CD47-Dependent Regulation of Immune Checkpoint Gene Expression and MYCN mRNA Splicing in Murine CD8 and Jurkat T Cells.

Elevated expression of CD47 in some cancers is associated with poor survival related to its function as an innate immune checkpoint when expressed on tumor cells. In contrast, elevated CD47 expression in cutaneous melanomas is associated with improved survival. Previous studies implicated protective functions of CD47 expressed by immune cells in the melanoma tumor microenvironment. RNA sequencing analysis of responses induced by CD3 and CD28 engagement on wild type and CD47-deficient Jurkat T lymphoblast cells identified additional regulators of T cell function that were also CD47-dependent in mouse CD8 T cells. MYCN mRNA expression was upregulated in CD47-deficient cells but downregulated in CD47-deficient cells following activation. CD47 also regulated alternative splicing that produces two N-MYC isoforms. The CD47 ligand thrombospondin-1 inhibited expression of these MYCN mRNA isoforms, as well as induction of the oncogenic decoy MYCN opposite strand (MYCNOS) RNA during T cell activation. Analysis of mRNA expression data for melanomas in The Cancer Genome Atlas identified a significant coexpression of MYCN with CD47 and known regulators of CD8 T cell function. Thrombospondin-1 inhibited the induction of TIGIT, CD40LG, and MCL1 mRNAs following T cell activation in vitro. Increased mRNA expression of these T cell transcripts and MYCN in melanomas was associated with improved overall survival.

Bambino: a variant detector and alignment viewer for next-generation sequencing data in the SAM/BAM format.

SUMMARY: Bambino is a variant detector and graphical alignment viewer for next-generation sequencing data in the SAM/BAM format, which is capable of pooling data from multiple source files. The variant detector takes advantage of SAM-specific annotations, and produces detailed output suitable for genotyping and identification of somatic mutations. The assembly viewer can display reads in the context of either a user-provided or automatically generated reference sequence, retrieve genome annotation features from a UCSC genome annotation database, display histograms of non-reference allele frequencies, and predict protein-coding changes caused by SNPs. AVAILABILITY: Bambino is written in platform-independent Java and available from https://cgwb.nci.nih.gov/goldenPath/bamview/documentation/index.html, along with documentation and example data. Bambino may be launched online via Java Web Start or downloaded and run locally.

Genetic variations at loci involved in the immune response are risk factors for hepatocellular carcinoma.

UNLABELLED: Primary liver cancer is the third most common cause of cancer-related death worldwide, with a rising incidence in Western countries. Little is known about the genetic etiology of this disease. To identify genetic factors associated with hepatocellular carcinoma (HCC) and liver cirrhosis (LC), we conducted a comprehensive, genome-wide variation analysis in a population of unrelated Asian individuals. Copy number variation (CNV) and single nucleotide polymorphisms (SNPs) were assayed in peripheral blood with the high-density Affymetrix SNP6.0 microarray platform. We used a two-stage discovery and replication design to control for overfitting and to validate observed results. We identified a strong association with CNV at the T-cell receptor gamma and alpha loci (P < 1 × 10(-15)) in HCC cases when contrasted with controls. This variation appears to be somatic in origin, reflecting differences between T-cell receptor processing in lymphocytes from individuals with liver disease and healthy individuals that is not attributable to chronic hepatitis virus infection. Analysis of constitutional variation identified three susceptibility loci including the class II MHC complex, whose protein products present antigen to T-cell receptors and mediate immune surveillance. Statistical analysis of biologic networks identified variation in the "antigen presentation and processing" pathway as being highly significantly associated with HCC (P = 1 × 10(-11)). SNP analysis identified two variants whose allele frequencies differ significantly between HCC and LC. One of these (P = 1.74 × 10(-12)) lies in the PTEN homolog TPTE2. CONCLUSION: Combined analysis of CNV, individual SNPs, and pathways suggest that HCC susceptibility is mediated by germline factors affecting the immune response and differences in T-cell receptor processing.

Alview: Portable Software for Viewing Sequence Reads in BAM Formatted Files.

The name Alview is a contraction of the term Alignment Viewer. Alview is a compiled to native architecture software tool for visualizing the alignment of sequencing data. Inputs are files of short-read sequences aligned to a reference genome in the SAM/BAM format and files containing reference genome data. Outputs are visualizations of these aligned short reads. Alview is written in portable C with optional graphical user interface (GUI) code written in C, C++, and Objective-C. The application can run in three different ways: as a web server, as a command line tool, or as a native, GUI program. Alview is compatible with Microsoft Windows, Linux, and Apple OS X. It is available as a web demo at https://cgwb.nci.nih.gov/cgi-bin/alview. The source code and Windows/Mac/Linux executables are available via https://github.com/NCIP/alview.

Genome-wide transcriptional sequencing identifies novel mutations in metabolic genes in human hepatocellular carcinoma.

We report on next-generation transcriptome sequencing results of three human hepatocellular carcinoma tumor/tumor-adjacent pairs. This analysis robustly examined ∼12,000 genes for both expression differences and molecular alterations. We observed 4,513 and 1,182 genes demonstrating 2-fold or greater increase or decrease in expression relative to their normal, respectively. Network analysis of expression data identified the Aurora B signaling, FOXM1 transcription factor network and Wnt signaling pathways pairs being altered in HCC. We validated as differential gene expression findings in a large data set containing of 434 liver normal/tumor sample pairs. In addition to known driver mutations in TP53 and CTNNB1, our mutation analysis identified non-synonymous mutations in genes implicated in metabolic diseases, i.e. diabetes and obesity: IRS1, HMGCS1, ATP8B1, PRMT6 and CLU, suggesting a common molecular etiology for HCC of alternative pathogenic origin.

Genetic alterations activating kinase and cytokine receptor signaling in high-risk acute lymphoblastic leukemia.

Genomic profiling has identified a subtype of high-risk B-progenitor acute lymphoblastic leukemia (B-ALL) with alteration of IKZF1, a gene expression profile similar to BCR-ABL1-positive ALL and poor outcome (Ph-like ALL). The genetic alterations that activate kinase signaling in Ph-like ALL are poorly understood. We performed transcriptome and whole genome sequencing on 15 cases of Ph-like ALL and identified rearrangements involving ABL1, JAK2, PDGFRB, CRLF2, and EPOR, activating mutations of IL7R and FLT3, and deletion of SH2B3, which encodes the JAK2-negative regulator LNK. Importantly, several of these alterations induce transformation that is attenuated with tyrosine kinase inhibitors, suggesting the treatment outcome of these patients may be improved with targeted therapy.

Systematic analysis of genetic alterations in tumors using Cancer Genome WorkBench (CGWB).

Systematic investigations of genetic changes in tumors are expected to lead to greatly improved understanding of cancer etiology. To meet the analytical challenges presented by such studies, we developed the Cancer Genome WorkBench (http://cgwb.nci.nih.gov), the first computational platform to integrate clinical tumor mutation profiles with the reference human genome. A novel heuristic algorithm, IndelDetector, was developed to automatically identify insertion/deletion (indel) polymorphisms as well as indel somatic mutations with high sensitivity and accuracy. It was incorporated into an automated pipeline that detects genetic alterations and annotates their effects on protein coding and 3D structure. The ability of the system to facilitate identifying genetic alterations is illustrated in three projects with publicly accessible data. Mutagenesis in tumor DNA replication leading to complex genetic changes in the EGFR kinase domain is suggested by a novel deletion-insertion combination observed in paired tumor-normal lung cancer resequencing data. Automated analysis of 152 genes resequenced by the SeattleSNPs group was able to identify 91% of the 1251 indel polymorphisms discovered by SeattleSNPs. In addition, our system discovered 518 novel indels in this data set, 451 of which were found to be valid by manual inspection of sequence traces. Our experience demonstrates that CGWB not only greatly improves the productivity and the accuracy of mutation identification, but also, through its data integration and visualization capabilities, facilitates identification of underlying genetic etiology.

Detecting false expression signals in high-density oligonucleotide arrays by an in silico approach.

High-density oligonucleotide arrays have become a popular assay for concurrent measurement of mRNA expression at the genome scale. Much effort has been devoted to the development of statistical analysis tools aimed at reducing experimental noise and normalizing experimental variation in gene expression analysis. However, these investigations do not detect or catalog systematic problems associated with specific oligonucleotide probes. Here, we present an investigation of problematic probes that yield consistent but inaccurate signals across multiple experiments. By evaluating data integrity among gene, probe sequence, and genomic structure we identified a total of 20,696 (10.5%) nonspecific probes that could cross-hybridize to multiple genes and a total of 18,363 (9.3%) probes that miss the target transcript sequences on the Affymetrix GeneChip U95A/Av2 array. The numbers of nonspecific and mistargeted probes on the U133A array are 29,405 (12.1%) and 19,717 (8.0%), respectively. The poor performance of the mistargeted probes was confirmed in two GeneChip experiments, in which these probes showed a 20-30% decrease in detecting present signals compared with normal probes. Comparison of qualitative expression signals obtained from SAGE and EST data with those from GeneChip arrays showed that the consistency of the two platforms is 30% lower in problematic probes than in normal probes. A Web application was developed to apply our results for improving the accuracy of expression analysis.

A high-resolution multistrain haplotype analysis of laboratory mouse genome reveals three distinctive genetic variation patterns.

Understanding of the structure and the origin of genetic variation patterns in the laboratory inbred mouse provides insight into the utility of the mouse model for studying human complex diseases and strategies for disease gene mapping. In order to address this issue, we have constructed a multistrain, high-resolution haplotype map for the 99-Mb mouse Chromosome 16 using approximately 70,000 single nucleotide polymorphism (SNP) markers derived from whole-genome shotgun sequencing of five laboratory inbred strains. We discovered that large polymorphic blocks (i.e., regions where only two haplotypes, thus one SNP conformation, are found in the five strains), large monomorphic blocks (i.e., regions where the five strains share the same haplotype), and fragmented blocks (i.e., regions of greater complexity not resembling at all the first two categories) span 50%, 18%, and 32% of the chromosome, respectively. The haplotype map has 98% accuracy in predicting mouse genotypes in two other studies. Its predictions are also confirmed by experimental results obtained from resequencing of 40-kb genomic sequences at 21 distinct genomic loci in 13 laboratory inbred strains and 12 wild-derived strains. We demonstrate that historic recombination, intra-subspecies variations and inter-subspecies variations have all contributed to the formation of the three distinctive genetic signatures. The results suggest that the controlled complexity of the laboratory inbred strains may provide a means for uncovering the biological factors that have shaped genetic variation patterns.