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Nanopore Sensitivity/Specificity details

All supplementary data except table S3A and S3B

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.

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.

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.

BackgroundPediatric and adult high-grade glioma (HGG) frequently harbor PDGFRA alterations. We hypothesized that co-treatment with everolimus may improve the efficacy of dasatinib in PDGFRα-driven glioma through combinatorial synergism and increased tumor accumulation of dasatinib.MethodsDose response, synergism studies, P-gp inhibition and pharmacokinetic studies were performed on in vitro and in vivo human and mouse models of HGG. Six patients with recurrent PDGFRα-driven glioma were treated with dasatinib and everolimus.ResultsDasatinib effectively inhibited the proliferation of mouse and human primary HGG cells with a variety of PDGFRA alterations. Dasatinib exhibited synergy with everolimus in the treatment of HGG cells at low nanomolar concentrations of both agents, with reduction in mTOR signaling that persists after dasatinib treatment alone. Prolonged exposure to everolimus significantly improved the CNS retention of dasatinib and extended survival of PPK tumor bearing mice. Pediatric patients (n=6) with glioma tolerated this combination without significant adverse events. Recurrent patients (n=4) demonstrated median overall survival of 8.5 months.ConclusionEfficacy of dasatinib treatment of PDGFRα-driven HGG is improved with everolimus and suggests a promising route for improving targeted therapy for this patient population.Trial RegistrationClinicalTrials.gov NCT03352427FundingThe authors thank the patients and their families for participation in this study. CK is supported by NIH/NINDS K08-NS099427-01, the University of Michigan Chad Carr Pediatric Brain Tumor Center, the Chad Tough Foundation, Hyundai Hope on Wheels, Catching up With Jack, Prayers from Maria Foundation, U CAN-CER VIVE FOUNDATION, Morgan Behen Golf Classic, and the DIPG Collaborative. The PEDS-MIONCOSEQ study was supported by grant 1UM1HG006508 from the National Institutes of Health Clinical Sequencing Exploratory Research Award (PI: Arul Chinnaiyan).

Diffuse midline glioma (DMG) with H3K27M mutation is a lethal childhood brain cancer, with limited means of monitoring beyond serial MRI scans. We conducted a multi-site Phase 1 trial of the imipridone ONC201 for children with H3K27M-mutant glioma (NCT03416530). Patients on Arm D of the trial (n=24) underwent serial lumbar puncture (baseline, 2 and 6-months) for cell-free tumor DNA (cf-tDNA) analysis at time of MRI. Additionally, patients on all arms of the trial at the University of Michigan underwent serial plasma collection. We collected a total of 96 plasma-samples and 53 CSF-samples from 29 patients. We performed ddPCR analysis of cf-tDNA samples and compared variant allele fraction (VAF) to radiographic change (maximal tumor area on MRI). For our H3F3A-mutated (K27M) patients, cf-tDNA was positive in 53/62 plasma samples (sensitivity 85.4%) and 28/29 CSF samples (sensitivity 96.5%) and overall specificity of 100%. There was no direct correlation between percent-change in tumor-area and plasma (p=0.47) or CSF VAF (p=0.89), implying that VAF provided information supplemental to radiographic assessments. “Spikes” in plasma cf-tDNA VAF (increase of ≥25%) co-occurred with progression in 2/9 (22%) cases and preceded progression in 5/9 cases (55%) by an average of 1.22 months. In CSF, spikes preceded progression in 4/6 cases (66%) by an average of 1.8 months. Two patients had increases in tumor-area with no increase in plasma VAF; both were later confirmed as pseudo-progressors, suggesting additional potential utility of cf-tDNA VAF monitoring. A 14yo male with spinal cord glioma received concurrent bevacizumab with ONC201, which resulted in a decrease in tumor area but continued increase in plasma VAF, predicting radiologic progression at the next time. In summary, we present data which suggests monitoring serial CSF/plasma H3K27M tDNA is a promising clinical tool. Changes in cf-tDNAVAF over time appear to correlate with response, predict progression, and differentiate pseudo-progression.

Diffuse midline glioma (DMG) is a highly morbid pediatric brain tumor. Up to 80% of DMGs harbor mutations in histone H3-encoding genes, associated with poor prognosis. We previously showed the feasibility of detecting H3 mutations in circulating tumor DNA (ctDNA) in the liquid biome of children diagnosed with DMG. However, detection of low levels of ctDNA is highly dependent on platform sensitivity and sample type. To address this, we optimized ctDNA detection sensitivity and specificity across two commonly used digital droplet PCR (ddPCR) platforms (RainDance and BioRad), and validated methods for detecting H3F3A c.83A > T (H3.3K27M) mutations in DMG CSF, plasma, and primary tumor specimens across three different institutions. DNA was extracted from H3.3K27M mutant and H3 wildtype (H3WT) specimens, including H3.3K27M tumor tissue (n = 4), CSF (n = 6), plasma (n = 4), and human primary pediatric glioma cells (H3.3K27M, n = 2; H3WT, n = 1). ctDNA detection was enhanced via PCR pre-amplification and use of distinct custom primers and fluorescent LNA probes for c.83 A > T H3F3A mutation detection. Mutation allelic frequency (MAF) was determined and validated through parallel analysis of matched H3.3K27M tissue specimens (n = 3). We determined technical nuances between ddPCR instruments, and optimized sample preparation and sequencing protocols for H3.3K27M mutation detection and quantification. We observed 100% sensitivity and specificity for mutation detection in matched DMG tissue and CSF across assays, platforms and institutions. ctDNA is reliably and reproducibly detected in the liquid biome using ddPCR, representing a clinically feasible, reproducible, and minimally invasive approach for DMG diagnosis, molecular subtyping and therapeutic monitoring.

Diffuse midline glioma (DMG) with the H3K27M mutation is a lethal childhood brain cancer, with patients rarely surviving 2 years from diagnosis. We conducted a multi-site Phase 1 trial of the imipridone ONC201 for children with H3K27M-mutant glioma ({"type":"clinical-trial","attrs":{"text":"NCT03416530","term_id":"NCT03416530"}}NCT03416530). Patients enrolled on Arm D of the trial (n=24) underwent serial lumbar puncture (baseline, 2, 6 months) for cell-free tumor DNA (cf-tDNA) analysis at time of MRI. Additionally, patients on all arms of the trial at the University of Michigan underwent serial plasma collection. CSF collection was feasible in this cohort, with no procedural complications. We collected 96 plasma samples and 53 CSF samples from 29 patients, including those with H3F3A (H3.3) (n=13), HIST13HB (H3.1) (n= 4), and unknown H3 status/not biopsied (n=12) [range of 0–8 CSF samples and 0–10 plasma samples]. We performed digital droplet polymerase chain reaction (ddPCR) analysis and/or amplicon-based electronic sequencing (Oxford Nanopore) of cf-tDNA samples and compared variant allele fraction (VAF) to radiographic change (maximal 2D tumor area on MRI). Preliminary analysis of samples demonstrates a correlation between changes in tumor size and H3K27M cf-tDNA VAF, when removing samples with concurrent bevacizumab. In multiple cases, early reduction in CSF cf-tDNA predicts long-term clinical response (>1 year) to ONC201, and does not increase in cases of later-defined pseudo-progression (radiation necrosis). For example, a now 9-year old patient with thalamic H3K27M-mutant DMG underwent treatment with ONC201 after initial radiation and developed increase in tumor size at 4 months post-radiation (124% baseline) of unclear etiology at the time. Meanwhile, her ddPCR declined from baseline 6.76% VAF to

Background Diffuse midline glioma (DMG) is a highly morbid pediatric brain tumor. Up to 80% of DMGs harbor mutations in histone H3-encoding genes, associated with poorer prognosis. We previously showed the feasibility of detecting H3 mutations in circulating tumor DNA (ctDNA) in the liquid biome of children diagnosed with DMG. However, detection of low ctDNA concentrations is highly dependent on platform sensitivity and sample type. To address this, we optimized ctDNA detection sensitivity and specificity across two commonly used digital droplet PCR (ddPCR) platforms (RainDance and BioRad), and validated methods for detecting H3F3A mutations in DMG CSF, plasma, and primary tumor specimens across three different institutions. Methods DNA was extracted from H3.3K27M mutant and H3 wildtype (H3WT) specimens, including H3.3K27M tumor tissue (n=4), CSF (n=6), plasma (n=4), and human primary pediatric glioma cells (H3.3K27M, n=2; H3WT, n=1). ctDNA detection was enhanced via PCR pre-amplification and use of distinct custom primers and fluorescent LNA probes for c.83 AàT H3F3A mutation detection. Mutation allelic frequency (MAF) was determined and validated through parallel analysis of matched H3.3K27M tissue specimens (n=3). Results We determined technical nuances between ddPCR instruments, and optimized sample preparation and sequencing protocols for H3F3A mutation detection and quantification. We observed 100% sensitivity and specificity for mutation detection in matched DMG tissue and CSF across assays, platforms and institutionsConclusion Our study demonstrates that ctDNA is reliably and reproducibly detected in ctDNA using ddPCR, representing a clinically feasible and reproducible minimally invasive approach for DMG diagnosis, molecular subtyping and therapeutic monitoring.

Patients with diffuse midline glioma (DMG) harboring H3 K27M mutation have no proven therapies beyond radiation. ONC201, a DRD2 antagonist and mitochondrial ClpP agonist, has induced early responses in patients with H3 K27M-mutant DMG. We performed an integrated pre-clinical and clinical assessment of ONC201 treatment, in order to define response rates in H3 K27M-mutant DMG patients and to clarify predictors of response. ONC201 was effective in murine H3 K27M-mutant gliomas with excellent CNS penetration and survival benefit. H3 K27M-mutant DMG patients treated with ONC201 on active clinical trials (n=50) showed significant survival benefit in recurrent and non-recurrent settings, with multiple sustained responses. Tumor sequencing from treated patients demonstrates an EGFR/FOXG1-driven telencephalic gene regulatory network that imparts a critical resistance phenotype to ONC201. Genetic and pharmacologic knockdown of EGFR in H3 K27M-mutant cell cultures results in improved sensitivity to ONC201 and reduced FOXG1 enhancer binding, suggesting possible future combinatorial opportunities.

The original version of this review article unfortunately contained a mistake in the author group section.

PURPOSE OF REVIEW: H3K27M is a frequent histone mutation within diffuse midline gliomas and is associated with a dismal prognosis, so much so that the 2016 CNS WHO classification system created a specific category of “Diffuse Midline Glioma, H3K27M-mutant”. Here we outline the latest pre-clinical data and ongoing current clinical trials that target H3K27M, as well as explore diagnosis and treatment monitoring by serial liquid biopsy. RECENT FINDINGS: Multiple epigenetic compounds have demonstrated efficacy and on-target effects in pre-clinical models. The imipridone ONC201 and the IDO1 inhibitor indoximod have demonstrated early clinical activity against H3K27M-mutant gliomas. Liquid biopsy of cerebrospinal fluid has shown promise for clinical use in H3K27M-mutant tumors for diagnosis and monitoring treatment response. SUMMARY: While H3K27M has elicited a widespread platform of pre-clinical therapies with promise, much progress still needs to be made to improve outcomes for diffuse midline glioma patients. We present current treatment and monitoring techniques as well as novel approaches in identifying and targeting H3K27M-mutant gliomas.

2012 Background: Diffuse midline glioma (DMG) with the H3K27M mutation is a lethal childhood brain cancer, with patients rarely surviving 2 years from diagnosis. There are few available means of monitoring the disease beyond serial MRI scans, making clinical decision making slow, difficult, and often reactive. 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 (baseline, 2 and 6 months) for cell-free tumor DNA (cf-tDNA) analysis at time of MRI. Additionally, patients on all arms of the trial at the University of Michigan underwent serial plasma collection. CSF collection was feasible in this cohort, with no procedural complications. We collected a total of 96 plasma samples and 53 CSF samples from 29 patients, including those with H3F3A (H3.3) (n=13), HIST13HB (H3.1) (n= 4), and unknown H3 status/not biopsied (n=12) [range of 0-8 CSF samples and 0-10 plasma samples]. We performed digital droplet polymerase chain reaction (ddPCR) analysis and/or amplicon-based electronic sequencing (Oxford Nanopore) of cf-tDNA samples and compared variant allele fraction (VAF) to radiographic change (maximal 2D tumor area on MRI). Results: Preliminary analysis of samples (n=58) demonstrates a correlation between changes in tumor size and H3K27M cf-tDNA VAF, when removing samples with concurrent bevacizumab. Analysis of remaining CSF and plasma samples is ongoing, including analysis of novel biomarkers of response. In multiple cases, early reduction in CSF cf-tDNA predicts long-term clinical response (>1 year) to ONC201 and does not increase in cases of later-defined pseudo-progression (radiation necrosis). For example, a now 9-year old patient with thalamic H3K27M-mutant DMG underwent treatment with ONC201 after initial radiation and developed an increase in tumor size at 4 months post-radiation (124% baseline) of unclear etiology at the time. Meanwhile, her ddPCR declined from baseline 6.76% VAF to

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.

ONC201, a bitopic DRD2 antagonist and allosteric ClpP agonist, has shown encouraging efficacy in H3 K27M-mutant glioma. Given that the thalamus has the highest extra-striatal expression of DRD2, we performed an integrated preclinical and clinical analysis of ONC201 in thalamic H3 K27M-mutant glioma. ONC201 was effective in mouse intra-uterine electroporation (IUE)-generated H3 K27M-mutant gliomas, with an in vitro IC50 of 500 nM and 50% prolongation of median survival in vivo (p=0.02, n=14). We analyzed thalamic H3 K27M-mutant glioma patients treated with ONC201 on active clinical trials as of 5/22/19 enrollment (n=19 recurrent and 10 post-radiation, non-recurrent; 5–70 years old). As of 12/18/2019, PFS6 and OS12 are 26.3% and 36.8%, respectively, in the recurrent group. For non-recurrent patients, with median follow up of 21.9 months (8.6–26.6) from diagnosis, median PFS or OS have not been reached. This surpasses historical OS of 13.5 months. Best response by RANO includes 1 CR, 3 PR, 4 SD, 8 PD for recurrent patients and 2 PR, 4 SD, 1 PD for non-recurrent patients (4 on-trial patients experienced regressions that are yet unconfirmed responses). Median duration of response for recurrent patients is 14.0 months (2.0–33.1). Furthermore, H3 K27M cell-free tumor DNA in plasma and CSF correlated with MRI response. In summary, single agent ONC201 administered at recurrence, or adjuvantly following radiation, demonstrates promising clinical efficacy in thalamic H3 K27M-mutant glioma patients who currently have no effective treatments following radiation. Investigations are ongoing to assess whether micro-environmental DRD2 expression explains the early exceptional responses in thalamic H3 K27M-mutant glioma.

Primary brain tumors and CNS metastases shed circulating tumor DNA (ctDNA) into the CSF, which can be assessed for tumor-associated mutations. Thus far, there have been no extensive studies using droplet digital PCR (ddPCR) to detect and quantify ctDNA in the CSF of pediatric high-grade brain tumor patients. There are also gaps in our knowledge, including the potential dependence of ctDNA amount on location of sample collection and whether ctDNA can be used to quantify tumor growth and treatment response. To address these questions, we developed a novel H3F3A K27M ddPCR assay and applied it to four pediatric patients with H3F3A K27M-mutant DIPG and GBM. We found that ddPCR was able to detect the K27M mutation in patient CSF and that the closest relation emerged between mutant K27M copies per ng of total DNA (henceforth K27M copies) and contrast-enhancing tumor area on MRI. Multi-focal CSF sampling at autopsy of a DIPG patient exhibited differences in K27M copies by proximity to the tumor. To better understand changes in K27M copies in response to both growth and treatment of DIPG, we developed an in vitro system comprised of astrocytes (NHAs) co-cultured with luciferase-expressing human DIPG cell line DIPG007 as a means to simulate ctDNA release into the CSF. We found that DIPG007 cells released ctDNA into culture media in proportion to their proliferation, even when the media was changed frequently to approximate the constant production and resorption of CSF. Irradiation with 8 Gy resulted in a spike in mutant ctDNA 72–120 hours post-radiotherapy before decreasing. In summary, our study suggests that H3F3A K27M copies in the CSF of children with high-grade brain tumors have a linear relation with contrast-enhancing tumor area and that ddPCR can be used to follow treatment response including ctDNA release shortly after effective therapies.