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H3 K27M-altered diffuse midline gliomas (DMGs) are highly malignant tumours that arise in the midline structures of the CNS. Most DMGs carry an H3 K27M-mutation in one of the genes encoding for histone H3. Recent studies suggested that epigenetic subgroups of DMGs can be distinguished based on alterations in the MAPK-signalling pathway, tumour localisation, mutant H3-gene, or overall survival (OS). However, as these parameters were studied individually, it is unclear how they collectively influence survival. Hence, we analysed dependencies between different parameters, to define novel epigenetic, clinically meaningful subgroups of DMGs. We collected a multifaceted cohort of 149 H3 K27M-mutant DMGs, also incorporating data of published cases. DMGs were included in the study if they could be clearly allocated to the spinal cord (n = 31; one patient with an additional sellar tumour), medulla (n = 20), pons (n = 64) or thalamus (n = 33), irrespective of further known characteristics. We then performed global genome-wide DNA methylation profiling and, for a subset, DNA sequencing and survival analyses. Unsupervised hierarchical clustering of DNA methylation data indicated two clusters of DMGs, i.e. subtypes DMG-A and DMG-B. These subtypes differed in mutational spectrum, tumour localisation, age at diagnosis and overall survival. DMG-A was enriched for DMGs with MAPK-mutations, medullary localisation and adult age. 13% of DMG-A had a methylated MGMT promoter. Contrarily, DMG-B was enriched for cases with TP53-mutations, PDGFRA-amplifications, pontine localisation and paediatric patients. In univariate analyses, the features enriched in DMG-B were associated with a poorer survival. However, all significant parameters tested were dependent on the cluster attribution, which had the largest effect on survival: DMG-A had a significantly better survival compared to DMG-B (p < 0.001). Hence, the subtype attribution based on two methylation clusters can be used to predict survival as it integrates different molecular and clinical parameters., (© 2024. The Author(s).)

Background: Extracellular vesicles (EVs) obtained by noninvasive liquid biopsy from patient blood can serve as biomarkers. Here, we investigated the potential of circulating plasma EVs to serve as an indicator in the diagnosis, prognosis, and treatment response of glioblastoma patients., Methods: Plasma samples were collected from glioblastoma patients at multiple timepoints before and after surgery. EV concentrations were measured by nanoparticle tracking analysis and imaging flow cytometry. Tumor burden and edema were quantified by 3D reconstruction. EVs and tumors were further monitored in glioma-bearing mice., Results: Glioblastoma patients displayed a 5.5-fold increase in circulating EVs compared to healthy donors (P < .0001). Patients with higher EV levels had significantly shorter overall survival and progression-free survival than patients with lower levels, and the plasma EV concentration was an independent prognostic parameter for overall survival. EV levels correlated with the extent of peritumoral fluid-attenuated inversion recovery hyperintensity but not with the size of the contrast-enhancing tumor, and similar findings were obtained in mice. Postoperatively, EV concentrations decreased rapidly back to normal levels, and the magnitude of the decline was associated with the extent of tumor resection. EV levels remained low during stable disease, but increased again upon tumor recurrence. In some patients, EV resurgence preceded the magnetic resonance imaging detectability of tumor relapse., Conclusions: Our findings suggest that leakiness of the blood-brain barrier may primarily be responsible for the high circulating EV concentrations in glioblastoma patients. Elevated EVs reflect tumor presence, and their quantification may thus be valuable in assessing disease activity., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Genome-wide DNA methylation profiling has recently been developed into a tool that allows tumor classification in central nervous system tumors. Extracellular vesicles (EVs) are released by tumor cells and contain high molecular weight DNA, rendering EVs a potential biomarker source to identify tumor subgroups, stratify patients and monitor therapy by liquid biopsy. We investigated whether the DNA in glioblastoma cell-derived EVs reflects genome-wide tumor methylation and mutational profiles and allows noninvasive tumor subtype classification., Methods: DNA was isolated from EVs secreted by glioblastoma cells as well as from matching cultured cells and tumors. EV-DNA was localized and quantified by direct stochastic optical reconstruction microscopy. Methylation and copy number profiling was performed using 850k arrays. Mutations were identified by targeted gene panel sequencing. Proteins were differentially quantified by mass spectrometric proteomics., Results: Genome-wide methylation profiling of glioblastoma-derived EVs correctly identified the methylation class of the parental cells and original tumors, including the MGMT promoter methylation status. Tumor-specific mutations and copy number variations (CNV) were detected in EV-DNA with high accuracy. Different EV isolation techniques did not affect the methylation profiling and CNV results. DNA was present inside EVs and on the EV surface. Proteome analysis did not allow specific tumor identification or classification but identified tumor-associated proteins that could potentially be useful for enriching tumor-derived circulating EVs from biofluids., Conclusions: This study provides proof of principle that EV-DNA reflects the genome-wide methylation, CNV, and mutational status of glioblastoma cells and enables their molecular classification., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Up to 40% of advance lung, melanoma and breast cancer patients suffer from brain metastases (BM) with increasing incidence. Here, we assessed whether circulating tumor cells (CTCs) in peripheral blood can serve as a disease surrogate, focusing on CD44 and CD74 expression as prognostic markers for BM. We show that a size-based microfluidic approach in combination with a semi-automated cell recognition system are well suited for CTC detection in BM patients and allow further characterization of tumor cells potentially derived from BM. CTCs were found in 50% (7/14) of breast cancer, 50% (9/18) of non-small cell lung cancer (NSCLC) and 36% (4/11) of melanoma patients. The next-generation sequencing (NGS) analysis of nine single CTCs from one breast cancer patient revealed three different CNV profile groups as well as a resistance causing ERS1 mutation. CD44 and CD74 were expressed on most CTCs and their expression was strongly correlated, whereas matched breast cancer BM tissues were much less frequently expressing CD44 and CD74 (negative in 46% and 54%, respectively). Thus, plasticity of CD44 and CD74 expression during trafficking of CTCs in the circulation might be the result of adaptation strategies.

The molecular biology of ependymomas is not well understood and this is particularly true for ependymoma relapses. We aimed at finding out if and to which extent, relapses differ from their corresponding primary tumors on the morphological, chromosomal and epigenetic level. We investigated 24 matched ependymoma primary and relapsed tumor samples and, as a first step, compared cell density, necrosis, vessel proliferation, Ki67 proliferative index, trimethylation at H3K27 and expression of CXorf67. For the investigation of global methylation profiles, we used public data in order to analyze copy number variation profiles, differential methylation, methylation status and fractions of hypo- and hypermethylated CpGs in different epigenomic substructures. Morphologically, we found a significant increase with relapse in cell density and proliferation. H3K27 trimethylation and CXorf67 expression remained stable between primary and relapse tumor samples, and the analysis of DNA methylation profiles neither revealed significant differences in copy number variations nor differentially methylated regions. Significant differences in the methylation status were found for CpG islands, but also in N Shelves or S Shelves, depending on the molecular subgroup. The fraction of probes changing their methylation in the epigenomic substructures appeared subgroup-specific. Most changes occur in CpG islands, for which relapsed tumors demonstrate higher methylation values than primary tumors. The morphological differences reflect increased aggressiveness upon ependymoma relapse, but, despite slight changes, this observation does not appear to be sufficiently explained by epigenetic changes., (© 2020 The Authors. Brain Pathology published by John Wiley & Sons Ltd on behalf of International Society of Neuropathology.)

Purpose: ERGO2 is the first randomized clinical trial on a calorically restricted ketogenic diet (KD) and intermittent fasting (KD-IF) in addition to reirradiation for recurrent malignant gliomas., Methods and Materials: Fifty patients were randomized 1:1 to reirradiation combined with either a calorically unrestricted diet or KD-IF. The KD-IF schedule included 3 days of KD (21-23 kcal/kg/d), followed by 3 days of fasting and again 3 days of KD. Primary endpoint was progression-free survival (PFS) at 6 months (PFS6). Secondary endpoints were PFS, local PFS, overall survival (OS), frequency of epileptic seizures, rate of ketosis and quality of life., Results: Four patients quit the trial before treatment and 3 patients stopped KD-IF prematurely. Of the 20 patients who completed KD-IF, 17 patients developed ketosis at day 6 and glucose levels declined significantly. KD-IF was well-tolerated with a modest weight loss of -2.1 ± 1.8 kg. No severe adverse events attributable to the diet occurred. PFS6 was not significantly different between the 2 groups (KD-IF: 20%; calorically unrestricted diet: 16%). Similarly, no difference in PFS, local PFS6, or OS was observable. Explorative analysis revealed that patients in the KD-IF group who had a glucose level of less than the median (83.5 mg/dL) on day 6 had significantly longer PFS and OS compared with those above the median (P < .05)., Conclusions: KD-IF is feasible and effective in inducing ketosis in heavily pretreated patients with recurrent glioma. However, the short schedule reported here failed to increase the efficacy of reirradiation. CLINICALTRIALS., Gov Number: NCT01754350., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Brain metastasis (BM) in non-small-cell lung cancer (NSCLC) has a very poor prognosis. Recent studies have demonstrated the importance of cell adhesion molecules in tumor metastasis. The aim of our study was to investigate the role of activated leukocyte cell adhesion molecule (ALCAM) in BM formation in NSCLC., Methods: Immunohistochemical analysis was performed on 143 NSCLC primary tumors and BM. A correlation between clinicopathological parameters and survival was developed. Biological properties of ALCAM were assessed in vitro by gene ablation using CRISPR/Cas9 technology in the NCI-H460 NSCLC cell line and in vivo by intracranial and intracardial cell injection of NCI-H460 cells in NMRI-Foxn1nu/nu mice., Results: ALCAM expression was significantly upregulated in NSCLC brain metastasis (P = 0.023) with a de novo expression of ALCAM in 31.2% of BM. Moderate/strong ALCAM expression in both primary NSCLC and brain metastasis was associated with shortened survival. Functional analysis of an ALCAM knock-out (KO) cell line showed a significantly decreased cell adhesion capacity to human brain endothelial cells by 38% (P = 0.045). In vivo studies showed significantly lower tumor cell dissemination in mice injected with ALCAM-KO cells in both mouse models, and both the number and size of BM were significantly diminished in ALCAM depleted tumors., Conclusions: Our findings suggest that elevated levels of ALCAM expression promote BM formation in NSCLC through increased tumor cell dissemination and interaction with the brain endothelial cells. Therefore, ALCAM could be targeted to reduce the occurrence of BM., Key Points: 1. ALCAM expression associates with poor prognosis and brain metastasis in NSCLC.2. ALCAM mediates interaction of NSCLC tumor cells with brain vascular endothelium.3. ALCAM might represent a novel preventive target to reduce the occurrence of BM in NSCLC., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

According to the WHO classification, ependymal tumors are classified as subependymomas, myxopapillary ependymomas, classic ependymomas, anaplastic ependymomas, and RELA-fusion-positive ependymomas (RELA-EPN). Among classic ependymomas, the WHO defines rare histological variants, i.e., the clear cell, papillary, and tanycytic ependymoma. In parallel, global DNA methylation patterns distinguish nine molecular groups, some of which tightly overlap with histopathological subgroups. However, the match of the aforementioned histological variants to DNA methylation classes remains unclear. We analyzed histomorphology, clinical parameters, and global DNA methylation of tumors with the initial histological diagnoses of tanycytic (n = 12), clear cell (n = 14), or papillary ependymoma (n = 19). Forty percent of these tumors did not match to the epigenetic profile of ependymomas, using a previously published DNA methylation-based classifier for brain tumors. Instead, they were classified as low-grade glioma (n = 3), plexus tumor (n = 2), CNS high-grade neuroepithelial tumor with MN1 alteration (n = 2), papillary tumor of the pineal region (n = 2), neurocytoma (n = 1), or did not match to any known brain tumor methylation class (n = 8). Overall, integrated diagnosis had to be changed in 35.6% of cases as compared to the initial diagnosis. Among the tumors molecularly classified as ependymoma (27/45 cases), tanycytic ependymomas were mostly located in the spine (5/7 cases) and matched to spinal or myxopapillary ependymoma. 6/8 clear cell ependymomas were found supratentorially and fell into the methylation class of RELA-EPN. Papillary ependymomas with a positive ependymoma match (12/19 cases) showed either a "papillary" (n = 5), a "trabecular" (n = 1), or a "pseudo-papillary" (n = 6) growth pattern. The papillary growth pattern was strongly associated with the methylation class B of posterior fossa ependymoma (PFB, 5/5 cases) and tumors displayed DNA methylation sites that were significantly different when compared to PFB ependymomas without papillary growth. Tumors with pseudo-papillary histology matched to the methylation class of myxopapillary ependymoma (4/6 cases), whereas the trabecular case was anatomically and molecularly a spinal ependymoma. Our results show that the diagnosis of histological ependymoma variants is challenging and epigenetic profiles may improve diagnostic accuracy of these cases. Whereas clear cell and papillary ependymomas display correlations between localization, histology, and methylation, tanycytic ependymoma does not represent a molecularly distinct subgroup.

Background: The incidence of brain metastases in breast cancer (BCBM) patients is increasing. These patients have a very poor prognosis, and therefore, identification of blood-based biomarkers, such as circulating tumor cells (CTCs), and understanding the genomic heterogeneity could help to personalize treatment options., Methods: Both EpCAM-dependent (CellSearch® System) and EpCAM-independent Ficoll-based density centrifugation methods were used to detect CTCs from 57 BCBM patients. DNA from individual CTCs and corresponding primary tumors and brain metastases were analyzed by next-generation sequencing (NGS) in order to evaluate copy number aberrations and single nucleotide variations (SNVs)., Results: CTCs were detected after EpCAM-dependent enrichment in 47.7% of the patients (≥ 5 CTCs/7.5 ml blood in 20.5%). The CTC count was associated with ERBB2 status (p = 0.029) of the primary tumor as well as with the prevalence of bone metastases (p = 0.021). EpCAM-independent enrichment revealed CTCs in 32.6% of the patients, especially among triple-negative breast cancer (TNBC) patients (70.0%). A positive CTC status after enrichment of either method was significantly associated with decreased overall survival time (p < 0.05). Combining the results of both enrichment methods, 63.6% of the patients were classified as CTC positive. In three patients, the matched tumor tissue and single CTCs were analyzed by NGS showing chromosomal aberrations with a high genomic clonality and mutations in pathways potentially important in brain metastasis formation., Conclusion: The detection of CTCs, regardless of the enrichment method, is of prognostic relevance in BCBM patients and in combination with molecular analysis of CTCs can help defining patients with higher risk of early relapse and suitability for targeted treatment.

Metastatic seeding is driven by cell-intrinsic and environmental cues, yet the contribution of biomechanics is poorly known. We aim to elucidate the impact of blood flow on the arrest and the extravasation of circulating tumor cells (CTCs) in vivo. Using the zebrafish embryo, we show that arrest of CTCs occurs in vessels with favorable flow profiles where flow forces control the adhesion efficacy of CTCs to the endothelium. We biophysically identified the threshold values of flow and adhesion forces allowing successful arrest of CTCs. In addition, flow forces fine-tune tumor cell extravasation by impairing the remodeling properties of the endothelium. Importantly, we also observe endothelial remodeling at arrest sites of CTCs in mouse brain capillaries. Finally, we observed that human supratentorial brain metastases preferably develop in areas with low perfusion. These results demonstrate that hemodynamic profiles at metastatic sites regulate key steps of extravasation preceding metastatic outgrowth., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Despite improvement of therapeutic treatments for breast cancer, the development of brain metastases has become a major limitation to life expectancy for many patients. Brain metastases show very commonly alterations in EGFR and HER2 driven pathways, of which PTEN is an important regulator. Here, we analyzed PTEN expression in 111 tissue samples of breast cancer brain metastases (BCBM). Loss of PTEN was found in a substantial proportion of BCBM samples (48.6%) and was significantly associated with triple-negative breast cancer (67.5%, p = 0.001) and a shorter survival time after surgical resection of brain metastases (p = 0.048). Overexpression of PTEN in brain-seeking MDA-MB-231 BR cells in vitro reduced activation of the AKT pathway, notably by suppression of Akt1 kinase activity. Furthermore, the migration of MDA-MB-231 BR cells in vitro was promoted by co-culturing with both astrocytes and microglial cells. Interestingly, when PTEN was overexpressed the migration was significantly inhibited. Moreover, in an ex vivo organotypic brain slice model, PTEN overexpression reduced invasion of tumor cells. This was accompanied by reduced astrocyte activation that was mediated by autocrine and paracrine activation of GM-CSF/ CSF2RA and AKT/ PTEN pathways. In conclusion, loss of PTEN is frequently detected in triple-negative BCBM patients and associated with poor prognosis. The findings of our functional studies suggest that PTEN loss promotes a feedback loop between tumor cells and glial cells, which might contribute to disease progression.

Purpose: The aim of this study was to investigate the epidermal growth factor receptor (EGFR) status in ependymoma specimens, as there is a need for new prognostic and druggable targets in this disease., Methods: Ependymomas (WHO grade II, n = 40; WHO grade III, n = 15) located spinal (n = 35), infratentorial (n = 14), and supratentorial (n = 6) of 53 patients with a median age of 40 (range, 2-79) years were analyzed for Ki-67, p53, and EGFR expression by immunohistochemistry using a tissue microarray and for EGFR gene copy number alterations/mutations. Results were correlated to clinical data., Results: EGFR overexpression was found in 30/60% of ependymomas depending on the antibody used and was more pronounced in WHO grade III. High EGFR gene copy number gains were found in 6 (11%) ependymomas with half of them being amplifications. EGFR amplified ependymomas displayed an EGFR overexpression with both antibodies in two of three cases. A missense mutation in exon 20 of EGFR (S768I) was detected in one amplified case., Conclusions: EGFR is frequently overexpressed in ependymomas. Other mechanisms than amplification of the EGFR gene appear to contribute to EGFR overexpression in most cases. EGFR mutations may be present in a small subset of ependymomas.

The activation of immune cells by targeting checkpoint inhibitors showed promising results with increased patient survival in distinct primary cancers. Since only limited data exist for human brain metastases, we aimed at characterizing tumor infiltrating lymphocytes (TILs) and expression of immune checkpoints in the respective tumors. Two brain metastases cohorts, a mixed entity cohort (n = 252) and a breast carcinoma validation cohort (n = 96) were analyzed for CD3+, CD8+, FOXP3+, PD-1+ lymphocytes and PD-L1+ tumor cells by immunohistochemistry. Analyses for association with clinico-epidemiological and neuroradiological parameters such as patient survival or tumor size were performed. TILs infiltrated brain metastases in three different patterns (stromal, peritumoral, diffuse). While carcinomas often show a strong stromal infiltration, TILs in melanomas often diffusely infiltrate the tumors. Highest levels of CD3+ and CD8+ lymphocytes were seen in renal cell carcinomas (RCC) and strongest PD-1 levels on RCCs and melanomas. High amounts of TILs, high ratios of PD-1+/CD8+ cells and high levels of PD-L1 were negatively correlated with brain metastases size, indicating that in smaller brain metastases CD8+ immune response might get blocked. PD-L1 expression strongly correlated with TILs and FOXP3 expression. No significant association of patient survival with TILs was observed, while high levels of PD-L1 showed a strong trend towards better survival in melanoma brain metastases (Log-Rank p = 0.0537). In summary, melanomas and RCCs seem to be the most immunogenic entities. Differences in immunotherapeutic response between tumor entities regarding brain metastases might be attributable to this finding and need further investigation in larger patient cohorts.

Glioblastoma multiforme (GBM) is the most frequent and aggressive brain tumor in adults. The dogma that GBM spread is restricted to the brain was challenged by reports on extracranial metastases after organ transplantation from GBM donors. We identified circulating tumor cells (CTCs) in peripheral blood (PB) from 29 of 141 (20.6%) GBM patients by immunostaining of enriched mononuclear cells with antibodies directed against glial fibrillary acidic protein (GFAP). Tumor cell spread was not significantly enhanced by surgical intervention. The tumor nature of GFAP-positive cells was supported by the absence of those cells in healthy volunteers and the presence of tumor-specific aberrations such as EGFR gene amplification and gains and losses in genomic regions of chromosomes 7 and 10. Release of CTCs was associated with EGFR gene amplification, suggesting a growth potential of these cells. We demonstrate that hematogenous GBM spread is an intrinsic feature of GBM biology., (Copyright © 2014, American Association for the Advancement of Science.)

Breast cancer brain metastases (BCBM) are detected with increasing incidence. In order to detect potential genes involved in BCBM, we first screened for genes down-regulated by methylation in cell lines with site-specific metastatic ability. The expression of five genes, CADM1, SPARC, RECK, TNFAIP3 and CXCL14, which were also found down-regulated in gene expression profiling analyses of BCBM tissue samples, was verified by qRT-PCR in a larger patient cohort. CADM1 was chosen for further down-stream analyses. A higher incidence of CADM1 methylation, correlating with lower expression levels, was found in BCBM as compared to primary BC. Loss of CADM1 protein expression was detected most commonly among BCBM samples as well as among primary tumors with subsequent brain relapse. The prognostic role of CADM1 expression was finally verified in four large independent breast cancer cohorts (n=2136). Loss of CADM1 protein expression was associated with disease stage, lymph node status, and tumor size in primary BC. Furthermore, all analyses revealed a significant association between loss of CADM1 and shorter survival. In multivariate analyses, survival was significantly shorter among patients with CADM1-negative tumors. Loss of CADM1 expression is an independent prognostic factor especially associated with the development of brain metastases in breast cancer patients.

Current standard systemic therapies for treating breast cancer patients with brain metastases are inefficient. Targeted therapies against human epidermal growth factor receptors are of clinical interest because of their alteration in a subset of breast cancers (BCs). We analyzed copy number, mutation status, and protein expression of epidermal growth factor receptor (EGFR), human epidermal growth factor 2 (HER2), phosphatase and tensin homologue (PTEN), and PI3K catalytic subunit (PIK3CA) in 110 ductal carcinoma in situ, primary tumor, and metastatic BC samples. Alterations in EGFR, HER2, and PTEN, alone or in combination, were found in a significantly larger fraction of breast cancer brain metastases tumor tissue compared with samples from primary tumors with good prognosis, bone relapse, or other distant metastases (all P < 0.05). Primary tumor patients with a subsequent brain relapse showed almost equally high frequencies of especially EGFR and PTEN alteration as the breast cancer brain metastases patients. PIK3CA was not associated with an increased risk of brain metastases. Genetic alterations in both EGFR and PTEN were especially common in triple-negative breast cancer patients and rarely were seen among HER2-positive patients. In conclusion, we identified two independent high-risk primary BC subgroups for developing brain metastases, represented by genetic alterations in either HER2 or EGFR/PTEN-driven pathways. In contrast, none of these pathways was associated with an increased risk of bone metastasis. These findings highlight the importance of both pathways as possible targets in the treatment of brain metastases in breast cancer., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Diffuse infiltration remains the fulcrum of glioblastoma's incurability, leading inevitably to recurrence. Therefore, uncovering the pathological mechanism is imperative. Because signal transducer and activator of transcription 3 (STAT3) correlates with glioma malignancy and predicts poor clinical outcome, we determined its role in glioma single cell infiltration and tumor growth., Methods: STAT3 was silenced in Tu-2449 glioma cells via lentiviral gene transfer. Target gene expression was measured by real-time reverse transcription PCR, Western blotting, and immunohistochemistry. Microvilli were visualized by staining with wheat germ agglutinin. Migration and invasion were measured by Scratch and Matrigel chamber assays. Diffuse infiltration was studied in 350-μm-thick organotypic tissue cultures over 14 days using cells tagged with enhanced green fluorescent protein and live confocal laser scanning microscopy. Survival of tumor-bearing syngeneic, immunocompetent B6C3F1 mice was analyzed by Kaplan-Meier plots., Results: STAT3 silencing reduced cell migration and invasion in vitro and stopped single cell infiltration ex vivo, while STAT3-expressing cells disseminated through the neuropil at ∼100 µm/day. STAT3 silencing reduced transcription of several tumor progression genes. Mice with intracranial STAT3 knockdown tumors had a significant (P< .0007) survival advantage over controls, yielding 27% long-term survival. STAT3 knockdown reduced podoplanin expression 50-fold and inhibited concurrent microvilli formation. STAT3 knockdown tumors exhibited a weaker podoplanin immunoreactivity compared with controls. Podoplanin staining was diffuse, preferentially at tumor margins, and absent in normal brain., Conclusions: Our results show compelling evidence that STAT3 is a key driver of diffuse infiltration and glioma growth and might therefore represent a promising target for an anti-invasive therapy.

Introduction: With the improvement of therapeutic options for the treatment of breast cancer, the development of brain metastases has become a major limitation to life expectancy in many patients. Therefore, our aim was to identify molecular markers associated with the development of brain metastases in breast cancer., Methods: Patterns of chromosomal aberrations in primary breast tumors and brain metastases were compared with array-comparative genetic hybridization (CGH). The most significant region was further characterized in more detail by microsatellite and gene-expression analysis, and finally, the possible target gene was screened for mutations., Results: The array CGH results showed that brain metastases, in general, display similar chromosomal aberrations as do primary tumors, but with a notably higher frequency. Statistically significant differences were found at nine different chromosomal loci, with a gain and amplification of EGFR (7p11.2) and a loss of 10q22.3-qter being among the most significant aberrations in brain metastases (P < 0.01; false discovery rate (fdr) < 0.04). Allelic imbalance (AI) patterns at 10q were further verified in 77 unmatched primary tumors and 21 brain metastases. AI at PTEN loci was found significantly more often in brain metastases (52%) and primary tumors with a brain relapse (59%) compared with primary tumors from patients without relapse (18%; P = 0.003) or relapse other than brain tumors (12%; P = 0.006). Loss of PTEN was especially frequent in HER2-negative brain metastases (64%). Furthermore, PTEN mRNA expression was significantly downregulated in brain metastases compared with primary tumors, and PTEN mutations were frequently found in brain metastases., Conclusions: These results demonstrate that brain metastases often show very complex genomic-aberration patterns, suggesting a potential role of PTEN and EGFR in brain metastasis formation.

Glioblastomas belong to the most devastating cancer diseases. For this reason, polysorbate 80 (Tween 80)-coated poly(isohexyl cyanoacrylate) (PIHCA) (Monorex) nanoparticles loaded with doxorubicin were developed and tested for their use for the treatment of glioblastomas. The preparation of the nanoparticles resulted in spherical particles with high doxorubicin loading. The physico-chemical properties and the release of doxorubicin from the PIHCA-nanoparticles were analysed, and the influence on cell viability of the rat glioblastoma 101/8-cell line was investigated. In vitro, the empty nanoparticles did not show any toxicity, and the anti-cancer effects of the drug-loaded nanoparticles were increased in comparison to doxorubicin solution, represented by IC(50) values. The in vivo efficacy was then tested in intracranially glioblastoma 101/8-bearing rats. Rats were treated with 3 × 1.5mg/kg doxorubicin and were sacrificed 18 days after tumour transplantation. Histological and immunohistochemical analyses were carried out to assess the efficacy of the nanoparticles. Tumour size, proliferation activity, vessel density, necrotic areas, and expression of glial fibrillary acidic protein demonstrated that doxorubicin-loaded PIHCA-nanoparticles were much more efficient than the free drug. The results suggest that poly(isohexyl cyanoacrylate) nanoparticles hold great promise for the non-invasive therapy of human glioblastomas., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Purpose: Glioblastomas are the most common and most deadly primary brain tumors. Here, we evaluated the chemotherapeutic effect of the natural polyphenol curcumin on glioma cells in vitro and in vivo using an immunocompetent orthotopic mouse model., Experimental Design: Curcumin's effects on proliferation, cell cycle, migration, invasion, JAK/STAT3 signaling, STAT3 target gene expression, and STAT3C rescue experiments were determined in murine glioma cell lines in vitro. Therapeutic effects of curcumin in vivo were evaluated in tumor-bearing mice fed a Western-type diet fortified with curcumin (0.05%, w/w) and in control animals. Tumor growth patterns and survival were evaluated by immunohistochemistry, morphometric analyses, and Kaplan-Meier plots., Results: In vitro, curcumin inhibited JAK1,2/STAT3 tyrosine-phosphorylation in a dose-dependent fashion in murine glioma cell lines. Real-time RT-PCR revealed that curcumin downregulated transcription of the STAT3 target genes c-Myc, MMP-9, Snail, and Twist, and of the proliferation marker Ki67. Curcumin dose-dependently suppressed cell proliferation by inducing a G2/M phase arrest. In wound healing and Matrigel invasion assays, curcumin treatment resulted in a dose-dependent attenuation of the glioma cells' migratory and invasive behavior, which could be rescued by constitutively active STAT3C. In vivo, curcumin intake reduced the growth and midline crossing of intracranially implanted tumors and proliferation of tumor cells ensuing in significant long-term survival compared with control diet., Conclusion: This preclinical study shows that curcumin is capable of suppressing malignant glioma growth in vitro and in vivo. Our data suggest that the pharmacologically safe agent curcumin holds promise for clinical application in glioma therapy., (©2010 AACR.)

The objective of this study was to investigate the therapeutic effects of doxorubicin bound to polysorbate-coated nanoparticles that had previously been shown to significantly enhance survival in the orthotopic rat 101/8 glioblastoma model. Tumor-bearing animals were subjected to chemotherapy using doxorubicin in solution (Dox-sol) or doxorubicin bound to polysorbate 80-coated poly(butyl cyanoacrylate) nanoparticles (Dox-np) injected intravenously on Days 2, 5 and 8 post tumor implantation. The antitumor effect was assessed on Days 10, 14 and 18 post tumor implantation. Tumors showed signs of malignancy including invasion of brain tissue, brisk mitotic activity, microvascular proliferation, necrosis and increased proliferation resembling human glioblastoma. Dox-np produced a considerably more pronounced antitumor effect exhibited as a reduced tumor size, lower proliferation, and a decreased necrotic area compared to Dox-sol and to untreated control groups. A drastic effect of Dox-np on vascularization indicated an antiangiogenic mode of action.

Purpose: Bone marrow is a common homing organ for early disseminated tumor cells (DTC) and their presence can predict the subsequent occurrence of overt metastasis and survival in lung cancer. It is still unclear whether the shedding of DTC from the primary tumor is a random process or a selective release driven by a specific genomic pattern., Experimental Design: DTCs were identified in bone marrow from lung cancer patients by an immunocytochemical cytokeratin assay. Genomic aberrations and expression profiles of the respective primary tumors were assessed by microarrays and fluorescence in situ hybridization analyses. The most significant results were validated on an independent set of primary lung tumors and brain metastases., Results: Combination of DNA copy number profiles (array comparative genomic hybridization) with gene expression profiles identified five chromosomal regions differentiating bone marrow-negative from bone marrow-positive patients (4q12-q32, 10p12-p11, 10q21-q22, 17q21, and 20q11-q13). Copy number changes of 4q12-q32 were the most prominent finding, containing the highest number of differentially expressed genes irrespective of chromosomal size (P=0.018). Fluorescence in situ hybridization analyses on further primary lung tumor samples confirmed the association between loss of 4q and bone marrow-positive status. In bone marrow-positive patients, 4q was frequently lost (37% versus 7%), whereas gains could be commonly found among bone marrow-negative patients (7% versus 17%). The same loss was also found to be common in brain metastases from both small and non-small cell lung cancer patients (39%)., Conclusions: Thus, our data indicate, for the first time, that early hematogenous dissemination of tumor cells might be driven by a specific pattern of genomic changes.

Human low-grade astrocytomas frequently recur and progress to states of higher malignancy. During tumor progression TP53 alterations are among the first genetic changes, while derangement of the p16/p14ARF/RB-1 system occurs later. To probe the pathogenetic significance of TP53 and RB-1 alterations, we introduced a v-src transgene driven by glial fibrillary acidic protein (GFAP) regulatory elements (which causes preneoplastic astrocytic lesions and stochastically astrocytomas of varying degrees of malignancy) into TP53+/- or RB-1+/- mice. Hemizygosity for TP53 or RB-1 did not increase the incidence or shorten the latency of astrocytic tumors in GFAP-v-src mice over a period of up to 76 weeks. Single strand conformation analysis of exons 5 to 8 of non-ablated TP53 alleles revealed altered migration patterns in only 3/16 tumors analyzed. Wild-type RB-1 alleles were retained in all RB-1+/-GFAP-v-src mice-derived astrocytic tumors analyzed, and pRb immunostaining revealed protein expression in all tumors. Conversely, the GFAP-v-src transgene did not influence the development of extraneural tumors related to TP53 or RB-1 hemizygosity. Therefore, the present study indicates that neither loss of RB-1 nor of TP53 confer a growth advantage in vivo to preneoplastic astrocytes expressing v-src, and suggests that RB-1 and TP53 belong to one single complementation group along with v-src in this transgenic model of astrocytoma development. The stochastic development of astrocytic tumors in GFAP-v-src, TP53+/- GFAP-v-src, and RB-1+/- GFAP-v-src transgenic mice indicates that additional hitherto unknown genetic lesions of astrocytes contribute to tumorigenesis, whose elucidation may prove important for our understanding of astrocytoma initiation and progression.