Klinisch-experimentelle Analyse zur Expression von IDO1 und TREM2 in Blut- und Gewebeproben von Patienten mit oralem Plattenepithelkarzinom

Hintergrund: Trotz multimodaler Therapiekonzepte hat die 5-Jahres-Überlebensrate des oralen Plattenepithelkarzinoms (oral squamous cell carcinoma, OSCC) in den letzten Jahrzehnten bei 53,7% stagniert. Das Hauptaugenmerk der aktuellen onkologischen Grundlagenforschung liegt daher auf der Identifizierung tumorspezifischer Marker, die für die Entwicklung von neuen diagnostischen und individualisierten, zielgerichteten therapeutischen Strategien eingesetzt werden können. Im Rahmen dieser Arbeit wurde die Bedeutung von Indolamin-2,3-dioxygenase 1 (IDO1) und Triggering receptor expressed on myeloid cells type 2 (TREM2) beim OSCC untersucht, denen beiden ein hoher Stellenwert in der tumorinitiierten Immunsuppression zugeschrieben wird. Methodik: Im ersten Teil dieser Arbeit erfolgten immunhistochemische Färbungen zum Nachweis der Expression von IDO1 und TREM2 im Tumorgewebe sowie korrespondierenden Lymphknotenmetastasen, um diese auf eine potentielle Korrelation mit klinischen und histopathologischen Parametern sowie eine mögliche prognostische Relevanz zu untersuchen. Im zweiten Schritt galt es, Makrophagen auf eine mögliche Expression von IDO1 und TREM2 zu analysieren. Hierfür wurde Tumor- sowie gesundes Nachbargewebe dissoziiert und in Einzelzellsuspensionen überführt sowie periphere mononukleäre Zellen (peripheral blood mononuclear cells, PBMCs) isoliert und anschließend mittels Durchflusszytometrie auf die Expression der Makrophagenmarker CD68, CD163 und CD206 überprüft. Soluble TREM2 (sTREM2, Shedding- bzw. alternatives Splicingprodukt von TREM2) und die IDO1-Aktivität (gemessen als Kynurenin/Tryptophan-Ratio) wurden als serologisch bestimmbare Tumormarker untersucht. Ergebnisse: Eine hohe epitheliale IDO1-Expression in Primärtumoren zeigte eine signifikante Assoziation mit dem weiblichen Geschlecht (Spearman‘s rank correlation coefficient (SRC), p=0,046), einem Alter ≤ 75 Jahre (SRC, p=0,017), den niedrigen Union internationale contre le cancer (UICC)-Stadien (SRC, p<0,001) sowie dem Auftreten eines Rezidivs (SRC, p=0,021). Eine hohe IDO1-Expression in Immunzellen der Primärtumore korrelierte signifikant mit dem weiblichen Geschlecht (SRC, p=0,006), einem Alter > 75 Jahre (SRC, p=0,019) sowie hohen UICC-Stadien (SRC, p=0,026). Die IDO1-Expression in Epithelzellen der Primärtumore zeigte in der Univariat-Analyse einen signifikanten Einfluss auf das progressionsfreie Überleben (Hazard Ratio (HR): 2,544; 95% confidence interval (9%CI): 1,259-5,139; p=0,009) und das Gesamtüberleben (HR: 2,817; 95%CI: 1,428-5,558; p=0,009). In der Multivariat-Analyse konnte ein annähernd signifikanter Einfluss auf die Rezidivwahrscheinlichkeit festgestellt werden (HR: 2,073; 95%CI: 0,959-4,479; p=0,064). Die IDO1-Expression in Immunzellen der Primärtumore wies in der Univariat-Analyse hingegen einen signifikanten Einfluss auf das progressionsfreie Überleben (HR: 1,483; 95%CI: 1,029-2,137; p=0,029) sowie einen annähernd signifikanten Einfluss auf das Gesamtüberleben auf (HR: 1,428; 95%CI: 0,985-2,070; p=0,054). In der Multivariat-Analyse wurde die IDO1-Expression in Immunzellen als annähernd signifikanter unabhängiger Einflussfaktor auf das progressionsfreie Überleben bestätigt (HR: 1,416; 95%CI: 0,967-2,074; p=0,074). Bei der Auswertung der TREM2-Expression in Immunzellen der Primärtumore zeigte sich, dass diese positiv mit dem weiblichen Geschlecht (SRC, p=0,021), den UICC-Stadien (SRC, p=0,040) und der PD-L1-Expression korreliert (SRC, p 75 Jahre (SRC, p=0,042) und einer hohen PD-L1-Expression (SRC, p<0,001). Die TREM2-Expression in Immunzellen der Lymphknotenmetastasen zeigte in der Multivariat-Analyse hinsichtlich des progressionsfreien Überlebens einen annähernd signifikanten Einfluss (HR: 4,904; 95%CI: 1,003-23,987; p=0,050). Die IDO1- und TREM2-Expression auf Makrophagen der PBMCs nahm mit zunehmendem UICC-Stadium zu und war im Allgemeinen höher als in der Kontrollkohorte. Der Anteil IDO1-exprimierender Zellen an den CD68+- und CD163+-Zellen war im Tumorgewebe hochsignifikant höher als im gesunden Nachbargewebe (t-Test, p0,05). Die Kynurenin- und Tryptophan-Konzentrationen sowie ihre Ratio unterschieden sich signifikant zwischen Patienten mit und ohne Lymphknotenmetastasen (t-Test, p<0,05). Die Kynurenin/Tryptophan-Ratio korrelierte signifikant mit der IDO1-Expression der PBMCs (SRC, p<0,05). Die sTREM2-Konzentration korrelierte mit dem Fortschritt der malignen Erkrankung, ohne das Signifikanzniveau zu erreichen (SRC, UICC-Stadium: p=0,059). Schlussfolgerung: IDO1 und TREM2 konnten als Biomarker des OSCCs verifiziert werden und könnten im Rahmen der Therapieindividualisierung (personalisierte Medizin) zur Therapiestratifizierung und -optimierung beitragen. Hierbei könnten vor allem Patienten in fortgeschrittenen UICC-Stadien von einer gegen die Marker gerichteten Immuntherapie profitieren. Nach klinischer Revalidierung könnte die Kynurenin/Tryptophan-Ratio (als Surrogat der IDO1-Aktivität) zukünftig zur Therapiestratifizierung von Patienten mit lokoregionär metastasiertem OSCC unter Berücksichtigung individueller Risikoprofile in die Routinediagnostik implementiert werden.Background: Despite multimodal therapy concepts, the 5-year survival rate for oral squamous cell carcinoma (OSCC) has stagnated at 53.7% in recent decades. The focus of current oncological basic research is therefore on the identification of tumor-specific markers that can be used for the development of new diagnostic and individualized, targeted therapeutic strategies. In this work, the importance of indolamine-2,3-dioxygenase 1 (IDO1) and triggering receptor expressed on myeloid cells type 2 (TREM2) in OSCC was investigated, both of which are considered to be of great importance in tumor-initiated immunosuppression. Methods: In the first part of this work, immunohistochemical staining was performed to detect the expression of IDO1 and TREM2 in the tumor tissue and the corresponding lymph node metastases in order to identify a correlation with clinical and histopathological parameters and a potential prognostic relevance. The second step was to investigate a possible expression of IDO1 and TREM2 in macrophages. For this purpose, tumor and healthy adjacent tissue were dissociated and transferred into single cell suspensions, and peripheral blood mononuclear cells (PBMCs) were isolated and then investigated for the expression of the macrophage markers CD68, CD163 and CD206 by flow cytometry. Soluble TREM2 (sTREM2, shedding or alternative splicing product of TREM2) and IDO1 activity (measured as kynurenine/tryptophan ratio) were investigated as serologically determinable tumor markers. Results: Immunohistochemical staining showed that high epithelial IDO1 expression in the primary tumors was significantly associated with female sex (Spearman's rank correlation coefficient (SRC), p=0.046), age ≤ 75 years (SRC, p=0.017), the lower Union international contre le cancer (UICC) stages (SRC, p<0.001) and the risk of recurrence (SRC, p=0.021). High IDO1 expression in immune cells of the primary tumors correlated significantly with female sex (SRC, p=0.006), age > 75 years (SRC, p=0.019), and more advanced UICC stages (SRC, p=0.026). In univariate analysis, IDO1 expression in epithelial cells of the primary tumors showed a significant influence on progression-free survival (hazard ratio (HR): 2.544; 95% confidence interval (95%CI): 1.259-5.139; p=0.009) and overall survival (HR: 2.817, 95%CI: 1.428-5.558, p=0.009). In the multivariate analysis, it almost reached statistical significance on the probability of recurrence (HR: 2.073; 95%CI: 0.959-4.479; p=0.064). In the univariate analysis, however, IDO1 expression in immune cells of the primary tumors showed a significant influence on progression-free survival (HR: 1.483; 95%CI: 1.029-2.137; p=0.029) and an almost significant influence on overall survival (HR: 1.428, 95%CI: 0.985-2.070, p=0.054). In multivariate analysis, IDO1 expression was confirmed to be an approximately significant independent factor influencing progression-free survival (HR: 1.416; 95%CI: 0.967-2.074; p=0.074). Analysis of TREM2 expression in immune cells of the primary tumors showed a positive correlation with female gender (SRC, p=0.021), UICC stage (SRC, p=0.040) and PD-L1 expression (SRC, p 75 years (SRC, p=0.042), and high PD-L1 expression (SRC, p<0.001). In the multivariate analysis, TREM2 expression in immune cells of the lymph node metastases showed an almost significant influence on progression-free survival (HR: 4.904; 95%CI: 1.003-23.987; p=0.050). IDO1 and TREM2 expression on macrophages of the PBMCs increased with increasing UICC stage and was generally higher than in the control cohort. The proportion of IDO1+- cells in the CD68+- and CD163+-cells was significantly higher in the tumor tissue than in the healthy adjacent tissue (t-test, p0.05). The kynurenine and tryptophan concentrations as well as their ratio differed significantly between patients with and without lymph node metastases (t-test, p<0.05). The kynurenine/tryptophan ratio correlated significantly with the IDO1 expression of the PBMCs (SRC, p<0.05). The sTREM2 concentration correlated with the progression of the malignant disease, but the level of significance was just missed (SRC, UICC stage: p=0.059). Conclusion: IDO1 and TREM2 could be verified as biomarkers of OSCC and could be used in the context of therapy individualization (personalized medicine) for therapy stratification and optimization. Patients in advanced UICC stages in particular could benefit from an immunotherapy targeting IDO1 or TREM2. After clinical revalidation, the kynurenine/tryptophan ratio (as a surrogate of IDO1 activity) could be implemented in routine diagnostics for therapy stratification of patients with locoregional metastatic OSCC, taking into account individual risk profiles

Femtosekundenlaser-assistierte vs. mechanische Implantation von ICRS und MyoRing : eine Metaanalyse

Fragestellung: Klinische Studien haben gezeigt, dass die intrakornealen Ringsegmente (ICRS) und der MyoRing geeignete Behandlungsoptionen für Patienten mit Keratektasien sind. Aufgrund der sichereren und präziseren Tunnelkreation mithilfe des Femtosekunden (FS) Lasers ersetzt dieser zunehmend die mechanische Tunnelkreation. Das Ziel dieser Metaanalyse war es, die Ergebnisse von intrakornealen Ringsegmenten und MyoRing bei der Behandlung von Keratektasien zu evaluieren und die klinischen Ergebnisse und Komplikationsraten zwischen mechanischer und FS Laser-assistierter Tunnelkreation zu vergleichen. Methodik: Die elektronische Literaturrecherche erfolgte via Pubmed und Cochrane Library. Es wurden Studien, die die Behandlung von Patienten mit Keratokonus, pelluzider marginaler Hornhautdegeneration oder Post-LASIK-Ektasie mithilfe von Intacs, Intacs SK, Keraring, Ferrara Ring oder MyoRing untersuchten, eingeschlossen, sofern diese prä- und postoperative Messdaten mit Standardabweichung eindeutig differenziert zwischen FS Laser-Präparation und mechanischer Tunnelkreation zu den Zeitpunkten drei, sechs oder zwölf Monate post operationem zu einem oder mehreren der folgenden Parametern aufführten: Sphäre, Zylinder, Sphärisches Äquivalent, unkorrigierter Visus, korrigierter Visus, flacher, steiler und mittlerer Keratometriewert. Die Ergebnisse wurden mithilfe der gewichteten mittleren Differenz und des dazu gehörigen 95%-Konfidenzintervalls (95% CI) veranschaulicht. Bei der Berechnung der Komplikationsraten erfolgte eine Varianzstabilisierung mithilfe der Freeman-Tukey-Transformation. Ergebnisse: Von 1484 in die Vorauswahl miteinbezogenen Studien konnten letztendlich 115 Studien in die Metaanalyse eingeschlossen werden. Alle Ringarten zeigten postoperativ eine signifikante Verbesserung der klinischen Messparameter. Die Veränderungen waren zwischen beiden Präparationsarten vergleichbar. Die Komplikationsraten waren in der Gruppe der mechanischen Präparation ausnahmslos größer. Die Komplikationsraten betrugen für Dezentrierung 1,6% vs. 0,7%, superfizielle Deplatzierung 1,7% vs. 0%, Hornhautperforation 1,1% vs. 0,1%, Explantation 6,3% vs. 1,2%, Hornhauteinschmelzung 1,3% vs. 0,7%, Migration des Ringsegments 2,9% vs. 0,3%, korneale Neovaskularisation 2,3% vs. 0,7%, Ringextrusion 4,7% vs. 1,7% und infektiöse Keratitis 1,2% vs. 0,1%. Schlussfolgerungen: Die ICRS und der MyoRing sind geeignete Behandlungsoptionen für Patienten mit Keratektasien. Mit beiden Methoden können Verbesserungen des Visus, der Refraktion und der Keratometrie erreicht werden. Die intra- und postoperativen Komplikationsraten sind bei der mechanischen Tunnelpräparation im Vergleich zur FS Laser-assistierten Technik deutlich erhöht.Purpose: The aims of this meta-analysis were to evaluate the results of intracorneal ring segments (ICRS) and MyoRing in the management of corneal ectasia and to compare the clinical outcomes and complication rates between mechanical and femtosecond (FS) laserassisted surgery. Methods: An online electronic search was performed for pre-post studies published until April 2020. Studies were included if they dealt with the treatment of patients with keratoconus, pellucide marginal degeneration (PMD) or post-LASIK-ectasia with Intacs, Intacs SK, Keraring, Ferrara Ring or MyoRing implantation and reported the mean change of one or more outcome measures, including uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), sphere, cylinder, spherical equivalent (SE), steep (Ks), flat (Kf) and mean keratometry (Km) (with the standard deviation) at time point of 3, 6 or 12 months after implantation. Weighted mean difference (WMD) with 95% confidence interval was used as a pooled estimation of intervention efficacy. Complication rates were calculated using the Freeman-Tukey transformation. Results: Of 1484 potentially related studies, 115 studies were finally included in the metaanalysis. Findings of this meta-analysis demonstrated considerable improvement in visual, refractive and keratometric outcomes in all ICRS models and MyoRing. Intrastromal tunnel creation with both methods yielded similar results. Complication rates were without exception higher when mechanical dissection was used compared to FS laser-assisted dissection, with respectively: 1.6% vs. 0.7% decentration, 1.7% vs. 0% superficial placement, 1.1% vs. 0.1% corneal perforation, 6.3% vs. 1.2% explantation, 1.3% vs. 0.7% corneal melting, 2.9% vs. 0.3% ring segment migration, 2.3% vs. 0.7% corneal neovascularization, 4.7% vs. 1.7% ring extrusion and 1.2% vs. 0.1% keratitis. Conclusions: ICRS and MyoRing are appropriate treatment options for patients with corneal ectasia. Both techniques for tunnel creation are efficacious in achieving good visual, keratometric and refractive results. Mechanical intrastromal tunnel creation is associated with much higher complication rates when compared to FS laser-assisted technique

HLA-E and Its Soluble Form as Indicators of a Sex-Specific Immune Response in Patients with Oral Squamous Cell Carcinoma

The human leukocyte antigene E (HLA-E) is associated with tumorigenesis in various cancers. Immunoncology along with sex-specific aspects in cancer therapy are now in scientific focus. Therefore, immunohistochemical HLA-E expression was retrospectively analysed in a cohort of oral squamous cell carcinomas (OSCC) after surgical therapy. Then, serum concentration of HLA-E (sHLA-E) was quantified in a prospective cohort by enzyme-linked immunosorbent assay. High HLA-E expression was associated with advanced UICC stage (Spearman’s correlation: p = 0.002) and worse survival (Cox-regression: progression-free survival: hazard ratio (HR) 3.129, confidence range (CI) 1.443–6.787, p = 0.004; overall survival: HR 2.328, CI 1.071–5.060, p = 0.033). The sHLA-E concentration was significantly higher in the control group than in tumor group (Mann–Whitney U-test (MW-U): p = 0.021). Within the tumor group, women showed significantly higher sHLA-E levels than men (MW-U: p = 0.049). A closer look at the tumor group and the control group showed that gender-specific differences exist: while no differences in sHLA-E concentration were detectable between female subjects of tumor group and control group (MW-U: p = 0.916), male subjects of tumor group had a significantly lower sHLA-E concentration compared to those of control group (MW-U: p = 0.001). In summary, our results provide evidence for sex-specific differences in immune responses in OSCC. This fact should be considered regarding future immunotherapy regimens

TREM2 Is Associated with Advanced Stages and Inferior Prognosis in Oral Squamous Cell Carcinoma

Triggering receptor expressed on myeloid cells 2 (TREM2) is suggested to hamper antitumor immune response in multiple cancers. However, the role of TREM2 in oral squamous cell carcinoma (OSCC) and its expression in tumor-associated macrophages (TAMs) are unknown. In this study, TREM2 expression was analyzed in the primary tumors and corresponding lymph-node metastases of OSCC patients via immunohistochemistry on tissue microarrays. Human peripheral blood mononuclear cells (PBMCs) and single-cell suspensions of tumor and healthy adjacent tissues were analyzed for the presence of TREM2+ macrophages and TAMs using flow cytometry. The serum levels of soluble TREM2 (sTREM2) were quantified using an enzyme-linked immunosorbent assay. High TREM2 expression was associated with advanced UICC stages (Spearman’s rank correlation (SRC), p = 0.04) and significantly reduced survival rates in primary tumors (multivariate Cox regression, progression-free survival: hazard ratio (HR) of 2.548, 95% confidence interval (CI) of 1.089–5.964, p = 0.028; overall survival: HR of 2.17, 95% CI of 1.021–4.613, p = 0.044). TREM2 expression was significantly increased in the PBMCs of OSCC patients in UICC stage IV compared with healthy controls (ANOVA, p < 0.05). The serum levels of sTREM2 were higher in advanced UICC stages, but they narrowly missed significance (SRC, p = 0.059). We demonstrated that TREM2 was multi-factorially associated with advanced stages and inferior prognosis in OSCC patients and that it could serve as a prognostic biomarker in OSCC patients. Targeting TREM2 has the potential to reshape the local and systemic immune landscape for the potential enhancement of patients’ prognosis

Alterations in macrophage polarization in the craniofacial and extracranial skeleton after zoledronate application and surgical interventions – an in vivo experiment

PurposeMedication-related osteonecrosis occurs exclusively in the jaw bones. However, the exact pathogenesis of medication-related osteonecrosis of the jaw (MRONJ) and the unique predisposition of the jaw bones have not been elucidated, making its treatment a challenge. Recent evidence indicates that macrophages might play a pivotal role in MRONJ pathogenesis. The aim of the present study was to compare the macrophage populations between the craniofacial and extracranial skeleton and to investigate the changes induced by zoledronate (Zol) application and surgical interventions.Materials and methodsAn in vivo experiment was performed. 120 wistar rats were randomized to 4 groups (G1, G2, G3, G4). G1 served as an untreated control group. G2 and G4 received Zol injections for 8 weeks. Afterwards, the right lower molar of the animals from G3 and G4 was extracted and the right tibia osteotomized followed by osteosynthesis. Tissue samples were taken from the extraction socket and the tibia fracture at fixed time points. Immunohistochemistry was conducted to determine the labeling indexes of CD68+ and CD163+ macrophages.ResultsComparing the mandible and the tibia, we observed a significantly higher number of macrophages and a heightened pro-inflammatory environment in the mandible compared to the tibia. Tooth extraction caused an increase of the overall number of macrophages and a shift toward a more pro-inflammatory microenvironment in the mandible. Zol application amplified this effect.ConclusionOur results indicate fundamental immunological differences between the jaw bone and the tibia, which might be a reason for the unique predisposition for MRONJ in the jaw bones. The more pro-inflammatory environment after Zol application and tooth extraction might contribute to the pathogenesis of MRONJ. Targeting macrophages might represent an attractive strategy to prevent MRONJ and improve therapy. In addition, our results support the hypothesis of an anti-tumoral and anti-metastatic effect induced by BPs. However, further studies are needed to delineate the mechanisms and specify the contributions of the various macrophage phenotypes

IDO1 is highly expressed in macrophages of patients in advanced tumour stages of oral squamous cell carcinoma

Purpose Strategies for Indolamine-2,3-dioxygenase 1 (IDO1) inhibition in cancer immunotherapy once produced encouraging results, but failed in clinical trials. Recent evidence indicates that immune cells in the tumour microenvironment, especially macrophages, contribute to immune dysregulation and therefore might play a critical role in drug resistance. Methods In this study, we investigated the signifcance of IDO1 expressing immune cells in primary tumours and corresponding lymph node metastases (LNMs) in oral squamous cell carcinoma (OSCC) by immunohistochemistry. The link between IDO1 and macrophages was investigated by fow cytometry in tumour tissue, healthy adjacent tissue and peripheral blood mononuclear cells (PBMCs). IDO1 activity (measured as Kynurenine/Tryptophan ratio) was assessed by ELISAs. Results High IDO1 expression in tumour-infltrating immune cells was signifcantly correlated with advanced stages [Spearman’s rank correlation (SRC), p=0.027] and reduced progression-free survival (multivariate Cox regression, p=0.034). IDO1 was signifcantly higher expressed in PBMCs of patients in advanced stages than in healthy controls (ANOVA, p<0.05) and IDO1+ macrophages were more abundant in intratumoural areas than peritumoural (t test, p<0.001). IDO1 expression in PBMCs was signifcantly correlated with IDO1 activity in serum (SRC, p<0.05). IDO1 activity was signifcantly higher in patients with LNMs (t test, p<0.01). Conclusion All in all, IDO1 expressing immune cells, especially macrophages, are more abundant in advanced stages of OSCC and are associated with reduced progression-free survival. Further investigations are needed to explore their role in local and systemic immune response. The IDO1 activity might be a suitable biomarker of metastasis in OSCC patients

The Immune Checkpoint Receptor CD96: A Local and Systemic Immune Modulator in Oral Cancer?

Background: As immunotherapy of oral squamous cell carcinomas (OSCCs), using PD1 inhibitors, is only efficient in a small proportion of patients, additional immune checkpoints need to be identified as potential therapeutic targets. There is evidence that a blockade of CD96 might positively affect the anti-tumor immune response. The aim of this study was to analyze the gene and protein expression of CD96 in the tissue and peripheral blood of OSCC patients compared to healthy controls, while also checking for potential associations with a differential expression to the histomorphological parameters. In addition, possible correlations with the expression of PD1 and PD-L1 as well as the macrophage markers CD68 and CD163 should be tested to obtain further insights into the potential effectiveness of combined checkpoint blockage. Material and Methods: For real-time quantitative polymerase chain reaction (RT-qPCR), a total of 183 blood and tissue samples, divided into a patient and a control group, were included. Additionally, 141 tissue samples were examined by immunohistochemistry (IHC). The relative expression differences between the groups were calculated using statistical tests including the Mann–Whitney U test and AUC method. The Chi-square test was used to determine whether CD96 overexpression in individual samples is associated with malignancy. Correlation analysis was performed using the Spearman correlation test. Results: There was a significant CD96 mRNA and protein overexpression in the OSCC group compared to the controls (p = 0.001). In contrast, CD96 mRNA expression in the peripheral blood of the OSCC patients was significantly lower compared to the control group (p = 0.007). In the Chi-square test, the OSCC tissue samples showed a highly significant upregulation of CD96 mRNA expression (p p = 0.005) compared to the healthy mucosa. CD96 mRNA and protein expression correlated significantly (p = 0.005). In addition, there was a significant positive correlation of CD96 expression with PD1 (p ≤ 0.001), PD-L1 (p ≤ 0.001), and CD163 (p = 0.006) at the mRNA level. Conclusions: CD96 expression in the tumor tissue and peripheral blood of OSCC patients is differentially regulated and appears to be a relevant immune checkpoint