BIOM-12. DEVELOPMENT OF 5-ALA BASED LIQUID BIOPSY FOR THE NON-INVASIVE DIAGNOSIS OF GLIOBLASTOMA

Abstract

Abstract INTRODUCTION Tumor specificity of 5-aminolevulinic acid (5-ALA) induced protoporphyrin IX (PpIX) fluorescence is widely applied for fluorescence-guided surgery (FGS) in gliomas. We recently showed the feasibility of detecting tumour-specific fluorescent PpIX extracellular vesicles (EVs) derived from the plasma of glioblastoma (GBM) patients undergoing 5-ALA based fluorescence-guided surgery. Here, we further develop methods to characterize, sort and study fluorescent PpIX EVs in plasma of patients with glioma. METHODS We used imaging flow cytometry and Astrios EQ nanoFACS to characterize and sort PpIX EVs, respectively. Downstream RNA analysis utilized transcriptome sequencing analysis and droplet digital PCR (EGFRvIII mRNA). RESULTS All GBM cell lines (Gli36vIII, U87, Gli36 WT) dosed with 5-ALA demonstrated PpIX fluorescence, and released PpIX positive EVs. There was a high correlation between fluorescence in cells and the number of PpIX EVs released (r2=0.9). We sorted 100,000 PpIX EVs from Gli36vIII cells dosed with 5-ALA and detected 65 copies and 24 copies of mutant EGFRvIII mRNA and wildtype EGFR mRNA per 100,000 EVs, respectively. RNAseq analysis of the sorted PpIX EVs showed expression patterns reflective of parent cells. Furthermore, 100,000 sorted PpIX EVs from the plasma of a patient with EGFRvIII glioma yielded 22 copies of EGFRvIII mRNA while < 5 copies were detected in 1ml of plasma and healthy control plasma, demonstrating the tumor-specific nature of PpIX EVs. Finally, we performed transcriptome analysis on 250,000 PpIX EVs each from 8 patients undergoing 5-ALA based FGS. We identified several mRNAs including Gli3, STAG2, ELF3, PHLPP1 which play an important role in cancer. CONCLUSION The ability to sort and characterize tumor specific PpIX EVs following 5-ALA administration opens new avenues for liquid biopsy-based glioma diagnosis.