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Background Molecular brain tumor classification using DNA methylation profiling has revealed that the methylation-class of pleomorphic xanthoastrocytoma (mcPXA) comprised a substantial portion of divergent initial diagnoses, which had been established based on histology alone. This study aimed to characterize the survival outcome in patients with mcPXAs—in light of the diverse selected treatment regimes. Methods A retrospective cohort of adult mcPXAs were analyzed in regard to their progression-free survival following surgical resection and postoperative radiotherapy. Radiotherapy treatment plans were correlated with follow-up images to characterize the pattern of relapse. Treatment toxicities and molecular tumor characteristics were further analyzed. Results Divergent initial histological diagnoses were encountered in 40.7%. There was no significant difference in local progression-free (PFS) and overall survival (OS) following gross total or subtotal resection. Postoperative radiotherapy was completed in 81% (22/27) following surgical intervention. Local PFS was 54.4% (95% CI: 35.3–84.0%) and OS was 81.3% (95% CI: 63.8–100%) after 3 years following postoperative radiotherapy. Initial relapses post-radiotherapy were primarily located in the previous tumor location and/or the planning target volume (PTV) (12/13). All patients in our cohort demonstrated the prognostically favorable pTERT-wildtype mcPXA. Conclusion Our study demonstrated that adult patients with mcPXAs display a worse progression-free survival compared to the reported WHO grade 2 PXAs. Future matched-pair analyses are required with a non-irradiated cohort to elucidate the benefit of postoperative radiotherapy in adult patients with mcPXAs.

The aim of this study was to assess peripheral nerve involvement in patients with multiple sclerosis (MS) at first clinical presentation using quantitative magnetic resonance (MR) neurography in correlation with clinical, laboratory, electrophysiological, and central nervous MR imaging data.In this prospective monocentric study, 30 patients first diagnosed with MS according to the McDonald criteria (19 women; mean age, 32.4 ± 8.8 years) and 30 age- and sex-matched healthy volunteers were examined with high-resolution 3 T MR neurography using a dual-echo T2-relaxometry sequence covering the tibial and peroneal nerves from proximal thigh to distal calf. Magnetic resonance biomarkers of T2 relaxation time (T2 app ), proton spin density (PSD), and nerve cross-sectional area (CSA) were correlated with clinical symptoms, intrathecal immunoglobulin (Ig) synthesis, nerve conduction study, and lesion load on brain and spine MR imaging. The diagnostic accuracy of MR biomarkers was assessed using receiver-operating characteristic curves.Diffuse nerve changes were detected along the tibial and peroneal nerves in MS patients, who showed decreased PSD ( P0.001), increased T2 app ( P0.001), and smaller tibial nerve CSA ( P0.001) compared with healthy subjects. Tibial PSD was identified as best parameter separating patients from controls (area under the curve = 0.876). Intrathecal IgG and IgM synthesis correlated with PSD values ( r = -0.44, P = 0.016, and r = -0.42, P = 0.022). Contrast-enhancement of brain or spine lesions was related to larger tibial and peroneal CSA ( P0.001, P = 0.033). Abnormal electrophysiology correlated with higher tibial and peroneal T2 app ( P0.001 and P = 0.033), lower tibial and peroneal PSD ( P = 0.018 and P = 0.002), and smaller peroneal CSA ( P0.001).Quantitative MR neurography reveals peripheral nerve changes in patients with initial diagnosis of MS. Correlation of imaging findings with intrathecal immunoglobulin synthesis may indicate a primary coaffection of the peripheral nervous system in MS.

Background In patients with recurrent glioblastoma, corticosteroids are frequently used to mitigate intracranial pressure and to improve patient neurological functioning. To date, in these patients, no systematic studies have been performed to assess neurocognitive functioning (NCF) in relation to corticosteroid treatment. Methods Using baseline data (ie, prior to randomization) of European Organization for Research and Treatment of Cancer (EORTC) trial 26101, we performed regression analysis to assess the predictive value of corticosteroid intake on performance of the EORTC brain tumor clinical trial NCF test battery. The battery is comprised of the Hopkins Verbal Learning Test—Revised (HVLT-R), Controlled Oral Word Association Test (COWA), and Trail Making Test (A and B). Results Out of 321 patients, 148 (46.1%) were not using corticosteroids, and 173 were using dexamethasone (34.3%), methylprednisolone (9.7%), or other corticosteroids (9.9%). Patients on corticosteroids had worse performance on all neurocognitive tests. Regression analyses demonstrated a negative association between corticosteroids use and the HVLT-R free recall score (R2 change = 0.034, F change (1, 272) = 13.392, P < .001) and HVLT-R Delayed Recall score (R2 change = 0.028, F change (1, 270) = 10.623, P = .002). No statistically significant association was found for HVLT-R Delayed recognition, COWA, TMT part A and TMT part B (P > .05). Conclusions Glioblastoma patients prescribed with corticosteroids show poorer memory functions, expressive language, visual-motor scanning speed, and executive functioning than patients not using corticosteroids. Furthermore, we found a negative association between corticosteroid intake and memory functions. The possibility of deleterious effects of corticosteroids on NCF should be considered during clinical decision making.

Background The current CNS WHO classification of brain tumors distinguishes three malignancy grades in meningiomas, with increasing risk of recurrence from CNS WHO grade 1 to 3. Radiotherapy is recommended by current EANO guidelines for patients not safely amenable to surgery or after incomplete resection in higher grades. Despite adequately predicting recurrence probability for the majority of CNS WHO grade 2 meningioma patients, a considerable subset of patients demonstrates an unexpectedly early tumor recurrence following radiotherapy. Methods A retrospective cohort of 44 patients with CNS WHO grade 2 meningiomas was stratified into 3 risk groups (low, intermediate, high) using an integrated morphological, CNV- and methylation family-based classification. Local progression-free survival (lPFS) following radiotherapy (RT) was analyzed and total dose of radiation was correlated with survival outcome. Radiotherapy treatment plans were correlated with follow-up images to characterize the pattern of relapse. Treatment toxicities were further assessed. Results Risk stratification of CNS WHO grade 2 meningioma into integrated risk groups demonstrated a significant difference in 3-year lPFS following radiotherapy between the molecular low- and high-risk group. Recurrence pattern analysis revealed that 87.5 % of initial relapses occurred within the RT planning target volume or resection cavity. Conclusion Integrated risk scoring can identify CNS WHO grade 2 meningioma patients at risk or relapse and dissemination following radiotherapy. Therapeutic management of CNS WHO grade 2 meningiomas and future clinical trials should be adjusted according to the molecular risk-groups, and not rely on conventional CNS WHO grading alone.

Background IDH-mutant gliomas are separate based on the codeletion of the chromosomal arms 1p and 19q into oligodendrogliomas IDH-mutant 1p/19q-codeleted and astrocytomas IDH-mutant. While nuclear loss of ATRX expression excludes 1p/19q codeletion, its limited sensitivity prohibits to conclude on 1p/19q status in tumors with retained nuclear ATRX expression. Methods Employing mass spectrometry based proteomic analysis in a discovery series containing 35 fresh frozen and 72 formalin fixed and paraffin embedded tumors with established IDH and 1p/19q status, potential biomarkers were discovered. Subsequent validation immunohistochemistry was conducted on two independent series (together 77 oligodendrogliomas IDH-mutant 1p/19q-codeleted and 92 astrocytomas IDH-mutant). Results We detected highly specific protein patterns distinguishing oligodendroglioma and astrocytoma. In these patterns, high HIP1R and low vimentin levels were observed in oligodendroglioma while low HIP1R and high vimentin levels occurred in astrocytoma. Immunohistochemistry for HIP1R and vimentin expression in 35 cases from the FFPE discovery series confirmed these findings. Blinded evaluation of the validation cohorts predicted the 1p/19q status with a positive and negative predictive value as well as an accuracy of 100% in the first cohort and with a positive predictive value of 83%; negative predictive value of 100% and an accuracy of 92% in the second cohort. Nuclear ATRX loss as marker for astrocytoma increased the sensitivity to 96% and the specificity to 100%. Conclusions We demonstrate that immunohistochemistry for HIP1R, vimentin, and ATRX predict 1p/19q status with 100% specificity and 95% sensitivity and therefore, constitutes a simple and inexpensive approach to the classification of IDH-mutant glioma.

Supplementary Figures and Figure legends, Supplementary Video legends and supplementary materials and methods

Crossing the blood–brain barrier is a crucial, rate-limiting step of brain metastasis. Understanding of the mechanisms of cancer cell extravasation from brain microcapillaries is limited as the underlying cellular and molecular processes cannot be adequately investigated using in vitro models and endpoint in vivo experiments. Using ultrastructural and functional imaging, we demonstrate that dynamic changes of activated brain microcapillaries promote the mandatory first steps of brain colonization. Successful extravasation of arrested cancer cells occurred when adjacent capillary endothelial cells (EC) entered into a distinct remodeling process. After extravasation, capillary loops were formed, which was characteristic of aggressive metastatic growth. Upon cancer cell arrest in brain microcapillaries, matrix-metalloprotease 9 (MMP9) was expressed. Inhibition of MMP2/9 and genetic perturbation of MMP9 in cancer cells, but not the host, reduced EC projections, extravasation, and brain metastasis outgrowth. These findings establish an active role of ECs in the process of cancer cell extravasation, facilitated by cross-talk between the two cell types. This extends our understanding of how host cells can contribute to brain metastasis formation and how to prevent it.Significance:Tracking single extravasating cancer cells using multimodal correlative microscopy uncovers a brain seeding mechanism involving endothelial remodeling driven by cancer cell–derived MMP9, which might enable the development of approaches to prevent brain metastasis.See related commentary by McCarty, p. 1167

Supplementary Data - Figure Legends

Specific biological properties of those circulating cancer cells that are the origin of brain metastases (BM) are not well understood. Here, single circulating breast cancer cells were fate-tracked during all steps of the brain metastatic cascade in mice after intracardial injection over weeks. A novel in vivo two-photon microscopy methodology was developed that allowed to determine the specific cellular and molecular features of breast cancer cells that homed in the brain, extravasated, and successfully established a brain macrometastasis. Those BM-initiating breast cancer cells (BMIC) were mainly originating from a slow-cycling subpopulation that included only 16% to 20% of all circulating cancer cells. BMICs showed enrichment of various markers of cellular stemness. As a proof of principle for the principal usefulness of this approach, expression profiling of BMICs versus non-BMICs was performed, which revealed upregulation of NDRG1 in the slow-cycling BMIC subpopulation in one BM model. Here, BM development was completely suppressed when NDRG1 expression was downregulated. In accordance, in primary human breast cancer, NDRG1 expression was heterogeneous, and high NDRG1 expression was associated with shorter metastasis-free survival. In conclusion, our data identify temporary slow-cycling breast cancer cells as the dominant source of brain and other metastases and demonstrates that this can lead to better understanding of BMIC-relevant pathways, including potential new approaches to prevent BM in patients.Implications:Cancer cells responsible for successful brain metastasis outgrowth are slow cycling and harbor stemness features. The molecular characteristics of these metastasis-initiating cells can be studied using intravital microscopy technology.

Percentage growth change of individual nsNSCLC metastases during bevacizumab treatment

Supplementary Figure 3 shows PKH26 intensity and depth of the cell in relation to the successful establishment of a metastasis. Further, the brain metastatic cascade for PKH26 low (slow cycling) and PKH26 (high) (ultra slow cycling) cells is illustrated.

Supplementary Figure 4 shows IVIS results.

Supplementary Figure 6 shows NDRG1 expression in several cell lines.

Supplementary Figure 1 A-C shows loss of PKH26 over time. Supplementary Figure 1 D shows an additional example of a slow cycling cells with remaining PKH26 staining in vivo

Supplementary Figure 5 shows cell cycle analysis.

Supplementary Figure 1: Detection of asunercept/ APG101 in brains of treated mice. Supplementary Figure 2: qPCR for MMP2 (A) or MMP9 (B) and representative images of human microglia after 12 h treatment with vehicle (I), 20 µg/ml APG101 (II), 50 U/ml CD95L (III) and both 20 µg/ml APG101 and 50 U/ml CD95L (IV). Supplementary Figure 3: APG101 is effective in murine cells. Supplementary Figure 4: Radiation effects on CD95L expression.

Time from randomization to new brain lesions in AVAiL Trial

Supplementary Figure 2 cell index of PKH26 stained cells, the overlap of PKH26 loss and 2 other methods of cell division analysis as well as the reversion of a slow cycling cell population to a mixed cycling population.

Synergistic effects of trametinib and RG7388 treatment.

Purpose: Rechallenge with temozolomide (TMZ) at first progression of glioblastoma after temozolomide chemoradiotherapy (TMZ/RT→TMZ) has been studied in retrospective and single-arm prospective studies, applying temozolomide continuously or using 7/14 or 21/28 days schedules. The DIRECTOR trial sought to show superiority of the 7/14 regimen.Experimental Design: Patients with glioblastoma at first progression after TMZ/RT→TMZ and at least two maintenance temozolomide cycles were randomized to Arm A [one week on (120 mg/m2 per day)/one week off] or Arm B [3 weeks on (80 mg/m2 per day)/one week off]. The primary endpoint was median time-to-treatment failure (TTF) defined as progression, premature temozolomide discontinuation for toxicity, or death from any cause. O6-methylguanine DNA methyltransferase (MGMT) promoter methylation was prospectively assessed by methylation-specific PCR.Results: Because of withdrawal of support, the trial was prematurely closed to accrual after 105 patients. There was a similar outcome in both arms for median TTF [A: 1.8 months; 95% confidence intervals (CI), 1.8–3.2 vs. B: 2.0 months; 95% CI, 1.8–3.5] and overall survival [A: 9.8 months (95% CI, 6.7–13.0) vs. B: 10.6 months (95% CI, 8.1–11.6)]. Median TTF in patients with MGMT-methylated tumors was 3.2 months (95% CI, 1.8–7.4) versus 1.8 months (95% CI, 1.8–2) in MGMT-unmethylated glioblastoma. Progression-free survival rates at 6 months (PFS-6) were 39.7% with versus 6.9% without MGMT promoter methylation.Conclusions: Temozolomide rechallenge is a treatment option for MGMT promoter-methylated recurrent glioblastoma. Alternative strategies need to be considered for patients with progressive glioblastoma without MGMT promoter methylation. Clin Cancer Res; 21(9); 2057–64. ©2015 AACR.

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Purpose: Preclinical data indicate anti-invasive activity of APG101, a CD95 ligand (CD95L)–binding fusion protein, in glioblastoma.Experimental Design: Patients (N = 91) with glioblastoma at first or second progression were randomized 1:2 between second radiotherapy (rRT; 36 Gy; five times 2 Gy per week) or rRT+APG101 (400 mg weekly i.v.). Patient characteristics [N = 84 (26 patients rRT, 58 patients rRT+APG101)] were balanced.Results: Progression-free survival at 6 months (PFS-6) rates were 3.8% [95% confidence interval (CI), 0.1–19.6] for rRT and 20.7% (95% CI, 11.2–33.4) for rRT+APG101 (P = 0.048). Median PFS was 2.5 (95% CI, 2.3–3.8) months and 4.5 (95% CI, 3.7–5.4) months with a hazard ratio (HR) of 0.49 (95% CI, 0.27–0.88; P = 0.0162) adjusted for tumor size. Cox regression analysis adjusted for tumor size revealed a HR of 0.60 (95% CI, 0.36–1.01; P = 0.0559) for rRT+APG101 for death of any cause. Lower methylation levels at CpG2 in the CD95L promoter in the tumor conferred a stronger risk reduction (HR, 0.19; 95% CI, 0.06–0.58) for treatment with APG101, suggesting a potential biomarker.Conclusions: CD95 pathway inhibition in combination with rRT is an innovative concept with clinical efficacy. It warrants further clinical development. CD95L promoter methylation in the tumor may be developed as a biomarker. Clin Cancer Res; 20(24); 6304–13. ©2014 AACR.

Purpose: The role of blood–brain barrier (BBB) integrity for brain tumor biology and therapy is a matter of debate.Experimental Design: We developed a new experimental approach using in vivo two-photon imaging of mouse brain metastases originating from a melanoma cell line to investigate the growth kinetics of individual tumor cells in response to systemic delivery of two PI3K/mTOR inhibitors over time, and to study the impact of microregional vascular permeability. The two drugs are closely related but differ regarding a minor chemical modification that greatly increases brain penetration of one drug.Results: Both inhibitors demonstrated a comparable inhibition of downstream targets and melanoma growth in vitro. In vivo, increased BBB permeability to sodium fluorescein was associated with accelerated growth of individual brain metastases. Melanoma metastases with permeable microvessels responded similarly to equivalent doses of both inhibitors. In contrast, metastases with an intact BBB showed an exclusive response to the brain-penetrating inhibitor. The latter was true for macro- and micrometastases, and even single dormant melanoma cells. Nuclear morphology changes and single-cell regression patterns implied that both inhibitors, if extravasated, target not only perivascular melanoma cells but also those distant to blood vessels.Conclusions: Our study provides the first direct evidence that nonpermeable brain micro- and macrometastases can effectively be targeted by a drug designed to cross the BBB. Small-molecule inhibitors with these optimized properties are promising agents in preventing or treating brain metastases in patients. Clin Cancer Res; 22(24); 6078–87. ©2016 AACR.See related commentary by Steeg et al., p. 5953

supplemental legend

Supplementary Figure S1. Geometric mean plasma concentration-time profiles of BAY1436032 at dose levels of 150 mg to 1500 mg.

Purpose:BAY1436032, an inhibitor of mutant isocitrate dehydrogenase 1 (mIDH1), was active against multiple IDH1-R132X solid tumors in preclinical models. This first-in-human study was designed to determine the safety and pharmacokinetics of BAY1436032, and to evaluate its potential pharmacodynamics and antitumor effects.Patients and Methods:The study comprised of dose escalation and dose expansion cohorts. BAY1436032 tablets were orally administered twice daily on a continuous basis in subjects with mIDH1 solid tumors.Results:In dose escalation, 29 subjects with various tumor types were administered BAY1436032 across five doses (150–1,500 mg twice daily). BAY1432032 exhibited a relatively short half-life. Most evaluable subjects experienced target inhibition as indicated by a median maximal reduction of plasma R-2-hydroxyglutarate levels of 76%. BAY1436032 was well tolerated and an MTD was not identified. A dose of 1,500 mg twice daily was selected for dose expansion, where 52 subjects were treated in cohorts representing four different tumor types [lower grade glioma (LGG), glioblastoma, intrahepatic cholangiocarcinoma, and a basket cohort of other tumor types]. The best clinical outcomes were in subjects with LGG (n = 35), with an objective response rate of 11% (one complete response and three partial responses) and stable disease in 43%. As of August 2020, four of these subjects were in treatment for >2 years and still ongoing. Objective responses were observed only in LGG.Conclusions:BAY1436032 was well tolerated and showed evidence of target inhibition and durable objective responses in a small subset of subjects with LGG.

Supplementary Table S12. NGS analysis of select tumor-associated genes in archival tumor tissue obtained from a subset of subjects.

IGFBP1 mRNA expression after treatment with trametinib or IGFBP1 knockdown.

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Maintenance of RG7388 resistance after withdrawal of permanent RG7388 exposure.

Supplementary Figure 1. MMSE assessment at study entry, during treatment, and at follow-up

Supplementary Data from Temozolomide and Radiotherapy versus Radiotherapy Alone in Patients with Glioblastoma, IDH-wildtype: Post Hoc Analysis of the EORTC Randomized Phase III CATNON Trial

A, Metastasis sizes in the different treatment groups at start of treatment (p=0.596, ANOVA on Ranks). B, Quantifications of the mean grey values of the sodium fluorescein signal, normalized to background signal. Note that all metastases could clearly be attributed to be either permeable vs. non-permeable. There were no statistical significant differences between single metastases within the permeable (p=0.244, ANOVA) and the non-permeable (p=0.106, ANOVA) subgroups, when the different treatment groups were compared to each other. C, Quantification of growth rates per day after / before start of MCT control treatment (p=0.412, Mann-Whitney Rank Sum test).