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Background: The tumor microenvironment is being increasingly recognized as an important determinant of tumor progression as well as of therapeutic response. We investigated oncolytic virus (OV) therapy-induced changes in tumor blood vessels and the impact of modulating tumor vasculature on the efficacy of oncolytic virus therapy.Methods: Rat glioma cells (D74/HveC) were implanted intracranially in immune-competent rats. Seven days later, the rats (groups of 3-7 rats) were treated with oncolytic virus (hrR3), and, 3 days later, brains were harvested for evaluation. Some rats were treated with angiostatic cRGD peptide 4 days before oncolytic virus treatment. Some rats were treated with cyclophosphamide (CPA), an immunosuppressant, 2 days before oncolytic virus treatment. Changes in tumor vascular perfusion were evaluated by magnetic resonance imaging of live rats and by fluorescence microscopy of tumor sections from rats perfused with Texas red-conjugated lectin immediately before euthanasia. Leukocyte infiltration in tumors was evaluated by anti-CD45 immunohistochemistry, and the presence of oncolytic virus in tumors was evaluated by viral titration. Changes in cytokine gene expression in tumors were measured by quantitative real-time polymerase chain reaction-based microarrays. Survival was analyzed by the Kaplan-Meier method. All statistical tests were two-sided.Results: Oncolytic virus treatment of experimental rat gliomas increased tumor vascular permeability, host leukocyte infiltration into tumors, and intratumoral expression of inflammatory cytokine genes, including interferon gamma (IFN-gamma). The increase in vascular permeability was suppressed in rats pretreated with cyclophosphamide. Compared with rats treated with hrR3 alone, rats pretreated with a single dose of cRGD peptide before hrR3 treatment had reduced tumor vascular permeability, leukocyte infiltration, and IFN-gamma protein levels (mean IFN-gamma level for hrR3 versus hrR3 + cRGD = 203 versus 65.6 microg/mg, difference = 137 microg/mg, 95% confidence interval = 72.7 to 202.9 microg/mg, P = .006); increased viral titers in tumor tissue; and longer median survival (21 days versus 17 days, P<.001).Conclusions: A single dose of angiostatic cRGD peptide treatment before oncolytic virus treatment enhanced the antitumor efficacy of oncolytic virus. [ABSTRACT FROM AUTHOR]

Background: Intravascular lymphoma (IVL) is a rare non-Hodgkin's lymphoma with relative predilection for the central nervous system. In the absence of extraneural manifestations, the disease is not recognised until autopsy in the majority of cases underlining the need for new clinical markers.Methods: This is a retrospective series of five patients with IVL seen at a single institution over three years. An advanced magnetic resonance imaging (MRI) protocol was performed at various time points prior to diagnosis and during treatment.Results: MRI revealed multiple lesions scattered throughout the cerebral hemispheres; the brainstem, cerebellum, and spinal cord were less frequently involved. On initial presentation, hyperintense lesions were seen on diffusion weighted images suggestive of ischaemia in three of four patients in whom the images were obtained at that time point. In four patients lesions were also identifiable as hyperintense areas on fluid attenuated inversion recovery (FLAIR) sequences. Initial contrast enhancement was encountered in three cases. Diffusion weighted imaging lesions either vanished or followed the typical pattern of an ischaemic small vessel stroke with evolution of abnormal FLAIR signal followed by enhancement with gadolinium in the subacute stage and tissue loss in the chronic stage. Diffusion weighted imaging and FLAIR abnormalities proved to be partially reversible, correlating with the response to chemotherapy.Conclusion: We provide the first detailed description of the dynamic pattern of diffusion weighted MRI in IVL. These patterns in combination with systemic findings may facilitate early diagnosis and serve as a new tool to monitor treatment response. [ABSTRACT FROM AUTHOR]

The emerging field of liquid biopsy stands at the forefront of novel diagnostic strategies for cancer and other diseases. Liquid biopsy allows minimally invasive molecular characterization of cancers for diagnosis, patient stratification to therapy, and longitudinal monitoring. Liquid biopsy strategies include detection and monitoring of circulating tumor cells, cell-free DNA, and extracellular vesicles. In this review, we address the current understanding and the role of existing liquid-biopsy-based modalities in cancer diagnostics and monitoring. We specifically focus on the technical and clinical challenges associated with liquid biopsy and biomarker development being addressed by the Liquid Biopsy Consortium, established through the National Cancer Institute. The Liquid Biopsy Consortium has developed new methods/assays and validated existing methods/technologies to capture and characterize tumor-derived circulating cargo, as well as addressed existing challenges and provided recommendations for advancing biomarker assays., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Malignant gliomas are rapidly progressive brain tumors with high mortality. Fluorescence guided surgery (FGS) with 5-aminolevulinic acid (5-ALA) provides fluorescent delineation of malignant tissue, which helps achieve maximum safe resection. 5-ALA-based fluorescence is due to preferential accumulation of the fluorophore protoporphyrin-IX (PpIX) in malignant glioma tissue. Additionally, gliomas cells release extracellular vesicles (EVs) which carry biomarkers of disease. Herein, we performed animal and human studies to investigate whether 5-ALA dosed glioma cells, in vitro and in vivo, release PpIX positive EVs in circulation which can be captured and analyzed., Methods: We used imaging flow cytometry (IFC) to characterize PpIX-positive EVs released from 5-ALA-dosed glioma cells, glioma-bearing xenograft models, as well as patients with malignant glioma undergoing FGS., Findings: We first show that glioma cells dosed with 5-ALA release 247-fold higher PpIX positive EVs compared to mock dosed glioma cells. Second, we demonstrate that the plasma of glioma-bearing mice (n = 2) dosed with 5-ALA contain significantly higher levels of circulating PpIX-positive EVs than their pre-dosing background (p = 0.004). Lastly, we also show that the plasma of patients with avidly fluorescent tumors (n = 4) undergoing FGS contain circulating PpIX-positive EVs at levels significantly higher than their pre-dosing background (p = 0.00009) and this rise in signal correlates with enhancing tumor volumes (r 2   = 0.888)., Interpretation: Our findings highlight the potential of plasma-derived PpIX-positive EV-based diagnostics for malignant gliomas, offering a novel liquid biopsy platform for confirming and monitoring tumor status., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The 2016 World Health Organization Classification of Central Nervous System Tumors categorizes gliomatosis cerebri growth pattern (GC) as a subgroup of diffuse infiltrating gliomas, defined by extent of brain involvement on magnetic resonance imaging (MRI). Clinical and radiographic features in GC patients are highly heterogeneous; however, prognosis has historically been considered poor., Subjects, Materials, and Methods: We performed a retrospective search for patients at our institution meeting radiographic criteria of primary, type I GC (defined as diffuse tumor infiltration without associated tumor mass and contrast enhancement on MRI) and analyzed their clinical, imaging, and histopathologic features., Results: A total of 34 patients met radiographic criteria of primary, type I GC, and 33 had a confirmed histologic diagnosis of an infiltrating glial neoplasm. Age >47 years at diagnosis was associated with worse overall survival (OS) compared with age ≤47 years (hazard ratio [HR] 1.04, 95% confidence interval [CI] 1.01-1.07, p = .003). Patients with grade 2 tumors demonstrated a trend for improved OS compared with those with grade 3 tumors (HR 2.65, 95% CI 0.99-7.08, p = .051). Except for brainstem involvement, extent or location of radiographic involvement did not detectably affect clinical outcome. IDH mutation status identified a subgroup of GC patients with particularly long survival up to 25 years and was associated with longer time to progression (HR 4.81, 95% CI 0.99-23.47, p = .052)., Conclusion: Patients with primary, type I GC do not uniformly carry a poor prognosis, even in the presence of widespread radiographic involvement. Consistent with other reports, IDH mutation status may identify patients with improved clinical outcome. Molecular characterization, rather than MRI features, may be most valuable for prognostication and management of GC patients., Implications for Practice: Patients with gliomatosis cerebri growth pattern (GC) constitute a challenge to clinicians, given their wide range of clinical, histologic, and radiographic presentation, heterogeneous outcome patterns, and the lack of consensus on a standardized treatment approach. This study highlights that radiographic extent of disease-albeit category-defining-does not detectably influence survival and that IDH mutations may impact clinical outcome. Practicing oncologists should be aware that select GC patients may demonstrate exceptionally favorable survival times and prognosticate patients based on molecular markers, rather than imaging features alone., Competing Interests: Disclosures of potential conflicts of interest may be found at the end of this article., (© AlphaMed Press 2018.)

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

This article aims to document the growth in extracellular vesicle (EV) research. Here, we report the growth in EV-related studies, patents, and grants as well as emerging companies with major intent on exosomes. Four different databases were utilized for electronic searches of published literature: two general databases - Scopus/Elsevier and Web of Science (WoS), as well as two specialized US government databases - the USA Patent and Trademark Office and National Institutes of Health (NIH) of the Department of Health and Human Services. The applied combination of key words was carefully chosen to cover the most commonly used terms in titles of publications, patents and grants dealing with conceptual areas of EVs. Within the time frame from 1 January 2000 to 31 December 2016, limited to articles published in English, we identified output using search strategies based upon Scopus/Elsevier and WoS, patent filings and NIH Federal Reports of funded grants. Consistently, USA and UK universities are the most frequent among the top 15 affiliations/organizations of the authors of the identified records. There is clear evidence of upward streaming of EV-related publications. By documenting the growth of the EV field, we hope to encourage a roster of independent authorities skilled to provide peer review of manuscripts, evaluation of grant applications, support of foundation initiatives and corporate long-term planning. It is important to encourage EV research to further identify biomarkers in diseases and allow for the development of adequate diagnostic tools that could distinguish disease subpopulations and enable personalized treatment of patients., Competing Interests: Dr. Hochberg is President of CASCENT Inc. and TAVEC Inc (both exosome therapeutic companies without conflict).

Extracellular vesicles (EVs) carry RNA, DNA, proteins, and lipids. Specifically, tumor-derived EVs have the potential to be utilized as disease-specific biomarkers. However, a lack of methods to isolate tumor-specific EVs has limited their use in clinical settings. Here we report a sensitive analytical microfluidic platform ( EV HB-Chip) that enables tumor-specific EV-RNA isolation within 3 h. Using the EV HB-Chip, we achieve 94% tumor-EV specificity, a limit of detection of 100 EVs per μL, and a 10-fold increase in tumor RNA enrichment in comparison to other methods. Our approach allows for the subsequent release of captured tumor EVs, enabling downstream characterization and functional studies. Processing serum and plasma samples from glioblastoma multiforme (GBM) patients, we can detect the mutant EGFRvIII mRNA. Moreover, using next-generation RNA sequencing, we identify genes specific to GBM as well as transcripts that are hallmarks for the four genetic subtypes of the disease.

We examined the extracellular vesicle (EV) and RNA composition of pooled normal cerebrospinal fluid (CSF) samples and CSF from five major neurological disorders: Alzheimer's disease (AD), Parkinson's disease (PD), low-grade glioma (LGG), glioblastoma multiforme (GBM), and subarachnoid haemorrhage (SAH), representing neurodegenerative disease, cancer, and severe acute brain injury. We evaluated: (I) size and quantity of EVs by nanoparticle tracking analysis (NTA) and vesicle flow cytometry (VFC), (II) RNA yield and purity using four RNA isolation kits, (III) replication of RNA yields within and between laboratories, and (IV) composition of total and EV RNAs by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RNA sequencing (RNASeq). The CSF contained ~106 EVs/μL by NTA and VFC. Brain tumour and SAH CSF contained more EVs and RNA relative to normal, AD, and PD. RT-qPCR and RNASeq identified disease-related populations of microRNAs and messenger RNAs (mRNAs) relative to normal CSF, in both total and EV fractions. This work presents relevant measures selected to inform the design of subsequent replicative CSF studies. The range of neurological diseases highlights variations in total and EV RNA content due to disease or collection site, revealing critical considerations guiding the selection of appropriate approaches and controls for CSF studies.

Background: Vestibular schwannoma (VS) is a tumor of the vestibular nerve that transmits balance information from the inner ear to the brain. Sensorineural hearing loss occurs in 95% of patients with these tumors, but the cause of this loss is not well understood. We posit a role of VS-secreted extracellular vesicles (EVs) as a major contributing factor in cochlear nerve damage., Methods: Using differential centrifugation, we isolated EVs from VS cell line HEI-193 and primary cultured human VS cells from patients with good hearing or poor hearing. The EVs were characterized using a Nanosight device and transmission electron microscopy and by extracting their RNA content. The EVs' effects on cultured murine spiral ganglion cells and organotypic cochlear cultures were studied using a transwell dual-culture system and by direct labeling of EVs with PKH-67 dye. EV-induced changes in cochlear cells were quantified using confocal immunohistochemistry. Transfection of VS cells with a green fluorescent protein-containing plasmid was confirmed with reverse transcription PCR., Results: Human VS cells, from patients with poor hearing, produced EVs that could damage both cultured murine cochlear sensory cells and neurons. In contrast, EVs derived from VS cells from patients with good hearing did not damage the cultured cochlear cells., Conclusions: This is the first report on EVs derived from VSs and on the capacity of EVs from VSs from patients with hearing loss to selectively damage cochlear cells, thereby identifying a potential novel mechanism of VS-associated sensorineural hearing loss., (© The Author(s) 2016. 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.)

Patients with high-grade gliomas and glioblastomas (GBMs) have poor survival despite optimal surgical and drug therapy. Minimally invasive diagnostic biomarkers would enable early diagnosis and tumor-specific treatments for 'personalized targeted' therapy, and would create the basis for response tracking in patients with GBM. Extracellular vesicles (EVs) isolated from cerebrospinal fluid and blood contain glioma-specific molecules, including tumor-derived EV RNAs that are detectable in small copy numbers in these biofluids. EV RNA mutations or expression changes are also detectable, the analysis of which gives rise to 'liquid biopsy' tumor profiling.

Extracellular vesicles (EVs) have emerged as a promising biomarker platform for glioblastoma patients. However, the optimal method for quantitative assessment of EVs in clinical bio-fluid remains a point of contention. Multiple high-resolution platforms for quantitative EV analysis have emerged, including methods grounded in diffraction measurement of Brownian motion (NTA), tunable resistive pulse sensing (TRPS), vesicle flow cytometry (VFC), and transmission electron microscopy (TEM). Here we compared quantitative EV assessment using cerebrospinal fluids derived from glioblastoma patients using these methods. For EVs <150 nm in diameter, NTA detected more EVs than TRPS in three of the four samples tested. VFC particle counts are consistently 2-3 fold lower than NTA and TRPS, suggesting contribution of protein aggregates or other non-lipid particles to particle count by these platforms. While TEM yield meaningful data in terms of the morphology, its particle count are consistently two orders of magnitude lower relative to counts generated by NTA and TRPS. For larger particles (>150 nm in diameter), NTA consistently detected lower number of EVs relative to TRPS. These results unveil the strength and pitfalls of each quantitative method alone for assessing EVs derived from clinical cerebrospinal fluids and suggest that thoughtful synthesis of multi-platform quantitation will be required to guide meaningful clinical investigations.

Treatments of brain tumor associated edema with systemically delivered dexamethasone, the standard of care, and cediranib, a novel anti-edema agent, are associated with systemic toxicities in brain tumor patients. A tunable, reservoir-based drug delivery device was developed to investigate the effects of delivering dexamethasone and cediranib locally in the brain in an intracranial 9L gliosarcoma rat model. Reproducible, sustained releases of both dexamethasone and solid dispersion of cediranib in polyvinylpyrrolidone (AZD/PVP) from these devices were achieved. The water-soluble AZD/PVP, which exhibited similar bioactivity as cediranib, was developed to enhance the release of cediranib from the device. Local and systemic administration of both dexamethasone and cediranib was equally efficacious in alleviating edema but had no effect on tumor growth. Edema reduction led to modest but significant improvement in survival. Local delivery of dexamethasone prevented dexamethasone-induced weight loss, an adverse effect seen in animals treated with systemic dexamethasone. Local deliveries of dexamethasone and cediranib via these devices used only 2.36% and 0.21% of the systemic doses respectively, but achieved similar efficacy as systemic drug deliveries without the side effects associated with systemic administration. Other therapeutic agents targeting brain tumor can be delivered locally in the brain to provide similar improved treatment outcomes., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Real-time monitoring of drug efficacy in glioblastoma multiforme (GBM) is a major clinical problem as serial re-biopsy of primary tumours is often not a clinical option. MGMT (O(6)-methylguanine DNA methyltransferase) and APNG (alkylpurine-DNA-N-glycosylase) are key enzymes capable of repairing temozolomide-induced DNA damages and their levels in tissue are inversely related to treatment efficacy. Yet, serial clinical analysis remains difficult, and, when done, primarily relies on promoter methylation studies of tumour biopsy material at the time of initial surgery. Here we present a microfluidic chip to analyse mRNA levels of MGMT and APNG in enriched tumour exosomes obtained from blood. We show that exosomal mRNA levels of these enzymes correlate well with levels found in parental cells and that levels change considerably during treatment of seven patients. We propose that if validated on a larger cohort of patients, the method may be used to predict drug response in GBM patients.

Although the association between cancer and venous thromboembolism (VTE) has long been known, the mechanisms are poorly understood. Circulating tissue factor-bearing extracellular vesicles have been proposed as a possible explanation for the increased risk of VTE observed in some types of cancer. The International Society for Extracellular Vesicles (ISEV) and International Society on Thrombosis and Haemostasis (ISTH) held a joint Educational Day in April 2014 to discuss the latest developments in this field. This review discusses the themes of that event and the ISEV 2014 meeting that followed.

Targeting specific molecular alterations in glioblastoma (GBM) might more effectively kill tumor cells and increase survival. Vandetanib inhibits epidermal growth factor receptor and vascular endothelial growth factor receptor 2. Sirolimus inhibits mammalian target of rapamycin (mTOR), a member the phosphoinositide 3-Kinase signaling pathway. We sought to determine the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of vandetanib combined with sirolimus. Twenty-two patients (14 men; 8 women) with recurrent GBM enrolled. Median age and KPS were 52.5 years and 90 %, respectively. Patients were naive to anti-VEGF and anti-EGF therapy and mTOR inhibitors, and not on CYP3A4-inducing drugs. Vandetanib and sirolimus were orally administered on a continuous daily dosing schedule in escalating dose cohorts. Ten patients enrolled in the dose escalation phase. Twelve more enrolled at the MTD to explore progression-free survival at 6 months (PFS6) in a single arm, single stage phase II-type design. In total, 19 patients received at least one dose at the MTD, and 15 completed at least 1 cycle at MTD. MTD was 200 mg vandetanib plus 2 mg sirolimus. The DLT was elevated AST/SGOT. The most common toxicities were lymphopenia, fatigue, rash, and hypophosphatemia. For 19 patients who received at least one dose at the MTD, including seven from the phase I group, two had a partial response [10.5 %; 95 % CI (1, 33 %)] and PFS6 was 15.8 % [95 % CI (3.9, 34.9 %)]. Vandetanib and sirolimus can be safely co-administered on a continuous, daily dosing schedule.

Glioma is the most common brain tumor. For the more aggressive form, glioblastoma, standard treatment includes surgical resection, irradiation with adjuvant temozolomide and, on recurrence, experimental chemotherapy. However, the survival of patients remains poor. There is a critical need for minimally invasive biomarkers for diagnosis and as measures of response to therapeutic interventions. Glioma shed extracellular vesicles (EVs), which invade the surrounding tissue and circulate within both the cerebrospinal fluid and the systemic circulation. These tumor-derived EVs and their content serve as an attractive source of biomarkers. In this review, we discuss the current state of the art of biomarkers for glioma with emphasis on their EV derivation.

The treatment of metastatic brain lesions remains a central challenge in oncology. Because most chemotherapeutic agents do not effectively cross the blood-brain barrier, it is widely accepted that radiation remains the primary modality of treatment. The mode by which radiation should be delivered has, however, become a source of intense controversy in recent years. The controversy involves whether patients with a limited number of brain metastases should undergo whole brain radiation therapy (WBRT) or stereotactic radiosurgery (SRS) delivered only to the radiographically visible tumours. Survival is comparable for patients treated with either modality. Instead, the controversy involves the neurocognitive function (NCF) of radiating cerebrum that appeared radiographically normal relative to effects of the growth from micro-metastatic foci. A fundamental question in this debate involves quantifying the effect of WBRT in patients with cerebral metastasis. To disentangle the effects of WBRT on neurocognition from the effects inherent to the underlying disease, we analysed the results from randomised controlled studies of prophylactic cranial irradiation in oncology patients as well as studies where patients with limited cerebral metastasis were randomised to SRS versus SRS+WBRT. In aggregate, these results suggest deleterious effects of WBRT in select neurocognitive domains. However, there are insufficient data to resolve the controversy of upfront WBRT versus SRS in the management of patients with limited cerebral metastases.

Antiangiogenic therapy has shown clear activity and improved survival benefit for certain tumor types. However, an incomplete understanding of the mechanisms of action of antiangiogenic agents has hindered optimization and broader application of this new therapeutic modality. In particular, the impact of antiangiogenic therapy on tumor blood flow and oxygenation status (i.e., the role of vessel pruning versus normalization) remains controversial. This controversy has become critical as multiple phase III trials of anti-VEGF agents combined with cytotoxics failed to show overall survival benefit in newly diagnosed glioblastoma (nGBM) patients and several other cancers. Here, we shed light on mechanisms of nGBM response to cediranib, a pan-VEGF receptor tyrosine kinase inhibitor, using MRI techniques and blood biomarkers in prospective phase II clinical trials of cediranib with chemoradiation vs. chemoradiation alone in nGBM patients. We demonstrate that improved perfusion occurs only in a subset of patients in cediranib-containing regimens, and is associated with improved overall survival in these nGBM patients. Moreover, an increase in perfusion is associated with improved tumor oxygenation status as well as with pharmacodynamic biomarkers, such as changes in plasma placenta growth factor and sVEGFR2. Finally, treatment resistance was associated with elevated plasma IL-8 and sVEGFR1 posttherapy. In conclusion, tumor perfusion changes after antiangiogenic therapy may distinguish responders vs. nonresponders early in the course of this expensive and potentially toxic form of therapy, and these results may provide new insight into the selection of glioblastoma patients most likely to benefit from anti-VEGF treatments.

Glioblastoma cells secrete extra-cellular vesicles (EVs) containing microRNAs (miRNAs). Analysis of these EV miRNAs in the bio-fluids of afflicted patients represents a potential platform for biomarker development. However, the analytic algorithm for quantitative assessment of EV miRNA remains under-developed. Here, we demonstrate that the reference transcripts commonly used for quantitative PCR (including GAPDH, 18S rRNA, and hsa-miR-103) were unreliable for assessing EV miRNA. In this context, we quantitated EV miRNA in absolute terms and normalized this value to the input EV number. Using this method, we examined the abundance of miR-21, a highly over-expressed miRNA in glioblastomas, in EVs. In a panel of glioblastoma cell lines, the cellular levels of miR-21 correlated with EV miR-21 levels (p<0.05), suggesting that glioblastoma cells actively secrete EVs containing miR-21. Consistent with this hypothesis, the CSF EV miR-21 levels of glioblastoma patients (n=13) were, on average, ten-fold higher than levels in EVs isolated from the CSF of non-oncologic patients (n=13, p<0.001). Notably, none of the glioblastoma CSF harbored EV miR-21 level below 0.25 copies per EV in this cohort. Using this cut-off value, we were able to prospectively distinguish CSF derived from glioblastoma and non-oncologic patients in an independent cohort of twenty-nine patients (Sensitivity=87%; Specificity=93%; AUC=0.91, p<0.01). Our results suggest that CSF EV miRNA analysis of miR-21 may serve as a platform for glioblastoma biomarker development.

Development of biofluid-based molecular diagnostic tests for cancer is an important step towards tumor characterization and real-time monitoring in a minimally invasive fashion. Extracellular vesicles (EVs) are released from tumor cells into body fluids and can provide a powerful platform for tumor biomarkers because they carry tumor proteins and nucleic acids. Detecting rare point mutations in the background of wild-type sequences in biofluids such as blood and cerebrospinal fluid (CSF) remains a major challenge. Techniques such as BEAMing (beads, emulsion, amplification, magnetics) PCR and droplet digital PCR (ddPCR) are substantially more sensitive than many other assays for mutant sequence detection. Here, we describe a novel approach that combines biofluid EV RNA and BEAMing RT-PCR (EV-BEAMing), as well droplet digital PCR to interrogate mutations from glioma tumors. EVs from CSF of patients with glioma were shown to contain mutant IDH1 transcripts, and we were able to reliably detect and quantify mutant and wild-type IDH1 RNA transcripts in CSF of patients with gliomas. EV-BEAMing and EV-ddPCR represent a valuable new strategy for cancer diagnostics, which can be applied to a variety of biofluids and neoplasms.Molecular Therapy-Nucleic Acids (2013) 2, e109; doi:10.1038/mtna.2013.28; published online 23 July 2013.

The discovery that tumor-derived proteins and nucleic acids can be detected in nano-sized vesicles in the plasma and cerebrospinal fluid of patients afflicted with brain tumors has expanded opportunities for biomarker and therapeutic discovery. Through delivery of their contents to surrounding cells, exosomes, microvesicles, and other nano-sized extracellular vesicles secreted by tumors modulate their environment to promote tumor growth and survival. In this review, we discuss the biological processes mediated by these extracellular vesicles and their applications in terms of brain tumor diagnosis, monitoring, and therapy. We review the normal physiology of these extracellular vesicles, their pertinence to tumor biology, and directions for research in this field.

Object: The authors' goal was to review the current understanding of the underlying molecular and genetic mechanisms involved in low-grade glioma development and how these mechanisms can be targets for detection and treatment of the disease and its recurrence., Methods: On October 4, 2012, the authors convened a meeting of researchers and clinicians across a variety of pertinent medical specialties to review the state of current knowledge on molecular genetic mechanisms of low-grade gliomas and to identify areas for further research and drug development., Results: The meeting consisted of 3 scientific sessions ranging from neuropathology of IDH1 mutations; CIC, ATRX, and FUBP1 mutations in oligodendrogliomas and astrocytomas; and IDH1 mutations as therapeutic targets. Sessions consisted of a total of 10 talks by international leaders in low-grade glioma research, mutant IDH1 biology and its application in glioma research, and treatment., Conclusions: The recent discovery of recurrent gene mutations in low-grade glioma has increased the understanding of the molecular mechanisms involved in a host of biological activities related to low-grade gliomas. Understanding the role these genetic alterations play in brain cancer initiation and progression will help lead to the development of novel treatment modalities than can be personalized to each patient, thereby helping transform this now often-fatal malignancy into a chronic or even curable disease.

Glioblastomas shed large quantities of small, membrane-bound microvesicles into the circulation. Although these hold promise as potential biomarkers of therapeutic response, their identification and quantification remain challenging. Here, we describe a highly sensitive and rapid analytical technique for profiling circulating microvesicles directly from blood samples of patients with glioblastoma. Microvesicles, introduced onto a dedicated microfluidic chip, are labeled with target-specific magnetic nanoparticles and detected by a miniaturized nuclear magnetic resonance system. Compared with current methods, this integrated system has a much higher detection sensitivity and can differentiate glioblastoma multiforme (GBM) microvesicles from nontumor host cell-derived microvesicles. We also show that circulating GBM microvesicles can be used to analyze primary tumor mutations and as a predictive metric of treatment-induced changes. This platform could provide both an early indicator of drug efficacy and a potential molecular stratifier for human clinical trials.

Microvesicles are nano-sized lipid vesicles released by all cells in vivo and in vitro. They are released physiologically under normal conditions but their rate of release is higher under pathological conditions such as tumors. Once released they end up in the systemic circulation and have been found and characterized in all biofluids such as plasma, serum, cerebrospinal fluid, breast milk, ascites, and urine. Microvesicles represent the status of the donor cell they are released from and they are currently under intense investigation as a potential source for disease biomarkers. Currently, the "gold standard" for isolating microvesicles is ultracentrifugation, although alternative techniques such as affinity purification have been explored. Viscosity is the resistance of a fluid to a deforming force by either shear or tensile stress. The different chemical and molecular compositions of biofluids have an effect on its viscosity and this could affect movements of the particles inside the fluid. In this manuscript we addressed the issue of whether viscosity has an effect on sedimentation efficiency of microvesicles using ultracentrifugation. We used different biofluids and spiked them with polystyrene beads and assessed their recovery using the Nanoparticle Tracking Analysis. We demonstrate that MVs recovery inversely correlates with viscosity and as a result, sample dilutions should be considered prior to ultracentrifugation when processing any biofluids.

Background: RNA from exosomes and other microvesicles contain transcripts of tumour origin. In this study we sought to identify biomarkers of glioblastoma multiforme in microvesicle RNA from serum of affected patients., Methods: Microvesicle RNA from serum from patients with de-novo primary glioblastoma multiforme (N = 9) and normal controls (N = 7) were analyzed by microarray analysis. Samples were collected according to protocols approved by the Institutional Review Board. Differential expressions were validated by qRT-PCR in a separate set of samples (N = 10 in both groups)., Results: Expression profiles of microvesicle RNA correctly separated individuals in two groups by unsupervised clustering. The most significant differences pertained to down-regulated genes (121 genes > 2-fold down) in the glioblastoma multiforme patient microvesicle RNA, validated by qRT-PCR on several genes. Overall, yields of microvesicle RNA from patients was higher than from normal controls, but the additional RNA was primarily of size < 500 nt. Gene ontology of the down-regulated genes indicated these are coding for ribosomal proteins and genes related to ribosome production., Conclusions: Serum microvesicle RNA from patients with glioblastoma multiforme has significantly down-regulated levels of RNAs coding for ribosome production, compared to normal healthy controls, but a large overabundance of RNA of unknown origin with size < 500 nt.

Primary central nervous system non-Hodgkin lymphoma (PCNSL) carries a poor prognosis and, although it responds to chemotherapy, fewer than 20% of patients are long-term disease-free survivors. Secondary CNS non-Hodgkin lymphoma (SCNSL) has an even worse prognosis with a median survival of only months and very few reported long-term survivors. For both of these groups of patients, there has been interest in using high-dose chemotherapy with autologous stem cell transplantation (ASCT) following conditioning with thiotepa, busulfan, and cyclophosphamide (TBC). We performed a retrospective review (from 2006-2010) of 32 patients from the Dana-Farber Cancer Institute and Massachusetts General Hospital with PCNSL or SCNSL who underwent ASCT with TBC conditioning. Of the 32 patients, 56% received TBC/ASCT after achieving brain magnetic resonance imaging (MRI) and/or cerebrospinal fluid complete response in brain, and 44% of patients were treated with TBC/ASCT in the setting of measurable CNS disease. The 100-day transplant-related mortality rate was only 3%. The most common nonhematologic grade 3 or 4 toxicity was mucositis, which occurred in 73% of patients. Notably, there was only 1 patient with prolonged significant neurologic toxicity that manifested as ataxia and dysphagia. The 1-year OS estimate is 93% (95% confidence interval [CI]: 75%-98%), and the 1-year progression-free survival (PFS) estimate is 90% (95% CI: 72%-96%) from the date of transplantation. Although these outcomes are encouraging, longer follow-up is required and comparison with other traditional ASCT regimens used for patients with non-Hodgkin lymphoma (NHL) is warranted., (Copyright © 2012 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

The central nervous system (CNS) manifestations of Waldenström's macroglobulinemia (WM) are known as the Bing-Neel syndrome (BNS). Patients with BNS can be classified into Group A and Group B based on the presence of lymphoplasmacytoid (LMP) cells within the brain parenchyma, leptomeninges, dura, and/or cerebrospinal fluid (CSF). To identify characteristic imaging findings for both Group A and Group B patients, we reviewed all 36 cases (26 referenced, 10 unreported) of proven WM with CNS symptoms, CSF analysis and/or biopsy, and magnetic resonance imaging (MRI) of the brain and/or spinal cord. Enhancement on MRI suggests invasion of the central neuraxis by LMP cells, and can help distinguish between Group A and Group B patients. In addition to differentiating true WM lesions in the CNS from ischemia, hyperviscosity events, and demyelinating lesions, evaluation of brain and spinal cord with gadolinium-enhanced MRI has the potential to guide management., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Bing-Neel syndrome (BNS) is defined as intracranial involvement of Waldenström macroglobulinemia (WM). Few cases of orbital involvement have been reported. A 51-year-old man with a history of WM developed bilateral orbitopathy and optic neuropathy. Orbital biopsy, cerebrospinal fluid studies, and neuroimaging confirmed the diagnosis of BNS involving the orbital soft tissues, optic nerves, meninges, and cauda equina. The neuro-ophthalmic manifestations resolved after parenteral and intrathecal chemotherapy in addition to autologous stem cell transplantation. The rare neuro-ophthalmic manifestations of BNS may require a multifaceted approach to therapy.

Purpose: Glioblastoma is an incurable solid tumor characterized by increased expression of vascular endothelial growth factor (VEGF). We performed a phase II study of cediranib in patients with recurrent glioblastoma., Methods: Cediranib, an oral pan-VEGF receptor tyrosine kinase inhibitor, was administered (45 mg/d) until progression or unacceptable toxicity to patients with recurrent glioblastoma. The primary end point was the proportion of patients alive and progression free at 6 months (APF6). We performed magnetic resonance imaging (MRI) and plasma and urinary biomarker evaluations at multiple time points., Results: Thirty-one patients with recurrent glioblastoma were accrued. APF6 after cediranib was 25.8%. Radiographic partial responses were observed by MRI in 17 (56.7%) of 30 evaluable patients using three-dimensional measurements and in eight (27%) of 30 evaluable patients using two-dimensional measurements. For the 15 patients who entered the study taking corticosteroids, the dose was reduced (n = 10) or discontinued (n = 5). Toxicities were manageable. Grade 3/4 toxicities included hypertension (four of 31; 12.9%); diarrhea (two of 31; 6.4%); and fatigue (six of 31; 19.4%). Fifteen (48.4%) of 31 patients required at least one dose reduction and 15 patients required temporary drug interruptions due to toxicity. Drug interruptions were not associated with outcome. Changes in plasma placental growth factor, basic fibroblast growth factor, matrix metalloproteinase (MMP) -2, soluble VEGF receptor 1, stromal cell-derived factor-1alpha, and soluble Tek/Tie2 receptor and in urinary MMP-9/neutrophil gelatinase-associated lipocalin activity after cediranib were associated with radiographic response or survival., Conclusion: Cediranib monotherapy for recurrent glioblastoma is associated with encouraging proportions of radiographic response, 6-month progression-free survival, and a steroid-sparing effect with manageable toxicity. We identified early changes in circulating molecules as potential biomarkers of response to cediranib. The efficacy of cediranib and the predictive value of these candidate biomarkers will be explored in prospective trials.

Vitreoretinal lymphoma is the most common type of intraocular lymphoma. It is mostly a high-grade B-cell malignancy with a poor prognosis, and is often associated with primary central nervous system lymphoma. Since intraocular lymphoma was first recognized almost 60 years ago, its treatment has gradually evolved. In the early years enucleation was often performed. Since that time, radiation therapy alone, systemic chemotherapy alone, or a combination of the two have been used extensively Because of the limited intraocular penetration of drugs administered systemically, the systemic and local toxicity of chemotherapy and radiation therapy and the high rate of recurrence, intravitreous chemotherapy, mainly using methotrexate, has become popular in the last decade, with encouraging results. More recently, biological treatment with intravitreal injections of rituximab has been investigated, with good results and minimal side effects. This review summarizes the present knowledge on vitreoretinal lymphoma therapy, with an eye to future molecular approaches.

The introduction of methotrexate (MTX)-based chemotherapy has improved median survival for patients with primary CNS lymphoma (PCNSL). Older age is a negative prognostic marker in patients with PCNSL and may increase the likelihood of MTX toxicity. We studied the response and adverse effects of intravenous high-dose MTX in patients who were 70 or more years of age at the time of diagnosis. We identified 31 patients at our institution diagnosed with PCNSL at age > or =70 years (median, 74 years) who were treated with high-dose MTX (3.5-8 g/m(2)) as initial therapy from 1992 through 2006. The best response to MTX was determined by contrast-enhanced MRI. Toxicity was analyzed by chart review. These 31 patients received a total of 303 cycles of MTX (median, eight cycles per patient). Overall, 87.9% of the cycles required dose reduction because of impaired creatinine clearance. In 30 evaluable patients, the overall radiographic response rate was 96.7%, with 18 complete responses (60%) and 11 partial responses (36.7%). Progression-free survival and overall survival were 7.1 months and 37 months, respectively. Grade I-IV toxicities were observed in 27 of 31 patients and included gastrointestinal disturbances in 58% (3.2% grade III), hematological complications in 80.6% (6.5% grade III), and renal toxicity in 29% (0% grade III/IV). High-dose MTX is associated with a high proportion of radiographic responses and a low proportion of grade III/IV toxicity in patients 70 or more years of age. High-dose MTX should be considered as a feasible treatment option in elderly patients with PCNSL.

Waldenström macroglobulinemia, a condition that most commonly occurs in lymphoplasmacytic lymphoma, typically manifests with diffuse lymphadenopathies, cytopenias, and a markedly elevated erythrocyte sedimentation rate. Peripheral neuropathy occurs in nearly half of patients with this condition, and hyperviscosity-related nervous system disorders are encountered in up to a third. Other neurological complications, such as encephalopathy or myelopathy caused by direct tumor infiltration, paraprotein deposition or autoimmune phenomena, are rare. Diagnosis of Waldenström macroglobulinemia requires identification of monoclonal IgM protein in the serum, bone marrow biopsy, and appropriate neurological testing (e.g. imaging studies of affected areas of the central neuraxis, electrophysiological studies). Treatment options, which should address both the paraprotein burden and the lymphoplasmacytic clone, include plasmapheresis and chemotherapy with alkylating agents, nucleoside analogs, and rituximab. As the disease is incurable and its course indolent, these treatments are only provided to symptomatic patients.

Purpose: Temozolomide (TMZ), given concurrently with radiotherapy (RT) and as adjuvant monotherapy afterwards, has led to improved survival in glioblastoma multiforme (GBM). However, it is unclear whether its primary mechanism of action is through enhancement of radiation response, independent cytotoxicity, or both. We sought to determine the relative contribution of concomitant temozolomide in patients treated by concurrent and adjuvant TMZ versus adjuvant TMZ alone in the setting of newly diagnosed GBM., Methods and Materials: We identified patients diagnosed with GBM and treated with surgery, involved-field radiotherapy, and chemotherapy at MGH between 2002 and 2004. Eligible patients received either adjuvant temozolomide alone (group 1) or temozolomide concurrently with RT followed by adjuvant TMZ (group 2). The primary endpoint of this retrospective analysis was overall survival (OS)., Results: Forty-three patients (group 1, n = 21; group 2, n = 22) were included in this study. The median follow-up was 33.7 months for surviving patients. There were no significant differences in baseline characteristics between these two groups. On univariate analysis, patients who received concurrent and adjuvant temozolomide experienced a 2-year OS of 51% and median survival of 25.5 months, compared with a 2-year OS of 36% and median survival of 15.6 months for group 1 patients (P < 0.05). On multivariable analysis, the hazard ratio (HR) favoring concurrent TMZ trended towards significance (HR = 0.51, P = 0.08) despite modest patient numbers., Conclusions: Concurrent and adjuvant TMZ was associated with improved survival compared to adjuvant TMZ alone, highlighting the potentiation of radiation effect by temozolomide in the clinical setting.

Clinical trials have proven oncolytic virotherapy to be safe but not effective. We have shown that oncolytic viruses (OV) injected into intracranial gliomas established in rodents are rapidly cleared, and this is associated with up-regulation of markers (CD68 and CD163) of cells of monocytic lineage (monocytes/microglia/macrophages). However, it is unclear whether these cells directly impede intratumoral persistence of OV through phagocytosis and whether they infiltrate the tumor from the blood or the brain parenchyma. To investigate this, we depleted phagocytes with clodronate liposomes (CL) in vivo through systemic delivery and ex vivo in brain slice models with gliomas. Interestingly, systemic CL depleted over 80% of peripheral CD163+ macrophages in animal spleen and peripheral blood, thereby decreasing intratumoral infiltration of these cells, but CD68+ cells were unchanged. Intratumoral viral titers increased 5-fold. In contrast, ex vivo CL depleted only CD68+ cells from brain slices, and intratumoral viral titers increased 10-fold. These data indicate that phagocytosis by both peripheral CD163+ and brain-resident CD68+ cells infiltrating tumor directly affects viral clearance from tumor. Thus, improved therapeutic efficacy may require modulation of these innate immune cells. In support of this new therapeutic paradigm, we observed intratumoral up-regulation of CD68+ and CD163+ cells following treatment with OV in a patient with glioblastoma.

Non-Hodgkin's lymphoma invades the brain, the vitreous body and nerves of the eye, the meninges, and the nerve roots of brain and spine, leading to the development of a primary CNS lymphoma. The mechanism of involvement of these locations by malignant B lymphocytes is unknown, but it might involve molecular targeting of lymphoma cells generated at cryptic systemic sites. The diagnosis of primary CNS lymphoma has been facilitated by advances in imaging techniques and the discovery of molecular markers. Methotrexate-based regimens, even when radiation is deferred, prolong overall survival to over 5 years, but relapses eventually occur in most cases. Better tools for earlier diagnosis and monitoring of treatment response will emerge from molecular studies of therapeutic targets.

Objective: To determine the sensitivity and specificity of clonal immunoglobulin heavy chain gene rearrangement (IGHR) analysis in the distinction of benign and malignant lymphoproliferative diseases., Methods: A retrospective analysis was conducted of patients in whom a malignant lymphoproliferative process was suspected. Cells of CSF samples were collected by centrifugation, resuspended in 100 microl of the supernatant and boiled. A 10 microl aliquot of this lysate served as template for semi-nested polymerase chain reaction using variable and joining region consensus primers. PCR products were analyzed by polyacrylamide gel electrophoresis. Cytopathological diagnosis and flow cytometry results were recorded. Sensitivity and specificity of IGHR analysis, cytopathology and flow cytometry were calculated., Results: Eleven patients (12 specimens) had involvement of leptomeninges at the time of lumbar puncture. Another 25 cases (27 specimens) had normal CSF findings or were diagnosed with benign lymphoproliferative conditions. Sensitivity of CSF cytopathology, flow cytometry and IGHR analysis were 0.27 [95% confidence interval 0.06, 0.61], 0.1 [0.003, 0.45] and 0.58 [0.28, 0.85]. Specificity was 1 [0.86, 1], 0.95 [0.77, 1.0] and 0.85 [0.66, 0.96]., Interpretation: IGHR analysis appears to be a useful addition to morphological and flow cytometry analysis of cerebrospinal fluid in the evaluation of CNS lymphoproliferative processes.

Peripheral neuropathies occur in 5% of patients with Non-Hodgkin Lymphoma and represent the effects of therapy, direct compression or nerve infiltration by tumor, or paraneoplastic effects. Multifocal motor neuropathy with conduction block (MMNCB) is a rare demyelinating disorder of unknown etiology characterized by progressive, distal, asymmetric weakness mostly of the upper limbs with minimal or no sensory loss. We report a patient, who developed MMNCB at the time of isolated CNS relapse from a diffuse large B-cell lymphoma. Marked neurological improvement was achieved using intravenous immunoglobulin treatment. To our knowledge, MMNCB has thus far not been described as part of the spectrum of lymphoma-related peripheral neuropathies.

The study protocol was approved by the institutional review board and was in full compliance with HIPAA guidelines. Informed consent was obtained from all patients. The purpose of this study was to prospectively compare intra- and intersubject variability of manual versus automatic magnetic resonance (MR) imaging section prescription. In two examinations, T2-weighted series were acquired with both methods. All intrasubject and three of six intersubject section prescription variances were significantly higher for manual prescription (P < .01). Root mean square errors confirmed better coregistration of the automated approach (P < .001). Automatic section prescription leads to improved reproducibility of imaging orientations for intra- and intersubject series in clinical practice.

A 46-year-old man with a 1-year history of distal paresthesias and mild distal weakness subacutely developed paralysis of the left hand. Electrodiagnostic evaluation revealed a demyelinating peripheral neuropathy that met criteria for chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). Magnetic resonance imaging of the brain revealed a mass that enhanced with contrast, but revealed focal myelin loss with intense macrophage activity and axonal preservation on biopsy. The mass and hand weakness improved following steroid therapy. The combination of CIDP and central demyelination with mass effect broadens the spectrum of demyelinating disease in association with CIDP.

The Response Evaluation Criteria in Solid Tumors, or RECIST criteria (one-dimensional [1D] measurement), are widely used to measure response in tumors, but there are few studies evaluating these criteria in brain tumors. We compared linear and volumetric measurements in adult high-grade supratentorial enhancing gliomas to determine the agreement between measurements, in defining responses and in their subsequent relation to survival. We hypothesized that the 1D RECIST criteria maybe suitable for response assessment in adult high-grade gliomas. Tumor size on MRI scans in 104 patients with high-grade enhancing gliomas treated on clinical trial protocols was measured by using 1D (greatest length), 2D (two-dimensional: product of the two longest perpendicular diameters), 3D (three dimensional: product of the longest perpendicular diameters in one plane and the longest orthogonal diameter to that plane), enhancing volume (EV), and total volume (TV). A total of 388 T1 postgadolinium MRI scans (104 baseline and 284 follow-up scans) were evaluated. Volumetric analysis (EV and TV) was performed with commercially available software. Intraobserver and interobserver correlations (rho) were high for all modalities (rho > 0.92 and rho > 0.71, respectively). Correlation was excellent (rho > 0.9) among all modalities except for 3D (rho < 0.6). Patient response rates ranged from 12% to 26%. Median progression-free survival (mPFS) and six-month progression-free survival (6mPFS) were not significantly different among the methods (range, 5.3 months to 5.9 months and 42% to 48%, respectively). Landmark analyses of response at two months using linear methods predicted overall survival with hazard ratios of 0.19 to 0.29 (P < 0.005). These results suggest high concordance among 1D, 2D, TV, and EV, but not 3D, methods in assessing enhancing tumor progression and in estimating mPFS and 6mPFS in adult brain tumor patients. The tumor response at two months assessed by linear methods correlated better with overall survival. Thus, linear methods are comparable to volumetric methods, but simpler to implement for routine clinical use and for designing clinical trials of brain tumors.