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Abstract
Diffuse midline glioma (DMG) is a high-risk pediatric brain tumor that has primarily been diagnosed and monitored via medical imaging (MRI) due to risks associated with re-biopsying midline brain structures. Recent advances in molecular diagnostics have improved classification and molecular characterization of DMG; these tumors are in dire need of non-invasive, molecular-based diagnostic tools to supplement serial assessments of radiographic response. Our group recently developed a single-molecule imaging platform, Epigenetics of Plasma Isolated Nucleosomes (EPINUC), to profile histone modification patterns in cell-free tumor DNA (ctDNA) from plasma of cancer patients. We developed a novel strategy for single-molecule detection of the histone mutation H3K27M, a key DMG genetic driver, by capturing mutant nucleosomes on a PEG-streptavidin coated surface and detecting them with fluorescently labeled antibodies targeting histone modifications enriched on these mutant nucleosomes. We applied this method to 46 H3K27M and 10 H3 wildtype DMG samples and found significantly higher signal in K27M samples. Using this data, we generated a predictive model of H3 status with an AUC of 0.81, sensitivity 90%, specificity 71%, and precision 73%. This test was found to be highly reproducible and rapid (60 minutes) and required less than 100 uL of plasma. We also analyzed mutant p53 protein in plasma samples from 11 p53-mutant and 4 p53-wildtype DMG patients, seeing significant separation between the two groups. We overlayed H3K27M and p53 cell-free protein results with H3.3K27M and TP53 ctDNA variant allele fraction (VAF) by digital droplet PCR (ddPCR) in serial plasma samples from n = 3 DMG patients undergoing experimental therapy. These data highlighted unique patterns of disease response by (1) MRI, (2) plasma tumor protein, and (3) plasma ctDNA. Overall, these results demonstrate the clinical potential of our platform for non-invasive and accurate detection and monitoring of levels of key oncoproteins in plasma of DMG patients.