A proteogenomic surfaceome study identifies DLK1 as an immunotherapeutic target in neuroblastoma.

Cancer immunotherapies produce remarkable results in B cell malignancies; however, optimal cell surface targets for many solid cancers remain elusive. Here, we present an integrative proteomic, transcriptomic, and epigenomic analysis of tumor and normal tissues to identify biologically relevant cell surface immunotherapeutic targets for neuroblastoma, an often-fatal childhood cancer. Proteogenomic analyses reveal sixty high-confidence candidate immunotherapeutic targets, and we prioritize delta-like canonical notch ligand 1 (DLK1) for further study.

Characterization of aberrant splicing in pediatric central nervous system tumors reveals as a candidate oncogenic dependency.

Pediatric brain cancer is the leading cause of disease-related mortality in children, and many aggressive tumors still lack effective treatment strategies. We characterized aberrant alternative splicing across pediatric brain tumors, identifying pediatric high-grade gliomas (HGGs) among the most heterogeneous. Annotating these events with UniProt, we identified 11,940 splice events in 5,368 genes leading to potential protein function changes.

AutoGVP: a dockerized workflow integrating ClinVar and InterVar germline sequence variant classification.

Summary: With the increasing rates of exome and whole genome sequencing, the ability to classify large sets of germline sequencing variants using up-to-date American College of Medical Genetics-Association for Molecular Pathology (ACMG-AMP) criteria is crucial. Here, we present Automated Germline Variant Pathogenicity (AutoGVP), a tool that integrates germline variant pathogenicity annotations from ClinVar and sequence variant classifications from a modified version of InterVar (PVS1 strength adjustments, removal of PP5/BP6). This tool facilitates large-scale, clinically focused classification of germline sequence variants in a research setting.

A proteogenomic surfaceome study identifies DLK1 as an immunotherapeutic target in neuroblastoma.

Unlabelled: Cancer immunotherapies have produced remarkable results in B-cell malignancies; however, optimal cell surface targets for many solid cancers remain elusive. Here, we present an integrative proteomic, transcriptomic, and epigenomic analysis of tumor specimens along with normal tissues to identify biologically relevant cell surface proteins that can serve as immunotherapeutic targets for neuroblastoma, an often-fatal childhood cancer of the developing nervous system. We apply this approach to human-derived cell lines (N=9) and cell/patient-derived xenograft (N=12) models of neuroblastoma.

AutoGVP: a dockerized workflow integrating ClinVar and InterVar germline sequence variant classification.

With the increasing rates of exome and whole genome sequencing, the ability to classify large sets of germline sequencing variants using up-to-date American College of Medical Genetics - Association for Molecular Pathology (ACMG-AMP) criteria is crucial. Here, we present Automated Germline Variant Pathogenicity (AutoGVP), a tool that integrates germline variant pathogenicity annotations from ClinVar and sequence variant classifications from a modified version of InterVar (PVS1 strength adjustments, removal of PP5/BP6). This tool facilitates large-scale, clinically-focused classification of germline sequence variants in a research setting.

OpenPBTA: The Open Pediatric Brain Tumor Atlas.

Pediatric brain and spinal cancers are collectively the leading disease-related cause of death in children; thus, we urgently need curative therapeutic strategies for these tumors. To accelerate such discoveries, the Children's Brain Tumor Network (CBTN) and Pacific Pediatric Neuro-Oncology Consortium (PNOC) created a systematic process for tumor biobanking, model generation, and sequencing with immediate access to harmonized data. We leverage these data to establish OpenPBTA, an open collaborative project with over 40 scalable analysis modules that genomically characterize 1,074 pediatric brain tumors.

The children's brain tumor network (CBTN) - Accelerating research in pediatric central nervous system tumors through collaboration and open science.

Pediatric brain tumors are the leading cause of cancer-related death in children in the United States and contribute a disproportionate number of potential years of life lost compared to adult cancers. Moreover, survivors frequently suffer long-term side effects, including secondary cancers. The Children's Brain Tumor Network (CBTN) is a multi-institutional international clinical research consortium created to advance therapeutic development through the collection and rapid distribution of biospecimens and data via open-science research platforms for real-time access and use by the global research community.

annoFuse: an R Package to annotate, prioritize, and interactively explore putative oncogenic RNA fusions.

Background: Gene fusion events are significant sources of somatic variation across adult and pediatric cancers and are some of the most clinically-effective therapeutic targets, yet low consensus of RNA-Seq fusion prediction algorithms makes therapeutic prioritization difficult. In addition, events such as polymerase read-throughs, mis-mapping due to gene homology, and fusions occurring in healthy normal tissue require informed filtering, making it difficult for researchers and clinicians to rapidly discern gene fusions that might be true underlying oncogenic drivers of a tumor and in some cases, appropriate targets for therapy.

Results: We developed annoFuse, an R package, and shinyFuse, a companion web application, to annotate, prioritize, and explore biologically-relevant expressed gene fusions, downstream of fusion calling.

Integrated Proteogenomic Characterization across Major Histological Types of Pediatric Brain Cancer.

We report a comprehensive proteogenomics analysis, including whole-genome sequencing, RNA sequencing, and proteomics and phosphoproteomics profiling, of 218 tumors across 7 histological types of childhood brain cancer: low-grade glioma (n = 93), ependymoma (32), high-grade glioma (25), medulloblastoma (22), ganglioglioma (18), craniopharyngioma (16), and atypical teratoid rhabdoid tumor (12). Proteomics data identify common biological themes that span histological boundaries, suggesting that treatments used for one histological type may be applied effectively to other tumors sharing similar proteomics features. Immune landscape characterization reveals diverse tumor microenvironments across and within diagnoses.

Detection of circulating extracellular mRNAs by modified small-RNA-sequencing analysis.

Extracellular mRNAs (ex-mRNAs) potentially supersede extracellular miRNAs (ex-miRNAs) and other RNA classes as biomarkers. We performed conventional small-RNA-sequencing (sRNA-seq) and sRNA-seq with T4 polynucleotide kinase (PNK) end-treatment of total exRNA isolated from serum and platelet-poor EDTA, ACD, and heparin plasma to study the effect on ex-mRNA capture. Compared to conventional sRNA-seq PNK-treatment increased the detection of informative ex-mRNAs reads up to 50-fold.

Author Correction: Integrated molecular subtyping defines a curable oligometastatic state in colorectal liver metastasis.

In the originally published version of this Article, the affiliation details for Kevin P. White inadvertently omitted 'Tempus Labs, Chicago, IL, 60654, USA'. This has now been corrected in both the PDF and HTML versions of the Article.

Human Organoids Share Structural and Genetic Features with Primary Pancreatic Adenocarcinoma Tumors.

Patient-derived pancreatic ductal adenocarcinoma (PDAC) organoid systems show great promise for understanding the biological underpinnings of disease and advancing therapeutic precision medicine. Despite the increased use of organoids, the fidelity of molecular features, genetic heterogeneity, and drug response to the tumor of origin remain important unanswered questions limiting their utility. To address this gap in knowledge, primary tumor- and patient-derived xenograft (PDX)-derived organoids, and 2D cultures for in-depth genomic and histopathologic comparisons with the primary tumor were created.

Integrated molecular subtyping defines a curable oligometastatic state in colorectal liver metastasis.

The oligometastasis hypothesis suggests a spectrum of metastatic virulence where some metastases are limited in extent and curable with focal therapies. A subset of patients with metastatic colorectal cancer achieves prolonged survival after resection of liver metastases consistent with oligometastasis. Here we define three robust subtypes of de novo colorectal liver metastasis through integrative molecular analysis.

Characterizing Expression and Processing of Precursor and Mature Human tRNAs by Hydro-tRNAseq and PAR-CLIP.

The participation of tRNAs in fundamental aspects of biology and disease necessitates an accurate, experimentally confirmed annotation of tRNA genes and curation of tRNA sequences. This has been challenging because RNA secondary structure, nucleotide modifications, and tRNA gene multiplicity complicate sequencing and mapping efforts. To address these issues, we developed hydro-tRNAseq, a method based on partial alkaline RNA hydrolysis that generates fragments amenable for sequencing.

Comparative RNA-sequencing analysis of myocardial and circulating small RNAs in human heart failure and their utility as biomarkers.

Heart failure (HF) is associated with high morbidity and mortality and its incidence is increasing worldwide. MicroRNAs (miRNAs) are potential markers and targets for diagnostic and therapeutic applications, respectively. We determined myocardial and circulating miRNA abundance and its changes in patients with stable and end-stage HF before and at different time points after mechanical unloading by a left ventricular assist device (LVAD) by small RNA sequencing.

Human CLP1 mutations alter tRNA biogenesis, affecting both peripheral and central nervous system function.

CLP1 is a RNA kinase involved in tRNA splicing. Recently, CLP1 kinase-dead mice were shown to display a neuromuscular disorder with loss of motor neurons and muscle paralysis. Human genome analyses now identified a CLP1 homozygous missense mutation (p.

Identification of distinct miRNA target regulation between breast cancer molecular subtypes using AGO2-PAR-CLIP and patient datasets.

Background: Various microRNAs (miRNAs) are up- or downregulated in tumors. However, the repression of cognate miRNA targets responsible for the phenotypic effects of this dysregulation in patients remains largely unexplored. To define miRNA targets and associated pathways, together with their relationship to outcome in breast cancer, we integrated patient-paired miRNA-mRNA expression data with a set of validated miRNA targets and pathway inference.

Mammalian miRNA curation through next-generation sequencing.

Characteristic small RNA biogenesis processing patterns are used for the discovery of novel microRNAs (miRNAs) from next-generation sequencing data. Here, we highlight and discuss key criteria for mammalian - specifically human - miRNA database curation based on small RNA sequencing data. Sequence reads obtained from small RNA cDNA libraries are aligned to reference genomic regions, and miRNA genes are revealed by their distinct read length and bimodal read frequency distribution, the predicted secondary structure of the deduced miRNA stem-loop precursor molecule, and, to a lesser degree, based on evolutionary conservation of small RNAs from other vertebrates.

Multicolor microRNA FISH effectively differentiates tumor types.

MicroRNAs (miRNAs) are excellent tumor biomarkers because of their cell-type specificity and abundance. However, many miRNA detection methods, such as real-time PCR, obliterate valuable visuospatial information in tissue samples. To enable miRNA visualization in formalin-fixed paraffin-embedded (FFPE) tissues, we developed multicolor miRNA FISH.

Identification of mRNAs bound and regulated by human LIN28 proteins and molecular requirements for RNA recognition.

Human LIN28A and LIN28B are RNA-binding proteins (RBPs) conserved in animals with important roles during development and stem cell reprogramming. We used Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in HEK293 cells and identified a largely overlapping set of ∼3000 mRNAs at ∼9500 sites located in the 3' UTR and CDS. In vitro and in vivo, LIN28 preferentially bound single-stranded RNA containing a uridine-rich element and one or more flanking guanosines and appeared to be able to disrupt base-pairing to access these elements when embedded in predicted secondary structure.

Comprehensive profiling of circulating microRNA via small RNA sequencing of cDNA libraries reveals biomarker potential and limitations.

We profiled microRNAs (miRNAs) in cell-free serum and plasma samples from human volunteers using deep sequencing of barcoded small RNA cDNA libraries. By introducing calibrator synthetic oligonucleotides during library preparation, we were able to calculate the total as well as specific concentrations of circulating miRNA. Studying trios of samples from newborn babies and their parents we detected placental-specific miRNA in both maternal and newborn circulations and quantitated the relative contribution of placental miRNAs to the circulating pool of miRNAs.

Genome-wide annotation and analysis of zebra finch microRNA repertoire reveal sex-biased expression.

Background: MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post-transcriptionally in a wide range of biological processes. The zebra finch (Taeniopygia guttata), an oscine songbird with characteristic learned vocal behavior, provides biologists a unique model system for studying vocal behavior, sexually dimorphic brain development and functions, and comparative genomics.

Results: We deep sequenced small RNA libraries made from the brain, heart, liver, and muscle tissues of adult male and female zebra finches.

Bioinformatic analysis of barcoded cDNA libraries for small RNA profiling by next-generation sequencing.

The characterization of post-transcriptional gene regulation by small regulatory RNAs of 20-30 nt length, particularly miRNAs and piRNAs, has become a major focus of research in recent years. A prerequisite for the characterization of small RNAs is their identification and quantification across different developmental stages, normal and diseased tissues, as well as model cell lines. Here we present a step-by-step protocol for the bioinformatic analysis of barcoded cDNA libraries for small RNA profiling generated by Illumina sequencing, thereby facilitating miRNA and other small RNA profiling of large sample collections.