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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.

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.

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.

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.

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.

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.

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.

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.

Object: In mouse models of prostate and breast cancer therapeutic effects are enhanced when adenoviral HSV TK gene therapy is combined with ionizing radiation. In the present study, we adopted this approach for the treatment of human glioblastoma xenografts in an athymic mouse model and assessed treatment results as well as toxic side effects., Methods: About 72 nude mice received intracerebral inoculations of 2 x 10(5) U87deltaEGFR cells. On day 7 after tumor implantation the study population was randomized into six treatment arms: (1) intratumoral buffer inoculation on day 7, (2) intratumoral adenoviral vector injection (2 x 10(9) vp) on day 7, (3) single dose radiation (2.1 Gy) on day 9, (4) adenoviral injection + radiation, (5) adenoviral injection + ganciclovir (GCV) (20 microg/g twice daily from day 8 to 17), (6) adenoviral injection + GCV + radiation. On day 21 half of the animals were sacrificed for histological evaluation of the brain tumors, the other half was assessed for survival., Results: This study showed significantly prolonged median survival time of 5 days for the GCV treated groups. The addition of radiation decreased the frequency of neurological symptoms and delayed the onset of deficits without altering the expression of thymidine kinase in the tumor cells., Conclusions: We conclude that adenoviral HSV TK gene therapy in combination with adjuvant radiotherapy does not generate increased toxic side effects in glioblastoma treatment. The prolonged survival time of animals receiving gene therapy and the reduced occurrence of neurological symptoms in irradiated mice constitute promising features of the combined treatment.

Suramin is a polysulfonated naphthylurea that inhibits the function of growth factors and growth factor receptors implicated in glioma progression, angiogenesis, and radioresistance. The safety and benefits of combining inhibitors of angiogenesis and growth factors with cytotoxic therapies in patients with neoplasms of the central nervous system remain unclear. The objectives of this phase 2 study were to determine the safety of administering suramin with standard cranial radiotherapy (RT) and to estimate survival using this approach in patients with newly diagnosed glioblastoma multiforme (GBM). Fifty-five patients with newly diagnosed GBM (Karnofsky performance status >or= 60) were enrolled in this multicenter phase 2 study. Patients received suramin by a conventional intermittent fixed-dosing regimen for 1 week prior to and during cranial RT (60 Gy in 30 fractions, weeks 2-7). Patients with stable or responsive disease at week 18 received an additional 4 weeks of suramin (weeks 19-22). The median survival for suramin-treated patients was 11.6 months, with 1-year and 18-month survival rates of 49% (95% confidence interval [CI], 36%-62%) and 18% (95% CI, 8%-28%), respectively. Overall, 55% of the patients (30/55) had greater than grade 2 toxicity at least possibly related to suramin therapy. Two patients died of possibly related neurologic events (i.e., stroke, elevated intracranial pressure). Otherwise, toxicities were generally transient and self-limited. Administration of suramin using an intermittent fixed-dosing regimen during cranial RT was generally well tolerated. However, overall survival is not significantly improved when compared with the New Approaches to Brain Tumor Therapy GBM database or other comparable patient populations.

Object: After conventional doses of 55 to 65 Gy of fractionated irradiation, glioblastoma multiforme (GBM) usually recurs at its original location. This institutional phase II study was designed to assess whether dose escalation to 90 cobalt gray equivalent (CGE) with conformal protons and photons in accelerated fractionation would improve local tumor control and patient survival., Methods: Twenty-three patients were enrolled in this study. Eligibility criteria included age between 18 and 70 years, Karnofsky Performance Scale score of greater than or equal to 70, residual tumor volume of less than 60 ml, and a supratentorial, unilateral tumor. Actuarial survival rates at 2 and 3 years were 34% and 18%, respectively. The median survival time was 20 months, with four patients alive 22 to 60 months postdiagnosis. Analysis by Radiation Therapy Oncology Group prognostic criteria or Medical Research Council indices showed a 5- to 11-month increase in median survival time over those of comparable conventionally treated patients. All patients developed new areas of gadolinium enhancement during the follow-up period. Histological examination of tissues obtained at biopsy, resection, or autopsy was conducted in 15 of 23 patients. Radiation necrosis only was demonstrated in seven patients, and their survival was significantly longer than that of patients with recurrent tumor (p = 0.01). Tumor regrowth occurred most commonly in areas that received doses of 60 to 70 CGE or less; recurrent tumor was found in only one case in the 90-CGE volume., Conclusions: A dose of 90 CGE in accelerated fractionation prevented central recurrence in almost all cases. The median survival time was extended to 20 months, likely as a result of central control. Tumors will usually recur in areas immediately peripheral to this 90-CGE volume, but attempts to extend local control by enlarging the central volume are likely to be limited by difficulties with radiation necrosis.

The biopsy specimens of 342 patients with malignant glioma were evaluated to determine the extent and prognostic significance of round cell infiltration within these representative tissues: (a) tumor, (b) peritumoral, (c) hypervascular, (d) necrotic, and (e) normal tissue. Thirty-six percent of all biopsy specimens showed at least one tissue area with round cell infiltration. Patients in age groups 0 to 25 years and 71 years and older tended to show less round cell infiltration than did patients 41 to 55 years old. The presence of round cell infiltration in tissue was associated with a poor prognosis. Patients showing any infiltration had mean survival times of 8.4 months as opposed to 11.9 months for those showing no infiltration. The relationship between round cell infiltration and poor prognosis is true, irrespective of postoperative therapy, sex, and age.

A retrospective evaluation of the quality and length of survival of 74 patients nonrandomly receiving lomustine, 100 to 110 mg/sq m, following craniotomy and irradiation for glioblastoma multiforme was performed. After surgery all patients were capable of at least partial self-care. Patients receiving postoperative irradiation and lomustine had a median survival of 11.5 months. While receiving chemotherapy, 40% of these patients were capable of at least partial employment; 75% were able to care for themselves. These levels of function were stable for 70% of the average postoperative course (8.0 months), following which a decline ensued.

The rationale for, methodology of, and experience with intra-arterial BCNU infusion therapy of malignant glioma are described. This approach achieves tumor levels of drug four times greater than equal doses infused intravenously, and has been used to treat 79 patients over the course of 4 years. The drug was given in 192 infraophthalmic and 66 supraophthalmic carotid artery infusions. Patients who were treated via infraophthalmic carotid artery infusion following tumor recurrence (after both operation and irradiation) survived 54 additional weeks (92 weeks after initial diagnosis). Patients who were treated with BCNU immediately after initial irradiation therapy survived 64 weeks (infraophthalmic carotid artery infusion) and 49.5 weeks (supraophthalmic carotid artery infusion). The major ocular complications (pain and diminished visual acuity) associated with infraophthalmic carotid artery infusion are avoided by selective balloon-guided supraophthalmic carotid artery administration. However, both approaches were associated with white-matter changes, seen as diminished absorption on computerized tomography scans, in 20% of patients treated following irradiation therapy. This toxicity appears to preclude intra-arterial BCNU treatment in the immediate postirradiation period. Better results are being achieved with our current therapy, which involves four infusions of BCNU (400 mg every 4 weeks) into the infraophthalmic or supraophthalmic carotid artery in advance of irradiation. Cisplatin infusions (60 to 90 mg/sq m every 5 weeks) are offered for recurrent glioblastoma.

In the light of advances in computerized tomography (CT), we have retrospectively evaluated the assumptions that underlie the radiation therapy of glioblastoma: (1) No neuroradiologic technique provides an accurate delineation of tumor bulk and location, (2) glioblastoma is commonly multicentric, and (3) a major source of therapeutic failure is recurrence beyond radiotherapy fields. 1. CT scans, performed on glioblastoma patients within 2 months of postmortem examination, defined both gross and microscopic tumor extent (within a 2-cm margin) in all but 6 of 35 patients evaluated. The major source of error was subependymal spread (four patients). 2. Multicentricity occurred in only 4% of untreated and 6% of treated (radiotherapy with or without chemotherapy) patients. All multicentric lesions were identified on CT scans. 3. Serial CT scans on 42 patients revealed that glioblastoma recurred within a 2-cm margin of the primary site in 90%. Occurrences outside this margin were accurately delineated by CT in all instances. Because most patients show recurrence within or in close proximity to the original site, current radiation doses would appear to be inadequate for therapy of the primary tumor. CT scan accuracy may permit smaller-field and higher-dose irradiation therapy for glioblastoma.

Eleven patients with recurrent malignant glioma were treated with single high doses of BCNU ranging from 600 to 1400 mg/sq m. To prevent the characteristic late myelosuppression observed after conventional doses of BCNU, autologous bone marrow harvested just before drug treatment was infused 24 to 36 hours after therapy. Higher doses of BCNU causes earlier and more profound myelosuppression; one patient died on pancytopenia, breakdown of the gut epithelium, and Clostridium septicemia 10 days after receiving 1400 mg/sq m of BCNU. All patients experienced transient emesis; four developed transient elevation of hepatic enzymes, two reversible interstitial pulmonary infiltrates, and two who received 1400 mg/sq m BCNU suffered irreversible cortical damage. Eight patients receiving 600 to 1200 mg/sq m demonstrated reconstitution of polymorphonuclear leukocytes an platelets within at least 30 days after treatment. With a follow-up time of up to 19 months, four patients improved, three stabilized, and three deteriorated and died. The median survival time was 7 months. Computerized tomography performed on patients receiving constant corticosteroids showed diminished contrast enhancement and mass effect in eight patients. High-dose BCNU at doses up to 1200 mg/sq m with marrow rescue is a feasible approach to the treatment of patients with glioblastoma.

Thirteen patients selected for long-term survival with primary astrocytic tumor (who failed to return to premorbid educational or vocational levels) were examined by neuropsychologic tests of specific and generalized higher cortical functions. In the absence of tumor regrowth or other neurologic disorders, each demonstrated difficulty in problem solving or coping with novel situations when previously acquired abilities, overlearned material, and psychometric intelligence appeared consistent with their premorbid level. The diffuse difficulties were unrelated to tumor type or location, and were not explicable by existing focal deficits, psychotic or depressive thought disorders, metabolic difficulties, or hydrocephalus. These examinations explained in part why these patients failed to resume active social lives or premorbid employment. The diffuse cortical dysfunction was most notable on the Category Test, Trails B, and Localization component of the Tactual Performance Test.

High-dose 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) infusion into the internal carotid artery following cranial irradiation in the treatment of glioblastoma multiforme is accompanied by evidence of leukoencephalopathy in a significant number of patients. In an attempt to avoid this problem, a phase I trial was performed using intracarotid BCNU infusion before irradiation. Twenty-eight patients with grade III/III astrocytoma (World Health Organization Classification, equivalent to Kernohan grade IV) received a 400-mg infusion of BCNU into the infraophthalmic carotid artery. The treatment was repeated every 4 weeks for a total of four cycles prior to cranial irradiation (5500 to 6000 cGy). The major toxic sequelae included nausea and vomiting (24%), decreased visual acuity (14%), transient cerebral ischemia (3.5%), and thrombocytopenia (3.5%). Fatal leukoencephalopathy occurred in two patients. The median survival time was 37 weeks for all evaluable patients and 56+ weeks for those completing the protocol. The tumor response to drug infusion as judged by computerized tomography (CT) was complete in 22% of patients and partial in 22%; 56% showed no CT tumor response. Pre-irradiation intracarotid artery BCNU benefits a very small group of patients with grade III/III astrocytoma. The associated severe leukoencephalopathy makes this mode of therapy unacceptable for a phase III trial.