Purpose: Liquid biopsy offers a minimally invasive tool to diagnose and monitor the heterogeneous molecular landscape of tumors over time and therapy. Detection of TERT promoter mutations (C228T, C250T) in cfDNA has been successful for some systemic cancers but has yet to be demonstrated in gliomas, despite the high prevalence of these mutations in glioma tissue (>60% of all tumors). Experimental Design: Here, we developed a novel digital droplet PCR (ddPCR) assay that incorporates features to improve sensitivity and allows for the simultaneous detection and longitudinal monitoring of two TERT promoter mutations (C228T and C250T) in cfDNA from the plasma of patients with glioma. Results: In baseline performance in tumor tissue, the assay had perfect concordance with an independently performed clinical pathology laboratory assessment of TERT promoter mutations in the same tumor samples [95% confidence interval (CI), 94%–100%]. Extending to matched plasma samples, we detected TERT mutations in both discovery and blinded multi-institution validation cohorts with an overall sensitivity of 62.5% (95% CI, 52%–73%) and a specificity of 90% (95% CI, 80%–96%) compared with the gold-standard tumor tissue–based detection of TERT mutations. Upon longitudinal monitoring in 5 patients, we report that peripheral TERT-mutant allele frequency reflects the clinical course of the disease, with levels decreasing after surgical intervention and therapy and increasing with tumor progression. Conclusions: Our results demonstrate the feasibility of detecting circulating cfDNA TERT promoter mutations in patients with glioma with clinically relevant sensitivity and specificity.

Over the last 20 years, gliomas have made up over 89% of malignant CNS tumor cases in the American population (NIH SEER). Within this, glioblastoma is the most common subtype, comprising 57% of all glioma cases. Being highly aggressive, this deadly disease is known for its high genetic and phenotypic heterogeneity, rendering a complicated disease course. The current standard of care consists of maximally safe tumor resection concurrent with chemoradiotherapy. However, despite advances in technology and therapeutic modalities, rates of disease recurrence are still high and survivability remains low. Given the delicate nature of the tumor location, remaining margins following resection often initiate disease recurrence. Photodynamic therapy (PDT) is a therapeutic modality that, following the administration of a non-toxic photosensitizer, induces tumor-specific anti-cancer effects after localized, wavelength-specific illumination. Its effect against malignant glioma has been studied extensively over the last 30 years, in pre-clinical and clinical trials. Here, we provide a comprehensive review of the three generations of photosensitizers alongside their mechanisms of action, limitations, and future directions.

The last decade has seen a rapid expansion of interest in extracellular vesicles (EVs) released by cells and proposed to mediate intercellular communication in physiological and pathological conditions. Considering that the genetic content of EVs reflects that of their respective parent cell, many researchers have proposed EVs as a source of biomarkers in various diseases. So far, the question of heterogeneity in given EV samples is rarely addressed at the experimental level. Because of their relatively small size, EVs are difficult to reliably isolate and detect within a given sample. Consequently, standardized protocols that have been optimized for accurate characterization of EVs are lacking despite recent advancements in the field. Continuous improvements in pre-analytical parameters permit more efficient assessment of EVs, however, methods to more objectively distinguish EVs from background, and to interpret multiple single-EV parameters are lacking. Here, we review EV heterogeneity according to their origin, mode of release, membrane composition, organelle and biochemical content, and other factors. In doing so, we also provide an overview of currently available and potentially applicable methods for single EV analysis. Finally, we examine the latest findings from experiments that have analyzed the issue at the single EV level and discuss potential implications.

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. 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. 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). 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.

Metabolic mapping of D-2-hydroxyglutarate and tumor metabolism in isocitrate dehydrogenase 1-mutated human gliomas was performed with a resolution of 1.7 × 1.7 × 3 mm by using super-resolution upsa... Background Isocitrate dehydrogenase (IDH) mutations are highly frequent in glioma, producing high levels of the oncometabolite D-2-hydroxyglutarate (D-2HG). Hence, D-2HG represents a valuable imagi...