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• eMERGE Geisinger eGenomic Medicine (GeM) Abdominal Aortic Aneurysm Project (AAAP)

  A large research cohort of Geisinger Abdominal Aortic Aneurysm (AAA) patients was created by enrolling and consenting patients of the Geisinger Department of Vascular Surgery. Consented patients provide blood, serum and DNA samples for research and authorize use of data in their medical record for research. They also complete a data questionnaire that asks information about family history of AAA and other vascular diseases, as well as information on known or suspected AAA risk factors, including smoking history, body mass index, hypertension, type 2 diabetes, and atherosclerotic disease. The AAA cases are 78% male, with a mean age of 74 years; 39% have undergone surgical or endovascular repair; more than 85% or current or past smokers; approximately 20% of cases report a positive family history of AAA. Geisinger AAA patients undergo regular imaging studies, typically every 6 months, to monitor the progression of aneurysm expansion. This allows the growth rates of their aneurysm to be calculated. DNA samples from a total of 910 AAA patients were used for whole genome genotyping; these results are included in the dbGaP.

  Study phs000387

• Genomic Analysis of Head and Neck Cancers

  These aggregate studies focus on specimens from head and neck squamous cell carcinoma (HNSCC) patients who were treated with standard of care or window of opportunity clinical trial approaches. Specimens were interrogated with exome and RNA-Seq analysis. Patient derived xenografts (PDXs) represent a robust platform for performing co-clinical trial approaches. In the first study we generated a large panel of PDXs and compared genomic profiles between the primary tumor and matched PDX with the patients blood serving as a source of somatic variants. In the second study, we performed a window of opportunity clinical trial in oral cavity squamous cell carcinoma patients using the MEK inhibitor trametinib. In addition, PDX models were generated from these specimens also and analyzed as in the first study. Finally, the third approach used pembrolizumab, an anti-PD1 blocking antibody, in a window of opportunity setting in HNSCC patients. For the latter two studies genomic matched sample analysis was performed to define response and resistance mechanisms. Together, these genomic data will inform several therapeutic approaches for HNSCC patients.

  Study phs001623

• Functionally Active Copy Number Variants Associated with Prostate Cancer Risk

  Prostate cancer is a leading cause of cancer death in males throughout the world and has the largest estimated effect of heritability among the most common tumor types. Copy Number Variants (CNVs) are a recently recognized class of human germline polymorphisms (Iafrate AJ, et al. (2004) Nat Genet 36, 949-951; Sebat J, et al. (2004) Science 305, 525-528.) and are associated with a variety of human diseases, including cancer. This study characterized 1,903 individuals from the Tyrol Early Prostate Cancer Detection Program (Bartsch G, et al. (2008) BJU Int 101, 809-816) using a computational framework (Banerjee S, et al. (2011) PLoS One 29;6(3)). The study results establish non-coding and coding germline CNVs as significant risk factors for prostate cancer susceptibility and implicate their role in disease development and progression. The study "Identification of functionally active, low frequency copy number variants at 15q21.3 and 12q21.31 associated with prostate cancer risk," Demichelis F, Setlur SR, Proc Natl Acad Sci U S A. 2012 Apr 24;109(17):6686-91.

  Study phs000487

• A Pharmcogenetic Study of Bipolar Disorder in a Taiwanese Han Chinese Population (TWBP)

  Objective: To identify genetic variants associated with response to lithium prophylaxis treatment. Materials: 294 bipolar I (BPI) patients receiving lithium treatment, a subset of 1761 bipolar I (BPI) patients recruited by the Taiwan Bipolar Consortium. Lithium response was assessed based on the Alda Scale. Methods: Responders to lithium treatment were defined if the patients' Alda Scale > 4, 5, 6, and 7. Genome-wide association (GWA) analysis was carried out on the 294 BPI patients for the 4 definitions of responders. The SNPs showing strongest association in the GWAS were then tested for association in a replication sample of 100 patients and further tested in a prospective follow-up series of 24 patients. Results: Two SNPs on GADL1 showed the strongest associations in the four GWA analyses and in the replication set of 100 patients. These two SNPs had a sensitivity of 0.93 for predicting lithium response and differentiated between the good and poor responders in the follow-up cohort. Summary: These genetic variants are associated with response to lithium prophylaxis treatment for BPI.

  Study phs000692

• HiDEF-seq Single-Molecule Sequencing of Single-Strand Mismatches and Damage

  Our study sought to resolve, with single-molecule resolution, the mismatches and damage events that precede DNA mutations. Using a novel single-molecule, long-read sequencing method (HiDEF-seq) we detected base substitutions when present in either one or both DNA strands. We compared the fidelity for dsDNA mutations to that of the same samples profiled by NanoSeq duplex sequencing. We also detected cytosine deamination, a common type of DNA damage, with single-molecule resolution. We profiled 134 samples from diverse tissues, including from individuals with cancer predisposition syndromes. These samples revealed single-strand mismatch and damage signatures. Since double-strand DNA mutations are only the endpoint of the mutation process, our approach enables new studies of how mutations arise in a variety of contexts, especially in cancer and aging.The data available via dbGaP includes raw HiDEF-seq sequencing files (Pacific Biosciences subreads in BAM format), raw NanoSeq sequencing files (Illumina FASTQ format), and raw germline sequencing files (Illumina FASTQ format).The HiDEF-seq main publication contains a supplementary table with a list of accession IDs for each sample's files.

  Study phs003604

• Mutant KIT as imatinib-sensitive target in metastatic sinonasal carcinoma (H021)

  Sinonasal carcinomas (SNCs) comprise various rare tumor types that are characterized by marked histologic diversity and largely unknown molecular profiles, yet share an overall poor prognosis owing to an aggressive clinical course and frequent late-stage diagnosis. The lack of effective systemic therapies for locally advanced or metastatic SNC poses a major challenge to therapeutic decision making for individual patients. Here, we used whole-exome and transcriptome sequencing to identify a KIT exon 11 mutation (c.1733_1735del, p.578_579del) as actionable target in a patient with metastatic SNC whose tumor, despite all diagnostic efforts, could not be assigned to any known SNC category and was refractory to multimodal therapy. Molecularly guided treatment with imatinib resulted in a dramatic and durable response with remission of nearly all tumor manifestations, indicating a dominant driver function of mutant KIT in this tumor. This observation highlights the potential of unbiased genomic profiling for uncovering the vulnerabilities of individual malignancies, particularly in rare and unclassifiable tumors, and underscores that KIT exon 11 mutations represent tractable therapeutic targets across different histologies.H021

  Study EGAS00001001845

• Spatial transcriptomics analysis of triple negative breast cancers

  While triple negative breast cancer (TNBC) is known to be heterogeneous at the genomic and transcriptomic levels, spatial information on tumor organization and cell composition is still lacking. Here, we investigated TNBC tumor architecture including its microenvironment using spatial transcriptomics on a series of 92 patients. We performed an in-depth characterization of tumor and stroma organization and composition using an integrative approach combining histomorphological and spatial transcriptomics. Furthermore, a detailed molecular characterization of tertiary lymphoid structures led to identify a gene signature strongly associated to disease outcome and response to immunotherapy in several tumor types beyond TNBC. A stepwise clustering analysis identified nine TNBC ecotypes, further validated in external datasets. Several ecotypes were associated with disease outcome and characterized by potentially actionable features. In this work, we provideThis is the largest TNBC series providing a comprehensive insight into the complexity of TNBC ecosystem with potential clinical relevance, opening avenues for treatment tailoring including immunotherapy. All the processed data needed to replicate the results from the publication (counts, images, clinical data, etc) are available on Zenodo: https://zenodo.org/records/14204217

  Study EGAS50000000475

• Interactions between the tumor and the systemic response of breast cancer patients

  Breast cancer (BC) research has largely focused on the molecular properties of the tumor proper. However, to understand how cancer progresses and ultimately modulates patient outcome, we also require an understanding of the molecular changes in the patient systemic response (SR). Toward this end, we generated and analyzed RNA profiles from tumor and matched blood samples in 173 BC patients. We designed a system (MIxT) to explore and link tightly co-expressed gene sets (modules) across matched tissues. The gene composition of modules, and their expression, largely vary across tissues. Distinct patterns of expression in the SR are predominantly detected in highly immunogenic BC subtypes. We also find that the expression pattern of a module in one tissue is correlated to the expression pattern of a module in the other tissue in a fashion that depends on subtype. For example, systemic immunosuppression is detected in basalL patients in a fashion that is proportional to the level of expression of several tumor modules highlighting immune evasion mechanisms active in these particular cancers.

  Study EGAS00001001804

• Multi-omics analysis defines core genomic alterations in pheochromocytomas and paragangliomas

  Pheochromocytomas (PCC) and paragangliomas (PGL) are neural crest-derived tumors. We performed the integrated genomic analysis of a collection of PCC/PGL from the French COMETE network using SNP arrays, whole-exome sequencing, DNA methylation arrays, mRNA expression arrays and miRNA sequencing. We found that PCC/PGL are characterized by a low mutation rate, with few recurrent somatic mutations and focal copy number alterations (CNAs). In contrast, we detected recurrent broad CNAs strongly associated with known molecular groups, often leading to the biallelic inactivation of the known susceptibility genes of PCC/PGL. We also identified DNA methylation changes and miRNA expression clusters strongly associated with molecular groups. Of note, overexpression of the miRNA cluster 182/96/183 is specific of SDHB-mutated tumors and induces invasive traits, whereas silencing of the imprinted DLK1-MEG3 miRNA cluster due to a loss of heterozygosity of the 14q32.2 locus is a potential driver in a subset of sporadic tumors. This comprehensive analysis illustrates the functional interdependence between genomic and epigenomic dysregulations underlying PCC/PGL.

  Study EGAS00001000933

• FetalQuant-SD: Accurate quantification of fetal DNA fraction by shallow-depth sequencing of maternal plasma DNA

  Noninvasive prenatal testing using massively parallel sequencing of maternal plasma DNA has been rapidly adopted in clinical use worldwide. Fetal DNA fraction in a maternal plasma sample is an important parameter for accurate interpretations of these tests. However, there is a lack of methods involving low-sequencing depth and yet would allow a robust and accurate determination of fetal DNA fraction in maternal plasma for all pregnancies. In this study, we have developed a new method to accurately quantify the fetal DNA fraction by analysing the maternal genotypes and sequencing data of maternal plasma DNA. Fetal DNA fraction was calculated based on the proportion of non-maternal alleles at single-nucleotide polymorphisms where the mother is homozygous. This new approach achieves a median deviation of 0.6% between predicted fetal DNA fraction and the actual fetal DNA fraction using as low as 0.03-fold sequencing coverage of the human genome. We believe that this method will further enhance the clinical interpretations of noninvasive prenatal testing using genome-wide random sequencing.

  Study EGAS00001001611

• Genomic landscape of human diversity across Madagascar

  Although situated ~400 km from the east coast of Africa, Madagascar exhibits cultural, linguistic, and genetic traits from both Southeast Asia and Eastern Africa. The settlement history remains contentious; we therefore used a grid-based approach to sample at high-resolution the genomic diversity (including maternal lineages, paternal lineages, and genome-wide data) across 257 villages and 2704 Malagasy individuals. We find a common Bantu and Austronesian descent for all Malagasy individuals with a limited paternal contribution from Europe and the Middle East. Admixture and demographic growth happened recently, suggesting a rapid settlement of Madagascar during the last millennium. However, the distribution of African and Asian ancestry across the island reveals that the admixture was sex biased and happened heterogeneously across Madagascar, suggesting independent colonization of Madagascar from Africa and Asia rather than settlement by an already-admixed population. In addition, there are geographic influences on the present genomic diversity, independent of the admixture, showing that a few centuries is sufficient to produce detectable genetic structure in human populations.

  Study EGAS00001002549

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

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

  Study EGAS00001002895

• Long-read-transcriptome-sequencing of CLL and MDS patients uncovers common molecular effects of SF3B1 mutations

  Mutations in SF3B1 occur frequently in patients with chronic lymphocytic leukemia (CLL) and myelodysplastic syndromes (MDS), and a full-length transcriptome approach can expand our current knowledge on SF3B1 mutation effects on RNA splicing. We applied long-read-transcriptome-sequencing (LRTS) to 44 MDS and CLL patients with and without SF3B1 mutations and found a large fraction (>60%) of novel isoforms. Furthermore, we revealed that mutation effects on alternative splicing were largely common in both cancer types and specifically altered the usage of introns as well as 3’-splice-sites. We combined the LRTS with genome-wide SF3B1-RNA binding maps and show multimodal binding at 3’-splice-sites highlighting a window of 12-21nt upstream of the canonical 3’-splice-site in which a dynamic switch in splice site usage is observable in patients carrying SF3B1 mutations. Our work presents the hitherto most complete LRTS study in CLL and MDS and provides a resource for further research on aberrant splicing in cancer.

  Study EGAS50000000053

• Targeted sequencing (paired) of HR genes in primary and metastatic patient-derived xenografts (PDXs) of colorectal cancer (CRC)

  Chemotherapy is the standard-of-care treatment for metastatic colorectal cancer (mCRC) and benefits some patients, but what distinguishes responders from non-responders is unclear. In this study, we leveraged a comprehensive collection of 69 molecularly annotated patient-derived xenografts to uncover functional predictors of response to 5-FU and irinotecan combination therapy (FOLFIRI) in mCRC. Genetic analyses revealed that treatment sensitivity was marked by genomic scars indicative of BRCAness, suggesting homologous recombination (HR) deficiency as a key determinant. Accordingly, we surveyed a manually curated panel of 44 genes with a documented role in HR for the potential presence of pathogenic mutations. We did not observe a specific enrichment of HR gene mutations based on response to FOLFIRI. This result, combined with the absence of widespread biallelic inactivation of the analyzed genes and the predominance of mutations categorized as variants of unknown significance, suggests that FOLFIRI sensitivity is not primarily governed by underlying mutations in HR genes responsible for mitigating the genotoxic effects of this therapeutic regimen.

  Study EGAS50000000075

• Shallow Whole Genome Sequencing of Patient Derived Xenografts

  Chemotherapy is the standard-of-care treatment for metastatic colorectal cancer (mCRC) and benefits some patients, but what distinguishes responders from non-responders is unclear. In this study, we leveraged a comprehensive collection of 27 molecularly annotated patient-derived xenografts to uncover functional predictors of response to 5-FU and irinotecan combination therapy (FOLFIRI) in mCRC. Genetic analyses revealed that treatment sensitivity was marked by genomic scars indicative of BRCAness, suggesting homologous recombination (HR) deficiency as a key determinant. Accordingly, we surveyed a manually curated panel of 44 genes with a documented role in HR for the potential presence of pathogenic mutations. We did not observe a specific enrichment of HR gene mutations based on response to FOLFIRI. This result, combined with the absence of widespread biallelic inactivation of the analyzed genes and the predominance of mutations categorized as variants of unknown significance, suggests that FOLFIRI sensitivity is not primarily governed by underlying mutations in HR genes responsible for mitigating the genotoxic effects of this therapeutic regimen.

  Study EGAS50000000191

• Whole genome, transcriptome and methylome profiling enhances actionable target discovery in high-risk paediatric cancer

  Zero Childhood Cancer is a precision medicine program to benefit children with poor-outcome, rare, relapsed or refractory cancer. Utilising tumour and germline whole genome sequencing (WGS) and RNA sequencing (RNAseq) across 252 tumours from high-risk paediatric cancer patients, we identified 968 reportable molecular aberrations (39.9% in WGS and RNAseq, 35.1% WGS-only, 25.0% RNAseq-only). 93.7% of patients had at least one germline or somatic aberration, 71.4% had therapeutic targets and 5.2% had a change in diagnosis. WGS identified pathogenic cancer-predisposing variants in 16.2% of patients. In 76 CNS tumours, methylome analysis confirmed diagnosis in 71.1% and contributed to a change of diagnosis in two cases (2.6%). To date 43 patients have received a recommended therapy, 38 of whom could be evaluated. A small but encouraging proportion of 31% showed some evidence of clinical benefit. Comprehensive molecular profiling resolved the molecular basis of virtually all high-risk cancers, which in some cases has significant clinical impact.

  Study EGAS00001004572

• Somatic_mutation_profiling_of_intestinal_crypts_from_IBD

  In this project we want to investigate the role that somatic mutations in intestinal stem cells play in IBD. We will use laser capture microdissection to ascertain epithelial cells from an individual crypts/vilus. We will extract DNA from these and use a new low-input material library prep methods to make accurate libraries for sequencing. In this pilot phase of the project we will perform 15X whole-genome sequencing of a small number of crypts biopsied from four patients. Biopsies have been taken from inflammed and noninflammed patches to compare mutation rates and mutational signatures. We will also test if crypts from inflammed patches of a single patient are clonal (either within a given inflammed patch or across inflammed patches). We will quantify the rate of cancer driver mutations in inflammed and non-inflammed crypts. 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 EGAS00001002896

• Exome Sequecning of MDS xenografted samples

  Pre-clinical research of Myelodysplastic Syndromes (MDS) is hampered by a lack of feasible disease models. Previously, we have established a robust patient-derived xenograft (PDX) model for MDS. Here, we demonstrate for the first time that this model is applicable as a pre-clinical platform to address pending clinical questions by interrogating the efficacy and safety of the thrombopoietin receptor agonist eltrombopag. Our pre-clinical study included n=49 xenografts generated from n=9 MDS patient samples. Substance efficacy was evidenced by FACS-based human platelet quantification, and clonal bone marrow evolution was reconstructed by serial whole exome sequencing of the PDX samples. In contrast to clinical trials in humans, this experimental setup allowed vehicle- and replicate-controlled analyses on a patient-individual level deciphering substance-specific effects from natural disease progression. We found that eltrombopag effectively stimulated thrombopoiesis in MDS PDX without adversely affecting the patients’ clonal composition. In conclusion, our MDS PDX model is a useful tool for testing new therapeutic concepts in MDS preceding clinical trials.

  Study EGAS00001005329

• Automated image-based profiling of complex drug induced phenotypes in patient-derived organoids

  We established a platform for high-throughput confocal fluorescence microscopy and automated image analysis to perform large-scale drug response profiling with patient derived organoids (PDOs). Using PDO lines newly established from colorectal cancer patients with a standardized workflow, we performed chemical perturbations with more than 500 experimental and clinically used compounds. Subsequently, we captured approx. six million images by confocal microscopy. To analyze drug-induced PDO re-organization on a single organoid level we developed SCOPE, a software framework for automated PDO high-throughput image analysis. We observed a rich variety of divergent and reoccurring compound induced phenotypes in organoids. Perturbation of cellular processes, including signaling by MEK, GSK3β, cyclin dependent kinases (CDKs) and others, led to recurring and distinct architectural changes. Highlighting the clinical relevance, PDO viability was heterogeneous after perturbation with anticancer drugs and matched clinical response of corresponding colorectal cancer patients. Our work opens new avenues for image-based profiling for accelerated drug discovery and clinical decision-making.

  Study EGAS00001003140

• Sensitive detection of tumor mutations from blood and its application to immunotherapy prognosis

  Cell-free DNA (cfDNA) is attractive for many applications, including cancer detection, locating, and monitoring. A fundamental task underlying these applications is the SNV calling from cfDNA, which, however, faces a new challenge, namely, the generally very low tumor content in cfDNA. Thus all existing callers fail to achieve satisfactory performance. Here we present cfSNV, a method incorporating multi-layer error suppression and hierarchical mutation calling, to address this important challenge. Furthermore, by leveraging cfDNA’s comprehensive coverage of clonal landscape, for the first time cfSNV can profile mutations even in subclones. In both simulated and real patient data, cfSNV vastly outperforms existing tools, showing tens of times increase in sensitivity in detecting mutations with low allele frequency while maintaining high precision. cfSNV can enhance the clinical utilities of cfDNA by dramatically reducing the required sequencing depth and therefore reduce the cost by magnitudes, and further make the Whole-Exome-Sequencing of cfDNA a viable option. As an example, we demonstrate that cfDNA-WES allows a new biomarker to effectively select patients for immunotherapy.

  Study EGAS00001004373

• An Isogenic Cell Line Panel for Sequence-based Screening of Targeted Anti-cancer Drugs

  We describe the creation and characterization of an isogenic cell line panel representing common cancer pathways, with multiple features optimized for high-throughput screening. More than 1,800 cell lines from three normal human cells were generated using CRISPR-technologies. Surprisingly, we discovered most of these lines did not result in complete gene inactivation, despite integration of sgRNA at the desired genomic site. However, a subset of the lines harbored true, biallelic disruptions of the targeted tumor suppressor gene, yielding a final panel of 100 well-characterize lines covering 19 pathways frequently subject to loss of function in cancers. This panel included genetic markers optimized for sequence-based ratiometric assays for drug-based screening assays. To illustrate the potential utility of this panel, we developed a multiplexed high-throughput screen that identified Wee1 inhibitor MK-1775 as a selective growth inhibitor of cells with inactivation of TP53. These cell lines and screening approach should prove useful for researchers studying a variety of cellular and biochemical phenomena

  Study EGAS00001005974

• The miR-185/PAK6 Axis Predicts Therapy Response and Regulates Survival of Drug-Resistant Leukemic Stem Cells in Chronic Myeloid Leukemia

  Overcoming drug resistance and targeting cancer stem cells remain challenges for curative cancer treatment. To investigate the role of miRNAs in regulating drug resistance and leukemic stem cell (LSCs) fate, we performed global transcriptome profiling in treatment-naïve chronic myeloid leukemia (CML) stem/progenitor cells and identified that miR-185 levels anticipate their response to ABL tyrosine kinase inhibitors (TKIs). miR-185 functions as a tumor suppressor; its restored expression impaired survival of drug-resistant cells, sensitized them to TKIs in vitro, and markedly eliminated long-term repopulating LSCs and infiltrating blast cells, conferring a survival advantage in pre-clinical xenotransplantation models. Integrative analysis with mRNA profiles uncovered PAK6 as a crucial target of miR-185 and pharmacological inhibition of PAK6 perturbed the RAS/MAPK pathway and mitochondrial activity, sensitizing therapy-resistant cells to TKIs. Thus, miR-185 presents as a potential predictive biomarker, and dual targeting of miR-185-mediated PAK6 activity and BCR-ABL may provide a valuable strategy for overcoming drug resistance in patients.

  Study EGAS00001004153

• RNA sequencing data of 257 samples from 106 patients with HR+/HER2- breast cancer treated with AC plus paclitaxel or letrozole plus ribociclib (SOLTI-1402 CORALLEEN trial)

  Early-stage Luminal B breast cancer is frequent and is a major cause of breast cancer death due to its poor prognosis. Our proposal aims to study the biology behind the sensitivity and resistance of Luminal B breast cancer to chemotherapy (CHT) or a non-CHT regimen composed of hormone therapy in combination with ribociclib, a CDK4/6 inhibitor. To accomplish this, we first completed the SOLTI-1402 CORALLEEN phase II trial, a study where 106 patients with early-stage Luminal B breast cancer were randomized to standard neoadjuvant CHT for 6 months, or neoadjuvant letrozole and ribociclib for 6 months. After treatment, patients underwent surgery. The primary results of the study, which showed that the response rate to letrozole+ribociclib was similar to CHT, was reported (Prat et al; Lancet Oncol). Tumor biopsies were available at baseline, week 3 and surgery. A total of 257 samples were analyzed using the Illumina TruSeq Stranded Total RNA w/Ribo Zero Gold with MiSeq in TGL (Sequencer NovaSeq S4/PE/100x).

  Study EGAS00001007060

• Modulation of the peripheral blood immune cell transcriptome by vitamin D3 supplementation in people with a first demyelinating event: a randomized placebo-controlled trial

  Vitamin D deficiency is a risk factor for developing multiple sclerosis (MS). Both in vitro and animal studies suggest an immunomodulatory effect of vitamin D. The PrevANZ trial, a phase IIb randomized placebo-controlled trial of oral vitamin D3 supplementation in people with a first demyelinating event (FDE), was conducted to determine if supplementation can prevent recurrent disease activity in this cohort at high risk of developing definite MS. As a sub-study of this trial, we used whole blood transcriptomic analyses to investigate the effect of vitamin D3 supplementation on peripheral immune cells in people with an FDE, and to gain insight into potential mechanisms by which vitamin D3 may regulate MS risk and disease activity. The PrevANZ trial randomized participants to 1000 IU, 5000 IU or 10,000 IU daily of oral vitamin D3 or placebo. Peripheral blood was collected at baseline and 12 weeks in PAXgene Blood RNA tubes. Transcriptomic datasets were generated by RNA sequencing.

  Study EGAS00001007346

• Family Investigation of Nephropathy and Diabetes (FIND) Study

  The Family Investigation of Nephropathy and Diabetes (FIND) is a multicenter study designed to identify genetic determinants of diabetic kidney disease. Study subjects were recruited from eleven centers and in many ethnic groups throughout the United States. A genome-wide association study (GWAS) was conducted with the Affymetrix 6.0 chip. Subjects (index cases) with diabetes and kidney disease were initially recruited, and their parents and siblings were invited to participate. Genetic material from these participants was used to genotype markers throughout the genome. For association-based testing, a case-control design was implemented with study subjects selected primarily from the index cases of the families. Unrelated controls were selected from families where a case was not already selected. Several study sites also contributed non-FIND subjects, both cases and controls (consent forms for the release of FIND and non-FIND subjects/samples are included in this dbGaP release). Cases were selected if they met study criteria for diabetic nephropathy or met inclusion criteria based on elevated serum creatinine levels and abnormal urine protein excretion. Similarly, controls were long-term diabetics with otherwise normal kidney function. See inclusion/exclusion criteria section for a detailed description for the FIND study as a whole and this GWAS. The goal of the FIND study is to identify genes that influence susceptibility to diabetic kidney disease, leading to a better understanding of how kidney disease develops. In the long run, this may lead to improved treatment and prevention of diabetic kidney disease.

  Study phs000333

• Common Variation in Candidate Genes in the Diabetes Prevention Program

  The Diabetes Prevention Program (DPP) was a multicenter controlled clinical trial examining the efficacy of an intensive lifestyle intervention or metformin to prevent or delay the development of diabetes in a population selected to be at high risk due to the presence of impaired glucose tolerance (IGT) and obesity. Development of diabetes, defined by 1997 American Diabetes Association (ADA) criteria, was the primary outcome while cardiovascular disease and its risk factors were important secondary outcomes. The pharmacological intervention was double blinded and placebo controlled. After randomization, participants had quarterly clinical evaluations and had, in addition, a fasting plasma glucose at semi-annual visits and a 75 gram oral glucose tolerance test at annual visits. Volunteers were recruited from populations known to be at particularly high risk for impaired glucose tolerance and type 2 diabetes including the following: persons with a family history of NIDDM, the elderly, overweight individuals, women with a history of diabetes during pregnancy ("gestational diabetes"), and minority populations including African Americans, Hispanic Americans, Asian and Pacific Island Americans, and Native Americans. The primary focus of the genetic investigations have been on candidate genes, including candidates derived from GWAS of diabetes, lipid and glycemia-related phenotypes. Please note more phenotype data for the DPPG cohort is available through the NIDDK Data Repository. Information on obtaining this phenotype data can be found by going to https://www.niddkrepository.org/studies/dppos/?query=DPP and https://www.niddkrepository.org/studies/dpp/?query=DPP. Data will need to be requested from both data sets. A linking file will be available to the NIDDK Data Repository.

  Study phs000681

• Genetics of Mammographic Density in Ashkenazi Jews

  Mammographic density (MD) is a strong risk factor for breast cancer and is also a highly heritable trait with ~60-70% of the variance due to genetic factors, based on twin studies. MD is also higher in families with a strong history of breast cancer. Genome-wide association studies (GWAS), which focus on common genetic variants, have identified several single nucleotide polymorphisms (SNPs) associated with MD. However, these SNPs explain a very small fraction of the variance of MD, suggesting many other genes are involved. Thus, the vast majority of the heritability of mammographic density remains unexplained and may be explained, at least in part, by rare variants. Ashkenazi Jewish women are a founder population; founder populations frequently have alleles affecting phenotypes which may be unique and/or extremely rare in other populations. Prior reports have identified an association between higher mammographic density and Ashkenazi Jewish ancestry. Therefore, we developed a study of mammographic density in AJ women. We combined datasets from several different cohorts including (a) the California Pacific Medical Center Research Institute (b) the Athena Breast Health Cohort and (c) the Marin Women's study. In each study, we identified women who reported AJ ancestry (determined by self-report or by genetic analysis). We identified mammograms by linking the women to the San Francisco Mammography Registry. We retrieved digital mammographic results and used software to infer volumetric density and percent volumetric density for each woman. We then performed genome-wide genotyping of the samples using the Illumina MEGA array.

  Study phs001857

• National Eye Institute (NEI) Primary Open-Angle African American Glaucoma Genetics (POAAGG) Study

  The purpose of the POAAGG study is to investigate the genetic architecture of primary open-angle glaucoma (POAG) in African Americans. This population is up to eight times more likely to develop glaucoma than non-Hispanic whites, yet no large-scale genetics studies have specifically addressed this ethnic group. The POAAGG study aims to enroll 7765 patients, consisting of a discovery cohort of 5500 subjects (2000 cases and 3500 controls) and a validation cohort of 2265 subjects (1000 cases and 1265 controls). The study population consists of self-identified blacks (African Americans, African descent, or African Caribbean), 35 years or older, identified from the Philadelphia region. Subjects are recruited from the Scheie Eye Institute and its research sites in Philadelphia, PA, as well as from the Penn Medicine Biobank. Fellowship-trained glaucoma specialists classify each subject as a case, control, or glaucoma suspect based on specified criteria. As of 02/01/2017, a total of 8187 subjects have been recruited to the POAAGG study, including 2522 cases, 4545 controls, and 1120 suspects. A comprehensive genetic analysis of POAG, including a genome-wide association analysis, is currently underway. The GWAS was conducted on the Illumina Infinium Multi Ethnic Genotyping Array (MEGA), which contained custom content tailored to POAG and African Americans. A comprehensive genetic analysis of quantitative traits associated with POAG is also in progress. Over 90% of cases have full phenotypic information, including intraocular pressure, cup-to-disc ratio, central corneal thickness, retinal nerve fiber layer thickness, visual acuity, and visual fields.

  Study phs001312

• Bayesian-Based Noninvasive Prenatal Diagnosis of Single-Gene Disorders

  In the last decade, non-invasive prenatal diagnosis (NIPD) has emerged as an effective procedure for early detection of inherited diseases during pregnancy. This technique is based on using cell-free DNA (cfDNA) and fetal cfDNA (cffDNA) in maternal blood, and hence, has minimal risk for the mother and fetus compared with invasive techniques. NIPD is used today for identifying chromosomal abnormalities (in some instances) and for single-gene disorders (SGDs) of paternal origin. However, for SGDs of maternal origin, sensitivity poses a challenge that limits the testing to one genetic disorder at a time. Here we present a Bayesian method for the NIPD of monogenic diseases that is independent of the mode of inheritance and parental origin. Furthermore, we show that accounting for differences in the fragment length distribution of fetal- and maternal-derived cfDNA results in increased accuracy. Our model is the first to predict inherited insertions-deletions (indels). The method described can serve as a general framework for the NIPD of SGDs; this will facilitate easy integration of further improvements. One such improvement that is presented in the current study is a machine learning model that corrects errors based on patterns found in previously processed data. Overall, we show that next generation sequencing (NGS) can be used for the NIPD of a wide range of monogenic diseases, simultaneously. We believe that our study will lead to the achievement of a comprehensive NIPD for monogenic diseases. (Reprinted from Bayesian-based noninvasive prenatal diagnosis of single-gene disorders, with permission from Genome Research)

  Study phs001659

• Transposable Elements in FTLD-TDP and ALS-TDP

  Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease involving the rapid and progressive loss of motor neurons. While a number of inherited mutations have been identified as causing ALS, the vast majority of cases are sporadic with no family history of disease. This has led to the model that ALS may be a complex syndrome with an unknown number of causal molecular pathways. However, nearly all ALS patient tissues display aggregates of the RNA binding protein TDP-43 (TARDBP/TAR DNA-binding protein), and mutations in the gene encoding this protein are known to cause ALS. We show that one of the normal functions of TDP-43 is to bind and regulate retrotransposons, repetitive sequences in the genome that are normally silent, due in part to the function of TDP-43. Furthermore, we demonstrate that many ALS patients with TDP-43 pathology also display de-silencing of retrotransposon RNAs, consistent with a loss of normal TDP-43 function. Those ALS patients that exhibit high levels of retrotransposon expression form a distinct subset of ALS subjects that also show other correlated alterations to the transcriptome. Using unsupervised machine learning algorithms, we identified three distinct molecular subtypes from the whole genomes and transcriptomes of a large cohort of 148 ALS patient samples. The retrotransposon re-activated subset formed 20% of ALS samples; two additional groups displayed signatures of oxidative stress (61%), and glial dysfunction (19%), respectively. Together these results demonstrate that retrotransposon re-activation is common in a subset of ALS patients with TDP-43 pathology.

  Study phs001889

• Women's Ischemia Syndrome Evaluation (WISE-BioLINCC)

  Data Access NOTE: Please refer to the "Authorized Access" section below for information about how access to the data from this accession differs from many other dbGaP accessions. Objectives: This is a National Heart, Lung and Blood Institute sponsored, four-center study designed to: 1) optimize symptom evaluation and diagnostic testing for ischemic heart disease; 2) explore mechanisms for symptoms and myocardial ischemia in the absence of epicardial coronary artery stenoses, and 3) evaluate the influence of reproductive hormones on symptoms and diagnostic test response.Background: Women tend to have a higher prevalence of chest pain than men, yet a lower prevalence of epicardial coronary stenoses, and diagnostic tests such as exercise-induced ECG changes tend to have a higher false-positive rate in women than in men. Thus, accurate diagnosis of ischemic heart disease in women is a major challenge to physicians. In addition, prognosis in women with abnormal diagnostic tests is largely unknown and the role reproductive hormones play in this diagnostic uncertainty is unexplored. Moreover, the significance and pathophysiology of ischemia in the absence of significant epicardial coronary stenoses is unknown.Participants: In Phase I (1996-1997), a pilot phase, 256 women were studied. During Phase II (1997-1999) angiographic and baseline data were collected on an additional 680 women to bring total enrollment to 936. Baseline data was also collected on a reference population of 70 women.Conclusions: Among women without CAD, abnormal Magnetic Resonance Spectroscopy (MRS) consistent with myocardial ischemia predicted cardiovascular outcome, notably higher rates of anginal hospitalization, repeat catherization, and greater treatment costs.

  Study phs004310

• Search for new loci and low-frequency variants influencing glioma risk by exome-array analysis

  To identify protein altering variants (PAVs) for glioma we analysed Illumina HumanExome BeadChip exome array data on 1,882 glioma cases and 8,079 controls from three independent European populations. In addition to single variant tests we incorporated information on the predicted functional consequences of PAVs and analysed sets of genes with a higher likelihood of having a role in glioma on the basis of the profile of somatic mutations documented by large-scale sequencing initiatives. Globally tThere was a strong relationship between effect size and SNPs predicted to be damaging (P=2.29x10-49); however, these variants which are most likely to impact on risk, are rare (MAF<5%). While no single variant showed an association which was statistically significant at the genomewide threshold a number of represented promising Notable associations - were seen with the BRCA2:c.9976A>T, p.(Lys3326Ter)BRCA2 p.Lys332X variant, which has been shown documented to influence breast and lung cancer risks (OR=2.3, P=4.00x10-4 for glioblastoma [GBM]) and IDH2:c.782G>A, p.(Arg261His)IDH2 p.Arg261His (OR=3.21, P=7.67x10-3, for non-GBM). Additionally, gene burden tests revealed a statistically significant association for HARS2 and risk of GBM (P=2.20x10-6). Genome scans of low frequency PAVs represent a complementary strategy to identify disease-causing variants compared with scans based on tagSNPs. Strategies to lessen the multiple testing burden by restricting analysis to PAVs with higher priors affords an opportunity to maximise study power.

  Study EGAS00001001258

• Recurrent epimutations activate gene body promoters in primary glioblastoma

  Aberrant DNA hypomethylation may play an important role in the growth rate of glioblastoma (GBM), but the functional impact on transcription remains poorly understood. We assayed the GBM methylome with MeDIP-seq and MRE-seq, adjusting for copy number differences, in a small set of non-glioma CpG island methylator phenotype (non-G-CIMP) primary tumors. Recurrent hypomethylated loci were enriched within a region of chromosome 5p15 that is specified as a cancer amplicon and also encompasses TERT, encoding telomerase reverse transcriptase, which plays a critical role in tumorigenesis. Overall, 76 gene body promoters were recurrently hypomethylated, including TERT and the oncogenes GLI3 and TP73. Recurring hypomethylation also affected previously unannotated alternative promoters, and luciferase reporter assays for three of four of these promoters confirmed strong promoter activity in GBM cells. Histone H3 lysine 4 trimethylation (H3K4me3) ChIP-seq on tissue from the GBMs uncovered peaks that coincide precisely with tumor-specific decrease of DNA methylation at 200 loci, 133 of which are in gene bodies. Detailed investigation of TP73 and TERT gene body hypomethylation demonstrated increased expression of corresponding alternate transcripts, which in TP73 encodes a truncated p73 protein with oncogenic function and in TERT encodes a putative reverse transcriptase-null protein. Our findings suggest that recurring gene body promoter hypomethylation events, along with histone H3K4 trimethylation, alter the transcriptional landscape of GBM through the activation of a limited number of normally silenced promoters within gene bodies, in at least one case leading to expression of an oncogenic protein.

  Study EGAS00001000685

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

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

  Study EGAS00001002687

• Sequencing-based counting and size profiling of plasma Epstein-Barr virus DNA enhance population screening of nasopharyngeal carcinoma.

  Circulating tumor-derived DNA testing for cancer screening has recently been demonstrated in a prospective study on identification of nasopharyngeal carcinoma (NPC) among 20,174 asymptomatic individuals. Plasma EBV DNA, a marker for NPC, was detected using real-time PCR. While plasma EBV DNA was persistently detectable in 97.1% of the NPCs identified, ∼5% of the general population had transiently detectable plasma EBV DNA. We hypothesized that EBV DNA in plasma of subjects with or without NPC may have different molecular characteristics. We performed target-capture sequencing of plasma EBV DNA and identified differences in the abundance and size profiles of EBV DNA molecules within plasma of NPC and non-NPC subjects. NPC patients had significantly higher amounts of plasma EBV DNA, which showed longer fragment lengths. Cutoff values were established from an exploratory dataset and tested in a validation sample set. Adopting an algorithm that required a sample to concurrently pass cutoffs for EBV DNA counting and size measurements, NPCs were detected at a positive predictive value (PPV) of 19.6%. This represented superior performance compared with the PPV of 11.0% in the prospective screening study, which required participants with an initially detectable plasma EBV DNA result to be retested within 4 weeks. The observed differences in the molecular nature of EBV DNA molecules in plasma of subjects with or without NPC were successfully translated into a sequencing-based test that had a high PPV for NPC screening and achievable through single time-point testing.

  Study EGAS00001002707

• A_systems_biology_approach_to_understand_immunity_and_pathogenesis_of_malaria_in_children_exposed_to_endemic_Plasmodium_falciparum_transmission

  From long-term studies of children in Kenya, we know that some children have numerous malaria episodes through to older age, while other children become immune more rapidly. Our hypothesis is that these children with frequent malaria episodes are caught in a vicious circle, whereby malaria episodes lead to impaired immunity to malaria, which in turn leads to further episodes of malaria. We will therefore investigate immune responses in children with different life histories of malaria episodes. We will also include a group of children living in similar environmental conditions but who are completely unexposed to malaria. Aims: Using RNAseq we will characterise the transcriptome of a group of children with frequent and febrile malaria or infrequent/asymptomatic. These data will be combined with cytokine profiling data (NIMR) and used to build predictive models (Exeter). The predictions will be validated against a similar second group. A single snap shot of immune responses may represent only the endpoint of many immune processes and may not reflect those that are causally related to differences in malaria outcome. Therefore, we will collect and store samples from a larger group of children who will then be followed up for over 3-4 years. This extended follow up will allow us to identify the same groups as described above, and then examine samples taken before the development of those outcomes. 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 EGAS00001002978

• Cerebrospinal fluid cfDNA sequencing for classification of central nervous system glioma

  Primary central nervous system (CNS) gliomas can be classified by characteristic genetic alterations. In addition to solid tissue obtained by surgery or biopsy, cell-free DNA (cfDNA) derived from cerebrospinal fluid (CSF) is an alternative source of material for genomic analyses. Experimental design: We performed targeted next-generation sequencing (NGS) of CSF cfDNA in a representative cohort of 85 patients presenting with with suspicion of primary or recurrent glioma at two neurooncological centers. Copy-number variation (CNV) profiles, single nucleotide variants (SNVs), and small insertions/ deletions (indels) were combined into a molecular-guided tumor classification. Comparison with the solid tumor was performed for 38 cases with matching solid tissue available. Results: Cases were stratified into four groups: glioblastoma (n = 32), other glioma (n = 19), non-malignant (n = 17) and no confirmed diagnosis available (n = 17). We introduce a molecular-guided tumor classification, enabling identification of tumor entities and/ or cancer specific alterations in 75.0 % (n = 24) of glioblastoma and 52.6 % (n = 10) of other glioma cases. The overlap between CSF and matching solid tissue was highest for CNVs (26-48 %) and SNVs at pre-defined gene loci (44 %), followed by SNVs/indels identified via uninformed variant calling (8-14 %). A molecular-guided tumor classification was made 23.5 % (n = 4) of cases with no confirmed diagnosis available. Conclusions: We developed a workflow for targeted sequencing of CSF cfDNA as well as a strategy for interpretation and reporting of sequencing results based on a molecular-guided tumor classification in glioma.

  Study EGAS50000000060

• Circulating tumor DNA dynamics reveal KRAS G12C mutation heterogeneity and response to treatment with the KRAS G12C inhibitor divarasib in solid tumors

  Purpose: To inform prognosis, treatment response, disease biology, and KRAS G12C mutation heterogeneity, we conducted exploratory circulating tumor DNA (ctDNA) profiling on 134 patients with solid tumors harboring a KRAS G12C mutation treated with single-agent divarasib (GDC-6036) in a phase 1 study. Experimental design: Plasma samples were collected for serial ctDNA profiling at baseline (Cycle 1 Day 1 prior to treatment) and multiple on-treatment time points (Cycle 1 Day 15 and Cycle 3 Day 1). Results: KRAS G12C ctDNA was detectable from plasma samples in 72.9% (43/59) and 92.6% (50/54) of patients with non-small cell lung cancer and colorectal cancer, respectively, the majority of whom were eligible for study participation based on a local test detecting the KRAS G12C mutation in tumor tissue. Baseline ctDNA tumor fraction was associated with tumor type, disease burden, and metastatic sites. A decline in ctDNA level was observed as early as Cycle 1 Day 15. Serial assessment showed a decline in ctDNA tumor fraction associated with response and progression-free survival. Except for a few cases of KRAS G12C sub-clonality, on-treatment changes in KRAS G12C variant allele frequency mirrored changes in the overall ctDNA tumor fraction. Conclusion: Across tumor types, the KRAS G12C mutation likely represents a truncal mutation in the majority of patients. Rapid and deep decline in ctDNA tumor fraction was observed in patients responding to divarasib treatment. Early on-treatment dynamics of ctDNA were associated with patient outcomes and tumor response to divarasib treatment.

  Study EGAS50000000315

• Surgery in combination with immune checkpoint therapy as an effective treatment for patients with metastatic cancer.

  Abstract Surgical removal of primary tumors was shown to reverse tumor-mediated immune suppression in pre-clinical models with metastatic disease. However, how cytoreductive surgery in the metastatic setting modulates the immune responses in patients, especially in the context of immune checkpoint therapy (ICT)-containing treatments is not understood. Here, we report the first prospective, non-comparative clinical trial (N=104) using three different ICT-containing strategies plus cytoreductive or “debulking” surgery to remove the primary tumor-bearing kidney or a metastatic lesion as a treatment for patients (N=43) with metastatic clear cell renal cell carcinoma (mccRCC). For those patients (N=61) who were not candidates for cytoreductive surgery, a biopsy was obtained instead for correlative biological studies. Our data demonstrated that the combination of ICT with cytoreductive surgery was safe and feasible in patients with mccRCC. The 2-year overall survival was 84% with a median OS of 54.7 months for patients who received ICT containing regimens plus surgery. Immune-monitoring studies with co-detection by indexing (CODEX) identified distinct tumor spatial conformation of cellular subsets as a novel and improved predictor of response to ICT. Importantly, single-cell RNA-sequencing data demonstrated that surgical removal of the tumor increased antigen-presenting dendritic cell population with a concurrent reduction in KDM6B expressing immune-suppressive myeloid cells in the peripheral blood. Together, this study highlighted the feasibility of combining ICT with cytoreductive surgery in a metastatic setting and demonstrated potential enhancement of immune responses following ICT plus cytoreductive surgery in patients with metastatic disease.

  Study EGAS00001005667

• Molecular classification improves risk assessment in adult B-lineage ALL: Patients on the international UKALLXII-ECOG2993 trial.

  Acute lymphoblastic leukemia (ALL) in adults is a rare disease with outcomes that remain significantly inferior to those of children with ALL. While genomic classification of pediatric B-lineage ALL (B-ALL) has led to improved risk assignment and therapeutic paradigms, similar advances in adults have been lacking. The international UKALLXII/E2993 (NCT00002514) trial accrued over 2000 adult (17-64 years old) patients over 13 years, including 1229 BCR/ABL negative B-ALLs. While 93% of B-ALL achieved a remission, 41% relapsed at a median time of 13 months (Range) and overall survival (OS) at 5 years was 42% (CI). Older age and higher white blood cell (WBC) count at presentation negatively affected OS. Patients with a sibling donor assigned to allogeneic transplantation had superior OS compared to patients without donor. Randomization of patients without a donor showed an advantage of chemotherapy over autologous transplantation in first remission. Transcriptome sequencing of 94% of E2993 patients enabled genomic subtyping. We found that one-third of patients belonged to favorable genotypes (DUX4-rearranged, ETV6-RUNX1, TCF3-PBX1, PAX5 P80R, high hyperdiploidy). Of 63.4% of patients classified as high-risk based age and WBC count at diagnosis, almost 40% harbored subtype-defining genetic associations associated with favorable- or intermediate outcomes. These patients did significantly better than expected from their protocol-defined risk assignment. Our data in a large adult B-ALL cohort treated on a single international trial show the important prognostic value of genomic analysis and the potential benefit for many of the novel genotypes.

  Study EGAS00001004638

• shallow WGS and deep targeted panel on ctDNA in rhabdomyosarcomas

  The genomic spectrum of rhabdomyosarcoma (RMS) progression from primary to relapse is not fully understood. In this study we investigate 35 patients with relapsed RMS from two contributing institutions, 18 fusion-positive (FP-RMS) and 17 fusion-negative RMS (FN-RMS). Targeted DNA or whole exome sequencing (WES) was used to detect alterations in paired primary/relapsed samples. In 10 cases, circulating tumor DNA (ctDNA) from multiple timepoints through clinical care and progression was analyzed for feasibility of liquid biopsy in monitoring treatment response/relapse. ctDNA alterations were evaluated using a targeted custom RMS panel (36 genes) at high coverage for single nucleotide variation and fusion detection, and a shallow whole genome sequencing for copy number variation. FP-RMS had a stable genome with relapse, with the most common secondary alterations : CDKN2A/B, MYCN and CDK4 alterations, being already present at diagnosis and impacting overall survival. FP-RMS lacking major secondary events at baseline acquired recurrent MYCN and AKT1 alterations. FN-RMS acquired a higher number of new alterations, most commonly SMARCA2 missense mutations. ctDNA analyses detected pathognomonic variants in all RMS patients at diagnosis, regardless of FP/FN or type of alterations, while at relapse selected alterations were confirmed in 86% of FP-RMS and 100% FN-RMS. Moreover, a higher number of fusion reads was detected with increased disease burden and at relapse in patients following a fatal outcome. These results underscore patterns of tumor progression within a relatively stable genomic landscape and provide rationale for using liquid biopsy to monitor treatment response.

  Study EGAS00001007399

• The University of Hong Kong Intestinal Metaplasia Organoids Study

  Background: Gastric intestinal metaplasia (IM) is a pre-cancerous stage spanning a morphological spectrum that is poorly represented by human cell line models. Objective: We aim to establish and characterize human IM cell models to better understand IM progression along the cancer spectrum. Design: A large human gastric IM organoid (IMO) cohort (n=28), their clonal derivatives and normal gastric organoids (n=42) for comparison were established. Comprehensive multi-omics profiling and functional characterization were performed. Results: Single-cell transcriptomes revealed IMO cells spanning a spectrum from hybrid gastric/intestinal to advanced intestinal differentiation. Their lineage trajectories connected different cycling and quiescent stem and progenitors, highlighting differences in gastric to IM transition and the potential origin of IM from STMN1 cycling isthmus stem cells. Hybrid IMOs showed impaired differentiation potential, high lineage plasticity beyond gastric or intestinal fates, and reactivation of a fetal gene program. Cell populations in gastric IM and cancer tissues were highly similar to those derived from IMOs and exhibited a fetal signature. Genomically, IMOs showed elevated mutation burden, frequent chromosome 20 gain, and epigenetic de-regulation of many intestinal and gastric genes. Functionally, IMOs were FGF10 independent and showed downregulated FGFR2. Several IMOs exhibited a cell-matrix adhesion independent (CMi) subpopulation that displayed chromosome 20 gain but lacked key cancer driver mutations, potentially representing the earliest neoplastic precursor of IM-induced gastric cancer. Conclusions: Overall, our IM organoid biobank captured the heterogeneous nature of IM, revealing mechanistic insights on IM pathogenesis and progression, offering an ideal platform for studying early gastric neoplastic transformation and chemoprevention.

  Study EGAS00001007899

• NHLBI and NIDDK Sponsored GWAS in Benign Ethnic Neutropenia/Leukopenia (BEN) in African-Americans (age >45 yrs old) from the REGARDS

  Benign ethnic neutropenia (BEN) is a clinical condition more commonly observed in African-Americans. It is characterized by a relative reduction in neutrophil count by about 1000 cells per microliter, leading to a decrease in total leukocyte count by similar decrement. Previous reports of this condition showed that there was neither higher frequency nor increased severity of infections in affected individuals. Bone marrow examinations showed normal white cell maturation; and ex vivo culture of marrow cells showed low normal or slightly reduced number of myeloid colonies. Under physiologic stress, the increases in neutrophil and leukocyte counts of BEN individuals are slightly lower, compared to normal African-Americans or Caucasians. These clinical observations suggest that BEN results from a lower 'set point' for cell number in the marrow. Additionally, case reports of familial BEN, the persistence of BEN over many decades in the US, UK, and Africa, and the recent report of Duffy antigen and chemokine receptor (DARC) being associated with neutropenia, all suggest a strong genetic association to neutropenia/leukopenia. Our initial look into microarray analyses in a pilot trial of subjects showed that there were no significant differences in mRNA signals between BEN and normal subjects. Therefore, we are now proposing a larger study, utilizing Illumina Omni Express chips, to look for genetic associations. We have partnered with the Reasons for Geographic and Racial Differences in Stroke study (REGARDS), where nearly half of the cohort are African-Americans. This will be one of the few GWAS being performed in only African-Americans, and will provide valuable genetic information to link with neutropenia and possibly other conditions/diseases. Genotyping was performed by the Johns Hopkins University Center for Inherited Disease Research (CIDR). Quality control of the genotypic and phenotypic data was performed through a collaboration between CIDR and the Genetics Coordinating Center, Department of Biostatistics at the University of Washington, which is funded by a federal contract supported by 14 NIH Institutes (HHSN268200782096C).

  Study phs000507

• Center for Common Disease Genomics [CCDG] - Cardiovascular: TexGen

  Cardiovascular disease (CVD), especially coronary heart disease, heart failure and cerebrovascular disease remain the leading causes of death in men and women across all race groups in the United States. Substantial evidence exists for genetic factors underlying CVD risk, and their discovery offers an opportunity to enhance understanding of disease mechanisms, to provide specific diagnostic and prognostic indicators, and to identify novel therapeutic targets. The Centers for Common Disease Genomics (CCDG) are a large-scale genome sequencing effort undertaken as a collaboration to identify rare risk and protective alleles for common chronic diseases, with a current focus on early onset heart disease, hemorrhagic stroke, and autism. In this study, whole genome sequencing (WGS) was performed in 6,146 individuals with any personal or family history of CVD from the TexGen study who were recruited from several institutions in the Texas Medical Center located in Houston, Texas. The participants included patients who were admitted with a variety of diagnoses including acute coronary syndromes, strokes, and transient ischemic attacks, or who underwent percutaneous coronary interventions or coronary artery bypass grafting (CABG) (Virani SS et al. Am J Cardiology 107:1504-1509, 2011). Patients in the TexGen cohort with a confirmed diagnosis of acute coronary syndrome (ACS) or CABG when discharged from the hospital between 2001-2008 were eligible for a follow-up study of recurrent myocardial infarction (Virani SS et al. Circulation J 76:950-956, 2012), while those presenting with ACS beginning in 2007 were recruited for an investigation of the role of depressive symptoms in the risk of major adverse coronary events (Sanner J et al. Biol Res Nurs 20:168-176, 2018). In addition, the initial criterion for enrollment in a third TexGen sub-study undertaken between 2007 and 2013 was the occurrence of premature acute myocardial infarction in men ≤ 50 years and women ≤ 55 years in order to establish a blood sample collection resource with the intention of localizing genes that contribute to disease risk. This definition was later expanded to include individuals with a clinical history of premature coronary heart disease such as CABG or percutaneous coronary interventions.

  Study phs003010

• Temporal Lobe Epilepsy and Retrotransposons

  Idiopathic (cryptogenic) epilepsy is a common, chronic group of brain disorders, affecting 1-2% of the US population, creating a significant public health problem because of the associated morbidity and mortality. One subtype of this complex group of brain disorders, temporal lobe epilepsy (TLE), is particularly burdensome, because a substantial fraction of patients do not respond to anticonvulsant medications. If the seizure focus can be localized by imaging and/or EEG, the TLE patient often elects to have a partial resection of the affected temporal lobe, because they have daily seizures, despite multiple pharmacotherapy regimens. Although genetic risk is thought to play a role in TLE, identification of common risk alleles has had limited success. In the past 5 years, data have accumulated to prove that neuronal embryogenesis is accompanied by activation of LINE1 (L1) retrotransposons (RTPs) to an unexpected degree, such that developing neurons may accumulate de novo L1 insertions. This results in a substantial mosaicism within populations of CNS neurons. While most of these somatic de novo L1s will have little effect on neuronal function (perhaps because they occur in gene deserts or in large introns or in genes not required for that cell's function), some may interfere with normal neuronal activity because they have inserted into a gene needed by that particular neuron for normal function. If one or more functional de novo L1s occur early in CNS development, all the daughter neurons that derive from that neuronal precursor will also carry the L1 insertion, perhaps leading to a dysfunctional population of neurons destined to become an epileptic focus. This study leveraged CNS tissue (obtained at craniotomy) from patients with intractable TLE, who have undergone a therapeutic partial resection of one temporal lobe. Using fluorescence-assisted cell sorting of temporal lobe neuronal nuclei, followed by high-throughput sequencing and alignment to the reference genome, detection of de novo L1s will be done. In this manner, it is expected that de-novo intragenic L1s, contributing to idiopathic TLE, will be discovered.

  Study phs002067

• Identification of Modifiers of 22q11.2 Deletion Syndrome in Whole Genome Sequence - CIDR

  This project aims to identify genetic modifiers of congenital heart disease (CHD), which occurs among 22q11.2 deletion syndrome (22q11.2DS; also known as DiGeorge syndrome/velo-cardio-facial syndrome) patients. In contrast to the prevalence of ~0.8% in the general population, about 60%-70% of 22q11.2DS patients have CHD, of which most are conotruncal heart defects, making 22q11.2DS a very ideal model to study the genetic underpins of CHD. The chromosome 22q11.2 region is susceptible to meiotic chromosome rearrangements leading to hemizygous deletions, because there are large blocks of low copy repeats, termed LCR22s, that are dispersed in the region. Non-allelic homologous recombination during meiosis can lead to specific-sized deletions. Approximately 90% have a typical hemizygous 3 million base pair (Mb) deletion between LCR22-A and -D, while some have nested deletions between LCR22-A and B or LCR22-A and C. Our goal is to perform a case-control association study, in which cases have CHD, while controls have normal heart structures, all with 22q11.2DS. All individuals are unrelated and have been collected from collaborators in the US, Canada and Europe. All subjects received a clinical diagnosis of 22q11.2DS and signed informed consent to allow a blood sample to be collected for research. The DNA samples were de-identified in 2 steps to protect their privacy. We then determined deletion type using multiplex ligation-dependent probe amplification (MLPA). This information is provided for the great majority of subjects. We also obtained cardiac phenotype information from echocardiogram reports as well as reported race and ethnicity (Hispanic vs. non-Hispanic information), which is required for the analysis. We will obtain 30X whole-genome sequence data from NIH CIDR. We will first annotate the sequence variants identified to uncover rare ( For genotype data, we will provide the raw fastq data from whole-genome sequencing as well as the called variants file in a VCF file. For phenotype data, we will provide the sample relationship files (ped files), CHD affection status, deletion type, and race.

  Study phs002514

• Parallel Genomic Alterations of Antigen and Payload Targets Mediate Polyclonal Acquired Clinical Resistance to the Antibody Drug Conjugate Sacituzumab Govitecan

  Sacituzumab Govitecan (SG), a novel antibody drug conjugate (ADC) incorporating the anti-TROP2 antibody hRS7 conjugated to a topoisomerase-1 inhibitor (SN-38) payload, is the first ADC to be approved for advanced triple negative breast cancer (TNBC). However, mechanisms governing therapeutic resistance to SG are not known. We sought to identify mechanisms of de novo and acquired resistance to SG through unbiased whole-exome sequencing (WES) and RNA sequencing analysis of pre-treatment and multi-site post-progression (autopsy) tumor specimens. We examined three metastatic TNBC cases exhibiting (1) de novo progression, (2) stable disease, and (3) a deep response followed by progression, then mapped the temporal and spatial genomic evolution of acquired resistance in the responding patient. We then conducted additional pre-clinical experiments to validate the observed resistance mechanisms. TROP2 RNA and gene copy number were associated with de novo resistance, as case (1) was found to have absent TROP2 expression in all specimens, case (2) expressed TROP2, while case (3) exhibited both expression and focal genomic amplification of the TACSTD2/TROP2 locus. The genomic phylogeny tree inferred from case (3) post-progression specimens revealed one branch harboring an acquired canonical E418K resistance mutation in TOP1 (encoding topoisomerase 1) and a subsequent sub-clonal TOP1 inactivating frameshift mutation, while a distinct branch exhibited acquisition of a novel T256R missense mutation in TACSTD2 (encoding TROP2). Both the TOP1- and TACSTD2-mutant clones seeded multiple distinct metastatic sites. Through reconstitution experiments in TROP2 negative cells we found that TROP2 T256R is a stable protein with defective cell membrane localization and reduced cell surface binding by RS7 compared to wild-type TROP2. Collectively, these findings underscore TROP2 as a determinant of initial response to SG, and they reveal parallel and mutually exclusive polyclonal molecular mechanisms of acquired resistance involving the direct antibody target and drug payload target in distinct metastatic subclones of a single patient. While further research is needed to extend these novel findings, this study highlights the specificity of SG and illustrates how identifying such mechanisms will inform rational therapeutic strategies to overcome ADC resistance.  

  Study phs002555

• Personalized Medicine Strategy for MPNSTs: Using Precision Oncology on PDOX Models to Inform Tumor Boards

  Background: Malignant peripheral nerve sheath tumors (MPNSTs) are a heterogeneous group of aggressive soft tissue sarcomas with poor prognosis. Currently there is a lack of effective treatments for MPNSTs. Here, we propose a personalized medicine approach that integrates a precision oncology strategy guided by MPNST genomic analysis, with a functional validation of treatment response in an orthotopic xenograft model (PDOX) derived from the same MPNST. Methods: Comprehensive whole genome sequencing analysis was performed in primary MPNSTs, relapses and (in one case) metastases, following disease progression in two independent individuals. Matched MPNST PDOX models were generated by orthotopically implanting tumor fragments near the sciatic nerve of immunodeficient mice. Candidate targeted combination therapies were prioritized based on genomic alterations and tested in vivo in the PDOX models. Results: The feasibility of the developed strategy is illustrated for two MPNST patients, one Neurofibromatosis type 1 (NF1) individual that developed two independent MPNSTs and another sporadic MPNST case with multiple metastatic relapses. Genomic analysis revealed a remarkable degree of genomic stability across primary MPNSTs and their successive relapses in each patient, and even metastases in one individual. While based on a small number of cases requiringing additional analyses, this finding aligns with previous evidence suggesting a fair genomic conservation throughout tumor evolution. This stability supports the identification of consistent therapeutic vulnerabilities throughout disease progression. Among the therapies tested, co-treatment of MEK inhibitor (MEKi) plus bromodomain inhibitor (BETi) elicited the highest antitumor activity, resulting in approximately 60% tumor volume reduction in the sporadic MPNST PDOX model, whose patient has been receiving this therapy for eight months with sustained remission. Conclusions: This study demonstrates the feasibility and clinical utility of integrating genomic-driven precision oncology with PDOX-based functional testing for MPNSTs. This strategy may support molecular tumor boards (MTBs) in their treatment decisions. The observed genomic stability supports the use of longitudinal tumor profiling to guide treatment, and the success of MEKi+BETi highlights its potential as a combination therapy for MPNSTs.

  Study EGAS50000001502

• Therapeutic Resistance to PI3K-alpha Inhibitors

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

  Study EGAS00001000991

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

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

  Study EGAS00001006703