Extracellular vesicles (EVs) play an important role in cell-cell communication, and tumor-derived EVs circulating in patient blood can serve as biomarkers. Here, we investigated the potential role of plasma EVs in meningioma patients for tumor detection and determined whether EVs secreted by meningioma cells reflect epigenetic, genomic, and proteomic alterations of original tumors.EV concentrations were quantified in patient plasma (n = 46). Short-term meningioma cultures were established (n = 26) and secreted EVs were isolated. Methylation and copy number profiling was performed using 850k arrays, and mutations were identified by targeted gene panel sequencing. Differential quantitative mass spectrometry was employed for proteomic analysis.Levels of circulating EVs were elevated in meningioma patients compared to healthy individuals, and the plasma EV concentration correlated with malignancy grade and extent of peritumoral edema. Postoperatively, EV counts dropped to normal levels, and the magnitude of the postoperative decrease was associated with extent of tumor resection. Methylation profiling of EV-DNA allowed correct tumor classification as meningioma in all investigated cases, and accurate methylation subclass assignment in almost all cases. Copy number variations present in tumors, as well as tumor-specific mutations were faithfully reflected in meningioma EV-DNA. Proteomic EV profiling did not permit original tumor identification but revealed tumor-associated proteins that could potentially be utilized to enrich meningioma EVs from biofluids.Elevated EV levels in meningioma patient plasma could aid in tumor diagnosis and assessment of treatment response. Meningioma EV-DNA mirrors genetic and epigenetic tumor alterations and facilitates molecular tumor classification.
Abstract INTRODUCTION Extracellular vesicles (EVs) carry biological information from their cell of origin that is useful for non-invasive detection of tumor biomarkers and disease monitoring. In glioblastoma (GBM), blood circulating EVs are elevated and carry GBM-associated proteins. However, it is still challenging to analyze tumor derived EVs for translational purposes. Here, we used imaging flow cytometry (IFCM) as a robust strategy to perform phenotyping of EVs with GBM related surface markers in human plasma. METHODS EVs were isolated via differential ultracentrifugation from plasma of (a) 40 GBM patients, pre- and post-surgery, (b) 11matched GBM relapses and (c) 12 healthy donors (HD). EV sizes and concentrations were evaluated by NTA. EV markers (CD9,CD63 and CD81) together with glioma-related markers (integrin beta-1 [ITGB1], tenascin C [TNC], Profilin-1 [PFN1], CD44,GPNMB, SPARC, HLA-II or CD133) were analyzed by IFCM. EV percentages and objects/mL plasma were compared among the groups and correlated with clinical parameters. RESULTS CD9 was the predominant tetraspanin in all groups (15-96%), while CD63 had the lowest levels (0-33%) and the strongestdecrease in GBM patients after surgery (fold change [FC]=-5.4, p<0.01). Among the glioma-related markers, ITGB1 and TNC displayed the most significant differences between the analyzed groups, especially the double positives ITGB1+/CD63+and TNC+/CD63+, which decreased in patients after tumor removal (FC=-3.5 and -12, respectively; p<0.001). Meanwhile,ITGB1+/CD9+and TNC+/CD9+EVs exhibited the highest levels in GBM when compared to HD subjects (FC=8.6 and 17.4;p<0.001) and upon tumor recurrence (FC=3.7 and 10.9, respectively; p<0.01). SUMMARY/CONCLUSION We identified EV surface antigens with potential clinical utility as GBM biomarkers. Among them, we highlight ITGB1 and TNC as the most promising markers.
Abstract Background Extracellular vesicles (EVs) obtained by noninvasive liquid biopsy from patient blood can serve as biomarkers. Here, we investigated the potential of circulating plasma EVs to serve as an indicator in the diagnosis, prognosis, and treatment response of glioblastoma patients. Methods Plasma samples were collected from glioblastoma patients at multiple timepoints before and after surgery. EV concentrations were measured by nanoparticle tracking analysis and imaging flow cytometry. Tumor burden and edema were quantified by 3D reconstruction. EVs and tumors were further monitored in glioma-bearing mice. Results Glioblastoma patients displayed a 5.5-fold increase in circulating EVs compared to healthy donors (P < .0001). Patients with higher EV levels had significantly shorter overall survival and progression-free survival than patients with lower levels, and the plasma EV concentration was an independent prognostic parameter for overall survival. EV levels correlated with the extent of peritumoral fluid-attenuated inversion recovery hyperintensity but not with the size of the contrast-enhancing tumor, and similar findings were obtained in mice. Postoperatively, EV concentrations decreased rapidly back to normal levels, and the magnitude of the decline was associated with the extent of tumor resection. EV levels remained low during stable disease, but increased again upon tumor recurrence. In some patients, EV resurgence preceded the magnetic resonance imaging detectability of tumor relapse. Conclusions Our findings suggest that leakiness of the blood-brain barrier may primarily be responsible for the high circulating EV concentrations in glioblastoma patients. Elevated EVs reflect tumor presence, and their quantification may thus be valuable in assessing disease activity.
Abstract Extracellular vesicles (EVs) transport biological and specific information from tumors into the bloodstream, enabling non-invasive detection of tumor material and disease monitoring. Based on a proteomics screen, we performed immunophenotyping of eight glioma-related antigens (tenascin-C (TNC), integrin-beta 1 (ITGB1), profilin-1 (PFN1), CD44, CD133, GPNMB, HLA-II, SPARC) and tetraspanins (CD9, CD63 and CD81) in plasma EVs from GBM patients (before and after surgery (n = 38)), from matched GBM relapse patients (n = 11), and from healthy donors (HD, n = 12) using imaging flow cytometry. Double-positive TNC+/CD9+ EVs showed the strongest differences per mL of plasma in primary (FC = 7.6, p< .0001, ROC analysis AUC = 81%) and relapsed GBM (FC = 16.5, p< .0001; AUC = 90%) compared to HD subjects. High TNC signals were also observed in GBM-EVs by immunogold electron microscopy compared to HD-EVs. In paired analysis, TNC+/CD9+ EVs showed a 3.9-fold decrease after tumor removal (p< .001) and re-increased at GBM recurrence in these patients (FC = 8.4, p< .05; AUC = 84%). In tissue samples, TNC levels were 5.4-fold higher in GBM patients than in non-neoplastic cortex controls (p< .01) measured by immunohistochemistry and correlated positively with plasma TNC+/CD9+ EV levels in RTK-I/II GBM patients (r = 0.42, p< .05). Accordingly, spatial transcriptomics of GBM tissue sections revealed that TNC is specifically overexpressed in GBM cells. Furthermore, magnetic sorting of TNC in plasma EVs allowed detection of GBM-specific TERT*C228T mutations by digital droplet PCR. Mutated TERT DNA was enriched in TNC+ EVs (n = 13) compared to TNC- EVs (FC = 110, p< 0.0001), total EV DNA (FC = 36.7, p< 0.01), and cfDNA (FC = 7.2, p < 0.05). In conclusion, we identified TNC as a biomarker in circulating EVs from GBM patients that can be isolated and used for tumor-specific mutation analysis.
Seizures can present at any time before or after the diagnosis of a glioma. Roughly, 25%-30% of glioblastoma (GBM) patients initially present with seizures, and an additional 30% develop seizures during the course of the disease. Early studies failed to show an effect of general administration of antiepileptic drugs for glioblastoma patients, since they were unable to stratify patients into high- or low-risk seizure groups.111 patients, who underwent surgery for a GBM, were included. Genome-wide DNA methylation profiling was performed, before methylation subclasses and copy number changes inferred from methylation data were correlated with clinical characteristics. Independently, global gene expression was analyzed in GBM methylation subclasses from TCGA datasets (n = 68).Receptor tyrosine Kinase (RTK) II GBM showed a significantly higher incidence of seizures than RTK I and mesenchymal (MES) GBM (P < .01). Accordingly, RNA expression datasets revealed an upregulation of genes involved in neurotransmitter synapses and vesicle transport in RTK II glioblastomas. In a multivariate analysis, temporal location (P = .02, OR 5.69) and RTK II (P = .03, OR 5.01) were most predictive for preoperative seizures. During postoperative follow-up, only RTK II remained significantly associated with the development of seizures (P < .01, OR 8.23). Consequently, the need for antiepileptic medication and its increase due to treatment failure was highly associated with the RTK II methylation subclass (P < .01).Our study shows a strong correlation of RTK II glioblastomas with preoperative and long-term seizures. These results underline the benefit of molecular glioblastoma profiling with important implications for postoperative seizure control.
Abstract Standard monitoring after meningioma resection relies on serial MRI examinations, which are time-consuming, expensive and provide no information on molecular alterations that may indicate progression towards a more aggressive tumor. Extracellular vesicles (EVs) secreted by tumor cells play an important role in cell-cell communication, and tumor-derived EVs circulating in patient blood can serve as biomarkers. We investigated the potential role of plasma EVs in meningioma patients for tumor detection and determined whether EVs secreted by meningioma cells reflect epigenetic, genomic and proteomic alterations of original tumors. EV concentrations were quantified in patient plasma (n = 46). Short-term meningioma cultures were established (n = 26) and secreted EVs were isolated. Methylation and copy number profiling was performed using 850k arrays, and mutations were identified by targeted gene panel sequencing. Differential quantitative mass spectrometry was employed for proteomic analysis. We found that the levels of circulating EVs were significantly elevated in meningioma patients compared to healthy individuals, and that plasma EV concentrations correlated with malignancy grade and extent of peritumoral edema. Postoperatively, EV counts dropped to normal levels, and the magnitude of the postoperative decrease was associated with extent of tumor resection (Simpson grade). Methylation profiling of EV-DNA allowed correct tumor classification as meningioma in all investigated cases, and accurate methylation subclass assignment in nearly all cases. Copy number variations present in tumors, as well as tumor-specific mutations were faithfully reflected in meningioma EV-DNA. Proteomic EV profiling did not permit original tumor identification but revealed tumor-associated proteins such as desmoplakin that could potentially be utilized to enrich meningioma EVs from biofluids. In conclusion, elevated EV levels in meningioma patient plasma may aid in tumor diagnosis and assessment of treatment response. Meningioma EV-DNA mirrors genetic and epigenetic tumor alterations and facilitates molecular tumor classification.
Abstract The majority of meningiomas are benign but approximately 20% of display an aggressive behavior, resulting in significant patient morbidity and mortality. Standard monitoring after meningioma resection relies on serial MRI examinations, which are time-consuming, expensive and provide no information on molecular alterations that may indicate progression towards a more aggressive tumor. Extracellular vesicles (EVs) are released by tumor cells and contain high molecular weight DNA, rendering circulating EVs a potential biomarker source for non-invasive disease monitoring and for obtaining information on genetic and epigenetic alterations. We quantified EVs in plasma of 46 meningioma patients (n = 29 M1, 12 M2, 5 M3) by nanoparticle tracking analysis and detected significantly higher levels compared to age-matched healthy donors (n = 18). EV concentrations correlated with malignancy grade (p = 0.0049) and with the extent of peritumoral edema (p = 0.0031). Comparisons between paired pre- and postoperative samples revealed that EV levels counts dropped significantly the day after tumor resection and were reduced to normal levels after about one week. Completely resected patients (Simpson grade I) displayed a greater reduction of postoperative EV concentrations than incompletely resected patients. DNA methylation profiling was performed on EVs secreted by cultured meningioma cells, as well as matched cells and original tumors using 850k arrays (n = 7 M1, 5 M2, 3 M3). All EV samples were correctly identified as meningiomas by the Heidelberg classifier, and methylation subclasses were also correctly assigned in almost all cases. t-SNE analysis showed that EVs mapped in close proximity to their corresponding parental cells and tumor tissue. Tumor specific mutations and copy number variations were detected in EV-DNA with high accuracy. Differential quantitative proteomic analysis of EVs, cells and tumors identified shared proteins that could potentially be useful for enriching tumor-derived circulating EVs from biofluids.
Abstract OBJECTIVE Extracellular vesicles (EVs) represent a population of lipid bilayer nanoparticles released by all cell types, including tumor cells that can serve as a noninvasive source for liquid biopsy. To date, MRI images have been the established method for monitoring treatment efficacy in brain tumor patients. We investigated the potential of pure EV count for diagnosis, prognosis, and treatment monitoring in gliomas. METHODS Plasma samples, differential blood counts, at multiple timepoints before and after surgery of glioblastoma patients (n=101) were collected. Follow-up samples were obtained every 3 months. Healthy donors served as controls (n=29). Plasma EVs concentration was measured by Nanoparticle Tracking Analysis (NTA). EVs were characterized by electron microscopy and imaging flow cytometry. Tumor burden was measured by MRI images. Clinical characteristics were prospectively recorderd. In addition plasma EVs from Mut3 tumor bearing mice were analysed at d3, d5, d7, d10, d12 after tumor injection (n=20). MRI images and differential blood counts were analyzed. RESULTS Glioblatoma patients have a 5-fold increase of plasma EVs compared to HD; p < 0.0001). Circulating EVs counts correlated only with FLAIR hyperintensity and with no other MRI or blood-based parameter. Similar results were obtained from Mut3 tumor mice. Dichotomisation of GBM patients in EVhigh and low revealed a significant overall survival and progression free survival benefit for EVlow patients (p=0.004). After surgery, EVs decreased significantly (5-fold, p< 0.0001). A massive drop in EVs was associated with gross-total resection (p < 0.05). At the time of tumor recurrence, the number of circulating EVs increased in all patients during a follow-up (9 months). CONCLUSION Our findings highlight the potential of circulating EVs as a biomarker tool for diagnosis, prognosis and treatment monitoring in GBM patietns, as they seem to reflect the presence of a tumor mass and thus may assist in clinical decision making.
