INNV-06. DETECTION OF MUTANT P53 POSITIVE GLIOBLASTOMA CELLS IN CEREBROSPINAL FLUID (CSF) USING A MICROFLUIDIC BASED IMMUNOASSAY

Abstract

Abstract BACKGROUND Metastatic involvement of the CSF by non-CNS neoplasms surpasses that of primary brain tumors, although conventional glioblastoma (GBM) can occasionally be identified in the CSF. Here, we apply Biocept’s CNSide test to examine a patient CSF sample with GBM, verified with an antibody against mutant p53. The p53 pathway is deregulated in 84% of glioblastoma patients and point mutations lead to expression of mutant p53 protein. METHODS A CSF sample from 65-year-old male patient, with GBM diagnosed by MRI & CT, and prior identified p53 mutation on R273C by NGS was collected into Biocept’s CSF collection tubes at Providence Saint John’s Health Center. CSF-cells were incubated, 2 days post collection, with a proprietary antibody cocktail, including anti-CD9, followed with a biotinylated secondary, which enables enrichment of capture antibody labeled cells in a streptavidin coated microfluidic device. Cells were fixed and permeabilized immunofluorescence was performed against mutant p53, CD45, fluorescently labeled streptavidin, for capture antibody detection, and DAPI. Microfluidic chips were scanned and analyzed on a Bioview system. The specificity of the mutant p53 antibody was verified using the same test on tumor cells with either wildtype p53 vs various p53 mutations. RESULTS CSF analysis by the CNSide platform detected 3 tumor cells / mL. Immunofluorescence confirmed strong expression of mutant p53 protein on about 1/3 of cells identified as tumor cells. Besides the patient R273C mutation, we demonstrated detection of P223L/V274F, E285K and Q331R mutations on tumor cell lines. DISCUSSION Our work suggests that a microfluidic based GBM capture test paired with appropriate biomarkers can be used as sensitive means to detect GBM cells in CSF and may be useful for diagnostics and treatment monitoring. Further, we demonstrated the utility of a mutant p53 antibody to identify tumor cells. Technical and clinical studies are needed to substantiate this hypothesis.