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Liquid Biopsy Using Fluorescent Extracellular Vesicles Shows Promise for Diagnosis of Glioma

Key findings

  • Fluorescence-guided resection of malignant glioma requires a patient to ingest a 5-aminolevulinic acid (5-ALA) oral solution, which results in fluorescent protoporphyrin IX (PpIX) accumulating in glioma tumor cells and not in normal cells
  • This study investigated whether 5-ALA might also facilitate as a liquid biopsy of glioma
  • When exposed to 5-ALA, human glioma cells secreted fluorescent extracellular vesicles both in vitro and in vivo
  • In plasma samples from four patients with avidly fluorescent tumors, the level of PpIX-positive extracellular vesicles was significantly greater after 5-ALA dosing than before, and the increase was highly correlated with tumor volume
  • Analysis of tumor-specific fluorescent extracellular vesicles derived from the plasma of patients dosed with 5-ALA may allow diagnosis of malignant glioma as well as monitoring of recurrence

5-aminolevulinic acid (5-ALA) oral solution is approved in the U.S. to improve the resection of malignant glioma. After a patient ingests 5-ALA, a naturally occurring fluorescent molecule called protoporphyrin IX (PpIX) accumulates in glioma tumor cells and not in normal cells. Under blue light microscopy, PpIX glows pink, which allows the neurosurgeon to distinguish tumor from normal brain.

Bob S. Carter, MD, PhD, chief of the Department of Neurosurgery and neurosurgical oncologist in the Mass General Cancer CenterLeonora Balaj, PhD, investigator in neurosurgery, Pamela S. Jones, MD, neurosurgeon, and colleagues have published evidence that 5-ALA might also be useful for minimally invasive diagnosis of glioma and monitoring its response to treatment. Their report appears in EBioMedicine.

Examining Extracellular Vesicles

The research team investigated whether the administration of PpIX would cause fluorescence not just of tumor tissue but also of extracellular vesicles (EVs). These lipid-covered particles are secreted by organ-specific cell populations into the blood and other biofluids, where they can be captured and analyzed. They carry genetic material from their cells of origin and therefore serve as biomarkers—and in the case of glioma, they are biomarkers of disease burden.

A major problem in studying EVs is their tiny size, 30 to 1,000 nm in diameter. The research team used state-of-the-art imaging flow cytometry, which can detect even single EVs in plasma. The ability to study glioma at the single-EV level is important because different subpopulations of tumor cells exist within an individual tumor, both at a single time point and over the course of treatment.

Laboratory Results

  • A human glioma cell line dosed with 5-ALA released fluorescent EVs, and normal endothelial cells did not
  • In xenograft models, fluorescent EVs were present in the plasma of glioma-bearing mice after dosing with 5-ALA and were absent from normal control mice

Results in Patients

Finally, the researchers tested their liquid biopsy in six patients with glioma who underwent 5-ALA fluorescence-guided surgery at Mass General. They collected plasma samples before 5-ALA intake and during surgery.

Imaging flow cytometry of the samples showed that:

  • In four patients with avidly fluorescent tumors — During surgery (after dosing of 5-ALA), the level of PpIX-positive EVs was significantly greater than the pre-dosing level, and the increase was highly correlated with tumor volume
  • In two patients with minimally fluorescent tumors — There was no rise in PpIX-positive EV levels after 5-ALA administration

Work Continues

The researchers are still collecting blood samples from patients who undergo fluorescence-guided glioma resection at Mass General and certain other institutions. They hope to validate the evidence for using 5-ALA in first-time glioma diagnosis as well as monitoring patients for recurrence.

Refer a patient to the Department of Neurosurgery

Learn about the Stephen E. and Catherine Pappas Center for Neuro-Oncology

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