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.

Curated single cell multimodal landmark datasets for R/Bioconductor.

BACKGROUND: The majority of high-throughput single-cell molecular profiling methods quantify RNA expression; however, recent multimodal profiling methods add simultaneous measurement of genomic, proteomic, epigenetic, and/or spatial information on the same cells. The development of new statistical and computational methods in Bioconductor for such data will be facilitated by easy availability of landmark datasets using standard data classes. RESULTS: We collected, processed, and packaged publicly available landmark datasets from important single-cell multimodal protocols, including CITE-Seq, ECCITE-Seq, SCoPE2, scNMT, 10X Multiome, seqFISH, and G&T. We integrate data modalities via the MultiAssayExperiment Bioconductor class, document and re-distribute datasets as the SingleCellMultiModal package in Bioconductor's Cloud-based ExperimentHub. The result is single-command actualization of landmark datasets from seven single-cell multimodal data generation technologies, without need for further data processing or wrangling in order to analyze and develop methods within Bioconductor's ecosystem of hundreds of packages for single-cell and multimodal data. CONCLUSIONS: We provide two examples of integrative analyses that are greatly simplified by SingleCellMultiModal. The package will facilitate development of bioinformatic and statistical methods in Bioconductor to meet the challenges of integrating molecular layers and analyzing phenotypic outputs including cell differentiation, activity, and disease.

Enter the Matrix: Factorization Uncovers Knowledge from Omics.

Omics data contain signals from the molecular, physical, and kinetic inter- and intracellular interactions that control biological systems. Matrix factorization (MF) techniques can reveal low-dimensional structure from high-dimensional data that reflect these interactions. These techniques can uncover new biological knowledge from diverse high-throughput omics data in applications ranging from pathway discovery to timecourse analysis. We review exemplary applications of MF for systems-level analyses. We discuss appropriate applications of these methods, their limitations, and focus on the analysis of results to facilitate optimal biological interpretation. The inference of biologically relevant features with MF enables discovery from high-throughput data beyond the limits of current biological knowledge - answering questions from high-dimensional data that we have not yet thought to ask.

MOGSA: Integrative Single Sample Gene-set Analysis of Multiple Omics Data.

Gene-set analysis (GSA) summarizes individual molecular measurements to more interpretable pathways or gene-sets and has become an indispensable step in the interpretation of large-scale omics data. However, GSA methods are limited to the analysis of single omics data. Here, we introduce a new computation method termed multi-omics gene-set analysis (MOGSA), a multivariate single sample gene-set analysis method that integrates multiple experimental and molecular data types measured over the same set of samples. The method learns a low dimensional representation of most variant correlated features (genes, proteins, etc.) across multiple omics data sets, transforms the features onto the same scale and calculates an integrated gene-set score from the most informative features in each data type. MOGSA does not require filtering data to the intersection of features (gene IDs), therefore, all molecular features, including those that lack annotation may be included in the analysis. Using simulated data, we demonstrate that integrating multiple diverse sources of molecular data increases the power to discover subtle changes in gene-sets and may reduce the impact of unreliable information in any single data type. Using real experimental data, we demonstrate three use-cases of MOGSA. First, we show how to remove a source of noise (technical or biological) in integrative MOGSA of NCI60 transcriptome and proteome data. Second, we apply MOGSA to discover similarities and differences in mRNA, protein and phosphorylation profiles of a small study of stem cell lines and assess the influence of each data type or feature on the total gene-set score. Finally, we apply MOGSA to cluster analysis and show that three molecular subtypes are robustly discovered when copy number variation and mRNA data of 308 bladder cancers from The Cancer Genome Atlas are integrated using MOGSA. MOGSA is available in the Bioconductor R package "mogsa."

BRCA1-IRIS promotes human tumor progression through PTEN blockade and HIF-1α activation.

BRCA1 is an established breast and ovarian tumor suppressor gene that encodes multiple protein products whose individual contributions to human cancer suppression are poorly understood. BRCA1-IRIS (also known as "IRIS"), an alternatively spliced BRCA1 product and a chromatin-bound replication and transcription regulator, is overexpressed in various primary human cancers, including breast cancer, lung cancer, acute myeloid leukemia, and certain other carcinomas. Its naturally occurring overexpression can promote the metastasis of patient-derived xenograft (PDX) cells and other human cancer cells in mouse models. The IRIS-driven metastatic mechanism results from IRIS-dependent suppression of phosphatase and tensin homolog (PTEN) transcription, which in turn perturbs the PI3K/AKT/GSK-3ß pathway leading to prolyl hydroxylase-independent HIF-1α stabilization and activation in a normoxic environment. Thus, despite the tumor-suppressing genetic origin of IRIS, its properties more closely resemble those of an oncoprotein that, when spontaneously overexpressed, can, paradoxically, drive human tumor progression.

Dimension reduction techniques for the integrative analysis of multi-omics data.

State-of-the-art next-generation sequencing, transcriptomics, proteomics and other high-throughput 'omics' technologies enable the efficient generation of large experimental data sets. These data may yield unprecedented knowledge about molecular pathways in cells and their role in disease. Dimension reduction approaches have been widely used in exploratory analysis of single omics data sets. This review will focus on dimension reduction approaches for simultaneous exploratory analyses of multiple data sets. These methods extract the linear relationships that best explain the correlated structure across data sets, the variability both within and between variables (or observations) and may highlight data issues such as batch effects or outliers. We explore dimension reduction techniques as one of the emerging approaches for data integration, and how these can be applied to increase our understanding of biological systems in normal physiological function and disease.

Public data and open source tools for multi-assay genomic investigation of disease.

Molecular interrogation of a biological sample through DNA sequencing, RNA and microRNA profiling, proteomics and other assays, has the potential to provide a systems level approach to predicting treatment response and disease progression, and to developing precision therapies. Large publicly funded projects have generated extensive and freely available multi-assay data resources; however, bioinformatic and statistical methods for the analysis of such experiments are still nascent. We review multi-assay genomic data resources in the areas of clinical oncology, pharmacogenomics and other perturbation experiments, population genomics and regulatory genomics and other areas, and tools for data acquisition. Finally, we review bioinformatic tools that are explicitly geared toward integrative genomic data visualization and analysis. This review provides starting points for accessing publicly available data and tools to support development of needed integrative methods.

Molecular Subtypes Improve Prognostic Value of International Metastatic Renal Cell Carcinoma Database Consortium Prognostic Model.

INTRODUCTION: Gene-expression signatures for prognosis have been reported in localized renal cell carcinoma (RCC). The aim of this study was to test the predictive power of two different signatures, ClearCode34, a 34-gene signature model [Eur Urol 2014;66:77-84], and an 8-gene signature model [Eur Urol 2015;67:17-20], in the setting of systemic therapy for metastatic disease. MATERIALS AND METHODS: Metastatic RCC (mRCC) patients from five institutions who were part of TCGA were identified and clinical data were retrieved. We trained and implemented each gene model as described by the original study. The latter was demonstrated by faithful regeneration of a figure and results from the original study. mRCC patients were dichotomized to good or poor prognostic risk groups using each gene model. Cox proportional hazard regression and concordance index (C-Index) analysis were used to investigate an association between each prognostic risk model and overall survival (OS) from first-line therapy. RESULTS: Overall, 54 patients were included in the final analysis. The primary endpoint was OS. Applying the ClearCode34 model, median survival for the low-risk-ccA (n = 17)-and the high-risk-ccB (n = 37)-subtypes were 27.6 and 22.3 months (hazard ratio (HR): 2.33; p = .039), respectively. ClearCode34 ccA/ccB and International Metastatic Renal Cell Carcinoma Database Consortium (IMDC) classifications appear to represent distinct risk criteria in mRCC, and we observed no significant overlap in classification (p > .05, chi-square test). On multivariable analyses and adjusting for IMDC groups, ccB remained independently associated with a worse OS (p = .044); the joint model of ccA/ccB and IMDC was significantly more accurate in predicting OS than a model with IMDC alone (p = .045, F-test). This was also observed in C-Index analysis; a model with both ccA and ccB subtypes had higher accuracy (C-Index 0.63, 95% confidence interval [CI] = 0.51-0.75) and 95% CIs of the C-Index that did not include the null value of 0.5 in contrast to a model with IMDC alone (0.60, CI = 0.47-0.72). The 8-gene signature molecular subtype model was a weak but insignificant predictor of survival in this cohort (p = .13). A model that included both the 8-gene signature and IMDC (C-Index 0.62, CI = 0.49-0.76) was more prognostic than IMDC alone but did not reach significance, as the 95% CI included the null value of 0.5. These two genomic signatures share no genes in common and are enriched in different biological pathways. The ClearCode34 included genes ARNT and EPAS1 (also known as HIF2a), which are involved in regulation of gene expression by hypoxia-inducible factor. CONCLUSION: The ClearCode34 but not the 8-gene molecular model improved the prognostic predictive power of the IMDC model in this cohort of 54 patients with metastatic clear cell RCC. The Oncologist 2017;22:286-292 IMPLICATIONS FOR PRACTICE: The clinical and laboratory factors included in the International Metastatic Renal Cell Carcinoma Database Consortium model provide prognostic information in metastatic renal cell carcinoma (mRCC). The present study shows that genomic signatures, originally validated in localized RCC, may add further complementary prognostic information in the metastatic setting. This study may provide new insights into the molecular basis of certain mRCC subgroups. The integration of clinical and molecular data has the potential to redefine mRCC classification, enhance the understanding of mRCC biology, and potentially predict response to treatment in the future.

Palb2 synergizes with Trp53 to suppress mammary tumor formation in a model of inherited breast cancer.

Germ-line mutations in PALB2 lead to a familial predisposition to breast and pancreatic cancer or to Fanconi Anemia subtype N. PALB2 performs its tumor suppressor role, at least in part, by supporting homologous recombination-type double strand break repair (HR-DSBR) through physical interactions with BRCA1, BRCA2, and RAD51. To further understand the mechanisms underlying PALB2-mediated DNA repair and tumor suppression functions, we targeted Palb2 in the mouse. Palb2-deficient murine ES cells recapitulated DNA damage defects caused by PALB2 depletion in human cells, and germ-line deletion of Palb2 led to early embryonic lethality. Somatic deletion of Palb2 driven by K14-Cre led to mammary tumor formation with long latency. Codeletion of both Palb2 and Tumor protein 53 (Trp53) accelerated mammary tumor formation. Like BRCA1 and BRCA2 mutant breast cancers, these tumors were defective in RAD51 focus formation, reflecting a defect in Palb2 HR-DSBR function, a strongly suspected contributor to Brca1, Brca2, and Palb2 mammary tumor development. However, unlike the case of Brca1-mutant cells, Trp53bp1 deletion failed to rescue the genomic instability of Palb2- or Brca2-mutant primary lymphocytes. Therefore, Palb2-driven DNA damage control is, in part, distinct from that executed by Brca1 and more similar to that of Brca2. The mechanisms underlying Palb2 mammary tumor suppression functions can now be explored genetically in vivo.

A multivariate approach to the integration of multi-omics datasets.

BACKGROUND: To leverage the potential of multi-omics studies, exploratory data analysis methods that provide systematic integration and comparison of multiple layers of omics information are required. We describe multiple co-inertia analysis (MCIA), an exploratory data analysis method that identifies co-relationships between multiple high dimensional datasets. Based on a covariance optimization criterion, MCIA simultaneously projects several datasets into the same dimensional space, transforming diverse sets of features onto the same scale, to extract the most variant from each dataset and facilitate biological interpretation and pathway analysis. RESULTS: We demonstrate integration of multiple layers of information using MCIA, applied to two typical "omics" research scenarios. The integration of transcriptome and proteome profiles of cells in the NCI-60 cancer cell line panel revealed distinct, complementary features, which together increased the coverage and power of pathway analysis. Our analysis highlighted the importance of the leukemia extravasation signaling pathway in leukemia that was not highly ranked in the analysis of any individual dataset. Secondly, we compared transcriptome profiles of high grade serous ovarian tumors that were obtained, on two different microarray platforms and next generation RNA-sequencing, to identify the most informative platform and extract robust biomarkers of molecular subtypes. We discovered that the variance of RNA-sequencing data processed using RPKM had greater variance than that with MapSplice and RSEM. We provided novel markers highly associated to tumor molecular subtype combined from four data platforms. MCIA is implemented and available in the R/Bioconductor "omicade4" package. CONCLUSION: We believe MCIA is an attractive method for data integration and visualization of several datasets of multi-omics features observed on the same set of individuals. The method is not dependent on feature annotation, and thus it can extract important features even when there are not present across all datasets. MCIA provides simple graphical representations for the identification of relationships between large datasets.

RamiGO: an R/Bioconductor package providing an AmiGO visualize interface.

SUMMARY: The R/Bioconductor package RamiGO is an R interface to AmiGO that enables visualization of Gene Ontology (GO) trees. Given a list of GO terms, RamiGO uses the AmiGO visualize API to import Graphviz-DOT format files into R, and export these either as images (SVG, PNG) or into Cytoscape for extended network analyses. RamiGO provides easy customization of annotation, highlighting of specific GO terms, colouring of terms by P-value or export of a simplified summary GO tree. We illustrate RamiGO functionalities in a genome-wide gene set analysis of prognostic genes in breast cancer. AVAILABILITY AND IMPLEMENTATION: RamiGO is provided in R/Bioconductor, is open source under the Artistic-2.0 License and is available with a user manual containing installation, operating instructions and tutorials. It requires R version 2.15.0 or higher. URL: http://bioconductor.org/packages/release/bioc/html/RamiGO.html

Significance analysis of prognostic signatures.

A major goal in translational cancer research is to identify biological signatures driving cancer progression and metastasis. A common technique applied in genomics research is to cluster patients using gene expression data from a candidate prognostic gene set, and if the resulting clusters show statistically significant outcome stratification, to associate the gene set with prognosis, suggesting its biological and clinical importance. Recent work has questioned the validity of this approach by showing in several breast cancer data sets that "random" gene sets tend to cluster patients into prognostically variable subgroups. This work suggests that new rigorous statistical methods are needed to identify biologically informative prognostic gene sets. To address this problem, we developed Significance Analysis of Prognostic Signatures (SAPS) which integrates standard prognostic tests with a new prognostic significance test based on stratifying patients into prognostic subtypes with random gene sets. SAPS ensures that a significant gene set is not only able to stratify patients into prognostically variable groups, but is also enriched for genes showing strong univariate associations with patient prognosis, and performs significantly better than random gene sets. We use SAPS to perform a large meta-analysis (the largest completed to date) of prognostic pathways in breast and ovarian cancer and their molecular subtypes. Our analyses show that only a small subset of the gene sets found statistically significant using standard measures achieve significance by SAPS. We identify new prognostic signatures in breast and ovarian cancer and their corresponding molecular subtypes, and we show that prognostic signatures in ER negative breast cancer are more similar to prognostic signatures in ovarian cancer than to prognostic signatures in ER positive breast cancer. SAPS is a powerful new method for deriving robust prognostic biological signatures from clinically annotated genomic datasets.

GeneSigDB: a manually curated database and resource for analysis of gene expression signatures.

GeneSigDB (http://www.genesigdb.org or http://compbio.dfci.harvard.edu/genesigdb/) is a database of gene signatures that have been extracted and manually curated from the published literature. It provides a standardized resource of published prognostic, diagnostic and other gene signatures of cancer and related disease to the community so they can compare the predictive power of gene signatures or use these in gene set enrichment analysis. Since GeneSigDB release 1.0, we have expanded from 575 to 3515 gene signatures, which were collected and transcribed from 1604 published articles largely focused on gene expression in cancer, stem cells, immune cells, development and lung disease. We have made substantial upgrades to the GeneSigDB website to improve accessibility and usability, including adding a tag cloud browse function, facetted navigation and a 'basket' feature to store genes or gene signatures of interest. Users can analyze GeneSigDB gene signatures, or upload their own gene list, to identify gene signatures with significant gene overlap and results can be viewed on a dynamic editable heatmap that can be downloaded as a publication quality image. All data in GeneSigDB can be downloaded in numerous formats including .gmt file format for gene set enrichment analysis or as a R/Bioconductor data file. GeneSigDB is available from http://www.genesigdb.org.

Gene Expression Atlas update--a value-added database of microarray and sequencing-based functional genomics experiments.

Gene Expression Atlas (http://www.ebi.ac.uk/gxa) is an added-value database providing information about gene expression in different cell types, organism parts, developmental stages, disease states, sample treatments and other biological/experimental conditions. The content of this database derives from curation, re-annotation and statistical analysis of selected data from the ArrayExpress Archive and the European Nucleotide Archive. A simple interface allows the user to query for differential gene expression either by gene names or attributes or by biological conditions, e.g. diseases, organism parts or cell types. Since our previous report we made 20 monthly releases and, as of Release 11.08 (August 2011), the database supports 19 species, which contains expression data measured for 19,014 biological conditions in 136,551 assays from 5598 independent studies.

The Stem Cell Discovery Engine: an integrated repository and analysis system for cancer stem cell comparisons.

Mounting evidence suggests that malignant tumors are initiated and maintained by a subpopulation of cancerous cells with biological properties similar to those of normal stem cells. However, descriptions of stem-like gene and pathway signatures in cancers are inconsistent across experimental systems. Driven by a need to improve our understanding of molecular processes that are common and unique across cancer stem cells (CSCs), we have developed the Stem Cell Discovery Engine (SCDE)-an online database of curated CSC experiments coupled to the Galaxy analytical framework. The SCDE allows users to consistently describe, share and compare CSC data at the gene and pathway level. Our initial focus has been on carefully curating tissue and cancer stem cell-related experiments from blood, intestine and brain to create a high quality resource containing 53 public studies and 1098 assays. The experimental information is captured and stored in the multi-omics Investigation/Study/Assay (ISA-Tab) format and can be queried in the data repository. A linked Galaxy framework provides a comprehensive, flexible environment populated with novel tools for gene list comparisons against molecular signatures in GeneSigDB and MSigDB, curated experiments in the SCDE and pathways in WikiPathways. The SCDE is available at http://discovery.hsci.harvard.edu.

Immune-restoring CAR-T cells display antitumor activity and reverse immunosuppressive TME in a humanized ccRCC mouse model.

One of the major barriers that have restricted successful use of chimeric antigen receptor (CAR) T cells in the treatment of solid tumors is an unfavorable tumor microenvironment (TME). We engineered CAR-T cells targeting carbonic anhydrase IX (CAIX) to secrete anti-PD-L1 monoclonal antibody (mAb), termed immune-restoring (IR) CAR G36-PDL1. We tested CAR-T cells in a humanized clear cell renal cell carcinoma (ccRCC) orthotopic mouse model with reconstituted human leukocyte antigen (HLA) partially matched human leukocytes derived from fetal CD34+ hematopoietic stem cells (HSCs) and bearing human ccRCC skrc-59 cells under the kidney capsule. G36-PDL1 CAR-T cells, haploidentical to the tumor cells, had a potent antitumor effect compared to those without immune-restoring effect. Analysis of the TME revealed that G36-PDL1 CAR-T cells restored active antitumor immunity by promoting tumor-killing cytotoxicity, reducing immunosuppressive cell components such as M2 macrophages and exhausted CD8+ T cells, and enhancing T follicular helper (Tfh)-B cell crosstalk.

Identification of transcription factors associated with castration-resistance: is the serum responsive factor a potential therapeutic target?

BACKGROUND: Advanced prostate cancer is treated by hormone ablation therapy. However, despite an initial response, the majority of men relapse to develop castration-resistant disease for which there are no effective treatments. We have previously shown that manipulating individual proteins has only minor alterations on the resistant phenotype so we hypothesize that targeting the central transcription factors (TFs) would represent a better therapeutic approach. METHODS: We have undertaken a transcriptomic analysis of gene expression differences between the androgen-dependent LNCaP parental cells and its castration-resistant Abl and Hof sublines, revealing 1,660 genes associated with castration-resistance. Using effective bioinformatic techniques, these transcriptomic data were integrated with TF binding sites resulting in a list of TFs associated with the differential gene expression observed. RESULTS: Following validation of the gene-chip results, the serum response factor (SRF) was chosen for clinical validation and functional analysis due to its recent association with prostate cancer progression. SRF immunoreactivity in prostate tumor samples was shown for the first time to be associated with castration-resistance. SRF inhibition by siRNA and the small molecule inhibitor CCG-1423 resulted in decreased proliferation. CONCLUSION: SRF is a key TF by which resistant cells survive with depleted levels of androgens representing a target for therapeutic manipulation.

iBBiG: iterative binary bi-clustering of gene sets.

MOTIVATION: Meta-analysis of genomics data seeks to identify genes associated with a biological phenotype across multiple datasets; however, merging data from different platforms by their features (genes) is challenging. Meta-analysis using functionally or biologically characterized gene sets simplifies data integration is biologically intuitive and is seen as having great potential, but is an emerging field with few established statistical methods. RESULTS: We transform gene expression profiles into binary gene set profiles by discretizing results of gene set enrichment analyses and apply a new iterative bi-clustering algorithm (iBBiG) to identify groups of gene sets that are coordinately associated with groups of phenotypes across multiple studies. iBBiG is optimized for meta-analysis of large numbers of diverse genomics data that may have unmatched samples. It does not require prior knowledge of the number or size of clusters. When applied to simulated data, it outperforms commonly used clustering methods, discovers overlapping clusters of diverse sizes and is robust in the presence of noise. We apply it to meta-analysis of breast cancer studies, where iBBiG extracted novel gene set-phenotype association that predicted tumor metastases within tumor subtypes. AVAILABILITY: Implemented in the Bioconductor package iBBiG CONTACT: aedin@jimmy.harvard.edu.

Correspondence analysis for dimension reduction, batch integration, and visualization of single-cell RNA-seq data.

Effective dimension reduction is essential for single cell RNA-seq (scRNAseq) analysis. Principal component analysis (PCA) is widely used, but requires continuous, normally-distributed data; therefore, it is often coupled with log-transformation in scRNAseq applications, which can distort the data and obscure meaningful variation. We describe correspondence analysis (CA), a count-based alternative to PCA. CA is based on decomposition of a chi-squared residual matrix, avoiding distortive log-transformation. To address overdispersion and high sparsity in scRNAseq data, we propose five adaptations of CA, which are fast, scalable, and outperform standard CA and glmPCA, to compute cell embeddings with more performant or comparable clustering accuracy in 8 out of 9 datasets. In particular, we find that CA with Freeman-Tukey residuals performs especially well across diverse datasets. Other advantages of the CA framework include visualization of associations between genes and cell populations in a "CA biplot," and extension to multi-table analysis; we introduce corralm for integrative multi-table dimension reduction of scRNAseq data. We implement CA for scRNAseq data in corral, an R/Bioconductor package which interfaces directly with single cell classes in Bioconductor. Switching from PCA to CA is achieved through a simple pipeline substitution and improves dimension reduction of scRNAseq datasets.