A Set of Cell Lines Derived from a Genetic Murine Glioblastoma Model Recapitulates Molecular and Morphological Characteristics of Human Tumors

Simple Summary Glioblastoma (GBM) is a highly aggressive and almost inevitably lethal brain tumor. Animal models for GBM are crucial to study how the tumor evolves in vivo and to test novel treatment options. Most currently available models are based on the transplantation of human GBM cells into mice with a defective immune system. However, this approach does not allow to study the contribution of immune cells to GBM growth and to test immunotherapies. Transplantation of murine GBM cells overcomes this limitation, however, up to now, only a limited number, which mostly do not mimic important characteristics of human GBM, have been available. Via in vivo passaging, we established a set of m…

Abstract A110: Mutant MHC class II epitopes drive therapeutic immune responses to cancer

Abstract Mutations are regarded as ideal targets for cancer immunotherapy. As neoepitopes with strict lack of expression in any healthy tissue, they are expected to be safe and could bypass the central tolerance mechanisms. Recent advances in nucleic acid sequencing technologies have revolutionized the field of genomics, allowing the readily targeting of mutated neoantigens for personalized cancer vaccination. We demonstrated in three independent murine tumor models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that unexpectedly the immunogenic mutanome is pre-dominantly recognized by CD4+ T cells. RNA vaccination with such MHC class II restricted immuno…

Abstract CT202: IVAC MUTANOME: Individualized vaccines for the treatment of cancer

Abstract Cancer arises from the accumulation of genomic alterations and epigenetic changes that constitute a hallmark of cancer. Owing to the molecular heterogeneity in cancer, only a minor fraction of patients profit from approved therapies. Available targeted therapies can only address alterations common to a particular type of cancer and induce transient effects due to the generation of resistant sub-clones. In contrast, the IVAC MUTANOME project aims to immunologically target multiple cancer mutations uniquely expressed in a given patient's tumor. The IVAC MUTANOME approach should be applicable to the majority of patients irrespective of the tumor entity and offers the potential to expl…

A catalog of HLA type, HLA expression, and neo-epitope candidates in human cancer cell lines

Cancer cell lines are a tremendous resource for cancer biology and therapy development. These multipurpose tools are commonly used to examine the genetic origin of cancers, to identify potential novel tumor targets, such as tumor antigens for vaccine devel-opment, and utilized to screen potential therapies in preclinical studies. Mutations, gene expression, and drug sensitivity have been determined for many cell lines using next-generation sequencing (NGS). However, the human leukocyte antigen (HLA) type and HLA expression of tumor cell lines, characterizations necessary for the development of cancer vaccines, have remained largely incomplete and, such information, when available, has been …

Instruction of haematopoietic lineage choices, evolution of transcriptional landscapes and cancer stem cell hierarchies derived from an AML1-ETO mouse model.

The t(8;21) chromosomal translocation activates aberrant expression of the AML1-ETO (AE) fusion protein and is commonly associated with core binding factor acute myeloid leukaemia (CBF AML). Combining a conditional mouse model that closely resembles the slow evolution and the mosaic AE expression pattern of human t(8;21) CBF AML with global transcriptome sequencing, we find that disease progression was characterized by two principal pathogenic mechanisms. Initially, AE expression modified the lineage potential of haematopoietic stem cells (HSCs), resulting in the selective expansion of the myeloid compartment at the expense of normal erythro- and lymphopoiesis. This lineage skewing was foll…

Comprehensive Genomic and Transcriptomic Analysis of Three Synchronous Primary Tumours and a Recurrence from a Head and Neck Cancer Patient

Synchronous primary malignancies occur in a small proportion of head and neck squamous cell carcinoma (HNSCC) patients. Here, we analysed three synchronous primaries and a recurrence from one patient by comparing the genomic and transcriptomic profiles among the tumour samples and determining the recurrence origin. We found remarkable levels of heterogeneity among the primary tumours, and through the patterns of shared mutations, we traced the origin of the recurrence. Interestingly, the patient carried germline variants that might have predisposed him to carcinogenesis, together with a history of alcohol and tobacco consumption. The mutational signature analysis confirmed the impact of alc…

Improving mRNA-Based Therapeutic Gene Delivery by Expression-Augmenting 3' UTRs Identified by Cellular Library Screening.

Synthetic mRNA has emerged as a powerful tool for the transfer of genetic information, and it is being explored for a variety of therapeutic applications. Many of these applications require prolonged intracellular persistence of mRNA to improve bioavailability of the encoded protein. mRNA molecules are intrinsically unstable and their intracellular kinetics depend on the UTRs embracing the coding sequence, in particular the 3′ UTR elements. We describe here a novel and generally applicable cell-based selection process for the identification of 3′ UTRs that augment the expression of proteins encoded by synthetic mRNA. Moreover, we show, for two applications of mRNA therapeutics, namely, (1) …

ArtiFuse—computational validation of fusion gene detection tools without relying on simulated reads

Abstract Motivation Gene fusions are an important class of transcriptional variants that can influence cancer development and can be predicted from RNA sequencing (RNA-seq) data by multiple existing tools. However, the real-world performance of these tools is unclear due to the lack of known positive and negative events, especially with regard to fusion genes in individual samples. Often simulated reads are used, but these cannot account for all technical biases in RNA-seq data generated from real samples. Results Here, we present ArtiFuse, a novel approach that simulates fusion genes by sequence modification to the genomic reference, and therefore, can be applied to any RNA-seq dataset wit…

Integrative analysis of structural variations using short-reads and linked-reads yields highly specific and sensitive predictions.

Genetic diseases are driven by aberrations of the human genome. Identification of such aberrations including structural variations (SVs) is key to our understanding. Conventional short-reads whole genome sequencing (cWGS) can identify SVs to base-pair resolution, but utilizes only short-range information and suffers from high false discovery rate (FDR). Linked-reads sequencing (10XWGS) utilizes long-range information by linkage of short-reads originating from the same large DNA molecule. This can mitigate alignment-based artefacts especially in repetitive regions and should enable better prediction of SVs. However, an unbiased evaluation of this technology is not available. In this study, w…

In Silico HLA Typing Using Standard RNA-Seq Sequence Reads

Next-generation sequencing (NGS) enables high-throughput transcriptome profi ling using the RNA-Seq assay, resulting in billions of short sequence reads. Worldwide adoption has been rapid: many laboratories worldwide generate transcriptome sequence reads daily. Here, we describe methods for obtaining a sample’s human leukocyte antigen (HLA) class I and II types and HLA expression using standard NGS RNA- Seq sequence reads. We demonstrate the application using our algorithm, seq2HLA, and a publicly available RNA-Seq dataset from the Burkitt lymphoma cell line Raji.

Bioinformatics for Cancer Immunotherapy

Our immune system plays a key role in health and disease as it is capable of responding to foreign antigens as well as acquired antigens from cancer cells. Latter are caused by somatic mutations, the so-called neoepitopes, and might be recognized by T cells if they are presented by HLA molecules on the surface of cancer cells. Personalized mutanome vaccines are a class of customized immunotherapies, which is dependent on the detection of individual cancer-specific tumor mutations and neoepitope (i.e., prediction, followed by a rational vaccine design, before on-demand production. The development of next generation sequencing (NGS) technologies and bioinformatic tools allows a large-scale an…

IVAC MUTANOME: A first-in-human phase I clinical trial targeting individual mutant neoantigens for the treatment of melanoma

Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer

T cells directed against mutant neo-epitopes drive cancer immunity. However, spontaneous immune recognition of mutations is inefficient. We recently introduced the concept of individualized mutanome vaccines and implemented an RNA-based poly-neo-epitope approach to mobilize immunity against a spectrum of cancer mutations. Here we report the first-in-human application of this concept in melanoma. We set up a process comprising comprehensive identification of individual mutations, computational prediction of neo-epitopes, and design and manufacturing of a vaccine unique for each patient. All patients developed T cell responses against multiple vaccine neo-epitopes at up to high single-digit p…

Large-scale analysis of SARS-CoV-2 spike-glycoprotein mutants demonstrates the need for continuous screening of virus isolates

Due to the widespread of the COVID-19 pandemic, the SARS-CoV-2 genome is evolving in diverse human populations. Several studies already reported different strains and an increase in the mutation rate. Particularly, mutations in SARS-CoV-2 spike-glycoprotein are of great interest as it mediates infection in human and recently approved mRNA vaccines are designed to induce immune responses against it. We analyzed 1,036,030 SARS-CoV-2 genome assemblies and 30,806 NGS datasets from GISAID and European Nucleotide Archive (ENA) focusing on non-synonymous mutations in the spike protein. Only around 2.5% of the samples contained the wild-type spike protein with no variation from the reference. Among…

NeoFox: annotating neoantigen candidates with neoantigen features

Abstract Summary The detection and prediction of true neoantigens is of great importance for the field of cancer immunotherapy. Wesearched the literature for proposed neoantigen features and integrated them into a toolbox called NEOantigen Feature toolbOX (NeoFox). NeoFox is an easy-to-use Python package that enables the annotation of neoantigen candidates with 16 neoantigen features. Availability and implementation NeoFox is freely available as an open source Python package released under the GNU General Public License (GPL) v3 license at https://github.com/TRON-Bioinformatics/neofox. Supplementary information Supplementary data are available at Bioinformatics online.

HLA typing from RNA-Seq sequence reads.

We present a method, seq2HLA, for obtaining an individual's human leukocyte antigen (HLA) class I and II type and expression using standard next generation sequencing RNA-Seq data. RNA-Seq reads are mapped against a reference database of HLA alleles, and HLA type, confidence score and locus-specific expression level are determined. We successfully applied seq2HLA to 50 individuals included in the HapMap project, yielding 100% specificity and 94% sensitivity at a P-value of 0.1 for two-digit HLA types. We determined HLA type and expression for previously un-typed Illumina Body Map tissues and a cohort of Korean patients with lung cancer. Because the algorithm uses standard RNA-Seq reads and …

Mutant MHC class II epitopes drive therapeutic immune responses to cancer

Tumour-specific mutations are ideal targets for cancer immunotherapy as they lack expression in healthy tissues and can potentially be recognized as neo-antigens by the mature T-cell repertoire. Their systematic targeting by vaccine approaches, however, has been hampered by the fact that every patient's tumour possesses a unique set of mutations ('the mutanome') that must first be identified. Recently, we proposed a personalized immunotherapy approach to target the full spectrum of a patient's individual tumour-specific mutations. Here we show in three independent murine tumour models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that, unexpectedly, the …

T-cell Receptor Therapy Targeting Mutant Capicua Transcriptional Repressor in Experimental Gliomas

Abstract Purpose: Gliomas are intrinsic brain tumors with a high degree of constitutive and acquired resistance to standard therapeutic modalities such as radiotherapy and alkylating chemotherapy. Glioma subtypes are recognized by characteristic mutations. Some of these characteristic mutations have shown to generate immunogenic neoepitopes suitable for targeted immunotherapy. Experimental Design: Using peptide-based ELISpot assays, we screened for potential recurrent glioma neoepitopes in MHC-humanized mice. Following vaccination, droplet-based single-cell T-cell receptor (TCR) sequencing from established T-cell lines was applied for neoepitope-specific TCR discovery. Efficacy of intravent…

Myeloid Cell-Derived Reactive Oxygen Species Induce Epithelial Mutagenesis

Increased oxidative stress has been suggested to initiate and promote tumorigenesis by inducing DNA damage and to suppress tumor development by triggering apoptosis and senescence. The contribution of individual cell types in the tumor microenvironment to these contrasting effects remains poorly understood. We provide evidence that during intestinal tumorigenesis, myeloid cell-derived H2O2 triggers genome-wide DNA mutations in intestinal epithelial cells to stimulate invasive growth. Moreover, increased reactive oxygen species (ROS) production in myeloid cells initiates tumor growth in various organs also in the absence of a carcinogen challenge in a paracrine manner. Our data identify an i…

Confidence-based Somatic Mutation Evaluation and Prioritization

Next generation sequencing (NGS) has enabled high throughput discovery of somatic mutations. Detection depends on experimental design, lab platforms, parameters and analysis algorithms. However, NGS-based somatic mutation detection is prone to erroneous calls, with reported validation rates near 54% and congruence between algorithms less than 50%. Here, we developed an algorithm to assign a single statistic, a false discovery rate (FDR), to each somatic mutation identified by NGS. This FDR confidence value accurately discriminates true mutations from erroneous calls. Using sequencing data generated from triplicate exome profiling of C57BL/6 mice and B16-F10 melanoma cells, we used the exist…

Tick Salivary Sialostatin L Represses the Initiation of Immune Responses by Targeting IRF4-Dependent Transcription in Murine Mast Cells

Abstract Coevolution of ticks and the vertebrate immune system has led to the development of immunosuppressive molecules that prevent immediate response of skin-resident immune cells to quickly fend off the parasite. In this article, we demonstrate that the tick-derived immunosuppressor sialostatin L restrains IL-9 production by mast cells, whereas degranulation and IL-6 expression are both unaffected. In addition, the expression of IL-1β and IRF4 is strongly reduced in the presence of sialostatin L. Correspondingly, IRF4- or IL-1R–deficient mast cells exhibit a strong impairment in IL-9 production, demonstrating the importance of IRF4 and IL-1 in the regulation of the Il9 locus in mast cel…

Multi-Omics Characterization of the 4T1 Murine Mammary Gland Tumor Model

Background: Tumor models are critical for our understanding of cancer and the development of cancer therapeutics. The 4T1 murine mammary cancer cell line is one of the most widely used breast cancer models. Here, we present an integrated map of the genome, transcriptome, and immunome of 4T1. Results: We found Trp53 (Tp53) and Pik3g to be mutated. Other frequently mutated genes in breast cancer, including Brca1 and Brca2, are not mutated. For cancer related genes, Nav3, Cenpf, Muc5Ac, Mpp7, Gas1, MageD2, Dusp1, Ros, Polr2a, Rragd, Ros1, and Hoxa9 are mutated. Markers for cell proliferation like Top2a, Birc5, and Mki67 are highly expressed, so are markers for metastasis like Msln, Ect2, and P…