Die Analyse der ektopischen Sox9-Expression zu verschiedenen Zeitpunkten der Oligodendrozyten-Entwicklung im murinen ZNS

Repair in the adult brain is often dependent on the reactivation of processes usually only active during development. In the case of demyelinating diseases such as multiple sclerosis, repair requires the generation of new oligodendrocytes and myelin by NG2 glia. While early postnatal NG2 glia proliferate and differentiate efficiently and endogenously express the transcription factor Sox9, adult NG2 glia are unable to maintain this efficient differentiation ability and do not express endogenous Sox9. Whether differences in Sox9 expression determine the differentiation efficiency of NG2 glia is the center of the research shown in this thesis. The current study examines whether ectopic Sox9 expression impacts the differentiation of NG2 glia at different stages of mouse development and in the adult. First, overexpressing Sox9 in prenatal NG2 glia results in an increase in the oligodendroglia population, more specifically in both the NG2 glia and the differentiating oligodendrocytes. Both an early boost in proliferative cells and a later increase in apoptotic cells was observed. Analysis of RNA sequencing from these mice shows that that the expression of genes associated with the ensheathment of axons and the positive regulation of myelination is upregulated while the expression of genes related to negative regulation of cell proliferation is decreased. Second, overexpressing Sox9 in early postnatal NG2 glia shows a transient decline in the oligodendroglial population in the white matter, with the NG2 glia population remaining stable but the pre-myelinating and differentiating oligodendrocytes displaying a reduction in overall numbers. Decreases in proliferation are evident in P7 and P14 mice in the corpus callosum while young juvenile mice exhibit an upturn in proliferative cells. Furthermore, the number of apoptotic cells in the younger mice is unaffected while the number of apoptotic, mature oligodendrocytes is increased in juvenile mice. Third, overexpressing Sox9 in adult NG2 glia results in more efficient maturation compared to controls. The cell numbers of total oligodendroglia, NG2 glia, and mature oligodendrocytes all remain unchanged. Overall, this study reveals that Sox9 overexpression at different time points of NG2 glia development and in the adult has different consequences for the NG2 glia and oligodendroglia populations and indicates a role for Sox9 in NG2 glia maturation. Uncovering Sox9 as a potential key regulator of NG2 glia differentiation opens the door to future therapies for demyelinating diseases.Reparaturprozesse im adulten Gehirn sind oft abhängig von der Reaktivierung von Abläufen, die normalerweise während der Entwicklung stattfinden. Bei demyelinisierenden Erkrankungen wie der Multiplen Sklerose erfordert dies die Bildung neuer Oligodendrozyten und neuen Myelins durch die NG2-Glia. Während früh postnatale NG2-Glia effizient proliferieren, differenzieren, und den Transkriptionsfaktor Sox9 exprimieren, sind adulte NG2-Glia nicht in der Lage, die Differenzierungsfähigkeit aufrechtzuerhalten, und besitzen kein Sox9. Ob die Sox9-Expression ein Schlüsselfaktor für die Differenzierungsfähigkeit der NG2-Glia ist, ist die zentrale Frage dieser Arbeit. Die vorliegende Studie untersucht, wie die Expression von Sox9 die Differenzierung von NG2-Glia in verschiedenen Stadien der Mausentwicklung und im Adulten beeinflusst. Die Überexpression von Sox9 in pränatalen NG2-Glia resultiert in einer Zunahme der oligodendroglialen Population, und zwar sowohl der NG2-Glia- als auch der differenzierenden Oligodendrozyten. Es wurde früh ein Anstieg der Proliferation und später ein erhöhter Zelltod beobachtet. Die Analyse von RNA-Sequenzierungen, bei denen Sox9 in pränatalen NG2-Glia überexprimiert wird, zeigt, dass die Expression von Genen, die mit der Ummantelung von Axonen und der positiven Regulierung der Myelinisierung verbunden sind, erhöht wird. Gene für die negative Regulierung der Zellproliferation werden herunterreguliert. Eine Überexpression von Sox9 in früh postnatalen NG2-Glia verursacht eine transiente Verringerung der oligodendroglialen Population, wobei die NG2-Glia-Population stabil blieb, die prä-myelinisierenden und differenzierenden Oligodendrozyten eine Reduktion aufwiesen. An P7 und P14 wurde eine erniedrigte Proliferation der NG2 Glia im Corpus callosum von Mäusen nachgewiesen, wohingegen juvenile Mäuse mehr proliferative Zellen zeigten. Die Zahl der apoptotischen Zellen in jüngeren Mäusen blieb unbeeinflusst, während in juvenilen Mäusen die Anzahl apoptotischer reifer Oligodendrozyten anstieg. Bei Überexpression von Sox9 in adulten NG2-Glia ergibt sich eine effizientere Reifung dieser Zellen. Die Zahl der Oligodendroglia, NG2-Glia und reifen Oligodendrozyten bleibt unverändert. Zusammenfassend lässt sich sagen, dass die Überexpression von Sox9 zu verschiedenen Zeitpunkten der NG2-Glia-Entwicklung und im Adulten unterschiedliche Konsequenzen für die NG2-Glia- und die Oligodendroglia-Populationen hat. Die Datenweisen aber auf eine Rolle von Sox9 bei der NG2-Glia-Reifung. Die Entdeckung von Sox9 als potenzieller Schlüsselregulator der NG2-Glia-Differenzierung eröffnet die Möglichkeit für zukünftige Therapieansätze zur Behandlung neurologischer demyelinisierender Erkrankungen

Medical needs and therapeutic options for melanoma patients resistant to anti-PD-1-directed immune checkpoint inhibition

Available 4- and 5-year updates for progression-free and for overall survival demonstrate a lasting clinical benefit for melanoma patients receiving anti-PD-directed immune checkpoint inhibitor therapy. However, at least one-half of the patients either do not respond to therapy or relapse early or late following the initial response to therapy. Little is known about the reasons for primary and/or secondary resistance to immunotherapy and the patterns of relapse. This review, prepared by an interdisciplinary expert panel, describes the assessment of the response and classification of resistance to PD-1 therapy, briefly summarizes the potential mechanisms of resistance, and analyzes the medical needs of and therapeutic options for melanoma patients resistant to immune checkpoint inhibitors. We appraised clinical data from trials in the metastatic, adjuvant and neo-adjuvant settings to tabulate frequencies of resistance. For these three settings, the role of predictive biomarkers for resistance is critically discussed, as well as are multimodal therapeutic options or novel immunotherapeutic approaches which may help patients overcome resistance to immune checkpoint therapy. The lack of suitable biomarkers and the currently modest outcomes of novel therapeutic regimens for overcoming resistance, most of them with a PD-1 backbone, support our recommendation to include as many patients as possible in novel or ongoing clinical trials

Evaluating policy responses to noncommunicable diseases in seven Caribbean countries: challenges to addressing unhealthy diets and physical inactivity.

OBJECTIVE: To identify, assess, and compare existing policies on noncommunicable diseases (NCDs) in the Caribbean, gaps in policy responses, and the factors influencing successful policy development and implementation following the Port of Spain Declaration of 2007. Specifically, to examine policies that target the upstream determinants of two NCD risk factors-unhealthy diets and physical inactivity. METHODS: A total of 76 semi-structured interviews with 80 relevant stakeholders in government, the private sector, and civil society were complemented by policy document analysis. Interviews were analyzed pragmatically, framed by the CARICOM government commitments, the WHO NCD Action Plan, a Multiple Streams framework approach, and realist evaluation ideas. RESULTS: The most widely-reported policy successes involved health promotion activities (e.g., school meal programs) that leveraged multisectoral collaboration among government ministries, such as Health, Education, and Agriculture. Large policy gaps still exist around creating legislative, physical, and social environments to support healthy eating and physical activity at the population level. Multisectoral NCD commissions successfully reached across sectors, but had limited influence on policy development. Different policy levels emerged with national-level policies considered a lengthy process, while "On-the-ground" programming was considered faster to implement than national policies. External barriers included a reliance on food imports enabled by international trade agreements limited availability, quality, and affordability of healthy foods. International pushback limited legislation to reduce food imports and the absence of an international/regional framework, similar to the Framework Convention on Tobacco Control, further impedes efforts. CONCLUSIONS: Regional collaboration and political support across sectors are essential to accelerating the pace of action to support healthy eating and active living environments. Policy "blueprints" could accelerate the process of development. Regional "NCD champions" could spearhead such responses and approaches

Tolerability of BRAF and MEK Inhibitors for Metastasized Melanoma after Intra-Class Switch: A Multicenter, Retrospective Study

Simple Summary Patients suffering from metastasized melanoma can be treated with three different combinations of tablets (“targeted therapy”), with similar efficacy but different side effect profiles. When one of the combinations is not tolerated well, their physicians may switch to a different combination; however, it has not been evaluated whether the second combination is actually tolerated better. In this work, we collected data on 94 patients from six German cancer centers who received two different combinations of targeted melanoma therapy to figure out whether the second combination was tolerated better. We found that indeed many side effects did not occur again and the novel combination was overall tolerated better, which justifies switching the combination when a patient experiences severe side effects. However, new side effects may occur. We believe our results are important for oncologists to adequately counsel patients with poor tolerability of this commonly used melanoma treatment. Abstract Targeted therapy with BRAF and MEK inhibitors (BRAFi, MEKi) is one of the mainstays of melanoma treatment. When dose-limiting toxicity (DLT) is observed, an option represents the intra-class switch to a different BRAFi+MEKi combination. Currently, there is scarce evidence for this procedure. This is a multicenter, retrospective analysis from six German skin cancer centers of patients who received two different combinations of BRAFi and MEKi. In total, 94 patients were included: 38 patients (40%) were re-exposed with a different combination because of previous unacceptable toxicity, 51 (54%) were re-exposed after progression, and 5 (5%) were included for other reasons. Of the 44 patients with a DLT during their first BRAFi+MEKi combination, only five (11%) experienced the same DLT during their second combination. A new DLT was experienced by 13 patients (30%). Six patients (14%) had to discontinue the second BRAFi treatment due to its toxicity. Compound-specific adverse events were avoided in the majority of patients by switching to a different combination. Efficacy data were similar to historical cohorts of BRAFi+MEKi rechallenge, with an overall response rate of 31% for patients who had previously progressed to treatment. We conclude that switching to a different BRAFi+MEKi combination if dose-limiting toxicity occurs is a feasible and rational approach in patients with metastatic melanoma

Interleukin-4 and -13 Gene Expression Profiles in Immune-Related Bullous Pemphigoid Indicate Efficacy of IL-4/IL-13 Inhibitors

Simple Summary Immune checkpoint inhibitor therapies are effective in treating different types of cancer. However, they also induceimmune-related adverse events, such as immune-related bullous pemphigoid (irBP), a cutaneous autoimmune disease that leads to blistering. To identify the best treatment option for irBP, this multicenter study analyzed gene expression in skin biopsies from patients with irBP to understand the underlying pathomechanism. We found that, similar to the spontaneous form of bullous pemphigoid (BP), interleukin-4 (IL-4) and interleukin-13 (IL-13) were upregulated. Thus, for patients who develop irBP as a side effect of cancer immunotherapy, IL-4 and IL-13 inhibitors such as dupilumab, so far approved to treat atopic dermatitis and prurigo nodularis, appear to be a promising new treatment option.Background/Objectives: Cutaneous side effects are the most common immune-related adverse events (irAEs) caused by immune checkpoint inhibitors (ICIs) and affect 70–90% of patients. Besides diverse types of exanthema, rare skin toxicity includes bullous dermatoses in 0.3% of cases. Systemic steroids are the first-line treatment for immune-related bullous pemphigoid (irBP); however, some cases are corticosteroid-resistant. IrBP is one of the irAEs most frequently chronic and associated with long-term steroid use. However, steroids may interfere with tumor response. Therefore, alternative treatment strategies for irBP are desperately needed. Dupilumab, a monoclonal antibody blocking the receptor binding of interleukin-4 (IL-4) and interleukin-13 (IL-13), has been successfully used to treat spontaneous forms of bullous pemphigoid (BP). In this study, we analyzed the gene expression profiles of BP and irBP. Patients and Methods: A retrospective multicenter study evaluated the gene expression profiles of irBP and BP in comparison to healthy controls. Gene expression analyses of skin biopsies were performed using NanoString technology from patients with BP (n = 17), irBP (n = 19), and healthy skin (n = 24) after the patients had consented to participate in this study, and differentially expressed genes (DEGs) were determined using Rosalind software. Results: Compared to healthy skin, BP showed 167 DEGs, and irBP revealed 99 DEGs. Some of the DEGs from irBP and BP vs. healthy skin overlapped. Specifically, IL-4- and IL-13-associated genes were upregulated in both irBP and BP compared to healthy skin. Interestingly, expression profiles of BP vs. irBP also showed 13 DEGs. Conclusions: These findings suggest a possibility for therapeutic efficacy of IL-4 and IL-13 inhibitors in the treatment of irBP.This research was funded by BMBF Grant MelAutim ‘Systems medicine of melanoma and autoimmunity in the context of immunotherapy’ (01ZX1905E and 01ZX2205E).BMBF Grant MelAutim ‘Systems medicine of melanoma and autoimmunity in the context of immunotherapy

Clinical-scale, modular manufacturing of tumor-reactive TILs using a closed and automated culture system

Recent studies have revealed the potential of tumor-infiltrating lymphocytes (TILs) to treat solid tumors effectively and safely. However, the translation of TIL therapy for patients is still hampered by non-standardized and laborious manufacturing procedures that are expensive and produce highly variable cellular products. To address these limitations, the CliniMACS Prodigy® Tumor Reactive T cell (TRT) Process has been developed. The TRT Process allows the automated isolation, transduction, and expansion of tumor-reactive T cells in a clinically compliant and closed system under GMP conditions. The TRT Process can generate tumor-reactive T cells using several methodologies which reflect clinically relevant applications. It can manage an automated Rapid Expansion Protocol (REP) using GMP-compliant reagents to generate a TIL cell product from solid tumors, including melanoma. Additionally, the TRT Process automates the closed selection of CD137-expressing TILs directly from tumor digest followed by the direct expansion of selected cells. Enriched CD137+ TILs could be robustly expanded even when as few as 1x104 TILs were used to seed the REP phase. These data provide proof-of-concept for the isolation and expansion of tumor-reactive T cells from tumor digest in a closed, automated manner in the CliniMACS Prodigy, allowing for an efficient, simple, and reproducible manufacturing of TIL products. The direct selection of CD137+ TILs from tumor digest removes the need for the pre-REP phase, selects for therapeutically relevant cells, and can dramatically shorten the manufacturing time compared to conventional methods

Brain metastasis and survival outcomes after first-line therapy in metastatic melanoma: a multicenter DeCOG study on 1704 patients from the prospective skin cancer registry ADOREG

Background Despite the availability of effective systemic therapies, a significant number of advanced melanoma patients develops brain metastases. This study investigated differences in incidence and time to diagnosis of brain metastasis and survival outcomes dependent on the type of first-line therapy.Methods Patients with metastatic, non-resectable melanoma (AJCCv8 stage IIIC–V) without brain metastasis at start of first-line therapy (1L-therapy) were identified from the prospective multicenter real-world skin cancer registry ADOREG. Study endpoints were incidence of brain metastasis, brain metastasis-free survival (BMFS), progression-free survival (PFS), and overall survival (OS).Results Of 1704 patients, 916 were BRAF wild-type (BRAFwt) and 788 were BRAF V600 mutant (BRAFmut). Median follow-up time after start of 1L-therapy was 40.4 months. BRAFwt patients received 1L-therapy with immune checkpoint inhibitors (ICI) against CTLA-4+PD-1 (n=281) or PD-1 (n=544). In BRAFmut patients, 1L-therapy was ICI in 415 patients (CTLA-4+PD-1, n=108; PD-1, n=264), and BRAF+MEK targeted therapy (TT) in 373 patients. After 24 months, 1L-therapy with BRAF+MEK resulted in a higher incidence of brain metastasis compared with PD-1±CTLA-4 (BRAF+MEK, 30.3%; CTLA-4+PD-1, 22.2%; PD-1, 14.0%). In multivariate analysis, BRAFmut patients developed brain metastases earlier on 1L-therapy with BRAF+MEK than with PD-1±CTLA-4 (CTLA-4+PD-1: HR 0.560, 95% CI 0.332 to 0.945, p=0.030; PD-1: HR 0.575, 95% CI 0.372 to 0.888, p=0.013). Type of 1L-therapy, tumor stage, and age were independent prognostic factors for BMFS in BRAFmut patients. In BRAFwt patients, tumor stage was independently associated with longer BMFS; ECOG Performance status (ECOG-PS), lactate dehydrogenase (LDH), and tumor stage with OS. CTLA-4+PD-1 did not result in better BMFS, PFS, or OS than PD-1 in BRAFwt patients. For BRAFmut patients, multivariate Cox regression revealed ECOG-PS, type of 1L-therapy, tumor stage, and LDH as independent prognostic factors for PFS and OS. 1L-therapy with CTLA-4+PD-1 led to longer OS than PD-1 (HR 1.97, 95% CI 1.122 to 3.455, p=0.018) or BRAF+MEK (HR 2.41, 95% CI 1.432 to 4.054, p=0.001), without PD-1 being superior to BRAF+MEK.Conclusions In BRAFmut patients 1L-therapy with PD-1±CTLA-4 ICI resulted in a delayed and less frequent development of brain metastasis compared with BRAF+MEK TT. 1L-therapy with CTLA-4+PD-1 showed superior OS compared with PD-1 and BRAF+MEK. In BRAFwt patients, no differences in brain metastasis and survival outcomes were detected for CTLA-4+PD-1 compared with PD-1

Early Exanthema Upon Vemurafenib Plus Cobimetinib Is Associated With a Favorable Treatment Outcome in Metastatic Melanoma: A Retrospective Multicenter DeCOG Study

Background The combination of BRAF and MEK inhibitors has become standard of care in the treatment of metastatic BRAF V600-mutated melanoma. Clinical factors for an early prediction of tumor response are rare. The present study investigated the association between the development of an early exanthema induced by vemurafenib or vemurafenib plus cobimetinib and therapy outcome. Methods This multicenter retrospective study included patients with BRAF V600-mutated irresectable AJCC-v8 stage IIIC/D to IV metastatic melanoma who received treatment with vemurafenib (VEM) or vemurafenib plus cobimetinib (COBIVEM). The development of an early exanthema within six weeks after therapy start and its grading according to CTCAEv4.0 criteria was correlated to therapy outcome in terms of best overall response, progression-free (PFS), and overall survival (OS). Results A total of 422 patients from 16 centers were included (VEM, n=299; COBIVEM, n=123). 20.4% of VEM and 43.1% of COBIVEM patients developed an early exanthema. In the VEM cohort, objective responders (CR/PR) more frequently presented with an early exanthema than non-responders (SD/PD); 59.0% versus 38.7%; p=0.0027. However, median PFS and OS did not differ between VEM patients with or without an early exanthema (PFS, 6.9 versus 6.0 months, p=0.65; OS, 11.0 versus 12.4 months, p=0.69). In the COBIVEM cohort, 66.0% of objective responders had an early exanthema compared to 54.3% of non-responders (p=0.031). Median survival times were significantly longer for patients who developed an early exanthema compared to patients who did not (PFS, 9.7 versus 5.6 months, p=0.013; OS, not reached versus 11.6 months, p=0.0061). COBIVEM patients with a mild early exanthema (CTCAEv4.0 grade 1-2) had a superior survival outcome as compared to COBIVEM patients with a severe (CTCAEv4.0 grade 3-4) or non early exanthema, respectively (p=0.047). This might be caused by the fact that 23.6% of patients with severe exanthema underwent a dose reduction or discontinuation of COBIVEM compared to only 8.9% of patients with mild exanthema. Conclusions The development of an early exanthema within 6 weeks after treatment start indicates a favorable therapy outcome upon vemurafenib plus cobimetinib. Patients presenting with an early exanthema should therefore be treated with adequate supportive measures to provide that patients can stay on treatment

Improved survival of advanced melanoma patients receiving immunotherapy with concomitant antithrombotic therapy – a multicenter study on 2419 patients from the prospective skin cancer registry ADOReg

Background Cancer immunotherapy has revolutionized melanoma treatment, but the high number of non-responders still emphasizes the need for improvement of therapy. One potential avenue for enhancing anti-tumor treatment is through the modulation of coagulation and platelet activity. Both have been found to play an important role in the tumor microenvironment, tumor growth and metastasis. Preclinical studies indicate a beneficial effect, clinical data has been inconsistent. Methods We examined a cohort of advanced, non-resectable melanoma patients (n = 2419) derived from the German prospective multicenter skin cancer registry ADOReg, who were treated with immune checkpoint inhibitors (ICI). The patients were classified based on whether it was documented that they received platelet aggregation inhibition (PAI) (n = 137) (acetylsalicylic acid (ASA) or clopidogrel), anticoagulation (AC) (n = 185) (direct oral anticoagulation (DOAC), phenprocoumon, heparins) at the start of ICI or no antithrombotic medication (n = 2097) at any point during ICI treatment. The study endpoints were best overall response (BOR), progression-free survival (PFS) and overall survival (OS). Results A significantly improved PFS was observed in patients documented to receive ASA (15.1 vs 6.4 months, HR 0.67, 95 % CI: 0.5 to 0.88, p = 0.0047) as well as in patients to receive AC (15.1 vs. 6.4 months, HR 0.7, 95 % CI: 0.53 to 0.91, p = 0.01) compared to patients for whom no antithrombotic medication was documented. Multivariate analysis of OS showed significant risk reduction in patients who received DOAC (HR 0.68, 95 % CI: 0.49 to 0.92, p = 0.0170) or phenprocoumon (HR: 0.44, 95 % CI: 0.19 to 0.85, p = 0.0301). Conclusion Our study indicates a positive prognostic effect of anticoagulant and antiplatelet concomitant medication in melanoma patients receiving ICI. Further studies are needed to confrim the cancer-related benefit of adding anticoagulation or platelet inhibition to ICI treatment