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Background Three- and five-year progression-free survival (PFS) for low-risk meningioma managed with surgery and observation reportedly exceeds 90%. Herewith we summarize outcomes for low-risk meningioma patients enrolled on NRG/RTOG 0539. Methods This phase II trial allocated patients to one of three groups per World Health Organization grade, recurrence status, and resection extent. Low-risk patients had either gross total (GTR) or subtotal resection (STR) for a newly diagnosed grade 1 meningioma and were observed after surgery. The primary endpoint was 3-year PFS. Adverse events (AEs) were scored using Common Terminology Criteria for Adverse Events (CTCAE) version 3. Results Among 60 evaluable patients, the median follow-up was 9.1 years. The 3-, 5-, and 10-year rates were 91.4% (95% CI, 84.2 to 98.6), 89.4% (95% CI, 81.3 to 97.5), 85.0% (95% CI, 75.3 to 94.7) for PFS and 98.3% (95% CI, 94.9 to 100), 98.3%, (95% CI, 94.9 to 100), 93.8% (95% CI, 87.0 to 100) for overall survival (OS), respectively. With centrally confirmed GTR, 3/5/10y PFS and OS rates were 94.3/94.3/87.6% and 97.1/97.1/90.4%. With STR, 3/5/10y PFS rates were 83.1/72.7/72.7% and 10y OS 100%. Five patients reported one grade 3, four grade 2, and five grade 1 AEs. There were no grade 4 or 5 AEs. Conclusions These results prospectively validate high PFS and OS for low-risk meningioma managed surgically but raise questions regarding optimal management following STR, a subcohort that could potentially benefit from adjuvant therapy.

TGF beta1 increases GSC invasion via POSTN.

Supplementary figure legends

Univariate and multivariate survival analysis.

Array gene list by knock down of POSTN

S1 shows intratumor mutation heterogeneity between two specimens of one tumor. S2 describes mutational load of the patient cohort. S3 shows survival difference based on histology. S4 shows mutational signature.

Malignant gliomas are a group of intracranial cancers associated with disproportionately high mortality and morbidity. Here, we report ultradeep targeted sequencing of a prospective cohort of 237 tumors from 234 patients consisting of both glioblastoma (GBM) and lower-grade glioma (LGG) using our customized gene panels. We identified 2,485 somatic mutations, including single-nucleotide substitutions and small indels, using a validated in-house protocol. Sixty-one percent of the mutations were contributed by 12 hypermutators. The hypermutators were enriched for recurrent tumors and had comparable outcome, and most were associated with temozolomide exposure. TP53 was the most frequently mutated gene in our cohort, followed by IDH1 and EGFR. We detected at least one EGFR mutation in 23% of LGGs, which was significantly higher than 6% seen in The Cancer Genome Atlas, a pattern that can be partially explained by the different patient composition and sequencing depth. IDH hotspot mutations were found with higher frequencies in LGG (83%) and secondary GBM (77%) than primary GBM (9%). Multivariate analyses controlling for age, histology, and tumor grade confirm the prognostic value of IDH mutation. We predicted 1p/19q status using the panel sequencing data and received only modest performance by benchmarking the prediction to FISH results of 50 tumors. Targeted therapy based on the sequencing data resulted in three responders out of 14 participants. In conclusion, our study suggests ultradeep targeted sequencing can recapitulate previous findings and can be a useful approach in the clinical setting.

PDF file, 269K, Bevacizummab resistant cell lines are more invasive than their untreated controls.

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Purpose: Antiangiogenic therapy reduces vascular permeability and delays progression but may ultimately promote an aggressive treatment-resistant phenotype. The aim of the present study was to identify mechanisms responsible for glioblastoma resistance to antiangiogenic therapy.Experimental Design: Glioma stem cell (GSC) NSC11 and U87 cell lines with acquired resistance to bevacizumab were developed from orthotopic xenografts in nude mice treated with bevacizumab. Genome-wide analyses were used to identify changes in tumor subtype and specific factors associated with resistance.Results: Mice with established parental NSC11 and U87 cells responded to bevacizumab, whereas glioma cell lines derived at the time of acquired resistance to anti-VEGF therapy were resistant to bevacizumab and did not have prolongation of survival compared with untreated controls. Gene expression profiling comparing anti-VEGF therapy-resistant cell lines to untreated controls showed an increase in genes associated with a mesenchymal origin, cellular migration/invasion, and inflammation. Gene-set enrichment analysis showed that bevacizumab-treated tumors showed a highly significant correlation to published mesenchymal gene signatures. Mice bearing resistant tumors showed significantly greater infiltration of myeloid cells in NSC11- and U87-resistant tumors. Invasion-related genes were also upregulated in both NSC11 and U87 resistant cells which had higher invasion rates in vitro compared with their respective parental cell lines.Conclusions: Our studies identify multiple proinflammatory factors associated with resistance and identify a proneural to mesenchymal transition in tumors resistant to antiangiogenic therapy. Clin Cancer Res; 19(16); 4392–403. ©2013 AACR.

Purpose: Antiangiogenic therapy is effective in blocking vascular permeability, inhibiting vascular proliferation, and slowing tumor growth, but studies in multiple cancer types have shown that tumors eventually acquire resistance to blockade of blood vessel growth. Currently, the mechanisms by which this resistance occurs are not well understood.Experimental Design: In this study, we evaluated the effects of neutrophils on glioma biology both in vitro and in vivo and determined target genes by which neutrophils promote the malignant glioma phenotype during anti-VEGF therapy.Results: We found that an increase in neutrophil infiltration into tumors is significantly correlated with glioma grade and in glioblastoma with acquired resistance to anti-VEGF therapy. Our data demonstrate that neutrophils and their condition media increased the proliferation rate of glioblastoma-initiating cells (GIC). In addition, neutrophils significantly increased GICs Transwell migration compared with controls. Consistent with this behavior, coculture with neutrophils promoted GICs to adopt morphologic and gene expression changes consistent with a mesenchymal signature. Neutrophil-promoting tumor progression could be blocked by S100A4 downregulation in vitro and in vivo. Furthermore, S100A4 depletion increased the effectiveness of anti-VEGF therapy in glioma.Conclusions: Collectively, these data suggest that increased recruitment of neutrophils during anti-VEGF therapy promotes glioma progression and may promote treatment resistance. Tumor progression with mesenchymal characteristics is partly mediated by S100A4, the expression of which is increased by neutrophil infiltration. Targeting granulocytes and S100A4 may be effective approaches to inhibit the glioma malignant phenotype and diminish antiangiogenic therapy resistance. Clin Cancer Res; 20(1); 187–98. ©2013 AACR.

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Study Workflow Diagram. Key steps in the study are outlined, including the number of patients at each stage. BEV: bevacizumab.

PDF file, 809K, TGF-beta increases following the acquisition of bevacizumab resistance.

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Supplementary Materials and Methods

Purpose:Exploration of novel strategies to extend the benefit of PARP inhibitors beyond BRCA-mutant cancers is of great interest in personalized medicine. Here, we identified EGFR amplification as a potential biomarker to predict sensitivity to PARP inhibition, providing selection for the glioblastoma (GBM) patient population who will benefit from PARP inhibition therapy.Experimental Design:Selective sensitivity to the PARP inhibitor talazoparib was screened and validated in two sets [test set (n = 14) and validation set (n = 13)] of well-characterized patient-derived glioma sphere-forming cells (GSC). FISH was used to detect EGFR copy number. DNA damage response following talazoparib treatment was evaluated by γH2AX and 53BP1 staining and neutral comet assay. PARP–DNA trapping was analyzed by subcellular fractionation. The selective monotherapy of talazoparib was confirmed using in vivo glioma models.Results:EGFR-amplified GSCs showed remarkable sensitivity to talazoparib treatment. EGFR amplification was associated with increased reactive oxygen species (ROS) and subsequent increased basal expression of DNA-repair pathways to counterelevated oxidative stress, and thus rendered vulnerability to PARP inhibition. Following talazoparib treatment, EGFR-amplified GSCs showed enhanced DNA damage and increased PARP–DNA trapping, which augmented the cytotoxicity. EGFR amplification–associated selective sensitivity was further supported by the in vivo experimental results showing that talazoparib significantly suppressed tumor growth in EGFR-amplified subcutaneous models but not in nonamplified models.Conclusions:EGFR-amplified cells are highly sensitive to talazoparib. Our data provide insight into the potential of using EGFR amplification as a selection biomarker for the development of personalized therapy.

PDF file 71K, Microarray analysis by IPA for the characteristics changes of GIC11 cells when co-culture of MPRO cells

Related Article from High-Dose Antiangiogenic Therapy for Glioblastoma: Less May Be More?

PDF file, 854K, Bevacizumab resistance does not lead to an increase in cellular proliferation.

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PDF file 124K, Phosphorylated Erk, Akt (pErk, pAkt) and the total proteins were analysis in GSCs incubated with or without CM

Supplementary Figure Legends and Methods

PDF file 42K, MPO expression correlates with glioblastoma grades

Supplementary Figure 4

PDF file, 21K, Genes significantly increased following acquired resistance to bevacizumab.

Targeting angiogenesis in glioblastoma rapidly reduces vascular permeability and contrast enhancement on MRI and prolongs progression-free survival. The long-term efficacy of bevacizumab and other antiangiogenic agents is limited, however, because of the rapid development of resistance. Alternative dosing approaches may be one mechanism of prolonging therapeutic efficacy. Clin Cancer Res; 17(19); 6109–11. ©2011 AACR.

PDF file, 137K, Gene Set Enrichment Analysis of gene expression data of tumors before and after treatment with bevacizumab.

Purpose: VEGF and infiltrating myeloid cells are known regulators of tumor angiogenesis and vascular permeability in glioblastoma. We investigated potential blood-based markers associated with radiographic changes to aflibercept, which binds VEGF and placental growth factor (PlGF) in patients with recurrent glioblastoma.Experimental Design: In this single-arm phase II trial, aflibercept was given intravenously every two weeks until disease progression. Plasma and peripheral blood mononuclear cells were collected at baseline and 24 hours, 14 days, and 28 days posttreatment. Plasma cytokines and angiogenic factors were quantified by using ELISA and multiplex bead assays, and myeloid cells were assessed by flow cytometry in a subset of patients.Results: Circulating levels of VEGF significantly decreased 24 hours after treatment with aflibercept, coincident with radiographic response observed by MRI. PlGF initially decreased 24 hours posttreatment but increased significantly by days 14 and 28. Lower baseline levels of PlGF, elevated baseline levels of CTACK/CCL27, MCP3/CCL7, MIF, and IP-10/CXCL10, and a decrease in VEGFR1+ monocytes from baseline to 24 hours were all associated with improved response. Tumor progression was associated with increases in circulating matrix metalloproteinase 9.Conclusions: These data suggest that decreases in VEGF posttreatment are associated with radiographic response to aflibercept. Elevated baseline chemokines of monocyte lineage in responding patients supports a role for myeloid cells and chemokines as potential biomarkers and regulators of glioma angiogenesis. Clin Cancer Res; 17(14); 4872–81. ©2011 AACR.

Supplementary Figure 2

PDF file, 485K, HIF-1alpha is overexpressed following bevacizumab treatment

Supplementary Figure 1

Supplemental Figures and legends for Figure S1-S8

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PDF file, 425K, Bevacizumab resistance promotes the infiltration of myeloid cells from the bone marrow.

Purpose:Radiomics is the extraction of multidimensional imaging features, which when correlated with genomics, is termed radiogenomics. However, radiogenomic biological validation is not sufficiently described in the literature. We seek to establish causality between differential gene expression status and MRI-extracted radiomic-features in glioblastoma.Experimental Design:Radiogenomic predictions and validation were done using the Cancer Genome Atlas and Repository of Molecular Brain Neoplasia Data glioblastoma patients (n = 93) and orthotopic xenografts (OX; n = 40). Tumor phenotypes were segmented, and radiomic-features extracted using the developed radiome-sequencing pipeline. Patients and animals were dichotomized on the basis of Periostin (POSTN) expression levels. RNA and protein levels confirmed RNAi-mediated POSTN knockdown in OX. Total RNA of tumor cells isolated from mouse brains (knockdown and control) was used for microarray-based expression profiling. Radiomic-features were utilized to predict POSTN expression status in patient, mouse, and interspecies.Results:Our robust pipeline consists of segmentation, radiomic-feature extraction, feature normalization/selection, and predictive modeling. The combination of skull stripping, brain-tissue focused normalization, and patient-specific normalization are unique to this study, providing comparable cross-platform, cross-institution radiomic features. POSTN expression status was not associated with qualitative or volumetric MRI parameters. Radiomic features significantly predicted POSTN expression status in patients (AUC: 76.56%; sensitivity/specificity: 73.91/78.26%) and OX (AUC: 92.26%; sensitivity/specificity: 92.86%/91.67%). Furthermore, radiomic features in OX were significantly associated with patients with similar POSTN expression levels (AUC: 93.36%; sensitivity/specificity: 82.61%/95.74%; P = 02.021E−15).Conclusions:We determined causality between radiomic texture features and POSTN expression levels in a preclinical model with clinical validation. Our biologically validated radiomic pipeline also showed the potential application for human–mouse matched coclinical trials.

PDF file 429K, S100A4 expression in glioma tumors

Purpose: Glioblastoma is an aggressive and molecularly heterogeneous cancer with few effective treatment options. We hypothesized that next-generation sequencing can be used to guide treatment recommendations within a clinically acceptable time frame following surgery for patients with recurrent glioblastoma.Experimental Design: We conducted a prospective genomics-informed feasibility trial in adults with recurrent and progressive glioblastoma. Following surgical resection, genome-wide tumor/normal exome sequencing and tumor RNA sequencing were performed to identify molecular targets for potential matched therapy. A multidisciplinary molecular tumor board issued treatment recommendations based on the genomic results, blood–brain barrier penetration of the indicated therapies, drug–drug interactions, and drug safety profiles. Feasibility of generating genomics-informed treatment recommendations within 35 days of surgery was assessed.Results: Of the 20 patients enrolled in the study, 16 patients had sufficient tumor tissue for analysis. Exome sequencing was completed for all patients, and RNA sequencing was completed for 14 patients. Treatment recommendations were provided within the study's feasibility time frame for 15 of 16 (94%) patients. Seven patients received treatment based on the tumor board recommendations. Two patients reached 12-month progression-free survival, both adhering to treatments based on the molecular profiling results. One patient remained on treatment and progression free 21 months after surgery, 3 times longer than the patient's previous time to progression. Analysis of matched nonenhancing tissue from 12 patients revealed overlapping as well as novel putatively actionable genomic alterations.Conclusions: Use of genome-wide molecular profiling is feasible and can be informative for guiding real-time, central nervous system–penetrant, genomics-informed treatment recommendations for patients with recurrent glioblastoma. Clin Cancer Res; 24(2); 295–305. ©2017 AACR.See related commentary by Wick and Kessler, p. 256

PDF file 177K, S100A4 expression correlates with the survival curve of glioblatoma patients

Supplementary Figure 1 from Myeloid Biomarkers Associated with Glioblastoma Response to Anti-VEGF Therapy with Aflibercept

Purpose:Cytomegalovirus (CMV) antigens occur in glioblastoma but not in normal brains, making them desirable immunologic targets.Patients and Methods:Highly functional autologous polyclonal CMV pp65-specific T cells from patients with glioblastoma were numerically expanded under good manufacturing practice compliant conditions and administered after 3 weeks of lymphodepleting dose-dense temozolomide (100 mg/m2) treatment. The phase I component used a 3+3 design, ascending through four dose levels (5 × 106–1 × 108 cells). Treatment occurred every 6 weeks for four cycles. In vivo persistence and effector function of CMV-specific T cells was determined by dextramer staining and multiparameter flow cytometry in serially sampled peripheral blood and in the tumor microenvironment.Results:We screened 65 patients; 41 were seropositive for CMV; 25 underwent leukapheresis; and 20 completed ≥1 cycle. No dose-limiting toxicities were observed. Radiographic response was complete in 1 patient, partial in 2. Median progression-free survival (PFS) time was 1.3 months [95% confidence interval (CI), 0–8.3 months]; 6-month PFS was 19% (95% CI, 7%–52%); and median overall survival time was 12 months (95% CI, 6 months to not reached). Repeated infusions of CMV-T cells paralleled significant increases in circulating CMV+ CD8+ T cells, but cytokine production showing effector activity was suppressed, especially from T cells obtained directly from glioblastomas.Conclusions:Adoptive infusion of CMV-specific T cells after lymphodepletion with dose-dense temozolomide was well tolerated. But apparently CMV seropositivity does not guarantee tumor susceptibility to CMV-specific T cells, suggesting heterogeneity in CMV antigen expression. Moreover, effector function of these T cells was attenuated, indicating a requirement for further T-cell modulation to prevent their dysfunction before conducting large-scale clinical studies.

Supplementary Tables

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Isocitrate dehydrogenase (IDH) 1 and 2 mutations represent essential components for the diagnosis of diffuse astrocytic tumors and oligodendroglioma. IDH wild-type glial tumors include a wide spectrum of tumors with differences in prognosis and recommended therapeutic approaches. Tumors characterized as molecular glioblastoma in the World Health Organization 2021 classification should be treated according to the glioblastoma therapeutic principles and included in glioblastoma trials. Improving on existing treatments options including targeted and immunotherapy approaches is imperative for most patients with IDH wild-type glial tumors, and enrollment in clinical trials is encouraged.

Chimeric antigen receptor (CAR) T-cell therapy is a recently approved breakthrough treatment that has become a new paradigm in treatment of recurrent or refractory B-cell lymphomas and pediatric or adult acute lymphoid leukemia. CAR T cells are a type of cellular immunotherapy that artificially enhances T cells to boost eradication of malignancy through activation of the native immune system. The CAR construct is a synthetically created functional cell receptor grafted onto previously harvested patient T cells, which bind to preselected tumor-associated antigens and thereby activate host immune signaling cascades to attack tumor cells. Advantages include a single treatment episode of 2-3 weeks and durable disease elimination, with remission rates of over 80%. Responses to therapy are more rapid than with conventional chemotherapy or immunotherapy, with intervening short-interval edema. CAR T-cell administration is associated with therapy-related toxic effects in a large percentage of patients, notably cytokine release syndrome, immune effect cell-associated neurotoxicity syndrome, and infections related to immunosuppression. Knowledge of the expected evolution of therapy response and potential adverse events in CAR T-cell therapy and correlation with the timeline of treatment are important to optimize patient care. Some toxic effects are radiologically evident, and familiarity with their imaging spectrum is key to avoiding misinterpretation. Other clinical toxic effects may be occult at imaging and are diagnosed on the basis of clinical assessment. Future directions for CAR T-cell therapy include new indications and expanded tumor targets, along with novel ways to capture T-cell activation with imaging.

Durable objective response rate (ORR) remains a meaningful endpoint in recurrent cancer; however, the target ORR for single-arm recurrent glioblastoma trials has not been based on historic information or tied to patient outcomes. The current study reviewed 68 treatment arms comprising 4793 patients in past trials in recurrent glioblastoma in order to judiciously define target ORRs for use in recurrent glioblastoma trials. ORR was estimated at 6.1% [95% CI 4.23; 8.76%] for cytotoxic chemothera + pies (ORR = 7.59% for lomustine, 7.57% for temozolomide, 0.64% for irinotecan, and 5.32% for other agents), 3.37% for biologic agents, 7.97% for (select) immunotherapies, and 26.8% for anti-angiogenic agents. ORRs were significantly correlated with median overall survival (mOS) across chemotherapy (R2 = 0.4078, P < .0001), biologics (R2 = 0.4003, P = .0003), and immunotherapy trials (R2 = 0.8994, P < .0001), but not anti-angiogenic agents (R2 = 0, P = .8937). Pooling data from chemotherapy, biologics, and immunotherapy trials, a meta-analysis indicated a strong correlation between ORR and mOS (R2 = 0.3900, P < .0001; mOS [weeks] = 1.4xORR + 24.8). Assuming an ineffective cytotoxic (control) therapy has ORR = 7.6%, the average ORR for lomustine and temozolomide trials, a sample size of ≥40 patients with target ORR>25% is needed to demonstrate statistical significance compared to control with a high level of confidence (P < .01) and adequate power (>80%). Given this historic data and potential biases in patient selection, we recommend that well-controlled, single-arm phase II studies in recurrent glioblastoma should have a target ORR >25% (which translates to a median OS of approximately 15 months) and a sample size of ≥40 patients, in order to convincingly demonstrate antitumor activity. Crucially, this response needs to have sufficient durability, which was not addressed in the current study.