ABSTRACT
ATRX, a chromatin remodeler protein, is recurrently mutated in H3F3A-mutant pediatric glioblastoma (GBM) and isocitrate dehydrogenase (IDH)-mutant grade 2/3 adult glioma. Previous work has shown that ATRX-deficient GBM cells show enhanced sensitivity to irradiation, but the etiology remains unclear. We find that ATRX binds the regulatory elements of cell-cycle phase transition genes in GBM cells, and there is a marked reduction in Checkpoint Kinase 1 (CHEK1) expression with ATRX loss, leading to the early release of G2/M entry after irradiation. ATRX-deficient cells exhibit enhanced activation of master cell-cycle regulator ATM with irradiation. Addition of the ATM inhibitor AZD0156 doubles median survival in mice intracranially implanted with ATRX-deficient GBM cells, which is not seen in ATRX-wild-type controls. This study demonstrates that ATRX-deficient high-grade gliomas (HGGs) display Chk1-mediated dysregulation of cell-cycle phase transitions, which opens a window for therapies targeting this phenotype.
Subject(s)
Checkpoint Kinase 1/metabolism , Glioma/metabolism , X-linked Nuclear Protein/metabolism , Animals , Brain Neoplasms/metabolism , Cell Cycle/genetics , Cell Cycle Checkpoints/genetics , Cell Line, Tumor , Checkpoint Kinase 1/physiology , Female , Histones/metabolism , Humans , Isocitrate Dehydrogenase/genetics , Male , Mice , Mice, Inbred C57BL , Mutation , Neoplasm Recurrence, Local/metabolism , Primary Cell Culture , X-linked Nuclear Protein/geneticsABSTRACT
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.
Subject(s)
Biomarkers, Tumor/genetics , Brain Neoplasms/pathology , Circulating Tumor DNA/genetics , Electronics , Glioma/pathology , Mutation , Adolescent , Biomarkers, Tumor/cerebrospinal fluid , Brain Neoplasms/cerebrospinal fluid , Brain Neoplasms/genetics , Brain Neoplasms/surgery , Case-Control Studies , Child , Child, Preschool , Circulating Tumor DNA/cerebrospinal fluid , Female , Follow-Up Studies , Glioma/cerebrospinal fluid , Glioma/genetics , Glioma/surgery , Humans , Male , Polymerase Chain Reaction , PrognosisABSTRACT
Children's Oncology Group (COG) has been highly successful in improving childhood cancer survival through well-designed multi-institutional clinical trials. However, our center has recognized a decline in the number of enrollments on COG therapeutic clinical trials over recent years. Our single center, retrospective analysis evaluated in detail the patient enrollment rates, annual number of available clinical trials and reason for nonenrollment over the last decade. We found a 61% decrease in enrollment for phase II to III trials of newly diagnosed patients at our center (2011-2018) along a 29% decrease in the number of open COG studies annually. The primary reason for nonenrollment was unavailability of a suitable trial (76%). We also recognized a decrease in number of adolescent and young adult enrollment particularly in the last 8 years (2010-2018); however, the enrollment rate for adolescent and young adults was not substantially different than enrollment of children. The reasons for reduced enrollments are most likely multifactorial and complex. It is imperative that we continue to develop novel clinical studies using a portfolio of federal, investigator-initiated, and industry trials for pediatric oncology patients to continue to advance outcomes, study survivorship, and improve quality of life for these patients.
