BIOM-62. DETECTION OF EXCLUSIVE MUTATIONS AND GENE-GENE FUSIONS IN GLIOBLASTOMA THROUGH ANALYSIS OF CIRCULATING CELL-FREE DNA (CFDNA) AS AN EMERGING DIAGNOSTIC TOOL

Abstract

Abstract Tumor heterogeneity is a significant obstacle for developing tailored therapies for glioblastoma (GBM). We collected blood and tumors from 20 GBM patients at 4-time points and 20 blood samples from healthy controls. cfDNA was extracted from the plasma of GBM patients and healthy controls. Tumor DNA was extracted from fresh tumor samples. Extracted DNA was sequenced using a whole-genome sequencing procedure. We found that plasma cfDNA concentrations in GBM patients were significantly elevated (22.6 ± 5 ng·mL-1), as compared to healthy controls (1.4 ± 0.4 ng·mL-1). We identified unique mutations in cfDNA of GBM patients, including some of the most frequently mutated genes in GBM (TP53, 18.75%; EGFR, 37.5%; NF1, 12.5%; LRP1B, 25%; IRS4, 25%) and built a unique clinical panel for further patients monitoring. Using our gene fusion database, ChiTaRS 5.0, we identified gene fusions in both tumor DNA and cfDNA, such as KDR-PDGFRA and NCDN-PDGFRA, which correspond to previously reported alterations of PDGFRA in GBM (44% of all samples). Interestingly, the PDGFRA protein fusions can be targeted by tyrosine kinase inhibitors. We identified ROS1 fusions (CEP85L-ROS1 and GOPC-ROS1) in 8% of samples, and those might be targeted by crizotinib analogs. Moreover, we analyzed TCGA/ICGC data of 180 GBM samples and observed FGFR3-TACC3, EGFR-SEPT14 fusions in silico. Next, we measured cfDNA levels in vitro in response to Temozolomide treatment and discovered a positive correlation between unique 2000bp cfDNA fragments (R = 0.8961, P-value = 0.015632) and treatment responses in vitro. We extracted exosomes from the in-vitro experiments and validated the concentrations of both the encapsulated cfDNA as well as the free cfDNA. We found that half of cfDNA was exosome encapsulated and it varied according to the tumor stage. CONCLUSION: cfDNA may be used for the detection of exclusive patient mutations and gene fusions and is an emerging diagnostic tool in GBM.