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• SS18-SSX-mediated hijacking of BAF complexes drives synovial sarcoma

  Synovial sarcoma (SS) is defined by a recurrent t(x;18) chromosomal translocation, which produces the hallmark SS18-SSX oncogenic fusion. Incorporation of SS18-SSX into BAF complexes renders BAF complexes aberrant in two distinct manners: the addition of 78aa of SSX onto SS18, and concomitant loss of BAF47 assembly. However, the importance and functional contributions of each of these perturbations on BAF complex targeting and gene expression regulation remain unclear. Here we use an integrative set of genomic approaches in human cancer cell lines and primary tumor samples to define the mechanistic consequences of the SS18-SSX fusion oncoprotein. We find that SS18-SSX hijacks BAF complexes to broad polycomb domains to activate bivalent genes, driving a unique gene expression program distinct from other loss-of-function BAF complex malignancies. Importantly, restoration of BAF47 rescues enhancer activation but is dispensable for proliferative arrest in cell lines. These results demonstrate that gain-of-function SS18-SSX-mediated BAF complex targeting and gene activation is the driving event in SS, and present a mechanism by which distinct functions of BAF complexes can be co-opted to drive oncogenesis.

  Study EGAS00001002920

• Khoe-San genomes reveal unique variation and confirm deepest population divergence in Homo sapiens

  The southern African indigenous Khoe-San populations harbor the most divergent lineages of all living peoples. Exploring their genomes is key to understanding deep human history. We sequenced 25 full genomes from five Khoe-San populations, revealing many novel variants, that 25% of variants are unique to the Khoe-San, and that the Khoe-San group harbors the greatest level of diversity across the globe. In line with previous studies, we found several gene-regions with extreme values in genome-wide distributions, potentially caused by natural selection early in the modern human lineage and more recent in time. These gene-regions included immunity-, sperm-, brain-, diet- and muscle-related genes. When accounting for recent admixture, all Khoe-San groups display genetic diversity approaching the levels in other African groups and a reduction in effective population size starting around 100,000 years ago. Hence, all human groups show a reduction in effective population size commencing around the time of the Out-of-Africa migrations, which coincides with changes in the paleoclimate records, changes that potentially impacted all humans at the time.

  Study EGAS00001004459

• Patient-derived neuroblastoma model system OHC-NB1

  Accurate disease modeling presents a bottleneck against effective translation of novel neuroblastoma therapies into the clinic. Recently, considerable intra-tumor genetic heterogeneity has been observed in neuroblastoma, which calls for preclinical models that reflect this feature. Flexibility in a testing platform is also desirable to support assessment of different endpoints and tumor cell characteristics. We present the novel neuroblastoma model OHC-NB1, propagated from a bone marrow metastasis from a patient with INRG stage M, MYCN-amplified neuroblastoma at first diagnosis. To model different aspects of the disease, several preclinical models were developed from the bone marrow aspirate: a tissue plastic-adherent cell line, a 3-dimensional (3D) spheroid culture, and cell line-derived xenografts that were subcutaneously passaged in mice. Typical aspects of MYCN-amplified neuroblastoma, such as MYCN amplification in double minutes, 1p deletion, 17q gain, and a diploid karyotype, are present in OHC-NB1 and persist in all culture types. Whole exome sequencing revealed intra-tumor heterogeneity in the bone marrow metastasis reflected in the culture models. The set of OHC-NB1 cultures with a genetically heterogeneous MYCN-amplified neuroblastoma background provide a flexible preclinical testing system that recapitulates important features of metastatic neuroblastoma.

  Study EGAS00001003031

• A Single-cell Atlas of Progressive TKI Resistance in Chronic Myeloid Leukemia

  High-dimensional scRNA seq and CyTOF were used to identify features predictive of treatment outcome at diagnosis. An optimal TKI response was characterised by erythroid lineage skewing of the CD34+ HSPC compartment. While TKI-resistant CP was reminiscent of a BC-like state, where HSPCs were enriched for inflammation, stemness and quiescence. We designed a machine-learning approach which assessed the prognostic potential of all 32 sub-populations of our scRNA seq dataset. LSCs and NK populations harboured the highest prognostic power at diagnosis. Within the LSCs, erythroid lineage priming and MYC activation were features of an optimal and poor TKI responses, respectively. With the NK cell compartment, apart from a higher abundance of NK cells in TKI optimal responders, a memory-like HLA-DR+ adaptive NK and KLRC1+ NK populations were hallmarks of an optimal and poor TKI responses, respectively. Mechanistically, both cell intrinsic and extrinsic mechanisms contributed towards the higher sensitivity of EPs to TKI therapy. In summary, our high-dimensional single-cell atlas of TKI resistance highlights pivotal transcriptional features associated with TKI-resistance disease, some of which have the potential to be exploited as biomarkers.

  Study EGAS00001005509

• Transcriptomes of human CD4+ T lymphocytes - Metabolic project

  Human CD4+ T cells are essential mediators of immune responses. By altering the mitochondrial and metabolic states, we defined metabolic requirements of human CD4+ T cells for in vitro activation, expansion, and effector function. T cell activation and proliferation were reduced by inhibiting oxidative phosphorylation, while early cytokine production was maintained by either OXPHOS or glycolytic activity. Glucose deprivation in the presence of mild mitochondrial stress markedly reduced all three T cell functions, contrasting the exposure to resveratrol, an antioxidant and sirtuin-1 activator, which specifically inhibited cytokine production and T cell proliferation, but not T cell activation. Conditions that inhibited T cell activation were associated with the downregulation of 2′,5′-oligoadenylate synthetase genes via interferon response pathways. Our findings indicate that T cell function is grossly impaired by stressors combined with nutrient deprivation, suggesting that correcting nutrient availability, metabolic stress and/or the function of T cells in these conditions will improve the efficacy of T cell-based therapies.

  Study EGAS00001005565

• Clonal Decomposition and DNA Replication States Defined by Scaled Single-Cell Genome Sequencing

  Accurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations.

  Study EGAS00001003190

• Targeted sequencing of diffuse large B-cell lymphoma tumour samples from the UK's Haematological Malignancy Research Network

  The UK’s Haematological Malignancy Research Network (www.HMRN.org) was established in 2004 to provide robust generalizable data to inform clinical practice and research. HMRN is a collaboration between researchers in the Epidemiology & Statistics Group (ECSG) at the University of York, a unified Clinical Network operating across 14 hospitals, and an integrated Haematological Malignancy Diagnostic Service (HMDS) in Leeds. Covering a population of around 4 million, HMRN collects detailed information about all patients diagnosed with a haematological malignancy within the HMRN region, accruing around 2,400 new diagnoses each year. The population has a similar socio-demographic profile to the country as a whole, and HMRN’s maturing data present an increasingly valuable resource to address real questions of concern to haematologists, commissioners and health service researchers – locally, nationally and internationally. This study forms part of a larger project employing targeted exome sequencing on tumour samples from patients diagnosed with a variety of both lymphoid and myeloid malignancies. The results for samples collected from 928 diffuse large B-cell lymphoma patients, diagnosed within HMRN from 2005 to 2012, are provided.

  Study EGAS00001005953

• Allele-specific expression of GATA2 due to epigenetic dysregulation in double mutated CEBPA AML

  Transcriptional deregulation is a central event in the development of acute myeloid leukemia (AML). To identify potential disturbances in gene regulation, we conducted an unbiased screen of allele-specific expression (ASE) in 209 AML cases. The gene encoding GATA binding protein 2 (GATA2) displayed ASE more often than any other myeloid or cancer-related gene. GATA2 ASE was strongly associated with CEBPA double mutations (CEBPA DM), with 95% of cases presenting GATA2 ASE. In CEBPA DM AML with GATA2 mutations, the mutated allele was preferentially expressed. We found that GATA2 ASE is a somatic event lost in complete remission, supporting the notion that it plays a role in CEBPA DM AML. Acquisition of GATA2 ASE involved silencing of one allele via promoter methylation, compensated by overactivation of the other allele, thereby preserving expression levels. Notably, promoter methylation was also lost in remission together with GATA2 ASE. In summary, we propose that GATA2 ASE is acquired by epigenetic mechanisms and is a prerequisite for the development of AML with CEBPA DM. This finding constitutes a novel example of an epigenetic hit cooperating with a genetic hit in the pathogenesis of AML.

  Study EGAS00001004684

• Genomics-based personalized oncology in cancer of unknown primary (CUP, project H021)

  To perform a comprehensive genomic characterization of 70 patients suffering from cancer of unknown primary (CUP) we used whole-exome, whole-genome, transcriptome and methylome analysis. We detected a substantial mutational heterogeneity with genes most commonly affected by SNVs, indels and fusions being TP53, TTN, MUC16, ABCA13, COL6A3, KRAS, LRP1B, XIRP2 and CSMD3. The most common fusion involved FGFR2, the most common focal deletion affected CDKN2A. A molecular tumor board recommended genomics-based therapies in 56/70 (80%) patients which were applied in 20/56 (35.7%) cases. Entity predictions based on transcriptome and methylome data could be made in up to 62/70 (88.6%) cases but were conclusive in only 16/48 (33.3%) cases. Germline analysis revealed 6 (likely) pathogenic mutations in 5 patients. Recommended therapies translated into a mean PFS2/1 ratio of 3.61 (median=2.25) with a median PFS1 of 89 days (n=17) compared to a median PFS2 of 182.5 days (n=20). Our data emphasize the clinical benefit of comprehensive genetic approaches in diagnostic and therapeutic management and underline the need for innovative, mechanism-based clinical trials in this heterogeneous group of diseases.

  Study EGAS00001004786

• PDAC organoid genomic heterogeneity

  The establishment of patient-derived pancreatic cancer organoid culture in recent years creates an exciting opportunity for researchers to perform a wide range of in vitro studies on a model that closely recapitulates the tumor. Among the outstanding questions in pancreatic cancer biology are the causes and consequences of genomic heterogeneity observed in the disease. However, to use pancreatic cancer organoids as a model to study genomic variations, we need to first understand the degree of genomic heterogeneity and its stability within organoids. Here, we used single-cell whole-genome sequencing to investigate the genomic heterogeneity of two independent pancreatic cancer organoids, as well as their genomic stability with extended culture. Clonal populations with similar copy number profiles were observed within the organoids, and the proportion of these clones was shifted with extended culture, suggesting the growth advantage of some clones. However, sub-clonal genomic heterogeneity was also observed within each clonal population, indicating the genomic instability of the pancreatic cancer cells themselves. Furthermore, our transcriptomic analysis also revealed a positive correlation between copy number alterations and gene expression regulation, suggesting the “gene dosage” effect of these copy number alterations that translates to gene expression regulation.

  Study EGAS00001006782

• Clonal relationships of ductal carcinoma in situ and recurrent invasive breast cancers defined by genomic analysis

  Ductal Carcinoma In Situ (DCIS) is the most common form of pre-invasive breast cancer and despite treatment a small fraction (5-10%) of DCIS patients present with invasive disease many years later. A fundamental biologic question is whether the invasive disease recurring in the same breast is established by tumor cells in the initial DCIS or represents new unrelated disease. To address this question, we performed genomic analyses on the initial pure DCIS lesion and paired invasive recurrent tumors in 95 patients together with single cell DNA sequencing in a subset of cases. Our data shows that in 75% the invasive recurrence was clonally related to the initial DCIS, suggesting that the tumor cells were not eliminated during the initial treatment with surgery +/- radiotherapy. Surprisingly however, 18% were clonally unrelated to the DCIS, representing new independent lineages, and 7% of cases were ambiguous. Our findings show that although DCIS is often the precursor of invasive recurrence, a significant fraction of invasive recurrences are unrelated to the initial DCIS. This knowledge is essential for accurate risk evaluation of DCIS treatment de-escalation strategies and the identification of predictive biomarkers.

  Study EGAS00001006306

• DNA methylation landscape of prostate cancer

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

  Study EGAS00001006670

• RNAseq

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

  Study EGAS00001007165

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

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

  Study EGAS00001007563

• H3Africa - Deciphering Developmental Disorders in Africa

  Developmental disorders are typically rare conditions, causing an impairment in physical, learning, language, or behavioural areas. Genetic heterogeneity and highly variable clinical manifestations make diagnosing developmental disorders particularly challenging. Developmental disorders are increasing in prevalence in low- and middle-income countries, partly due to effective prevention and treatment of infectious diseases leading to a decrease in childhood mortality. New sequencing technologies are used effectively in the diagnosis of rare diseases and results can impact clinical practice significantly. Despite this international trend, very little implementation of these new approaches has taken place in low-resource settings, especially in Africa. The Deciphering Developmental Disorders in Africa (DDD-Africa) study aims to take advantage of an unbiased genotype-driven approach to improve healthcare services for patients. The study aims to recruit and perform detailed clinical phenotyping and exome sequencing (ES) on 500 African patients with a developmental disorder in whom a genetic diagnosis has not been confirmed. Where available, parents are also included in sequencing efforts. The project has two active research sites in Johannesburg (South Africa) and Kinshasa (Democratic Republic of Congo) representing diversity in geography, clinical, socio-economic, genetic and environmental factors.

  Study EGAS00001008319

• H3Africa - Identification and characterization of novel hereditary neurological disease genes in Mali.

  Despite the vast diversity of its populations, genetic studies in Africa have been limited. African populations, Malians in particular, have a high rate of intra-ethnic and consanguineous marriage, resulting in increased prevalence of autosomal recessive diseases. Family-based genetic studies can be limited in developed countries due to small sibships. The average fertility rate in Mali is over 6 births per woman, offering a unique opportunity to find new disease genes or mutations that can then be studied in other populations. Hereditary neurological diseases are very debilitating diseases, and developing countries, particularly in Africa, pay a high price in terms of disability-adjusted life years. Although most are currently untreatable, increasing awareness about hereditary neurological disorders can reduce this burden. This study will help to identify and characterize novel hereditary neurological disease genes in Mali. These genes are likely to be important in the normal function of the nervous system and to have important pathophysiological implications for African and other populations. This study will also train physicians and students in the characterization of neurodegenerative diseases as well as in genetic technology and molecular biology, and build a suitable research environment that will retain them.

  Study EGAS00001003016

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

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

  Study EGAS00001003635

• Center for Education and Drug Abuse Research (CEDAR)

  Under its programmatic name, the Center for Education and Drug Abuse research (CEDAR), this Center of Excellence project was completed in 2015. The CEDAR sample consists of 775 nuclear families comprising an adult male (proband), his spouse/mate, and their biological child who is 10-12 years of age at recruitment. This child is designated as index child (IC), and is followed through age 30. Minimal exclusionary criteria were applied to maximize the sample's representativeness. Follow-up evaluations on ICs were conducted at ages 14, 16, 19, 22, 25, 27 and 30. Interim questionnaires were mailed annually beginning at age 20. CEDAR utilizes the family/high-risk paradigm. The offspring thus comprise high average risk (HAR) and low average risk (LAR) groups, constituting the majority of the CEDAR children sample: 250 HAR males, 100 HAR females, 250 LAR males and 94 LAR females. The remainder of the IC sample, 50 males and 31 females, are the offspring of fathers who have a lifetime non-SUD psychiatric dis-order. The follow-up rate from baseline to the final assessment is ~65%. The cohort of children is 76% White and 22% Black. The representation of females among ICs is lower than males, due to the later start of recruit-ment (2nd funding cycle; the study originally focused on males). The CEDAR genetic study has been augmented by the inclusion of its sample into the whole-genome genotyping effort conducted under the Genes, Environment and Development Initiative (GEDI). DNA from the blood samples of target children (index cases) and their siblings submitted to the NIDA Genetics Depository a Rutgers University was obtained and genotyped on Illumina Human660W-Quad Beadchips, with a high average genotyping rate of 99.8%. The sample comprised 158 females and 271 males (37 and 63 %), ~70:30 European- and African- American, with approximately 1:1 ratio of children of affected fathers and normal controls.

  Study phs001649

• Emirati Genome Project subset of 43,608 WGS samples for population-scale variant discovery and allele frequency mapping.

  Here, we present a comprehensive genomic characterization of a cohort of 43,608 Emirati genomes sequenced as part of the Emirati Genome Program (EGP). This study identified more than 421 million single nucleotide variants and indels and more than 600 million copy number- and structural variants. Small variants had 756 million molecular effects annotated. Of the identified variants, 69.04% were observed with more than one allele. Of 7.7 million polymorphic variants having an allele frequency (AF) of more than 5% in EGP, 1,348 have a predicted deleterious effect on a protein. Characterization of global variation reveals that EGP represents a genetic continuum encompassing the range of African, Asian, and European populations. It is best described by two Arabian, an Eurasian, and an African component, with the predominant Arabian component linked to mitochondrial haplogroups J and T, which are commonly attributed to the Middle East. Various aneuploidies of sex chromosomes were detected in 93 individuals overall and of chromosome 21 in 41 individuals. The median inbreeding coefficient and cumulative runs of homozygosity (ROHs) lengths were increased due to extensive consanguinity, being largest in the groups with Arabian main ancestry components and higher than reported for Qatar. Families were identified based on genetic relatedness and classified into 264 families with unrelated parents and 247 families with third- and fourth-degree consanguineous parents. Representative consanguineous pedigrees of families in EGP were outlined. Cumulative ROHs were affected by the main ancestry component and significantly increased in offspring of consanguineous parents, with a pronounced difference between 3rd and 4th-degree relatedness. Investigation of cumulative AFs of variants causing Mendelian diseases highlighted genes related to alpha- and beta-thalassemia (HBB, HBA2). It showed a high burden of variants causing severe recessive diseases, metabolic and retinal disorders, and hearing loss. In summary, EGP represents a landmark effort in characterizing the genetic diversity of the Emirati population, leveraging the largest Middle Eastern cohort reported to date.

  Study EGAS50000001071

• Whole Genome Sequencing of Asian Lung Cancers: Second Hand Smoke is Not Responsible for Higher Incidence of Lung Cancer Among Asian Never-Smokers

  Background Asian nonsmoking populations have a higher incidence of lung cancer compared to their European counterparts. There is a long-standing hypothesis that the increase of lung cancer in Asian never-smokers is due to environmental factors such as second-hand smoke. Results We analyzed whole-genome sequencing of 30 Asian lung cancers. Unsupervised clustering of mutational signatures separated the patients into two categories: i) all of the never-smokers, and ii) only smokers or ex-smokers. Half of the ex-smokers / smokers were in the never-smoker-like cluster. The overall somatic variant profiles of Asian lung cancers were similar to that of European origin with G.C>T.A being predominant in smokers. We found EGFR and TP53 are the most frequently mutated genes with mutations in 50% and 27% of individuals, respectively. Among these Asian never-smokers, 71% had an EGFR mutation compared to 20% of the smokers in the smoking cluster. Other frequently mutated genes include RYR2, SATB2, C1orf88, FERMT1 and CTNNB1. Somatic alterations occurred in WNT signaling pathway genes, suggesting this pathway plays a role in both Western and Asian lung cancers. Conclusions Asian never-smokers have lung cancer signatures distinct from the smoker signature and their mutation profiles are similar to that of European never-smokers. The profiles of Asian and European smokers are also similar. This suggests that the same mutational mechanisms underlie the etiology for both ethnic groups, and the high incidence of lung cancer in Asian never-smokers might not be due to second hand smoke or other carcinogens that cause oxidative DNA damage. Half of the ex-smokers/smokers have a molecular phenotype similar to never-smokers; of which, 50% had EGFR mutations. This suggests that routine EGFR testing is warranted in the Asian population, regardless of smoking status.

  Study EGAS00001000621

• Substantial somatic genomic variation and selection for BCOR mutations in human induced pluripotent stem cells

  Human Induced Pluripotent Stem Cells (hiPSC) are an established patient-specific model system where opportunities are emerging for cell-based therapies. We compared and contrasted hiPSCs derived from different tissues, skin and blood, in the same individual. We show extensive single-nucleotide mutagenesis in all hiPSC lines, although fibroblast-derived hiPSCs (F-hiPSCs) are particularly heavily mutagenized by ultraviolet (UV)-related damage. We utilized genome sequencing data on 454 F-hiPSCs and 44 blood-derived hiPSCs (B-hiPSCs) to gain further insights. Across 324 whole genome sequenced (WGS) F-hiPSCs derived by the Human Induced Pluripotent Stem Cell Initiative (HipSci), UV-related damage is present in ~72% of cell lines, sometimes causing substantial mutagenesis (range 0.25-15 per Mb). Furthermore, we find remarkable genomic heterogeneity between independent F-hiPSC clones derived from the same reprogramming process in the same donor, due to oligoclonal populations within fibroblasts. Combining WGS and exome-sequencing data of 452 HipSci F-hiPSCs, we identify 272 predicted pathogenic mutations in cancer-related genes, of which 21 genes were hit recurrently three or more times, involving 77 (17%) lines. Notably, 151 of 272 mutations were present in starting fibroblast populations suggesting that more than half of putative driver events in F-hiPSCs were acquired in vivo. In contrast, B-hiPSCs reprogrammed from erythroblasts show lower levels of genome-wide mutations (range 0.28-1.4 per Mb), no UV damage, but a strikingly high prevalence of acquired BCOR mutations in ~57% of lines, indicative of strong selection pressure. All hiPSCs had otherwise stable, diploid genomes on karyotypic pre-screening, highlighting how copy-number-based approaches do not have the required resolution to detect widespread nucleotide mutagenesis. This work strongly suggests that models for cell-based therapies require detailed nucleotide-resolution characterization prior to clinical application.

  Dataset EGAD00001007029

• Dynamics of multiple resistance mechanisms in plasma DNA and their clinical implications for NSCLC patients receiving EGFR-targeted therapies

  Background:The development of resistance mechanisms to targeted therapies is complicated by underlying tumour heterogeneity. When multiple resistance mechanisms co-exist, identifying the dominant driver(s) is critical for selection of treatment. Patients and methods:We studied the relative dynamics of multiple oncogenic drivers and resistance mechanisms in 50 patients with EGFR-mutant non-small cell lung cancer (NSCLC), during treatment with gefitinib and hydroxychloroquine (NCT00809237). We performed digital PCR and targeted deep sequencing of cell-free DNA from longitudinal plasma samples from all patients, and shallow whole genome sequencing of serial samples from three patients who underwent histological transformation to small-cell lung cancer (SCLC). ResultsEGFR activating mutations were assessed in tumour samples and accurately identified in plasma samples of 95% of patients (41/43). We identified additional mutations including EGFR T790M (31/50, 62%), TP53 (23/50, 46%), PIK3CA (7/50, 14%), and PTEN (4/50, 8%), and tracked their relative levels in plasma. Patients with both TP53 and EGFR mutations detected in plasma pre-treatment tended to have worse overall survival than those where only EGFR was detected. The resistance-conferring EGFR T790M mutation was identified in 62% of the patients who progressed. Changes in relative levels of T790M and activating mutations in EGFR corresponded to clinical responses in two patients who were given sequential treatments. Patients who progressed without T790M detected in plasma had worse PFS during TKI continuation, and developed alternative resistance mechanisms, that could be identified in plasma DNA, including SCLC-associated copy number changes and TP53 mutations, that tracked with progression and response to subsequent therapies. Conclusion:Longitudinal analysis of multiple oncogenic drivers in ctDNA may help identifying dominant mechanisms of resistance to EGFR-targeted therapies, and highlight the importance of monitoring genetic events that are not direct drug targets but provide non-invasive molecular information that may guide clinical management.

  Study EGAS00001002908

• Deep single-cell RNA sequencing data for 11138 T cells from tumour, adjacent normal tissue and peripheral blood of treatment-naive CRC patients. The DATA ACCESS AGREEMENT is provided at https://github.com/zhangyybio/single-T-cell-data-access. Applicants can request access to the data by directly downloading it or by sending an email to cancerpku@pku.edu.cn. The process that is used to approve an application includes verifying the institution, participants and research purposes of the application. In general this process will take about two weeks. In principal, any scientific research program complying with the laws and bioethic regulation policies of China will be approved.

  T cells are central players in cancer immunotherapy1, yet some of their fundamental properties such as development and migration within tumours remain elusive. The enormous T cell receptor (TCR) repertoire, required for recognising foreign and self-antigens2,3, could serve as lineage tags to track these T cells in tumours4. Here, we obtained transcriptomes of 11,138 single T cells from 12 colorectal cancer (CRC) patients and developed STARTRAC (Single T-cell Analysis by Rna-seq and Tcr TRACking) indices to quantitatively analyse dynamic relationships among 20 identified T cell subsets with distinct functions and clonalities. While both CD8+ effector and “exhausted” T cells exhibited high clonal expansion, they were independently connected with tumour-resident CD8+ effector memory cells, implicating a TCR-based fate decision. Of the CD4+ T cells, the majority of tumour-infiltrating Tregs showed clonal exclusivity, whereas certain Treg clones were developmentally linked to multiple TH clones. Notably, we identified two IFNG+ TH1-like clusters in tumours, the GZMK+ TEM and CXCL13+ TH1-like clusters, which were associated with distinct IFN-γ-regulating transcription factors, EOMES/RUNX3 and BHLHE40, respectively. Only CXCL13+BHLHE40+ TH1-like cells were preferentially enriched in tumours of microsatellite-instable (MSI) patients, which might explain their favourable response rates to immune-checkpoint blockade. Furthermore, we found IGFLR1 to be highly expressed in both CXCL13+BHLHE40+ TH1-like and CD8+ exhausted T cells and possessed co-stimulatory functions. Our integrated STARTRAC analyses provides a powerful avenue to comprehensively dissect the T cell properties in CRC, which could shed new insights into the dynamic relationships of T cells in other cancers

  Study EGAS00001002791

• A circulating biomarker for severity of facioscapulohumeral muscular dystrophy

  Facioscapulohumeral muscular dystrophy (FSHD) is a prevalent, incurable skeletal myopathy. Clinical trials for FSHD are hindered by heterogeneous biomarkers poorly associated with clinical severity, requiring invasive muscle biopsy. Macroscopically FSHD presents with slow fatty replacement of muscle, rapidly accelerated by inflammation. Mis-expression of the transcription factor DUX4 is currently accepted to underlie FSHD pathogenesis and mechanisms including PAX7 target gene repression have been proposed. Here we perform RNA-sequencing on MRI guided inflamed (n=24) and isogenic non-inflamed (n=24) muscle biopsies from the same clinically characterised FSHD patients, alongside isogenic peripheral blood mononucleated cells from a subset of patients (n=13), and unaffected controls. We employ multivariate models to evaluate the clinical associations of 5 published FSHD transcriptomic biomarkers. We demonstrate that PAX7 target gene repression can discriminate control, inflamed and non-inflamed FSHD muscle independently of age and sex (p<0.013), while the discriminatory power of DUX4 target genes is limited to distinguishing FSHD muscle from control. Importantly, the level of PAX7 target gene repression in non-inflamed muscle associates with clinical assessments of FSHD severity (p=0.04). DUX4 target gene biomarkers in FSHD muscle show associations with lower limb fat fraction and D4Z4 array length, but not clinical assessment. Lastly, PAX7 target gene repression in FSHD muscle correlates with the level in isogenic peripheral blood mononucleated cells (p=0.002). A refined PAX7 target gene biomarker comprising 143/601 PAX7 target genes computed in peripheral blood (the FSHD muscle-blood biomarker) associates with clinical severity in FSHD patients (p<0.036). Our new circulating biomarker validates as a classifier of clinical severity in an independent data set of 54 FSHD patient blood samples, with improved power in older patients (p=0.03). In summary, we present the minimally invasive FSHD muscle-blood biomarker of FSHD clinical severity valid in patient muscle and blood, with potential utility in routine disease monitoring and clinical trials.

  Study EGAS00001007350

• Molecular Signatures of DCIS to Invasive Progression for Basal-Like Breast Cancers: An Integrated Mouse Model and Human Tumor Study

  We studied the genomic signatures of progression in breast ductal carcinoma in situ (DCIS) as it progresses towards triple negative invasive breast cancer (TNBC). Single cell RNA sequencing (scRNAseq) was performed on the C3Tag genetically engineered mouse model that forms human breast-like DCIS and TNBC. Bulk RNA sequencing was performed on RNA harvested from archival formalin-fixed, paraffin embedded (FFPE) pairs comprising of estrogen receptor (ER) positive (ER+) and negative (ER-) subsets of DCIS. 

  Study phs002443