Validation of genomic and transcriptomic models of homologous recombination deficiency in a real-world pan-cancer cohort.

BACKGROUND: With the introduction of DNA-damaging therapies into standard of care cancer treatment, there is a growing need for predictive diagnostics assessing homologous recombination deficiency (HRD) status across tumor types. Following the strong clinical evidence for the utility of DNA-sequencing-based HRD testing in ovarian cancer, and growing evidence in breast cancer, we present analytical validation of the Tempus HRD-DNA test. We further developed, validated, and explored the Tempus HRD-RNA model, which uses gene expression data from 16,750 RNA-seq samples to predict HRD status from formalin-fixed paraffin-embedded tumor samples across numerous cancer types. METHODS: Genomic and transcriptomic profiling was performed using next-generation sequencing from Tempus xT, Tempus xO, Tempus xE, Tempus RS, and Tempus RS.v2 assays on 48,843 samples. Samples were labeled based on their BRCA1, BRCA2 and selected Homologous Recombination Repair pathway gene (CDK12, PALB2, RAD51B, RAD51C, RAD51D) mutational status to train and validate HRD-DNA, a genome-wide loss-of-heterozygosity biomarker, and HRD-RNA, a logistic regression model trained on gene expression. RESULTS: In a sample of 2058 breast and 1216 ovarian tumors, BRCA status was predicted by HRD-DNA with F1-scores of 0.98 and 0.96, respectively. Across an independent set of 1363 samples across solid tumor types, the HRD-RNA model was predictive of BRCA status in prostate, pancreatic, and non-small cell lung cancer, with F1-scores of 0.88, 0.69, and 0.62, respectively. CONCLUSIONS: We predict HRD-positive patients across many cancer types and believe both HRD models may generalize to other mechanisms of HRD outside of BRCA loss. HRD-RNA complements DNA-based HRD detection methods, especially for indications with low prevalence of BRCA alterations.

Mixed graphical models for integrative causal analysis with application to chronic lung disease diagnosis and prognosis.

MOTIVATION: Integration of data from different modalities is a necessary step for multi-scale data analysis in many fields, including biomedical research and systems biology. Directed graphical models offer an attractive tool for this problem because they can represent both the complex, multivariate probability distributions and the causal pathways influencing the system. Graphical models learned from biomedical data can be used for classification, biomarker selection and functional analysis, while revealing the underlying network structure and thus allowing for arbitrary likelihood queries over the data. RESULTS: In this paper, we present and test new methods for finding directed graphs over mixed data types (continuous and discrete variables). We used this new algorithm, CausalMGM, to identify variables directly linked to disease diagnosis and progression in various multi-modal datasets, including clinical datasets from chronic obstructive pulmonary disease (COPD). COPD is the third leading cause of death and a major cause of disability and thus determining the factors that cause longitudinal lung function decline is very important. Applied on a COPD dataset, mixed graphical models were able to confirm and extend previously described causal effects and provide new insights on the factors that potentially affect the longitudinal lung function decline of COPD patients. AVAILABILITY AND IMPLEMENTATION: The CausalMGM package is available on http://www.causalmgm.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Data-Independent Acquisition Mass Spectrometry To Quantify Protein Levels in FFPE Tumor Biopsies for Molecular Diagnostics.

Mass spectrometry-based protein quantitation is currently used to measure therapeutically relevant protein biomarkers in CAP/CLIA setting to predict likely responses of known therapies. Selected reaction monitoring (SRM) is the method of choice due to its outstanding analytical performance. However, data-independent acquisition (DIA) is now emerging as a proteome-scale clinical assay. We evaluated the ability of DIA to profile the patient-specific proteomes of sample-limited tumor biopsies and to quantify proteins of interest in a targeted fashion using formalin-fixed, paraffin-embedded (FFPE) tumor biopsies ( n = 12) selected from our clinical laboratory. DIA analysis on the tumor biopsies provided 3713 quantifiable proteins including actionable biomarkers currently in clinical use, successfully separated two gastric cancers from colorectal cancer specimen solely on the basis of global proteomic profiles, and identified subtype-specific proteins with prognostic or diagnostic value. We demonstrate the potential use of DIA-based quantitation to inform therapeutic decision-making using TUBB3, for which clinical cutoff expression levels have been established by SRM. Comparative analysis of DIA-based proteomic profiles and mRNA expression levels found positively and negatively correlated protein-gene pairs, a finding consistent with previously reported results from fresh-frozen tumor tissues.

Learning mixed graphical models with separate sparsity parameters and stability-based model selection.

BACKGROUND: Mixed graphical models (MGMs) are graphical models learned over a combination of continuous and discrete variables. Mixed variable types are common in biomedical datasets. MGMs consist of a parameterized joint probability density, which implies a network structure over these heterogeneous variables. The network structure reveals direct associations between the variables and the joint probability density allows one to ask arbitrary probabilistic questions on the data. This information can be used for feature selection, classification and other important tasks. RESULTS: We studied the properties of MGM learning and applications of MGMs to high-dimensional data (biological and simulated). Our results show that MGMs reliably uncover the underlying graph structure, and when used for classification, their performance is comparable to popular discriminative methods (lasso regression and support vector machines). We also show that imposing separate sparsity penalties for edges connecting different types of variables significantly improves edge recovery performance. To choose these sparsity parameters, we propose a new efficient model selection method, named Stable Edge-specific Penalty Selection (StEPS). StEPS is an expansion of an earlier method, StARS, to mixed variable types. In terms of edge recovery, StEPS selected MGMs outperform those models selected using standard techniques, including AIC, BIC and cross-validation. In addition, we use a heuristic search that is linear in size of the sparsity value search space as opposed to the cubic grid search required by other model selection methods. We applied our method to clinical and mRNA expression data from the Lung Genomics Research Consortium (LGRC) and the learned MGM correctly recovered connections between the diagnosis of obstructive or interstitial lung disease, two diagnostic breathing tests, and cigarette smoking history. Our model also suggested biologically relevant mRNA markers that are linked to these three clinical variables. CONCLUSIONS: MGMs are able to accurately recover dependencies between sets of continuous and discrete variables in both simulated and biomedical datasets. Separation of sparsity penalties by edge type is essential for accurate network edge recovery. Furthermore, our stability based method for model selection determines sparsity parameters faster and more accurately (in terms of edge recovery) than other model selection methods. With the ongoing availability of comprehensive clinical and biomedical datasets, MGMs are expected to become a valuable tool for investigating disease mechanisms and answering an array of critical healthcare questions.

Learning subgroup-specific regulatory interactions and regulator independence with PARADIGM.

UNLABELLED: High-dimensional '-omics' profiling provides a detailed molecular view of individual cancers; however, understanding the mechanisms by which tumors evade cellular defenses requires deep knowledge of the underlying cellular pathways within each cancer sample. We extended the PARADIGM algorithm (Vaske et al., 2010, Bioinformatics, 26, i237-i245), a pathway analysis method for combining multiple '-omics' data types, to learn the strength and direction of 9139 gene and protein interactions curated from the literature. Using genomic and mRNA expression data from 1936 samples in The Cancer Genome Atlas (TCGA) cohort, we learned interactions that provided support for and relative strength of 7138 (78%) of the curated links. Gene set enrichment found that genes involved in the strongest interactions were significantly enriched for transcriptional regulation, apoptosis, cell cycle regulation and response to tumor cells. Within the TCGA breast cancer cohort, we assessed different interaction strengths between breast cancer subtypes, and found interactions associated with the MYC pathway and the ER alpha network to be among the most differential between basal and luminal A subtypes. PARADIGM with the Naive Bayesian assumption produced gene activity predictions that, when clustered, found groups of patients with better separation in survival than both the original version of PARADIGM and a version without the assumption. We found that this Naive Bayes assumption was valid for the vast majority of co-regulators, indicating that most co-regulators act independently on their shared target. AVAILABILITY: http://paradigm.five3genomics.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Efficient stochastic simulation of chemical kinetics networks using a weighted ensemble of trajectories.

We apply the "weighted ensemble" (WE) simulation strategy, previously employed in the context of molecular dynamics simulations, to a series of systems-biology models that range in complexity from a one-dimensional system to a system with 354 species and 3680 reactions. WE is relatively easy to implement, does not require extensive hand-tuning of parameters, does not depend on the details of the simulation algorithm, and can facilitate the simulation of extremely rare events. For the coupled stochastic reaction systems we study, WE is able to produce accurate and efficient approximations of the joint probability distribution for all chemical species for all time t. WE is also able to efficiently extract mean first passage times for the systems, via the construction of a steady-state condition with feedback. In all cases studied here, WE results agree with independent "brute-force" calculations, but significantly enhance the precision with which rare or slow processes can be characterized. Speedups over "brute-force" in sampling rare events via the Gillespie direct Stochastic Simulation Algorithm range from ~10(12) to ~10(18) for characterizing rare states in a distribution, and ~10(2) to ~10(4) for finding mean first passage times.

A Genomic Analysis of Esophageal Squamous Cell Carcinoma in Eastern Africa.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) comprises 90% of all esophageal cancer cases globally and is the most common histology in low-resource settings. Eastern Africa has a disproportionately high incidence of ESCC. METHODS: We describe the genomic profiles of 61 ESCC cases from Tanzania and compare them to profiles from an existing cohort of ESCC cases from Malawi. We also provide a comparison to ESCC tumors in The Cancer Genome Atlas (TCGA). RESULTS: We observed substantial transcriptional overlap with other squamous histologies via comparison with TCGA PanCan dataset. DNA analysis revealed known mutational patterns, both genome-wide as well as in genes known to be commonly mutated in ESCC. TP53 mutations were the most common somatic mutation in tumors from both Tanzania and Malawi but were detected at lower frequencies than previously reported in ESCC cases from other settings. In a combined analysis, two unique transcriptional clusters were identified: a proliferative/epithelial cluster and an invasive/migrative/mesenchymal cluster. Mutational signature analysis of the Tanzanian cohort revealed common signatures associated with aging and cytidine deaminase activity (APOBEC) and an absence of signature 29, which was previously reported in the Malawi cohort. CONCLUSIONS: This study defines the molecular characteristics of ESCC in Tanzania, and enriches the Eastern African dataset, with findings of overall similarities but also some heterogeneity across two unique sites. IMPACT: Despite a high burden of ESCC in Eastern Africa, investigations into the genomics in this region are nascent. This represents the largest comprehensive genomic analysis ESCC from sub-Saharan Africa to date.

A single-cell transcriptional gradient in human cutaneous memory T cells restricts Th17/Tc17 identity.

The homeostatic mechanisms that fail to restrain chronic tissue inflammation in diseases, such as psoriasis vulgaris, remain incompletely understood. We profiled transcriptomes and epitopes of single psoriatic and normal skin-resident T cells, revealing a gradated transcriptional program of coordinately regulated inflammation-suppressive genes. This program, which is sharply suppressed in lesional skin, strikingly restricts Th17/Tc17 cytokine and other inflammatory mediators on the single-cell level. CRISPR-based deactivation of two core components of this inflammation-suppressive program, ZFP36L2 and ZFP36, replicates the interleukin-17A (IL-17A), granulocyte macrophage-colony-stimulating factor (GM-CSF), and interferon gamma (IFNγ) elevation in psoriatic memory T cells deficient in these transcripts, functionally validating their influence. Combinatoric expression analysis indicates the suppression of specific inflammatory mediators by individual program members. Finally, we find that therapeutic IL-23 blockade reduces Th17/Tc17 cell frequency in lesional skin but fails to normalize this inflammatory-suppressive program, suggesting how treated lesions may be primed for recurrence after withdrawal of treatment.

Large scale, robust, and accurate whole transcriptome profiling from clinical formalin-fixed paraffin-embedded samples.

Transcriptome profiling can provide information of great value in clinical decision-making, yet RNA from readily available formalin-fixed paraffin-embedded (FFPE) tissue is often too degraded for quality sequencing. To assess the clinical utility of FFPE-derived RNA, we performed ribo-deplete RNA extractions on > 3200 FFPE slide samples; 25 of these had direct FFPE vs. fresh frozen (FF) replicates, 57 were sequenced in 2 different labs, 87 underwent multiple library analyses, and 16 had direct microdissected vs. macrodissected replicates. Poly-A versus ribo-depletion RNA extraction methods were compared using transcriptomes of TCGA cohort and 3116 FFPE samples. Compared to FF, FFPE transcripts coding for nuclear/cytoplasmic proteins involved in DNA packaging, replication, and protein synthesis were detected at lower rates and zinc finger family transcripts were of poorer quality. The greatest difference in extraction methods was in histone transcripts which typically lack poly-A tails. Encouragingly, the overall sequencing success rate was 81%. Exome coverage was highly concordant in direct FFPE and FF replicates, with 98% agreement in coding exon coverage and a median correlation of whole transcriptome profiles of 0.95. We provide strong rationale for clinical use of FFPE-derived RNA based on the robustness, reproducibility, and consistency of whole transcriptome profiling.

Single-Cell Profiling Reveals Divergent, Globally Patterned Immune Responses in Murine Skin Inflammation.

Inflammatory response heterogeneity has impeded high-resolution dissection of diverse immune cell populations during activation. We characterize mouse cutaneous immune cells by single-cell RNA sequencing, after inducing inflammation using imiquimod and oxazolone dermatitis models. We identify 13 CD45+ subpopulations, which broadly represent most functionally characterized immune cell types. Oxazolone pervasively upregulates Jak2/Stat3 expression across T cells and antigen-presenting cells (APCs). Oxazolone also induces Il4/Il13 expression in newly infiltrating basophils, and Il4ra and Ccl24, most prominently in APCs. In contrast, imiquimod broadly upregulates Il17/Il22 and Ccl4/Ccl5. A comparative analysis of single-cell inflammatory transcriptional responses reveals that APC response to oxazolone is tightly restricted by cell identity, whereas imiquimod enforces shared programs on multiple APC populations in parallel. These global molecular patterns not only contrast immune responses on a systems level but also suggest that the mechanisms of new sources of inflammation can eventually be deduced by comparison to known signatures.

PARP1 rs1805407 Increases Sensitivity to PARP1 Inhibitors in Cancer Cells Suggesting an Improved Therapeutic Strategy.

Personalized cancer therapy relies on identifying patient subsets that benefit from a therapeutic intervention and suggest alternative regimens for those who don't. A new data integrative approach, based on graphical models, was applied on our multi-modal -omics, and clinical data cohort of metastatic melanoma patients. We found that response to chemotherapy is directly linked to ten gene expression, four methylation variables and PARP1 SNP rs1805407. PARP1 is a DNA repair gene critical for chemotherapy response and for which FDA-approved inhibitors are clinically available (olaparib). We demonstrated that two PARP inhibitors (ABT-888 and olaparib) make SNP carrier cancer cells of various histologic subtypes more sensitive to alkylating agents, but they have no effect in wild-type cells. Furthermore, PARP1 inhibitors act synergistically with chemotherapy in SNP carrier cells (especially in ovarian cancer for which olaparib is FDA-approved), but they are additive at best in wild-type cancer cells. Taken together, our results suggest that the combination of chemotherapy and PARP1 inhibition may benefit the carriers of rs1805407 in the future and may be used in personalized therapy strategies to select patients that are more likely to respond to PARP inhibitors.

Regional Molecular Signature of the Symptomatic Atherosclerotic Carotid Plaque.

BACKGROUND: Many studies have explored molecular markers of carotid plaque development and vulnerability to rupture, usually having examined whole carotid plaques. However, there are regional differences in plaque morphology and known shear-related mechanisms in areas surrounding the lipid core. OBJECTIVE: To determine whether there are regional differences in protein expression along the long axis of the carotid plaque and how that might produce gaps in our understanding of the carotid plaque molecular signature. METHODS: Levels of 7 inflammatory cytokines (IL-1ß, IL-6, IL-8, IL-10, IL-12 p70, IFN-γ, and TNF-α) and caspase-3 were analyzed in prebifurcation, bifurcation, and postbifurcation segments of internal carotid plaques surgically removed from symptomatic and asymptomatic patients. Expression profiles of miRNAs and mRNAs were determined with microarrays for the rupture-prone postbifurcation segment for comparison with published whole plaque results. RESULTS: Expression levels of all proteins examined, except IL-10, were lowest in the prebifurcation segment and significantly higher in the postbifurcation segment. Patient group differences in protein expression were observed for the prebifurcation segment; however, no significant differences were observed in the postbifurcation segment between symptomatic and asymptomatic patients. Expression profiles from postbifurcation carotid plaques identified 4 novel high priority miRNAs differentially expressed between patient groups (miR-214, miR-484, miR-942, and miR-1287) and 3 high-confidence miRNA:mRNA targets, including miR-214:APOD, miR-484:DACH1, and miR-942:GPR56. CONCLUSION: The results demonstrate regional differences in protein expression for the first time and show that focus on the rupture-prone postbifurcation region leads to prioritization for further study of novel miRNA gene regulation mechanisms.

A20 and ABIN1 Suppression of a Keratinocyte Inflammatory Program with a Shared Single-Cell Expression Signature in Diverse Human Rashes.

Genetic variation in the NF-κB inhibitors, ABIN1 and A20, increase risk for psoriasis. While critical for hematopoietic immune cell function, these genes are believed to additionally inhibit psoriasis by dampening inflammatory signaling in keratinocytes. We dissected ABIN1 and A20's regulatory role in human keratinocyte inflammation using an RNA sequencing-based comparative genomic approach. Here we show subsets of the IL-17 and tumor necrosis factor-α signaling pathways are robustly restricted by A20 overexpression. In contrast, ABIN1 overexpression inhibits these genes more modestly for IL-17, and weakly for tumor necrosis factor-α. Our genome-scale analysis also indicates that inflammatory program suppression appears to be the major transcriptional influence of A20/ABIN1 overexpression, without obvious influence on keratinocyte viability genes. Our findings thus enable dissection of the differing anti-inflammatory mechanisms of two distinct psoriasis modifiers, which may be directly exploited for therapeutic purposes. Importantly, we report that IL-17-induced targets of A20 show similar aberrant epidermal layer-specific transcriptional upregulation in keratinocytes from diseases as diverse as psoriasis, atopic dermatitis, and erythrokeratodermia variabilis, suggesting a contributory role for epidermal inflammation in a broad spectrum of rashes.

Transcriptional Programming of Normal and Inflamed Human Epidermis at Single-Cell Resolution.

Perturbations in the transcriptional programs specifying epidermal differentiation cause diverse skin pathologies ranging from impaired barrier function to inflammatory skin disease. However, the global scope and organization of this complex cellular program remain undefined. Here we report single-cell RNA sequencing profiles of 92,889 human epidermal cells from 9 normal and 3 inflamed skin samples. Transcriptomics-derived keratinocyte subpopulations reflect classic epidermal strata but also sharply compartmentalize epithelial functions such as cell-cell communication, inflammation, and WNT pathway modulation. In keratinocytes, ∼12% of assessed transcript expression varies in coordinate patterns, revealing undescribed gene expression programs governing epidermal homeostasis. We also identify molecular fingerprints of inflammatory skin states, including S100 activation in the interfollicular epidermis of normal scalp, enrichment of a CD1C+CD301A+ myeloid dendritic cell population in psoriatic epidermis, and IL1ßhiCCL3hiCD14+ monocyte-derived macrophages enriched in foreskin. This compendium of RNA profiles provides a critical step toward elucidating epidermal diseases of development, differentiation, and inflammation.