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OBJECTIVE Resection of brain metastases (BMs) may be associated with increased risk of leptomeningeal disease (LMD). This study examined rates and predictors of LMD, including imaging subtypes, in patients who underwent resection of a BM followed by postoperative radiation. METHODS A retrospective, single-center study was conducted examining overall LMD, classic LMD (cLMD), and nodular LMD (nLMD) risk. Logistic regression, Cox proportional hazards, and random forest analyses were performed to identify risk factors associated with LMD. RESULTS Of the 217 patients in the cohort, 47 (21.7%) developed postoperative LMD, with 19 cases (8.8%) of cLMD and 28 cases (12.9%) of nLMD. Six-, 12-, and 24-month LMD-free survival rates were 92.3%, 85.6%, and 71.4%, respectively. Patients with cLMD had worse survival outcomes from the date of LMD diagnosis compared with nLMD (median 2.4 vs 6.9 months, p = 0.02, log-rank test). Cox proportional hazards analysis identified cerebellar/insular/occipital location (hazard ratio [HR] 3.25, 95% confidence interval [CI] 1.73–6.11, p = 0.0003), absence of extracranial disease (HR 2.49, 95% CI 1.27–4.88, p = 0.008), and ventricle contact (HR 2.82, 95% CI 1.5–5.3, p = 0.001) to be associated with postoperative LMD. A predictive model using random forest analysis with an area under the receiver operating characteristic curve of 0.87 in a test cohort identified tumor location, systemic disease status, and tumor volume as the most important factors associated with LMD. CONCLUSIONS Tumor location, absence of extracranial disease at the time of surgery, ventricle contact, and increased tumor volume were associated with LMD. Further work is needed to determine whether escalating therapies in patients at risk of LMD prevents disease dissemination.

Interfacility transfers represent a large proportion of neurosurgical admissions to tertiary care centers each year. In this study, the authors examined the impact of the COVID-19 pandemic on the number of transfers, timing of transfers, demographic profile of transfer patients, and clinical outcomes including rates of surgical intervention.A retrospective review of neurosurgical transfer patients at a single tertiary center was performed. Patients transferred from April to November 2020 (the "COVID Era") were compared with an institutional database of transfer patients collected before the COVID-19 pandemic (the "Pre-COVID Era"). During the COVID Era, both emergent and nonemergent neurosurgical services had resumed. A comparison of demographic and clinical factors between the 2 cohorts was performed.A total of 674 patients were included in the study (331 Pre-COVID and 343 COVID-Era patients). Overall, there was no change in the average monthly number of transfers (P = 0.66) or in the catchment area of referral hospitals. However, COVID-Era patients were more likely to be uninsured (1% vs. 4%), had longer transfer times (COVID vs. Pre-COVID Era: 18 vs. 9 hours; P0.001), required higher rates of surgical intervention (63% vs. 50%, P = 0.001), had higher rates of spine pathology (17% vs. 10%), and less frequently were admitted to the intensive care unit (34% vs. 52%, P0.001). Overall, COVID-Era patients did not experience delays to surgical intervention (3.1 days vs. 3.6 days, P = 0.2). When analyzing the subgroup of COVID-Era patients, COVID infection status did not impact the time of transfer or rates of operation, although COVID-infected patients experienced a longer time to surgery after admission (14 vs. 2.9 days, P0.001).The COVID-19 pandemic did not reduce the number of monthly transfers, operation rates, or catchment area for transfer patients. Transfer rates of uninsured patients increased during the COVID Era, potentially reflecting changes in access to community neurosurgery care. Shorter time to surgery seen in COVID-Era patients possibly reflects institutional policies that improved operating room efficiency to compensate for surgical backlogs. COVID status affeted time to surgery, reflecting the preoperative care that these patients require before intervention.

Table S6: Results of the in vitro drug screen.

Although tumor-propagating cells can be derived from glioblastomas (GBM) of the proneural and mesenchymal subtypes, a glioma stem-like cell (GSC) of the classic subtype has not been identified. It is unclear whether mesenchymal GSCs (mGSC) and/or proneural GSCs (pGSC) alone are sufficient to generate the heterogeneity observed in GBM. We performed single-cell/single-nucleus RNA sequencing of 28 gliomas, and single-cell ATAC sequencing for 8 cases. We found that GBM GSCs reside on a single axis of variation, ranging from proneural to mesenchymal. In silico lineage tracing using both transcriptomics and genetics supports mGSCs as the progenitors of pGSCs. Dual inhibition of pGSC-enriched and mGSC-enriched growth and survival pathways provides a more complete treatment than combinations targeting one GSC phenotype alone. This study sheds light on a long-standing debate regarding lineage relationships among GSCs and presents a paradigm by which personalized combination therapies can be derived from single-cell RNA signatures, to overcome intratumor heterogeneity.Significance:Tumor-propagating cells can be derived from mesenchymal and proneural glioblastomas. However, a stem cell of the classic subtype has yet to be demonstrated. We show that classic-subtype gliomas are comprised of proneural and mesenchymal cells. This study sheds light on a long-standing debate regarding lineage relationships between glioma cell types.See related commentary by Fine, p. 1650.This article is highlighted in the In This Issue feature, p. 1631

Supplementary Figure S2 shows that combination treatment with OS2966 and oHSV does not enhance oHSV replication in intracranial glioma-bearing mice in vivo.

Supplementary Table S1 shows sequences of primers used in this study.

Supplementary Figures

Integrin β1 receptor, expressed on the surface of tumor cells and macrophages in the tumor microenvironment (TME), has been implicated in both tumor progression and resistance to multiple modalities of therapy. OS2966 is the first clinical-ready humanized monoclonal antibody to block integrin β1 and was recently orphan designated by the FDA Office of Orphan Products Development. Here, we tested therapeutic potential of OS2966-mediated integrin β1 blockade to enhance the efficacy of oncolytic herpes simplex virus-1 (oHSV) through evaluation of virus replication, tumor cell killing efficiency, effect on the antiviral signaling pathway, co-culture assays of oHSV-infected cells with macrophages, and in vivo bioluminescence imaging on mammary fat pad triple-negative breast cancer xenograft and subcutaneous and intracranial glioma xenografts. OS2966 treatment decreased interferon signaling and proinflammatory cytokine induction in oHSV-treated tumor cells and inhibited migration of macrophages, resulting in enhanced oHSV replication and cytotoxicity. OS2966 treatment also significantly enhanced oHSV replication and oHSV-mediated antitumor efficacy in orthotopic xenograft models, including triple-negative breast cancer and glioblastoma. The results demonstrated the synergistic potential of the combinatory treatment approach with OS2966 to improve antitumor efficacy of conventional oHSV therapy.

Although overall survival (OS) has improved in patients with brain metastases (BMs), control of recurrent BMs remains a therapeutic challenge. Salvage surgery may achieve acceptable control rates in the setting of progression after previous stereotactic radiosurgery (SRS), yet it remains a question how additional adjuvant therapies may affect outcomes and how patient selection for salvage surgery may be optimized.Patients receiving salvage surgery for BM progression after previous SRS were retrospectively reviewed from a single center. Outcomes of interest included local tumor progression, leptomeningeal dissemination, and OS. Cox proportional hazard models and nominal logistic regression were applied to determine factors associated with outcomes of interest.A total of 43 patients with 50 BMs were included. After salvage surgery, local progression was observed for 17 BMs (34%), leptomeningeal dissemination was observed in 17 patients (39.5%), and censored median OS was 17.9 months. On multivariate analysis, use of brachytherapy was associated with improved local control (hazard ratio [HR], 0.15; 95% confidence interval [CI], 0.04-0.6; P = 0.008). For patients treated with SRS ≥4.5 months before salvage surgery, both brachytherapy (HR, 0.07; 95% CI, 0.01-0.39; P = 0.002) and postoperative adjuvant SRS (HR, 0.14; 95% CI, 0.02-1.00; P = 0.05) were associated with improved local control compared with no adjuvant radiation therapy. Presence of extracranial malignancy (HR, 6.70; 95% CI, 2.58-17.42; P0.0001) was associated with shorter survival. Graded prognostic assessment underestimated survival in 79.1% of patients, with a mean difference of 18.9 months between graded prognostic assessment-estimated and actual OS.In properly selected patients, salvage surgery may be an appropriate therapy for BM progression after previous SRS. Adjuvant brachytherapy and repeat SRS can offer significant benefit for local control with salvage resection.

Since its inception, greater than a century ago, neurosurgery has represented the fundamental trait-d'union between clinical management, scientific investigation, and therapeutic advancements in the field of brain tumors. During the years, oncological neurosurgery has evolved as a self-standing subspecialty, due to technical progress, equipment improvement, evolution of therapeutic paradigms, and the progressively crucial role that it plays in the execution of complex therapeutic strategies and modern clinical trials.

Supplementary Table S3 has primer sequences used to validate GBM subtype genes

PDF file - 3201K, Supplementary Figure S1. Real time RT PCR analysis of BRGs compared to paired pre-treatment GBMs; Supplementary Figure S2. FISH reveals no change in c-Met amplification after bevacizumab resistance as compared to before; Supplementary Figure S3. Changes in Pearson correlation coefficients to 3 previously described molecular GBM subtypes in BRGs and bevacizumab-na�ve GBMs after recurrence; Supplementary Figure S4. C-Met phosphorylation increases after bevacizumab resistance; Supplementary Figure S5. Increased expression of phosphorylated FAK, a downstream effector of c-Met, in bevacizumab-resistant glioblastomas (BRGs) compared to before treatment; Supplementary Figure S6. Increased expression of phosphorylated STAT3, a downstream effector of c-Met, in bevacizumab-resistant glioblastomas (BRGs) compared to before treatment; Supplementary Figure S7. Hypoxia after bevacizumab resistance versus bevacizumab-na�ve recurrence; Supplementary Figure S8. Endothelial cells do not contribute to increased c-Met expression after bevacizumab resistance; Supplementary Figure S9. Hypoxia increases HGF expression in U87-IgG and U87-BevR cells; Supplementary Figure S10. Xenografts established from BRGs analyzed by immunohistochemistry; Supplementary Figure S11. Immunofluorescent staining of xenografts established from bevacizumab-na�ve GBMs; Supplementary Figure S12. Hematoxylin and Eosin staining of xenografts established from bevacizumab-na�ve GBMs; Supplementary Figure S13. Comparison of xenografts from BRGs versus xenografts from bevacizumab-na�ve GBMs; Supplementary Figure S14. Murine MRI of intracranial BRG-derived xenografts reveals loss of the ability of VEGF blockade to reduce vascular permeability and fractional blood volume; Supplementary Figure S15. Reduced c-Met expression after transducing BRG-derived SF7796 xenograft cells with c-Met shRNA; Supplementary Figure S16. Effects of c-Met knockdown on brain slice invasion in U87-BevR cells; Supplementary Figure S17. Altered morphology in BRG cells after transduction with shRNA targeting c-Met; Supplementary Figure S18. C-Met knockdown alters brain slice invasion in BRG-derived SF7796 cells; Supplementary Figure S19. Hematoxylin and eosin stainings of intracranial SF7796/shControl and SF7796/shCmet1 xenografts; Supplementary Figure S20. Transduction of U87 with retrovirus derived from pBABE-Puro-Tpr-met leads to increased Tpr-met phosphorylation.

Correlation between duration of clinical benefit (DOCB) and overall survival (OS) for the standard RANO, iRANO, and mRANO criteria. (A) Correlation between radiographic DOCB using the standard RANO criteria applied to local measurements and OS for patients who died. (B) Correlation between radiographic DOCB using the iRANO criteria applied to local measurements and OS for patients who died. (C) Correlation between radiographic DOCB using the mRANO criteria applied to local measurements and OS for patients who died. (D) Correlation between radiographic DOCB using the standard RANO criteria applied to IRF-determined measurements and OS for patients who died. (E) Correlation between radiographic DOCB using the iRANO criteria applied to IRF-determined measurements and OS for patients who died. (F) Correlation between radiographic DOCB using the mRANO criteria applied to IRF-determined measurements and OS for patients who died. Solid circles = patients who progressed. Open circles = patients who were censored (not used in correlation analysis, only for visualization purposes).

Purpose: To identify mediators of glioblastoma antiangiogenic therapy resistance and target these mediators in xenografts.Experimental Design: We conducted microarray analysis comparing bevacizumab-resistant glioblastomas (BRG) with pretreatment tumors from the same patients. We established novel xenograft models of antiangiogenic therapy resistance to target candidate resistance mediator(s).Results: BRG microarray analysis revealed upregulation versus pretreatment of receptor tyrosine kinase c-Met, which underwent further investigation because of its prior biologic plausibility as a bevacizumab resistance mediator. BRGs exhibited increased hypoxia versus pretreatment in a manner correlating with their c-Met upregulation, increased c-Met phosphorylation, and increased phosphorylation of c-Met–activated focal adhesion kinase and STAT3. We developed 2 novel xenograft models of antiangiogenic therapy resistance. In the first model, serial bevacizumab treatment of an initially responsive xenograft generated a xenograft with acquired bevacizumab resistance, which exhibited upregulated c-Met expression versus pretreatment. In the second model, a BRG-derived xenograft maintained refractoriness to the MRI tumor vasculature alterations and survival-promoting effects of bevacizumab. Growth of this BRG-derived xenograft was inhibited by a c-Met inhibitor. Transducing these xenograft cells with c-Met short hairpin RNA inhibited their invasion and survival in hypoxia, disrupted their mesenchymal morphology, and converted them from bevacizumab-resistant to bevacizumab-responsive. Engineering bevacizumab-responsive cells to express constitutively active c-Met caused these cells to form bevacizumab-resistant xenografts.Conclusion: These findings support the role of c-Met in survival in hypoxia and invasion, features associated with antiangiogenic therapy resistance, and growth and therapeutic resistance of xenografts resistant to antiangiogenic therapy. Therapeutically targeting c-Met could prevent or overcome antiangiogenic therapy resistance. Clin Cancer Res; 19(7); 1773–83. ©2012 AACR.

PDF file - 1353K, Supplementary Table S1. Patient and tumor characteristics from the 22 bevacizumab-resistant versus 22 bevacizumab-na�ve glioblastomas from whom paired pre-treatment tissue was available. Supplementary Table S2. Altered transcripts in 15 bevacizumab-resistant glioblastomas compared to before treatment. Supplementary Table S3. Altered transcripts in 15 bevacizumab-resistant glioblastomas compared to before treatment. Supplementary Table S4. Altered transcripts in bevacizumab-naive recurrent glioblastomas compared to their paired glioblastomas at diagnosis. Supplemetary Table S5. FISH Results. Supplementary Table S6. Patient and tumor characteristics from 22 bevacizumab-na�ve recurrent glioblastomas (control group) from whom paired pre-treatment tissue was available.

Purpose: To identify mechanisms and mediators of resistance to antiangiogenic therapy in human glioblastoma.Experimental Design: We carried out microarray gene expression analysis and immunohistochemistry comparing 21 recurrent glioblastomas progressing during antiangiogenic treatment with VEGF neutralizing antibody bevacizumab to paired pretreatment tumors from the same patients.Results: Microarray analysis revealed that bevacizumab-resistant glioblastomas (BRG) had two clustering patterns defining subtypes that reflect radiographic growth patterns. Enhancing BRGs (EBRG) exhibited MRI enhancement, a long-established criterion for glioblastoma progression, and expressed mitogen-activated protein kinases, neural cell adhesion molecule-1 (NCAM-1), and aquaporin 4. Compared with their paired pretreatment tumors, EBRGs had unchanged vascularity and hypoxia, with increased proliferation. Nonenhancing BRGs (NBRG) exhibited minimal MRI enhancement but had FLAIR-bright expansion, a newer criterion for glioblastoma recurrence since the advent of antiangiogenic therapy, and expressed integrin α5, laminin, fibronectin1, and PDGFRβ. NBRGs had less vascularity, more hypoxia, and unchanged proliferation than their paired pretreatment tumors. Primary NBRG cells exhibited more stellate morphology with a 3-fold increased shape factor and were nearly 4-fold more invasive in Matrigel chambers than primary cells from EBRGs or bevacizumab-naive glioblastomas (P < 0.05).Conclusion: Using microarray analysis, we found two resistance patterns during antiangiogenic therapy with distinct molecular profiles and radiographic growth patterns. These studies provide valuable biologic insight into the resistance that has limited antiangiogenic therapy to date. Clin Cancer Res; 18(10); 2930–42. ©2012 AACR.

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XLSX file, 1707KB, This is a single excel file with 12 internal sections marked by tabs at the bottom of the file. These 12 tabs represent Supplementary Tables S1-S12, most of which is data generated from our microarray analysis.

Glioblastoma (GBM) responses to bevacizumab are invariably transient with acquired resistance. We profiled paired patient specimens and bevacizumab-resistant xenograft models pre- and post-resistance toward the primary goal of identifying regulators whose targeting could prolong the therapeutic window, and the secondary goal of identifying biomarkers of therapeutic window closure. Bevacizumab-resistant patient specimens and xenografts exhibited decreased vessel density and increased hypoxia versus pre-resistance, suggesting that resistance occurs despite effective therapeutic devascularization. Microarray analysis revealed upregulated mesenchymal genes in resistant tumors correlating with bevacizumab treatment duration and causing three changes enabling resistant tumor growth in hypoxia. First, perivascular invasiveness along remaining blood vessels, which co-opts vessels in a VEGF-independent and neoangiogenesis-independent manner, was upregulated in novel biomimetic 3D bioengineered platforms modeling the bevacizumab-resistant microenvironment. Second, tumor-initiating stem cells housed in the perivascular niche close to remaining blood vessels were enriched. Third, metabolic reprogramming assessed through real-time bioenergetic measurement and metabolomics upregulated glycolysis and suppressed oxidative phosphorylation. Single-cell sequencing of bevacizumab-resistant patient GBMs confirmed upregulated mesenchymal genes, particularly glycoprotein YKL-40 and transcription factor ZEB1, in later clones, implicating these changes as treatment-induced. Serum YKL-40 was elevated in bevacizumab-resistant versus bevacizumab-naïve patients. CRISPR and pharmacologic targeting of ZEB1 with honokiol reversed the mesenchymal gene expression and associated stem cell, invasion, and metabolic changes defining resistance. Honokiol caused greater cell death in bevacizumab-resistant than bevacizumab-responsive tumor cells, with surviving cells losing mesenchymal morphology. Employing YKL-40 as a resistance biomarker and ZEB1 as a target to prevent resistance could fulfill the promise of antiangiogenic therapy.Significance:Bevacizumab resistance in GBM is associated with mesenchymal/glycolytic shifts involving YKL-40 and ZEB1. Targeting ZEB1 reduces bevacizumab-resistant GBM phenotypes.

PDF file, 2979KB, This is a PDF file containing Supplementary Figures S1-S17 on 17 pages, with the figure legend on the same page as each Supplementary Figure. These Supplementary Figures are cited in the text of the manuscript.

Supplementary Figures S1-S23 show some extra data including immunohistochemical stains and bar graphs that could not fit in the main text.

Supplemental Figure Captions

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Medicenna Imaging Charter for Independent Radiologic Review of MDNA55-05.

Supplementary Methods describes details about methods including animal work, microarray analysis, neurosphere assays, and hyaluronic acid assays that could not fit in the main methods.

Purpose:The current study compared the standard response assessment in neuro-oncology (RANO), immunotherapy RANO (iRANO), and modified RANO (mRANO) criteria as well as quantified the association between progression-free (PFS) and overall survival (OS) in an immunotherapy trial in recurrent glioblastoma (rGBM).Patients and Methods:A total of 47 patients with rGBM were enrolled in a prospective phase II convection-enhanced delivery of an IL4R-targeted immunotoxin (MDNA55-05, NCT02858895). Bidirectional tumor measurements were created by local sites and centrally by an independent radiologic faculty, then standard RANO, iRANO, and mRANO criteria were applied.Results:A total of 41 of 47 patients (mean age 56 ± 11.7) were evaluable for response. PFS was significantly shorter using standard RANO compared with iRANO (log-rank, P < 0.0001; HR = 0.3) and mRANO (P < 0.0001; HR = 0.3). In patients who died and had confirmed progression on standard RANO, no correlation was observed between PFS and OS (local, P = 0.47; central, P = 0.34). Using iRANO, a weak association was observed between confirmed PFS and OS via local site measurements (P = 0.017), but not central measurements (P = 0.18). A total of 24 of 41 patients (59%) were censored using iRANO and because they lacked confirmation of progression 3 months after initial progression. A strong correlation was observed between mRANO PFS and OS for both local (R2 = 0.66, P < 0.0001) and centrally determined reads (R2 = 0.57, P = 0.0007).Conclusions:No correlation between radiographic PFS and OS was observed for standard RANO or iRANO, but a correlation was observed between PFS and OS using the mRANO criteria. Also, the iRANO criteria was difficult to implement due to need to confirm progression 3 months after initial progression, censoring more than half the patients.

MOV file - 1285K, Supplementary Movie 3. Epithelial Migration Of Bevacizumab-Na�ve Glioma Cells. Related to Figure 2. Timelapse phase-contrast imaging of N1 bevacizumab-na�ve (left) and BRG3 bevacizumab-resistant (right) cells on fibronectin. N1 cells demonstrated classic epithelial migration with broad lamellipodial extensions and slow rearward translocation with an overall polygonal morphology. In contrast, BRG3 cells display rapid, saltatory migration with spindle-like morphology. Movie images captured with a 40x objective representing approximately 14 hours of imaging, 35 minutes between each frame.

MOV file - 868K, Supplementary Movie 4. Inhibitory Anti-Beta1 Integrin mAb OS2966 Disrupts BRG3 Adhesion, Spreading, And Migration. Related to Figure 2. Timelapse phase-contrast imaging of BRG3 bevacizumab-resistant cells on fibronectin demonstrates mesenchymal migration of control (10 μg/ml IgG, left) cells into the wound in a scratch wound healing assay. In contrast, BRG3 cells in the treatment condition (10μg/ml OS2966, right) rapidly lose adhesive contacts to the substrate (within 20-30 mins) and are unable to migrate into the wound. Movie images captured with a 40x objective representing approximately 10 hours of imaging, 35 minutes between each frame.

PDF file - 915K, Supplementary Figure 1. Bevacizumab Treatment Promotes Invasiveness Of GBM. Supplementary Figure 2. Increased Expression Of Beta1 In The U87-BEV Cell Line. Supplementary Figure 3. Beta1 Integrin Expression In GBM Associated With Cellular Insults And Mesenchymal Phenotype. Supplementary Figure 4. Establishment And Characterization Of Stable Beta1 Knockdown Clones In The BRG3 Line. Supplementary Figure 5. Beta1 integrin knockdown in resistant GBM cells inhibits mesenchymal phenotype. Supplementary Figure 6. Inhibitory Anti-Beta1 Mab OS2966 Attenuates Adhesion And Proliferation Of BRG Cells. Supplementary Figure 7. A Role For Beta1 In Spheroidal Growth Of U87MG Glioma Cell Line. Supplementary Figure 8. Beta1 Inhibition In Xenograft Models.

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