Predicting patient outcomes after treatment with immune checkpoint blockade: A review of biomarkers derived from diverse data modalities.

Immune checkpoint blockade (ICB) therapy targeting cytotoxic T-lymphocyte-associated protein 4, programmed death 1, and programmed death ligand 1 has shown durable remission and clinical success across different cancer types. However, patient outcomes vary among disease indications. Studies have identified prognostic biomarkers associated with immunotherapy response and patient outcomes derived from diverse data types, including next-generation bulk and single-cell DNA, RNA, T cell and B cell receptor sequencing data, liquid biopsies, and clinical imaging. Owing to inter- and intra-tumor heterogeneity and the immune system's complexity, these biomarkers have diverse efficacy in clinical trials of ICB. Here, we review the genetic and genomic signatures and image features of ICB studies for pan-cancer applications and specific indications. We discuss the advantages and disadvantages of computational approaches for predicting immunotherapy effectiveness and patient outcomes. We also elucidate the challenges of immunotherapy prognostication and the discovery of novel immunotherapy targets.

Tutorial: integrative computational analysis of bulk RNA-sequencing data to characterize tumor immunity using RIMA.

RNA-sequencing (RNA-seq) has become an increasingly cost-effective technique for molecular profiling and immune characterization of tumors. In the past decade, many computational tools have been developed to characterize tumor immunity from gene expression data. However, the analysis of large-scale RNA-seq data requires bioinformatics proficiency, large computational resources and cancer genomics and immunology knowledge. In this tutorial, we provide an overview of computational analysis of bulk RNA-seq data for immune characterization of tumors and introduce commonly used computational tools with relevance to cancer immunology and immunotherapy. These tools have diverse functions such as evaluation of expression signatures, estimation of immune infiltration, inference of the immune repertoire, prediction of immunotherapy response, neoantigen detection and microbiome quantification. We describe the RNA-seq IMmune Analysis (RIMA) pipeline integrating many of these tools to streamline RNA-seq analysis. We also developed a comprehensive and user-friendly guide in the form of a GitBook with text and video demos to assist users in analyzing bulk RNA-seq data for immune characterization at both individual sample and cohort levels by using RIMA.

Hippo Signaling Pathway Regulates Cancer Cell-Intrinsic MHC-II Expression.

MHC-II is known to be mainly expressed on the surface of antigen-presenting cells. Evidence suggests MHC-II is also expressed by cancer cells and may be associated with better immunotherapy responses. However, the role and regulation of MHC-II in cancer cells remain unclear. In this study, we leveraged data mining and experimental validation to elucidate the regulation of MHC-II in cancer cells and its role in modulating the response to immunotherapy. We collated an extensive collection of omics data to examine cancer cell-intrinsic MHC-II expression and its association with immunotherapy outcomes. We then tested the functional relevance of cancer cell-intrinsic MHC-II expression using a syngeneic transplantation model. Finally, we performed data mining to identify pathways potentially involved in the regulation of MHC-II expression, and experimentally validated candidate regulators. Analyses of preimmunotherapy clinical samples in the CheckMate 064 trial revealed that cancer cell-intrinsic MHC-II protein was positively correlated with more favorable immunotherapy outcomes. Comprehensive meta-analyses of multiomics data from an exhaustive collection of data revealed that MHC-II is heterogeneously expressed in various solid tumors, and its expression is particularly high in melanoma. Using a syngeneic transplantation model, we further established that melanoma cells with high MHC-II responded better to anti-PD-1 treatment. Data mining followed by experimental validation revealed the Hippo signaling pathway as a potential regulator of melanoma MHC-II expression. In summary, we identified the Hippo signaling pathway as a novel regulator of cancer cell-intrinsic MHC-II expression. These findings suggest modulation of MHC-II in melanoma could potentially improve immunotherapy response.

Machine learning on syngeneic mouse tumor profiles to model clinical immunotherapy response.

Most patients with cancer are refractory to immune checkpoint blockade (ICB) therapy, and proper patient stratification remains an open question. Primary patient data suffer from high heterogeneity, low accessibility, and lack of proper controls. In contrast, syngeneic mouse tumor models enable controlled experiments with ICB treatments. Using transcriptomic and experimental variables from >700 ICB-treated/control syngeneic mouse tumors, we developed a machine learning framework to model tumor immunity and identify factors influencing ICB response. Projected on human immunotherapy trial data, we found that the model can predict clinical ICB response. We further applied the model to predicting ICB-responsive/resistant cancer types in The Cancer Genome Atlas, which agreed well with existing clinical reports. Last, feature analysis implicated factors associated with ICB response. In summary, our computational framework based on mouse tumor data reliably stratified patients regarding ICB response, informed resistance mechanisms, and has the potential for wide applications in disease treatment studies.

TISMO: syngeneic mouse tumor database to model tumor immunity and immunotherapy response.

Syngeneic mouse models are tumors derived from murine cancer cells engrafted on genetically identical mouse strains. They are widely used tools for studying tumor immunity and immunotherapy response in the context of a fully functional murine immune system. Large volumes of syngeneic mouse tumor expression profiles under different immunotherapy treatments have been generated, although a lack of systematic collection and analysis makes data reuse challenging. We present Tumor Immune Syngeneic MOuse (TISMO), a database with an extensive collection of syngeneic mouse model profiles with interactive visualization features. TISMO contains 605 in vitro RNA-seq samples from 49 syngeneic cancer cell lines across 23 cancer types, of which 195 underwent cytokine treatment. TISMO also includes 1518 in vivo RNA-seq samples from 68 syngeneic mouse tumor models across 19 cancer types, of which 832 were from immune checkpoint blockade (ICB) studies. We manually annotated the sample metadata, such as cell line, mouse strain, transplantation site, treatment, and response status, and uniformly processed and quality-controlled the RNA-seq data. Besides data download, TISMO provides interactive web interfaces to investigate whether specific gene expression, pathway enrichment, or immune infiltration level is associated with differential immunotherapy response. TISMO is available at http://tismo.cistrome.org.

Development of a plasminogen activator inhibitor (PAI-1) assay and comparison of plasma PAI-1 activity in hyperlipidemic/dyslipidemic dogs with either hyperadrenocorticism or diabetes mellitus, and healthy dogs.

Thrombosis is a serious complication of many canine diseases and may be related to decreased fibrinolytic potential. Plasminogen activator inhibitor-1 (PAI-1) is the key regulator of fibrinolysis with increased levels demonstrated in states of pro-thrombosis and abnormal lipid metabolism. Our objective was to develop and validate a canine PAI-1 activity assay and test whether dogs with hyperadrenocorticism or diabetes mellitus that are hyperlipidemic/dyslipidemic have increased plasma PAI-1 activity. Functionally active PAI-1 in the plasma sample was incubated with recombinant tissue plasminogen activator (tPA), allowing the formation of a 1:1 stoichiometric inactive complex. Residual unbound tPA was then reacted with excess plasminogen in the presence of a colorimetric plasmin substrate. Plasmin production is quantified by computing the area under the curve of time (x) vs optical density (y) plot and converted to tPA IU/mL by comparison to a calibration curve of tPA standards. PAI-1 activity was determined by calculating the proportion of exogeneous tPA suppressed by PAI-1 in plasma. Assay verification included assessment of linearity, specificity, precision, sensitivity, and stability. PAI-1 activity was increased in hyperlipidemic compared to healthy dogs, but there was no significant difference between dogs with hyperadrenocorticism and diabetes mellitus. A near significant decrease in activity was detected in thawed plasma stored for 20h at 4°C. Our successfully validated assay offers a new tool for investigating fibrinolysis in dogs. Investigation of PAI-1 activity in dogs with other diseases associated with an increased risk of thrombosis would be valuable. Future studies of PAI-1 activity should consider its lability.