Analytical and Clinical Validation of an H3K27M ctDNA Assay for Diffuse Midline Glioma (DMG) Surveillance

About This Grant

PROJECT SUMMARY / ABSTRACT Diffuse Midline Gliomas (DMGs) are a type of brain tumor with a survival rate of less than 10% beyond two years from diagnosis. Approximately two-thirds of DMGs have mutations in histone H3.3 in H3.3A (H3F3A K27M). Disease monitoring for DMG patients is generally limited to periodic MRI scans, from which progression is difficult to determine. Recent work indicates that circulating tumor DNA (cf-tDNA) in cerebrospinal fluid (CSF) and plasma of patients with H3K27M-DMG can be quantified using a droplet digital polymerase chain reaction (ddPCR) assay and can offer key information that supplements and often precedes a tumor’s response to treatment on MRI. This assay has established a low-level variant allele frequency (VAF) of 0.01% that can be validated as a cf-tDNA positive state (ctDNA+) to indicate diagnosis or earlier detection of progression. However, the novel H3K27M ddPCR assay remains “research grade” and not yet designed for clinical-grade repeatability and reliability. Currently, there are no Clinical Laboratory Improvement Amendments (CLIA)-certified molecular tests (academic or commercial) for H3K27M detection that are sensitive enough to track low levels of tumor DNA (<1%) in patient plasma. Therefore, there is a critical need to establish a clinical- grade H3K27M plasma assay with sensitivity for the diagnosis and therapeutic monitoring of H3K27M-DMG therapy. The central hypothesis is that the H3K27M cf-tDNA ddPCR assay (i) will meet the necessary assay performance milestones to advance our CLIA-compliant, analytically validated LDT into clinical testing (UH2) and (ii) will have a positive predictive value of 90%+ for a positive cf-tDNA result (>0.01%) to predict or correlate with radiographic progression within 2 months. Aim 1 (UH2): To analytically validate an H3K27M ctDNA plasma assay as a lab developed test. The objective of this aim is to systematically define the analytic performance characteristics of this high-performance H3K27M cf-tDNA assay using synthetic controls, true positives, and clinical specimens for analysis in a CLIA-compliant setting. Upon completion of this aim, the assay performance milestones necessary to advance this CLIA-compliant, analytically validated LDT into clinical testing (UH3) will be met. Aim 2 (UH3): To evaluate the predictive properties of an H3K27M ctDNA lab developed test for prediction of recurrence in a cohort of DMG patients. This team co-leads an ongoing phase 2 clinical trial to explore the benefit of ONC201 and paxalisib in H3K27M-DMG [PNOC22, NCT04773782]. This project will leverage retrospective-prospective H3K27M+ DMG clinical validation cohorts drawn from PNOC22, comprising 75 (training cohort) and 50 (test cohort) patients who have completed treatment for H3K27M-DMG and provided plasma specimens every two months during therapy. This project represents a critical and feasible step towards a liquid biomarker to supplement MRI monitoring in H3K27M- DMG.