Liquid Biopsy in H3K27M Diffuse Midline Glioma.

Diffuse midline glioma (DMG) with H3K27M mutation is an aggressive and difficult to treat pediatric brain tumor. Recurrent gain of function mutations in H3.3 (H3.3A) and H3.1 (H3C2) at the 27th lysine to methionine (H3K27M) are seen in over 2/3 of DMGs, and are associated with a worse prognosis. Due to the anatomical location of DMG, traditional biopsy carries risk for neurologic injury as it requires penetration of vital midline structures. Further, radiographic (MRI) monitoring of DMG often shows non-specific changes, which makes therapeutic monitoring difficult. This indicates a critical need for more minimally invasive methods, such as liquid biopsy, to understand, diagnose, and monitor H3K27M DMG. Here we review the use of all modalities to date to detect biomarkers of H3K27M in CSF, blood, and urine, and compare their effectiveness in detection, diagnosis, and monitoring treatment response. We provide specific detail of recent efforts to monitor CSF and plasma H3K27M cell-free DNA in patients undergoing therapy with the imipridone ONC201. Lastly, we discuss the future of therapeutic monitoring of H3K27M-DMG, including biomarkers such as mitochondrial DNA, mutant and modified histones, and novel sequencing-based approaches for improved detection methods.

Single-molecule systems for detection and monitoring of plasma circulating nucleosomes and oncoproteins in Diffuse Midline Glioma.

The analysis of cell-free tumor DNA (ctDNA) and proteins in the blood of cancer patients potentiates a new generation of non-invasive diagnostics and treatment monitoring approaches. However, confident detection of these tumor-originating markers is challenging, especially in the context of brain tumors, in which extremely low amounts of these analytes circulate in the patient's plasma. Here, we applied a sensitive single-molecule technology to profile multiple histone modifications on millions of individual nucleosomes from the plasma of Diffuse Midline Glioma (DMG) patients. The system reveals epigenetic patterns that are unique to DMG, significantly differentiating this group of patients from healthy subjects or individuals diagnosed with other cancer types. We further develop a method to directly capture and quantify the tumor-originating oncoproteins, H3-K27M and mutant p53, from the plasma of children diagnosed with DMG. This single-molecule system allows for accurate molecular classification of patients, utilizing less than 1ml of liquid-biopsy material. Furthermore, we show that our simple and rapid detection strategy correlates with MRI measurements and droplet-digital PCR (ddPCR) measurements of ctDNA, highlighting the utility of this approach for non-invasive treatment monitoring of DMG patients. This work underscores the clinical potential of single-molecule-based, multi-parametric assays for DMG diagnosis and treatment monitoring.

Rapid Real-time Squiggle Classification for Read until using RawMap.

ReadUntil enables Oxford Nanopore Technology's (ONT) sequencers to selectively sequence reads of target species in real-time. This enables efficient microbial enrichment for applications such as microbial abundance estimation and is particularly beneficial for metagenomic samples with a very high fraction of non-target reads (> 99% can be human reads). However, read-until requires a fast and accurate software filter that analyzes a short prefix of a read and determines if it belongs to a microbe of interest (target) or not. The baseline Read Until pipeline uses a deep neural network-based basecaller called Guppy and is slow and inaccurate for this task (~60% of bases sequenced are unclassified). We present RawMap, an efficient CPU-only microbial species-agnostic Read Until classifier for filtering non-target human reads in the squiggle space. RawMap uses a Support Vector Machine (SVM), which is trained to distinguish human from microbe using non-linear and non-stationary characteristics of ONT's squiggle output (continuous electrical signals). Compared to the baseline Read Until pipeline, RawMap is a 1327X faster classifier and significantly improves the sequencing time and cost, and compute time savings. We show that RawMap augmented pipelines reduce sequencing time and cost by ~24% and computing cost by 22%. Additionally, since RawMap is agnostic to microbial species, it can also classify microbial species it is not trained on. We also discuss how RawMap may be used as an alternative to the RT-PCR test for viral load quantification of SARS-CoV-2.

Ultra-rapid somatic variant detection via real-time targeted amplicon sequencing.

Molecular markers are essential for cancer diagnosis, clinical trial enrollment, and some surgical decision making, motivating ultra-rapid, intraoperative variant detection. Sequencing-based detection is considered the gold standard approach, but typically takes hours to perform due to time-consuming DNA extraction, targeted amplification, and library preparation times. In this work, we present a proof-of-principle approach for sub-1 hour targeted variant detection using real-time DNA sequencers. By modifying existing protocols, optimizing for diagnostic time-to-result, we demonstrate confirmation of a hot-spot mutation from tumor tissue in ~52 minutes. To further reduce time, we explore rapid, targeted Loop-mediated Isothermal Amplification (LAMP) and design a bioinformatics tool-LAMPrey-to process sequenced LAMP product. LAMPrey's concatemer aware alignment algorithm is designed to maximize recovery of diagnostically relevant information leading to a more rapid detection versus standard read alignment approaches. Using LAMPrey, we demonstrate confirmation of a hot-spot mutation (250x support) from tumor tissue in less than 30 minutes.

Cell-Free Tumor DNA (cf-tDNA) Liquid Biopsy: Current Methods and Use in Brain Tumor Immunotherapy.

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.

Serial H3K27M cell-free tumor DNA (cf-tDNA) tracking predicts ONC201 treatment response and progression in diffuse midline glioma.

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. Nonrecurrent patients that had a decrease in CSF VAF displayed a longer progression free survival (P = .0042). Decrease in plasma VAF displayed a similar trend (P = .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.

Liquid biopsy in pediatric brain tumors.

Malignant primary brain tumors are the most common cancer in children aged 0-14 years, and are the most common cause of death among pediatric cancer patients. Compared to other cancers, pediatric brain tumors have been difficult to diagnose and study given the high risk of intracranial biopsy penetrating through vital midline structures, where the majority of pediatric brain tumors originate (Ostrom et al., 2015). Furthermore, the vast majority of these tumors recur. With limitations in the ability to monitor using clinical and radiographic methods alone, minimally invasive methods such as liquid biopsy will be crucial to our understanding and treatment. Liquid biopsy of blood, urine, and cerebrospinal fluid (CSF) can be used to sample cfDNA, ctDNA, RNA, extracellular vesicles, and tumor-associated proteins. In the past year, four seminal papers have made significant advances in the use of liquid biopsy in pediatric brain tumor patients (Liu et al., 2021; Cantor et al., 2022; Miller et al., 2022; Pagès et al., 2022). In this review, we integrate the results of these studies and others to discuss how the newest technologies in liquid biopsy are being developed for molecular diagnosis and treatment response in pediatric brain tumors.

SquiggleNet: real-time, direct classification of nanopore signals.

We present SquiggleNet, the first deep-learning model that can classify nanopore reads directly from their electrical signals. SquiggleNet operates faster than DNA passes through the pore, allowing real-time classification and read ejection. Using 1 s of sequencing data, the classifier achieves significantly higher accuracy than base calling followed by sequence alignment. Our approach is also faster and requires an order of magnitude less memory than alignment-based approaches. SquiggleNet distinguished human from bacterial DNA with over 90% accuracy, generalized to unseen bacterial species in a human respiratory meta genome sample, and accurately classified sequences containing human long interspersed repeat elements.

Electronic DNA Analysis of CSF Cell-free Tumor DNA to Quantify Multi-gene Molecular Response in Pediatric High-grade Glioma.

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.