Identifizierung prädiktiver statischer und <em>on-treatment</em> Biomarker zur Vorhersage des Ansprechens auf Immuncheckpoint-Inhibition im Nierenzellkarzinom

Das übergeordnete Ziel dieser kumulativen Habilitationsschrift ist die Identifizierung von vielversprechenden prädiktiven Biomarkern, die das Ansprechen auf Immuntherapie für Patienten mit metastasiertem Nierenzellkarzinom (mRCC) vorhersagen können. Diese Zielsetzung ist von besonderer klinischer Relevanz, da aktuell unterschiedliche, aber als gleichwertig eingestufte anti-PD-1 basierte Kombinationstherapien (ICI+ICI versus ICI+TKI) in der Erstlinientherapie des mRCC eingesetzt werden. Da vergleichende, prospektive Studien noch ausstehend sind, ergibt sich für den klinischen Alltag daher zum jetzigen Zeitpunkt eine weitgehend unselektierte Erstlinientherapieentscheidung. Trotz intensiver Bemühungen in der translationalen Forschung gibt es bisher keinen robusten, prädiktiven, im klinischen Alltag eingesetzten Biomarker, der eine rationale Therapieentscheidungsfindung ermöglicht. In zwei der in dieser Habilitationsschrift zugrunde liegenden wissenschaftlichen Arbeiten wurden Methylierungssignaturen im therapienaiven Tumorgewebe untersucht, um das Therapieansprechen auf Immuntherapie vorherzusagen. So konnte eine Rationale für die Messung des LAG3-Promotormethylierungsstatus als möglicher prädiktiver Biomarker für eine anti-LAG3 Immuntherapie geschaffen werden. Zudem wurde der Promotormethylierungsstatus von CTLA4 (mCTLA4) untersucht, der das Ansprechen auf Immuntherapie bei Patienten mit mRCC zuverlässig vorhersagen konnte und hinsichtlich des prädiktiven Potenzials der PD-L1-Expression überlegen war. Inwieweit mCTLA4 zu einer verbesserten Erstlinientherapieentscheidung für Patienten mit mRCC beitragen kann, muss im Rahmen prospektiver Validierungsstudien untersucht werden. Um die Komplexität der Tumor-Immun-Interaktion genauer vorhersagen zu können, wurden zudem dynamische on-treatment Biomarker untersucht. Im Rahmen dieser Habilitationsschrift ist es gelungen, den prädiktiven Wert einer charakteristischen frühen on-treatment CRP-Kinetik nach Einleitung der Immuntherapie, welche im Januar 2021 durch Fukuda et al. erstbeschrieben wurde, sowohl für das mRCC und metastasierte Urothelkarzinom (mUC) zu bestätigen als auch erstmalig für das NSCLC mittels retrospektiver Entdeckungs- und einer prospektiver Validierungskohorte zu beweisen. Unserer Ergebnisse deuten darauf hin, dass die frühe on-treatment CRP-Kinetik ein biologisches Tumortyp-unabhängiges Phänomen zu sein scheint und somit in der Ära 30 der Immunonkologie auch für weitere Tumorentitäten ein prädiktives Potenzial aufweisen könnte. Ein besonders hervorzuhebender Vorteil für die Anwendung der frühen CRP-Kinetik als neuen prädiktiven on-treatment Biomarker ist, dass die Bestimmung der Serum CRP-Konzentration ein klinisch etablierter, kosteneffizienter und nicht-invasiver Biomarker ist, der somit rasch in den klinischen Alltag integriert werden könnte. Ein wesentliches Ergebnis dieser kumulativen Habilitationsarbeit ist, dass durch eine Modifikation der ursprünglichen CRP-Kinetik-Definition der klinische Wert dieses ontreatment Biomarkers signifikant gesteigert werden konnte. Durch Anwendung unserer modifizierten CRP-Kinetik Definition kann bereits vier Wochen nach Start der Immuntherapie das Therapieansprechen robust vorausgesagt werden. Da das erste Routine-Staging in der Regel 8-12 Wochen nach Therapiestart durchgeführt wird, öffnet sich durch unsere Anpassung der CRP-Kinetik Definition ein großes therapeutisches Fenster für rasche Therapieadjustierungen. Basierend auf unserer neuen CRP-Kinetik Definition könnten zukünftig CRP-Kinetik-stratifizierte Studien initiiert werden, in denen bereits vier Wochen nach Einleitung der Immuntherapie für den CRP-Non-Responder-Arm eine Therapieeskalation folgen könnte, wohingegen für den CRP-Flare-Responder-Arm auch eine Therapiedeeskalation ein rationales Therapiekonzept darstellen könnte. Zusammenfassend lässt sich sagen, dass die frühe on-treatment CRP-Kinetik ein biologisches Tumortyp-unabhängiges Phänomen zu sein scheint, welches ein großes Potenzial hat, das Therapieansprechen auf eine Immuntherapie vorherzusagen. Ob die frühe longitudinale CRP-Kinetik Definition den für diese Anwendung optimalen prädiktiven on-treatment Biomarker darstellt, müssen prospektive Untersuchungen zeigen. Die im Rahmen dieser Habilitationsarbeit erhobenen Daten stellen jedoch einen wichtigen Ausgangspunkt für die Untersuchung weiterer prädiktiver on-treatment Biomarkermodelle dar, welche zukünftig das Therapiemonitoring und damit auch die Therapieadjustierung einer Vielzahl von Tumorpatienten unter Immuntherapie verbessern könnte

C‐reactive protein flare‐response predicts long‐term efficacy to first‐line anti‐PD‐1‐based combination therapy in metastatic renal cell carcinoma

Objectives Immune checkpoint blockade (IO) has revolutionised the treatment of metastatic renal cell carcinoma (mRCC). Early C-reactive protein (CRP) kinetics, especially the recently introduced CRP flare-response phenomenon, has shown promising results to predict IO efficacy in mRCC, but has only been studied in second line or later. Here, we aimed to validate the predictive value of early CRP kinetics for 1st-line treatment of mRCC with αPD-1 plus either αCTLA-4 (IO+IO) or tyrosine kinase inhibitor (IO+TKI). Methods In this multicentre retrospective study, we investigated the predictive potential of early CRP kinetics during 1st-line IO therapy. Ninety-five patients with mRCC from six tertiary referral centres with either IO+IO (N = 59) or IO+TKI (N = 36) were included. Patients were classified as CRP flare-responders, CRP responders or non-CRP responders as previously described, and their oncological outcome was compared. Results Our data validate the predictive potential of early CRP kinetics in 1st-line immunotherapy in mRCC. CRP responders, especially CRP flare-responders, had significantly prolonged progression-free survival (PFS) compared with non-CRP responders (median PFS: CRP flare-responder: 19.2 months vs. responders: 16.2 vs. non-CRP responders: 5.6, P < 0.001). In both the IO+IO and IO+TKI subgroups, early CRP kinetics remained significantly associated with improved PFS. CRP flare-response was also associated with long-term response ≥ 12 months. Conclusions Early CRP kinetics appears to be a low-cost and easy-to-implement on-treatment biomarker to predict response to 1st-line IO combination therapy. It has potential to optimise therapy monitoring and might represent a new standard of care biomarker for immunotherapy in mRCC

MAGE expression in head and neck squamous cell carcinoma primary tumors, lymph node metastases and respective recurrences-implications for immunotherapy

Melanoma associated antigens (MAGE) are potential targets for immunotherapy and have been associated with poor overall survival (OS) in head and neck squamous cell carcinoma (HNSCC). However, little is known about MAGE in lymph node metastases (LNM) and recurrent disease (RD) of HNSCC. To assess whether MAGE expression increases with metastasis or recurrence, a tissue microarray (TMA) of 552 primary tumors (PT), 219 LNM and 75 RD was evaluated by immunohistochemistry for MAGE antigens using three monoclonal antibodies to multiple MAGE family members. Mean expression intensity (MEI) was obtained from triplicates of each tumor specimen. The median MEI compared between PT, LNM and RD was significantly higher in LNM and RD. In paired samples, MEI was comparable in PT to respective LNM, but significantly different from RD. Up to 25% of patients were negative for pan-MAGE or MAGE-A3/A4 in PT, but positive in RD. The prognostic impact of MAGE expression was validated in the TMA cohort and also in TCGA data (mRNA). OS was significantly lower for patients expressing pan-MAGE or MAGE-A3/A4 in both independent cohorts. MAGE expression was confirmed as a prognostic marker in HNSCC and may be important for immunotherapeutic strategies as a shared antigen

Immune signatures predict development of autoimmune toxicity in patients with cancer treated with immune checkpoint inhibitors

Background: Immune checkpoint inhibitors (ICIs) are among the most promising treatment options for melanoma and non-small cell lung cancer (NSCLC). While ICIs can induce effective anti-tumor responses, they may also drive serious immune-related adverse events (irAEs). Identifying biomarkers to predict which patients will suffer from irAEs would enable more accurate clinical risk-benefit analysis for ICI treatment and may also shed light on common or distinct mechanisms underpinning treatment success and irAEs. Methods: In this prospective multi-center study, we combined a multi-omics approach including unbiased single-cell profiling of over 300 peripheral blood mononuclear cell (PBMC) samples and high-throughput proteomics analysis of over 500 serum samples to characterize the systemic immune compartment of patients with melanoma or NSCLC before and during treatment with ICIs. Findings: When we combined the parameters obtained from the multi-omics profiling of patient blood and serum, we identified potential predictive biomarkers for ICI-induced irAEs. Specifically, an early increase in CXCL9/CXCL10/CXCL11 and interferon-γ (IFN-γ) 1 to 2 weeks after the start of therapy are likely indicators of heightened risk of developing irAEs. In addition, an early expansion of Ki-67+ regulatory T cells (Tregs) and Ki-67+ CD8+ T cells is also likely to be associated with increased risk of irAEs. Conclusions: We suggest that the combination of these cellular and proteomic biomarkers may help to predict which patients are likely to benefit most from ICI therapy and those requiring intensive monitoring for irAEs. Funding: This work was primarily funded by the European Research Council, the Swiss National Science Foundation, the Swiss Cancer League, and the Forschungsförderung of the Kantonsspital St. Gallen

Immune signatures predict development of autoimmune toxicity in patients with cancer treated with immune checkpoint inhibitors.

BACKGROUND Immune checkpoint inhibitors (ICIs) are among the most promising treatment options for melanoma and non-small cell lung cancer (NSCLC). While ICIs can induce effective anti-tumor responses, they may also drive serious immune-related adverse events (irAEs). Identifying biomarkers to predict which patients will suffer from irAEs would enable more accurate clinical risk-benefit analysis for ICI treatment and may also shed light on common or distinct mechanisms underpinning treatment success and irAEs. METHODS In this prospective multi-center study, we combined a multi-omics approach including unbiased single-cell profiling of over 300 peripheral blood mononuclear cell (PBMC) samples and high-throughput proteomics analysis of over 500 serum samples to characterize the systemic immune compartment of patients with melanoma or NSCLC before and during treatment with ICIs. FINDINGS When we combined the parameters obtained from the multi-omics profiling of patient blood and serum, we identified potential predictive biomarkers for ICI-induced irAEs. Specifically, an early increase in CXCL9/CXCL10/CXCL11 and interferon-γ (IFN-γ) 1 to 2 weeks after the start of therapy are likely indicators of heightened risk of developing irAEs. In addition, an early expansion of Ki-67+ regulatory T cells (Tregs) and Ki-67+ CD8+ T cells is also likely to be associated with increased risk of irAEs. CONCLUSIONS We suggest that the combination of these cellular and proteomic biomarkers may help to predict which patients are likely to benefit most from ICI therapy and those requiring intensive monitoring for irAEs. FUNDING This work was primarily funded by the European Research Council, the Swiss National Science Foundation, the Swiss Cancer League, and the Forschungsförderung of the Kantonsspital St. Gallen

Insights into Urologic Cancer

Collectively, urological malignancies account for a considerable proportion of cancer cases worldwide [...

CircEHD2, CircNETO2 and CircEGLN3 as Diagnostic and Prognostic Biomarkers for Patients with Renal Cell Carcinoma

Background: Circular RNA (circRNA) plays an important role in the carcinogenesis of various tumors. It is assumed that circRNAs have a high tissue and tumor specificity, thus they are discussed as cancer biomarkers. The knowledge about circRNAs in clear cell renal carcinoma (ccRCC) is limited so far, and thus we studied the expression profile of seven circRNAs (circCOL5A1, circEHD2, circEDEM2, circEGNL3, circNETO2, circSCARB1, circSOD2) in a cohort of ccRCC patients. Methods: Fresh-frozen normal and cancerous tissues were prospectively collected from patients with ccRCC undergoing partial/radical nephrectomy. Total RNA was isolated from 121 ccRCC and 91 normal renal tissues, and the circRNA expression profile was determined using quantitative real-time PCR. Results: circEHD2, circENGLN3, and circNETO2 were upregulated in ccRCC compared with non-malignant renal tissue. circENGLN3 expression was highly discriminative between normal and cancerous tissue. None of the circRNAs was correlated with clinicopathological parameters. High circEHD2 and low circNETO2 levels were an independent predictor of a shortened progression-free survival, cancer-specific survival, and overall survival in patients with ccRCC undergoing nephrectomy. Conclusions: The analysis of circRNAs may provide diagnostic and prognostic information. Thus, circRNAs could help to optimize the individual treatment and ultimately improve ccRCC patients’ survival

LAG3 (LAG-3, CD223) DNA methylation correlates with LAG3 expression by tumor and immune cells, immune cell infiltration, and overall survival in clear cell renal cell carcinoma

BackgroundLymphocyte activating 3 (LAG3, LAG-3, CD223) is a promising target for immune checkpoint inhibition in clear cell renal cell carcinoma (KIRC). The aim of this study was to investigate the epigenetic regulation of LAG3 in KIRC by methylation.MethodsWe correlated quantitative LAG3 methylation levels with transcriptional activity, immune cell infiltration, and overall survival in a cohort of n=533 patients with KIRC and n=160 normal adjacent tissue (NAT) samples obtained from The Cancer Genome Atlas (TCGA). Furthermore, we analyzed LAG3 methylation in peripheral blood mononuclear cells (PBMCs) and KIRC cell lines. We validated correlations between LAG3 expression, immune cell infiltrates, survival, and methylation in an independent KIRC cohort (University Hospital Bonn (UHB) cohort, n=118) by means of immunohistochemistry and quantitative methylation-specific PCR.ResultsWe found differential methylation profiles among PBMCs, NAT, KIRC cell lines, and KIRC tumor tissue. Methylation strongly correlated with LAG3 mRNA expression in KIRCs (TCGA cohort) and KIRC cell lines. In the UHB cohort, methylation correlated with LAG3-positive immune cells and tumor-intrinsic LAG3 protein expression. Furthermore, LAG3 methylation strongly correlated with signatures of distinct immune cell infiltrates, an interferon-y signature (TCGA cohort), and immunohistochemically quantified CD45+, CD8+, and CD4+ immune cell infiltrates (UHB cohort). LAG3 mRNA expression (TCGA cohort), methylation (both cohorts), and tumor cell-intrinsic protein expression (UHB cohort) was significantly associated with overall survival.ConclusionOur data suggest an epigenetic regulation of LAG3 expression in tumor and immune cells via DNA methylation. LAG3 expression and methylation is associated with a subset of KIRCs showing a distinct clinical course and immunogenicity. Our study provides rationale for further testing LAG3 DNA methylation as a predictive biomarker for response to LAG3 immune checkpoint inhibitors