Molecular subtyping of brain tumors using a blood-based multiplexed assay

SUMMARY Gliomas are typically characterized by high malignancy and poor prognosis. The most recent WHO classification of CNS neoplasms has advanced the diagnostic framework to stratify disease by incorporating tumor histological features and genetic/molecular mutations. The current standard of diagnosis and monitoring is neuroimaging followed by invasive tumor tissue biopsy. This modality, while beneficial to some, provides only a localized snapshot of tumor mutational burden and is often unrealistic for a large population of patients with tumors in complex anatomic locations. In the last decade, liquid biopsy has risen to the forefront of personalized, minimally-invasive disease diagnosis and monitoring, posing as a promising alternative to tumor tissue biopsy. Previous work on liquid biopsy in our lab achieved a significant breakthrough in the detection of rare mutations using a highly-sensitive digital PCR technology. Through rigorous testing, we developed highly sensitive and specific blood-based assays for three common mutations in gliomas: TERT C228T/C250T, EGFRvIII, IDH1.R132H. We have demonstrated the clinical significance of our tests which enable quantification of tumor mutational burden for accurate classification of the lesions and facilitate therapeutic decision-making as well as disease prognostication and monitoring. We now propose the development of GLIscan, a multi-analyte, multi-mutation blood-based assay for rapid, sensitive molecular subtyping of gliomas via mutation (TERT C228T/C250T, EGFRvIII, IDH1.R132H) and control gene (HPRT1) quantification in a single-use test. This multi-phase study will optimize, verify, and validate GLIscan in adherence with industry standards with the intention of establishing an assay for CLIA-validation and clinical implementation post- award. Phase 1 will consist of multiplexed assay optimization in tumor tissue and plasma spike-in experiments via comparison of multi-/single- assay performance and input analytes. Following rigorous testing to develop assay reproducibility, we will verify performance in a larger clinical cohort through inter- and intra-laboratory testing to establish a rapid tumor tissue test as well as a blood-based assay. At this stage, assay performance metrics of precision, accuracy, specificity, as well as the limits of detection, blank, and quantification, will be determined. The final phase of the study will consist of a large, blinded cohort validation in accordance with industry standards. The cumulative results from patient sample testing will be used to develop a machine learning algorithm to establish GLIscan as a highly sensitive and specific Multianalyte Liquid Biopsy Assay with Algorithmic Analysis. GLIscan can significantly impact the overall clinical care of gliomas, providing a modality for minimally-invasive disease diagnosis, stratification, and monitoring to guide clinical decision making, minimizing delays in intervention and improving patient overall survival. Founded on robust, industry- standard quality assurance measures, GLIscan will translate to industry validation for reliable and reproducible development of a novel, widely accessible tool for clinical application.