Kyle Hernandez

Density of immunogenic antigens does not explain the presence or absence of the T-cell–inflamed tumor microenvironment in melanoma

Significance The T-cell–inflamed tumor microenvironment correlates with efficacy of immunotherapy. It is critical to understand whether non–T-cell–inflamed tumors lack antigens for T-cell recognition. In melanoma, no difference between inflamed and noninflamed tumors for multiple antigen classes was observed. Synthesized peptides corresponding to predicted HLA-A2 binding epitopes showed no differences between inflamed and noninflamed tumors. Extrapolation of a T-cell signature across The Cancer Genome Atlas showed no correlation between gene expression and mutational burden in any cancer type. These results indicate that lack of spontaneous immune infiltration in solid tumors is unlikely to be due to lack of antigens. Rather, transcriptional profiling suggests lack of Batf3-lineage dendritic cells. Our data suggest that strategies to restore T-cell entry into noninflamed tumors should be developed. Melanoma metastases can be categorized by gene expression for the presence of a T-cell–inflamed tumor microenvironment, which correlates with clinical efficacy of immunotherapies. T cells frequently recognize mutational antigens corresponding to nonsynonymous somatic mutations (NSSMs), and in some cases shared differentiation or cancer–testis antigens. Therapies are being pursued to trigger immune infiltration into non–T-cell–inflamed tumors in the hope of rendering them immunotherapy responsive. However, whether those tumors express antigens capable of T-cell recognition has not been explored. To address this question, 266 melanomas from The Cancer Genome Atlas (TCGA) were categorized by the presence or absence of a T-cell–inflamed gene signature. These two subsets were interrogated for cancer–testis, differentiation, and somatic mutational antigens. No statistically significant differences were observed, including density of NSSMs. Focusing on hypothetical HLA-A2+ binding scores, 707 peptides were synthesized, corresponding to all identified candidate neoepitopes. No differences were observed in measured HLA-A2 binding between inflamed and noninflamed cohorts. Twenty peptides were randomly selected from each cohort to evaluate priming and recognition by human CD8+ T cells in vitro with 25% of peptides confirmed to be immunogenic in both. A similar gene expression profile applied to all solid tumors of TCGA revealed no association between T-cell signature and NSSMs. Our results indicate that lack of spontaneous immune infiltration in solid tumors is unlikely due to lack of antigens. Strategies that improve T-cell infiltration into tumors may therefore be able to facilitate clinical response to immunotherapy once antigens become recognized.

Genomics of Ovarian Cancer Progression Reveals Diverse Metastatic Trajectories Including Intraepithelial Metastasis to the Fallopian Tube

Accumulating evidence has supported the fallopian tube rather than the ovary as the origin for high-grade serous ovarian cancer (HGSOC). To understand the relationship between putative precursor lesions and metastatic tumors, we performed whole-exome sequencing on specimens from eight HGSOC patient progression series consisting of serous tubal intraepithelial carcinomas (STIC), invasive fallopian tube lesions, invasive ovarian lesions, and omental metastases. Integration of copy number and somatic mutations revealed patient-specific patterns with similar mutational signatures and copy-number variation profiles across all anatomic sites, suggesting that genomic instability is an early event in HGSOC. Phylogenetic analyses supported STIC as precursor lesions in half of our patient cohort, but also identified STIC as metastases in 2 patients. Ex vivo assays revealed that HGSOC spheroids can implant in the fallopian tube epithelium and mimic STIC lesions. That STIC may represent metastases calls into question the assumption that STIC are always indicative of primary fallopian tube cancers. SIGNIFICANCE We find that the putative precursor lesions for HGSOC, STIC, possess most of the genomic aberrations present in advanced cancers. In addition, a proportion of STIC represent intraepithelial metastases to the fallopian tube rather than the origin of HGSOC. Cancer Discov; 6(12); 1342-51. ©2016 AACR.See related commentary by Swisher et al., p. 1309This article is highlighted in the In This Issue feature, p. 1293.

GR and ER Coactivation Alters the Expression of Differentiation Genes and Associates with Improved ER+ Breast Cancer Outcome.

In estrogen receptor (ER)–negative breast cancer, high tumor glucocorticoid receptor (GR) expression has been associated with a relatively poor outcome. In contrast, using a meta-analysis of several genomic datasets, here we find that tumor GR mRNA expression is associated with improved ER+ relapse-free survival (RFS; independently of progesterone receptor expression). To understand the mechanism by which GR expression is associated with a better ER+ breast cancer outcome, the global effect of GR-mediated transcriptional activation in ER+ breast cancer cells was studied. Analysis of GR chromatin immunoprecipitation followed by high-throughput sequencing in ER+/GR+ MCF-7 cells revealed that upon coactivation of GR and ER, GR chromatin association became enriched at proximal promoter regions. Furthermore, following ER activation, increased GR chromatin association was observed at ER, FOXO, and AP1 response elements. In addition, ER associated with GR response elements, suggesting that ER and GR interact in a complex. Coactivation of GR and ER resulted in increased expression (relative to ER activation alone) of transcripts that encode proteins promoting cellular differentiation (e.g., KDM4B, VDR) and inhibiting the Wnt signaling pathway (IGFBP4). Finally, expression of these individual prodifferentiation genes was associated with significantly improved RFS in ER+ breast cancer patients. Together, these data suggest that the coexpression and subsequent activity of tumor cell GR and ER contribute to the less aggressive natural history of early-stage breast cancer by coordinating the altered expression of genes favoring differentiation. Implications: The interaction between ER and GR activity highlights the importance of context-dependent nuclear receptor function in cancer. Mol Cancer Res; 14(8); 707–19. ©2016 AACR.

Glucocorticoid receptor ChIP-sequencing of subcutaneous fat reveals modulation of inflammatory pathways.

To identify glucocorticoid receptor (GR)‐associated chromatin sequences and target genes in primary human abdominal subcutaneous fat.

Understanding the genetic architecture of complex traits using the function-valued approach.

The genetic architecture of a phenotype refers to its genetic basis, including the relevant genes, their allelic effects, and their interactions among loci and environments. While much is known about the genetic architecture of some traits like flowering time (e.g. Buckler et al., 2009), unraveling the genetic basis of other biologically or economically important traits remains a challenge for plant breeders, quantitative geneticists and evolutionary ecologists. Many of these traits are too complex to be described by a single measurement because they change over time and across environmental conditions. Such traits are often called functionvalued traits (FVTs; Pletcher & Geyer, 1999) or infinite-dimensional characters (Kirkpatrick &Heckman, 1989). Some examples of FVTs include plant growth/development over time or the response of a trait to environmental changes. Statistical models that explicitly incorporate the complex nature of FVTs generally have more power, provide greater insight, and are more statistically robust than the commonly used univariate and multivariate methods (Griswold et al., 2008). Themodeling and applications of FVTs have been extensively studied (e.g. see reviewby Stinchcombe et al., 2012) andoften involvemathematical and statisticalmethods that are unfamiliar to biologists. Despite their biological relevance and statistical advantages, FVTs are frequently discussed but rarely implemented in practice. In this issue of New Phytologist, Baker et al. (pp. 257–268) put the FVTapproach into practice through an exploration of the genetic architecture of leaf development in Brassica rapa and its relationships with organism-level performance.

Alternative haplotypes of antigen processing genes in zebrafish diverged early in vertebrate evolution

Significance Antigen presentation genes are exceptionally polymorphic, enhancing immune defense. Polymorphism within additional components of the MHC pathway, particularly the antigen processing genes, may also shape immune responses. Using transcriptome, exome, and whole-genome sequencing to examine immune gene variation in zebrafish, we uncovered several antigen processing genes not found in the reference genome clustered within a deeply divergent haplotype of the core MHC locus. Our data provide evidence that these previously undescribed antigen processing genes retain ancient alternative sequence lineages, likely derived during the formation of the adaptive immune system, and represent the most divergent collection of antigen processing and presentation genes yet identified. These findings offer insights into the evolution of vertebrate adaptive immunity. Antigen processing and presentation genes found within the MHC are among the most highly polymorphic genes of vertebrate genomes, providing populations with diverse immune responses to a wide array of pathogens. Here, we describe transcriptome, exome, and whole-genome sequencing of clonal zebrafish, uncovering the most extensive diversity within the antigen processing and presentation genes of any species yet examined. Our CG2 clonal zebrafish assembly provides genomic context within a remarkably divergent haplotype of the core MHC region on chromosome 19 for six expressed genes not found in the zebrafish reference genome: mhc1uga, proteasome-β 9b (psmb9b), psmb8f, and previously unknown genes psmb13b, tap2d, and tap2e. We identify ancient lineages for Psmb13 within a proteasome branch previously thought to be monomorphic and provide evidence of substantial lineage diversity within each of three major trifurcations of catalytic-type proteasome subunits in vertebrates: Psmb5/Psmb8/Psmb11, Psmb6/Psmb9/Psmb12, and Psmb7/Psmb10/Psmb13. Strikingly, nearby tap2 and MHC class I genes also retain ancient sequence lineages, indicating that alternative lineages may have been preserved throughout the entire MHC pathway since early diversification of the adaptive immune system ∼500 Mya. Furthermore, polymorphisms within the three MHC pathway steps (antigen cleavage, transport, and presentation) are each predicted to alter peptide specificity. Lastly, comparative analysis shows that antigen processing gene diversity is far more extensive than previously realized (with ancient coelacanth psmb8 lineages, shark psmb13, and tap2t and psmb10 outside the teleost MHC), implying distinct immune functions and conserved roles in shaping MHC pathway evolution throughout vertebrates.

Integrative genomics reveals hypoxia inducible genes that are associated with a poor prognosis in neuroblastoma patients

Neuroblastoma is notable for its broad spectrum of clinical behavior ranging from spontaneous regression to rapidly progressive disease. Hypoxia is well known to confer a more aggressive phenotype in neuroblastoma. We analyzed transcriptome data from diagnostic neuroblastoma tumors and hypoxic neuroblastoma cell lines to identify genes whose expression levels correlate with poor patient outcome and are involved in the hypoxia response. By integrating a diverse set of transcriptome datasets, including those from neuroblastoma patients and neuroblastoma derived cell lines, we identified nine genes (SLCO4A1, ENO1, HK2, PGK1, MTFP1, HILPDA, VKORC1, TPI1, and HIST1H1C) that are up-regulated in hypoxia and whose expression levels are correlated with poor patient outcome in three independent neuroblastoma cohorts. Analysis of 5-hydroxymethylcytosine and ENCODE data indicate that at least five of these nine genes have an increase in 5-hydroxymethylcytosine and a more open chromatin structure in hypoxia versus normoxia and are putative targets of hypoxia inducible factor (HIF) as they contain HIF binding sites in their regulatory regions. Four of these genes are key components of the glycolytic pathway and another three are directly involved in cellular metabolism. We experimentally validated our computational findings demonstrating that seven of the nine genes are significantly up-regulated in response to hypoxia in the four neuroblastoma cell lines tested. This compact and robustly validated group of genes, is associated with the hypoxia response in aggressive neuroblastoma and may represent a novel target for biomarker and therapeutic development.

ExScalibur: A High-Performance Cloud-Enabled Suite for Whole Exome Germline and Somatic Mutation Identification.

Whole exome sequencing has facilitated the discovery of causal genetic variants associated with human diseases at deep coverage and low cost. In particular, the detection of somatic mutations from tumor/normal pairs has provided insights into the cancer genome. Although there is an abundance of publicly-available software for the detection of germline and somatic variants, concordance is generally limited among variant callers and alignment algorithms. Successful integration of variants detected by multiple methods requires in-depth knowledge of the software, access to high-performance computing resources, and advanced programming techniques. We present ExScalibur, a set of fully automated, highly scalable and modulated pipelines for whole exome data analysis. The suite integrates multiple alignment and variant calling algorithms for the accurate detection of germline and somatic mutations with close to 99% sensitivity and specificity. ExScalibur implements streamlined execution of analytical modules, real-time monitoring of pipeline progress, robust handling of errors and intuitive documentation that allows for increased reproducibility and sharing of results and workflows. It runs on local computers, high-performance computing clusters and cloud environments. In addition, we provide a data analysis report utility to facilitate visualization of the results that offers interactive exploration of quality control files, read alignment and variant calls, assisting downstream customization of potential disease-causing mutations. ExScalibur is open-source and is also available as a public image on Amazon cloud.

Genetic analysis of impulsive personality traits: Examination of a priori candidates and genome-wide variation

Impulsive personality traits are heritable risk factors and putative endophenotypes for addiction and other psychiatric disorders involving disinhibition. This study examined the genetic basis of impulsive personality traits, defined as scores on the Barratt Impulsiveness Scale (BIS-11) and the UPPS-P Impulsive Behavior Scale (UPPS-P). In 983 healthy young adults of European ancestry, the study examined genetic variation in relation to a combined phenotype of seven subscales based on high phenotypic intercorrelations. The study first tested 14 a priori loci that have previously been associated impulsive personality traits or closely related constructs. Second, the study included an exploratory genome-wide scan (i.e., GWAS), acknowledging that only relatively large effects would be detectable in a sample size of ~ 1000. A priori SNP analyses revealed a significant association between the combined impulsivity phenotype and two SNPs within the 5-HT2a receptor gene (HTR2A; rs6313 and rs6311). Follow-up analyses suggested that the effects were specific to the Motor and Non-planning subscales on the BIS-11, and also that the two loci were in linkage disequilibrium. The GWAS yielded no statistically significant findings. This study further implicates loci within HTR2A with certain forms of self-reported impulsivity and identifies candidates for future investigation from the genome-wide analyses.

Hierarchical Investigation of Genetic Influences on Response Inhibition in Healthy Young Adults

Poor inhibitory control is a known risk factor for substance use disorders, making it a priority to identify the determinants of these deficits. The aim of the current study was to identify genetic associations with inhibitory control using the stop signal task in a large sample (n = 934) of healthy young adults of European ancestry. We genotyped the subjects genome-wide and then used a hierarchical approach in which we tested seven a priori single nucleotide polymorphisms (SNPs) previously associated with stop signal task performance, approximately 9,000 SNPs designated as high-value addiction (HVA) markers by the SmokeScreen array, and approximately five million genotyped and imputed SNPs, followed by a gene-based association analysis using the resultant p values. A priori SNP analyses revealed nominally significant associations between response inhibition and one locus in HTR2A (rs6313; p = .04, dominance model, uncorrected) in the same direction as prior findings. A nominally significant association was also found in one locus in ANKK1 (rs1800497; p = .03, uncorrected), although in the opposite direction of previous reports. After accounting for multiple comparisons, the HVA, genome-wide, and gene-based analyses yielded no significant findings. This study implicates variation in serotonergic and dopaminergic genes while underscoring the difficulty of detecting the influence of individual SNPs, even when biological information is used to prioritize testing. Although such small effect sizes suggest limited utility of individual SNPs in predicting risk for addiction or other impulse control disorders, they may nonetheless shed light on complex biological processes underlying poor inhibitory control.