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Abstract

Colorectal cancer (CRC) is the third most common cancer worldwide and the number of early onset CRCs in individuals younger than 50 years is rising. A genetic predisposition for cancer is identified in 20% of young onset CRC patients. The most common inherited CRC syndrome, estimated to affect around 25 million people worldwide, is Lynch syndrome (LS). LS carriers present with a germline variant in a DNA mismatch repair (MMR) gene and an elevated risk of developing cancer in different organ systems, most commonly in the colorectum and endometrium. Thus, timely diagnosis of LS is paramount for the initiation of preventive screening programs and reduction of cancer risk. However, current diagnostic strategies are mainly based on molecular screening of an already manifest cancer, rendering the identification of healthy LS carriers challenging. LS-associated cancers are characterized by MMR deficiency leading to the accumulation of numerous mutations in the entire genome, particularly at microsatellite regions and resulting in microsatellite instability (MSI). The MSI phenotype is associated with high immunogenicity due to the generation of immunogenic peptides, termed frameshift peptides (FSPs), upon mutations in coding microsatellites (cMS). Systemic FSP-specific immune responses have been identified in MSI cancer patients and healthy LS carriers. The presence of FSP-specific immune responses prior to cancer manifestation is thought to be attributable to LS-specific, MMR-deficient premalignant lesions in the colonic mucosa. The proposed immune activation in LS carriers may possess potential to be used diagnostically and to enable the identification of healthy LS carriers. The present thesis undertook the comprehensive characterization of local and systemic immune responses in LS individuals and the evaluation of their clinical potential. In addition, the suitability of plasma-derived extracellular vesicles (EVs) for tumor tissue-independent MSI testing was explored. First, the systematic analysis of local immune responses in LS-associated tumors and the normal colorectal mucosa formed the basis of the project. The qualitative and quantitative review of existing literature on immune infiltration and immune evasion indicated that the hereditary origin shapes the immune phenotype of MSI tumors. LS-associated tumors presented with a more pronounced immune infiltration and a higher frequency of B2M mutations, compared to sporadic cases. The observed immunological differences between hereditary and sporadic MSI tumors possibly reflect differences in their pathogenesis and point towards a life-long immune surveillance in LS carriers. As a consequence, the normal colorectal mucosa of LS carriers might already carry traces of the tumor-independent immune activation. Thus, the present thesis, for the first time, characterized the mucosal immune milieu of LS carriers. The quantification of different T cell subpopulations and gene expression analysis revealed distinct immune profiles in the normal colonic mucosa of LS carriers with and without cancer manifestation. Moreover, a positive correlation between T cell density in the rectal mucosa and time to tumor manifestation in LS was observed for the first time, pointing at a possible role of the mucosal immune status as a temporary or permanent cancer risk modifier in LS. Second and underpinned by the described local immunological alterations in LS carriers, systemic FSP-specific immune responses were characterized in LS carriers and MSI cancer patients. The analysis of FSP-specific T cell and antibody responses in healthy LS carriers enabled the identification of seven FSPs which were associated with significantly stronger immune responses in LS individuals, compared to healthy non-LS controls. The respective candidates may be suited for the immune-based identification of LS carriers prior to cancer development and establish the basis for prospective validation studies in larger cohorts. The analysis of FSP-specific immune responses in MSI cancer patients did not yield a clear picture and was possibly influenced by cancer- and therapy-mediated effects. The potential of FSP-specific T cell responses as immune checkpoint blockade (ICB) therapy response markers was descriptively assessed in MSI cancer patients. Therefore, the present thesis contributed to expanding available data on FSP-specific T cell responses under ICB therapy which are currently scarce. However, no substantial correlation between FSP-specific T cell responses and a patient's clinical course was observed, underscoring the need for larger clinically defined cohorts and a stringent patient follow-up. In addition, systemic FSP-specific T cell responses in MSI cancer patients were correlated with the tumor's cMS mutation pattern, providing evidence for strong peripheral T cell responses against cMS mutation-derived FSPs and the potential counterselection of highly immunogenic FSPs during tumor evolution. An additional aspect of the present project accounted for an individual's HLA type as a factor possibly influencing the immune response. The analysis of HLA-FSP epitope binding predictions generally supported the suitability of the used FSP panel. Third, the potential of plasma-derived vesicular DNA as a minimally invasive MSI diagnostic approach was explored. The present thesis, for the first time, demonstrated MSI in vesicular DNA from MSI cancer patients, implying that plasma EVs sustain the MSI phenotype of their parental cancer cell. Moreover, the vesicular MSI status was found to change with advancing ICB therapy, suggesting its suitability as therapy response marker and a possible link between MSI detectability in vesicular DNA and tumor burden. This correlation not only supported the clinical potential of EVs as cancer DNA carriers, but also underlined the dependence of successful sampling on the quantity of cancer-specific EVs in the plasma, thereby calling for the enrichment of specific EV populations. Respective enrichment strategies for colon-specific EVs were successfully established using an MSI CRC cell line and preliminary results indicate their feasibility for the application in plasma-derived EVs. The detection of MSI in plasma EVs opens doors for their versatile diagnostic and predictive application in the context of MSI cancers and LS. In summary, the present thesis demonstrated the significance of local and systemic immune responses in LS carriers and outlined novel approaches for using such in tumor-independent LS diagnostics. Moreover, the presented work provides an incentive for further research on EVs as MSI-specific biomarkers.