14459 results for "PLOS+ONE+Pennsylvania+German+ACS+language+code+pdc+open+access+study"

in 38.23 milliseconds.

• BCR Signaling in human BM PC

  The functions of bone marrow plasma cells (BMPC) beyond antibody production are not fully elucidated and distinct subsets of BMPC suggest potential different functions. Phenotypic differences were identified for human BMPC depending on CD19 expression. Since CD19 is a co-stimulatory molecule of the B-cell-receptor (BCR), and IgA+ and IgM+ BMPC express the BCR on their surface, we here studied whether CD19 expression affects cellular responses, such as BCR signaling and the expression of checkpoint molecules. We analyzed 132 BM samples from individuals undergoing routine total hip arthroplasty. We found that both CD19+ and CD19- BMPC expressed BCR signaling molecules. Notably, the BCR-associated kinase spleen tyrosine kinase (SYK) including pSYK was higher expressed in CD19+BMPC compared to CD19- BMPC. BCR stimulation also resulted in increased kinase phosphorylation downstream of the BCR while expression of CD19 remained stable afterwards. Interestingly, the BCR response was restricted to IgA+ BMPC independently of CD19 expression. With regard to the expression of checkpoint molecules, CD19- BMPC expressed higher levels of co-inhibitory molecule programmed cell death protein-1 (PD-1) than CD19+ BMPC. IgA+ BMPC characteristically upregulated PD-1 upon BCR stimulation in contrast to other PC subsets and inhibition of the kinase SYK abrogated PD-1 upregulation. In contrast, expression of PD-1 ligand, B and T lymphocyte attenuator (BTLA) and CD28 did not change upon BCR activation of IgA+ BMPC. Here, we identify a distinct functiotype of IgA+ BMPC that is independent of the phenotypic heterogeneity of the subsets according to their CD19 expression. The data suggest that IgA+ BMPC underlie different regulatory principles and/or exert distinct regulatory functions.

  Study EGAS00001004948

• Tracking the evolution of Therapy-Related Myeloid Neoplasms using chemotherapy signatures

  Patients treated with cytotoxic therapies, including autologous stem cell transplantation, are at risk for developing therapy-related myeloid neoplasms (tMN). Pre-leukemic clones (i.e., clonal hematopoiesis; CH) are detectable years before the development of these aggressive malignancies, though the genomic events leading to transformation and expansion are not well-defined. Here, leveraging distinctive chemotherapy-associated mutational signatures from whole-genome sequencing data and targeted sequencing of pre-chemotherapy samples, we reconstruct the evolutionary life-history of 39 therapy-related myeloid malignancies. A dichotomy is revealed, in which neoplasms with evidence of chemotherapy-induced mutagenesis from platinum and melphalan are hypermutated and enriched for complex structural variants (i.e., chromothripsis) while neoplasms with non-mutagenic chemotherapy exposures are genomically similar to de novo acute myeloid leukemia. Using chemotherapy-associated mutational signatures as temporal barcodes linked to a discrete clinical exposure in each patient’s life, we estimate that several complex events and genomic drivers are acquired after chemotherapy is administered. For patients with prior multiple myeloma who were treated with high-dose melphalan and autologous stem cell transplantation, we demonstrate that tMN can develop from either a reinfused CH clone that escapes chemotherapy exposure and is selected following reinfusion, or from TP53-mutant CH that survives direct myeloablative conditioning and acquires melphalan-induced DNA-damage. Overall, we reveal a novel mode of tMN progression that is not reliant on direct mutagenesis or even exposure to chemotherapy. Conversely, for tMN that evolve under the influence of chemotherapy-induced mutagenesis, distinct chemotherapies not only select pre-existing CH, but also promote the acquisition of recurrent genomic drivers.

  Study EGAS00001006903

• Genetic heritage of the Baphuthi highlights an over-ethnicised notion of 'Bushman' in the Maloti-Drakensberg, southern Africa

  Using contemporary people as proxies for ancient communities is a contentious but necessary practice in anthropology. In southern Africa, the distinction between the Cape KhoeSan and eastern KhoeSan remains unclear as ethnicity labels have been changed through time and most communities were decimated if not extirpated. The eastern KhoeSan may have had genetic distinctions from neighbouring communities who speak Bantu languages and KhoeSan further away, alternatively the identity may not have been tied to any notion of biology, instead denoting communities with a nomadic 'lifeway' distinct from African agro-pastoralism. The Baphuthi of the 1800s in the Maloti-Drakensberg, southern Africa had a substantial KhoeSan constituency and a lifeway of nomadism, cattle raiding, and horticulture. Baphuthi heritage could provide insights into the history of the eastern KhoeSan. We examine genetic affinities of 23 Baphuthi to discern whether the narrative of KhoeSan descent reflects distinct genetic ancestry. Genome-wide SNP data (Illumina GSA) were merged with 52 global populations, for 160K SNPs. Genetic analyses show no support for a unique eastern KhoeSan ancestry distinct from other KhoeSan or southern Bantu-speakers. The Baphuthi have strong affinities with early-arriving southern Bantu-speaking (Nguni) communities, as the later-arriving non-Nguni show strong evidence of recent African admixture possibly related to Late-Iron Age migrations. The references to communities as 'San',and 'Bushman' in historic literature has often been misconstrued as notions of ethnic/biological distinctions. The terms may have reflected ambiguous references to non-sedentary polities instead, as seems to be the case for the eastern ‘Bushman’ heritage of the Baphuthi.

  Study EGAS00001007080

• Analysis of T-cell receptor clonotypes in tumor micro-environment identifies shared cancer type-specific signatures

  Despite the conventional view that a truly random V(D)J recombination process should generate a highly diverse immune repertoire, emerging reports suggest that there is a certain bias towards the generation of shared/public immune receptor chains. These studies were performed in viral diseases where public T-cell receptors (TCR) appear to confer better protective responses. Selective pressures generating common TCR clonotypes are currently not well understood but it is believed that they confer a growth advantage. As very little is known about public TCR clonotypes in cancer, here we set out to determine the extent of shared TCR clonotypes in the intra-tumor microenvironments of virus- and non-virus- driven head and neck cancers using TCR sequencing. We report that tumor-infiltrating T-cell clonotypes were indeed shared across individuals with the same cancer type, where the majority of shared sequences were specific to the cancer type (ie viral versus non-viral). These shared clonotypes were not particularly enriched in EBV-associated nasopharynx cancer, but in both cancers, exhibited distinct characteristics, namely shorter CDR3 lengths, restricted V- and J-gene usages and also demonstrated convergent V(D)J recombination. Many of these shared TCRs were expressed in patients with a shared HLA background. Pattern recognition of CDR3 amino acid sequences revealed strong convergence to specific motifs that were unique to each cancer type, suggesting they may be enriched for specificity to common antigens found in the tumor microenvironment. The identification of shared TCRs in infiltrating tumor T-cells not only adds to our understanding of the tumor adaptive immune recognition but could also serve as disease-specific biomarkers and guide the development of future immunotherapies.

  Study EGAS00001005480

• IL7-receptor expression is frequent in T-cell acute lymphoblastic leukemia and predicts sensitivity to JAK-inhibition

  T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with a dismal prognosis related to refractory/relapsing diseases, raising the need for new targeted-therapies. Activating mutations of the IL7-receptor pathway genes (IL7Rp) play a proven leukemia-supportive role in T-ALL. JAK-inhibitors such as ruxolitinib have recently demonstrated preclinical efficacy. However, prediction markers for sensitivity to JAK-inhibitors are still lacking. Herein, we show that IL7R (CD127) expression is more frequent (~70%) than IL7Rp-mutations in T-ALL (~30%). We compared the so-called non-expressers (no IL7R-expression/IL7Rp-mutation), expressers (IL7R-expression without IL7Rp-mutation) and mutants (IL7Rp-mutations). Integrative multi-omics analysis outlined IL7R-deregulation in virtually all T-ALL subtypes, at the epigenetic-level in non-expressers, genetic-level in mutants, and post-transcriptional level in expressers. Ex-vivo data using primary-derived xenografts support that IL7Rp is functional whenever the IL7R is expressed, regardless of the IL7Rp mutational status. Consequently, ruxolitinib impaired T-ALL survival in both expressers and mutants. Interestingly, we show that expressers displayed ectopic IL7R-expression and IL7Rp-addiction conferring a deeper sensitivity to ruxolitinib. Conversely, mutants were more sensitive to venetoclax than expressers. Overall, combination of ruxolitinib and venetoclax resulted in synergistic effects in both groups. We illustrate the clinical relevance of this association by reporting achievement of complete remission in two patients with refractory/relapsed-T-ALL. This provides proof of concept for translation of this strategy into clinics as bridge to transplant. Altogether, IL7R-expression can be used as a biomarker for sensitivity to JAK-inhibition, thereby expanding the fraction of T-ALL patients eligible to ruxolitinib up to nearly ~70% of T-ALL.

  Study EGAS00001007144

• Whole genome sequencing of 108 epileptic patients from CENet cohort

  Epileptic patients have been sequenced as part of the CENet cohort which is composed of patients with Genetic Generalized Epilepsy (GGE) or Non-Acquired Focal Epilepsy (NAFE) collected in CHUM Research Center in Montreal. Patients were diagnosed by epileptologists. The clinical epilepsy phenotype was classified according to the current classification by the International League against Epilepsy (ILAE). Libraries preparation and whole-genome sequencing: gDNA was cleaned up using ZR-96 DNA Clean & ConcentratorTM-5 Kit (Zymo) prior to being quantified using the Quant-iTTM PicoGreen dsDNA Assay Kit (Life Technologies) and its integrity assessed on agarose gels. Libraries were generated using the TruSeq DNA PCR-Free Library Preparation Kit (Illumina) according to the manufacturer’s recommendations. Libraries were quantified using the Quant-iTTM PicoGreen dsDNA Assay Kit (Life Technologies) and the Kapa Illumina GA with Revised Primers-SYBR Fast Universal kit (Kapa Biosystems). Average size fragment was determined using a LabChip GX (PerkinElmer) instrument. The libraries were denatured in 0.05N NaOH and diluted to 8pM using HT1 buffer. The clustering was done on a Illumina cBot and the flowcell was ran on a HiSeq 2500 for 2x125 cycles (paired-end mode) using v4 chemistry and following the manufacturer's instructions. A phiX library was used as a control and mixed with libraries at 0.01 level. Bioinformatics: The Illumina control software was HCS 2.2.58, the real-time analysis program was RTA v. 1.18.64. Program bcl2fastq v1.8.4 was used to demultiplex samples and generate fastq reads. The filtered reads were aligned to reference Homo_sapiens assembly b37. Each readset was aligned to create a .cram file.

  Study EGAS00001007507

• Single-cell decoding of drug induced transcriptomic reprogramming in triple negative breast cancers

  Background The encoding of cell intrinsic drug resistance states in breast cancer reflects the contributions of genomic and non-genomic variations and requires accurate estimation of clonal fitness from co-measurement of transcriptomic and genomic data. Somatic copy number (CN) variation is the dominant mutational mechanism leading to transcriptional variation and notably contributes to platinum chemotherapy resistance cell states. Here, we deploy time series measurements of triple negative breast cancer (TNBC) single-cell transcriptomes, along with co-measured single-cell CN fitness, identifying genomic and transcriptomic mechanisms in drug associated transcriptional cell states. Results We present scRNA-seq data (53,641 filtered cells) from serial passaging TNBC patient-derived xenograft (PDX) experiments spanning 2.5 years, matched with genomic single-cell CN data from the same samples. Our findings reveal distinct clonal responses within TNBC tumors exposed to platinum. Clones with high drug fitness undergo clonal sweeps and show subtle transcriptional reversion, while those with weak fitness exhibit dynamic transcription upon drug withdrawal. Pathway analysis highlights convergence on epithelial-mesenchymal transition and cytokine signaling, associated with resistance. Furthermore, pseudotime analysis demonstrates hysteresis in transcriptional reversion, indicating generation of new intermediate transcriptional states upon platinum exposure. Conclusions Within a polyclonal tumor, clones with strong genotype-associated fitness under platinum remained fixed, minimizing transcriptional reversion upon drug withdrawal. Conversely, clones with weaker fitness display non-genomic transcriptional plasticity. This suggests CN-associated and CN-independent transcriptional states could both contribute to platinum resistance. The dominance of genomic or non-genomic mechanisms within polyclonal tumors has implications for drug sensitivity, restoration and re-treatment strategies.

  Study EGAS00001007242

• Elucidating Transcription Regulation by Epigenetics in Neuroblastoma

  PURPOSE Whole-genome profiles of the epigenetic modification 5-hydroxymethylcytosine (5-hmC) are robust diagnostic biomarkers in adult patients with cancer. We investigated if 5-hmC profiles would serve as novel prognostic markers in neuroblastoma, a clinically heterogeneous pediatric cancer. Because this DNA modification facilitates active gene expression, we hypothesized that 5-hmC profiles would identify transcriptomic networks driving the clinical behavior of neuroblastoma. PATIENTS AND METHODS Nano-hmC-Seal sequencing was performed on DNA from Discovery (n = 51), Validation (n = 38), and Children's Oncology Group (n = 20) cohorts of neuroblastoma tumors. RNA was isolated from 48 tumors for RNA sequencing. Genes with differential 5-hmC or expression between clusters were identified using DESeq2. A 5-hmC model predicting outcome in high-risk patients was established using linear discriminant analysis. RESULTS Comparison of low- versus high-risk tumors in the Discovery cohort revealed 577 genes with differential 5-hmC. Hierarchical clustering of tumors from the Discovery and Validation cohorts using these genes identified two main clusters highly associated with established prognostic markers, clinical risk group, and outcome. Genes with increased 5-hmC and expression in the favorable cluster were enriched for pathways of neuronal differentiation and KRAS activation, whereas genes involved in inflammation and the PRC2 complex were identified in the unfavorable cluster. The linear discriminant analysis model trained on high-risk Discovery cohort tumors was prognostic of outcome when applied to high-risk tumors from the Validation and Children's Oncology Group cohorts (hazard ratio, 3.8). CONCLUSION 5-hmC profiles may be optimal DNA-based biomarkers in neuroblastoma. Analysis of transcriptional networks regulated by these epigenomic modifications may lead to a deeper understanding of drivers of neuroblastoma phenotype. "Reprinted from Applebaum et. JCO Precision Oncology, 2019, with permission from creative commons license."

  Study phs001831

• Uncovering RNA Signatures of C9orf72-Linked ALS and Related Disorders

  Substantial clinical and pathological variability has been reported in patients carrying an expanded repeat in C9orf72, who are generally diagnosed with amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD). Emerging evidence suggests that C9orf72 expression, RNA foci, and dipeptide-repeat proteins contribute to C9orf72-related diseases; however, the majority of the clinicopathological variability remains unexplained. We hypothesize that RNA sequencing (RNA-Seq) might reveal individual genes or groups of highly correlated genes (modules) that could account for this variability, which may help to identify much-needed druggable targets and/or biomarkers. Our preliminary studies performed in the frontal cortex, where we compared C9orf72 expansion carriers to (disease) controls, demonstrated that the top differentially expressed gene was C9orf72 itself. We also noticed that differentially expressed genes were enriched for the endocytosis pathway, which is consistent with the reported function of the C9orf72 protein in vesicle trafficking. Furthermore, we detected a module enriched for vesicular pathways, and additionally, we discovered an association with survival after onset for several promising candidates involved in vesicular transport. To find new therapeutic targets and biomarkers, here we have completed RNA-Seq on an increased number of samples to examine another relevant region: the cerebellum. As such, we performed RNA-Seq on a well-characterized cohort of C9orf72 expansion carriers, patients without this expansion, and control subjects without neurological diseases. We used multivariable linear regression models for gene-level analysis, weighted correlation network analysis for module-level analysis, and leafcutter for splicing analysis. Using the cerebellar RNA-Seq data, we found a large number (>6,000) of differentially expressed genes, with a notable increase in expression of homeobox genes in C9orf72 expansion carriers, as well as many gene modules that were associated with C9orf72 expansion status. Additionally, using leafcutter, we detected over 1,000 differential splicing events and an increase in the number of cryptic splicing events in C9orf72 expansion carriers.

  Study phs003065

• Genetic defects in familial renal disorders

  Several different genes cause, when mutated, increased urinary phosphate excretion and hypophosphatemia leading to rickets/osteomalacia; however, the majority of phosphate-wasting disorders has not yet been defined at the molecular level. Identification of phosphate-regulating genes has provided important novel insights into the mechanism contributing to phosphate homeostasis, which remains incompletely understood. We therefore pursue exome-wide nucleotide sequence analysis to define the underlying mutations, which is expected to provide novel insights into the regulation of this important mineral. Virtually nothing is known about the genetic mutations that cause non-syndromic structural abnormalities involving the urinary tract, which represent about 50% of the causes of end-stage renal disease (ESRD). The completion of the human genome project and the technological advances that allow low cost whole exome sequencing have now made it feasible to readily search for the cause of different inherited disorders in humans, particularly if clinical information and genomic DNA is available from larger kindreds with multiple affected members that can be used for mapping the disease-causing genetic locus. Furthermore, catalogs were generated that document the expression profiles of numerous genes during embryonic, fetal, and postnatal development, thereby supporting the exploration of kidney involvement in animal models of different diseases and in human genetic syndromes. We therefore plan to define the mechanisms leading to inherited disorders of the urogenital tract and the kidneys by establishing the causative genetic defects through a combination of genetic mapping and whole exome sequencing. Hajdu-Cheney Syndrome (HCS) is a rare autosomal-dominant skeletal disorder characterized by severe osteoporosis, acroosteolysis of the distal phalanges, renal cysts and other abnormalities. Our goal is to identify the genetic cause of HCS using exome sequencing. Opsismodysplasia is a very rare recessive spondylometaphyseal dysplasia characterized by delayed epiphyseal ossification, shortness of bones and sometimes low serum phosphate levels. Discovery of the causing mutation will give us insights into the pathogenesis of this often lethal disease.

  Study phs000477

• CaBagE: a Cas9-Based Background Elimination Strategy for Targeted, Long-Read DNA Sequencing

  While repeat-expansion polymorphisms are known to underlie several developmental and neurological disorders, technological limitations have inhibited the ability to resolve accurate genotypes for these variants. A pertinent example is the (CCCCGG)n repeat expansion in C9orf72 that segregates with up to 40% of familial amyotrophic lateral sclerosis (ALS) cases. There is a clear relationship between this repeat expansion and neurodegeneration. However, widely-used short-read sequencing is ill-suited to characterize the C9orf72 repeat expansion due to polymerase slippage over low complexity sequences and the inherently limited mapping/haplotype resolution of redundant short sequences. Consequently, researchers and physicians must rely on crude variant characterization methods (e.g. Southern blots), which limit direct genotype-phenotype associations and create challenges for the diagnosis of disease in patients with atypical clinical presentation. In contrast, ultra-long (e.g., 10-150kb) sequencing reads generated by Oxford Nanopore Technologies (ONT) enable direct measurement of loci containing complex structures without being subject to amplification or alignment bias. We are therefore developed a molecular and computational framework for targeted sequencing and quantification of pathogenic repeat expansions by combining Cas9 and amplification-free sequence capture methods with Nanopore long-read sequencing technology. First, extracted high molecular weight DNA is bound by Cas9 on either side of a DNA target. Bound Cas9 protects the target DNA from challenge by processive exonucleases. Unprotected (off-target) DNA is degraded with exonucleases and the remaining sample is sequenced using the ONT MinION sequencer. Then, base-called reads are aligned to the target locus and repeat copy number genotypes are estimated using a Gaussian Mixture Model. We applied this molecular and computational framework to 2 ALS patients with C9orf72 repeat expansions with biospecimens available from the NINDS collection from Coriell. Genotype estimates produced with CaBagE were commensurate with genotypes derived from repeat-primed PCR for each individual.

  Study phs002368

• Prediction of Trastuzumab Benefit in Adjuvant Breast Cancer: Gene Expression Profiling in NSABP B31

  Background National Surgical Adjuvant Breast and Bowel Project (NSABP) trial B-31 suggested the efficacy of adjuvant trastuzumab, even in HER2-negative breast cancer. This finding prompted us to develop a predictive model for degree of benefit from trastuzumab using archived tumor blocks from B-31. Methods Case subjects with tumor blocks were randomly divided into discovery (n = 588) and confirmation cohorts (n = 991). A predictive model was built from the discovery cohort through gene expression profiling of 462 genes with nCounter assay. A predefined cut point for the predictive model was tested in the confirmation cohort. Gene-by-treatment interaction was tested with Cox models, and correlations between variables were assessed with Spearman correlation. Principal component analysis was performed on the final set of selected genes. All statistical tests were two-sided. Results Eight predictive genes associated with HER2 (ERBB2, c17orf37, GRB7) or ER (ESR1, NAT1, GATA3, CA12, IGF1R) were selected for model building. Three-dimensional subset treatment effect pattern plot using two principal components of these genes was used to identify a subset with no benefit from trastuzumab, characterized by intermediate-level ERBB2 and high-level ESR1 mRNA expression. In the confirmation set, the predefined cut points for this model classified patients into three subsets with differential benefit from trastuzumab with hazard ratios of 1.58 (95% confidence interval [CI] = 0.67 to 3.69; P = .29; n = 100), 0.60 (95% CI = 0.41 to 0.89; P = .01; n = 449), and 0.28 (95% CI = 0.20 to 0.41; P < .001; n = 442; P interaction between the model and trastuzumab < .001). Conclusions We developed a gene expression-based predictive model for degree of benefit from trastuzumab and demostrated that HER2-negative tumors belong to the moderate benefit group, thus providing justification for testing trastuzumab in HER2-negative patients (NSABP B-47). ** Reprinted from J Natl Cancer Inst;2013;105:1782-1788 with permission from Oxford University Press.

  Study phs000826

• Autism Sequencing Consortium (ASC)

  The ARRA Autism Sequencing Collaboration was created in 2010 bringing together expert large-scale sequencing center (at the Baylor College of Medicine, PI Richard Gibbs and the Board Institute of MIT and Harvard, PI Mark J. Daly) and a collaborative network of research labs focused on the genetics of autism (brought together by the Autism Genome Project and the Autism Consortium). These groups worked together to utilize dramatic new advances in DNA sequencing technology to reveal the genetic architecture of autism through comprehensive examination of the exotic sequence of all genes. The Autism Sequencing Consortium (ASC) was founded by Joseph D. Buxbaum and colleagues as an international group of scientists who share autism spectrum disorder (ASD) samples and genetic data. The PIs are Drs. Joseph D. Buxbaum (Icahn School of Medicine at Mount Sinai), Mark J. Daly (Broad Institute of MIT and Harvard), Bernie Devlin (University of Pittsburgh School of Medicine), Kathryn Roeder (Carnegie Mellon University, Matthew State and Stephan Sanders (University of California, San Francisco). The rationale for the ASC is described in Buxbaum et al. 2012, and this paper should be cited when referencing the data set. All shared data and analysis is hosted at a single site, which enables joint analysis of large-scale data from many groups. The ASC was first supported by a cooperative agreement grant to four lead sites funded by the National Institute of Mental Health (U01MH100233, U01MH100209, U01MH100229, U01MH100239), with additional support from the National Human Genome Research Institute. The NIMH recently renewed their support with a second grant (U01MH111661, U01MH111660, U01MH111658 and U01MH111662) to expand the project from 29,000 genomes to more than 50,000 exomes over the next 5 years. NHGRI provides ongoing sequencing support for the ASD through the Broad Center for Common Disease Genomics (UM1HG008895, Mark Daly, PI).

  Study phs000298

• Childhood Cancer Data Initiative (CCDI): Enhancement of Data Sharing in Pediatric, Adolescent and Young Adult Cancers

  NCI's Childhood Cancer Data Initiative (CCDI) is building a community centered around childhood cancer care and research data. Through enhanced data sharing, we can improve our understanding of cancer biology to improve preventive measures, treatment, quality of life, and survivorship, as well as ensure that researchers learn from every child with cancer. While childhood cancers represent the leading cause of death in children over the age of 1, they are collectively rare, comprising approximately 1%‒3% of cancers diagnosed annually in the United States. Information on diagnosis, treatment, and outcomes is often stored at the hospital or institution where a child is treated, making it difficult to answer scientific questions about childhood cancer. Sharing clinical care and research data generated by children's hospitals, clinics, or networks broadly with the community can help us learn faster and, on a scale much larger than any single institution caring for children can learn on its own. To exploit the rapid advances in high-throughput DNA sequencing technologies and realize the goals of precision cancer medicine, the UMICH Cancer Center established the Michigan Oncology Sequencing Center (MI-ONCOSEQ). This UMICH Cancer Center provisions genomic data derived from the Michigan Oncology Sequencing Center (MI-ONCOSEQ) clinical sequencing assay for patients that fall within the inclusion criteria of the CCDI data sharing initiative. An "integrative sequencing approach" carried out in a CLIA-certified laboratory is utilized to provide a comprehensive landscape of the genetic alterations in individual tumor specimens for the purpose of identifying informative and/or actionable mutations. Using a number of computational pipelines that we have developed in-house or have adapted from the public domain, candidate molecular aberrations are nominated and then analyzed by scientists for clinical significance and/or relevance. This approach enables the detection of point mutations, insertions/deletions, gene fusions and rearrangements, amplifications/deletions, and outlier expressed genes. Furthermore, we can identify certain germline alterations that may also be relevant.

  Study phs002431

• A Scalable, GMP-Compatible, Autologous Organotypic Cell Therapy for Dystrophic Epidermolysis Bullosa

  Gene editing in induced pluripotent stem (iPS) cells has been hailed for enabling new cell therapies for various monogenetic diseases including dystrophic epidermolysis bullosa (DEB). However, manufacturing, efficacy, and safety roadblocks have limited the development of genetically corrected, autologous iPS cell-based therapies. Dystrophic Epidermolysis Bullosa Cell Therapy (DEBCT), is a new generation, Good Manufacturing Practice-compatible (cGMP), reproducible, and scalable platform to produce autologous clinical-grade iPS cell-derived organotypic induced skin composite (iSC) grafts to treat incurable wounds of patients lacking type VII collagen (C7). DEBCT uses a single step, combined, high-efficiency reprogramming, and CRISPR-based genetic correction to generate genome scar-free, COL7A1 corrected clonal iPS cells from primary patient fibroblasts. Validated iPS cells are converted into epidermal, dermal, and melanocyte progenitors with a novel 2D organoid differentiation protocol, followed by CD49f enrichment and expansion to minimize maturation heterogeneity. iSC product characterization by single cell transcriptomics was followed by mouse xenografting for disease correcting activity at 1 month and toxicology analysis at 1-6 months. Culture-acquired mutations, potential CRISPR-off target effects, and cancer-driver variants were evaluated by targeted and whole genome sequencing. iPS cell-derived iSC grafts were reproducibly generated from four recessive DEB patients with different pathogenic mutations. Organotypic iSC grafts onto immune-compromised mice developed into stable stratified skin with functional C7 restoration. Single cell transcriptomic characterization of iSCs revealed prominent holoclone stem cell signatures in keratinocytes and the recently described Gibbin-dependent signature in dermal fibroblasts. The latter correlated with enhanced graftability. Multiple orthogonal sequencing and subsequent computational approaches identified random and non-oncogenic mutations introduced by the manufacturing process. Toxicology revealed no detectable tumors after 3-6 months in DEBCT-treated mice. In conclusion, DEBCT successfully overcomes previous roadblocks and establishes a robust, scalable, and safe cGMP manufacturing platform for the production of a CRISPR-corrected autologous organotypic skin graft to heal DEB patient wounds.

  Study phs003271

• Genomic Basis of Phenotypic Variability of Complex Disorders

  The 520-kbp deletion on chromosome 16p12.1 is associated with multiple neurodevelopmental outcomes, including intellectual disability/developmental delay (ID/DD), schizophrenia, autism, and epilepsy. This variant is inherited in >95% of cases from carrier parents manifesting neuropsychiatric features, while affected children with the deletion were more likely to carry another large Copy Number Variation (CNV) or rare deleterious mutation elsewhere in the genome, or "second-hits", compared to carrier parents. Thus, while the 16p12.1 deletion confers significant risk for a range of disorders, the ultimate phenotypic trajectory is determined by "second-hit" variants in the genetic background. However, outstanding questions remain about the genetic background in these disorders, including the functional effects of "second-hit" variants towards gene expression or developmental phenotypes, the full spectrum of "second-hit" variants, and the mechanisms driving accumulation of these variants.In version 1, we performed deep clinical phenotyping, whole-genome sequencing (WGS), SNP microarray analysis, and RNA-sequencing of lymphoblastoid cell lines of 32 individuals in five large families with the 16p12.1 deletion. We found that specific classes of rare single-nucleotide and structural variants in coding and non-coding regions contributed towards expression changes in affected children based on their inheritance patterns, including a subset of variants that acted synergistically with the deletion to alter expression. We further found that rare variant-mediated expression changes contribute to specific developmental phenotypes in the carrier children, suggesting a potential genetic mechanism for variable expressivity of the deletion.In version 2, we additionally analyzed genetic and phenotypic data from individuals with 16p12.1 deletion and their spouses to assess the role of assortative mating modulating phenotypic outcomes of the 16p12.1 deletion. We identified significant within- and cross- disorder phenotypic correlations between spouses, but observed no significant genetic correlations between spouses, in line with expectations.

  Study phs002450

• Spatial Transcriptomics of Vaccine Therapy With or Without Cyclophosphamide in Treating Patients Undergoing Chemotherapy and Radiation Therapy for Stage I or Stage II Pancreatic Cancer That Can Be Removed by Surgery

  Pancreatic ductal adenocarcinoma (PDAC) is a rapidly progressing cancer that responds poorly to immunotherapies. Intratumoral tertiary lymphoid structures (TLS) have been associated with rare long-term PDAC survivors, but the role of TLS in PDAC and their spatial relationships within the context of the broader tumor microenvironment remain unknown. Here, spatial transcriptomics data were generated from 16 pancreatic cancer patients treated with GVAX, whose tumors presented with induced intratumoral TLS. Resected tumor specimens were embedded in OCT media and freshly frozen. Sections were cut into 6.5 x 6.5 mm dimensions compatible with Visium spatial transcriptomics slides. TLS-positive tumor sections from 14 patients (14 tumor samples in total) and lymph nodes from remaining patients (2 lymph node samples in total) were selected by an expert pathologist for profiling. We used 4 Visium slides and covered the 16 capture areas with sections cut in a cryostat at 10 µm. The fresh frozen Visium protocol was followed according to manufacturer's recommendations (10x Genomics Spatial 3' v1). Tissue permeabilization and library construction was carried out according to the manufacturer's protocols. Permeabilization time was optimized to 30 minutes. The captured tissue sections were permeabilized to allow RNA to be captured onto the slide where it reverse-transcribed to cDNA. The cDNA was then amplified, and sequencing libraries were prepared using the 10x Genomics Visium Spatial Gene Expression Reagent Kit for Library Preparation. The libraries were then sequenced using an Illumina NovaSeq sequencer. The raw data obtained from the sequencing were processed using the SpaceRanger (spaceranger-1.2.0) pipeline for alignment onto the GRCh38-2020-A reference transcriptome quantification of gene expression. Unsupervised learning generated a TLS-specific spatial gene expression signature that significantly associates with improved survival in PDAC patients. These analyses demonstrate TLS-associated intratumoral B cell maturation.

  Study phs003862

• CEBP-Beta/IL-1-Beta/ TNF-alpha Feedback Loop Drives Drug Resistance to BCL2 and MDM2 Inhibitors in Monocytic Leukemia Cells

  To uncover the mechanisms of MDM2 inhibitor resistance that are mediated by IL-1, we performed RNAseq on three primary AML patient cells treated with idasanutlin in the presence or absence of IL-1β. These three leukemia samples were chosen because they exhibit high blast counts and are protected by IL-1β when treated with idasanutlin, as determined by apoptosis assay. We observed that a majority of the major p53 pathway targets remained upregulated in the presence of IL-1β. We next performed differential expressed gene (DEG) analysis following IL-1β treatment with and without idasanutlin. IL-1β treatment upregulated 3295, 1353, and 680 genes with ≥1 Log2FC, in each of the three patient samples, and a significant majority of these genes (82%, 62.4%, and 47.6%) remained upregulated in the presence of idasanutlin. Pathway analysis showed that IL-1β upregulated genes were associated with NF-κB, Interleukin-1 family signaling pathways, immune and inflammatory associated genes and myeloid maturation markers. To identify downstream targets that contribute to the protective effect of IL-1β during idasanutlin treatment, we performed DEG analysis on IL-1β + idasanutlin treatment compared to idasanutlin treatment alone. Interestingly, IL-1β induces its own expression and other pro-survival cytokines including TNF, CSF2 (GMCSF), IL-6, CSF3 (GCSF), as well as their respective receptors and/or coactivators, including IL1RL1 and multiple TNF receptors. We verified that IL-1β and TNF-α induce the upregulation of their own membrane-bound forms, their corresponding receptors, CSF2RB, and monocytic markers, as determined by flow cytometry immunophenotyping. Furthermore, IL-1β upregulated a number of pro-inflammatory chemokines, including CXCL13, CXCL2, etc. These findings highlight a pleiotropic role of IL-1β. The RNA expression of these three samples in the presence and absence of IL-1β and idasanutlin treatment will be deposited in dbGaP.

  Study phs003479

• Whole Exome Sequencing of Diffuse Large B-Cell Lymphoma

  We performed massively parallel whole exome sequencing of 55 primary tumor samples from patients with diffuse large B cell lymphoma (DLBCL) and matched normal tissue. We identified recurrent mutations in genes that are well known to be functionally relevant in DLBCL, as well as in genes for which a functional role in DLBCL has not been previously suspected. Significantly mutated genes include MYD88, CARD11, EZH2, CREBBP, as well as MEF2B, MLL2, BTG1, GNA13, ACTB, P2RY8, PCLO and TNFRSF14. Analysis of somatic mutations in patients with DLBCL also revealed that BCL2 mutations commonly occur in patients with BCL2/IgH rearrangements as a result of somatic hypermutation normally occurring at the IgH locus. The BCL2 point mutations are primarily synonymous, likely caused by activation-induced cytidine deaminase (AID)-mediated somatic hypermutation, and those non-synonymous mutations that are observed tend to be found outside of the functionally important BH domains of the protein, suggesting that strong negative selection against BCL2 loss-of-function mutations is at play. We further identified genes with mutations that are likely to be functionally relevant despite being mutated at a low rate. These genes include KRAS, BRAF and NOTCH1 as likely drivers of DLBCL pathogenesis in some patients.In order to facilitate additional in vitro and in vivo analyses of the discrete molecular clusters and associated targeted therapies in preclinical models, we also performed our subtyping analysis on 28 Diffuse Large B-Cell Lymphoma (DLBCL) cell lines. We performed whole exome sequencing (WES) on these cell lines to capture structural variants (SVs), from which we obtained full genetic characterization and segregation of these cell lines into our previously defined molecular clusters by DLBclass assignment. These analyses are available in an interactive DLBclass portal available to the public. Our data provide an unbiased view of the landscape of mutations in DLBCL. These data may point towards new targets for the treatment of DLBCL.

  Study phs000450

• Proteogenomic Characterization Unveils Biomarkers Associated With Chemoresistance in Muscle Invasive Bladder Cancer

  Cisplatin-based neoadjuvant chemotherapy (NAC) combined with radical cystectomy has been used as a standard of care for patients with clinically non-metastatic muscle invasive urothelial bladder cancer, but 40-50% patients will benefit from the treatment. Molecular subtypes of muscle invasive bladder cancer (MIBC) provide a possible biomarker roadmap that may help identify patients who may or may not respond to NAC. Recently, by integrating Mass Spectrometry-based proteomics, proteogenomic studies from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) have demonstrated the potential of this approach in prioritizing candidate driver genes and facilitating understanding of the chemotherapy resistance mechanisms. To demonstrate the proteogenomic workflow for investigating the mechanisms underlying chemotherapy resistance in MIBC, we conducted proteogenomic characterization of 46 pre- and 14 post- treatment tumors, including whole exome sequencing (WES), mRNA sequencing, Tandem Mass Tag (TMT)-proteomics, or TMT-phosphoproteomic. These samples were selected from an initial cohort of 143 MIBC tissues and filtered by tumor purity, clinical history, pathological response, and proteomic quality control metrics. The final cohort comprised samples from 53 patients, including 45 with both genomic and proteomic data and 8 with genomic data only.Our multi-omics-based clustering reveals subtypes associated with chemotherapy sensitivity. Protein abundance of a short isoform of ATAD1 and RAF family proteins were identified as biomarkers of chemosensitivity which highlights the importance of proteomics data. Integration of proteomic and phosphoproteomic data reveals Wnt signaling via GSK3B-S9 phosphorylation and the JAK/STAT pathway as potential targets to overcome chemoresistance. Correlations between PD-L1 and TROP-2/NECTIN-4 indicate an additive benefit of combination therapy targeting these proteins. Our data analysis clearly demonstrates that proteogenomic plays an important role in uncovering potential resistance mechanisms that operate only at protein and Post-Translational Modification (PTM) levels. Proteomics data can be accessed through PRIDE with ID PXD060290. RNA-seq and whole-exome sequencing (WES) FASTQ files are archived in the dbGaP database.

  Study phs004049

• Genomic and Immune Profiling of Breast Cancer Brain Metastases

  Previous work has shown that breast cancer brain metastases (BCBrM) can have unique molecular profiles compared to extracranial tumors (ECTs), resulting in some patients demonstrating subtype switching between primary/extracranial tumors and brain metastases. Description of the biology of BCBrM and associations between biological features and clinical outcomes, particularly between molecular intrinsic subtypes, will facilitate the design of rational therapies for patients with BCBrM. To better characterize the molecular profiles of BCBrM, we conducted whole exome sequencing (WES) and total RNA sequencing (RNA-Seq) on BCBrM tissues, regardless of clinical or molecular subtype, obtained from a cohort of 42 patients from Duke University Medical Center. This cohort adds valuable patient samples to the field and enables strengthened investigation of somatic mutations, copy number alterations, gene expression profiles, and immune populations within and between BCBrM patients and other similar cohorts. The Duke BCBrM cohort includes clinical and molecular annotation of the 42 patient specimens and their associated WES and RNA-Seq data. Clinical and molecular (PAM50) intrinsic subtypes in ECTs and brain metastases were largely concordant within patients. There were observed differences in intrinsic subtype calls between frozen and FFPE specimens of the same tissue, with discordance mostly observed with LumB calls. Copy number alterations in chromosomal regions included clinically relevant genes, and somatic alterations were largely conserved within patients across matched samples, with few genes, including TP53, being recurrently altered in the majority of patient specimens. In matched FFPE brain metastasis and ECT samples, we observed differentially expressed genes and pathways that had increased or decreased expression in brain metastasis, including in oxidative phosphorylation and DNA repair. Survival outcomes for patients differed by intrinsic molecular subtypes, with some association between expression of select gene pathways and survival. Data available through dbGaP will be the sequencing data and sample attributes from BCBrM tissues obtained from 42 patients from Duke University Medical Center.

  Study phs003673

• Human Dorsal Root Ganglion RNA Landscape Profiling for Neuropathic and Chronic Pain

  Bulk and spatial RNA sequencing were performed on human Dorsal Root Ganglia (DRGs), peripheral sensory nerves and nodose ganglia and relative gene abundances were calculated. Various analyses were performed: Human DRG gene expression profiles were contrasted with a panel of gene expression profiles of relevant tissues in human and mouse (integrating, among other sources, datasets from ENCODE and GTex) in order to identify. DRG-enriched gene expression, co-expression modules of DRG-expressed genes, and key transcriptional regulators in humans. Contrasting the human and mouse DRG transcriptomes to identify DRG-enriched gene expression patterns that were conserved between human and mouse, identifying putative cell types of expression of these genes, and potential known drugs that might target the corresponding gene products. Characterization of non-coding RNA profile of human and mouse DRGs. Characterization of DRG-enriched alternative splicing and alternative transcription start site usage based transcript variants in humans and mouse, and the overlap between these two species. Contrasting of human DRG and GTex human tibial nerve samples to identify putative axonally transported mRNAs in sensory neurons. Human DRG and peripheral nerves transcriptomes from donors suffering from neuropathic and/or chronic pain were contrasted with controls to identify. Differentially expressed genes, pathways and regulators path play a potential role in neuronal plasticity, electrophysiological activity, immune signaling and response. Predictive models (Random Forests) were built to jointly predict the sex and pain state of samples based on information contained solely in autosomal gene expression profile. Gene co-expression modules were identified and gene set enrichment analysis performed.to identify sample - pathway associations, and to broadly characterize plasticity in human DRG cell types. Spatial transcriptomics was performed on human DRGs and nodose ganglia to obtain near single-neuron resolution. We identified distinct clusters of human DRG sensory neurons. We characterized the expression of different gene families (e.g., GPCRs, sodium channels, potassium channels, etc.). We investigated differences between male and female neurons.

  Study phs001158

• Parallel CRISPR Editing of Familial ALS Mutations

  Induced pluripotent stem cells (iPSCs) provide a modeling system for human disease within the critical context of the human genome. CRISPR editing of iPSCs allows the construction of isogenic pairs where the cells have nearly identical genomes except for the desired edited change. Here, we used CRISPR editing on a healthy control line to generate three new isogenic iPSC lines that each contains a different familial amyotrophic lateral sclerosis (fALS) mutation (TARDBP[G298S/+], SOD1[G85R/+], and PFN1[G118V/+]). As controls for each mutation, we retained iPSC lines that went through the editing process but did not have the desired heterozygous mutation. We have also performed editing in a patient cell line with a hexanucleotide repeat in C9ORF72. This edit mutates a non-canonical start codon upstream of the hexanucleotide repeat. These edited lines have been used in three different studies:(1) In “Human sensorimotor organoids derived from healthy and amyotrophic lateral sclerosis stem cells form neuromuscular junctions” (PMID: 34362895), we performed exome sequencing to confirm the absence of off-target CRISPR events. We also performed single-cell sequencing on sensorimotor organoids from a second healthy control iPSC line, which is deposited alongside the exome sequencing data.(2) In “iPSC motor neurons, but not other derived cell types, capture gene expression changes in postmortem sporadic ALS motor neurons (PMID: 37651231), we differentiated these isogenic lines into motor neurons, sensory neurons, cortical neurons, and astrocytes and then performed bulk RNA-sequencing. We then assessed similarities between fALS mutations, differences between cell types, and the capacity of iPSC modeling to capture differentially expressed genes in postmortem sporadic ALS motor neurons.(3) In "Blocking RAN translation without altering RNA foci rescues C9ORF72-related ALS/FTD phenotypes" (Jiang et al. 2025), we mutate a non-canonical start codon upstream of a hexanucleotide repeat in C9ORF72. This mitigates several phenotypes related to neurodegeneration.

  Study phs002440

• Genomic features of Helicobacter pylori-na��ve diffuse-type gastric cancer.

  Helicobacter pylori (HP) is a major etiologic driver of diffuse-type gastric cancer (DGC). However, improvements in hygiene have led to an increase in the prevalence of HP-na��ve DGC; that is, DGC that occurs independent of HP. Although multiple genomic cohort studies for gastric cancer have been conducted, including studies for DGC, distinctive genomic differences between HP-exposed and HP-na��ve DGC remain largely unknown. Here, we employed exome and RNA sequencing with immunohistochemical analyses to perform binary comparisons between 36 HP-exposed and 27 HP-na��ve DGCs from sporadic, early-stage, and intramucosal or submucosal tumor samples. Among the samples, 33 HP-exposed and 17 HP-na��ve samples had been preserved as fresh-frozen samples. HP infection status was determined using stringent criteria. HP-exposed DGCs exhibited an increased single nucleotide variant burden (HP- exposed DGCs; 1.97 [0.48���7.19] and HP-na��ve DGCs; 1.09 [0.38���3.68] per megabase; p=0.0003) and a higher prevalence of chromosome arm-level aneuploidies (p<0.0001). CDH1 was mutated at similar frequencies in both groups, whereas RHOA���ARHGAP pathway misregulation was exclusive to HP-exposed DGCs (p=0.0167). HP-exposed DGCs showed gains in chromosome arms 8p/8q (p<0.0001), 7p (p=0.0035) and 7q (p=0.0354), and losses in 16q (p=0.0167). Immunohistochemical analyses revealed a higher expression of intestinal markers such as CD10 (p<0.0001) and CDX2 (p=0.0002) and a lower expression of the gastric marker, MUC5AC (p=0.0305) among HP-exposed DGCs. HP-na��ve DGCs, on the other hand, had a purely gastric marker phenotype. This work reveals that HP-na��ve and HP-exposed DGCs develop along different molecular pathways, which provide a basis for early detection strategies in high incidence settings.

  Study JGAS000575

• Molecular analysis of diffuse cerebellar gliomas

  Among diffuse gliomas, oligodendrogliomas show relatively better prognosis, respond well to radiotherapy and chemotherapy, and seldom progress to very aggressive tumors. To elucidate the genetic and epigenetic background for such behavior and tumor evolution during tumor relapse, we comparatively analyzed 12 pairs of primary and recurrent oligodendrogliomas with 1p/19q-codeletion. Initial treatment for these patients was mostly chemotherapy alone. Temozolomide was used for 3, and procarbazine, nimustine and vincristine (PAV chemotherapy) were used for 7 patients. World Health Organization histological grade at recurrence was mostly stable; it was increased in 2, the same in 9, and decreased in 1 cases. Whole-exome sequencing demonstrated that the rate of shared mutation between the primary and recurrent tumors was relatively low, ranging from 3.2-57.9% (average, 33.3%), indicating a branched evolutionary pattern. The trunk alterations that existed throughout the course were restricted to IDH1 mutation, 1p/19q-codeletion, and TERT promoter mutation, and mutation of the known candidate tumor suppressor genes CIC and FUBP1 were not consistently observed between primary and recurrent tumors. Multiple sampling from different regions within a tumor showed marked intratumoral heterogeneity. Notably, in general, the number of mutations was not significantly different after recurrence, remaining under 100, and no hypermutator phenotype was observed. FUBP1 mutation, loss of chr. 9p21, and TCF12 mutation were among a few recurrent de novo alterations that were found at recurrence, indicating that these events were clonally selected at recurrence but were not enough to enhance malignancy. Genome-wide methylation status, measured by Illumina 450 K arrays, was stable between recurrence and the primary tumor. In summary, although oligodendroglioma displays marked mutational heterogeneity, histological malignant transformation accompanying events such as considerable increase in mutation number and epigenetic profile change were not observed at recurrence, indicating that noticeable temporal and spatial genetic heterogeneity in oligodendrogliomas does not result in rapid tumor progression.

  Study JGAS000106

• High-throughput telomere length measurement at nucleotide resolution using the PacBio high fidelity sequencing platform

  Telomeres are specialized nucleoprotein structures at the ends of linear chromosomes that are essential for the stability as well as the complete replication of the human genome. The progressive shortening of steady-state telomere length in normal human somatic cells is a promising biomarker for age-associated diseases. However, there remain substantial challenges in quantifying telomere length in clinics and research laboratories due to the lack of accurate and high-throughput telomere length measurement method. Here, we describe a novel workflow to capture and analyze telomeres accurately at the single-nucleotide resolution. By using newly designed telobaits that are complementary to the single-stranded G-rich 3’ telomeric overhangs, we can specifically and efficiently capture full-length telomeres and subject them to single-molecule real-time (SMRT) sequencing. We show that the telomere length can be measured at nucleotide resolution using the PacBio high fidelity (HiFi) sequencing platform, which provides the necessary accuracy and throughput to uncover the steady-state telomere length distribution in human culture cell lines as well as clinical patient samples. This new method is a valuable tool not only in population-wide epidemiology studies but also for the longitudinal assessment of telomere attrition in individuals. Our results also reveal the extreme heterogeneity of telomeric variant sequences (TVSs) that is dispersed throughout the telomere repeat region. Importantly, the unique distribution pattern and sequence of TVSs in individuals may be used as a fingerprint for sample identification. In addition, the presence of TVSs disrupts the continuity of the canonical (5’-TTAGGG-3’)n telomere repeats, which could affect the binding of shelterin complexes at the chromosomal ends. Therefore, TVSs may act as a form of genetic polymorphisms that impairs telomere protection. Together with the absolute telomere length in each individual, TVSs may have profound implications in human aging and diseases.

  Study EGAS00001006103

• Successful immune checkpoint blockade in a patient with advanced stage microsatellite unstable biliary tract cancer (H021)

  Cancers acquire multiple somatic mutations that can lead to the generation of immunogenic mutation-induced neoantigens. These neoantigens can be recognized by the host’s immune system. However, continuous stimulation of immune cells against tumor antigens can lead to immune cell exhaustion, which allows uncontrolled outgrowth of tumor cells. Recently, immune checkpoint inhibitors have emerged as a novel approach to overcome immune cell exhaustion and re-activate anti-tumor immune responses. In particular, antibodies blocking the exhaustion-mediating programmed death receptor (PD-1)/PD-L1 pathway have shown clinical efficacy. The effects were particularly pronounced in tumors with DNA mismatch repair deficiency and a high mutational load, which typically occur in the colon and endometrium. Here, we report on a 24-year old woman diagnosed with extrahepatic cholangiocarcinoma who showed strong and durable response to the immune checkpoint inhibitor pembrolizumab, although treatment was initiated at an advanced stage of disease. The patient’s tumor displayed DNA mismatch repair deficiency and microsatellite instability (MSI), but lacked other features commonly discussed as predictors of response towards checkpoint blockade, such as PD-L1 expression or dense infiltration with cytotoxic T cells. Notably, high levels of HLA class I and II antigen expression were detected in the tumor, suggesting a potential causal relation between functionality of the tumor’s antigen presentation machinery and the success of immune checkpoint blockade. We suggest determining MSI status in combination with HLA class I and II antigen expression in tumors potentially eligible for immune checkpoint blockade even in the absence of conventional markers predictive for anti-PD-1/PD-L1 therapy and in entities not commonly linked to MSI phenotype. Further studies are required to determine the value of these markers for predicting the success of immune checkpoint blockade. (H021)

  Study EGAS00001002441

• H3Africa - Genomic and Environmental Risk Factors for Cardiometabolic Disease in Africans

  The long-term vision of the AWI-Gen Collaborative Centre (CC) is to build sustainable capacity in Africa for research that leads to an understanding of the interplay between genetic, epigenetic and environmental risk factors for obesity and related cardiometabolic diseases (CMD) in sub-Saharan Africa. The CC will be consolidated under the auspices of the University of the Witwatersrand (Wits) and the International Network for the Demographic Evaluation of Populations and Their Health in Developing Countries (INDEPTH). It will capitalize on the unique strengths of existing longitudinal cohorts, including the urban Soweto and rural Agincourt studies in South Africa (Wits based), and the well established INDEPTH demographic health and surveillance centers in Kenya, Ghana, Burkina Faso and South Africa. The centers offer established infrastructure, trained fieldworkers, long-standing community engagement, and detailed longitudinal phenotypic data, focusing on obesity and cardiometabolic health. Key strengths are harmonized phenotyping across sites, building on strong existing cohorts, and representation of the geographic and social variability of African populations. We aim to: 1. Build sustainable infrastructure (biobanks and laboratories) and capabilities (well characterized population cohorts, genotyping and bioinformatics) for genomic research on the African continent; 2. Understand the genomic architecture of sub-Saharan populations from west, east and south Africa to guide genomic studies (genome sequencing and high throughput SNP and CNV arrays using unrelated individuals and family trios to improve the accuracy of haplotype analyses) and; 3. Investigate the independent and synergistic genomic contributions to body fat distribution (BMI, hip/waist circumference, subcutaneous and visceral fat) in these populations considering the relevant environmental and social contexts (rural/urban communities, quickly transitioning obesity prevalence, differential HIV, TB, and malaria infection histories). We will investigate the effect of obesity and fat distribution on the risk for CMD in the longitudinal cohorts.

  Study EGAS00001002482

• Local In Time Statistics for processual research

  Background: Functional genomics in a processual analysis cover the time-dependent changes in transcriptomics and epigenetics before diagnosis of a disease, reflecting the changes in both life style and disease processes. The aim of this paper is to explore the dynamic, time-dependent mechanisms of the metastatic processes, using blood transcriptomics and including time in a continuous manner. For achieving this goal we develop new statistical methods based on statistics that are local in time. Methods: The new statistical method, Local In Time Statistics (LITS), is based on calculating statistics in moving windows and randomization. The method has been tested for the analysis of a dataset that collectively provides information on the blood transcriptome up to eight years before breast cancer diagnosis. The dataset from the NOWAC Post-genome Cohort consists of 467 case-control pairs matched on birth year and time of blood sampling. The data for a pair is the difference in log2 gene expression between the case and control. The stratified analyses are based on important biological differences like metastatic versus non-metastatic cancer, and the mode of cancer detection, i.e. screening detected versus clinically detected cancers. The dataset was used for examining whether the gene expression profile varies between cases and controls, with time, or between cases with and without metastases. Results: The null hypotheses of no differences between cases and controls, no time-dependent changes, and no differences between different strata were all rejected. For screening detected cancers the probability of correct prediction of metastasis status was best in year 1 before diagnosis compared to year 3 and 4 before diagnosis for clinically detected cancers. The predictor was not very sensitive to the number of genes included.Conclusions: Using a new statistical method, LITS, we have demonstrated time-dependent changes of the blood transcriptome up to eight years before breast cancer diagnosis.

  Study EGAS00001002520

• Genome_Diversity_in_Africa_Project__Uganda

  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 bioinformatics

  Study EGAS00001002523

• Probabilistic modeling of personalized drug combinations from integrated chemical screen and molecular data in sarcoma

  Background: Cancer patients with advanced disease exhaust available clinical regimens and lack actionable genomic medicine results, leaving a large patient population without effective treatments options when their disease inevitably progresses. To address the unmet clinical need for evidence-based therapy assignment when standard clinical approaches have failed, we have developed a probabilistic computational modeling approach which integrates sequencing data with functional assay data to develop patient-specific combination cancer treatments. Methods: Tissue taken from a murine model of alveolar rhabdomyosarcoma was used to perform single agent drug screening and DNA/RNA sequencing experiments; results integrated via our computational modeling approach identified a synergistic personalized two-drug combination. Cells derived from the tumor were allografted into mouse models and used to validate the personalized two-drug combination. Computational modeling of single agent drug screening and RNA sequencing of multiple heterogenous sites from a single patient’s epithelioid sarcoma identified a personalized two-drug combination effective across all tumor regions. The heterogeneity-consensus combination was validated in a xenograft model derived from the patient’s primary tumor. Cell cultures derived from human and canine undifferentiated pleomorphic sarcoma were assayed by drug screen; computational modeling identified a resistance-abrogating two-drug combination common to both cell cultures. This combination was validated in vitro via a cell regrowth assay. Results Our computational modeling approach addresses three major challenges in personalized cancer therapy: synergistic drug combination predictions (validated in vitro and in vivo in a genetically engineered murine cancer model), identification of unifying therapeutic targets to overcome intra-tumor heterogeneity (validated in vivo in a human cancer xenograft), and mitigation of cancer cell resistance and rewiring mechanisms (validated in vitro in a human and canine cancer model). Conclusions These proof-of-concept studies support the use of an integrative functional approach to personalized combination therapy prediction for the population of high-risk cancer patients lacking viable clinical options and without actionable DNA sequencing-based therapy.

  Study EGAS00001003564

• 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

• Assessing the impact of low frequency coding variants on disease risk using the Exomechip

  Following several rounds of Genome Wide Association (GWA) scans and subsequent meta-analyses of results multiple risk loci have been identified for many common diseases. However, in most instances the implicated common variants identified so far explain only a modest fraction of the genetic risk. In parallel to our efforts to identify the causative variants in the known loci it is necessary to assess the full spectrum of sequence variants for disease risk and in particular low frequency and rare variants that have not been tested in a comprehensive way so far. To this end an international effort by investigators who have performed whole exome sequencing in circa 12,000 individuals have assembled a set of ~250,000 exonic variants of low frequency – defined as seen in at least two studies and a minimum of three individuals (non-sysnonymous) or two individuals for SNPs altering splice sites / stop codons. The SNP content from the exome studies was complemented with additional interesting SNP sets totalling 25,000 markers (GWA tag SNPs, grid of common variants, HLA, Mitochondrial, Y-chromosome etc) and were used to generate a custom iSELECT array, the exome chip. Adequately powered association studies to test low frequency variants for association to disease risk are still very expensive if conducted by whole exome sequencing. The exome chip provides a cost efficient way to undertake such an experiment and the Wellcome Trust Cases Control Consortium will be applying this approach to eight diseases, type 1 and type 2 diabetes, coronary artery disease, hypertension, multiple sclerosis, rheumatoid arthritis, bipolar, and ankylosing spondylitis. The first objective will be to generate a large set of at least 10,000 common UK controls as analysis of lower frequency variants will require large sample sizes. This includes ~6000 samples from the 1958 Birth Cohort.

  Study EGAS00001000584

• 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

• UK10K_NEURO_ASD_FI

  In the UK10K project we propose a series of complementary genetic approaches to find new low frequency/rare variants contributing to disease phenotypes. These will be based on obtaining the genome wide sequence of 4000 samples from the TwinsUK and ALSPAC cohorts (at 6x sequence coverage), and the exome sequence (protein coding regions and related conserved sequence) of 6000 samples selected for extreme phenotypes. Our studies will focus primarily on cardiovascular-related quantitative traits, obesity and related metabolic traits, neurodevelopmental disorders and a limited number of extreme clinical phenotypes that will provide proof-of-concept for future familial trait sequencing. We will analyse directly quantitative traits in the cohorts and the selected traits in the extreme samples, and also use imputation down to 0.1% allele frequency to extend the analyses to further sample sets with genome wide genotype data. In each case we will investigate indels and larger structural variants as well as SNPs, and use statistical methods that combine rare variants in a locus or pathway as well as single-variant approaches.These samples are a subset of a nationwide collection of Finnish autism spectrum disorder (ASD) samples. The samples have been collected from Central Hospitals across Finland in collaboration with the University of Helsinki. The samples consist of 93 individuals with a diagnosis of autistic disorder or Asperger syndrome from 36 families with at least two affected individuals. Of these individuals, 16 can be genealogically connected to form two large pedigrees originating from Central Finland, suggesting possible genetic risk factors shared identical by descent within the pedigrees. All diagnoses are based on ICD-10 and DSM-IV diagnostic criteria for ASDs. Additional phenotypic data is available for a subset of the individuals.For further information with regard to this cohort please contact Aarno Palotie (Aarno.palotie@helsinki.fi).

  Study EGAS00001000110

• Genetic analysis of HLA and immune escape genes in Diffuse Large B-cell Lymphoma

  Fifty percent of diffuse large B-cell lymphoma (DLBCL) cases lack cell-surface expression of the class-I major histocompatibility complex (MHC-I), thereby escaping immune recognition by cytotoxic T cells. In order to comprehensively identify the mechanisms involved in MHC-I loss, we first performed immunophenotypic analysis of both MHC class-I and -II in 657 cases across the spectrum of B-NHL, revealing that loss of MHC-I, but not MHC-II, is preferentially restricted to DLBCL. We then used whole exome and targeted deep-sequencing to examine genes involved in MHC-I expression in 74 DLBCL samples representative of MHC-I positive and negative cases. We show here that somatic biallelic or monoallelic inactivation of B2M and/or HLA-I is present in 80.9% (34/42) of MHC-I negative tumors. Furthermore, 68.8% (22/32) of MHC-I positive DLBCLs also harbored monoallelic HLA-I genetic alterations (MHC-I positivemono) that lead to allelic imbalance, suggesting allele-specific inactivation. Both MHC-I negative and MHC-I positivemono cases showed a significantly higher mutational burden as well as a higher number of inferred neo-antigens, suggesting co-selection of HLA-I loss and sustained neo-antigen production. Interestingly, the analysis of > 500.000 individuals in two databases revealed homozygosity of germline HLA-I genes in 26-38% of DLBCL patients, a frequency significantly higher than that observed in any other cancer type. In mice, germinal-center B cells lacking HLA-I expression did not progress to lymphoma and were counterselected in the context of oncogene-driven lymphomagenesis, suggesting that additional events are needed to license immune evasion. These results suggest a multi-step process of HLA-I loss including both germ-line and somatic events in DLBCL development, and have direct implications for the pathogenesis and immunotherapeutic targeting of this disease.

  Study EGAS00001005054

• Same-day genomic and epigenomic diagnosis of brain tumors using realtime nanopore sequencing

  Molecular classification of cancer has entered clinical routine to inform diagnosis, prognosis and treatment decisions. At the same time, new tumor entities have been identified that cannot be defined histologically. For central nervous systems tumors, the current World Health Organization classification explicitly demands molecular testing, e.g. for 1p/19q- codeletion or IDH mutations, to make an integrated histomolecular diagnosis. However, a plethora of sophisticated technologies is currently needed to assess different genomic and epigenomic alterations and turnaround times are in the range of weeks, which makes standardized and widespread implementation difficult and hinders timely decision making. Here, we explored the potential of a pocket-size nanopore sequencing device for multimodal and rapid molecular diagnostics of cancer. Low-pass whole genome sequencing was used to simultaneously generate copy number (CN) and methylation profiles from native tumor DNA in the same sequencing run. Single nucleotide variants in IDH1, IDH2, TP53, H3F3A and the TERT promoter region were identified using deep amplicon sequencing. Nanopore sequencing yielded ~0.1X genome coverage within six hours and resulting CN and epigenetic profiles correlated well with matched microarray data. Diagnostically relevant alterations, such as 1p/19q codeletion, and focal amplifications could be recapitulated. Using ad hoc random forests, we could perform supervised pan-cancer classification to distinguish gliomas, medulloblastomas and brain metastases of different primary sites. Single nucleotide variants in IDH1, IDH2 and H3F3A were identified using deep amplicon sequencing within minutes of sequencing. Detection of TP53 and TERT promoter mutations shows that sequencing of entire genes and GC-rich regions is feasible. Nanopore sequencing allows same-day detection of structural variants, point mutations and methylation profiling using a single device with negligible capital cost. It outperforms hybridization-based and current sequencing technologies with respect to time-to diagnosis and required laboratory equipment and expertise, aiming to make precision medicine possible for every cancer patient, even in resource restricted settings.

  Study EGAS00001002213

• Short and long-read genome sequencing methodologies for somatic variant detection; genomic analysis of a patient with diffuse large B-cell lymphoma

  Recent advances in throughput and accuracy mean that the Oxford Nanopore Technologies (ONT) PromethION platform is a now a viable solution for WGS. New bioinformatic methods have been developed to take advantage of this long read data, however much of the validation of these tools has focussed on calling germline variants (both SNVs and structural variants). Somatic variants are outnumbered many-fold by germline variants and their detection is further complicated due to their frequency varying depending on tumour purity/subclonality. Here, we evaluate the extent to which Nanopore WGS enables genome-wide detection and analysis of somatic variation. We do this through sequencing tumour and germline genomes for a patient with diffuse B-cell lymphoma. We examine the capability of currently available tools for calling somatic variants in ONT data by comparing the data with results from 150bp short-read sequencing of the same samples. We then conduct a detailed analysis of the performance of multiple long-read mappers and structural variant callers for calling large, somatic structural variants (SVs) in ONT data. Our protocol achieved yields of up to 96 mapped Gb per PromethION flow cell with average read lengths of ~5kb. Calling germline SNVs from these data achieved good specificity and sensitivity. However, results of somatic SNV calling highlight the need for the development of specialized joint calling algorithms. Our analysis of structural variants shows that the comparative performance of different tools varies significantly between SV types, and suggest long reads are especially advantageous for calling large somatic deletions and duplications. Finally, we highlight the utility of long reads for phasing clinically relevant variants by using the ONT data to confirm that a somatic 1.6Mb deletion and a p.(Arg249Met) mutation involving TP53 are oriented in trans.

  Study EGAS00001004266

• GDAP___Genome_Diversity_in_Africa_Project

  The Genomic Diversity in Africa Project (GDAP) started with the plan to develop a genomic resource from African populations, characterise genomic diversity and population history, and facilitate clinical studies in Africa. Currently, 25 individuals from 24 ethnolinguistic groups have been whole-genome sequenced at high depth totalling 585 individuals. An additional 41 individuals have been sequenced with 10X Genomics libraries. At this stage, the initial curation of this dataset has been finished and we are performing the analysis in coordination with our collaborators. The current state of the GDAP represents a very diverse panel of African populations that maximizes geographical and ethnic variation and represents a great starting point to achieve the aforementioned goals. However, southern sub-Saharan countries, Bantu speakers and hunter gatherer groups are currently underrepresented, despite being crucial to understand the evolutionary history of the continent. After extensive effort to collate studies documentation, we finally have the opportunity to sequence 600 new individuals from these groups, including countries as Gabon, Rwanda and Zambia, and address these deficiencies. We aim to proceed with the same strategy: to sequence at high depth 25 individuals with standard PCR free libraries, with 2 additional individuals with 10X Genomics Chromium libraries per ethnolinguistic group. The former allows a good representation of variants down to low frequency in any given population, and the latter allows accurate phasing and the analysis of structural variation. By including these new populations, we want to investigate three crucial questions in African history in addition to the initial objectives: the Bantu expansion, the evolutionary history of hunter gatherers and the transatlantic slave trade. Additionally, the expanded dataset will help us better discover the genetic variation present in Africa and characterize the African pangenome. 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 EGAS00001003602

• Separation of chronic myeloid leukemia stem cells from normal hematopoietic stem cells at single-cell resolution

  Chronic myeloid leukemia (CML) is a clonal disease, initiated and sustained by a subpopulation of CML stem cells (CML-SC) containing the BCR::ABL1 oncogene. Genetic markers that enrich for CML-SC have been identified, including CD26 being most specific. Genome sequencing analysis remain yet to be performed at single cell level in order to determine differences and heterogeneity in the genetic profile of CML-SC versus normal hematopoietic stem cells (HSC). Here we show that CD26 and CD33 separate CML-SC from normal HSC in freshly isolated bone marrow samples from newly diagnosed CML patients. DNA sequencing confirms that single CML-SC derived colonies are positive for BCR::ABL1 and harbor a recurrent set of patient-specific clonal somatic mutations across different CML-SC derived colonies. In contrast, marker-negative (CD26-CD33-) HSC from the same patients are negative for the BCR::ABL1 oncogene and do not carry recurrent, clonal mutation across different colonies. These results support the concept that CD26/CD33 expression separates genetically defined, clonal mutant CML-SC from normal HSC that carry individual sets of random mutations in CML patients. We provide whole exome DNA sequencing data for 23 clinical samples of CML-SC plus two buccal swipes derived normal samples. CML samples are comprised of 4 to 8 replicates from two patients, at diagnosis and after treatment. Single CML stem cells before treatment and single non-transformed HSC at remission were selected from bone marrow samples by FACS, according to genetic markers CD33+CD26+ at diagnosis and CD33+CD26-/CD33-CD26- at remission. WES libraries of colony forming assays derived CML-SC and HSC populations were prepared using Agilent SureSelect Human All Exon V6 kit and sequenced running 150 cycles (2x 75bp paired-end) on an Illumina NextSeq 500 platform. Following GATK best practices for somatic mutation calling, novel CML variants and random mutation events during remission phase were obtained.

  Study EGAS00001006904

• High-throughput telomere length measurement at nucleotide resolution using the PacBio high fidelity sequencing platform, P49-P104 and run1 replicates

  Telomeres are specialized nucleoprotein structures at the ends of linear chromosomes that are essential for the stability as well as the complete replication of the human genome. The progressive shortening of steady-state telomere length in normal human somatic cells is a promising biomarker for age-associated diseases. However, there remain substantial challenges in quantifying telomere length in clinics and research laboratories due to the lack of accurate and high-throughput telomere length measurement method. Here, we describe a novel workflow to capture and analyze telomeres accurately at the single-nucleotide resolution. By using newly designed telobaits that are complementary to the single-stranded G-rich 3’ telomeric overhangs, we can specifically and efficiently capture full-length telomeres and subject them to single-molecule real-time (SMRT) sequencing. We show that the telomere length can be measured at nucleotide resolution using the PacBio high fidelity (HiFi) sequencing platform, which provides the necessary accuracy and throughput to uncover the steady-state telomere length distribution in human culture cell lines as well as clinical patient samples. This new method is a valuable tool not only in population-wide epidemiology studies but also for the longitudinal assessment of telomere attrition in individuals. Our results also reveal the extreme heterogeneity of telomeric variant sequences (TVSs) that is dispersed throughout the telomere repeat region. Importantly, the unique distribution pattern and sequence of TVSs in individuals may be used as a fingerprint for sample identification. In addition, the presence of TVSs disrupts the continuity of the canonical (5’-TTAGGG-3’)n telomere repeats, which could affect the binding of shelterin complexes at the chromosomal ends. Therefore, TVSs may act as a form of genetic polymorphisms that impairs telomere protection. Together with the absolute telomere length in each individual, TVSs may have profound implications in human aging and diseases.

  Study EGAS00001006595

• Whole genome and whole exome sequencing of serial biopsies of relapsed/refractory diffuse large B-cell lymphoma.

  Purpose: Diffuse large B-cell lymphoma (DLBCL) is cured in more than 60% of patients, but outcomes remain poor for patients experiencing disease progression or relapse (refractory or relapsed DLBCL [rrDLBCL]), particularly if these events occur early. Although previous studies examining cohorts of rrDLBCL have identified features that are enriched at relapse, few have directly compared serial biopsies to uncover biological and evolutionary dynamics driving rrDLBCL. Here, we sought to confirm the relationship between relapse timing and outcomes after second-line (immuno)chemotherapy and determine the evolutionary dynamics that underpin that relationship. Patients and methods: Outcomes were examined in a population-based cohort of 221 patients with DLBCL who experienced progression/relapse after frontline treatment and were treated with second-line (immuno)chemotherapy with an intention-to-treat with autologous stem-cell transplantation (ASCT). Serial DLBCL biopsies from a partially overlapping cohort of 129 patients underwent molecular characterization, including whole-genome or whole-exome sequencing in 73 patients. Results: Outcomes to second-line therapy and ASCT are superior for late relapse (>2 years postdiagnosis) versus primary refractory (<9 months) or early relapse (9-24 months). Diagnostic and relapse biopsies were mostly concordant for cell-of-origin classification and genetics-based subgroup. Despite this concordance, the number of mutations exclusive to each biopsy increased with time since diagnosis, and late relapses shared few mutations with their diagnostic counterpart, demonstrating a branching evolution pattern. In patients with highly divergent tumors, many of the same genes acquired new mutations independently in each tumor, suggesting that the earliest mutations in a shared precursor cell constrain tumor evolution toward the same genetics-based subgroups at both diagnosis and relapse. Conclusion: These results suggest that late relapses commonly represent genetically distinct and chemotherapy-naïve disease and have implications for optimal patient management.

  Study EGAS00001007053

• 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

• Dataset for Sarcoma RNA sequencing linked from study EGAS00001004813

  Sequencing data of 410 sarcoma tumor runs, which were uploaded to EGAS00001004813. The sequencing was always paired.

  Dataset EGAD00001010276

• Molecular characterization of Barrett’s esophagus at single cell resolution

  Backgrounds & Aims: Patient with Barrett’s esophagus (BE) have an increased risk to develop esophageal adenocarcinoma and are under periodical endoscopic surveillance for malignant transformation. Pathologists distinguish between different BE stages to identify high-risk patients, but this process remains difficult as stage-specific markers are still missing. We established BE organoid cultures, applied single cell sequencing approaches to identify differences in gene expression and genomic alterations, and validated candidate genes for their predictive value on histological sections. Methods: We collected 43 epithelial biopsies from 21 patients, established organoids for the clonal expansion of matching healthy and diseased tissue from two patients to identify genomic alterations by whole genome sequencing (WGS). We performed single cell DNA-sequencing (scDNAseq) to analyse DNA alterations within biopsies from 8 patients and single cell RNA-sequencing (scRNAseq) to identify gene expression differences between BE stages from 19 patients. Candidate marker genes were then validated by RNA in situ hybridization experiments on histological resection specimen. Results: Most BE biopsies were chromosomal stable (CS) and their single base substitution (SBS) signatures were indistinguishable from the healthy control tissues. Dysplastic BE cells contained areas of chromosomal instabilities (CIN), which differed by the presence of the SBS17 signatures from biopsy-matching CS cells or patient-matching healthy control cells. Such CIN areas affected efficient clustering of the scRNAseq data. We identified two sets of marker genes (SLC5A5, PSCA, LIPF and ANPEP, CEACAM6, REG4), which distinguish columnar BE epithelium from non-dysplastic/dysplastic stages. CLDN2 allowed the distinction between dysplastic and more advanced stages as its expression level increased and spread in dysplastic BE glands. Conclusions: Molecular characterization of BE samples identified a correlation between the presence of CIN and COSMIC signature 17, and we report seven marker genes that help in distinguishing different BE stages.

  Study EGAS00001005221

• Copy-Number Analysis of Understudied Black Women Ovarian Cancers

  Two general types of genetic alterations drive cancer progression: mutations and copy number alterations (CNAs). With the exception of p53, single nucleotide variant mutations (here, simply "mutations") are not drivers of 80% of tumors within the most frequent and deadly histotype of ovarian cancer: serous ovarian cancer (SOC). Rather, CNAs are more prevalent in SOC than any other cancer type studied by phs000178 The Cancer Genome Atlas (TCGA). Fully two-thirds of genes are altered by CNAs in the average SOC. Bioinformatic methods have shown that these CNAs modulate specific molecular pathways, such as autophagy, and this finding enabled effective pathway-targeted therapies1. Unfortunately, TCGA ovarian cancer data lacks racial diversity. Only 6% of SOC tumors (n=32) studied are from African origin. Black women diagnosed with ovarian cancer are more likely to die from their disease than white women. Five-year survival after diagnosis is 31% among blacks versus 42% among whites, and this disparity is seen in every age group and tumor stage distribution. Dr. Kelemen examined data from 365 white and 95 black ovarian cancer patients from the Hollings Cancer Center (HCC) Cancer Registry between 2000 and 2015 and found that black women had an 81% higher risk of death after diagnosis compared to white women (HR 1.81, 95% CI 1.35-2.43), independent of diagnosis center (98% were treated at HCC but half were diagnosed elsewhere), tumor stage, histology, extent of residual tumor remaining following surgery, insurance status, smoking, type of treatment for ovarian cancer, and age-adjusted Charlson comorbidity index. This disparity remained even when restricted to women who received the standard of care, surgery-chemotherapy sequence (HR 1.79, 95% CI 1.10-2.89). Despite receiving the same treatment, black race was still associated with inferior survival. A similar disparity has been reported by others, indicating that the HCC experience is representative of national data. Here, an additional set of high-grade serous ovarian cancer primary tumors were acquired from biorepositories with a focus on expanding data originating from African American women. Data were analyzed for copy-number alterations using exome sequencing and control-FREEC software.

  Study phs002313

• A New Reference Panel to Boost African American Genotype Imputation

  Modern genetic studies have been conducted predominantly in cohorts of individuals of European ancestry. By 2010, there were approximately ten times as many published genome wide association studies (GWAS) in people of European ancestry than studies in people of all other ancestries combined. This research disparity has led to an uneven understanding of the genetic basis underlying disease in Europeans and non-Europeans. 23andMe's web-based, large scale research model is ideal for scaling genetics research within non-European populations and thereby bringing more parity to genetics research. Our database is composed of genotypes and phenotypes of over 1,000,000 consenting customers, including over 200,000 individuals with non-European ancestry. The data derived from non-European individuals represent a particularly valuable resource for genetic discovery of novel variants that may not be found in the European population. However, research studies in non-European populations are weakened by the lack of availability of large-scale reference datasets and, in particular, genotype imputation panels. Genotype imputation is a statistical methodology that uses observations of genotypes in a large reference panel to infer unobserved genotypes in a target dataset. This methodology is widely used within GWAS, and allows novel genetic associations to be identified and refined. Due to this utility, very large reference panels have been constructed, containing thousands or tens of thousands of whole genome sequences. Unfortunately, the largest imputation panels are composed of predominantly European genomes, reflecting the modern bias towards European studies in GWAS. This proposal aims to address this imbalance by constructing an imputation panel specifically for the African American population. In doing so, we will expand 23andMe's ability to perform genetic discovery in non-European populations, and improve the understanding of global genetic variation underlying diseases and traits. Key commercial outcomes of the research include the identification of novel genetic targets for internal and external therapeutic development. The long-term aim is to improve understanding of disease in minority populations, which we hope may eventually lead to improved treatments of disease in these historically medically understudied groups.

  Study phs001798

• A Single Cell Atlas of Gene Regulatory Elements in the Human Heart

  Background: Cis-regulatory elements such as enhancers and promoters are crucial for directing gene expression in the human heart. Dysregulation of these elements can result in many cardiovascular diseases that are major leading causes of morbidity and mortality worldwide. In addition, genetic variants associated with cardiovascular disease risk are enriched within cis-regulatory elements. However, the location and activity of these cis-regulatory elements in individual cardiac cell types remains to be fully defined. Methods: We performed single nucleus ATAC-seq and single nucleus RNA-seq to define a comprehensive catalogue of candidate cis-regulatory elements (cCREs) and gene expression patterns for the distinct cell types comprising each chamber of four non-failing human hearts. We used this catalogue to computationally deconvolute dynamic enhancers in failing hearts and to assign cardiovascular disease risk variants to cCREs in individual cardiac cell types. Finally, we applied reporter assays, genome editing, and electrophysiogical measurements in in vitro differentiated human cardiomyocytes to validate the molecular mechanisms of cardiovascular disease risk variants. Results: We defined >287,000 candidate cis-regulatory elements (cCREs) in human hearts at single-cell resolution, which notably revealed gene regulatory programs controlling specific cell types in a cardiac region/structure-dependent manner and during heart failure. We further report enrichment of cardiovascular disease risk variants in cCREs of distinct cardiac cell types, including a strong enrichment of atrial fibrillation variants in cardiomyocyte cCREs, and reveal 38 candidate causal atrial fibrillation variants localized to cardiomyocyte cCREs. Two such risk variants residing within a cardiomyocyte-specific cCRE at the KCNH2/HERG locus resulted in reduced enhancer activity compared to the non-risk allele. Finally, we found that deletion of the cCRE containing these variants decreased KCNH2 expression and prolonged action potential repolarization in an enhancer dosage-dependent manner. Conclusion: This comprehensive atlas of human cardiac cCREs provides the foundation for not only illuminating cell type-specific gene regulatory programs controlling human hearts during health and disease, but also interpreting genetic risk loci for a wide spectrum of cardiovascular diseases.

  Study phs002204

• NSIGHT North Carolina Newborn Exome Sequencing for Universal Screening (NC NEXUS)

  Since newborn screening (NBS) began in the 1960s, technological advances have resulted in its use in an increasing number of disorders. Recent developments in whole-genome sequencing and its simpler corollary, whole-exome sequencing (WES), now afford the opportunity to comprehensively define the variation within an individual's genome in a rapid and affordable manner. Many challenges arise with the clinical application of genome-scale sequencing and in deriving practical benefits to infants and children. Its utility in NBS has yet to be demonstrated and its application in the pediatric population requires special examination, not only for potential clinical benefits but also for the unique ethical challenges it presents. In this proposal, we outline a highly interdisciplinary approach to identify, confront, and overcome the major challenges that must be met in order to implement deep sequencing technology to enhance current newborn screening in a diverse pediatric population. Overarching Aim 1 will evaluate the utility of WES as a diagnostic tool to extend the utility of current NBS. Using diverse cohorts of infants and young children with known conditions identified through NBS, we will examine the sensitivity and specificity of WES. We will also utilize WES in cohorts of children with known conditions not currently screened as potential candidates for NBS in the future. Overarching Aim 2 will develop and assess a framework for analyzing WES in a clinically oriented framework based on principles of ethics and evidence-based medicine. We will develop strategies to guide clinicians, clinical laboratories, and patients/families in their decisions regarding the inevitable incidental findings that will be detected, in ways that respect the child and protect his/her future autonomy, while also respecting parental interests and rights. Overarching Aim 3 will explore ethical, legal, and social issues (ELSI) involved in informed decision making and develop best practices regarding the return of results after testing. We will develop novel decision support tools and evaluate their usefulness in parental decision making, and examine the burdens placed on clinicians as this new technology is deployed in the vulnerable and special population that are newborns and their families.

  Study phs002095

• DNA-Sequencing of Baseline Plasma From the Phase III Alliance A031201 Trial

  Liquid biopsy assays tailored for metastatic castration-resistant prostate cancer (mCRPC) patients are needed to identify molecular alterations and predict survival in the context of androgen receptor (AR) inhibitors. We developed a novel plasma cell-free DNA (cfDNA) sequencing assay to detect circulating tumor DNA (ctDNA) and alterations in key mCRPC driver genes, and tested associations with clinical features and survival outcomes in a phase III trial. cfDNA was isolated from evaluable baseline plasma specimens of untreated mCRPC patients (n=790) enrolled in the Alliance A031201 randomized phase III trial (NCT01949337) prior to enzalutamide therapy with or without abiraterone acetate and prednisone. A customized targeted cfDNA sequencing assay was used to detect AR and non-AR alterations in mCRPC. For this, DNA-seq libraries were constructed from cfDNA using Takara ThruPLEX Tag-Seq or Plasma-Seq kits, and subjected to hybridization enrichment using a custom-designed Agilent SureSelect bait panel. Post-capture DNA-seq libraries were pooled and sequenced on an Illumina HiSeq 2500 using 2x150 bp settings. ctDNA positivity was defined as >20% ctDNA using an aneuploidy-based estimate derived from ichorCNA (Group 1) or a novel definition of ≤20% ctDNA, but with detection of any alteration in AR, or gain of MYC and/or MYCN (Group 2). cfDNA sequencing data was obtained from 776 patients. There were 480/776 (62%) ctDNA-positive patients, with 324 (42%) in Group 1 and an additional 156 (20%) in Group 2 with AR or MYC/MYCN alterations detected. ctDNA-positive patients in Groups 1 and 2 had similarly poor median overall survival (OS) of 25.4 months (95% CI: 21.8-29.0) and 29.7 months (95% CI: 27.3-35.5), respectively, as compared to ctDNA-negative patients who had a median OS of 49.1 months (95% CI: 42.3-53.5). Relative to ctDNA-negative patients, OS was inferior in ctDNA-positive patients in Group 1 (HR 2.3, 95% CI: 1.9-2.9, P AR inhibitors in a contemporary phase III trial.

  Study phs003325