTH1R. Genome-wide chromatin profiling demonstrated direct binding of FUS-TFCP2 to the ALK and TERT loci outside their regular promoters, which correlated with the expression of alternative transcript variants. Finally, FUS-TFCP2 inhibited the repair of DNA double-strand breaks in immortalized cells, rendering them sensitive to treatment with cisplatin. This study provides insight into the pathogenesis and therapeutic vulnerabilities of a new RMS subtype and illustrates the value of linking comprehensive molecular diagnostics and functional annotation within multicenter consortia to improve the understanding and clinical management of rare cancers.

  Study EGAS00001006939

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

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

  Study EGAS00001007077

• The Haemgen RBC study

  Anaemia is a major determinant of global ill-health. To refine our understanding of the genetic factors influencing red blood cell formation and function, we carried out a meta-analysis of genome-wide association studies (GWAS) for six red blood cell traits: haemoglobin (HB), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), mean cell volume (MCV), packed cell volume (PCV) and red blood cell count (RBC). We provide genome-wide association results for 62,553 people of European ancestry using up to 2,644,161 autosomal SNPs. Participants with extreme measurements (>+/-3SD from mean) were excluded on a per phenotype basis. Imputation was done using haplotypes from HapMap Phase 2. SNP associations with each phenotype were tested by linear regression using an additive genetic model. Associations were tested separately in each cohort, with principal components and other study specific factors as covariates to account of population substructure. We then carried out meta-analysis of results from the individual cohorts using z-scores weighted by square root of sample size. SNPs with MAF<1% (weighted average across cohorts) were removed, as were SNPs with weight <50% of phenotype sample size. Anaemia is a major determinant of global ill-health. To refine our understanding of the genetic factors influencing red blood cell formation and function, we carried out a meta-analysis of genome-wide association studies (GWAS) for six red blood cell traits: haemoglobin (HB), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), mean cell volume (MCV), packed cell volume (PCV) and red blood cell count (RBC). We provide genome-wide association results for 62,553 people of European ancestry using up to 2,644,161 autosomal SNPs. Participants with extreme measurements (>+/-3SD from mean) were excluded on a per phenotype basis. Imputation was done using haplotypes from HapMap Phase 2. SNP associations with each phenotype were tested by linear regression using an additive genetic model. Associations were tested separately in each cohort, with principal components and other study specific factors as covariates to account of population substructure. We then carried out meta-analysis of results from the individual cohorts using z-scores weighted by square root of sample size. SNPs with MAF<1% (weighted average across cohorts) were removed, as were SNPs with weight <50% of phenotype sample size.

  Study EGAS00000000132

• Warm_autopsy__mutational_signatures_and_clonal_units

  Aims and objectives 1. To produce a ‘first generation map’ of mutational burdens and mutational processes that operate in normal human tissues. 2. To examine clonal units and their distribution across normal human tissues with the emphasis on these particular areas: a) Tissues with an architecturally defined unit: Are architectural structures populated by a progeny derived from a single stem cell? Are clonal units confined to a given architectural structure? Examples of tissues where such approach will be used include prostate gland (acini and ducts), small bowel (crypts), thyroid gland (follicles) and others. b) Tissues without an architecturally defined unit: What is the average size of clonal populations? This group includes stratified/pseudostratified epithelia with a basal layer within which there are stem cells that give rise to a progeny of cells showing upwards migration and lateral expansion (e.g. urothelium, cervical squamous epithelium, respiratory epithelium). This approach can also be used in tissues with a sheet-like cell arrangement, for example brain and adrenal gland. Methods To answer these questions, we will use a range of normal human tissue samples which have already been collected from a single deceased individual. The samples are currently stored in a -80C freezer. Ethanol fixed frozen sections will be prepared from these biopsies. Putative clonal units will be identified and dissected out using laser capture microscopy. DNA extraction will be performed using the in-house (CGP) protease based method. In terms of sequencing methods, the project will be split into 2 phases (2 different approaches): - Phase 1: This part of the project will utilise a combination of low input library preparation (developed by Peter Ellis) and standard whole genome sequencing (HiSeqX). The phase has already started (December 2016). This approach will be particularly useful in investigation of mitotically active tissues. - Phase 2: This part of the project will utilise a combination of low input library method (Peter Ellis) and Bottle-neck sequencing (BotSeqS). BotSeqS is a type of duplex sequencing method that offers a more accurate variant calling and allows genome interrogation at a single cell level. This method is currently being developed and is expected to be implemented in 3-6 months (March-April 2017). This approach will be particularly useful for post-mitotic tissues and tissues without pre-defined architectural units.

  Study EGAS00001002216

• SABE_1171_WGS_GVCF_IDX

  Dataset EGAD00001008203

• CRCbiome: Gut metagenome of Norwegian screening participants using FIT sampling

  The study aims to characterize the gut metagenome of Norwegian screening participants and assess the ability of the gut metagenome to identify those at need for colonoscopy. Background: Colorectal cancer (CRC) screening reduces CRC incidence and mortality. However, current screening methods are either hampered by invasiveness or suboptimal performance, limiting their effectiveness as primary screening methods. To aid in the development of a non-invasive screening test with improved sensitivity and specificity, we have initiated a prospective biomarker study (CRCbiome), nested within a large randomized CRC screening trial in Norway. We aim to develop a microbiome-based classification algorithm to identify advanced colorectal lesions in screening participants testing positive for an immunochemical fecal occult blood test (FIT). We will also examine interactions with host factors, diet, lifestyle and prescription drugs. The prospective nature of the study also enables the analysis of changes in the gut microbiome following the removal of precancerous lesions. Methods: The CRCbiome study recruits participants enrolled in the Bowel Cancer Screening in Norway (BCSN) study, a randomized trial initiated in 2012 comparing once-only sigmoidoscopy to repeated biennial FIT, where women and men aged 50–74 years at study entry are invited to participate. Since 2017, participants randomized to FIT screening with a positive test result have been invited to join the CRCbiome study. Self-reported diet, lifestyle and demographic data are collected prior to colonoscopy after the positive FIT-test (baseline). Screening data, including colonoscopy findings are obtained from the BCSN database. Fecal samples for gut microbiome analyses are collected both before and 2 and 12 months after colonoscopy. Samples are analyzed using metagenome sequencing, with taxonomy profiles, and gene and pathway content as primary measures. CRCbiome data will also be linked to national registries to obtain information on prescription histories and cancer relevant outcomes occurring during the 10 year follow-up period. Discussion: The CRCbiome study will increase our understanding of how the gut microbiome, in combination with lifestyle and environmental factors, influences the early stages of colorectal carcinogenesis. This knowledge will be crucial to develop microbiome-based screening tools for CRC. By evaluating biomarker performance in a screening setting, using samples from the target population, the generalizability of the findings to future screening cohorts is likely to be high.

  Study EGAS50000000170

• Small RNA sequencing of endometrial cancer patients with samples collected prior to diagnosis and controls

  This dataset contains unpaired FASTQ files of 316 endometrial cancer cases and 316 matched controls representing circulating RNAs. RNA count files are provided as the output of sncRNA pipeline and represent 12 RNA types (isomiR, lncRNA, precursor miRNA, miRNA, miscRNA, mRNA, piRNA, scaRNA, snoRNA, snRNA, tRF, and tRNA). We also provide metadata of the samples.

  Dataset EGAD50000000391

• Identification of improved IL28B SNPs and haplotypes for prediction of drug response in treatment of hepatitis C using massively parallel sequencing in a cross-sectional European cohort

  The hepatitis C virus infects nearly 3% of the World’s population, causing severe liver disease in many. Standard of care therapy is currently pegylated interferon alpha and ribavirin (PegIFN/R), which is effective in less than half of those infected with the most common viral genotype. Two IL28B SNPs, rs8099917 and rs12979860, predict response to (PegIFN/R) therapy in treatment of hepatitis C virus infection. These SNPs were identified in genome wide analyses using Illumina genotyping chips. In people of European ancestry, there are 6 common (>1%) haplotypes for IL28B, one tagged by rs8099917 minor allele, four tagged by rs12979860. We used massive parallel sequencing of the IL28B and IL28A gene regions generated by PCR from pooled DNA samples from 100 responders and 99 non-responders to therapy, to identify common variants. Variants that had high odds ratios and were validated were then genotyped in a cohort of 905 responders and non-responders. Their predictive power was assessed, alone and in combination with HLA-C. Only SNPs in the IL28B linkage disequilibrium block predicted drug response. Eighteen SNPs were identified with evidence for association with drug response, and with a high degree of confidence in the sequence call. We found that two SNPs, rs4803221 (homozygote minor allele positive predictive value (PPV) of 77%) and rs7248668 (PPV 78%), predicted failure to respond better than the current best, rs8099917 (PPV 73%) and rs12979860 (PPV 68%) in this cross-sectional cohort. The best SNPs tagged a single common haplotype, haplotype 2. Genotypes predicted lack of response better than alleles. However, combination of IL28B haplotype 2 carrier status with the HLA-C C2C2 genotype, which has previousl y been reported to improve prediction in combination with IL28B, provides the highest PPV (80%). The haplotypes present alternative putative transcription factor binding and methylation sites. Massive parallel sequencing allowed identification and comparison of the best common SNPs for identifying treatment failure in therapy for HCV SNPs tagging a single haplotype have the highest PPV, especially in combination with HLA-C. The functional basis for the association may be due to altered regulation of the gene. These approaches have utility in improving diagnostic testing and identifying causal haplotypes or SNPs.

  Study EGAS00001000096

• Sensitive and robust liquid biopsy-based detection of PIK3CA mutations

  Over the last years, the treatment of hormone receptor-positive metastatic breast cancer patients has dramatically changed. Combination strategies attempting to overcome resistance to endocrine therapy such as targeting the PI3 kinase pathway are gaining importance. The phase III SOLAR-1 trial demonstrated the benefit of the addition of alpelisib to fulvestrant, thereby providing further clinical evidence of the increasing importance of PIK3CA testing. Here, we performed a comparison of liquid biopsy and tissue-based detection of PIK3CA mutations in HR-positive metastatic breast cancer patients. Plasma samples and the latest available tumor tissue from 69 patients with metastatic hormone-receptor positive breast cancer were analyzed for PIK3CA hotspot mutations. A high-resolution NGS assay (SiMSen-Seq) was used for plasma samples covering 11 recurrent PIK3CA mutations, used for stratification in SOLAR-1. Additionally, mFAST-SeqS was used to estimate the tumor fractions in the plasma samples. For tumor tissue samples, targeted Ion Torrent NGS was employed. Matched tissue and plasma samples were available from 63 patients. PIK3CA mutations were detected in 46.1% of the tissue samples and in 50.7% of the plasma samples. The variant allele frequency (VAF) ranged from 4.36% to 72.93% with a median of 28.60% in tissue samples, and from 0.28% to 49.85% with a median of 1.17% in plasma samples. The most frequent variants in tissue and plasma were H1047R, E545K, and E542K. We detected at least one of 11 PIK3CA mutations from the SOLAR-1 trial in 51/63 patients, both in tissue and plasma. In 7 patients, such mutations were found only in plasma. In 5 patients, PIK3CA mutations found in tissue were not detectable in ctDNA, 2 of which had a low tumor fraction.Together, in 33/67 plasma samples without detectable PIK3CA mutations, 18 samples had elevated tumor fraction, implicating true negative results. In the remaining 15 patients with low tumor fraction, additional tissue analysis was needed. SiMSen-Seq-based detection of PIK3CA mutations in plasma shows advantageous concordance with the tissue analyses. A combination with an untargeted, mutation-independent approach for detecting ctDNA fractions may confirm a negative PIK3CA result and thereby enhance the performance of the SiMSen-Seq test. This combinatory approach for selecting suitable patients for alpelisib treatment should be validated in larger patient cohorts.

  Study EGAS00001004940

• Conjunctival fibrosis and the innate barriers to Chlamydia trachomatis intracellular infection: a genome wide association study

  This data comes from a genome-wide association study in 1090 cases of scarring trachoma and 1531 controls from a non-selected multi-ethnic sampling of The Gambia. The data constitutes hard base calls at 1457295 features from the HumanOmni2.5-8 (Omni2.5) BeadChip. Three genotype-calling algorithms were used : Illuminus (Bioinformatics 2007, 23:2741–6), GenCall (Patent US7035740, 2006) and GenoSNP (Bioinformatics 2008, 24:2209–14). Data from each calling algorithm was filtered to retain only SNPs with a call rate ≥ 0.98. To obtain a merged set of SNPs, all genotypes that matched across call-sets were retained, whilst those that mismatched between call-sets were set to missing. Genotypes that were present in one call set and missing in others were also retained. The merged SNP set contained 1467876 SNPs, which were filtered for call rate ≥ 0.99 and Hardy-Weinberg equilibrium P value &lt; 5 x 10-8. The case and control groups were approximately equivalent with respect to gender and ethnicity. 70% of cases and 63% of controls were female. Self-described ethnic composition of the cases was 29% Jola, 27% Mandinka, 21% Wolof, 10% Fula and 13% other/no data. In controls the composition was 25% Jola, 24% Mandinka, 21% Wolof, 8% Fula and 22% other/no data. Median age was 49 (range 32-60) in cases and 37 (range 12-52) in controls (t = -3.6019, P = 0.0003). This data set has substantial levels of population structure, which reflects extensive networks of pairwise kinship between participants. First pass tests of association in EMMAX (Nat Genet 2010, 42:348–54) led to genome-wide deflation of the test statistics (λ = 0.982 SE = 1.74e-05) that could be directly attributed to the differing age and gender distributions between cases and controls. Modelling the phenotype to adjust for age and gender successfully controlled for this and no genome-wide deviation from the null was subsequently observed (λ = 1.001, SE = 8.7 x 10-7). Neither principal components analysis (PCA) nor tests of proportional identity by state (IBS) variance in PLINK identified significant levels of within or between group genetic variance. The phenotypes submitted in the phenotypes file include both uncorrected (case/control status) and corrected (age and gender corrected case/control status) phenotypes, as well as age and self-described ethnicity.

  Study EGAS00001001516

• Clinical and Molecular Investigation of Familial CEBPA-mutated Acute Myeloid Leukaemia

  Myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML) are clonal diseases arising from the sequential acquisition of genetic aberrations in haematopoietic stem cells. Whilst the aetiology is predominantly sporadic, rare reports describe the autosomal dominant inheritance of MDS and AML (familial MDS/AML), often presenting in younger patients (&lt;40 years of age) with a germline mutation in one of three haematopoietic transcription factors: RUNX1, CEBPA and GATA2. The clinical recognition of familial disease can be challenging due to wide variations in phenotype, latency and transmission, complicated further by the patients’ awareness and reporting of affected relatives. Distinct clinical manifestations have however been reported for each genetic subtype and whilst RUNX1 and GATA2 mutations can harbour prolonged dysplastic or cytopenic phases, germline CEBPA mutations universally present with AML de novo. Our group were the first to describe germline CEBPA mutations in 3 related individuals, all of whom developed AML between 10-30 years of age. The initial pedigree together with those described in the intervening period, were predominantly isolated case reports with limited follow up, hence long term outcomes for this group of patients remain poorly defined. This contrasts with CEBPA mutations occurring in 10-15% of sporadic AML with normal karytoype (NK-AML), where double mutations (CEBPAdm, found in approximately 50% of all CEBPA-AML) are considered a unique molecular entity, associated with favourable clinical outcomes. In sporadic and familial CEBPAdm AML, N’ terminal mutations enforce translation of the shorter P30-kDa isoform and are typically accompanied by C’ terminal insertions or deletions (disrupting the DNA-binding and/or Leucine zipper domains). Definition of the full molecular landscape awaits extensive whole exome sequencing, although GATA2 mutations (targeting the Zinc Finger 1, ZF1, domain) have been reported in 20-40% of sporadic CEBPAdm AML and recent targeted resequencing analysis revealed a further association with WT1 mutations. With minimal clinical and molecular information available for familial CEBPA-AML, we are often consulted regarding the management of this unique patient population. To address this need, we have investigated a multi-centre cohort of families with germline CEBPA mutations, providing extended clinical follow up, whole exome profiling of secondary mutations and molecular analysis of relapsed disease.

  Study EGAS00001000949

• Molecular_characterization_of_invasive_lobular_carcinoma

  Invasive lobular carcinoma (ILC) is the second most common histological subtype of breast cancer accounting for 10-15% of cases. ILC differs from invasive ductal carcinoma (IDC)with respect to epidemiology, histology, and clinical presentation. Moreover, ILC is lesssensitive to chemotherapy, more frequently bilateral, and more prone to form gastrointestinal, peritoneal, and ovarian metastases than IDCs. In contrast to IDC, the prognostic value ofhistological grade (HG) in ILC is controversial. One of the three major components of histological grading (tubule formation) is missing in ILC which hinders the process of gradingin this histological subtype and results in the classification of approximately two thirds of ILC as HG 2.Over the last decade, a number of gene expression signatures have shed light onto breast cancer classification, allowing breast cancer care to become more personalized. Withrespect to the management of estrogen receptor (ER)-positive breast cancer, several gene expression signatures provide prognostic and/or predictive information beyond what is possible with current classical clinico-pathological parameters alone. Nevertheless, most studies using gene expression signature have not considered different histologic subtypesseparately. Recently, a comprehensive research program has elucidated some of the biological underpinnings of invasive lobular carcinoma. Genetic material extracted from 200 ILC tumor samples were studied using gene expression profiling and identified ILCmolecular subtypes. These proliferation-driven gene signatures of ILC appear to have prognostic significance. In particular, the Genomic Grade (GG) gene signature improved upon HG in ILC and added prognostic value to classic clinico-pathologic factors. In addition this study demonstrated that most ILC are molecularly characterized as luminal-A (~75%)followed by luminal-B (~20%) and HER2-positve tumors (~5%). Moreover, we investigated the prognostic value of known gene signatures/ gene modules in the same cohort of ILC. As a second step within the scope of this project, we aim to investigate the interactionsbetween somatic ILC tumor mutations to observed transcriptome findings. To this end, we aim to perform somatic mutation analysis for the ILC tumors for which Affymetrix gene expression profiling is available. To this end, we will use a gene screen assay, which specifically interrogates the mutational status of a few hundreds of cancer genes. We believe that this pioneering effort will be fundamental for a tailored treatment of ILC withimprovement in patients' outcome.

  Study EGAS00001000292

• Genome_Diversity_in_Africa_Project___GemCode_libraries

  Globally, human populations show structured genetic diversity as a result of geographical dispersion, selection and drift. Understanding this genetic variation can provide insights into the evolutionary processes that shape both human adaptation and variation in disease. Populations from Africa have the highest levels of genetic diversity. This characteristic, in addition to historical genetic admixture, can lead to complexities in the design of studies assessing the genetic determinants of disease and human variation. However, such studies of African populations are also likely to provide new opportunities to discover novel disease susceptibility loci and variants and refine gene–disease association signals. A systematic assessment of genetic diversity within Africa would facilitate genomic epidemiological studies in the region.The Genome Diversity in Africa Project (GDAP) will extend and expand the African Genome Variation (AGV) project, an international collaboration aimed to produce a comprehensive catalogue of human genetic variation in Sub-Saharan Africa (SSA). Using a sequencing-based approach, GDA project aims to capture human genetic variation in Africa, including single nucleotide polymorphisms (SNPs), structural variants, and haplotypes. This resource will make a substantial contribution to understanding patterns of genetic diversity within and among populations in Africa, as well as providing a global resource to help design, implement and interpret genomic studies in African populations and studies comprising globally diverse populations, complementing existing genomic resources. Specifically, we plan to carry out whole genome sequencing at high depth (30x) of up to 1000 individuals across Africa (25 individuals from each ethnolinguistic group). We have already completed sequencing from 5 ethno-linguistic groups and we are now adding others.Our scientific objectives are to: 1) develop a resource that provides a comprehensive catalogue of genetic variation in populations from Africa accessible to the global scientific community; 2) characterise population genetic diversity, structure, gene flow and admixture across Africa; 3) develop a cost-efficient, next-generation genotype array for diverse populations across Africa; and 4) facilitate whole genome-sequencing association studies of complex traits and diseases by developing a reference panel for imputation and resource for enhancing fine-mapping disease susceptibility loci. These scientific objectives will be supported by cross-cutting operational activities, including network and management of the consortium, research ethics, and research capacity building in statistical genetics and bioinformatics.

  Study EGAS00001001589

• Dissociation of solid tumour tissues with cold active protease for single-cell RNA-seq minimizes conserved collagenase-associated stress responses

  Background Single-cell RNA sequencing (scRNAseq) is a powerful tool for studying complex biological systems, such as tumour heterogeneity and tissue microenvironments. However, the sources of technical and biological variation in primary solid tumour tissues and patient-derived mouse xenografts for scRNAseq, are not well understood. Here, we used low temperature (6°C) protease and collagenase (37°C) to identify the transcriptional signatures associated with tissue dissociation across a diverse scRNAseq dataset comprising 128,481 cells from patient cancer tissues, patient-derived breast cancer xenografts and cancer cell lines.Results We observe substantial variation in standard quality control (QC) metrics of cell viability across conditions and tissues. From FACS sorted populations gated for cell viability, we identify a sub-population of dead cells that would pass standard data filtering practices, and quantify the extent to which their transcriptomes differ from live cells. We identify a further subpopulation of transcriptomically “dying” cells that exhibit up-regulation of MHC class I transcripts, in contrast with live and fully dead cells. From the contrast between tissue protease dissociation at 37°C or 6°C, we observe that collagenase digestion results in a stress response. We derive a core gene set of 512 heat shock and stress response genes, including FOS and JUN, induced by collagenase (37°C), which are minimized by dissociation with a cold active protease (6°C). While induction of these genes was highly conserved across all cell types, cell type-specific responses to collagenase digestion were observed in patient tissues. We observe that the yield of cancer and non-cancer cell types varies between tissues and dissociation methods.Conclusions The method and conditions of tumour dissociation influence cell yield and transcriptome state and are both tissue and cell type dependent. Interpretation of stress pathway expression differences in cancer single cell studies, including components of surface immune recognition such as MHC class I, may be especially confounded. We define a core set of 512 genes that can assist with identification of such effects in dissociated scRNA-seq experiments.

  Study EGAS00001003753

• Genome_Diversity_in_Africa_Project__Benin

  Globally, human populations show structured genetic diversity as a result of geographical dispersion, selection and drift. Understanding this genetic variation can provide insights into the evolutionary processes that shape both human adaptation and variation in disease. Populations from SSA have the highest levels of genetic diversity. This characteristic, in addition to historical genetic admixture, can lead to complexities in the design of studies assessing the genetic determinants of disease and human variation. However, such studies of African populations are also likely to provide new opportunities to discover novel disease susceptibility loci and variants and refine gene–disease association signals. A systematic assessment of genetic diversity within SSA would facilitate genomic epidemiological studies in the region.The Genome Diversity in Africa Project (GDAP) aims to produce a comprehensive catalogue of human genetic variation in SSA, including single nucleotide polymorphisms (SNPs), structural variants, and haplotypes. This resource will make a substantial contribution to understanding patterns of genetic diversity within and among populations in SSA, as well as providing a global resource to help design, implement and interpret genomic studies in SSA populations and studies comprising globally diverse populations, complementing existing genomic resources. Specifically, we plan to carry out high depth whole genome sequencing of up to 2000 individuals across Africa (25 individuals from each ethnolinguistic group).Our scientific objectives are to: 1) develop a resource that provides a comprehensive catalogue of genetic variation in populations from SSA accessible to the global scientific community; 2) characterise population genetic diversity, structure, gene flow and admixture across SSA; 3) develop a cost-efficient, next-generation genotype array for diverse populations across SSA; and 4) facilitate whole genome-sequencing association studies of complex traits and diseases by developing a reference panel for imputation and resource for enhancing fine-mapping disease susceptibility loci. These scientific objectives will be supported by cross-cutting operational activities, including network and management of the consortium, research ethics, and research capacity building in statistical genetics and bioinformaticsThis 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 EGAS00001001688

• UK10K RARE COLOBOMA

  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. Ocular coloboma is the most common significant developmental eye defect with an incidence of ~1 in 5,000 live births. It results from failure of optic fissure closure during embryogenesis. The position and extent of the fusion failure dictates the clinical appearance and functional effect. ~30% of coloboma cases are associated with other systemic malformations. These UK10K samples will mostly comprise isolated coloboma cases without systemic involvement (aka non-syndromal coloboma). There is strong evidence from family studies that coloboma has a major genetic component with autosomal dominance being the most common pattern of inheritance. However, many cases are isolated or show complex patterns of familial clustering. The genes responsible for isolated coloboma are largely unknown, but in a small number of families mutations in SHH, CHX10, and PAX6 have been identified indicating marked genetic heterogeneity. Thus in addition to the clinical benefits of achieving a molecular diagnosis there are also major scientific advantages to identifying coloboma genes, as these are likely to provide insights into the complex process of optic fissure closure, that is critical to normal eye development. In the longer term, understanding the molecular basis of the disease may provide clues to therapeutic strategies.For further information with regard to this cohort please contact David Fitzpatrick (david.fitzpatrick@ed.ac.uk).

  Study EGAS00001000127

• UK10K_OBESITY_SCOOP

  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 SCOOP samples are part of the Obesity group and will undergo exome sequencing. Severe Childhood Onset Obesity Project (SCOOP) is a sub-cohort of the Genetics Of Obesity Study (GOOS) cohort established by Sadaf Farooqi and Steve O’Rahilly at the University of Cambridge over the last 12 years. The GOOS cohort contains &gt;4,000 patients of diverse geographic origin, many of whom have monogenic and syndromic forms of obesity, and includes patients that are offspring of consanguineous union. SCOOP is a subset of &gt;1500 UK Caucasian patients with severe, early onset obesity (all patients have a BMI Standard Deviation Score (SDS) &gt; 3 and obesity onset before the age of 10 years), in whom all known monogenic causes of obesity have been excluded. GWAS data on the SCOOP cohort will be available (WTCCC2 independent study) at the time of the start of this study. Data from this cohort has demonstrated that the prevalence of the common obesity risk alleles in FTO, MC4R and NEGR1 are amongst the highest within SCOOP, demonstrating its value also in the study of genetic variants with an impact on more common obesity. For further information with regard to this cohort please contact Sadaf Farooqi (isf20@medschl.cam.ac.uk).

  Study EGAS00001000124

• Clinical outcomes in ctDNA-positive urothelial carcinoma patients treated with adjuvant immunotherapy

  Minimally invasive approaches to detect residual disease after surgery are urgently needed to select patients at highest risk for metastatic relapse for additional therapies. Circulating tumour DNA (ctDNA) holds promise as a biomarker for molecular residual disease (MRD) and relapse,1-3 but its clinical value has yet to be demonstrated in a randomised clinical trial. We evaluated outcomes in post-surgical ctDNA-positive (+) patients in a randomised phase III trial of adjuvant atezolizumab versus observation. IMvigor010 enrolled 809 patients with muscle-invasive urothelial carcinoma and did not meet its primary endpoint of disease-free survival (DFS) in the intent-to-treat population. Within the study, an exploratory planned analysis of prospectively collected plasma was performed, which tested the utility of ctDNA to identify patients who may benefit from adjuvant atezolizumab treatment. ctDNA was measured at the start of therapy (cycle 1 day 1; C1D1) and at week 6 (cycle 3 day 1; C3D1), and 581 patients were evaluable for ctDNA. The prevalence of ctDNA positivity at C1D1 was 37% (n=214), and ctDNA positivity identified patients with poor prognosis (observation arm DFS HR= 6.19 (4.29, 8.91), p&amp;lt;0.0001). Here we show that ctDNA(+) patients had improved DFS and overall survival (OS) with atezolizumab versus observation (DFS HR= 0.56 (0.41-0.77); p=0.0003 and OS HR= 0.58 (0.4-0.86); p=0.0063). No difference in DFS or OS between arms was noted for ctDNA-negative patients. The rate of ctDNA clearance was higher with atezolizumab (18%) versus observation (4%) (p=0.0041). Transcriptomic analysis revealed that tumours from ctDNA(+) patients had higher expression of cell cycle and keratin genes. Within the ctDNA(+) patient population in the atezolizumab arm, non-relapsing patients were further enriched in prominent immune response signatures including PD-L1, IFNG, CXCL9, and high tumour mutational burden, whereas relapse was associated with angiogenesis and fibroblast-transforming growth factor-beta signatures (F-TBRS). TCGA molecular subset analysis revealed increased efficacy of atezolizumab in patients with basal-squamous tumours, consistent with underlying tumour-immune contexture. Together these findings suggest that adjuvant atezolizumab may be associated with improved outcomes compared with observation in this high-risk ctDNA(+) population. These findings, if validated in other settings, would shift approaches to post-operative cancer care.

  Study EGAS00001004997

• RNA_Seq_in_Patients_with_Primordial_Dwarfism

  Transcriptome studies in patients with rare genetic diseases can potentially aid in theinterpretation of likely causal genetic variation through identification of altered transcriptabundance and/or structure. RNA-Seq is the most sensitive assay for both investigatingtranscript structure and abundanceThe primary aim of this pilot project is to investigate to what degree integrating exome-Seqand RNA-Seq data on the same individual can accelerate the identification of causal allelesfor rare genetic diseases. There are two main strands to this: (i) identifying which variantsdiscovered in exome-seq appear to be having a functional impact on transcripts, and (ii)identifying transcript outliers, especially among known causal genes, that may not necessarilyhave a causal variant identified from exome sequencing. The latter may identify the presenceof causal variants that lie far from coding regions (e.g. the formation of cryptic splice sitesdeep within introns, or loss of long range regulatory elements), which can be confirmed withfurther targeted genetic assays. Just over 50% of all disease-causing variants recorded in theHuman Gene Mutation Database (HGMD) affect transcript structure and abundance (e.g.nonsense SNVs, essential splice site SNVs, frameshifting indels, CNVs).This pilot project will study RNA from lymphoblastoid cell-lines from 12 patients withprimordial dwarfism syndromes, for 10 of these samples we have previously generate exomedata as part of our collaboration with the group of Prof Andrew Jackson. The two remainingsamples are positive controls where the causal mutation is known, and is known to affecttranscript structure and/or abundance.Primordial dwarfism is a prime candidate for these RNA-seq studies because all knowncausal mutations to date have key roles in DNA replication and thus, unsurprisingly, theproducts of the causal genes are typically ubiquitously expressed.Each RNA will be sequenced, with two technical replicates (independent RT-PCR and libraries) persample, and each replicate run in 1/2 of a HiSeq lane using 100bp paired reads. Samples preparation was as follows :The cells were grown to confluency, then pellets frozen at -80. RNA samples were prepared using the Qiagen RNeasy kit, then nanodropped and analyzed using the bioanalyzer to determine concentration and purity.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 EGAS00001000283

• Defective Homologous Recombination DNA Repair as Therapeutic Target in Advanced-Stage Chordoma (HIPO_021)

  HIPO project: HIPO_021 Importance: Chordomas are rare tumors of the axial skeleton and skull base with few therapeutic options and no clinically validated molecular drug targets. The value of comprehensive genomic analyses for guiding medical therapy of patients with advanced-stage chordoma is unknown.Objective: To identify therapeutically tractable genetic lesions in a cohort of chordoma patients within a genomics-guided precision oncology program and to document the outcome of individualized, molecularly targeted chordoma therapy.Design, Setting, and Participants: We performed whole-exome sequencing of tumor and matched germline control samples from seven patients with locally advanced or metastatic chordoma who were enrolled in a cross-institutional molecular stratification registry trial for younger adults with advanced-stage cancer across all histologies and patients with rare tumors. All patients were heavily pretreated and had progressive disease prior to molecular analysis.Interventions: Individualized medical therapy was administered according to the patients’ molecular profiles.Main Outcomes and Measures: Candidate therapeutic targets identified by whole-exome sequencing and response to genotype-directed therapy.Results: All patients harbored alterations of two or more genes known to be involved in DNA repair via homologous recombination (HR), including heterozygous deletions of ERCC6, FANC family members, RAD51L (n = 6), BRCA2 (n = 5), ATR, CHEK2, RAD18, RAD51B, and XRCC3 (n = 4); inactivating PTEN mutations coupled with loss of heterozygosity (n = 2); and pathogenic germline variants in BRCA2 (n = 1), NBN (n = 1), and CHEK2 (n = 1) that were accompanied by somatic deletion of the corresponding wild-type alleles. Consistently, a mutational signature associated with defective HR was enriched in all samples and co-occurred with extensive genomic instability, as evidenced by HR deficiency scores and high numbers of large-scale state transitions. These results prompted off-label treatment with the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib in a patient whose tumor was refractory to irradiation and systemic treatment with imatinib, which led to a prolonged response and substantial clinical improvement.Conclusions and Relevance: Advanced-stage chordomas are frequently characterized by genomic imprints of defective HR DNA repair. HR deficiency represents a new therapeutic opportunity in this intractable disease through repositioning of PARP inhibitors that warrants further exploration in clinical trials.

  Study EGAS00001002720

• Whole Genome Sequencing and Variant Calling for Autism Families

  We extracted DNA from whole blood or lymphoblast-derived cell lines and assessed the DNA quality with PicoGreenTM and gel electrophoresis. Whole genome sequencing was performed (Illumina HiSeq2000 and Illumina HiSeq X). WGS reads were mapped to the human reference genome assembly hg19 (GRCh37) using Burrows-Wheeler Aligner v.0.7.12 (TCAG) or Isaac v.2.0.13 (Macrogen). For each genome, we performed local realignment and quality recalibration and detected SNVs and small indels using GATK Haplotype Caller v.3.4.6 without genotype refinement. We detected CNVs using ERDS (estimation by read depth with single nucleotide variants) and CNVnator. We detected structural variants using Manta v.0.29.6. When available by the variant caller (i.e. GATK and Manta), trio-based joint variant calling was conducted for each family.

  Dataset EGAD00001008452