13380 results for ""pdc"+"heritage+language"+census+study"

in 152.10 milliseconds.

• Clonal fitness inferred from timeseries modeling of single cell cancer genomes

  Progress in defining genomic fitness landscapes in cancer by copy number alterations (CNA) has been impeded by lack of single cell and timeseries sampling. We generated 42,000 single cell whole genomes (scWGS) from breast epithelium and primary triple negative breast cancer (TNBC) patient-derived xenografts (PDX) collected during multi-year time series. Using a Wright-Fisher population genetics model, we inferred reproducible CNA-defined clonal fitness dynamics induced by TP53 mutation and cisplatin chemotherapy, with accurate forecasting of experimentally enforced clonal competition dynamics. Drug treatment in three long-term serially passaged TNBC PDX resulted in cisplatin resistant clones that had shown low fitness in the untreated setting. By contrast high fitness clones from treatment naive controls were eradicated reflecting an inversion of the fitness landscape. Upon drug release selective pressure dynamics were reversed indicating a fitness cost of treatment resistance. Taken together, our findings reveal clonal fitness dynamics linked to CNA and therapeutic resistance in polyclonal tumours.

  Study EGAS00001004448

• Versatile workflow for cell-type resolved transcriptional and epigenetic profiles from cryopreserved human lung

  The complexity of the lung microenvironment and changes in cellular composition during disease progression make it exceptionally hard to understand molecular mechanisms leading to the development of chronic lung diseases. Although recent advances in cell-type resolved and single-cell sequencing approaches hold great promise for studying complex diseases, their implementation relies on local access to fresh tissue, as traditional tissue storage methods do not allow viable cell isolation. To overcome these hurdles, we developed a novel, versatile workflow that allows long-term storage of human lung tissue with high cell viability, permits thorough sample quality check before cell isolation, and is compatible with sequencing-based profiling, including single-cell approaches. We demonstrate that cryopreservation is suitable for the isolation of multiple cell types from human lung and is applicable to both healthy and diseased tissue, including COPD and tumour samples. Basal cells isolated from cryopreserved airways retain the ability to differentiate, indicating that cellular identity is not altered by cryopreservation. Importantly, using RNA sequencing and EPIC Array, we show that genome-wide gene expression and DNA methylation signatures are preserved upon cryopreservation, emphasizing the suitability of our workflow for -omics profiling of human lung cells. In addition, we obtained high-quality single-cell RNA sequencing data of cells isolated from the cryopreserved human lung, demonstrating that cryopreservation empowers single-cell approaches. Overall, thanks to its simplicity, our cryopreservation workflow is well-suited for prospective tissue collection by academic collaborators and biobanks, opening worldwide access to human tissue.

  Study EGAS00001004477

• Open_Targets_020_Epigenomes_of_Cell_Lines

  There is currently a drive to establish cell based assay systems of greater human biological and disease relevance through the use of well characterised transformed cell lines, primary cells and complex cellular models (e.g. co-culture, 3D models). However, although the field is gaining valuable experience in running more non-standard & complex cell assays for target validation and compound pharmacology studies, there is the lack of a systematic approach to determine if this expansion in cell assay models is reflected in increased human biological and disease relevance. The increasing wealth of publically available transcriptomic, and epigenome (ENCODE and Epigenome Roadmap) data represents an ideal reference mechanism for determining the relationship between cell types used for target & compound studies to primary human cells and tissues from both healthy volunteers & patients. The CTTV020 epigenomes of cell line project aims to generate epigenetic and transcriptomic profiles of cell lines and compare these with existing and newly generated reference data sets from human tissue and cell types. The aim is to identify assay systems which will provide greater confidence in translating target biology and compound pharmacology to patients. Multiple cell types commonly used within research have been grouped according to biology. Examples include erythroid, lung epithelial, hepatocyte cell types and immortalised models of monocyte / macrophage biology. 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 EGAS00001003137

• Open_Targets_020_Epigenomes_of_Cell_Lines

  There is currently a drive to establish cell based assay systems of greater human biological and disease relevance through the use of well characterised transformed cell lines, primary cells and complex cellular models (e.g. co-culture, 3D models). However, although the field is gaining valuable experience in running more non-standard & complex cell assays for target validation and compound pharmacology studies, there is the lack of a systematic approach to determine if this expansion in cell assay models is reflected in increased human biological and disease relevance. The increasing wealth of publically available transcriptomic, and epigenome (ENCODE and Epigenome Roadmap) data represents an ideal reference mechanism for determining the relationship between cell types used for target & compound studies to primary human cells and tissues from both healthy volunteers & patients. The CTTV020 epigenomes of cell line project aims to generate epigenetic and transcriptomic profiles of cell lines and compare these with existing and newly generated reference data sets from human tissue and cell types. The aim is to identify assay systems which will provide greater confidence in translating target biology and compound pharmacology to patients. Multiple cell types commonly used within research have been grouped according to biology. Examples include erythroid, lung epithelial, hepatocyte cell types and immortalised models of monocyte / macrophage biology. 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 EGAS00001003139

• Capturing disease severity in LIS1-lissencephaly reveals proteostasis dysregulation in patient-derived forebrain organoids

  Lissencephaly is a developmental cortical malformation characterized by reduced to absent gyri and a disorganized cortex, often leading to severe impairments in affected individuals and a reduced life expectancy. Heterozygous mutations in the LIS1 gene, encoding a regulator of the microtubule motor dynein, cause lissencephaly with different clinical severities. While the clinical disease spectrum correlates with the degree of lissencephaly, location and type of mutation may not. We leveraged forebrain-type organoids from LIS1-lissencephaly patients, diagnosed with mild, moderate or severe lissencephaly to investigate, in a cytoarchitecture and multi-omics approach, how the severity degree in patients might relate to specific mutations in the LIS1 gene. We questioned which processes during cortical development might be differentially affected by severity grade, and whether they could be pharmacologically targeted. We found alterations in neurodevelopment often with a severity-dependent gradient. Specifically, we identified alterations of the cytoarchitecture, progenitor cell homeostasis and neurogenesis. Particularly important disease-linked molecular mechanisms were microtubule destabilization, WNT-signaling, and perturbed cadherin- and unfolded protein-binding. Some mechanisms exhibited a severity-dependent gradient, or were specific to a severe grade. We present strategies to reverse phenotypic changes in LIS1-patient organoids, and an in silico approach with therapeutic potential. Thus, we show that different LIS1-severity grades can be recapitulated in vitro, that there is a direct link between the phenotype and genotype, that organoid-based disease modeling can identify molecular underpinnings of malformations of cortical development and that organoids provide a valid platform to develop and test therapeutic strategies.

  Study EGAS00001008227

• The genomic landscape of pediatric acute lymphoblastic leukemia

  Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer and arises from B- or T-lineage lymphocyte precursors. ALL comprises dozens of subtypes, and genomic analyses have largely been performed within these subtypes. Here we analyze pediatric ALL genomes across all subtypes, including 768 whole genomes, 1,729 exomes, and 1,889 transcriptomes in 2,754 patients. Most ALL subtypes harbor 4 or more driver alterations per sample, similar to adult cancers, despite low mutation burdens. Hyperdiploid B-ALL copy gains are likely acquired early and synchronously, with copy gains occurring before ultraviolet-induced mutations. By contrast, ultraviolet-induced mutations precede copy gains in iAMP21 B-ALL. Overall, we identified 378 putative ALL driver genes. Most driver alterations vary in prevalence across ALL subtypes, with B-ALL enriched for Ras and B-lineage-related alterations, and T-ALL enriched for PI3K, JAK, and cell cycle alterations. Most B-ALL (54.3%) and T-ALL (51.2%) samples bear at least one rare driver gene alteration (present in less than 2% of samples), including 30 putative novel cancer driver genes associated with ubiquitination, SUMOylation, non-coding, and other functions. Known or novel alterations associated with poor outcomes in specific subtypes include TBL1XR1 alterations in ETV6-RUNX1 patients, CREBBP in Ph-like-CRLF2, SETD2 in hyperdiploid, and PTEN in TAL1 patients. Intriguingly, DUX4 and KMT2A subtypes separate into CEBPA/FLT3- or NFATC4-expressing subgroups with potential clinical implications. Together, these results deepen understanding of ALL etiology and outcomes, and facilitate ALL model development.

  Study EGAS00001005250

• Analysis of recent shared ancestry in a familial cohort identifies coding and noncoding autism spectrum disorder variants

  Autism spectrum disorder (ASD) is a collection of neurodevelopmental disorders characterized by deficits in social communication and restricted, repetitive patterns of behavior or interests. ASD is highly heritable, but genetically and phenotypically heterogeneous, reducing the power to identify causative genes. We performed whole genome sequencing (WGS) in an ASD cohort of 68 individuals from 22 families enriched for recent shared ancestry. We identified an average of 3.07 million variants per genome, of which an average of 112,512 were rare. We mapped runs of homozygosity (ROHs) in affected individuals and found an average genomic homozygosity of 9.65%, consistent with expectations for multiple generations of consanguineous unions. We identified potentially pathogenic rare exonic or splice site variants in 12 known (including KMT2C, SCN1A, SPTBN1, SYNE1, ZNF292) and 12 candidate (including CHD5, GRB10, PPP1R13B) ASD genes. Furthermore, we annotated noncoding variants in ROHs with brain-specific regulatory elements and identified putative disease-causing variants within brain-specific promoters and enhancers for 5 known ASD and neurodevelopmental disease genes (ACTG1, AUTS2, CTNND2, CNTNAP4, SPTBN4). We also identified copy number variants in two known ASD and neurodevelopmental disease loci in two affected individuals. In total we identified potentially etiological variants in known ASD or neurodevelopmental disease genes for ~61% (14/23) of affected individuals. We combined WGS with homozygosity mapping and regulatory element annotations to identify candidate ASD variants. Our analyses add to the growing number of ASD genes and variants and emphasize the importance of leveraging recent shared ancestry to map disease variants in complex neurodevelopmental disorders.

  Study EGAS00001006058

• Targeted and shallow whole genome sequencing identifies therapeutic opportunities in p53abn endometrial cancers

  Purpose: Shallow whole genome sequencing (sWGS) can detect copy number (CN) aberrations. In high-grade serous ovarian (HGSOC) sWGS identified CN signatures such as homologous recombination deficiency (HRD) to direct therapy. We applied sWGS with targeted sequencing to p53abn endometrial cancers (ECs) to identify additional prognostic stratification and therapeutic opportunities. Experimental Design: sWGS and targeted panel sequencing was performed on formalin-fixed paraffin-embedded p53abn ECs. CN alterations, mutational data and CN signatures were derived, and associations to clinicopathologic and outcomes data were assessed. Results: In 187 p53abn ECs, 5 distinct CN signatures were identified. Signature 5 was associated with BRCA1/2 CN loss with features similar to HGSOC HRD signature. 22% potential HRD cases were identified, 35 patients with signature 5, and 8 patients with BRCA1/2 somatic mutations. Signatures 3 and 4 were associated with whole genome duplication, and CCNE1, ERBB2 and MYC amplifications, with mutations in PIK3CA enriched in signature 3. We observed improved overall survival (OS) for patients with signature 2 and worse OS for signatures 1 and 3. 28% of patients had CCNE1 amplification and this subset was enriched with carcinosarcoma histotype. 34% of patients, across all histotypes, had ERBB2 amplification and/or HER2 overexpression on immunohistochemistry, which was associated with worse outcomes. Mutations in PPP2R1A (29%) and FBXW7 (16%) were among the top 5 most common mutations. Conclusions: sWGS and targeted sequencing identified therapeutic opportunities in 75% of p53abn EC patients. Further research is needed to determine the efficacy of treatments targeting these identified pathways within p53abn ECs.

  Study EGAS00001007661

• Robust methylation-based classification of brain tumours using nanopore sequencing

  Background: DNA methylation-based classification of cancer provides a comprehensive molecular approach to diagnose tumours. In fact, DNA methylation profiling of human brain tumours already profoundly impacts clinical neuro-oncology. However, current implementations using hybridization microarrays are time-consuming and costly. We recently reported on shallow nanopore whole-genome sequencing for rapid and cost-effective generation of genome-wide 5-methylcytosine profiles as input to supervised classification. Here, we demonstrate that this approach allows to discriminate a wide spectrum of primary brain tumours. Results: Using public reference data of 82 distinct tumour entities, we performed nanopore genome sequencing on N=382 tissue samples covering 46 brain tumour (sub)types. Using bootstrap sampling in a cohort of N = 56 cases, we find that a minimum set of 1,000 random CpG features is sufficient for high-confidence classification by ad hoc random forests. We implemented score recalibration as confidence measure for interpretation in a clinical context and empirically determined a platform-specific threshold in a randomly sampled discovery cohort (N = 185). Applying this cut-off to an independent validation series (N = 184) yielded 148 classifiable cases (sensitivity 80.4%) and demonstrated 100 % specificity. Cross-lab validation demonstrated robustness with concordant results across four laboratories in 10/11 (90.9%) cases. In a prospective benchmarking (N = 15), median time to results was 21.1 hours. Conclusions: In conclusion, nanopore sequencing allows robust and rapid methylation-based classification across the full spectrum of brain tumours. Platform-specific confidence scores facilitate clinical implementation for which prospective evaluation is warranted and ongoing.

  Study EGAS00001006540

• Transcriptional_reprogramming_from_innate_immune_functions_to_a_pro_thrombotic_signature_upon_SARS_CoV_2_sensing_by_monocytes_in_COVID_19

  Alterations in the myeloid immune compartment have been observed in COVID-19, but the specific mechanisms underlying these impairments are not completely understood. Here we examined the functionality of classical CD14+ monocytes as a main myeloid cell component in well-defined cohorts of patients with mild and moderate COVID-19 during the acute phase of infection and compared them to that of healthy individuals. We found that ex vivo isolated CD14+ monocytes from mild and moderate COVID-19 patients display specific expression patterns of costimulatory and inhibitory receptors that clearly distinguish them from healthy monocytes, as well as an altered metabolic profile. In addition, decreased NFB activation in COVID-19 monocytes ex vivo is accompanied by an intact type I IFN antiviral response. Secondary pathogen sensing ex vivo led to a state of functional unresponsiveness characterized by a defect in pro-inflammatory cytokine expression, NFB-driven cytokine responses and defective type I IFN response in moderate COVID-19 monocytes, together with defects in the metabolic reprogramming that innate immune cells usually undergo upon pathogen sensing. Transcriptionally and functionally, COVID-19 monocytes switched their gene expression signature from canonical innate immune functions to a pro-thrombotic phenotype characterized by enrichment of pathways involved in hemostasis, immunothrombosis, platelet aggregation and other accessory pathways to platelet activation, accumulation and clot formation, including extracellular matrix reorganization, integrin cell surface interactions and signaling by PDGF. These results provide a potential mechanism by which innate immune dysfunction in COVID-19 may contribute to disease pathology

  Study EGAS00001006788

• PD-L1 blockade in combination with carboplatin as immune induction in metastatic lobular breast cancer: the GELATO-trial

  Invasive lobular breast cancer (ILC) is the second most common histological breast cancer subtype but published data on trials specific for ILC are so far lacking. Translational research revealed that a subset of ILCs may be immune-related and more sensitive to DNA-damaging agents such as platinum. In murine ILC models, synergy between immune checkpoint blockade and platinum has been observed. Here, we tested this concept in the phase II, GELATO-trial (NCT03147040), in which patients with metastatic ILC were treated with weekly carboplatin (AUC 1.5) as immune induction treatment for 12 weeks and atezolizumab (PD-L1 blockade; every three weeks) from the third week onwards until disease progression. Four out of 23 evaluable patients had a partial response (17%, 95%CI 5-39%) and two patients had stable disease for at least 24 weeks, resulting in a clinical benefit rate of 26% (95%CI 10-48%). Out of these six patients, four patients had triple-negative ILC (TN-ILC). In serial biopsies of metastatic lesions, we observed higher CD8 T-cell infiltration, expression of immune checkpoints, and exhausted T cells upon carboplatin/PD-L1 blockade. This is the first report of a clinical trial specifically for ILC and we demonstrate promising anti-tumor activity of atezolizumab with carboplatin as immune induction, in particular for TN-ILC. While activity of carboplatin/PD-L1 blockade in classical ER+ ILC was limited, our translational data yield important insights for the design of highly needed clinical trials in ILC.

  Study EGAS00001006902

• Pediatric Papillary Thyroid Carcinoma Whole Exome Sequencing

  BACKGROUND: Pediatric papillary thyroid carcinoma (PPTC) is distinct from adult-onset disease. Despite higher rates of regional and distal metastases and more frequent recurrence, there is, paradoxically, a highly favourable prognosis. Historically, only 40-50% of childhood PTC are driven by genomic variants common to adult PTC, while the oncogenic drivers in the remainder are unknown. METHODS: Fifty-two PPTCs were genotyped by candidate gene testing, followed, in a subset (n=32), by whole exome (WES) and transcriptome sequencing (RNASeq) to identify oncogenic variants in driver-unknown tumors and to explore gene expression patterns. FINDINGS: Thirty-one tumors (60%) carried variants identified by candidate gene testing, specifically: BRAFV600E(22%), NRASQ61R(2%), RET-CCDC6(19%), RET-NCOA4(15%) and PAX8-PPARG. Among the 21 remaining tumors with negative first-line genotyping, oncogenic variants were identified in 19 (90%). These were enriched with oncogenic fusions, with 11 non-recurrent fusion transcripts identified, including two that had not been previously described: STRN-RET and TG-PBF. Most fusions were associated with 3’ receptor tyrosine kinase (RTK) moieties: RET, MET, ALK and NTRK3. Gene expression analysis defined three distinct clusters, which demonstrated differing expression of genes involved in thyroid differentiation and MAPK signaling. INTERPRETATION: Exome and transcriptome-wide sequencing identify genomic drivers of PPTC in 98% of tumors, whereas conventional genotyping leaves many tumors genetically unexplained. PPTC is driven, in the majority of cases, by oncogenic fusion transcripts involving one of a limited number of RTKs. These observations have broad implications for diagnostic testing and for therapeutic options in children with advanced disease.

  Study EGAS00001005187

• Immunodeficiency syndrome caused by LCP1 mutations

  Severe congenital neutropenia (SCN) is characterized by neutropenia, recurrent infections and an increased leukemia risk. Multiple genetic defects underlying SCN have been identified, but a genetic diagnosis is still lacking in a significant proportion of patients. Here, we report three independent pedigrees with autosomal dominant variants in LCP1. Variants c.740-1G>T and c.740-20_744del resulted in the same alternatively spliced RNA product, causing an in-frame deletion (p.A247_E254del). Variant c.509C>T in the third pedigree resulted in p.S170L. Variant carriers suffer from neutropenia, poor or complete lack of response to G-CSF treatment, and variable degrees of lymphopenia. Patients with A247_E254del presented with tetraploid cells in the bone marrow, indicative of disturbed cytokinesis. In one of these kindreds, two individuals developed acute leukemia. G-CSF non-responsiveness and defective cell cycling were repaired upon correction of the LCP1 A247_E254del variant in patient-derived induced pluripotent stem cells, supporting the monogenic origin of the disease. Indicative of their gain-of-function, both A247_E254del and S170L variants increased F-actin bundling and formation of abnormal protrusions. Single-cell transcriptome analysis of A247_E254del bone marrow-derived hematopoietic stem and progenitor cells (HSPCs) showed deregulation of signaling pathways controlling mitosis in multi-lineage and lymphoid-primed HSPC subsets. We conclude that activating LCP1 variants cause a new autosomal dominant hematopoietic disorder. Depending on the consequences of the LCP1 variants for protein structure, patients may suffer from G-CSF refractory severe neutropenia, lymphopenia, defective cytokinesis and predisposition to leukemia.

  Study EGAS00001008293

• Somatic mutations predict an aggressive phenotype in meningioma but do not drive grade progression

  A subset of meningiomas progress in histopathological grade and drivers of progression are poorly understood. We aimed to identify somatic mutations and copy number alterations (CNAs) associated with grade progression in a unique matched tumour dataset. Utilising a prospective database, we identified 10 patients with meningiomas that had undergone grade progression and for whom matched pre- and post-progression tissue (n=50 samples) was available for targeted next-generation sequencing. Mutations in NF2 were identified in 4/10 patients, 94% were non-skull base tumours. In one patient, three different NF2 mutations were identified in four tumours. NF2 mutated tumours showed large scale CNAs, with highly recurrent losses in 1p, 10, 22q, and frequent CNAs on chromosomes 2, 3 and 4. There was a correlation between grade and CNAs in a subset of patients. In two patients with sphenoid wing Grade III tumours without detected NF2 mutations, a STAT6-NAB2 inversion was identified. Two patients with tumours without detected NF2 mutations showed a combination of loss and high gain on chromosome 17q. Mutations in SETD2, TP53, TERT promoter and NF2 were not uniform across recurrent tumours and did not correspond with grade progression. Meningiomas that progress in grade generally have a mutational profile already detectable in the pre-progressed tumour, suggesting an aggressive phenotype . CNA profiling shows frequent alterations in NF2 mutated tumours compared to non NF2 mutated tumours and the pattern of CNAs may be associated with grade progression in a subset of cases.

  Study EGAS00001006585

• Spatiotemporal evolution and inter-patient heterogeneity in primary and recurrent/metastatic head and neck squamous cell carcinoma

  Head and neck cancer squamous cell carcinomas (HNSCCs) are heterogeneous in terms of origin and aetiology. In addition, there is uncertainty about the spatiotemporal genetic evolution from initial diagnosis to recurrence/metastasis (R/M) after primary treatments and further disease progression following systemic treatment. Changes in the genetic profile have implications on the selection of appropriate treatments for patients, especially in the era of targeted therapies and immunotherapies. Here, we analysed a cohort of 11 HNSCC patients with metachronous R/M, among whom nine had paired primary and R/M samples suitable for next-generation sequencing (multiple R/M samples collected at different time points were available for some of the patients). At the genomic level, the R/M samples shared a fraction of the somatic single nucleotide variants (SNVs) with the index primary tumours, but they also acquired many additional mutations, while losing only a few others. A similar behaviour was also observed when examining the changes of mutational signatures between primary and R/M samples. Overall, R/M appeared thus more genetically diverse than the respective primary tumours. The transcriptomic analysis showed that R/M samples had lower immune cell infiltration and several genes related to immune response were significantly downregulated compared to the primary samples. Our results underline the importance of analysing multiple samples per patient to obtain a more complete picture of the patient’s tumour and advocates a re-biopsy in the event of R/M and treatment failure, in order to select the most appropriate therapeutic strategy.

  Study EGAS00001007464

• Novel optineurin frameshift insertion causing familial frontotemporal dementia and parkinsonism without amyotrophic lateral sclerosis

  Frontotemporal Dementia (FTD) is a common cause of Young Onset Dementia and has diverse clinical manifestations involving behaviour, executive function, language and motor function, including parkinsonism. Up to 50% of FTD patients report a positive family history, supporting a strong genetic basis, particularly in cases with both FTD and amyotrophic lateral sclerosis (FTD-ALS). Mutations in three genes are associated with the majority of familial FTD (fFTD) cases - microtubule associated protein tau gene (MAPT), progranulin gene (PGRN), and hexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9orf72), whilst mutations in other genes such as optineurin (OPTN) have rarely been reported. Mutations in OPTN have been reported mostly in familial and sporadic cases of ALS, or in rare cases of FTD-ALS, but not in association with pure or predominant FTD and/or parkinsonian phenotype. Here, we report for the first time, a family from the Philippines with 4 affected members harbouring the same OPTN frameshift insertion, presenting with FTD-related phenotypes, including one sibling with predominant parkinsonism resembling corticobasal syndrome. Notably, none of the affected members showed any evidence of motor neuron disease or ALS at the time of writing, both clinically and on electrophysiological testing, expanding the phenotypic spectrum of OPTN mutations. Close follow-up of mutation carriers for the development of new clinical features and wider investigation of additional family members with further genetic analyses will be conducted to investigate the possibility of other genetic modifiers in this family which could explain phenotypic heterogeneity.

  Study EGAS00001005220

• Exome-wide mutation analysis of cell-free DNA to simultaneously monitor the full spectrum of cancer treatment outcomes

  Purpose: Cell-free DNA (cfDNA) offers a non-invasive approach to monitor cancer. Here we develop a method using whole-exome sequencing (WES) of cfDNA for simultaneously monitoring the full spectrum of cancer treatment outcomes, including MRD, recurrence, evolution, and second primary cancers. Experimental Design: Three simulation datasets were generated from 26 cancer patients to benchmark the detection performance of MRD/recurrence and second primary cancers. For further validation, cfDNA samples (n=76) from cancer patients (n=35) with six different cancer types were used for performance validation during various treatments.Results: We present a cfDNA-based cancer monitoring method, named cfTrack. Taking advantage of the broad genome coverage of WES data, cfTrack can sensitively detect MRD and cancer recurrence by integrating signals across known clonal tumor mutations of a patient. In addition, cfTrack detects tumor evolution and second primary cancers by de novo identifying emerging tumor mutations. A series of machine learning and statistical denoising techniques are applied to enhance the detection power. On the simulation data, cfTrack achieved an average AUC of 99% on the validation dataset and 100% on the independent dataset in detecting recurrence in samples with tumor fraction ≥0.05%. In addition, cfTrack yielded an average AUC of 88% in detecting second primary cancers in samples with tumor fraction ≥0.2%. On real data, cfTrack accurately monitors tumor evolution during treatment, which cannot be accomplished by previous methods.Conclusion: Our results demonstrated that cfTrack can sensitively and specifically monitor the full spectrum of cancer treatment outcomes using exome-wide mutation analysis of cfDNA.

  Study EGAS00001005906

• Transcriptome Sequencing PPGL

  RNA library preparation of samples with RNA Integrity Number (RIN) > 5.5 (median 7.1; range 5.6-8.9) was performed as described in the TruSeq Stranded mRNA Library Prep Kit (Illumina, RS-122-2101). Briefly, 0.7-1 µg total RNA PolyA+ fraction was purified and randomly fragmented, converted to double stranded cDNA and processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to adapters. Adapter-ligated library was completed by PCR with Illumina PE primers. The resulting purified cDNA library was applied to an Illumina flow cell for cluster generation and sequenced on an Illumina HiSeq2500 on a 51bp single-read format following manufacturer's protocols. cDNA libraries from FFPE tumors and low integrity RNA’s (RIN<5.5) from FF tumors (median 2.4; range 1-5.5) but % of fragments > 200 nucleotides higher than 20% (median 57.5; range 20-96) were prepared using QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen, 015) with a UMI Second Strand Synthesis Module for QuantSeq FWD (Lexogen, 081), vendor’s protocol for low input/ low quality/ FFPE RNA using 200-500 ng of total RNA, according to availability, was used as starting material. PCR Add-on Kit for Illumina (Lexogen, 020) was used to adjust the library amplification number of cycles. Libraries were applied to an Illumina flow cell for cluster generation and sequenced on NovaSeq6000. Image analysis, per-cycle basecalling and quality score assignment was performed with Illumina Real Time Analysis software. Demultiplexing of BCL files to FASTQ format was performed with the bcl2fastq Software (Illumina).

  Study EGAS00001006044

• Chronic lymphocytic leukemia IGHV somatic hypermutation detection by targeted capture next-generation sequencing

  Somatic hypermutation (SHM) status of the immunoglobulin heavy variable (IGHV) gene locus plays a crucial role in determining the prognosis and treatment of patients with chronic lymphocytic leukemia (CLL). A common approach for determining SHM status is multiplex PCR of the IGH gene followed by Sanger sequencing; however, this technique is low throughput, vulnerable to failure, and does not allow multiplexing with other molecular diagnostic assays. Here we designed and validated a DNA targeted capture approach that allows the assessment of IGHV SHM status as a sub-module of a larger next-generation sequencing (NGS) assay. We developed an end-to-end assay that takes as input raw FASTQ files and outputs a report containing IGHV SHM status and V-allele usage following the guidelines of the European Research Initiative on CLL (ERIC). We validated the approach on 35 CLL patient samples, 34 of which were previously characterized using Sanger sequencing. The targeted capture NGS panel identified the SHM status of 34 of 35 CLL patients. We showed 100% sensitivity and specificity among the 33 CLL samples with both NGS and Sanger calls. Furthermore, we demonstrated that this panel can be combined with additional targeted capture panels to detect common and prognostically important CLL single nucleotide variants (SNV) and insertion/deletion (indel) variants as well as copy number variants (del17p). Thus, a targeted capture approach to IGHV SHM detection can be easily integrated into broader sequencing panels, allowing broad CLL prognostication in a single molecular assay.

  Study EGAS00001006887

• A novel transcriptional signature identifies T-cell infiltration in high-risk paediatric cancer

  Background Molecular profiling of the tumour immune microenvironment (TIME) has enabled the rational choice of immunotherapies in some adult cancers. In contrast, the TIME of paediatric cancers is relatively unexplored. We speculated that a more refined appreciation of the TIME in childhood cancers, rather than a reliance on commonly used biomarkers such as tumour mutation burden (TMB), neoantigen load and PD-L1 expression, is an essential prerequisite for improved immunotherapies in childhood solid cancers. Methods We combined immunohistochemistry (IHC) with RNA-sequencing and whole genome sequencing across a diverse spectrum of high-risk paediatric cancers to develop an alternative, expression-based signature associated with CD8+ T-cell infiltration of the TIME. Further, we explored transcriptional features of immune archetypes, T-cell receptor sequencing diversity, assessed the relationship between CD8+ and CD4+ abundance by IHC and deconvolution predictions, and assessed common adult biomarkers such as neoantigen load and TMB. Results A novel 15-gene immune signature, Immune Paediatric Signature Score (IPASS), was identified. Using this signature, we estimate up to 31% of high-risk cancers harbour infiltrating T-cells. In addition, we showed that PD-L1 protein expression is poorly correlated with PD-L1 RNA expression and TMB and neoantigen load are not predictive of T-cell infiltration in paediatrics. Furthermore, deconvolution algorithms are only weakly correlated with IHC measurements of T-cells. Conclusions Our data provides new insights into the variable immune-suppressive mechanisms dampening responses in paediatric solid cancers. Effective immune-based interventions in high-risk paediatric cancer will require individualised analysis of the TIME.

  Study EGAS00001007029

• Treatment stratification and biomarker validation using patient-derived head and neck cancer organoids

  Background Organoids are three-dimensional structures that can be grown from patient-derived material including carcinomas. These in vitro models can be cryopreserved to generate “living” biobanks that reflect patient heterogeneity. Head and neck cancer (HNC) is a collective term used for tumors arising in the head and neck area, including squamous cell carcinomas (HNSCC) and adenocarcinoma of the salivary gland. Methods to culture patient-derived HNC organoids have previously been described. Methods Organoids were derived from resection or biopsy material of patients included between 2016 and 2022. Organoids were characterised with immunohistochemistry and whole exome sequencing (WES) and exposed to chemo-, radio- and targeted-therapies. CRISPR/Cas9-based gene editing was applied to validate proposed biomarkers. Results We have extended our previously published organoid biobank to a total of 110 organoid models, some of which were characterised by histology and WES. Organoids recapitulated both single nucleotide variants and copy number alterations as found in patient HNSCC samples. Organoid and patient response to radiotherapy (RT; primary (n=6) and adjuvant (n=15)) could be compared in 21 HNC cases. In the adjuvant RT group, organoids derived from patients that relapsed were more resistant to RT in vitro, showing potential for guiding treatment options in this subset of patients. Conclusion Taken together, these results indicate illustrate the potential of organoids as a diagnostic tool in personalised medicine for HNC and indicate the potential of this technology for biomarker discovery and validation within the context of relevant patient heterogeneity.

  Study EGAS00001007076

• Whole Exome Sequencing data of six chRCC tumors corresponding to three patients

  Mammalian target of rapamycin (mTOR) is a central regulator of mammalian metabolism and physiology. Aberrant hyperactivation of the mTOR pathway promotes tumor growth and metastasis, and can also promote tumor resistance to chemotherapy and cancer drugs; this makes mTOR an attractive cancer therapeutic target. mTOR inhibitors have been approved to treat cancer; however, the mechanisms underlying drug sensitivity remain poorly understood. Here, whole exome sequencing of three chromophobe renal cell carcinoma (chRCC) patients with exceptional mTOR inhibitor sensitivity revealed that all three patients shared somatic mutations in the deubiquitinase USP9X. The clonal characteristics of the mutations, which were amassed by studying multiple patients’ primary and metastatic samples from various years, together with the low USP9X mutation rate in unselected chRCC series, reinforced a causal link between USP9X and mTOR inhibitor sensitivity. Rapamycin treatment of USP9X-depleted HeLa and renal cancer 786-O cells, along with the pharmacological inhibition of USP9X, confirmed that this protein plays a role in patients’ sensitivity to mTOR inhibition. USP9X was not found to exert a direct effect on mTORC1, but subsequent ubiquitylome analyses identified p62 as a direct USP9X target. Increased p62 ubiquitination and the augmented rapamycin effect upon bortezomib treatment, together with the results of p62 and LC3 immunofluorescence assays, suggested that dysregulated autophagy in USP9X-depleted cells can have a synergistic effect with mTOR inhibitors. In summary, we show that USP9X constitutes a potential novel marker of sensitivity to mTOR inhibitors in chRCC patients, and represents a clinical strategy for increasing the sensitivity to these drugs.

  Study EGAS00001007104

• scRNAseq for patients with immunodeficiency and HCs

  The NFAT family of transcription factors plays central roles in adaptive immunity in murine models, however, their contribution to human immune homeostasis remains poorly defined. In a multigenerational pedigree, we identified three patients carrying germline biallelic missense variants in NFATC1, presenting with recurrent infections, hypogammaglobulinemia and decreased antibody responses. The compound heterozygous NFATC1 variants identified in the patients caused decreased stability and reduced binding of DNA and interacting proteins. We observed defects in early activation and proliferation of T and B cells from these patients, amenable to reconstitution upon genetic rescue. Following stimulation, T-cell activation and proliferation were impaired, reaching that of healthy controls with delay indicative of an adaptive capacity of the cells. Assessment of the metabolic capacity of patient T cells, revealed that NFATc1-dysfunction rendered T cells unable to engage in glycolysis following stimulation, although oxidative metabolic processes were intact. We hypothesized that NFATc1-mutant T cells could compensate for the energy deficit due to defective glycolysis by enhanced lipid metabolism as an adaptation, leading to a delayed, but not lost activation responses. Indeed, we observed increased 13C-labelled palmitate incorporation into citrate indicating higher fatty acid oxidation and we demonstrated that metformin and rosiglitazone improved patient T-cell effector functions. Collectively, enabled by our molecular dissection of NFATC1 mutations and extending the role of NFATc1 in human immunity beyond receptor signaling, and reveal evidence of metabolic plasticity in the context of impaired glycolysis observed in patient T cells to remedy delayed effector responses.

  Study EGAS00001007271

• Efficacy of nivolumab in pediatric cancers with high mutation burden and mismatch-repair deficiency

  Purpose: Checkpoint-inhibitors have limited efficacy for children with unselected solid and brain tumors. We report the first prospective pediatric trial (NCT02992964) using nivolumab exclusively for refractory non-hematological cancers harboring tumor mutation burden (TMB) ≥5 mutations/megabase (mut/Mb) and/or mismatch-repair deficiency (MMRD). Patients and methods: Twenty patients were screened, and ten ultimately included in the response cohort of whom nine had TMB >10mut/Mb (three initially eligible based on MMRD) and one patient had TMB between 5-10 mut/Mb. Results: Delayed immune responses contributed to best overall response of 50%, improving on initial objective responses (20%) and leading to 2-year overall survival (OS) of 50% (95% CI; 27, 93). Four children, including three with refractory malignant gliomas are in complete remission at a median follow-up of 37-months (range: 32.4-60), culminating in 2-year OS of 43% (95% CI; 18.2, 100). Biomarker analyses confirmed benefit in children with germline MMRD, microsatellite instability, higher activated and lower regulatory circulating T-cells. Stochastic mutation accumulation driven by underlying germline MMRD impacted the tumor microenvironment, contributing to delayed responses. No benefit was observed in the single patient with a MMR-proficient tumour and TMB 7.4 mut/Mb. Conclusions: Nivolumab resulted in durable responses and prolonged survival for the first time in a pediatric trial of refractory hypermutated cancers including malignant gliomas. Novel biomarkers identified here need to be translated rapidly to clinical care to identify children who can benefit from checkpoint-inhibitors, including for upfront management of their cancers.

  Study EGAS00001007393

• Cohesin Mutations in AML

  Cohesin shapes the nuclear chromatin architecture, including enhancer-promoter interactions, and its components, of which especially STAG2 and RAD21, are frequently mutated in myeloid malignancies. To elucidate mechanisms of leukemogenesis associated with cohesin mutations in humans, we comprehensively characterized genetic, epigenetic, transcriptional, and chromatin conformational changes in acute myeloid leukemia (AML). To corroborate our findings, we performed complementary siRNA-mediated depletion of STAG2, its paralogue STAG1 or RAD21 in cord blood-derived CD34+ primary human hematopoietic stem and progenitor cells (HSPCs). We show that STAG2 mutations consistently lead to the loss of STAG2 protein and are associated with a specific set of co-occurring mutations, while STAG1 was never mutated in AML. Loss of STAG2 was frequently compensated by STAG1. Still, specific loci displayed altered cohesin occupancy, gene expression and corresponding changes in local chromatin activation as measured by H3K27ac enrichment and chromatin accessibility. High-throughput chromosome conformation capture (in-situ Hi-C) revealed significantly altered chromatin looping in cohesin-mutated AMLs, including weakened enhancer-promoter contacts with reduced, cohesin-dependent promoter activity. In HSPCs, we detected transcriptomic and epigenetic effects overlapping STAG2-mutant AML-specific changes following STAG2 knockdown (KD), that were not invoked by the depletion of STAG1. We also found that STAG2 loss in cultured HSPCs impaired the differentiation capacity, especially erythroid colony formation which maintained HSPC-like gene expression. This work establishes STAG2 as a key regulator of cohesin-associated chromatin architecture, gene expression and differentiation in the human hematopoietic system and identifies candidate target genes that may be implicated in leukemogenesis.

  Study EGAS00001007405