Before and After: Comparison of Legacy and Harmonized TCGA Genomic Data Commons' Data.

We present a systematic analysis of the effects of synchronizing a large-scale, deeply characterized, multi-omic dataset to the current human reference genome, using updated software, pipelines, and annotations. For each of 5 molecular data platforms in The Cancer Genome Atlas (TCGA)-mRNA and miRNA expression, single nucleotide variants, DNA methylation and copy number alterations-comprehensive sample, gene, and probe-level studies were performed, towards quantifying the degree of similarity between the 'legacy' GRCh37 (hg19) TCGA data and its GRCh38 (hg38) version as 'harmonized' by the Genomic Data Commons. We offer gene lists to elucidate differences that remained after controlling for confounders, and strategies to mitigate their impact on biological interpretation. Our results demonstrate that the hg19 and hg38 TCGA datasets are very highly concordant, promote informed use of either legacy or harmonized omics data, and provide a rubric that encourages similar comparisons as new data emerge and reference data evolve.

The Immune Landscape of Cancer.

We performed an extensive immunogenomic analysis of more than 10,000 tumors comprising 33 diverse cancer types by utilizing data compiled by TCGA. Across cancer types, we identified six immune subtypes-wound healing, IFN-γ dominant, inflammatory, lymphocyte depleted, immunologically quiet, and TGF-ß dominant-characterized by differences in macrophage or lymphocyte signatures, Th1:Th2 cell ratio, extent of intratumoral heterogeneity, aneuploidy, extent of neoantigen load, overall cell proliferation, expression of immunomodulatory genes, and prognosis. Specific driver mutations correlated with lower (CTNNB1, NRAS, or IDH1) or higher (BRAF, TP53, or CASP8) leukocyte levels across all cancers. Multiple control modalities of the intracellular and extracellular networks (transcription, microRNAs, copy number, and epigenetic processes) were involved in tumor-immune cell interactions, both across and within immune subtypes. Our immunogenomics pipeline to characterize these heterogeneous tumors and the resulting data are intended to serve as a resource for future targeted studies to further advance the field.

Driver Fusions and Their Implications in the Development and Treatment of Human Cancers.

Gene fusions represent an important class of somatic alterations in cancer. We systematically investigated fusions in 9,624 tumors across 33 cancer types using multiple fusion calling tools. We identified a total of 25,664 fusions, with a 63% validation rate. Integration of gene expression, copy number, and fusion annotation data revealed that fusions involving oncogenes tend to exhibit increased expression, whereas fusions involving tumor suppressors have the opposite effect. For fusions involving kinases, we found 1,275 with an intact kinase domain, the proportion of which varied significantly across cancer types. Our study suggests that fusions drive the development of 16.5% of cancer cases and function as the sole driver in more than 1% of them. Finally, we identified druggable fusions involving genes such as TMPRSS2, RET, FGFR3, ALK, and ESR1 in 6.0% of cases, and we predicted immunogenic peptides, suggesting that fusions may provide leads for targeted drug and immune therapy.

Systematic Analysis of Splice-Site-Creating Mutations in Cancer.

For the past decade, cancer genomic studies have focused on mutations leading to splice-site disruption, overlooking those having splice-creating potential. Here, we applied a bioinformatic tool, MiSplice, for the large-scale discovery of splice-site-creating mutations (SCMs) across 8,656 TCGA tumors. We report 1,964 originally mis-annotated mutations having clear evidence of creating alternative splice junctions. TP53 and GATA3 have 26 and 18 SCMs, respectively, and ATRX has 5 from lower-grade gliomas. Mutations in 11 genes, including PARP1, BRCA1, and BAP1, were experimentally validated for splice-site-creating function. Notably, we found that neoantigens induced by SCMs are likely several folds more immunogenic compared to missense mutations, exemplified by the recurrent GATA3 SCM. Further, high expression of PD-1 and PD-L1 was observed in tumors with SCMs, suggesting candidates for immune blockade therapy. Our work highlights the importance of integrating DNA and RNA data for understanding the functional and the clinical implications of mutations in human diseases.

Mapping genetic variations to three-dimensional protein structures to enhance variant interpretation: a proposed framework.

The translation of personal genomics to precision medicine depends on the accurate interpretation of the multitude of genetic variants observed for each individual. However, even when genetic variants are predicted to modify a protein, their functional implications may be unclear. Many diseases are caused by genetic variants affecting important protein features, such as enzyme active sites or interaction interfaces. The scientific community has catalogued millions of genetic variants in genomic databases and thousands of protein structures in the Protein Data Bank. Mapping mutations onto three-dimensional (3D) structures enables atomic-level analyses of protein positions that may be important for the stability or formation of interactions; these may explain the effect of mutations and in some cases even open a path for targeted drug development. To accelerate progress in the integration of these data types, we held a two-day Gene Variation to 3D (GVto3D) workshop to report on the latest advances and to discuss unmet needs. The overarching goal of the workshop was to address the question: what can be done together as a community to advance the integration of genetic variants and 3D protein structures that could not be done by a single investigator or laboratory? Here we describe the workshop outcomes, review the state of the field, and propose the development of a framework with which to promote progress in this arena. The framework will include a set of standard formats, common ontologies, a common application programming interface to enable interoperation of the resources, and a Tool Registry to make it easy to find and apply the tools to specific analysis problems. Interoperability will enable integration of diverse data sources and tools and collaborative development of variant effect prediction methods.

The ISB Cancer Genomics Cloud: A Flexible Cloud-Based Platform for Cancer Genomics Research.

The ISB Cancer Genomics Cloud (ISB-CGC) is one of three pilot projects funded by the National Cancer Institute to explore new approaches to computing on large cancer datasets in a cloud environment. With a focus on Data as a Service, the ISB-CGC offers multiple avenues for accessing and analyzing The Cancer Genome Atlas, TARGET, and other important references such as GENCODE and COSMIC using the Google Cloud Platform. The open approach allows researchers to choose approaches best suited to the task at hand: from analyzing terabytes of data using complex workflows to developing new analysis methods in common languages such as Python, R, and SQL; to using an interactive web application to create synthetic patient cohorts and to explore the wealth of available genomic data. Links to resources and documentation can be found at www.isb-cgc.org Cancer Res; 77(21); e7-10. ©2017 AACR.

Learning a weighted sequence model of the nucleosome core and linker yields more accurate predictions in Saccharomyces cerevisiae and Homo sapiens.

DNA in eukaryotes is packaged into a chromatin complex, the most basic element of which is the nucleosome. The precise positioning of the nucleosome cores allows for selective access to the DNA, and the mechanisms that control this positioning are important pieces of the gene expression puzzle. We describe a large-scale nucleosome pattern that jointly characterizes the nucleosome core and the adjacent linkers and is predominantly characterized by long-range oscillations in the mono, di- and tri-nucleotide content of the DNA sequence, and we show that this pattern can be used to predict nucleosome positions in both Homo sapiens and Saccharomyces cerevisiae more accurately than previously published methods. Surprisingly, in both H. sapiens and S. cerevisiae, the most informative individual features are the mono-nucleotide patterns, although the inclusion of di- and tri-nucleotide features results in improved performance. Our approach combines a much longer pattern than has been previously used to predict nucleosome positioning from sequence-301 base pairs, centered at the position to be scored-with a novel discriminative classification approach that selectively weights the contributions from each of the input features. The resulting scores are relatively insensitive to local AT-content and can be used to accurately discriminate putative dyad positions from adjacent linker regions without requiring an additional dynamic programming step and without the attendant edge effects and assumptions about linker length modeling and overall nucleosome density. Our approach produces the best dyad-linker classification results published to date in H. sapiens, and outperforms two recently published models on a large set of S. cerevisiae nucleosome positions. Our results suggest that in both genomes, a comparable and relatively small fraction of nucleosomes are well-positioned and that these positions are predictable based on sequence alone. We believe that the bulk of the remaining nucleosomes follow a statistical positioning model.

Transmembrane topology and signal peptide prediction using dynamic bayesian networks.

Hidden Markov models (HMMs) have been successfully applied to the tasks of transmembrane protein topology prediction and signal peptide prediction. In this paper we expand upon this work by making use of the more powerful class of dynamic Bayesian networks (DBNs). Our model, Philius, is inspired by a previously published HMM, Phobius, and combines a signal peptide submodel with a transmembrane submodel. We introduce a two-stage DBN decoder that combines the power of posterior decoding with the grammar constraints of Viterbi-style decoding. Philius also provides protein type, segment, and topology confidence metrics to aid in the interpretation of the predictions. We report a relative improvement of 13% over Phobius in full-topology prediction accuracy on transmembrane proteins, and a sensitivity and specificity of 0.96 in detecting signal peptides. We also show that our confidence metrics correlate well with the observed precision. In addition, we have made predictions on all 6.3 million proteins in the Yeast Resource Center (YRC) database. This large-scale study provides an overall picture of the relative numbers of proteins that include a signal-peptide and/or one or more transmembrane segments as well as a valuable resource for the scientific community. All DBNs are implemented using the Graphical Models Toolkit. Source code for the models described here is available at http://noble.gs.washington.edu/proj/philius. A Philius Web server is available at http://www.yeastrc.org/philius, and the predictions on the YRC database are available at http://www.yeastrc.org/pdr.

Mesolimbic dopamine in desire and dread: enabling motivation to be generated by localized glutamate disruptions in nucleus accumbens.

An important issue in affective neuroscience concerns the role of mesocorticolimbic dopamine systems in positive-valenced motivation (e.g., reward) versus negative-valenced motivation (e.g., fear). Here, we assessed whether endogenous dopamine receptor stimulation in nucleus accumbens contributes to both appetitive behavior and fearful behavior that is generated in keyboard manner by local glutamate disruptions at different sites in medial shell. 6,7-Dinitroquinoxaline-2,3(1H,4H)-dione (DNQX) microinjections (450 ng) locally disrupt glutamate signals in <4 mm(3) of nucleus accumbens, and generate either desire or fear (or both) depending on precise rostrocaudal location in medial shell. At rostral shell sites, local AMPA/kainate blockade generates positive ingestive behavior, but the elicited motivated behavior becomes incrementally more fearful as the same microinjection is moved caudally. A dopamine-blocking mixture of D(1) and D(2) antagonists (raclopride and SCH-23390 [R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5,-tetrahydro-1H-3-benzazepine hydrochloride]) was combined here in the same microinjection with DNQX to assess the role of endogenous local dopamine in mediating the DNQX-motivated behaviors. We report that local dopamine blockade prevented DNQX microinjections from generating appetitive behavior (eating) in rostral shell, and equally prevented DNQX from generating fearful behavior (defensive treading) in caudal shell. We conclude that local dopamine is needed to enable disruptions of corticolimbic glutamate signals in shell to generate either positive incentive salience or negative fearful salience (valence depending on site and other conditions). Thus, dopamine interacts with localization of valence-biased glutamate circuits in medial shell to facilitate keyboard stimulation of both appetitive and fearful motivations.

Modeling peptide fragmentation with dynamic Bayesian networks for peptide identification.

MOTIVATION: Tandem mass spectrometry (MS/MS) is an indispensable technology for identification of proteins from complex mixtures. Proteins are digested to peptides that are then identified by their fragmentation patterns in the mass spectrometer. Thus, at its core, MS/MS protein identification relies on the relative predictability of peptide fragmentation. Unfortunately, peptide fragmentation is complex and not fully understood, and what is understood is not always exploited by peptide identification algorithms. RESULTS: We use a hybrid dynamic Bayesian network (DBN)/support vector machine (SVM) approach to address these two problems. We train a set of DBNs on high-confidence peptide-spectrum matches. These DBNs, known collectively as Riptide, comprise a probabilistic model of peptide fragmentation chemistry. Examination of the distributions learned by Riptide allows identification of new trends, such as prevalent a-ion fragmentation at peptide cleavage sites C-term to hydrophobic residues. In addition, Riptide can be used to produce likelihood scores that indicate whether a given peptide-spectrum match is correct. A vector of such scores is evaluated by an SVM, which produces a final score to be used in peptide identification. Using Riptide in this way yields improved discrimination when compared to other state-of-the-art MS/MS identification algorithms, increasing the number of positive identifications by as much as 12% at a 1% false discovery rate. AVAILABILITY: Python and C source code are available upon request from the authors. The curated training sets are available at http://noble.gs.washington.edu/proj/intense/. The Graphical Model Tool Kit (GMTK) is freely available at http://ssli.ee.washington.edu/bilmes/gmtk.

Emotional environments retune the valence of appetitive versus fearful functions in nucleus accumbens.

The nucleus accumbens mediates both appetitive motivation for rewards and fearful motivation toward threats, which are generated in part by glutamate-related circuits organized in a keyboard fashion. At rostral sites of the medial shell, localized glutamate disruptions typically generate intense appetitive behaviors in rats, but the disruption incrementally generates fearful behaviors as microinjection sites move more caudally. We found that exposure to stressful environments caused caudal fear-generating zones to expand rostrally, filling approximately 90% of the shell. Conversely, a preferred home environment caused fear-generating zones to shrink and appetitive-generating zones to expand caudally, filling approximately 90% of the shell. Thus, the emotional environments retuned the generation of motivation in corticolimbic circuits.

Independent and complementary methods for large-scale structural analysis of mammalian chromatin.

The fundamental building block of chromatin, the nucleosome, occupies 150 bp of DNA in a spaced arrangement that is a primary determinant in regulation of the genome. The nucleosomal organization of some regions of the human genome has been described, but mapping of these regions has been limited to a few kilobases. We have explored two independent and complementary methods for the high-throughput analysis of mammalian chromatin structure. Through adaptations to a protocol used to map yeast chromatin structure, we determined sites of nucleosomal protection over large regions of the mammalian genome using a tiling microarray. By modifying classical primer extension methods, we localized specific internucleosomally cleaved mammalian genomic sequences using a capillary electrophoresis sequencer in a manner that allows high-throughput nucleotide-resolution characterization of nucleosome protection patterns. We developed algorithms for the automated and unbiased analysis of the resulting data, a necessary step toward large-scale analysis. We validated these assays using the known positions of nucleosomes on the mouse mammary tumor virus LTR, and additionally, we characterized the previously unreported chromatin structure of the LCMT2 gene. These results demonstrate the effectiveness of the combined methods for reliable analysis of mammalian chromatin structure in a high-throughput manner.

Neurotensin antagonist acutely and robustly attenuates locomotion that accompanies stimulation of a neurotensin-containing pathway from rostrobasal forebrain to the ventral tegmental area.

Neurotensin exerts complex effects on the mesolimbic dopamine system that alter motivation and contribute to neuroadaptations associated with psychostimulant drug administration. Activation of abundant neurotensin receptors in the ventral tegmental area (VTA) enhances dopamine neuron activity and associated release of dopamine in the nucleus accumbens (Acb) and cortex. In view of recent anatomical studies demonstrating that 70% of all neurotensin-containing neurons projecting to the VTA occupy the lateral preoptic area-rostral lateral hypothalamus (LPH) and lateral part of the medial preoptic area (MPOA), the present study examined functionality in the LPH-MPOA neurotensinergic pathway in the rat. Disinhibition (resulting ultimately in stimulation-like effects) of LPH-MPOA neurons with microinjected bicuculline (50 or 100 ng in 0.25 microL) produced locomotor activation that was considerably attenuated by systemic administration of the neurotensin antagonist SR 142948 A (0.03 and 0.1 mg/kg). In contrast, locomotion elicited in this manner was completely blocked by SR 142948 A infused directly into the VTA (5.0 and 15.0 ng in 0.25 microL). Baseline locomotion was unaffected by systemic or intra-VTA administration of SR 142948 A and LPH-MPOA-elicited locomotion was unaffected by infusion of SR 142948 A into the substantia nigra pars compacta and sites rostral and dorsal to the VTA. Locomotion was not elicited by infusions of bicuculline into the lateral hypothalamus at sites caudal to the LPH-MPOA, where neurotensin neurons projecting to the VTA are fewer. The results demonstrate the capacity of a neurotensin-containing pathway from LPH-MPOA to VTA to modulate locomotion. This pathway may be important in linking hippocampal and mesolimbic mechanisms in normal behaviour and drug addiction.

Specificity in the projections of prefrontal and insular cortex to ventral striatopallidum and the extended amygdala.

The basal forebrain functional-anatomical macrosystems, ventral striatopallidum, and extended amygdala are innervated by substantially coextensive distributions of neurons in the prefrontal and insular cortex. This suggests two alternative organizational schemes: convergent, in which a given cortical area projects exclusively to only one of these macrosystems and divergent, in which a given cortical area innervates both forebrain macrosystems. To examine the underlying organization and possibly discriminate between these alternatives, rats were injected with two retrograde tracers in different parts of ventral striatopallidum or extended amygdala (homotypic injection pairs) or with one tracer in each macrosystem (heterotypic). The prefrontal and insular cortex was evaluated microscopically for overlap of retrograde labeling and double labeling of neurons. Homotypic injection pairs in the ventral striatum and extended amygdala produced extensive overlap of retrogradely labeled neurons and significant double labeling, suggesting that cortical projections spread broadly within macrosystems. In contrast, heterotypic injection pairs produced significant overlap of retrograde labeling but negligible double labeling, indicating that ventral striatopallidum and extended amygdala receive inputs from separate sets of prefronto- and insular cortical neurons. The caudomedial shell of the nucleus accumbens, a supposed "transition" zone between striatopallidum and extended amygdala, had extended amygdala-like afferents but produced few double-labeled neurons and these only when paired with ventral striatopallidum. The data suggest that a modular organization of the basal forebrain, with postulated independent information processing by the ventral striatopallidal and extended amygdala macrosystems, is reflected in a corresponding segregation of output neurons in the prefrontal and insular cortices.

Endogenous opioids are necessary for benzodiazepine palatability enhancement: naltrexone blocks diazepam-induced increase of sucrose-'liking'.

Opioid agonists and benzodiazepine agonists each increase food intake. Both also increase hedonic 'liking' reactions to sweet tastes in rats. Do opioids and benzodiazepines share overlapping mechanisms of hedonic impact? Or are benzodiazepine and opioid effects on hedonic impact mediated by independent mechanisms? The present study examined whether blockade of opioid receptors prevents benzodiazepine-induced enhancement of taste palatability, as assessed by the affective taste reactivity test. Rats were implanted with oral cannulae, and prior to an oral infusion of bittersweet quinine-sucrose solution, all received i.p. injections of either vehicle, or diazepam alone (5 mg/kg diazepam+0 mg/kg naltrexone), naltrexone alone (1 mg/kg naltrexone+0 mg diazepam), or both diazepam plus naltrexone (5 mg/kg diazepam+1mg/kg naltrexone). Videotaped hedonic ('liking') and aversive ('disliking') orofacial reactions elicited by sucrose/quinine taste were compared across drug conditions. Diazepam administration alone more than doubled hedonic 'liking' reactions to the bittersweet taste, while reducing 'disliking' in half, compared to vehicle levels. Naltrexone by itself had little effect on taste-elicited affective reactions, and only marginally increased aversive gapes. However, naltrexone completely blocked diazepam's enhancement of positive hedonic 'liking' reactions, and naltrexone similarly disrupted diazepam-reduction of aversive 'disliking' taste reactions. These results indicate that endogenous opioid neurotransmission may be crucial to benzodiazepine enhancement of hedonic 'liking' for natural taste reward.

Glutamate motivational ensembles in nucleus accumbens: rostrocaudal shell gradients of fear and feeding.

This study demonstrates that microinjection of an AMPA/kainate glutamate antagonist elicits motivated fear and feeding behaviour mapped along rostrocaudal gradients of positive-to-negative valence in nucleus accumbens shell (similar to rostrocaudal shell gradients recently reported for GABA agonist microinjections). Rats received rostral or caudal microinjections of the glutamate AMPA/kainate receptor antagonist DNQX (0, 50, 450 or 850 ng in 0.5 micro L) or the NMDA receptor antagonist MK-801 (0, 0.5, 1 or 2 micro g in 0.5 micro L), into medial accumbens shell prior to behavioural tests for fear, feeding or conditioning of place preference or avoidance. Another group received rostral or caudal microinjections of DNQX in nucleus accumbens core. Rostral shell DNQX microinjections potently increased appetitive food intake and established only weak conditioned place avoidance. Caudal shell DNQX microinjections elicited defensive treading behaviour, caused rats to defensively bite the experimenter and emit fearful distress vocalizations when handled, and established strong conditioned place avoidance. By contrast, no rostrocaudal gradients of motivational bivalence were produced by microinjections of the glutamate AMPA/kainate receptor antagonist DNQX into the core, or by microinjections of the NMDA antagonist MK-801 into the shell. Our results indicate that appetitive and aversive motivation is carried in anatomically differentiated channels by mesocorticolimbic glutamate signals to microcircuits in the medial shell. Hyperpolarization of local shell ensembles by AMPA/kainate glutamate receptor blockade elicits fear and feeding behaviours mapped along distinct positive-to-negative rostrocaudal gradients.

Positive and negative motivation in nucleus accumbens shell: bivalent rostrocaudal gradients for GABA-elicited eating, taste "liking"/"disliking" reactions, place preference/avoidance, and fear.

Microinjection of the GABA(A) agonist muscimol in the rostral medial accumbens shell in rats elicits appetitive eating behavior, but in the caudal shell instead elicits fearful defensive treading behavior. To further test the hypothesis that rostral shell muscimol microinjections produce positive motivational states, whereas caudal shell muscimol produces negative states, we measured behavioral place preference/avoidance conditioning and affective hedonic and aversive orofacial expressions of taste-elicited "liking" and "disliking" (gapes, etc.) in addition to fear and feeding behaviors. Farthest rostral muscimol microinjections (75 ng) caused increased eating behavior and also caused positive conditioned place preferences and increased positive hedonic reactions to the taste of sucrose. By contrast, caudal shell microinjections elicited negative defensive treading and caused robust negative conditioned place avoidance and negative aversive reactions to sucrose or quinine tastes. Intermediate rostral microinjections elicited effects of mixed positive/negative valence (positive appetitive eating behavior but negative place avoidance and negative taste reactions at mid-rostral sites, and sometimes positive eating simultaneously with fearful defensive treading more caudally). These results indicate that GABAergic neurotransmission in local microcircuits in nucleus accumbens mediates motivated/affective behavior that is bivalently organized along rostrocaudal gradients.