Library size confounds biology in spatial transcriptomics data.

Spatial molecular data has transformed the study of disease microenvironments, though, larger datasets pose an analytics challenge prompting the direct adoption of single-cell RNA-sequencing tools including normalization methods. Here, we demonstrate that library size is associated with tissue structure and that normalizing these effects out using commonly applied scRNA-seq normalization methods will negatively affect spatial domain identification. Spatial data should not be specifically corrected for library size prior to analysis, and algorithms designed for scRNA-seq data should be adopted with caution.

In situ single-cell profiling sheds light on IFI27 localisation during SARS-CoV-2 infection.

The utilization of single-cell resolved spatial transcriptomics to delineate immune responses during SARS-CoV-2 infection was able to identify M1 macrophages to have elevated expression of IFI27 in areas of infection.

vissE: a versatile tool to identify and visualise higher-order molecular phenotypes from functional enrichment analysis.

Functional analysis of high throughput experiments using pathway analysis is now ubiquitous. Though powerful, these methods often produce thousands of redundant results owing to knowledgebase redundancies upstream. This scale of results hinders extensive exploration by biologists and can lead to investigator biases due to previous knowledge and expectations. To address this issue, we present vissE, a flexible network-based analysis and visualisation tool that organises information into semantic categories and provides various visualisation modules to characterise them with respect to the underlying data, thus providing a comprehensive view of the biological system. We demonstrate vissE's versatility by applying it to three different technologies: bulk, single-cell and spatial transcriptomics. Applying vissE to a factor analysis of a breast cancer spatial transcriptomic data, we identified stromal phenotypes that support tumour dissemination. Its adaptability allows vissE to enhance all existing gene-set enrichment and pathway analysis workflows, empowering biologists during molecular discovery.

standR: spatial transcriptomic analysis for GeoMx DSP data.

To gain a better understanding of the complexity of gene expression in normal and diseased tissues it is important to account for the spatial context and identity of cells in situ. State-of-the-art spatial profiling technologies, such as the Nanostring GeoMx Digital Spatial Profiler (DSP), now allow quantitative spatially resolved measurement of the transcriptome in tissues. However, the bioinformatics pipelines currently used to analyse GeoMx data often fail to successfully account for the technical variability within the data and the complexity of experimental designs, thus limiting the accuracy and reliability of the subsequent analysis. Carefully designed quality control workflows, that include in-depth experiment-specific investigations into technical variation and appropriate adjustment for such variation can address this issue. Here, we present standR, an R/Bioconductor package that enables an end-to-end analysis of GeoMx DSP data. With four case studies from previously published experiments, we demonstrate how the standR workflow can enhance the statistical power of GeoMx DSP data analysis and how the application of standR enables scientists to develop in-depth insights into the biology of interest.

Discovery of a highly potent, selective, orally bioavailable inhibitor of KAT6A/B histone acetyltransferases with efficacy against KAT6A-high ER+ breast cancer.

KAT6A, and its paralog KAT6B, are histone lysine acetyltransferases (HAT) that acetylate histone H3K23 and exert an oncogenic role in several tumor types including breast cancer where KAT6A is frequently amplified/overexpressed. However, pharmacologic targeting of KAT6A to achieve therapeutic benefit has been a challenge. Here we describe identification of a highly potent, selective, and orally bioavailable KAT6A/KAT6B inhibitor CTx-648 (PF-9363), derived from a benzisoxazole series, which demonstrates anti-tumor activity in correlation with H3K23Ac inhibition in KAT6A over-expressing breast cancer. Transcriptional and epigenetic profiling studies show reduced RNA Pol II binding and downregulation of genes involved in estrogen signaling, cell cycle, Myc and stem cell pathways associated with CTx-648 anti-tumor activity in ER-positive (ER+) breast cancer. CTx-648 treatment leads to potent tumor growth inhibition in ER+ breast cancer in vivo models, including models refractory to endocrine therapy, highlighting the potential for targeting KAT6A in ER+ breast cancer.

Implementation of DNA Methylation Array Profiling in Pediatric Central Nervous System Tumors: The AIM BRAIN Project: An Australian and New Zealand Children's Haematology/Oncology Group Study.

DNA methylation array profiling for classifying pediatric central nervous system (CNS) tumors is a valuable adjunct to histopathology. However, unbiased prospective and interlaboratory validation studies have been lacking. The AIM BRAIN diagnostic trial involving 11 pediatric cancer centers in Australia and New Zealand was designed to test the feasibility of routine clinical testing and ran in parallel with the Molecular Neuropathology 2.0 (MNP2.0) study at Deutsches Krebsforschungszentrum (German Cancer Research Center). CNS tumors from 269 pediatric patients were prospectively tested on Illumina EPIC arrays, including 104 cases co-enrolled on MNP2.0. Using MNP classifier versions 11b4 and 12.5, we report classifications with a probability score ≥0.90 in 176 of 265 (66.4%) and 213 of 269 (79.2%) cases, respectively. Significant diagnostic information was obtained in 130 of 176 (74%) for 11b4, and 12 of 174 (7%) classifications were discordant with histopathology. Cases prospectively co-enrolled on MNP2.0 gave concordant classifications (99%) and score thresholds (93%), demonstrating excellent test reproducibility and sensitivity. Overall, DNA methylation profiling is a robust single workflow technique with an acceptable diagnostic yield that is considerably enhanced by the extensive subgroup and copy number profile information generated by the platform. The platform has excellent test reproducibility and sensitivity and contributes significantly to CNS tumor diagnosis.

vissE.cloud: a webserver to visualise higher order molecular phenotypes from enrichment analysis.

Gene-set analysis (GSA) dominates the functional interpretation of omics data and downstream hypothesis generation. Despite its ability to summarise thousands of measurements into semantically interpretable components, GSA often results in hundreds of significantly enriched gene-sets. However, summarisation and effective visualisation of GSA results to facilitate hypothesis generation is still lacking. While some webservers provide gene-set visualization tools, there is still a need for tools that can effectively summarize and guide exploration of GSA results. To enable versatility, webservers accept gene lists as input, however, none provide end-to-end solutions for emerging data types such as single-cell and spatial omics. Here, we present vissE.Cloud, a webserver for end-to-end gene-set analysis, offering gene-set summarisation and highly interactive visualisation. vissE.Cloud uses algorithms from our earlier R package vissE to summarise GSA results by identifying biological themes. We maintain versatility by allowing analysis of gene lists, as well as, analysis of raw single-cell and spatial omics data, including CosMx and Xenium data, making vissE.Cloud the first webserver to provide end-to-end gene-set analysis of sub-cellular localised spatial data. Structuring the results hierarchically allows swift interactive investigations of results at the gene, gene-set, and clusters level. vissE.Cloud is freely available at https://www.vissE.Cloud.

Spatial transcriptomic analysis of Sonic hedgehog medulloblastoma identifies that the loss of heterogeneity and promotion of differentiation underlies the response to CDK4/6 inhibition.

BACKGROUND: Medulloblastoma (MB) is a malignant tumour of the cerebellum which can be classified into four major subgroups based on gene expression and genomic features. Single-cell transcriptome studies have defined the cellular states underlying each MB subgroup; however, the spatial organisation of these diverse cell states and how this impacts response to therapy remains to be determined. METHODS: Here, we used spatially resolved transcriptomics to define the cellular diversity within a sonic hedgehog (SHH) patient-derived model of MB and show that cells specific to a transcriptional state or spatial location are pivotal for CDK4/6 inhibitor, Palbociclib, treatment response. We integrated spatial gene expression with histological annotation and single-cell gene expression data from MB, developing an analysis strategy to spatially map cell type responses within the hybrid system of human and mouse cells and their interface within an intact brain tumour section. RESULTS: We distinguish neoplastic and non-neoplastic cells within tumours and from the surrounding cerebellar tissue, further refining pathological annotation. We identify a regional response to Palbociclib, with reduced proliferation and induced neuronal differentiation in both treated tumours. Additionally, we resolve at a cellular resolution a distinct tumour interface where the tumour contacts neighbouring mouse brain tissue consisting of abundant astrocytes and microglia and continues to proliferate despite Palbociclib treatment. CONCLUSIONS: Our data highlight the power of using spatial transcriptomics to characterise the response of a tumour to a targeted therapy and provide further insights into the molecular and cellular basis underlying the response and resistance to CDK4/6 inhibitors in SHH MB.

Transcriptomic profiling of cardiac tissues from SARS-CoV-2 patients identifies DNA damage.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is known to present with pulmonary and extra-pulmonary organ complications. In comparison with the 2009 pandemic (pH1N1), SARS-CoV-2 infection is likely to lead to more severe disease, with multi-organ effects, including cardiovascular disease. SARS-CoV-2 has been associated with acute and long-term cardiovascular disease, but the molecular changes that govern this remain unknown. In this study, we investigated the host transcriptome landscape of cardiac tissues collected at rapid autopsy from seven SARS-CoV-2, two pH1N1, and six control patients using targeted spatial transcriptomics approaches. Although SARS-CoV-2 was not detected in cardiac tissue, host transcriptomics showed upregulation of genes associated with DNA damage and repair, heat shock, and M1-like macrophage infiltration in the cardiac tissues of COVID-19 patients. The DNA damage present in the SARS-CoV-2 patient samples, were further confirmed by γ-H2Ax immunohistochemistry. In comparison, pH1N1 showed upregulation of interferon-stimulated genes, in particular interferon and complement pathways, when compared with COVID-19 patients. These data demonstrate the emergence of distinct transcriptomic profiles in cardiac tissues of SARS-CoV-2 and pH1N1 influenza infection supporting the need for a greater understanding of the effects on extra-pulmonary organs, including the cardiovascular system of COVID-19 patients, to delineate the immunopathobiology of SARS-CoV-2 infection, and long term impact on health.

Computational Screening of Anti-Cancer Drugs Identifies a New BRCA Independent Gene Expression Signature to Predict Breast Cancer Sensitivity to Cisplatin.

The development of therapies that target specific disease subtypes has dramatically improved outcomes for patients with breast cancer. However, survival gains have not been uniform across patients, even within a given molecular subtype. Large collections of publicly available drug screening data matched with transcriptomic measurements have facilitated the development of computational models that predict response to therapy. Here, we generated a series of predictive gene signatures to estimate the sensitivity of breast cancer samples to 90 drugs, comprising FDA-approved drugs or compounds in early development. To achieve this, we used a cell line-based drug screen with matched transcriptomic data to derive in silico models that we validated in large independent datasets obtained from cell lines and patient-derived xenograft (PDX) models. Robust computational signatures were obtained for 28 drugs and used to predict drug efficacy in a set of PDX models. We found that our signature for cisplatin can be used to identify tumors that are likely to respond to this drug, even in absence of the BRCA-1 mutation routinely used to select patients for platinum-based therapies. This clinically relevant observation was confirmed in multiple PDXs. Our study foreshadows an effective delivery approach for precision medicine.

Profiling of lung SARS-CoV-2 and influenza virus infection dissects virus-specific host responses and gene signatures.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which emerged in late 2019 has spread globally, causing a pandemic of respiratory illness designated coronavirus disease 2019 (COVID-19). A better definition of the pulmonary host response to SARS-CoV-2 infection is required to understand viral pathogenesis and to validate putative COVID-19 biomarkers that have been proposed in clinical studies. METHODS: Here, we use targeted transcriptomics of formalin-fixed paraffin-embedded tissue using the NanoString GeoMX platform to generate an in-depth picture of the pulmonary transcriptional landscape of COVID-19, pandemic H1N1 influenza and uninfected control patients. RESULTS: Host transcriptomics showed a significant upregulation of genes associated with inflammation, type I interferon production, coagulation and angiogenesis in the lungs of COVID-19 patients compared to non-infected controls. SARS-CoV-2 was non-uniformly distributed in lungs (emphasising the advantages of spatial transcriptomics) with the areas of high viral load associated with an increased type I interferon response. Once the dominant cell type present in the sample, within patient correlations and patient-patient variation, had been controlled for, only a very limited number of genes were differentially expressed between the lungs of fatal influenza and COVID-19 patients. Strikingly, the interferon-associated gene IFI27, previously identified as a useful blood biomarker to differentiate bacterial and viral lung infections, was significantly upregulated in the lungs of COVID-19 patients compared to patients with influenza. CONCLUSION: Collectively, these data demonstrate that spatial transcriptomics is a powerful tool to identify novel gene signatures within tissues, offering new insights into the pathogenesis of SARS-COV-2 to aid in patient triage and treatment.

Systems pharmacogenomics identifies novel targets and clinically actionable therapeutics for medulloblastoma.

BACKGROUND: Medulloblastoma (MB) is the most common malignant paediatric brain tumour and a leading cause of cancer-related mortality and morbidity. Existing treatment protocols are aggressive in nature resulting in significant neurological, intellectual and physical disabilities for the children undergoing treatment. Thus, there is an urgent need for improved, targeted therapies that minimize these harmful side effects. METHODS: We identified candidate drugs for MB using a network-based systems-pharmacogenomics approach: based on results from a functional genomics screen, we identified a network of interactions implicated in human MB growth regulation. We then integrated drugs and their known mechanisms of action, along with gene expression data from a large collection of medulloblastoma patients to identify drugs with potential to treat MB. RESULTS: Our analyses identified drugs targeting CDK4, CDK6 and AURKA as strong candidates for MB; all of these genes are well validated as drug targets in other tumour types. We also identified non-WNT MB as a novel indication for drugs targeting TUBB, CAD, SNRPA, SLC1A5, PTPRS, P4HB and CHEK2. Based upon these analyses, we subsequently demonstrated that one of these drugs, the new microtubule stabilizing agent, ixabepilone, blocked tumour growth in vivo in mice bearing patient-derived xenograft tumours of the Sonic Hedgehog and Group 3 subtype, providing the first demonstration of its efficacy in MB. CONCLUSIONS: Our findings confirm that this data-driven systems pharmacogenomics strategy is a powerful approach for the discovery and validation of novel therapeutic candidates relevant to MB treatment, and along with data validating ixabepilone in PDX models of the two most aggressive subtypes of medulloblastoma, we present the network analysis framework as a resource for the field.

Stable gene expression for normalisation and single-sample scoring.

Gene expression signatures have been critical in defining the molecular phenotypes of cells, tissues, and patient samples. Their most notable and widespread clinical application is stratification of breast cancer patients into molecular (PAM50) subtypes. The cost and relatively large amounts of fresh starting material required for whole-transcriptome sequencing has limited clinical application of thousands of existing gene signatures captured in repositories such as the Molecular Signature Database. We identified genes with stable expression across a range of abundances, and with a preserved relative ordering across thousands of samples, allowing signature scoring and supporting general data normalisation for transcriptomic data. Our new method, stingscore, quantifies and summarises relative expression levels of signature genes from individual samples through the inclusion of these 'stably-expressed genes'. We show that our list of stable genes has better stability across cancer and normal tissue data than previously proposed gene sets. Additionally, we show that signature scores computed from targeted transcript measurements using stingscore can predict docetaxel response in breast cancer patients. This new approach to gene expression signature analysis will facilitate the development of panel-type tests for gene expression signatures, thus supporting clinical translation of the powerful insights gained from cancer transcriptomic studies.

Using singscore to predict mutation status in acute myeloid leukemia from transcriptomic signatures.

Advances in RNA sequencing (RNA-seq) technologies that measure the transcriptome of biological samples have revolutionised our ability to understand transcriptional regulatory programs that underpin diseases such as cancer. We recently published singscore - a single sample, rank-based gene set scoring method which quantifies how concordant the transcriptional profile of individual samples are relative to specific gene sets of interest. Here we demonstrate the application of singscore to investigate transcriptional profiles associated with specific mutations or genetic lesions in acute myeloid leukemia. Using matched genomic and transcriptomic data available through the TCGA we show that scoring of appropriate signatures can distinguish samples with corresponding mutations, reflecting the ability of these mutations to drive aberrant transcriptional programs involved in leukemogenesis. We believe the singscore method is particularly useful for studying heterogeneity within a specific subsets of cancers, and as demonstrated, we show the ability of singscore to identify where alternative mutations appear to drive similar transcriptional programs.

Differential co-expression-based detection of conditional relationships in transcriptional data: comparative analysis and application to breast cancer.

BACKGROUND: Elucidation of regulatory networks, including identification of regulatory mechanisms specific to a given biological context, is a key aim in systems biology. This has motivated the move from co-expression to differential co-expression analysis and numerous methods have been developed subsequently to address this task; however, evaluation of methods and interpretation of the resulting networks has been hindered by the lack of known context-specific regulatory interactions. RESULTS: In this study, we develop a simulator based on dynamical systems modelling capable of simulating differential co-expression patterns. With the simulator and an evaluation framework, we benchmark and characterise the performance of inference methods. Defining three different levels of "true" networks for each simulation, we show that accurate inference of causation is difficult for all methods, compared to inference of associations. We show that a z-score-based method has the best general performance. Further, analysis of simulation parameters reveals five network and simulation properties that explained the performance of methods. The evaluation framework and inference methods used in this study are available in the dcanr R/Bioconductor package. CONCLUSIONS: Our analysis of networks inferred from simulated data show that hub nodes are more likely to be differentially regulated targets than transcription factors. Based on this observation, we propose an interpretation of the inferred differential network that can reconstruct a putative causal network.

Single sample scoring of molecular phenotypes.

BACKGROUND: Gene set scoring provides a useful approach for quantifying concordance between sample transcriptomes and selected molecular signatures. Most methods use information from all samples to score an individual sample, leading to unstable scores in small data sets and introducing biases from sample composition (e.g. varying numbers of samples for different cancer subtypes). To address these issues, we have developed a truly single sample scoring method, and associated R/Bioconductor package singscore ( https://bioconductor.org/packages/singscore ). RESULTS: We use multiple cancer data sets to compare singscore against widely-used methods, including GSVA, z-score, PLAGE, and ssGSEA. Our approach does not depend upon background samples and scores are thus stable regardless of the composition and number of samples being scored. In contrast, scores obtained by GSVA, z-score, PLAGE and ssGSEA can be unstable when less data are available (NS < 25). The singscore method performs as well as the best performing methods in terms of power, recall, false positive rate and computational time, and provides consistently high and balanced performance across all these criteria. To enhance the impact and utility of our method, we have also included a set of functions implementing visual analysis and diagnostics to support the exploration of molecular phenotypes in single samples and across populations of data. CONCLUSIONS: The singscore method described here functions independent of sample composition in gene expression data and thus it provides stable scores, which are particularly useful for small data sets or data integration. Singscore performs well across all performance criteria, and includes a suite of powerful visualization functions to assist in the interpretation of results. This method performs as well as or better than other scoring approaches in terms of its power to distinguish samples with distinct biology and its ability to call true differential gene sets between two conditions. These scores can be used for dimensional reduction of transcriptomic data and the phenotypic landscapes obtained by scoring samples against multiple molecular signatures may provide insights for sample stratification.