The Role of Fibroblast Activation Protein in Glioblastoma and Gliosarcoma: A Comparison of Tissue, 68Ga-FAPI-46 PET Data, and Survival Data.

Despite their unique histologic features, gliosarcomas belong to the group of glioblastomas and are treated according to the same standards. Fibroblast activation protein (FAP) is a component of a tumor-specific subpopulation of fibroblasts that plays a critical role in tumor growth and invasion. Some case studies suggest an elevated expression of FAP in glioblastoma and a particularly strong expression in gliosarcoma attributed to traits of predominant mesenchymal differentiation. However, the prognostic impact of FAP and its diagnostic and therapeutic potential remain unclear. Here, we investigate the clinical relevance of FAP expression in gliosarcoma and glioblastoma and how it correlates with 68Ga-FAP inhibitor (FAPI)-46 PET uptake. Methods: Patients diagnosed with gliosarcoma or glioblastoma without sarcomatous differentiation with an overall survival of less than 2.5 y were enrolled. Histologic examination included immunohistochemistry and semiquantitative scoring of FAP (0-3, with higher values indicating stronger expression). Additionally, 68Ga-FAPI-46 PET scans were performed in a subset of glioblastomas without sarcomatous differentiation patients. The clinical SUVs were correlated with FAP expression levels in surgically derived tumor tissue and relevant prognostic factors. Results: Of the 61 patients who were enrolled, 13 of them had gliosarcoma. Immunohistochemistry revealed significantly more FAP in gliosarcomas than in glioblastomas without sarcomatous differentiation of tumor tissue (P < 0.0001). In the latter, FAP expression was confined to the perivascular space, whereas neoplastic cells additionally expressed FAP in gliosarcoma. A significant correlation of immunohistochemical FAP with SUVmean and SUVpeak of 68Ga-FAPI-46 PET indicates that clinical tracer uptake represents FAP expression of the tumor. Although gliosarcomas express higher levels of FAP than do glioblastomas without sarcomatous differentiation, overall survival does not significantly differ between the groups. Conclusion: The analysis reveals a significant correlation between SUVmean and SUVpeak in 68Ga-FAPI-46 PET and immunohistochemical FAP expression. This study indicates that FAP expression is much more abundant in the gliosarcoma subgroup of glioblastomas. This could open not only a diagnostic but also a therapeutic gap, since FAP could be explored as a theranostic target to enhance survival in a distinct subgroup of high-risk brain tumor patients with poor survival prognosis.

Histopathological Diagnosis of Primary Central Nervous System Lymphoma after Therapy with Corticosteroids or Anticoagulants.

In patients with primary central nervous system lymphoma (PCNSL), the choice of surgical strategy for histopathologic assessments is still controversial, particularly in terms of preoperative corticosteroid (CS) therapy. To provide further evidence for clinical decision-making, we retrospectively analyzed data from 148 consecutive patients who underwent surgery at our institution. Although patients treated with corticosteroids preoperatively were significantly more likely to require a second or third biopsy (p = 0.049), it was only necessary in less than 10% of the cases with preoperative (but discontinued) corticosteroid treatment. Surprisingly, diagnostic accuracy was significantly lower when patients were treated with anticoagulation or dual antiplatelet therapy (p = 0.015). Preoperative CSF sampling did not provide additional information but was associated with delayed surgery (p = 0.02). In conclusion, preoperative CS therapy can challenge the histological diagnosis of PCNSL. At the same time, our data suggest that preoperative CS treatment only presents a relative contraindication for early surgical intervention. If a definitive diagnosis cannot be made after the first surgical intervention, the timing of a repeat biopsy after the discontinuation of CS remains a case-by-case decision. The effect of anticoagulation and dual antiplatelet therapy on diagnostic accuracy might have been underestimated and should be examined closely in future investigations.

Feasibility and tolerability of trofosfamide and etoposide in progressive glioblastoma.

Background: Standard of care treatment options at glioblastoma relapse are still not well defined. Few studies indicate that the combination of trofosfamide plus etoposide may be feasible in pediatric glioblastoma patients. In this retrospective analysis, we determined tolerability and feasibility of combined trofosfamide plus etoposide treatment at disease recurrence of adult glioblastoma patients. Methods: We collected clinicopathological data from adult progressive glioblastoma patients treated with the combination of trofosfamide and etoposide for more than four weeks (one course). A cohort of patients receiving empiric treatment at the investigators' discretion balanced for tumor entity and canonical prognostic factors served as control. Results: A total of n = 22 progressive glioblastoma patients were eligible for this analysis. Median progression-free survival (3.1 vs 2.3 months, HR: 1.961, 95% CI: 0.9724-3.9560, P = .0274) and median overall survival (9.0 vs 5.7 months, HR: 4.687, 95% CI: 2.034-10.800, P = .0003) were significantly prolonged compared to the control cohort (n = 17). In a multivariable Cox regression analysis, treatment with trofosfamide plus etoposide emerged as a significant prognostic marker regarding progression-free and overall survival. We observed high-grade adverse events in n = 16/22 (73%) patients with hematotoxicity comprising the majority of adverse events (n = 15/16, 94%). Lymphopenia was by far the most commonly observed hematotoxic adverse event (n = 11/15, 73%). Conclusions: This study provides first indication that the combination of trofosfamide plus etoposide is safe in adult glioblastoma patients. The observed survival outcomes might suggest potential beneficial effects. Our data provide a reasonable rationale for follow-up of a larger cohort in a prospective trial.

Telemedicine in Neuro-Oncology-An Evaluation of Remote Consultations during the COVID-19 Pandemic.

In order to minimize the risk of infections during the COVID-19 pandemic, remote video consultations (VC) experienced an upswing in most medical fields. However, telemedicine in neuro-oncology comprises unique challenges and opportunities. So far, evidence-based insights to evaluate and potentially customize current concepts are scarce. To fill this gap, we analyzed >3700 neuro-oncological consultations, of which >300 were conducted as VC per patients' preference, in order to detect how both patient collectives distinguished from one another. Additionally, we examined patients' reasons, suitable/less suitable encounters, VC's benefits and disadvantages and future opportunities with an anonymized survey. Patients that participated in VC had a worse clinical condition, higher grade of malignancy, were more often diagnosed with glioblastoma and had a longer travel distance (all p < 0.01). VC were considered a fully adequate alternative to face-to-face consultations for almost all encounters that patients chose to participate in (>70%) except initial consultations. Most participants preferred to alternate between both modalities rather than participate in one alone but preferred VC over telephone consultation. VC made patients feel safer, and participants expressed interest in implementing other telemedicine modalities (e.g., apps) into neuro-oncology. VC are a promising addition to patient care in neuro-oncology. However, patients and encounters should be selected individually.

Metastasis Associated in Colorectal Cancer 1 (MACC1) mRNA Expression Is Enhanced in Sporadic Vestibular Schwannoma and Correlates to Deafness.

Vestibular schwannoma (VS) are benign cranial nerve sheath tumors of the vestibulocochlear nerve. Their incidence is mostly sporadic, but they can also be associated with NF2-related schwannomatosis (NF2), a hereditary tumor syndrome. Metastasis associated in colon cancer 1 (MACC1) is known to contribute to angiogenesis, cell growth, invasiveness, cell motility and metastasis of solid malignant cancers. In addition, MACC1 may be associated with nonsyndromic hearing impairment. Therefore, we evaluated whether MACC1 may be involved in the pathogenesis of VS. Sporadic VS, recurrent sporadic VS, NF2-associated VS, recurrent NF2-associated VS and healthy vestibular nerves were analyzed for MACC1 mRNA and protein expression by quantitative polymerase chain reaction and immunohistochemistry. MACC1 expression levels were correlated with the patients' clinical course and symptoms. MACC1 mRNA expression was significantly higher in sporadic VS compared to NF2-associated VS (p < 0.001). The latter expressed similar MACC1 concentrations as healthy vestibular nerves. Recurrent tumors resembled the MACC1 expression of the primary tumors. MACC1 mRNA expression was significantly correlated with deafness in sporadic VS patients (p = 0.034). Therefore, MACC1 might be a new molecular marker involved in VS pathogenesis.

BRMS1 in Gliomas-An Expression Analysis.

The metastatic suppressor BRMS1 interacts with critical steps of the metastatic cascade in many cancer entities. As gliomas rarely metastasize, BRMS1 has mainly been neglected in glioma research. However, its interaction partners, such as NFκB, VEGF, or MMPs, are old acquaintances in neurooncology. The steps regulated by BRMS1, such as invasion, migration, and apoptosis, are commonly dysregulated in gliomas. Therefore, BRMS1 shows potential as a regulator of glioma behavior. By bioinformatic analysis, in addition to our cohort of 118 specimens, we determined BRMS1 mRNA and protein expression as well as its correlation with the clinical course in astrocytomas IDH mutant, CNS WHO grade 2/3, and glioblastoma IDH wild-type, CNS WHO grade 4. Interestingly, we found BRMS1 protein expression to be significantly decreased in the aforementioned gliomas, while BRMS1 mRNA appeared to be overexpressed throughout. This dysregulation was independent of patients' characteristics or survival. The protein and mRNA expression differences cannot be finally explained at this stage. However, they suggest a post-transcriptional dysregulation that has been previously described in other cancer entities. Our analyses present the first data on BRMS1 expression in gliomas that can provide a starting point for further investigations.

First multicentric real-life experience with the combination of CCNU and temozolomide in newly diagnosed MGMT promoter methylated IDH wildtype glioblastoma.

Background: The randomized phase 3 CeTeG/NOA-09 trial assessed whether CCNU plus temozolomide was superior to temozolomide alone in newly diagnosed MGMT promoter methylated glioblastoma patients. Survival was significantly improved from 31.4 months (temozolomide) to 48.1 months (CCNU plus temozolomide). In view of this encouraging data, we assessed safety and efficacy of this regimen under real-life conditions. Methods: We retrospectively collected clinical and radiographic data from adult newly diagnosed MGMT promoter methylated IDH wildtype glioblastoma patients from five neuro-oncology centers in Germany. For inclusion in our analysis, treatment with CCNU and temozolomide had to be performed for at least six weeks (one course). Results: Seventy patients were included. Median progression-free survival was 14.4 months and median overall survival 33.8 months. Patients with TTFields treatment for at least 8 weeks and CCNU plus temozolomide (n = 22, 31%) had a prolonged progression-free survival compared to those with TTFields treatment for less than eight weeks (n = 48, 69%) (21.5 versus 11.2 months; P = .0105). In a multivariable Cox regression analysis, TTFields treatment for eight weeks or longer together with CCNU plus temozolomide and a Karnofsky performance score ≥ 90% were independent prognostic factors for progression-free and overall survival. Pseudoprogression occurred in n = 16 (33%) of investigated n = 49 (70%) patients. In n = 31 (44%) patients high-grade hematotoxicity was observed. Conclusions: The results from this multicentric trial indicate that-under real-life conditions-toxicity and survival estimates are comparable to the CeTeG/NOA-09 trial. TTFields therapy for at least eight weeks in combination with this regimen was independently associated with prolonged survival.

Protocadherin Gamma C3 (PCDHGC3) Is Strongly Expressed in Glioblastoma and Its High Expression Is Associated with Longer Progression-Free Survival of Patients.

Protocadherins (PCDHs) belong to the cadherin superfamily and represent the largest subgroup of calcium-dependent adhesion molecules. In the genome, most PCDHs are arranged in three clusters, α, ß, and γ on chromosome 5q31. PCDHs are highly expressed in the central nervous system (CNS). Several PCDHs have tumor suppressor functions, but their individual role in primary brain tumors has not yet been elucidated. Here, we examined the mRNA expression of PCDHGC3, a member of the PCDHγ cluster, in non-cancerous brain tissue and in gliomas of different World Health Organization (WHO) grades and correlated it with the clinical data of the patients. We generated a PCDHGC3 knockout U343 cell line and examined its growth rate and migration in a wound healing assay. We showed that PCDHGC3 mRNA and protein were significantly overexpressed in glioma tissue compared to a non-cancerous brain specimen. This could be confirmed in glioma cell lines. High PCDHGC3 mRNA expression correlated with longer progression-free survival (PFS) in glioma patients. PCDHGC3 knockout in U343 resulted in a slower growth rate but a significantly faster migration rate in the wound healing assay and decreased the expression of several genes involved in WNT signaling. PCDHGC3 expression should therefore be further investigated as a PFS-marker in gliomas. However, more studies are needed to elucidate the molecular mechanisms underlying the PCDHGC3 effects.

Effects of Long-Term Temozolomide Treatment on Glioblastoma and Astrocytoma WHO Grade 4 Stem-like Cells.

Glioblastoma leads to a fatal course within two years in more than two thirds of patients. An essential cornerstone of therapy is chemotherapy with temozolomide (TMZ). The effect of TMZ is counteracted by the cellular repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). The MGMT promoter methylation, the main regulator of MGMT expression, can change from primary tumor to recurrence, and TMZ may play a significant role in this process. To identify the potential mechanisms involved, three primary stem-like cell lines (one astrocytoma with the mutation of the isocitrate dehydrogenase (IDH), CNS WHO grade 4 (HGA)), and two glioblastoma (IDH-wildtype, CNS WHO grade 4) were treated with TMZ. The MGMT promoter methylation, migration, proliferation, and TMZ-response of the tumor cells were examined at different time points. The strong effects of TMZ treatment on the MGMT methylated cells were observed. Furthermore, TMZ led to a loss of the MGMT promoter hypermethylation and induced migratory rather than proliferative behavior. Cells with the unmethylated MGMT promoter showed more aggressive behavior after treatment, while HGA cells reacted heterogenously. Our study provides further evidence to consider the potential adverse effects of TMZ chemotherapy and a rationale for investigating potential relationships between TMZ treatment and change in the MGMT promoter methylation during relapse.

Analysis of microRNAs in Exosomes of Breast Cancer Patients in Search of Molecular Prognostic Factors in Brain Metastases.

Brain metastases are the most severe tumorous spread during breast cancer disease. They are associated with a limited quality of life and a very poor overall survival. A subtype of extracellular vesicles, exosomes, are sequestered by all kinds of cells, including tumor cells, and play a role in cell-cell communication. Exosomes contain, among others, microRNAs (miRs). Exosomes can be taken up by other cells in the body, and their active molecules can affect the cellular process in target cells. Tumor-secreted exosomes can affect the integrity of the blood-brain barrier (BBB) and have an impact on brain metastases forming. Serum samples from healthy donors, breast cancer patients with primary tumors, or with brain, bone, or visceral metastases were used to isolate exosomes and exosomal miRs. Exosomes expressed exosomal markers CD63 and CD9, and their amount did not vary significantly between groups, as shown by Western blot and ELISA. The selected 48 miRs were detected using real-time PCR. Area under the receiver-operating characteristic curve (AUC) was used to evaluate the diagnostic accuracy. We identified two miRs with the potential to serve as prognostic markers for brain metastases. Hsa-miR-576-3p was significantly upregulated, and hsa-miR-130a-3p was significantly downregulated in exosomes from breast cancer patients with cerebral metastases with AUC: 0.705 and 0.699, respectively. Furthermore, correlation of miR levels with tumor markers revealed that hsa-miR-340-5p levels were significantly correlated with the percentage of Ki67-positive tumor cells, while hsa-miR-342-3p levels were inversely correlated with tumor staging. Analysis of the expression levels of miRs in serum exosomes from breast cancer patients has the potential to identify new, non-invasive, blood-borne prognostic molecular markers to predict the potential for brain metastasis in breast cancer. Additional functional analyzes and careful validation of the identified markers are required before their potential future diagnostic use.

Simultaneous paraneoplastic cerebellar degeneration, Lambert-Eaton syndrome and neuropathy associated with AGNA/anti-SOX1 and VGCC antibodies.

Anti-glial nuclear antibody (AGNA) is an onconeuroal antibody targeting the nuclei of Bergmann glia in the cerebellum and Anti-SRY-related HMG-box 1 (SOX1). It is highly specific for small cell lung cancer (SCLC) and correlates to the appearance of paraneoplastic neurological syndromes such as Lambert-Eaton myasthenic syndrome (pLEMS) and paraneoplastic cerebellar degeneration (PCD) amongst others. Herein, we present a SCLC patient with rapidly progressive PCD, LEMS and axonal polyneuropathy associated with AGNA/SOX1-antibodies, successfully treated with plasma-exchange (PLEX).

Monopolar Spindle 1 Kinase (MPS1/TTK) mRNA Expression is Associated with Earlier Development of Clinical Symptoms, Tumor Aggressiveness and Survival of Glioma Patients.

Inhibition of the protein kinase MPS1, a mitotic spindle-checkpoint regulator, reinforces the effects of multiple therapies against glioblastoma multiforme (GBM) in experimental settings. We analyzed MPS1 mRNA-expression in gliomas WHO grade II, III and in clinical subgroups of GBM. Data were obtained by qPCR analysis of tumor and healthy brain specimens and correlated with the patients' clinical data. MPS1 was overexpressed in all gliomas on an mRNA level (ANOVA, p < 0.01) and correlated with tumor aggressiveness. We explain previously published conflicting results on survival: high MPS1 was associated with poorer long term survival when all gliomas were analyzed combined in one group (Cox regression: t < 24 months, p = 0.009, Hazard ratio: 8.0, 95% CI: 1.7-38.4), with poorer survival solely in low-grade gliomas (LogRank: p = 0.02, Cox regression: p = 0.06, Hazard-Ratio: 8.0, 95% CI: 0.9-66.7), but not in GBM (LogRank: p > 0.05). This might be due to their lower tumor volume at the therapy start. GBM patients with high MPS1 mRNA-expression developed clinical symptoms at an earlier stage. This, however, did not benefit their overall survival, most likely due to the more aggressive tumor growth. Since MPS1 mRNA-expression in gliomas was enhanced with increasing tumor aggressiveness, patients with the worst outcome might benefit best from a treatment directed against MPS1.

Ribosomal Protein S27/Metallopanstimulin-1 (RPS27) in Glioma-A New Disease Biomarker?

Despite its significant overexpression in several malignant neoplasms, the expression of RPS27 in the central nervous system (CNS) is widely unknown. We identified the cell types expressing RPS27 in the CNS under normal and disease conditions. We acquired specimens of healthy brain (NB), adult pilocytic astrocytoma (PA) World Health Organization (WHO) grade I, anaplastic PA WHO grade III, gliomas WHO grade II/III with or without isocitrate dehydrogenase (IDH) mutation, and glioblastoma multiforme (GBM). RPS27 protein expression was examined by immunohistochemistry and double-fluorescence staining and its mRNA expression quantified by RT-PCR. Patients' clinical and tumor characteristics were collected retrospectively. RPS27 protein was specifically expressed in tumor cells and neurons, but not in healthy astrocytes. In tumor tissue, most macrophages were positive, while this was rarely the case in inflamed tissue. Compared to NB, RPS27 mRNA was in mean 6.2- and 8.8-fold enhanced in gliomas WHO grade II/III with (p < 0.01) and without IDH mutation (p = 0.01), respectively. GBM displayed a 4.6-fold increased mean expression (p = 0.02). Although RPS27 expression levels did not affect the patients' survival, their association with tumor cells and tumor-associated macrophages provides a rationale for a future investigation of a potential function during gliomagenesis and tumor immune response.

Serum-derived factors of breast cancer patients with brain metastases alter permeability of a human blood-brain barrier model.

BACKGROUND: The most threatening metastases in breast cancer are brain metastases, which correlate with a very poor overall survival, but also a limited quality of life. A key event for the metastatic progression of breast cancer into the brain is the migration of cancer cells across the blood-brain barrier (BBB). METHODS: We adapted and validated the CD34+ cells-derived human in vitro BBB model (brain-like endothelial cells, BLECs) to analyse the effects of patient serum on BBB properties. We collected serum samples from healthy donors, breast cancer patients with primary cancer, and breast cancer patients with, bone, visceral or cerebral metastases. We analysed cytokine levels in these sera utilizing immunoassays and correlated them with clinical data. We used paracellular permeability measurements, immunofluorescence staining, Western blot and mRNA analysis to examine the effects of patient sera on the properties of BBB in vitro. RESULTS: The BLECs cultured together with brain pericytes in transwells developed a tight monolayer with a correct localization of claudin-5 at the tight junctions (TJ). Several BBB marker proteins such as the TJ proteins claudin-5 and occludin, the glucose transporter GLUT-1 or the efflux pumps PG-P and BCRP were upregulated in these cultures. This was accompanied by a reduced paracellular permeability for fluorescein (400 Da). We then used this model for the treatment with the patient sera. Only the sera of breast cancer patients with cerebral metastases had significantly increased levels of the cytokines fractalkine (CX3CL1) and BCA-1 (CXCL13). The increased levels of fractalkine were associated with the estrogen/progesterone receptor status of the tumour. The treatment of BLECs with these sera selectively increased the expression of CXCL13 and TJ protein occludin. In addition, the permeability of fluorescein was increased after serum treatment. CONCLUSION: We demonstrate that the CD34+ cell-derived human in vitro BBB model can be used as a tool to study the molecular mechanisms underlying cerebrovascular pathologies. We showed that serum from patients with cerebral metastases may affect the integrity of the BBB in vitro, associated with elevated concentrations of specific cytokines such as CX3CL1 and CXCL13.

Changes of O6-Methylguanine DNA Methyltransferase (MGMT) Promoter Methylation in Glioblastoma Relapse-A Meta-Analysis Type Literature Review.

Methylation of the O6-methylguanine DNA methyltransferase (MGMT) promoter has emerged as strong prognostic factor in the therapy of glioblastoma multiforme. It is associated with an improved response to chemotherapy with temozolomide and longer overall survival. MGMT promoter methylation has implications for the clinical course of patients. In recent years, there have been observations of patients changing their MGMT promoter methylation from primary tumor to relapse. Still, data on this topic are scarce. Studies often consist of only few patients and provide rather contrasting results, making it hard to draw a clear conclusion on clinical implications. Here, we summarize the previous publications on this topic, add new cases of changing MGMT status in relapse and finally combine all reports of more than ten patients in a statistical analysis based on the Wilson score interval. MGMT promoter methylation changes are seen in 115 of 476 analyzed patients (24%; CI: 0.21-0.28). We discuss potential reasons like technical issues, intratumoral heterogeneity and selective pressure of therapy. The clinical implications are still ambiguous and do not yet support a change in clinical practice. However, retesting MGMT methylation might be useful for future treatment decisions and we encourage clinical studies to address this topic.

Expression of activating transcription factor 5 (ATF5) is increased in astrocytomas of different WHO grades and correlates with survival of glioblastoma patients.

BACKGROUND: ATF5 suppresses differentiation of neuroprogenitor cells and is overexpressed in glioblastoma (GBM). A reduction of its expression leads to apoptotic GBM cell death. Data on ATF5 expression in astrocytoma WHO grade II (low-grade astrocytoma [LGA]) are scarce and lacking on recurrent GBM. PATIENTS AND METHODS: ATF5 mRNA was extracted from frozen samples of patients' GBM (n=79), LGA (n=40), and normal brain (NB, n=10), quantified by duplex qPCR and correlated with retrospectively collected clinical data. ATF5 protein expression was evaluated by measuring staining intensity on immunohistochemistry. RESULTS: ATF5 mRNA was overexpressed in LGA (sevenfold, P<0.001) and GBM (tenfold, P<0.001) compared to NB, which was confirmed on protein level. Although ATF5 mRNA expression in GBM showed a considerable fluctuation range, groups of varying biological behavior, that is, local/multifocal growth or primary tumor/relapse and the tumor localization at diagnosis, were not significantly different. ATF5 mRNA correlated with the patients' age (r=0.339, P=0.028) and inversely with Ki67-staining (r=-0.421, P=0.007). GBM patients were allocated to a low and a high ATF5 expression group by the median ATF5 overexpression compared to NB. Kaplan-Meier analysis and Cox regression indicated that ATF5 mRNA expression significantly correlated with short-term survival (t,12 months, median survival 18 vs 13 months, P=0.022, HR 2.827) and progression-free survival (PFS) (12 vs 6 months, P=0.024). This advantage vanished after 24 months (P=0.084). CONCLUSION: ATF5 mRNA expression could be identified as an additional, though not independent factor correlating with overall survival and PFS. Since its inhibition might lead to the selective death of glioma cells, it might serve as a potential ubiquitous therapeutic target in astrocytic tumors.