Background Diffuse Midline Glioma (DMG) with the H3K27M mutation is a lethal childhood brain cancer, with patients rarely surviving 2 years from diagnosis. Methods We conducted a multi-site Phase 1 trial of the imipridone ONC201 for children with H3K27M-mutant glioma (NCT03416530). Patients enrolled on Arm D of the trial (n=24) underwent serial lumbar puncture for cell-free tumor DNA (cf-tDNA) analysis and patients on all arms at the University of Michigan underwent serial plasma collection. We performed digital droplet polymerase chain reaction (ddPCR) analysis of cf-tDNA samples and compared variant allele fraction (VAF) to radiographic change (maximal 2D tumor area on MRI). Results Change in H3.3K27M VAF over time (“VAF delta”) correlated with prolonged PFS in both CSF and plasma samples. Non-recurrent patients that had a decrease in CSF VAF displayed a longer progression free survival (p=0.049). Decrease in plasma VAF displayed a similar trend (p=0.085). VAF “spikes” (increase of at least 25%) preceded tumor progression in 8/16 cases (50%) in plasma and 5/11 cases (45.4%) in CSF. In individual cases, early reduction in H3K27M VAF predicted long-term clinical response (>1 year) to ONC201, and did not increase in cases of later-defined pseudo-progression. Conclusion Our work demonstrates the feasibility and potential utility of serial cf-tDNA in both plasma and CSF of DMG patients to supplement radiographic monitoring. Patterns of change in H3K27M VAF over time demonstrate clinical utility in terms of predicting progression and sustained response and possible differentiation of pseudo-progression and pseudo-response.

Purpose: Pediatric high-grade glioma (pHGG) diagnosis portends poor prognosis and therapeutic monitoring remains difficult. Tumors release cell-free tumor DNA (cf-tDNA) into cerebrospinal fluid (CSF), allowing for potential detection of tumor-associated mutations by CSF sampling. We hypothesized that direct, electronic analysis of cf-tDNA with a handheld platform (Oxford Nanopore MinION) could quantify patient-specific CSF cf-tDNA variant allele fraction (VAF) with improved speed and limit of detection compared with established methods. Experimental Design: We performed ultra-short fragment (100–200 bp) PCR amplification of cf-tDNA for clinically actionable alterations in CSF and tumor samples from patients with pHGG (n = 12) alongside nontumor CSF (n = 6). PCR products underwent rapid amplicon-based sequencing by Oxford Nanopore Technology (Nanopore) with quantification of VAF. Additional comparison to next-generation sequencing (NGS) and droplet digital PCR (ddPCR) was performed. Results: Nanopore demonstrated 85% sensitivity and 100% specificity in CSF samples (n = 127 replicates) with 0.1 femtomole DNA limit of detection and 12-hour results, all of which compared favorably with NGS. Multiplexed analysis provided concurrent analysis of H3.3A (H3F3A) and H3C2 (HIST1H3B) mutations in a nonbiopsied patient and results were confirmed by ddPCR. Serial CSF cf-tDNA sequencing by Nanopore demonstrated correlation of radiological response on a clinical trial, with one patient showing dramatic multi-gene molecular response that predicted long-term clinical response. Conclusions: Nanopore sequencing of ultra-short pHGG CSF cf-tDNA fragments is feasible, efficient, and sensitive with low-input samples thus overcoming many of the barriers restricting wider use of CSF cf-tDNA diagnosis and monitoring in this patient population.

Pediatric high-grade glioma (pHGG), including both diffuse midline glioma (DMG) and non-midline tumors, continues to be one of the deadliest oncologic diagnoses (both henceforth referred to as “pHGG”). Targeted therapy options aimed at key oncogenic receptor tyrosine kinase (RTK) drivers using small-molecule RTK inhibitors has been extensively studied, but the absence of proper in vivo modeling that recapitulate pHGG biology has historically been a research challenge. Thankfully, there have been many recent advances in animal modeling, including Cre-inducible transgenic models, as well as intra-uterine electroporation (IUE) models, which closely recapitulate the salient features of human pHGG tumors. Over 20% of pHGG have been found in sequencing studies to have alterations in platelet derived growth factor-alpha (PDGFRA), making growth factor modeling and inhibition via targeted tyrosine kinases a rich vein of interest. With commonly found alterations in other growth factors, including FGFR, EGFR, VEGFR as well as RET, MET, and ALK, it is necessary to model those receptors, as well. Here we review the recent advances in murine modeling and precision targeting of the most important RTKs in their clinical context. We additionally provide a review of current work in the field with several small molecule RTK inhibitors used in pre-clinical or clinical settings for treatment of pHGG.

Gliomas are tumors derived from mutations in glial brain cells. Gliomas cause significant morbidity and mortality and development of precision diagnostics and novel targeted immunotherapies are critically important. Radiographic imaging is the most common technique to diagnose and track response to treatment, but is an imperfect tool. Imaging does not provide molecular information, which is becoming critically important for identifying targeted immunotherapies and monitoring tumor evolution. Furthermore, immunotherapy induced inflammation can masquerade as tumor progression in images (pseudoprogression) and confound clinical decision making. More recently, circulating cell free tumor DNA (cf-tDNA) has been investigated as a promising biomarker for minimally invasive glioma diagnosis and disease monitoring. cf-tDNA is shed by gliomas into surrounding biofluids (e.g. cerebrospinal fluid and plasma) and, if precisely quantified, might provide a quantitative measure of tumor burden to help resolve pseudoprogression. cf-tDNA can also identify tumor genetic mutations to help guide targeted therapies. However, due to low concentrations of cf-tDNA, recovery and analysis remains challenging. Plasma cf-tDNA typically represents <1% of total cf-DNA due to the blood-brain barrier, limiting their usefulness in practice and motivating the development and use of highly sensitive and specific detection methods. This mini review summarizes the current and future trends of various approaches for cf-tDNA detection and analysis, including new methods that promise more rapid, lower-cost, and accessible diagnostics. We also review the most recent clinical case studies for longitudinal disease monitoring and highlight focus areas, such as novel accurate detection methodologies, as critical research priorities to enable translation to clinic.

BACKGROUND For pediatric high-grade glioma (pHGG), non-invasive methods for diagnosis and surveillance are needed. Tumors release DNA (tDNA) into cerebrospinal fluid (CSF), allowing for detection of tumor-associated mutations by CSF sampling. We hypothesized that direct, electronic analysis of tDNA with a novel, hand-held platform (Oxford Nanopore MinION) could quantify patient-specific CSF tDNA variant allele fraction (VAF) with improved speed and limit of detection compared to established methods. METHODS We integrated required multi-timepoint (0, 2, and 6 months) correlate lumbar punctures (LP) in two ongoing pHGG clinical trials. Using Nanopore technology, we performed amplicon-based PCR on CSF tDNA for recurrent mutations from patient samples (n=19) and normal controls. VAF were determined via MinKNOW, Guppy, MiniMap2, and Integrated Genome Browser. RESULTS Nanopore CSF tDNA demonstrated improved sensitivity (91%) when compare to NGS sequencing (50%). Nanopore analysis of serially diluted CSF sample demonstrated significantly lower limit of detection (attomolar) than typical NGS sample requirement (nanomolar). H3K27M mutation was reliably detected with 1,000x depth sequencing, which was achieved in less than 15 minutes of sequencing after amplification. Multiplexed Nanopore analysis of H3F3A and HIST1H3B was employed when H3 status was unknown. Serial CSF tDNA analysis confirmed multi-gene (H3F3A K27M, PIK3CA, and TP53) molecular remission in a 17-year-old with thalamic diffuse midline glioma that correlated with sustained clinical response to ONC201 (14 months and ongoing). CONCLUSIONS Use of a hand-held, electronic DNA analysis platform allows quantification of multi-gene molecular response with improved speed and limit of detection in the CSF of children with high-grade glioma.