Beta-2-microglobulin Mutations Are Linked to a Distinct Metastatic Pattern and a Favorable Outcome in Microsatellite-Unstable Stage IV Gastrointestinal Cancers

Immune checkpoint blockade (ICB) shows remarkable clinical effects in patients with metastatic microsatellite-unstable (MSI) cancer. However, markers identifying potential non-responders are missing. We examined the prevalence of Beta-2-microglobulin (B2M) mutations, a common immune evasion mechanism, in stage IV MSI gastrointestinal cancer and its influence on metastatic pattern and patients’ survival under ICB. Twentyfive patients with metastatic, MSI gastrointestinal adenocarcinoma were included. Eighteen patients received ICB with pembrolizumab and one patient with nivolumab/ ipilimumab. Sequencing was performed to determine B2M mutation status. B2M mutations and loss of B2M expression were detected in 6 out of 25 stage IV MSI cancers. B2M mutations were strongly associated with exclusively peritoneal/peritoneal and lymph node metastases (p=0.0055). However, no significant differences in therapy response (25% vs. 46.6%, p>0.99) and survival (median PFS: 19.5 vs 33.0 months, p=0.74; median OS 39 months vs. not reached, p>0.99) were observed between B2Mmutant and B2M-wild type tumor patients. Among metastatic MSI GI cancers, B2Mmutant tumors represent a biologically distinct disease with distinct metastatic patterns. To assess ICB response in B2M-mutant MSI cancer patients, future studies need to account for the fact that baseline survival of patients with B2M-mutant MSI cancer may be longer than of patients with B2M-wild type MSI cancer

Beta-2-microglobulin Mutations Are Linked to a Distinct Metastatic Pattern and a Favorable Outcome in Microsatellite-Unstable Stage IV Gastrointestinal Cancers

Immune checkpoint blockade (ICB) shows remarkable clinical effects in patients with metastatic microsatellite-unstable (MSI) cancer. However, markers identifying potential non-responders are missing. We examined the prevalence of Beta-2-microglobulin (B2M) mutations, a common immune evasion mechanism, in stage IV MSI gastrointestinal cancer and its influence on metastatic pattern and patients’ survival under ICB. Twenty-five patients with metastatic, MSI gastrointestinal adenocarcinoma were included. Eighteen patients received ICB with pembrolizumab and one patient with nivolumab/ipilimumab. Sequencing was performed to determine B2M mutation status. B2M mutations and loss of B2M expression were detected in 6 out of 25 stage IV MSI cancers. B2M mutations were strongly associated with exclusively peritoneal/peritoneal and lymph node metastases (p=0.0055). However, no significant differences in therapy response (25% vs. 46.6%, p>0.99) and survival (median PFS: 19.5 vs 33.0 months, p=0.74; median OS 39 months vs. not reached, p>0.99) were observed between B2M-mutant and B2M-wild type tumor patients. Among metastatic MSI GI cancers, B2M-mutant tumors represent a biologically distinct disease with distinct metastatic patterns. To assess ICB response in B2M-mutant MSI cancer patients, future studies need to account for the fact that baseline survival of patients with B2M-mutant MSI cancer may be longer than of patients with B2M-wild type MSI cancer

The coding microsatellite mutation profile of PMS2-deficient colorectal cancer

Lynch syndrome (LS) is caused by a pathogenic heterozygous germline variant in one of the DNA mismatch repair (MMR) genes: MLH1, MSH2, MSH6 or PMS2. LS-associated colorectal carcinomas (CRCs) are characterized by MMR deficiency and by accumulation of multiple insertions/deletions at coding microsatellites (cMS). MMR deficiency-induced variants at defined cMS loci have a driver function and promote tumorigenesis. Notably, PMS2 variant carriers face only a slightly increased risk of developing CRC. Here, we investigate whether this lower penetrance is also reflected by differences in molecular features and cMS variant patterns. Tumor DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue cores or sections (n = 90). Tumors originated from genetically proven germline pathogenic MMR variant carriers (including 14 PMS2-deficient tumors). The mutational spectrum was analyzed using fluorescently labeled primers specific for 18 cMS previously described as mutational targets in MMR-deficient tumors. Immune cell infiltration was analyzed by immunohistochemical detection of T-cells on FFPE tissue sections. The cMS spectrum of PMS2-deficient CRCs did not show any sig-nificant differences from MLH1/MSH2-deficient CRCs. PMS2-deficient tumors, however, displayed lower CD3-positive T-cell infiltration compared to other MMR-deficient cancers (28.00 vs. 55.00 per 0.1 mm(2), p = 0.0025). Our study demonstrates that the spectrum of potentially immunogenic cMS variants in CRCs from PMS2 gene variant carriers is similar to that observed in CRCs from other MMR gene variant carriers. Lower immune cell infiltration observed in PMS2-deficient CRCs could be the result of alternative mechanisms of immune evasion or immune cell exclusion, similar to those seen in MMR-proficient tumors.Hereditary cancer genetic

A simple approach for detecting HLA-A02 alleles in archival formalin-fixed paraffin-embedded tissue samples and an application example for studying cancer immunoediting

The HLA system represents a central component of the antigen presentation machinery. As every patient possesses a defined set of HLA molecules, only certain antigens can be presented on the cell surface. Thus, studying HLA type-dependent antigen presentation can improve the understanding of variation in susceptibility to various diseases, including infectious diseases and cancer. In archival formalin-fixed paraffin-embedded (FFPE) tissue, the HLA type is difficult to analyze because of fragmentation of DNA, hindering the application of commonly used assays that rely on long DNA stretches. Addressing these difficulties, we present a refined approach for characterizing presence or absence of HLA-A*02, the most common HLA-A allele in the Caucasian population, in archival samples. We validated our genotyping strategy in a cohort of 90 samples with HLA status obtained by an NGS-based method. 90% (n = 81) of the samples could be analyzed with the approach. For all of them, the presence or absence of HLA-A*02 alleles was correctly determined with the method, demonstrating 100% sensitivity and specificity (95% CI: 91.40%-100% and 91.19%-100%). Furthermore, we provide an example of application in an independent cohort of 73 FFPE microsatellite-unstable (MSI) colorectal cancer samples. As MSI cancer cells encompass a high number of mutations in coding microsatellites, leading to the generation of highly immunogenic frameshift peptide antigens, they are ideally suited for studying relations between the mutational landscape of tumor cells and interindividual differences in the immune system, including the HLA genotype. Overall, our method can help to promote studying HLA type-dependency during the pathogenesis of a wide range of diseases, making archival and historic tissue samples accessible for identifying HLA-A*02 alleles.Peer reviewe

Distinct Mutational Profile of Lynch Syndrome Colorectal Cancers Diagnosed under Regular Colonoscopy Surveillance

Regular colonoscopy even with short intervals does not prevent all colorectal cancers (CRC) in Lynch syndrome (LS). In the present study, we asked whether cancers detected under regular colonoscopy surveillance (incident cancers) are phenotypically different from cancers detected at first colonoscopy (prevalent cancers). We analyzed clinical, histological, immunological and mutational characteristics, including panel sequencing and high-throughput coding microsatellite (cMS) analysis, in 28 incident and 67 prevalent LS CRCs (n total = 95). Incident cancers presented with lower UICC and T stage compared to prevalent cancers (p < 0.0005). The majority of incident cancers (21/28) were detected after previous colonoscopy without any pathological findings. On the molecular level, incident cancers presented with a significantly lower KRAS codon 12/13 (1/23, 4.3% vs. 11/21, 52%; p = 0.0005) and pathogenic TP53 mutation frequency (0/17, 0% vs. 7/21, 33.3%; p = 0.0108,) compared to prevalent cancers; 10/17 (58.8%) incident cancers harbored one or more truncating APC mutations, all showing mutational signatures of mismatch repair (MMR) deficiency. The proportion of MMR deficiency-related mutational events was significantly higher in incident compared to prevalent CRC (p = 0.018). In conclusion, our study identifies a set of features indicative of biological differences between incident and prevalent cancers in LS, which should further be monitored in prospective LS screening studies to guide towards optimized prevention protocols.Peer reviewe

Is HLA type a possible cancer risk modifier in Lynch syndrome?

Lynch syndrome (LS) is the most common inherited cancer syndrome. It is inherited via a monoallelic germline variant in one of the DNA mismatch repair (MMR) genes. LS carriers have a broad 30% to 80% risk of developing various malignancies, and more precise, individual risk estimations would be of high clinical value, allowing tailored cancer prevention and surveillance. Due to MMR deficiency, LS cancers are characterized by the accumulation of frameshift mutations leading to highly immunogenic frameshift peptides (FSPs). Thus, immune surveillance is proposed to inhibit the outgrowth of MMR-deficient cell clones. Recent studies have shown that immunoediting during the evolution of MMR-deficient cancers leads to a counter-selection of highly immunogenic antigens. The immunogenicity of FSPs is dependent on the antigen presentation. One crucial factor determining antigen presentation is the HLA genotype. Hence, a LS carrier's HLA genotype plays an important role in the presentation of FSP antigens to the immune system, and may influence the likelihood of progression from precancerous lesions to cancer. To address the challenge of clarifying this possibility including diverse populations with different HLA types, we have established the INDICATE initiative (Individual cancer risk by HLA type, ), an international network aiming at a systematic evaluation of the HLA genotype as a possible cancer risk modifier in LS. Here we summarize the current knowledge on the role of HLA type in cancer risk and outline future research directions to delineate possible association in the scenario of LS with genetically defined risk population and highly immunogenic tumors.Peer reviewe

Comprehensive characterization of local and systemic neoantigen-specific immune responses and biomarkers in Lynch syndrome

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

Implications of Hereditary Origin on the Immune Phenotype of Mismatch Repair-Deficient Cancers: Systematic Literature Review

Microsatellite instability (MSI) represents one of the major types of genomic instability in human cancers and is most common in colorectal cancer (CRC) and endometrial cancer (EC). MSI develops as a consequence of DNA mismatch repair (MMR) deficiency, which can occur sporadically or in the context of Lynch syndrome (LS), the most common inherited tumor syndrome. MMR deficiency triggers the accumulation of high numbers of somatic mutations in the affected cells, mostly indel mutations at microsatellite sequences. MSI tumors are among the most immunogenic human tumors and are often characterized by pronounced local immune responses. However, so far, little is known about immunological differences between sporadic and hereditary MSI tumors. Therefore, a systematic literature search was conducted to comprehensively collect data on the differences in local T cell infiltration and immune evasion mechanisms between sporadic and LS-associated MSI tumors. The vast majority of collected studies were focusing on CRC and EC. Generally, more pronounced T cell infiltration and a higher frequency of B2M mutations were reported for LS-associated compared to sporadic MSI tumors. In addition, phenotypic features associated with enhanced lymphocyte recruitment were reported to be specifically associated with hereditary MSI CRCs. The quantitative and qualitative differences clearly indicate a distinct biology of sporadic and hereditary MSI tumors. Clinically, these findings underline the need for differentiating sporadic and hereditary tumors in basic science studies and clinical trials, including trials evaluating immune checkpoint blockade therapy in MSI tumors

A Trickster in Disguise: Hyaluronan’s Ambivalent Roles in the Matrix

Hyaluronan (HA) is a simple but diverse glycosaminoglycan. It plays a major role in aging, cellular senescence, cancer, and tissue homeostasis. In which way HA affects the surrounding tissues greatly depends on the molecular weight of HA. Whereas high molecular weight HA is associated with homeostasis and protective effects, HA fragments tend to be linked to the pathologic state. Furthermore, the interaction of HA with its binding partners, the hyaladherins, such as CD44, is essential for sustaining tissue integrity and is likewise related to cancer. The naked mole rat, a rodent species, possesses a special form of very high molecular weight (vHMW) HA, which is associated with the extraordinary cancer resistance and longevity of those animals. This review addresses HA and its diverse facets: from HA synthesis to degradation, from oligomeric HA to vHMW-HA and from its beneficial properties to the involvement in pathologies. We further discuss the functions of HA in the naked mole rat and compare them to human conditions. Though intensively researched, this simple polymer bears some secrets that may hold the key for a better understanding of cellular processes and the development of diseases, such as cancer

The coding microsatellite mutation profile of PMS2-deficient colorectal cancer

Lynch syndrome (LS) is caused by a pathogenic heterozygous germline variant in one of the DNA mismatch repair (MMR) genes: MLH1, MSH2, MSH6 or PMS2. LS-associated colorectal carcinomas (CRCs) are characterized by MMR deficiency and by accumulation of multiple insertions/deletions at coding microsatellites (cMS). MMR deficiency-induced variants at defined cMS loci have a driver function and promote tumorigenesis. Notably, PMS2 variant carriers face only a slightly increased risk of developing CRC. Here, we investigate whether this lower penetrance is also reflected by differences in molecular features and cMS variant patterns. Tumor DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue cores or sections (n = 90). Tumors originated from genetically proven germline pathogenic MMR variant carriers (including 14 PMS2-deficient tumors). The mutational spectrum was analyzed using fluorescently labeled primers specific for 18 cMS previously described as mutational targets in MMR-deficient tumors. Immune cell infiltration was analyzed by immunohistochemical detection of T-cells on FFPE tissue sections. The cMS spectrum of PMS2-deficient CRCs did not show any significant differences from MLH1/MSH2-deficient CRCs. PMS2-deficient tumors, however, displayed lower CD3-positive T-cell infiltration compared to other MMR-deficient cancers (28.00 vs. 55.00 per 0.1 mm2, p = 0.0025). Our study demonstrates that the spectrum of potentially immunogenic cMS variants in CRCs from PMS2 gene variant carriers is similar to that observed in CRCs from other MMR gene variant carriers. Lower immune cell infiltration observed in PMS2-deficient CRCs could be the result of alternative mechanisms of immune evasion or immune cell exclusion, similar to those seen in MMR-proficient tumors.</p