Key Parameters of Tumor Epitope Immunogenicity Revealed Through a Consortium Approach Improve Neoantigen Prediction.

Many approaches to identify therapeutically relevant neoantigens couple tumor sequencing with bioinformatic algorithms and inferred rules of tumor epitope immunogenicity. However, there are no reference data to compare these approaches, and the parameters governing tumor epitope immunogenicity remain unclear. Here, we assembled a global consortium wherein each participant predicted immunogenic epitopes from shared tumor sequencing data. 608 epitopes were subsequently assessed for T cell binding in patient-matched samples. By integrating peptide features associated with presentation and recognition, we developed a model of tumor epitope immunogenicity that filtered out 98% of non-immunogenic peptides with a precision above 0.70. Pipelines prioritizing model features had superior performance, and pipeline alterations leveraging them improved prediction performance. These findings were validated in an independent cohort of 310 epitopes prioritized from tumor sequencing data and assessed for T cell binding. This data resource enables identification of parameters underlying effective anti-tumor immunity and is available to the research community.

Non-viral precision T cell receptor replacement for personalized cell therapy.

T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells1-3. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRß). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.

Rigorous benchmarking of T-cell receptor repertoire profiling methods for cancer RNA sequencing.

The ability to identify and track T-cell receptor (TCR) sequences from patient samples is becoming central to the field of cancer research and immunotherapy. Tracking genetically engineered T cells expressing TCRs that target specific tumor antigens is important to determine the persistence of these cells and quantify tumor responses. The available high-throughput method to profile TCR repertoires is generally referred to as TCR sequencing (TCR-Seq). However, the available TCR-Seq data are limited compared with RNA sequencing (RNA-Seq). In this paper, we have benchmarked the ability of RNA-Seq-based methods to profile TCR repertoires by examining 19 bulk RNA-Seq samples across 4 cancer cohorts including both T-cell-rich and T-cell-poor tissue types. We have performed a comprehensive evaluation of the existing RNA-Seq-based repertoire profiling methods using targeted TCR-Seq as the gold standard. We also highlighted scenarios under which the RNA-Seq approach is suitable and can provide comparable accuracy to the TCR-Seq approach. Our results show that RNA-Seq-based methods are able to effectively capture the clonotypes and estimate the diversity of TCR repertoires, as well as provide relative frequencies of clonotypes in T-cell-rich tissues and low-diversity repertoires. However, RNA-Seq-based TCR profiling methods have limited power in T-cell-poor tissues, especially in highly diverse repertoires of T-cell-poor tissues. The results of our benchmarking provide an additional appealing argument to incorporate RNA-Seq into the immune repertoire screening of cancer patients as it offers broader knowledge into the transcriptomic changes that exceed the limited information provided by TCR-Seq.

A Spatial Omnibus Test (SPOT) for Spatial Proteomic Data.

MOTIVATION: Spatial proteomics can reveal the spatial organization of immune cells in the tumor immune microenvironment. Relating measures of spatial clustering, such as Ripley's K or Besag's L, to patient outcomes may offer important clinical insights. However, these measures require pre-specifying a radius in which to quantify clustering, yet no consensus exists on the optimal radius which may be context-specific. RESULTS: We propose a SPatial Omnibus Test (SPOT) which conducts this analysis across a range of candidate radii. At each radius, SPOT evaluates the association between the spatial summary and outcome, adjusting for confounders. SPOT then aggregates results across radii using the Cauchy combination test, yielding an omnibus p-value characterizing the overall degree of association. Using simulations, we verify that the type I error rate is controlled and show SPOT can be more powerful than alternatives. We also apply SPOT to an ovarian and lung cancer study. AVAILABILITY: An R package and tutorial is provided at https://github.com/sarahsamorodnitsky/SPOT.

CIViCdb 2022: evolution of an open-access cancer variant interpretation knowledgebase.

CIViC (Clinical Interpretation of Variants in Cancer; civicdb.org) is a crowd-sourced, public domain knowledgebase composed of literature-derived evidence characterizing the clinical utility of cancer variants. As clinical sequencing becomes more prevalent in cancer management, the need for cancer variant interpretation has grown beyond the capability of any single institution. CIViC contains peer-reviewed, published literature curated and expertly-moderated into structured data units (Evidence Items) that can be accessed globally and in real time, reducing barriers to clinical variant knowledge sharing. We have extended CIViC's functionality to support emergent variant interpretation guidelines, increase interoperability with other variant resources, and promote widespread dissemination of structured curated data. To support the full breadth of variant interpretation from basic to translational, including integration of somatic and germline variant knowledge and inference of drug response, we have enabled curation of three new Evidence Types (Predisposing, Oncogenic and Functional). The growing CIViC knowledgebase has over 300 contributors and distributes clinically-relevant cancer variant data currently representing >3200 variants in >470 genes from >3100 publications.

ERK mediates interferon gamma-induced melanoma cell death.

BACKGROUND: Interferon-gamma (IFNγ) exerts potent growth inhibitory effects on a wide range of cancer cells through unknown signaling pathways. We pursued complementary screening approaches to characterize the growth inhibition pathway. METHODS: We performed chemical genomics and whole genome targeting CRISPR/Cas9 screens using patient-derived melanoma lines to uncover essential nodes in the IFNγ-mediated growth inhibition pathway. We used transcriptomic profiling to identify cell death pathways activated upon IFNγ exposure. Live imaging experiments coupled with apoptosis assays confirmed the involvement of these pathways in IFNγ-mediated cell death. RESULTS: We show that IFNγ signaling activated ERK. Blocking ERK activation rescued IFNγ-mediated apoptosis in 17 of 23 (~ 74%) cell lines representing BRAF, NRAS, NF1 mutant, and triple wild type subtypes of cutaneous melanoma. ERK signaling induced a stress response, ultimately leading to apoptosis through the activity of DR5 and NOXA proteins. CONCLUSIONS: Our results provide a new understanding of the IFNγ growth inhibition pathway, which will be crucial in defining mechanisms of immunotherapy response and resistance.

Elucidating the role of long intergenic non-coding RNA 339 in human endometrium and endometriosis.

Endometriosis is a complex disease, influenced by genetic factors. Genetic markers associated with endometriosis exist at chromosome 1p36.12 and lead to altered expression of the long intergenic non-coding RNA 339 (LINC00339), however, the role of LINC00339 in endometriosis pathophysiology remains unknown. The aim of this work was to characterize the expression patterns of LINC00339 mRNA in endometrium and endometriotic lesions in situ and to determine the functional role of LINC00339 in human endometrium. We employed RNA-sequencing (RNA-seq), quantitative RT-PCR and in situ hybridization to investigate the abundance of LINC00339 transcripts in endometrium and endometrial cell lines and to describe the pattern and localization of LINC00339 expression in endometrium and endometriotic lesions. LINC00339 mRNA expression was manipulated (overexpressed and silenced) in endometrial stromal cell lines and RNA-seq data from overexpression models were analysed using online bioinformatics platforms (STRING and Ingenuity Pathway Analysis) to determine functional processes. We demonstrated the expression of LINC00339 in endometriotic lesions for the first time; we found LINC00339 expression was restricted to the lesion foci and absent in surrounding non-lesion tissue. Furthermore, manipulation of LINC00339 expression in endometrial stromal cell lines significantly impacted the expression of genes involved in immune defence pathways. These studies identify a novel mechanism for LINC00339 activity in endometrium and endometriosis, paving the way for future work, which is essential for understanding the pathogenesis of endometriosis.

Perspectives in immunotherapy: meeting report from the "Immunotherapy Bridge" (December 4th-5th, 2019, Naples, Italy).

Over the last few years, numerous clinical trials and real-world experience have provided a large amount of evidence demonstrating the potential for long-term survival with immunotherapy agents across various malignancies, beginning with melanoma and extending to other tumours. The clinical success of immune checkpoint blockade has encouraged increasing development of other immunotherapies. It has been estimated that there are over 3000 immuno-oncology trials ongoing, targeting hundreds of disease and immune pathways. Evolving topics on cancer immunotherapy, including the state of the art of immunotherapy across various malignancies, were the focus of discussions at the Immunotherapy Bridge meeting (4-5 December, 2019, Naples, Italy), and are summarised in this report.

Standard operating procedure for somatic variant refinement of sequencing data with paired tumor and normal samples.

PURPOSE: Following automated variant calling, manual review of aligned read sequences is required to identify a high-quality list of somatic variants. Despite widespread use in analyzing sequence data, methods to standardize manual review have not been described, resulting in high inter- and intralab variability. METHODS: This manual review standard operating procedure (SOP) consists of methods to annotate variants with four different calls and 19 tags. The calls indicate a reviewer's confidence in each variant and the tags indicate commonly observed sequencing patterns and artifacts that inform the manual review call. Four individuals were asked to classify variants prior to, and after, reading the SOP and accuracy was assessed by comparing reviewer calls with orthogonal validation sequencing. RESULTS: After reading the SOP, average accuracy in somatic variant identification increased by 16.7% (p value = 0.0298) and average interreviewer agreement increased by 12.7% (p value < 0.001). Manual review conducted after reading the SOP did not significantly increase reviewer time. CONCLUSION: This SOP supports and enhances manual somatic variant detection by improving reviewer accuracy while reducing the interreviewer variability for variant calling and annotation.

Bags versus flasks: a comparison of cell culture systems for the production of dendritic cell-based immunotherapies.

In recent years, cell-based therapies targeting the immune system have emerged as promising strategies for cancer treatment. This review summarizes manufacturing challenges related to production of antigen presenting cells as a patient-tailored cancer therapy. Understanding cell-material interactions is essential because in vitro cell culture manipulations to obtain mature antigen-producing cells can significantly alter their in vivo performance. Traditional antigen-producing cell culture protocols often rely on cell adhesion to surface-treated hydrophilic polystyrene flasks. More recent commercial and investigational cancer immunotherapy products were manufactured using suspension cell culture in closed hydrophobic fluoropolymer bags. The shift to closed cell culture systems can decrease risks of contamination by individual operators, as well as facilitate scale-up and automation. Selecting closed cell culture bags over traditional open culture systems entails different handling procedures and processing controls, which can affect product quality. Changes in culture vessels also entail changes in vessel materials and geometry, which may alter the cell microenvironment and resulting cell fate decisions. Strategically designed culture systems will pave the way for the generation of more sophisticated and highly potent cell-based cancer vaccines. As an increasing number of cell-based therapies enter the clinic, the selection of appropriate cell culture vessels and materials becomes a critical consideration that can impact the therapeutic efficacy of the product, and hence clinical outcomes and patient quality of life.

DGIdb 2.0: mining clinically relevant drug-gene interactions.

The Drug-Gene Interaction Database (DGIdb, www.dgidb.org) is a web resource that consolidates disparate data sources describing drug-gene interactions and gene druggability. It provides an intuitive graphical user interface and a documented application programming interface (API) for querying these data. DGIdb was assembled through an extensive manual curation effort, reflecting the combined information of twenty-seven sources. For DGIdb 2.0, substantial updates have been made to increase content and improve its usefulness as a resource for mining clinically actionable drug targets. Specifically, nine new sources of drug-gene interactions have been added, including seven resources specifically focused on interactions linked to clinical trials. These additions have more than doubled the overall count of drug-gene interactions. The total number of druggable gene claims has also increased by 30%. Importantly, a majority of the unrestricted, publicly-accessible sources used in DGIdb are now automatically updated on a weekly basis, providing the most current information for these sources. Finally, a new web view and API have been developed to allow searching for interactions by drug identifiers to complement existing gene-based search functionality. With these updates, DGIdb represents a comprehensive and user friendly tool for mining the druggable genome for precision medicine hypothesis generation.

GenVisR: Genomic Visualizations in R.

UNLABELLED: Visualizing and summarizing data from genomic studies continues to be a challenge. Here, we introduce the GenVisR package to addresses this challenge by providing highly customizable, publication-quality graphics focused on cohort level genome analyses. GenVisR provides a rapid and easy-to-use suite of genomic visualization tools, while maintaining a high degree of flexibility by leveraging the abilities of ggplot2 and Bioconductor. AVAILABILITY AND IMPLEMENTATION: GenVisR is an R package available via Bioconductor (https://bioconductor.org/packages/GenVisR) under GPLv3. Support is available via GitHub (https://github.com/griffithlab/GenVisR/issues) and the Bioconductor support website. CONTACTS: obigriffith@wustl.edu or mgriffit@wustl.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

RNAi screen in Drosophila larvae identifies histone deacetylase 3 as a positive regulator of the hsp70 heat shock gene expression during heat shock.

The transcription regulation of the Drosophila hsp70 gene is a complex process that involves the regulation of multiple steps, including the establishment of paused Pol II and release of Pol II into elongation upon heat shock activation. While the major players involved in the regulation of gene expression have been studied in detail, additional factors involved in this process continue to be discovered. To identify factors involved in hsp70 expression, we developed a screen that capitalizes on a visual assessment of heat shock activation using a hsp70-beta galactosidase reporter and publicly available RNAi fly lines to deplete candidate proteins. We validated the screen by showing that the depletion of HSF, CycT, Cdk9, Nurf 301, or ELL prevented the full induction of hsp70 by heat shock. Our screen also identified the histone deacetylase HDAC3 and its associated protein SMRTER as positive regulators of hsp70 activation. Additionally, we show that HDAC3 and SMRTER contribute to hsp70 gene expression at a step subsequent to HSF-mediated activation and release of the paused Pol II that resides at the promoter prior to heat shock induction.

Choosing Wisely audit: CT KUB ordering in emergency department renal colic presentations.

OBJECTIVE: Our aim was to safely reduce unnecessary CT KUBs (kidneys, ureters, bladder) in patients with renal colic. METHODS: This was a before and after intervention observational study of 74 patients in April 2023 and 57 patients in October 2023. RESULTS: Seventy-five per cent of patients with suspected renal colic underwent a CT KUB in the pre-audit period. Following education, an update in the ED Renal Colic Policy, electronic medical record ordering and short stay pathway, a re-audit was undertaken in October 2023 resulting in an absolute reduction of 15% of CT KUBs ordered. CONCLUSIONS: Audit interventions can reduce unnecessary CT KUBs in renal colic.

A Spatial Omnibus Test (SPOT) for Spatial Proteomic Data.

Spatial proteomics can reveal the spatial organization of immune cells in the tumor immune microenvironment. Relating measures of spatial clustering, such as Ripley's K or Besag's L, to patient outcomes may offer important clinical insights. However, these measures require pre-specifying a radius in which to quantify clustering, yet no consensus exists on the optimal radius which may be context-specific. We propose a SPatial Omnibus Test (SPOT) which conducts this analysis across a range of candidate radii. At each radius, SPOT evaluates the association between the spatial summary and outcome, adjusting for confounders. SPOT then aggregates results across radii using the Cauchy combination test, yielding an omnibus p-value characterizing the overall degree of association. Using simulations, we verify that the type I error rate is controlled and show SPOT can be more powerful than alternatives. We also apply SPOT to an ovarian cancer study. An R package and tutorial is provided at https://github.com/sarahsamorodnitsky/SPOT.

Human mesenchymal stromal cell adhesion and expansion on fluoropolymer surfaces modified with oxygen and nitrogen-rich plasma polymers.

Fluorinated ethylene propylene (FEP) vessels are of significant interest for therapeutic cell biomanufacturing applications due to their chemical inertness, hydrophobic surface, and high oxygen permeability. However, these properties also limit the adhesion and survival of anchorage-dependent cells. Here, we develop novel plasma polymer coatings to modify FEP surfaces, enhancing the adhesion and expansion of human mesenchymal stromal cells (hMSCs). Similar to commercially available tissue culture polystyrene vessels, oxygen-rich or nitrogen-rich surface chemistries can be achieved using this approach. While steam sterilization increased the roughness of the coatings and altered the surface chemistry, the overall wettability and oxygen or nitrogen-rich nature of the coatings were maintained. In the absence of proteins during initial cell attachment, cells adhered to surfaces even in the presence of chelators, whereas adhesion was abrogated with chelator in a protein-containing medium, suggesting that integrin-mediated adhesion predominates over physicochemical tethering in normal protein-containing cell seeding conditions. Albumin adsorption was more elevated on nitrogen-rich coatings compared to the oxygen-rich coatings, which was correlated with a higher extent of hMSC expansion after 3 days. Both the oxygen and nitrogen-rich coatings significantly improved hMSC adhesion and expansion compared to untreated FEP. FEP surfaces with nitrogen-rich coatings were practically equivalent to commercially available standard tissue culture-treated polystyrene surfaces in terms of hMSC yields. Plasma polymer coatings show significant promise in expanding the potential usage of FEP-based culture vessels for cell therapy applications.

Opposing tumor-cell-intrinsic and -extrinsic roles of the IRF1 transcription factor in antitumor immunity.

Type I interferon (IFN-I) and IFN-γ foster antitumor immunity by facilitating T cell responses. Paradoxically, IFNs may promote T cell exhaustion by activating immune checkpoints. The downstream regulators of these disparate responses are incompletely understood. Here, we describe how interferon regulatory factor 1 (IRF1) orchestrates these opposing effects of IFNs. IRF1 expression in tumor cells blocks Toll-like receptor- and IFN-I-dependent host antitumor immunity by preventing interferon-stimulated gene (ISG) and effector programs in immune cells. In contrast, expression of IRF1 in the host is required for antitumor immunity. Mechanistically, IRF1 binds distinctly or together with STAT1 at promoters of immunosuppressive but not immunostimulatory ISGs in tumor cells. Overexpression of programmed cell death ligand 1 (PD-L1) in Irf1-/- tumors only partially restores tumor growth, suggesting multifactorial effects of IRF1 on antitumor immunity. Thus, we identify that IRF1 expression in tumor cells opposes host IFN-I- and IRF1-dependent antitumor immunity to facilitate immune escape and tumor growth.

Integrative systems biology reveals NKG2A-biased immune responses correlate with protection in infectious disease, autoimmune disease, and cancer.

Infection, autoimmunity, and cancer are principal human health challenges of the 21st century. Often regarded as distinct ends of the immunological spectrum, recent studies hint at potential overlap between these diseases. For example, inflammation can be pathogenic in infection and autoimmunity. T resident memory (TRM) cells can be beneficial in infection and cancer. However, these findings are limited by size and scope; exact immunological factors shared across diseases remain elusive. Here, we integrate large-scale deeply clinically and biologically phenotyped human cohorts of 526 patients with infection, 162 with lupus, and 11,180 with cancer. We identify an NKG2A+ immune bias as associative with protection against disease severity, mortality, and autoimmune/post-acute chronic disease. We reveal that NKG2A+ CD8+ T cells correlate with reduced inflammation and increased humoral immunity and that they resemble TRM cells. Our results suggest NKG2A+ biases as a cross-disease factor of protection, supporting suggestions of immunological overlap between infection, autoimmunity, and cancer.

Antitumor Immune Responses in B2M-Deficient Cancers.

ß2-microglobulin (B2M) is a critical component of the MHC class I molecule and is required to present tumor antigens to T cells. Its loss results in acquired resistance to immune checkpoint blockade (ICB) therapies. However, there have been well-documented cases of B2M-inactivated tumors responding to ICB, justifying investigation of how an antitumor immune response can be generated to tumors without surface MHC class I. We knocked out B2M in three murine models with varying baseline MHC class I expression and sensitivity to anti-programmed death receptor (PD-1) therapy and analyzed the immune responses. MC38 and YUMMER2.1 without B2M responded to anti-PD-1 alone or with an IL2 agonist, and this was mediated by CD4+ T cells and natural killer (NK) cells. The more aggressive B16 without B2M expression only partially responded to the IL2 agonist, and this was dependent on NK cells. When analyzing nearly 300 pretreatment biopsies from patients with melanoma receiving PD-1 blockade-based therapies, we found infrequent B2M mutations or homozygous loss but more frequent LOH or copy-number gains. B2M LOH was enriched in biopsies from patients without response to therapy, and these biopsies were more frequently infiltrated by activated NK cells. We conclude that in the absence of B2M, activation of CD4+ T cells and NK cells can mediate responses to murine models of PD-1 blockade therapy. In addition, in human melanoma, the intratumoral presence of activated NK cells upon partial B2M loss likely selects against tumor escape through low surface MHC class I expression.