In this chapter, we presented the comprehensive gene set analysis framework GeneTrail. Although the competition in this field is immense, we were able to establish GeneTrail in this area as demonstrated by the usage statistics and the citations of external groups. The continuous development and extensions lead to a powerful C++ framework that is not only useful for gene set analyses, but also builds the basis to answer more complex questions when using the network topology. Over the years, we integrated many ideas and suggestions of users and enhanced the functionality and the user-friendliness of the web- application. In summary, GeneTrail presents one of the most powerful non-commercial gene set analysis tools that are available for the research community.
In the next three chapters, we further demonstrate the usefulness of GeneTrail by perform- ing comprehensive analyses for different fields of cancer research comprising the analysis of characteristics of tumor associated antigens, the putative target pathways of miRNAs, and differential network analyses concerning glioma versus normal and BRCA1 mutation carriers versus non-mutation carriers.
TUMOR
ASSOCIATED
ANTIGENS
Tumor associated antigens (TAAs) are capable to elicit an immune response in cancer patients. Since these antigens stem for example from proteins which are also expressed under normal conditions in healthy tissues, there must exists reasons why they become immunogenic in cancer tissue. In cancer prognosis/diagnosis some TAAs are already ap- plied as biological marker (e.g. PSA, prostate specific antigen) [30]. Although the detection and usage of TAAs is already widely possible, the mechanisms which lead to a humoral immune response against these antigens are for the most part elusive. In this work, we will try to shed some light into this topic by testing different hypotheses for immunogenicity. First, we give a short overview concerning immune response in general and the discussed mechanisms for eliciting an immune response in autoimmune diseases and cancer. Sec- ond, we describe the experimental methods available for detecting antigens and the data sets used in this work. Third, we apply bioinformatics approaches for verifying if the stated hypotheses can be generalized for TAAs.
3.1 Immune response and autoimmunity
The defense mechanisms of the immune system of higher multicellular organisms origi- nate from the fact that their bodies provide an optimal environment for the reproduction of microorganisms such as bacteria, viruses, and parasites. In general, the immune system can be divided into two types of defense mechanisms: the innate and the adaptive immune system [98].
The innate immune system encompasses unchanging mechanisms that are continuously in force, as for example the skin as a physical barrier, which pathogens have to overcome. These non-specific mechanisms contribute to a basic resistance of an organism against
foreign pathogens. In contrast to the innate immune system, the adaptive immune sys- tem is characterized by a high degree of specificity. The reaction of the adaptive immune system is elicited by the recognition of specific molecules called antigens, or rather by the recognition of some specific surface structures of the antigen, called epitopes. The recognition of pathogen specific antigens is a multi-step process, which starts with the di- gestion of pathogens by macrophages or immature dendritic cells. Subsequently, these cells become activated or mature to so-called antigen presenting cells (APCs). The di- gested antigens are fragmented to peptides of about 9-15 amino acids, which are pre- sented to T-helper cells by means of major histocompatibility complexes (MHCs) on the surface of the APCs (Figure 3.1). T-helper cells specific for recognizing the peptide:MHC structure become activated and start to secrete cytokines, which activate in turn cytotoxic T-lymphocytes, antibody-secreting B-cells, macrophages, etc. resulting in the activation of the humoral and/or cellular immune response. The function of the humoral immunity is to recognize and to destroy extracellular pathogens and foreign substances. B-cells activated by their corresponding antigen and the cytokines of the CD4 T-helper cells will start to pro- liferate and differentiate into antibody secreting plasma cells. The antibodies secreted by the plasma cells bind to their specific epitope on the antigen, thereby disabling the anti- gen, and mark it for processes leading to its destruction. By contrast, the function of the cellular immunity is to detect intracellular pathogens. The main components of the cellular immunity are CD8 T-helper cells and cytotoxic T-lymphocytes (CTLs). To distinguish nor- mal cells from modified cells, a mechanism is necessary that reports the cells’ state. The proteins expressed in a cell are again decomposed to some extent. The protein fragments or peptides are presented on the cell surface by MHC class I molecules. CTLs can rec- ognize cells presenting non-self peptides like virus-infected cells or tumor cells expressing modified proteins and induce cell death by secreting toxins.
The reasons for the loss of the so-called self-tolerance in autoimmune diseases or can- cer, which results in the activation of the humoral immune response against self-antigens, are still elusive for the most part and can have many potential causes, some related to the immune system itself, and some related to the antigen targets. For some autoim- mune diseases the loss of self-tolerance originates from the similarity of self proteins to pathogenic antigens, which is called molecular mimicry. This theory proposes that the im- mune reaction initially elicited by a foreign antigen, which is structurally similar to a human protein, can result in a cross-reaction against the human protein [99]. While the loss of self-tolerance often comes along with autoimmunity, the immune response in cancer pa- tients may be initiated by alterations in the tumor itself. Such alterations comprise, e.g.,
T ll MHC class II T‐cell receptor T‐cell receptor Ag MHC class II
Figure 3.1: The humoral immune response. Antigen processing cells (APCs) ingest and process an antigen. The processed antigen is presented by MHC class II molecules to CD4 T- cells. The activation of antigen-specific CD4 T-cells leads to lymphoproliferation and cy- tokine secretion. The activation of a B-cell comprises several steps. Antigen-antibody complexes on the surface of the B-cell are internalized by receptor-mediated endocyto- sis and degraded to peptides. These are presented by MHC class II on the membrane to CD4 T-helper cells. Specific T-helper cells recognize the peptide:MHC structure and additional co-stimulatory signals, which lead to the activation of the T-helper cell. The activated T-helper cell secretes cytokines that help the B-cell to differentiate into an antibody secreting plasma cell.
mutated proteins or differential expression that may result in an increased immunogenicity of self-antigens [100].