Search Results - EGA European Genome-Phenome Archive

16S rRNA Rectal Mucus

16S rRNA sequencing of rectal mucus samples from patients suspected to have colorectal cancer. This is 1 of 4 sequencing experiments on the same sample type.

Study EGAS50000001262

Res1_HT29_exp2_MC_03_03_22

1 sample is pure plasmid DNA and 10 samples are cell pellets for genomic DNA extraction. CRISPR PCR1 and PCR2 indexing - Please use standard Kozuke primers.

Study EGAS00001006093

Res1_H23_exp1_MC_04_03_22

1 sample is pure plasmid DNA and 8 samples are cell pellets for genomic DNA extraction. CRISPR PCR1 and PCR2 indexing - Please use standard Kozuke primers.

Study EGAS00001006091

Res1_HT29_exp1_MC_02_03_22

1 sample is pure plasmid DNA and 10 samples are cell pellets for genomic DNA extraction. CRISPR PCR1 and PCR2 indexing - Please use standard Kozuke primers.

Study EGAS00001006092

HTAN MCL Pre-Cancer Atlas Pilot Project - Targeted Sequencing Development Study

The HTAN-MCL Pre-Cancer Atlas Pilot Project (PCAPP) is the result of a collaboration between the seven members of the MCL consortium. Across four organ types, PCAPP's goal is to collect and profile pre-malignant lesions for gene expression, DNA mutations, single-cell gene expression and immune-environment. Most PML are small in size and only available come from formalin fixed paraffin embedded archived tissue. The primary goal of PCAPP is to 1) understand the logistical challenges of PML specimen collection, 2) document technical limitations of the assays that are specific to the PML and 3) overcome them to support the generation of a more comprehensive Pre-Cancer Atlas in the future. The current upload provides RNA and DNA sequencing from participants with DCIS who were studied at the University of San Diego and the University of Vermont. Description of the overall study: A. Background/Significance One of the critical barriers to developing new approaches for cancer detection and prevention is the lack of understanding of the key molecular and cellular changes that cause cancer initiation and progression. Unlike the extensive work that has been done profiling advanced stage tumors, few studies have comprehensively profiled the molecular alterations found in precancerous tissues. Premalignant lesions are currently characterized by histologic changes that precede the development of invasive carcinoma1,2.These lesions can often be identified in regions surrounding an invasive tumor or in biopsies taken from patients undergoing diagnostic evaluation for suspicion of cancer. Currently, limited metrics exist to identify lesions that will likely progress to carcinoma and require intervention from those that will naturally regress or remain stable3,4. Characterization of the molecular alterations in premalignant lesions and the corresponding changes in the microenvironment would hasten the development of biomarkers for early detection and risk stratification as well as suggest preventive interventions to reverse or delay the development of cancer. Our pilot study will establish the feasibility of transcriptomic, genomic and immune profiling of FFPE premalignant lesions from multiple organ sites, collected and profiled with uniform SOPs across multiple institutions within the MCL consortium. We will characterize the molecular alterations in precancerous lesions and the corresponding microenvironment in four major organ sites, in order to uncover the molecular and cellular determinants of premalignancy, and establish standardized sequencing and immunohistochemistry protocols on FFPE precancerous tissue. We will also evaluate the technical feasibility of single nuclei sequencing of small FFPE pre-cancer lesions. Successful completion of the proposed pilot study will set the stage for expansion and development of a comprehensive Pre-Cancer Atlas (PCA) as part of the NCI's moonshot.B. Specific Aims Aim 1: Collect premalignant lesions (PML) and their associated microenvironment via LCM from FFPE tissue across four organ sites (breast, lung, pancreas & prostate). Aim 2: Perform bulk RNA and DNA seq on premalignant FFPE samples (and flash frozen tissue where available) and compare the genomic/transcriptomic alterations within and across organ sites. C. ApproachAim 1: Collect premalignant lesions (PML) and their associated microenvironment via LCM from FFPE tissue across four organ sites (breast, lung, pancreas & prostate). MethodsI. Patient Population/Sample Collection: Overview of the sites collecting PML tissue from the respective organs is provided in Table 1 and a full description of the biospecimens to be obtained is described in detail for each organ type below. Table 1. Breakdown of cohort by tissue type and collection site.Organ siteBreastLungPancreasProstateType of PMLDCISAAH, Squamous Dysplasia/CISIPMNsPINCollection of PatientsUCSF/UCSDUVMBU*/UCLAVanderbilt/MoffittMDACC*JHUStanford*# of Patients201920 (10 of each type)20 (10 of each type)242020Total patients per Organ39402440Note: single nuclei/cell RNA-Seq will be performed on 4-5 FFPE samples from each of the organ types 1. DCIS lesions from breast tissue: DCIS lesions will be collected from 39 patients (20 from UCSF/UCSD & 19 from UVM) with primary low or high-grade DCIS diagnosed from a breast core biopsy. Subsequent resected lumpectomy or mastectomy tissues will be prospectively sampled in the vicinity of the prior biopsy site using multiple approaches: 1) Live cells (heterogeneous mix) will be obtained as a cell scrape slurry from the lesion surface or by fine needle aspirate (FNA); 2) For a subset of specimens where size is sufficient, a block of breast tissue with DCIS will be fresh-frozen; 3) The remainder of the specimen will be taken for routine formalin-fixation and paraffin-embedding (FFPE). The FFPE sample will be annotated to identify the matched FFPE tissue block adjacent to the fresh-frozen sample and will be sectioned for use in bulk and single nuclei sequencing . We will dissect DCIS, adjacent normal and when available, associated carcinoma. In addition, when possible, normal tissue will be collected from a tissue block lacking lesions as well as collection of blood. A subset of patients (n = 5 | FFPE, flash frozen and fresh) will be sent to the Broad Institute for single nuclei/cell sequencing.2. AAH and squamous dysplastic/CIS lesions from airway and lung tissue: For squamous cell lung cancer, we will collect endobronchial biopsies from abnormal airway regions identified on autofluroscence bronchoscopy or identify PMLs in the margins of resected lung tissue. We will study 20 patients (5 each from BU/UCLA/Vanderbilt/Moffitt) with pre-invasive squamous lesions (moderate-severe dysplasia or carcinoma in situ (CIS)) identified on pathologic examination. LCM of the premalignant region and adjacent normal epithelium will be performed as well as the invasive tumor for those collected from the resection margin (n=5 from UCLA). On a subset of lesions collected at bronchoscopy (n=5), we will collect additional biopsies that will be flash frozen and fresh for single nuclei and cell sequencing, respectively, performed at the Broad Institute. In parallel to the work at the Broad, BU will perform single cell RNA-seq on these freshly cell sorted tissues (n = 5). Blood will be collected on all patients for genomic studies. For lung adenocarcinoma, we will collect resected FFPE lung tissues from 20 patients (10 from UCLA and 10 from Vanderbilt/Moffitt) with early stage lung adenocarcinoma that harbor atypical adenomatous (AAH) premalignant lesions in the resection margin. We will LCM multiple AAH regions (3-5 per patient) as well as adjacent regions of normal epithelium and invasive adenocarcinoma. In addition, blood will be collected on all patients for genomic studies. 3. IPMNs from pancreatic tissue: For pancreatic cancer PML, we will collect low and high grade lesions from 24 patients representing macroscopic Intraductal Papillary Mucinous Neoplasms (IPMN) (n=24) from surgically resected specimens along with blood samples. Archival FFPE specimens of microscopic PanIN lesions, occurring multi-focally adjacent to invasive PDAC, and archival IPMN lesions (with or without associated invasive cancer), along with the adjacent normal tissue, will undergo LCM and utilized for bulk DNA and RNA sequencing. If matched frozen tissues are available for a subset of these FFPE samples, we will bank for comparison of profiles. Because IPMNs are macroscopic lesions, they provide an opportunity for obtaining the samples fresh and therefore can be used for single cell sequencing (in contrast to PanINs). Therefore, 5 freshly obtained IPMNs will be used for the single cell RNA sequencing studies performed at both the Broad Institute and MDACC, and the matched FFPE and/or frozen sections from these lesions (obtained from the adjacent PML) will be sent to Broad Institute as a pilot to assess "single nuclei" RNA sequencing.4. PINs from prostate tissue: For prostate cancer PML, there will be 40 samples of Prostatic Intraepithelial Neoplasia (PIN) collected between the Stanford and JHU sites (20 cases per site). At the Stanford site, 20 prostate specimens detected by PSA screening who have/will undergo surgery (radical prostatectomy) for clinically localized disease will make up the final cohort. The age range of the participants would be 40-75, and we anticipate that 18 will be Caucasian, 1 Asian and 1 Latino or African American based on the practice demographics practice at Stanford. Clinical and MRI data will also be collected for these samples. We will collect low grade (e.g. Gleason score of 6/Grade group 1; n=10) and high grade (Gleason score 4+3=7 or higher/Grade group 3 or higher; n=10) PINs from FFPE samples that have prostate carcinoma. In addition to obtaining LCM archival samples of low and high grade PIN, we will also obtain normal prostatic epithelial from the peripheral, central and transition zones as well as multiple samples of prostate carcinoma in order to obtain the spectrum of Gleason grades in the carcinoma as needed. LCM samples will be used for bulk DNA and RNA sequencing. In addition, single cells will be dissected from FFPE samples to prepare single cell RNA seq libraries using techniques developed at Stanford, and FFPE tissue will be sent to the Broad for single nuclei sequencing. When available, flash frozen and fresh samples from these prostates will be archived and prepared for single nuclei and cell sequencing, respectively, at the Broad Institute and at Stanford (single cell only). JHU will also capture 10 cases (5 grade group 1 and 5 grade group 2) of high grade PIN, normal and invasive adenocarcinoma using frozen sections from fresh frozen tissues. When possible these will be from the same patients as the FFPE samples. Since frozen sections can be quite challenging to morphologically determine high grade PIN from normal epithelium, for these samples we will perform a number of additional tissue-based characterizations. These will include a multicolor combined basal cells (p63 and CK903) and PIN/carcinoma markers (AMACR) referred to in the cocktail as "PIN4", c-MYC (referred to as MYC) protein5, by IHC and mRNA by in situ hybridization (AM De Marzo, Q Zheng unpublished observations), telomere length by in situ hybridization6 and the 5'ETS/45S rRNA7. For these slides, the whole slides will be scanned with a Hammamatsu Nanozoomer with a 40x objective and regions of interest will be annotated as a guide for LCM.II. Laser-capture microdissection (LCM): FFPE tissue blocks will be sectioned at 7μm thickness and serial sections will be stained with H&E. LCM will be performed utilizing standard LCM systems, such as Leica LMD7000 and ArcturusXT at each site. Regions of premalignancy will be dissected and RNA/DNA will be extracted from microdissected cells using the Qiagen All Prep DNA/RNA FFPE Kit. Aim 2: Perform bulk RNA and DNA seq on premalignant FFPE samples and compare the genomic/transcriptomic alterations within and across organ sites. Rationale: There have been limited studies characterizing the genomic and transcriptomic landscape of premalignant lesions associated with breast, pancreatic, lung or prostate cancers. Characterizing the molecular determinants of premalignant disease that are unique and shared across multiple organs will enable new candidate biomarkers for early detection and novel therapeutic strategies for early intervention. MethodsBulk RNA-seq of LCM FFPE tissue: All participating sites will perform bulk RNA-seq in accordance with SOPs developed at BU. In brief, total RNA will be isolated from LCM'd lesion and associated microenvironment tissue using the Qiagen All Prep DNA/RNA FFPE Kit and quality will be assessed with the Agilent Bioanalyzer. Libraries will be generated with the Illumina TruSeq Access kit (for FFPE samples). They will be sequenced on the Illumina HiSeq2500 with 75base-pair paired-end reads. Quality of FASTQ files will be assessed with FastQC. Reads will be aligned to the human genome with STAR and gene-level and isoform-level expression will be quantified with RSEM. Splice junction saturation, transcript integrity, and biotype distributions will be calculated for each sample with RSeQC. DESeq2 and EdgeR will be used to identify associations between gene expression profiles and clinical variables while controlling for confounding covariates. BU will serve as an RNA-seq Core to assess reproducibility of FFPE RNA-seq methods across sites. We will perform RNA-seq according to the SOP listed above on a subset of samples for each organ type (total n ~ 20). Bulk seq of DNA from FFPE tissue: All participating sites will perform targeted or whole exome-seq (WES) in accordance with SOPs. In brief, DNA from laser captured material will be isolated using the Qiagen All Prep DNA/RNA FFPE Kit and undergo stringent quality control to ensure high quality input material for genomic profiling. Purified DNA (ideally 100-200 ng) will be used for library preparation and amplification, followed by next generation sequencing using standard protocols distributed by CDMG. Exome-seq methods are considered standardized, thus we will not need a DNA-seq Core to assess reproducibility across sites. We anticipate local centers will use Illumina paired end reads, following the following general approach. 1) DNA library preparation: Paired-end libraries will be prepared following the manufacturer's protocols (Illumina and Agilent), fragmented to 150-200 bp 2) Capture of targeted exome: Whole exome capture will be carried out using the protocol for Agilent's SureSelect Human All Exon kit. Purified capture products will be amplified using the SureSelect GA PCR primers (Agilent) for 12 cycles. 3) Sequencing will be carried out for the captured libraries using at least 100 bp paired-end reads. To achieve high level sensitivity and accuracy for detecting all the mutations in the whole exome, each sample will be sequenced at 200X mean depth. 4) Read mapping and alignment and variant analysis: Sequence short reads will be aligned to a reference genome (NCBI human genome assembly build 38) using BWA-MEM. Local realignment of aligned reads will be performed using Genome Analysis Toolkit (GATK).Data QC: To ensure scientific rigor and consistency among sites in RNA and DNA processing we will include a preliminary analysis of steps in processing and analysis. Protocols for extraction of high quality RNA and DNA from formalin fixed paraffin embedded (FFPE) tissues, which will be used extensively in these studies continue to improve and may have variable implementation among the sites participating in this study. To evaluate consistency of preliminary steps in processing and downstream analyses, we will initially distribute slides from one large FFPE fixed cancer of origin from prostate, breast, lung and pancreatic cancer. Analysis of these samples will allow us to review the DNA and RNA characteristics (yield, purity and strand length) among sites. Downstream analysis of these same samples will also allow us to compare among sites the consistency of variant calls among centers. We will be able to identify if there are some times of calls (such as small insertion deletions) that are more variable among centers versus other types of calls (such as relative gene expression or single base pair substitutions) that we expect to be less variable and to characterize the reliability of findings across sites. We are also including a 5% blind duplicate analysis of RNA sequencing. Samples will be analysed by the participating genomics cores without knowledge of the phenotype. RNA seq and CNA analyses are normalized for batch effects. We will also compare the observed sex to the self-reported sex as based on RNA profiles and exome sequencing of X chromosome genes as another check for processing accuracy and sample management. D. References 1. Wacholder, S. Precursors in Cancer Epidemiology: Aligning Definition and Function. Cancer Epidemiol. Prev. Biomark. 22, 521-527 (2013). PMID: 23549395.2. Berman, J. J. Precancer: The Beginning and the End of Cancer. (Jones & Bartlett Learning, 2011).3. Nasiell, K., Nasiell, M. & Vaćlavinková, V. Behavior of moderate cervical dysplasia during long-term follow-up. Obstet. Gynecol. 61, 609-614 (1983). PMID: 6835614.4. Merrick, D. T. et al. Persistence of Bronchial Dysplasia Is Associated with Development of Invasive Squamous Cell Carcinoma. Cancer Prev. Res. (Phila. Pa.) 9, 96-104 (2016). PMID: 26542061.5. Gurel, B. et al. Nuclear MYC protein overexpression is an early alteration in human prostate carcinogenesis. Mod. Pathol. Off. J. U. S. Can. Acad. Pathol. Inc 21, 1156-1167 (2008). PMID: 18567993.6. Meeker, A. K. et al. Telomere shortening is an early somatic DNA alteration in human prostate tumorigenesis. Cancer Res. 62, 6405-6409 (2002). PMID: 12438224.7. Guner, G. et al. Novel Assay to Detect RNA Polymerase I Activity In Vivo. Mol. Cancer Res. MCR 15, 577-584 (2017). PMID: 28119429.

Study phs002225

BRCA1 secondary splice-site mutations

BRCA1 splice isoforms d11 and d11q can contribute to PARP inhibitor (PARPi) resistance by splicing-out mutation-containing exons, producing truncated, partially-functional proteins. However, the clinical impact and underlying drivers of BRCA1 exon skipping remain undetermined. We analyzed nine ovarian and breast cancer patient derived xenografts (PDX) with BRCA1 exon 11 frameshift mutations for splice isoform expression and therapy response.

Study EGAS50000000022

Luminal progenitor cell line iHBEC(CD117)

Basal-like breast cancer originates in luminal progenitors, frequently with an altered PI3K pathway, and focally in close association with genetically altered myoepithelial cells at the site of tumor initiation. The exact trajectory behind this bi-lineage phenomenon remains poorly understood. Here we used a breast cancer relevant transduction protocol including hTERT, shp16, shp53, and PIK3CA(H1047R) to immortalize FACS isolated luminal cells, and we identified a candidate multipotent progenitor. We found that the apparent luminal phenotype of these oncogene transduced progenitors was metastable giving rise to basal-like cells dependent on culture conditions. After culturing the cells for more than 60 passages, cells were subjected to scRNA-seq as well as bulk RNA sequencing of two subpopulations (CD271+ and CD271-).

Dataset EGAD50000000723

NHLBI GO-ESP: Heart Cohorts Exome Sequencing Project (Ischemic Stroke Genetic Study, ISGS)

The NHLBI "Grand Opportunity" Exome Sequencing Project (GO-ESP), a signature project of the NHLBI Recovery Act investment, was designed to identify genetic variants in coding regions (exons) of the human genome (the "exome") that are associated with heart, lung and blood diseases. These and related diseases that are of high impact to public health and individuals from diverse racial and ethnic groups will be studied. These data may help researchers understand the causes of disease, contributing to better ways to prevent, diagnose, and treat diseases, as well as determine whether to tailor prevention and treatments to specific populations. This could lead to more effective treatments and reduce the likelihood of side effects. GO-ESP is comprised of five collaborative components: 3 cohort consortia - HeartGO, LungGO, and WHISP - and 2 sequencing centers - BroadGO and SeattleGO. The Ischemic Stroke Genetics Study (ISGS) is a study of newly onset cases (~600) with ischemic stroke (a stroke due to sudden interruption of blood flow to a part of the brain) compared with sex- and age-matched non-stroke participants. The study was conducted to determine the genes and their variants that contribute to an individual's risk of developing an ischemic stroke. The coordination of the recruitment and flow of the samples occurred at the Mayo Clinic, Jacksonville, FL, under the direction of James F. Meschia, MD. The University of Virginia (Stephen S. Rich, PhD) served as the analytic site for the genetic data. All GWAS data on ISGS participants have been deposited into dbGaP. As part of the NHLBI Exome Sequencing Project, DNA from a subset of ISGS participants will undergo exome sequencing. For the NHLBI ESP, a subset of 92 individuals with lacunar (small vessel) or atherosclerotic (large vessel) TOAST subtypes were selected from among all ISGS participants, excluding those individuals with TOAST subtypes of stroke of other etiology or of stroke with undetermined etiology. All 92 samples pass initial quality control metrics and 89 samples completed exome sequencing. A total of 75 participants with appropriate consent and variant calls had their genetic and phenotypic data deposited into dbGaP.

Study phs000546

Resuscitation Outcomes Consortium (ROC) Amiodarone, Lidocaine or Neither for Out-Of-Hospital Cardiac Arrest Due to Ventricular Fibrillation or Ventricular Tachycardia (ALPS)(ROC-ALPS-BioLINCC)

Accessing Data Please refer to “Authorized Access” below regarding accessing data through the BioData Catalyst ecosystem. The data from this accession is not available for download through dbGaP. Objective To compare the effects of amiodarone, lidocaine, and placebo on survival to hospital discharge after out-of-hospital cardiac arrest due to shock-refractory ventricular fibrillation or pulseless ventricular tachycardia. Background Ventricular fibrillation (VF) or pulseless ventricular tachycardia (VT) are common causes of out-of-hospital cardiac arrest, but are considered the most responsive to shock and therefore the most treatable. Nonetheless, most defibrillation attempts do not result in sustained return of spontaneous circulation, and VF or VT may persist or recur after shock. There is also evidence that longer durations of VF or VT are associated with decreases in the likelihood of resuscitation. Amiodarone and lidocaine are commonly used to promote successful defibrillation of shock-refractory VF or VT and prevent recurrences. Previous trials have shown amiodarone to be more effective than placebo or lidocaine for return of spontaneous circulation and survival at hospital admittance. This study sought to further extend the research and examine whether amiodarone would improve survival to hospital discharge and neurologic outcomes, as compared to placebo or lidocaine. Participants 3,026 eligible participants were enrolled, with 974 assigned to amiodarone, 993 assigned to lidocaine, and 1,059 assigned to placebo. An additional 1,627 participants that received a study intervention, but did not meet eligibility criteria, were included in analysis of the intention-to-treat population. Design The study interventions (amiodarone, lidocaine, and saline) were packaged in indistinguishable sealed kits and randomly distributed in to Emergency Medical Services (EMS) providers in a 1:1:1 ratio, stratified by participating site and agency. Each kit contained three syringes, and each syringe held 3 ml of colorless fluid containing 150 mg of amiodarone, 60 mg of lidocaine, or normal saline. Participants with out-of-hospital cardiac arrest were treated in accordance with local EMS protocols, in compliance with American Heart Association (AHA) guidelines. If VF or VT persisted or recurred after one or more shocks, eligible participants received a vasopressor and the masked kit containing amiodarone, lidocaine, or placebo. Approximating current clinical practice, the initial dose consisted of two syringes administered by rapid bolus. If the estimated body weight of the patient was less than 100 lbs., then one syringe was used. If VF or VT persisted, standard resuscitation measures, additional shocks, and an additional syringe of the study drug were administered. At that point the trial interventions were completed and standard interventions for advanced life support were employed. Upon arrival at the hospital, providers were notified of the patient's enrollment in the trial and encouraged to provide usual care in accordance with AHA guidelines, including open-label amiodarone or lidocaine if necessary. Components of hospital care were monitored but not standardized by the trial protocol. Participants, providers, and trial personnel were blinded to the trial drug assignments, with the exception of treating physicians if emergency un-blinding was required for care. Data from pre-hospital patient care records, CPR process measures, and hospital medical records were collected. The primary outcome of the trial was survival to hospital discharge, and the secondary outcome was survival with favorable neurologic status at discharge, defined as a score on the modified Rankin scale of 3 or less. Conclusions Neither amiodarone nor lidocaine resulted in a significantly higher rate of survival to hospital discharge or favorable neurologic outcome, as compared to placebo, among participants with out-of-hospital cardiac arrest due to initial shock-refractory ventricular fibrillation or pulseless ventricular tachycardia.

Study phs003784

Error-Corrected Next-Generation Sequencing Rectal Mucus

Error-corrected next-generation sequencing of rectal mucus samples from patients suspected to have colorectal cancer. This is 1 of 4 sequencing experiments on the same sample type.

Study EGAS50000001398

Genetic Determinants of Viral Clearance in HCV-Infected Populations

The World Health Organization estimates there are 170 million persons with HCV infection worldwide, and an estimated 4 million persons are infected in the United States. Persistent HCV infection can cause cirrhosis and hepatocellular cancer. Long term disease consequences do not occur in persons who spontaneously recover. It is not known why 10-40% recover from hepatitis C (and are not at increased risk of cirrhosis and cancer), but that knowledge would contribute to efforts to treat and/or prevent HCV infection. The overall purpose of this study is to investigate the host genetic basis for recovery from hepatitis C virus (HCV) infection. There is strong evidence that host genetic differences determine HCV recovery. After accidental exposure to the same HCV inoculum, some persons recover while others develop persistent infection. Recovery from HCV infection is an outstanding phenotype for genetic studies since both viral exposure and viral recovery can be ascertained with high sensitivity and specificity and since there are no known viral or environmental determinants. The study group is comprised of an ethnically and geographically diverse international population. All cohorts comprising this study group of 3350 individuals are well characterized. This case-control study is comprised of approximately 40% clearance individuals who are matched by age and HIV status within their respective cohorts at a ratio of 1:1 or 1:2 with HCV chronically infected individuals.

Study phs000248

Kids First: Genomics of Orofacial Cleft Birth Defects in Latin American Families

The Gabriella Miller Kids First Pediatric Research Program) (Kids First) is a trans-NIH effort initiated in response to the 2014 Gabriella Miller Kids First Research Act and supported by the NIH Common Fund. This program focuses on gene discovery in pediatric cancers and structural birth defects and the development of the Gabriella Miller Kids First Pediatric Data Resource (Kids First Data Resource). Both childhood cancers and structural birth defects are critical and costly conditions associated with substantial morbidity and mortality. Elucidating the underlying genetic etiology of these diseases has the potential to profoundly improve preventative measures, diagnostics, and therapeutic interventions. All of the WGS and phenotypic data from this study are accessible through dbGaP and https://kidsfirstdrc.org, where other Kids First datasets can also be accessed. The Kids First study of nonsyndromic orofacial cleft (OFC) birth defects in Latin American families is a whole genome sequencing study of 283 Latin-American parent-case trios drawn from ongoing collaborations led by Dr. Mary L. Marazita of the University of Pittsburgh Center for Craniofacial and Dental Genetics, and including a collaboration with Dr. Lina Moreno Uribe and Dr. Andrew Lidral of the University of Iowa. All families were ascertained through the Clinica Noel where patients with OFCs receive care from the Antioquia University School of Dentistry in Medellin, Colombia (key on-site colleagues included Dr. Luz Consuelo Valencia-Ramirez and Dr. Mauricio Arcos-Burgos). Genetic studies have shown that this population is comprised of an admixture of immigrant male Caucasians (mainly Spaniards and Basques) and native Amerindian females. Every subject has had a genetic evaluation, including a pedigree analysis for a family history of clefting and other birth defects, a pregnancy history for environmental exposures, and a complete physical exam to rule out suspected or known syndromes or environmental phenocopies. Sequencing was done by the Broad Institute sequencing center funded by the Kids First program (grant number U24-HD090743). The case in each of the Kids First OFC trios has cleft lip (CL, Figure A below), cleft palate (CP, Figure B), or both (CL+CP, Figure C): OFCs are genetically complex structural birth defects caused by genetic factors, environmental exposures, and their interactions. OFCs are the most common craniofacial anomalies in humans, affecting approximately 1 in 700 newborns, and are one of the most common structural birth defects worldwide. On average a child with an OFC initially faces feeding difficulties, undergoes 6 surgeries, spends 30 days in hospital, receives 5 years of orthodontic treatment, and participates in ongoing speech therapy, leading to an estimated total lifetime treatment cost of about $200,000. Further, individuals born with an OFC have higher infant mortality, higher mortality rates at all other stages of life, increased incidence of mental health problems, and higher risk for other disorders (notably including breast, brain, and colon cancers). Prior genome-wide linkage and association studies have now identified at least 18 genomic regions likely to contribute to the risk for nonsyndromic OFCs. Despite this substantial progress, the functional/pathogenic variants at OFC-associated regions are mostly still unknown. Because previous OFC genomic studies (genome-wide linkage, genome-wide association studies (GWAS), and targeted sequencing) are based on relatively sparse genotyping data, they cannot distinguish between causal variants and variants in linkage disequilibrium with unobserved causal variants. Moreover, it is unknown whether the association or linkage signals are due to single common variants, haplotypes of multiple common variants, clusters of multiple rare variants, or some combination. Finally, we cannot yet attribute specific genetic risk to individual cases and case families. Therefore, the goal of the current study is identify specific OFC risk variants in Latin American families by performing whole genome sequencing of parent-case trios.

Study phs001420

Clonal selection after gene therapy in sickle cell disease

While gene therapy (GT) provides a potentially curative treatment option for patients with sickle cell disease (SCD), the occurrence of myeloid malignancies in clinical trials has prompted concern. To interrogate potential mechanisms underlying increased cancer risk, we used hematopoietic stem cell (HSC) clonal tracking by whole genome sequencing (WGS) to map the somatic mutation and clonal landscape of 2,592 gene modified as well as unmodified single stem and progenitor cells from six SCD patients undergoing gene therapy (7-26 years old, average 12.7× depth). Pre-GT phylogenetic trees in SCD were highly polyclonal and mutation burdens per cell were elevated in some, but not all, patients. Post-GT, no clonal expansions were identified. However, an increased frequency of driver mutations associated with myeloid neoplasms or clonal hematopoiesis (DNMT3A- and EZH2-mutated clones in particular) were seen in both genetically modified and unmodified cells suggested positive selection of mutant clones during gene therapy. This work sheds light on the mutation landscape and HSC clonal dynamics in gene therapy for SCD and highlights enhanced fitness of some HSCs harboring pre-existing driver mutations following gene therapy. Future studies should define the long-term fate of mutant clones including any contribution to expansions associated with myeloid neoplasms.

Dataset EGAD00001010913

Salivary Gland Cancer TSO500 dataset

Salivary gland cancer (SGC) is a rare cancer for which systemic treatment options are limited. Therefore, it is important to characterize its genetic landscape in search for actionable aberrations. This research aimed to identify these actionable aberrations by NGS-based analysis of DNA (single and multiple nucleotide variants, copy number variants, microsatellite instability and tumor mutational burden) in a large cohort of SGC patients. DNA was extracted from archival tissue of 119 patients with various SGC subtypes and sequenced using a targeted NGS panel encompassing 523 cancer related genes (TruSight Oncology 500, TSO500). The 119 patients included 46 AdCC patients, 43 SDC patients, 15 MEC patients, 9 AciCC patients and 6 patients with other subtypes (1 secretory carcinoma, 1 polymorphic adenocarcinoma (PAC), 1 adenocarcinoma NOS, 1 myoepithelial carcinoma, 1 epithelial/myoepithelial carcinoma and 1 mixed PAC/myoepithelial carcinoma). Median exon coverage ranged from 80-904 (median 308) unique reads and the median percentage of exon coverage with at least 100 unique reads was median 96.6% in these 119 samples.

Study EGAS00001006232

Dissecting_global_protective_immune_response_to_dengue_virus_at_a_single_cell_resolution

1) 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 please see http://www.sanger.ac.uk/datasharing/

Study EGAS00001004980

The Genetic Basis of Aggressive Prostate Cancer, The Role of Rare Variation

In this study, we will apply a multi-staged approach to reveal genes harboring rare variants that are associated with aggressive PCa. Whole-exome sequencing (Aim 1a) of 2,774 aggressive cases and 2,776 non-aggressive cases of European ancestry will be conducted followed by rare variant analysis of single sites and gene burden testing to identify novel susceptibility loci/genes for aggressive disease. We will validate the most significantly associated genes (~500) through targeted sequencing in an additional 6,415 aggressive and 5,586 non-aggressive cases and 1,890 controls (Aim 1b). Next, we will investigate the clinical predictive utility of the genes/variants identified in 2,291 cases in the STHM3 trial who are undergoing biopsy based on PSA and genetic risk score stratification (Aim 2). Through this tiered approach we expect to significantly advance knowledge of aggressive PCa etiology and health disparities as well as guide the development of early detection and prognostic strategies for the subset of men who are most susceptible to this fatal form of disease. In this case-case study of aggressive vs non aggressive prostate cancer, aggressive cases are defined as prostate cancer as cause of death, (T4 disease or T3 disease) and Gleason 8+. Non-aggressive cases are men with T1/2 disease and Gleason ACKNOWLEDGMENTS and CONTRIBUTING SITES CAPS, PROCAP, STHM1, STHM2: Swedish Cancer Society (CAN 2016/818), Swedish Research Council (2014/2269).STHM3: Stockholm County Council (Stockholms Läns Landsting).MEC: Funding provided by the National Cancer Institute: Understanding Ethnic Differences in Cancer, 2U01CA164973 and The Genetic Basis of Aggressive Prostate Cancer, The Role of Rare Variation, 5R01CA196931-02.ATBC: The ATBC Study is supported by the Intramural Research Program of the U.S. National Cancer Institute, National Institutes of Health, and by U.S. Public Health Service contract HHSN261201500005C from the National Cancer Institute, Department of Health and Human Services.COSM: The Swedish Research Council/National Research Infrastructure Grant (VR 2014/6397; VR 2015/5997) The Swedish Cancer Foundation (CAN 2013/456; CAN 2016/727)CPSII: The authors express sincere appreciation to all Cancer Prevention Study II participants and to each member of the study and biospecimen management group. The American Cancer Society funds the creation, maintenance, and updating of the Cancer Prevention Study-II cohort.MCCS/APCS/PCFS: The Melbourne Collaborative Cohort Study (MCCS) recruitment was funded by VicHealth and Cancer Council Victoria and further supported by Australian National Health and Medical Research Council (NHMRC) grants 209057 and 396414. The Aggressive Prostate Cancer Case-Control Study (APCS) was funded by NHMRC grant 623204. The Prostate Cancer Family Study (PCFS) was fully funded by Cancer Council Victoria. Cancer Council Victoria funds the continuing maintenance and updating of the MCCS, APCS and PCFS. Cases and their vital status are ascertained and followed up through the Victorian Cancer Registry and the Australian Institute of Health and Welfare, including the National Death Index and the Australian Cancer Database.PLCO: The Prostate Lung Colorectal Ovarian Cancer Screening Trial (PLCO) was supported by the Intramural Research Program of the Division of Cancer Epidemiology and Genetics and by contracts from the Division of Cancer Prevention, National Cancer Institute, US National Institutes of Health, Department of Health and Human Services. EPIC: The coordination of EPIC is financially supported by International Agency for Research on Cancer (IARC) and also by the Department of Epidemiology and Biostatistics, School of Public Health, Imperial College London which has additional infrastructure support provided by the NIHR Imperial Biomedical Research Centre (BRC). The national cohorts are supported by: Danish Cancer Society (Denmark); Ligue Contre le Cancer, Institut Gustave Roussy, Mutuelle Générale de l'Education Nationale, Institut National de la Santé et de la Recherche Médicale (INSERM) (France); German Cancer Aid, German Cancer Research Center (DKFZ), German Institute of Human Nutrition Potsdam- Rehbruecke (DIfE), Federal Ministry of Education and Research (BMBF) (Germany); Associazione Italiana per la Ricerca sul Cancro-AIRC-Italy, Compagnia di SanPaolo and National Research Council (Italy); Dutch Ministry of Public Health, Welfare and Sports (VWS), Netherlands Cancer Registry (NKR), LK Research Funds, Dutch Prevention Funds, Dutch ZON (Zorg Onderzoek Nederland), World Cancer Research Fund (WCRF), Statistics Netherlands (The Netherlands); Health Research Fund (FIS) - Instituto de Salud Carlos III (ISCIII), Regional Governments of Andalucía, Asturias, Basque Country, Murcia and Navarra, and the Catalan Institute of Oncology - ICO (Spain); Swedish Cancer Society, Swedish Research Council and County Councils of Skåne and Västerbotten (Sweden); Cancer Research UK (14136 to EPIC-Norfolk; C8221/A19170 and C8221/A29017 to EPIC-Oxford), Medical Research Council (1000143 to EPIC-Norfolk; MR/M012190/1 to EPIC-Oxford). (United Kingdom). DFCI: Linda and Arthur Gelb and Rebecca and Nathan Milikowsky. HPFS and PHS: The Health Professionals Follow-up Study was supported by U01 167552 and P01 CA228696 from the National Cancer Institute, and with support from the Prostate Cancer Foundation. The Physicians' Health Study was supported by grants CA34944, CA40360, CA097193, HL26490 and HL34595.Northwestern: P50CA180995 (Catalona) 08/01/15 – 07/31/20 NIH/NCI SPORE in Prostate Cancer; The Urological Research FoundationPROMPT: MRC UK - Project reference G0500966, Cambridge BRC infrastructure funding, Cambridge Biomedical Research Campus (BRC-1215-20014), CRUK Cambridge Cancer Centre infrastructure funding (they are requesting this statement is written in blue for publications).ICR: This work was supported by the NIH R01 grant 5R01CA196931-02. The samples from the UK were from UKGPCS and PrompT. The UKGPCS study was supported by Cancer Research UK (grant numbers C5047/A7357, C1287/A10118, C1287/A5260, C5047/A3354, C5047/A10692, C16913/A6135 and C16913/A6835). We would like to acknowledge the NCRN nurses and Consultants for their work in the UKGPCS study. We thank all the patients who took part in this study. We also acknowledge The Institute of Cancer Research, The National Cancer Research Network UK, The National Cancer Research Institute (NCRI) UK for their ongoing support. We are grateful for support of NIHR funding to the NIHR Biomedical Research Centre at The Institute of Cancer Research and The Royal Marsden NHS Foundation Trust.Funding:CIDR grant X01HG008336

Study phs001524

Method to Assess Lung Water Accumulation During Exercise

The goal of this non-significant risk medical device study is to test the initial technical feasibility of a 0.55T MRI in healthy volunteers by technical optimization of scanner protocols, and to test accuracy of standard MR measurements in adult patients with known stable disease. Exercise-induced lung water is a key feature in heart failure. Dynamic quantitative lung water imaging during exercise stress is therefore of interest to unmask latent heart failure. We developed a novel magnetic resonance imaging (MRI) method using a proton density weighted ultrashort echo time MRI sequence along with a time-resolved and respiratory motion compensated image reconstruction for exercise lung water imaging. We validated our method in a porcine model of dynamic lung water accumulation (n=5), and tested its feasibility for use in humans using 12 healthy subjects, 1 patient with heart failure with preserved ejection fraction, and 1 patient with heart failure with reduced ejection fraction. Data suggest the novel imaging method is capable of dynamically imaging lung water accumulation during exercise stress. Reference 1 (PMID: 37288601) contains the main results for this study.

Study phs003346

Genetic Basis of Early Onset Bicuspid Aortic Valve Disease

The study protocol was approved by the Committee for the Protection of Human Subjects at the University of Texas Health Science Center at Houston (HSC-MS-11-0185). Study recruitment began on July 1, 2017, and concluded on March 30, 2022. After written informed consent, we enrolled probands with early onset BAV disease (EBAV), which we defined as individuals with BAV who were under the age of 30 at the time of first clinical event. Clinical events were defined as aortic replacement, aortic valve surgery, aortic dissection, moderate or severe aortic stenosis or aortic regurgitation, large aneurysm (Z > 4.5), or intervention for BAV-related conditions. Those with hypoplastic left heart, known genetic mutations, genetic syndromes, or complex congenital heart disease were excluded. Samples were collected and genotyped as previously reported. For comparison, we analyzed a cohort of older individuals of European ancestry with sporadic BAV disease selected from the International BAV Consortium (BAVGWAS). Phenotypes were derived from record review with confirmation of image data whenever possible [25-26]. The computational pipeline for CNV analysis of Illumina single nucleotide polymorphism (SNP) array data included three independent CNV detection algorithms.GenomeStudio was used to exclude samples with indeterminate sex or more than 5% missing genotypes, and single nucleotide polymorphisms (SNPs) with GenTrain = 0. Principal component analysis was used to remove outliers that did not cluster with European ancestry. Prior to CNV analysis, each dataset was trimmed by selecting a common set of 650,000 SNPs that were genotyped on each of the microarrays used in this study.Three independent algorithms (PennCNV, cnvPartition, and QuantiSNP) were used to generate CNV calls and sample-level quality statistics from SNP intensity data. PennCNV and QuantiSNP were run on Unix clusters and cnvPartition data were exported from GenomeStudio. The analysis was run using default configurations. PennCNV was used to generate QC data and remove CNV calls that intersect with polymorphic genomic regions. Samples that met any of the following criteria were excluded, standard deviation of the LogR ratio (obtained from PennCNV) > 0.35 or number of CNVs > 2 standard deviations above the mean for each data set. CNV calls less than 20 Kilobases in length and/or spanned by fewer than 6 probes were excluded. The overlap function for rare CNVs in PLINK was used to construct CNV regions (CNVRs) after adjacent regions were merged using PennCNV. LogR ratio (LRR) and B allele frequency (BAF) data at CNVRs and calls of interest were visualized in GenomeStudio for validation. For segregation analysis, GenomeStudio was used to determine the presence of CNVs in relatives.A total of 22,014 unselected control Illumina Genotypes obtained from the Database of Genotypes and Phenotypes were analyzed using identical methods (S1 Table). The Wisconsin Longitudinal Study (WLS) includes data on a cohort of 10,300 individuals who graduated from Wisconsin high schools in 1957. The Health and Retirement Study (HRS) includes data on 37,000 individuals aged 50 above from 23,000 households across the United States. Principal component analysis was used to select European ancestry genotypes from these datasets for analysis. Datasets were paired for case-control analysis based on the concordance of log-transformed sample-level quality control statistics (number of CNV calls and standard deviation of logR ratios). Chi-squared or Fisher exact tests were used to compare CNV frequencies in cases and controls.Rare CNV functions in PLINK (v1.7) were used to perform permutation-based burden tests or gene set-based enrichment tests. Case control burden tests were restricted to CNVs that were longer than 110 Kb and less than 0.1% in frequency. CNV overlap functions in PLINK were used to identify rare CNVs that intersect between datasets or involve specific BAV or CHD genes . The list of candidate genes included 190 CHD genes that have strong cumulative evidence to cause BAV or related congenital malformations from human or animal model data. Genome Reference Consortium Human Build 37 was used for CNV annotation [34].

Study phs003705

EWS-WT1 Fusion Isoforms Establish Oncogenic Programs and Therapeutic Vulnerabilities in Desmoplastic Small Round Cell Tumors

Desmoplastic Small Round Cell Tumor (DSRCT) is an aggressive mesenchymal tumor driven by fusions between the disordered domain of the Ewing sarcoma RNA binding protein 1 (EWSR1) and the developmental transcription factor Wilms tumor 1 (WT1). We used genome-wide chromatin profiling to identify EWSR1-WT1-dependent gene regulatory networks and target genes. Our studies show that EWS-WT1 is a powerful activator of distal regulatory elements and controls an oncogenic gene expression program that characterizes primary DSRCTs. ChIP-seq profiles for histone marks in primary DSRCT samples are available through dbGaP.

Study phs003682

Transcriptome sequencing across a prostate cancer cohort identifies PCAT-1, an unannotated lincRNA implicated in disease progression

Noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease and to reveal uncharacterized aspects of tumor biology. Here we discover 121 unannotated prostate cancer-associated ncRNA transcripts (PCATs) by ab initio assembly of high-throughput sequencing of polyA+ RNA (RNA-Seq) from a cohort of 102 prostate tissues and cells lines. We characterized one ncRNA, PCAT-1, as a prostate-specific regulator of cell proliferation and show that it is a target of the polycomb repressive complex 2 (PRC2). We further found that patterns of PCAT-1 and PRC2 expression stratified patient tissues into molecular subtypes distinguished by expression signatures of PCAT-1-repressed target genes. Taken together, our findings suggest that PCAT-1 is a transcriptional repressor implicated in a subset of prostate cancer patients. These findings establish the utility of RNA-Seq to identify disease-associated ncRNAs that may improve the stratification of cancer subtypes.

Study phs000443

NHLBI TOPMed: SubPopulations and InteRmediate Outcome Measures In COPD Study (SPIROMICS)

Subpopulations and Intermediate Outcome Measures in COPD Study Description Subpopulations and intermediate outcome measures in COPD study (SPIROMICS) supports the prospective collection and analysis of phenotypic, biomarker, genetic, genomic, and clinical data from subjects with COPD for the purpose of identifying subpopulations and intermediate outcome measures. It is funded by the National Heart, Lung, and Blood Institute and is coordinated by the University of North Carolina at Chapel Hill. Molecular fingerprinting and extensive subject phenotyping will be performed to identify disease subpopulations and to identify and validate surrogate markers of disease severity, which will be useful as intermediate outcome measures for future clinical trials. Secondary aims are to clarify the natural history of COPD, to develop bioinformatic resources that will enable the utilization and sharing of data in studies of COPD and related diseases, and to create a collection of clinical, biomarker, radiographic, and genetic data that can be used by external investigators for other studies of COPD. Participating Institutions: Research participants for SPIROMICS will be enrolled, phenotyped, and followed at twelve SPIROMICS Clinical Centers: Columbia University, Temple University, Johns Hopkins University, Wake Forest University, University of Michigan, University of Illinois at Chicago, University of Iowa, University of Utah, National Jewish Health, University of California at San Francisco, and University of California at Los Angeles. The University of North Carolina at Chapel Hill serves as the Genomics and Informatics Center. The Radiology Reading Center is based at the University of Iowa. The PFT Reading Center is based at the University of California at Los Angeles. Study Design: SPIROMICS is a prospective cohort study that enrolled approximately 2,981 participants at twelve clinical centers over five years. Participants are distributed across four enrollment strata (i.e., Never-smokers, Smokers without COPD, Mild/Moderate COPD, and Severe COPD). Study Visits: Participants have up to four in-person visits (Baseline and Annual Clinic Visits at years 1, 2, 3 after Baseline). Study questionnaires and spirometry are completed at all main study visits. Blood and urine samples are collected at visits 1, 2, and 4. Sputum samples are collected at Visit 1. The CT scans are completed and Baseline and Visit 2. Participants also receive quarterly follow-up calls to assess health status and determine if an exacerbation has occurred. Substudies Bronchoscopy and Immunophenotyping Substudy The Bronchoscopy Substudy will enroll 50 subjects per site, for a total of 300 participants. These participants will be recruited across all four study strata. This substudy includes two study visits. During the first visit, sputum samples are collected for Immunophenotyping analysis at the Immunophenotyping Core Lab based at the University of Michigan. Participants then return for a second visit during which samples are collected via bronchoscopy, including bronchoalveolar lavage, epithelial brushings and bronchial biopsies. Immunophenotyping analysis is also conducted on BAL and blood collected during the bronchoscopy study visit. Repeatability Substudy The entire baseline clinic visit was repeated on 98 volunteers to determine reliability of measurement procedures. All baseline study-related procedures and questionnaires, including the CT scan, were re-administered and new samples of blood, urine, saliva, and sputum were collected. Field center staff processed these biospecimen samples according to the same protocol. Exacerbation Substudy The Exacerbation Substudy is a prospective, longitudinal observational study of up to 400 participants, which will allow the assessment of participant-driven health care utilization (HCU) events and symptom-defined exacerbation events over time. HCU-driven events are defined by change in medical treatment in response to a perceived COPD Exacerbation. Symptom-based events will be defined by using EXACT-PRO (EXacerbations of Chronic Pulmonary Disease Tool - Patient Reported Outcome), a daily symptom diary developed to measure the frequency, severity, and duration of exacerbations in clinical trials. All participants are provided with a PDA on which to complete the diary. Participants reporting a possible COPD exacerbation will be brought into the study clinic for a study visit to collect biological specimens and questionnaire data. The overall objectives of the Exacerbation Substudy are to: Obtain clinical data and specimens on SPIROMICS participants before and during an acute COPD exacerbation defined by Health care utilization triggered by the subject, or Symptomatic change (triggered by an EXACT defined threshold) Describe symptomatic changes occurring around HCU and symptom-defined (EXACT) events and their relationship to clinical and specimen data, Characterize the non-exacerbation "stable" state in COPD using the EXACT, including: Symptom variability (EXACT), Clinical data and specimen parameters during a stable state (baseline), The relationship between clinical and specimen data and symptom severity and variability. Explore the characteristics of stable patients, defined as those who do not experience HCU or symptom-defined (EXACT) events during the sub-study period, using baseline clinical data and specimens, and compare these characteristics with patients who experience HCU and/or symptom-defined events. Examine the relationship between HCU and symptom-defined exacerbation frequency during the first one-year period of follow-up for exacerbations and health outcomes, including 12-month change in lung function and COPD health status, and longer-term morbidity and mortality, with the latter derived from long-term data from the larger SPIROMICS study.

Study phs001927

Transcriptional Profiling of Macrophages In Situ in Metastatic Melanoma Reveals Localization-Dependent Phenotypes and Function

Therapeutic interventions modulating immune function at the tumor site could improve outcomes in cancer. Here we analyzed metastatic melanoma, a tumor type highly responsive to T-cell based therapies, and found that the tumor-infiltrating T cells are localized closely to CD14+ monocytes/macrophages rather than to melanoma cells. Using novel immunofluorescence-guided laser capture micro-dissection, we analyzed transcriptomes of CD3+ T cells, CD14+ monocytes/macrophages and melanoma cells in non-dissociated tissue. CD14+ cells localized in tumors beds displayed specific transcriptional signatures distinct from CD14+ cells that were localized in tumor stroma.The 8 distinct metastatic melanoma samples are from the Cooperative Human Tissue Network (CHTN). 64 RNA-seq files are available from 16 intraCD14+, 16 stromaCD14+, 16 cancer cells, 8 intraCD3+ and 8 stromaCD3+ cells. 2 LCM harvests were completed for the CD14+ and cancer cells on two sections of the samples. One harvest was done for the CD3+ samples.The processed data (TPM/raw counts tables) are available on GEO under GSE180124. 

Study phs002564

Genomic Profiling of Sequentially Acquired Metastatic Sites from an "Exceptional Responder" Lung Adenocarcinoma Patient Reveals Extensive Genomic Heterogeneity and Novel Somatic Variants

We employed next-generation sequencing to identify somatic alterations in multiple metastatic sites from an "exceptional responder" lung adenocarcinoma patient during his seven year course of ERBB2-directed therapies. The degree of heterogeneity was unprecedented, with ~1% similarity between somatic alterations of the lung and lymph nodes. One novel translocation, PLAG1-ACTA2, present in both sites, up-regulated ACTA2 expression. ERBB2, the predominant driver oncogene, was amplified in both sites, more pronounced in the lung, and harbored an L869R mutation in the lymph node. Functional studies demonstrated increased proliferation, migration, metastasis, and resistance to ERBB2-directed therapy due to L869R mutation and increased migration due to ACTA2 overexpression. Within the lung, a nonfunctional CDK12, due to a novel G879V mutation, correlated with down-regulation of DNA damage response genes, causing genomic instability, and sensitivity to chemotherapy. We propose a model whereby a sub-clone metastasized early from the primary site and evolved independently in lymph nodes.

Study phs001159

WES in HCCOs with varying Doxorubicin resistance

We aimed to identify somatic mutations that could explain the resistance to Doxorubicin. Therefor, we purified Exomes from liver biopsies and HCC organoids using Agilent SureSelectXT (clinical research exome, HS2 V8, or Human all Exon V6 Cosmic) and performed WES. Matched non-tumoral liver biopsies served as the control for germline mutations. 12 of these samples were previously reported. Sequence reads were aligned to the reference human genome GRCh38 using Burrows-Wheeler Aligner (BWA, v0.7.17). Duplicate removal, base quality adjustment, mutation calling (Mutect2, intervals: SureSelectXT_HS_V8_S33266340, germline-recourse: gnomad.exomes.r2.1.1.sites.liftover_grch38, Panel-of-Normal: 1000g_pon.hg38), filtering of Mutect calls, and functional annotation (Funcotator, funcotator_dataSources.v1.7.20200521g) were performed using the Genome Analysis Toolkit (GATK, v4.4) according to the Broad Institute best practices in Somatic short variant discovery workflows. Mutations with a functional annotation of Deletion, Insertion, Non-stop, Splice-Site, Start-Codon and Missense mutations were kept. A list of 43 commonly mutated genes in HCC was analyzed in the HCC organoids [TCGA, Comprehensive and Integrative Genomic Characterization of Hepatocellular Carcinoma. Cell, 2017. 169(7): p. 1327-1341.e23].

Study EGAS50000000043

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

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