Pediatric Diffuse Midline Glioma Research Articles

BMP2 and BMP7 cooperate with H3.3K27M to promote quiescence and invasiveness in pediatric diffuse midline gliomas.

Pediatric diffuse midline gliomas (pDMG) are an aggressive type of childhood cancer with a fatal outcome. Their major epigenetic determinism has become clear, notably with the identification of K27M mutations in histone H3. However, the synergistic oncogenic mechanisms that induce and maintain tumor cell phenotype have yet to be deciphered. In 20 to 30% of cases, these tumors have an altered BMP signaling pathway with an oncogenic mutation on the BMP type I receptor ALK2, encoded by ACVR1. However, the potential impact of the BMP pathway in tumors non-mutated for ACVR1 is less clear. By integrating bulk, single-cell, and spatial transcriptomic data, we show here that the BMP signaling pathway is activated at similar levels between ACVR1 wild-type and mutant tumors and identify BMP2 and BMP7 as putative activators of the pathway in a specific subpopulation of cells. By using both pediatric isogenic glioma lines genetically modified to overexpress H3.3K27M and patients-derived DIPG cell lines, we demonstrate that BMP2/7 synergizes with H3.3K27M to induce a transcriptomic rewiring associated with a quiescent but invasive cell state. These data suggest a generic oncogenic role for the BMP pathway in gliomagenesis of pDMG and pave the way for specific targeting of downstream effectors mediating the K27M/BMP crosstalk.

BMP2 and BMP7 cooperate with H3.3K27M to promote quiescence and invasiveness in pediatric diffuse midline gliomas

Pediatric diffuse midline gliomas (pDMG) are an aggressive type of childhood cancer with a fatal outcome. Their major epigenetic determinism has become clear, notably with the identification of K27M mutations in histone H3. However, the synergistic oncogenic mechanisms that induce and maintain tumor cell phenotype have yet to be deciphered. In 20 to 30% of cases, these tumors have an altered BMP signaling pathway with an oncogenic mutation on the BMP type I receptor ALK2, encoded by ACVR1. However, the potential impact of the BMP pathway in tumors non-mutated for ACVR1 is less clear. By integrating bulk, single-cell, and spatial transcriptomic data, we show here that the BMP signaling pathway is activated at similar levels between ACVR1 wild-type and mutant tumors and identify BMP2 and BMP7 as putative activators of the pathway in a specific subpopulation of cells. By using both pediatric isogenic glioma lines genetically modified to overexpress H3.3K27M and patients-derived DIPG cell lines, we demonstrate that BMP2/7 synergizes with H3.3K27M to induce a transcriptomic rewiring associated with a quiescent but invasive cell state. These data suggest a generic oncogenic role for the BMP pathway in gliomagenesis of pDMG and pave the way for specific targeting of downstream effectors mediating the K27M/BMP crosstalk.

Combination of locoregional radiotherapy with a TIM-3 aptamer improves survival in diffuse midline glioma models.

Pediatric diffuse midline gliomas (DMG) with altered H3-K27M are aggressive brain tumors that arise during childhood. Despite advances in genomic knowledge and the significant number of clinical trials testing new targeted therapies, patient outcomes are still poor. Immune checkpoint blockades with small molecules, such as aptamers, are opening new therapeutic options that represent hope for this orphan disease. Here, we demonstrated that a TIM-3 aptamer (TIM-3 Apt) as monotherapy increased the immune infiltration and elicited a strong specific immune response with a tendency to improve the overall survival of treated DMG-bearing mice. Importantly, combining TIM-3 Apt with radiotherapy increased the overall median survival and led to long-term survivor mice in 2 pediatric DMG orthotopic murine models. Interestingly, TIM-3 Apt administration increased the number of myeloid populations and the proinflammatory CD8-to-Tregs ratios in the tumor microenvironment as compared with nontreated groups after radiotherapy. Importantly, the depletion of T cells led to a major loss of the therapeutic effect achieved by the combination. This work uncovers TIM-3 targeting as an immunotherapy approach to improve the radiotherapy outcome in DMGs and offers a strong foundation for propelling a phase I clinical trial using radiotherapy and TIM-3 blockade combination as a treatment for these tumors.

DIPG-08. PROBING THE BLOOD-BRAIN-TUMOR-BARRIER WITH A MICROVASCULAR MODEL OF DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Pediatric diffuse midline glioma (DMG) is a uniformly fatal brain tumor in children, with most patients surviving less than 12 months. Current treatment for DMG is limited, and outcomes for this tumor have not improved. The blood-brain-barrier (BBB) remains a key challenge for DMG therapeutics, preventing most drugs from entering brain tissue. Functional characterization of the blood-brain-tumor-barrier (BBTB) – the interaction between tumors and BBB vasculature – remains largely unexplored for DMG. There is a need for a preclinical model of DMG that can probe the functional, physiological, and biological properties of the BBTB in vitro as a platform to evaluate therapeutic efficacy that is representative of human physiology. METHODS To generate an in vitro model of DMG with human microvasculature (DMG-BBTB model), we co-cultured DMG neurospheres, human brain microvascular endothelial cells, human brain vascular pericytes, and human astrocytes. Cells are simultaneously seeded into microfluidic devices at varied concentrations within a fibrin matrix. Self-assembled microvascular networks are formed within 7-8 days and used for downstream functional and phenotypic assays. RESULTS Vascular morphology and solute permeability in microvascular networks are influenced by cell identity, growth pattern, and relative concentration. We show that DMG neurospheres exhibit different patterns of growth when co-cultured as uniform spheroids versus single cells in suspension. Devices representing each growth pattern are further characterized for paracellular diffusion and permeability compared to devices without DMG cells through perfusion of high molecular weight dextran. Permeability values are correlated to biologic features such as the abundance and localization of junctional proteins and transporters. CONCLUSIONS We developed a platform to evaluate the microvasculature of the DMG BBTB as a tool to develop new therapies informed by vascular biology. We probed the functional, physiological, and biological properties of the human DMG BBTB in vitro, and in ongoing work are assessing approved and experimental therapeutics.

DIPG-03. VENTRICULAR CEREBROSPINAL FLUID SAMPLING IN PEDIATRIC DIFFUSE MIDLINE GLIOMA PATIENTS: INSTITUTIONAL EXPERIENCE AND REVIEW OF THE LITERATURE

Abstract BACKGROUND Increasing evidence suggests that circulating biomarkers may serve diagnostic and longitudinal monitoring purposes in pediatric neuro-oncology. Mutant tumor DNA is detectable in the cerebrospinal fluid (CSF) of pediatric diffuse midline glioma (DMG) patients and quantity can reflect disease burden. CSF sampling (“liquid biopsy”) via a CSF access device could therefore play a role in DMG management. Therefore, we set to evaluate the incidence of hydrocephalus (HCP) in DMG patients, and to characterize ventricular reservoir placement and access practices. METHODS A single institution retrospective review of DMG patients <21-years-old was performed (1984-2019). MEDLINE searches for reports of ventricular reservoir or shunt placement in DMG, and reservoir access for intraventricular chemotherapy (IVC) were performed. RESULTS At our institution, 62.6% of DMG patients (67/108) required intervention for HCP: 19.4% provided transient CSF access (ETV alone n=3, EVD n=8, unspecified n=2), and 80.6% permanent CSF access (ETV + reservoir n=13, shunt n=41). Further, 22/34 patients with initially transient CSF devices required conversion to a permanent device. Five devices were revised for malfunction, one for infection. Seventeen articles cited HCP in 22% to 100% of DMG patients. IVC administration was described in 632 patients (seven articles), with 42 infectious and 63 non-infectious complications. CONCLUSIONS Management of HCP is often necessary in children with DMG. Given the low rate of clinical risk associated with VAD placement and access, and the potential utility of longitudinal disease monitoring via CSF analysis, VAD placement could be considered in future clinical trials to guide DMG treatment.

MDB-61. STARVING TUMORS OF POLYAMINES: A NOVEL APPROACH IN TREATING AGGRESSIVE PEDIATRIC MEDULLOBLASTOMA

Abstract BACKGROUND Polyamines, essential intracellular polycations, govern fundamental cell processes and drive oncogenesis. Our research unveiled the dependency of pediatric Diffuse Midline Gliomas (DMG) on the polyamine pathway (Khan et al.,2021). Disruption of this pathway with the synthesis inhibitor DFMO, and the transport inhibitor AMXT1501, potently inhibited DMG tumors, yielding unprecedented survival in-vivo. We hypothesized that dual polyamine pathway targeting could similarly suppress growth in other aggressive pediatric brain tumors. METHODS/RESULTS We analyzed the expression of polyamine pathway regulators in high-risk pediatric brain cancers ZERO precision medicine platform and found elevated expression in medulloblastoma. ODC1, a polyamine synthesis driver, was found to have significant correlation in high-grade gliomas with four key functional tumor states—stemness, cell cycle, hypoxia, and angiogenesis. Medulloblastoma cells were sensitive to treatment with DFMO but showed a compensatory increase in polyamine transporter expression. Conversely, treatment with AMXT1501 increased ODC1 expression. Dual targeting of the polyamine pathway potently and synergistically inhibited cell proliferation and clonogenic potential in Group-3 medulloblastoma cells. Flow cytometric analysis of Annexin V-stained cells showed that treatment with DFMO and AMXT1501 in combination led to induction of apoptosis. Critically, dual polyamine inhibition potently sensitized medulloblastoma cells to the topoisomerase-I inhibitor, SN-38, leading to complete eradication of medulloblastoma cell proliferation and colony formation at nanomolar chemotherapeutic concentrations, with the effect most pronounced in Group-3 medulloblastoma. In triple combination-treated cells, over-representation analysis revealed heightened mitotic DNA damage, macroautophagy, and apoptosis, coupled with diminished neuron/synapse development, glycosylation, and cellular signaling pathways. We are currently testing this treatment strategy in aggressive orthotopic medulloblastoma models. CONCLUSION These findings suggest targeting the polyamine pathway as a promising strategy to sensitize aggressive pediatric medulloblastoma tumors to chemotherapy. DFMO/AMXT1501 is in Phase I evaluation for adults (NCT05500508) and advancing to a Phase I/II trial for children through the CONNECT consortium.

IMG-25. PREDICTING ANATOMICAL TUMOR GROWTH IN PEDIATRIC HIGH-GRADE GLIOMAS VIA DENOISING DIFFUSION MODELS

Abstract BACKGROUND Pediatric diffuse midline glioma (DMG) has a poor prognosis with radiotherapy as the standard of palliative care. Radiation strategies using individual geometric doseshaping have the potential to maximize patient benefit. This study aimed to establish a proof-of-principle to generate clinically relevant anatomical representations of the progression of pediatric DMG magnetic resonance image (MRI) slices through denoising diffusion implicit models (DDIM). METHODS Based on the BraTS23 data, we analyzed the generation of MRIs with enlarged tumor sizes starting from a baseline scan. Models were pre-trained on adult glioblastoma (GBM) prior to transfer learning on pediatric high-grade glioma. Test set performance was evaluated using the Frechet Inception Distance (FID), and Structural Similarity Index (SSIM). Qualitative evaluation involved visual classification of real vs generated images by two independent radiology trainees. The optimized network was tested to forecast anatomical tumor growth in five DMG patients from an independent dataset comprising longitudinal T2-FLAIR images. RESULTS We obtain high-fidelity generated images, supported by an FID of 12.4 and 0.8 SSIM on the adult test set. Despite the smaller pediatric BraTS dataset yielding a 34.8 FID and 0.84 SSIM, human observers failed to distinguish generated from real MRIs (mean recall and precision of 0.51). The generated anatomical predictions also closely resemble the observed longitudinal tumor progression in the pediatric DMG subset. Notably, the direction and extent of tumor growth align while the surrounding brain anatomy is preserved. Quantitatively, the mean DICE score between segmentations of generated and real tumors was 0.8. CONCLUSIONS Our model produces high-quality images that conform to growth patterns of pediatric DMGs. Further quantitative validation on larger longitudinal datasets is required, to ensure the method’s robustness. The proposed method enables a personalized approach for defining target regions in radiation therapy which could translate to improved clinical outcomes.

DIPG-45. TARGETING HYPERACETYLATION IN PEDIATRIC DIFFUSE MIDLINE GLIOMAS USING ONCOLYTIC ADENOVIRUSES

Abstract BACKGROUND H3K27-altered pediatric diffuse midline glioma (DMG) is a highly aggressive type of brain tumor with a 5-year survival rate of less than 2%. Conventional treatments are restricted to palliative radiation therapy. Therefore, effective treatments are urgently needed. Results from a recent phase I clinical trial for Diffuse Intrinsic Pontine Glioma showed that intratumoral administration of Delta-24-RGD oncolytic adenovirus in combination with radiation improves survival without unwanted toxicity. Based on the rationale that, during infection, the early viral protein E1A binds P300, we hypothesize that combining oncolytic adenoviruses with p300 inhibitors will induce a robust modification of the post-translational landscape of DMGs and increase the anti-cancer effect of the oncolytic virus. METHODS Studies on Delta-24-RGD infectivity and replication were performed in a panel of clinically significant DMG cells. Modification of the acetylation levels of acid-extracted histones after Delta-24-RGD was analyzed using Western blot. Cell Titer Blue was utilized to measure cell viability, and ZIP-synergy scores were calculated using SynergyFinder. RESULTS We observed a time-dependent reduction in H3K27ac in human DMG cells following Delta-24-RGD infection with an 89% reduction in H3K27ac at 48h post-infection. Delta-24-RGD treatment of DMG cells resulted in optimum viral infectivity and replication. Combining Delta-24-RGD with the p300 inhibitor C646 resulted in enhanced cell death in vitro. Using a panel of oncolytic viruses that induce differential enzymatic and binding functions of P300, we showed that Delta-24-RGD mediated alteration of P300 activity is required for hypoacetylation in DMGs. We characterized the in vivo dynamics of K27M-H3.1 and K27M-H3.3 DMG-derived tumors in the brain stem of syngeneic animal models. In vivo experiments are in progress to examine the combinatorial anti-glioma effect of Delta-24-RGD and P300 inhibitors. CONCLUSION Collectively, our studies provide a strong mechanistic rationale for combining oncolytic adenoviruses and p300 inhibitors and should propel the eventual clinical testing of this approach in DMG patients.

IMG-02. USING MRI RADIOMICS AND MACHINE LEARNING TO PREDICT OVERALL SURVIVAL FOR PEDIATRIC DIFFUSE MIDLINE GLIOMAS

Abstract BACKGROUND Pediatric diffuse midline gliomas (DMG) are aggressive central nervous system tumors with a median overall survival (OS) of less than one year from diagnosis. There is no curative therapy for DMG, and radiation therapy (RT) is the standard treatment. Magnetic resonance imaging (MRI) is the common noninvasive tool for DMG diagnosis and monitoring of tumor response to therapy. We developed an automatic pipeline to segment subregions of DMG and select MRI features that predict patient OS. METHODS We acquired diagnostic and post-RT multisequence MRI (T1, T1ce, T2, T2 FLAIR) and manual segmentations of pediatric DMG patients from two centers: 53 from Children’s National Hospital (internal) and 16 from Children’s Hospital of Philadelphia (external). We pretrained a deep learning model on a public adult brain tumor dataset, and finetuned the model to segment tumor core (TC) and whole tumor (WT) of DMG. We used PyRadiomics and sequential feature selection to select discriminative features based on the segmented volumes. Two machine learning models were trained on our internal cohort to predict patient 1-year survival from diagnosis. One model used only diagnostic tumor features and the other used both diagnostic and post-RT features. RESULTS For segmentation, Dice score (mean [median]±SD) was 0.91 (0.94)±0.12 and 0.74 (0.83)±0.32 for TC, and 0.88 (0.91)±0.07 and 0.86 (0.89)±0.06 for WT for internal and external cohorts, respectively. For OS prediction, accuracy was 77% and 81% at time of diagnosis, and 85% and 78% post-RT for internal and external cohorts, respectively. Heterogeneous WT intensity in baseline T2 FLAIR and smaller post-RT TC/WT volume ratio indicate longer OS. CONCLUSIONS Our method accurately predict OS for DMG patients and can be extended to other rare pediatric brain tumors. With automated and standardized analysis, early prognostication of OS can guide patient risk stratification and clinical decisions.

NFKBIA deletions reshape the epigenome antithetical to the IDH mutation and indication of prognosis in diffuse gliomas.

2083 Background: Genetic alterations help predict the clinical behavior of diffuse gliomas, but some variability remains uncorrelated. The NFKBIA gene at 14q13.2 encodes IkBa, a protein that regulates the activity of NF-kB in the cytoplasm. Evidence that, in chromatin, nuclear NFKBIA dynamically interacts with histones H2A and H4 to regulate polycomb-dependent transcriptional repression and, thus, stem cell maturation and lineage specification and our characterization of NFKBIA as a tumor suppressor in glioblastoma prompted our investigation of the relationship of NFKBIA deletions to other genetic markers, alterations in the methylome, and the clinical course of gliomas. Methods: We analyzed the genetic profiles of 2,343 WHO grade 2 to 4 diffuse gliomas in multiple clinically well-characterized patient populations, including national randomized phase III consortium trial RTOG 9802, to determine whether incorporation of NFKBIA deletions could enhance the prognostic value of current molecular descriptions. Results: We demonstrate that haploinsufficient deletions of NFKBIA display distinct patterns of occurrence in relation to other genetic markers, including driver mutations IDH, TERT, ATRX, and PTEN, 1p19q codeletion, and CDKN2A/B deletion, and are disproportionally present at recurrence. NFKBIA haploinsufficiency is associated with unfavorable patient outcomes, independent of genetic and clinicopathologic predictors. NFKBIA deletions reshape the DNA and histone methylome antipodal to the IDH mutation. The methylome changes facilitated through NFKBIA deletions engender a transcriptome landscape partly reminiscent of H3K27M mutant pediatric gliomas and are highly enriched for neural stem cell and neurogenesis genes bearing repressive H3K27 marks. Our findings imply that the NFKBIA deletion in adult gliomas and the H3K27M mutation in pediatric diffuse midline gliomas define aggressive disease subtypes that may share an epigenetic state reflecting a common PRC2 loss-of-function phenotype antipodal to the quasi PRC2 gain-of-function phenotype of IDH mutant gliomas and their favorable clinical course. Lower-grade gliomas harboring NFKBIA deletions behave much like high-grade gliomas. In IDH mutant gliomas, NFKBIA deletions are common in tumors with a clinical course similar to that of IDH wildtype tumors. A sparse, easily interpretable, and externally validated nomogram model for estimating individual patient survival in IDH mutant gliomas, incorporating NFKBIA deletion and current best-established genetic and clinicopathologic factors, confirms that NFKBIA deletions predict comparatively brief survival. Conclusions: NFKBIA haploinsufficiency aligns with distinct epigenome changes, portends a poor prognosis, and should be incorporated into clinical models predicting the disease fate of diffuse gliomas.

Pediatric Diffuse Midline Glioma H3K27-Altered: From Developmental Origins to Therapeutic Challenges.

Diffuse intrinsic pontine glioma (DIPG), now referred to as diffuse midline glioma (DMG), is a highly aggressive pediatric cancer primarily affecting children aged 4 to 9 years old. Despite the research and clinical trials conducted to identify a possible treatment for DIPG, no effective drug is currently available. These tumors often affect deep midline brain structures in young children, suggesting a connection to early brain development's epigenetic regulation targets, possibly affecting neural progenitor functions and differentiation. The H3K27M mutation is a known DIPG trigger, but the exact mechanisms beyond epigenetic regulation remain unclear. After thoroughly examining the available literature, we found that over 85% of DIPG tumors contain a somatic missense mutation, K27M, in genes encoding histone H3.3 and H3.1, leading to abnormal gene expression that drives tumor growth and spread. This mutation impacts crucial brain development processes, including the epithelial-mesenchymal transition (EMT) pathway, and may explain differences between H3K27M and non-K27M pediatric gliomas. Effects on stem cells show increased proliferation and disrupted differentiation. The genomic organization of H3 gene family members in the developing brain has revealed variations in their expression patterns. All these observations suggest a need for global efforts to understand developmental origins and potential treatments.

Abstract 3173: Impact of hypoxia on the integrated stress response activated by imipridones ONC201 and ONC206 in pediatric diffuse midline glioma cells

Abstract Diffuse midline gliomas (DMGs) are highly aggressive, high-grade gliomas which typically arise in children and young adults. With currently approved treatments the median survival from time of diagnosis for pediatric DMG is 8-10 months with only 10% of children living to 2 years post diagnosis. Despite being only 15% of the cases it makes up 40% of the pediatric brain cancer deaths making it the leading cause of death for all pediatric glioma cases. Two recent clinical studies, NCT03416530 and NCT03134131, have shown the clinical efficacy of Dordaviprone (ONC201/TIC10) for the treatment of DMG; increasing median survival to 22 months in patients following radiation treatment prior to recurrence. Imipridone ONC206, a chemical derivative of ONC201, is under clinical development for treatment of pediatric and adult patients with primary brain tumors (NCT04732065 and NCT04541082). To further improve treatment, we must continue to study how the tumor microenvironment impacts the efficacy of imipridones. One crucial aspect of all brain cancers is hypoxia, so the IC50s of the SU-DIGP-25, SU-DIPG-XIII and SU-DIPGIV pediatric DMG cell lines treated with ONC201 or ONC206 were measured using the CellTiter-Glo assay under conditions of hypoxia and normoxia. Imipridones mediate apoptosis through the upregulation of the TRAIL death receptor DR5 and the activation of the integrated stress response (ISR), so western blots were used to show changes in the ISR protein expression in ONC201 treated DMG cells at multiple different degrees of hypoxia. To understand how hypoxia inducible factors (HIFs) play a role in resistance and susceptibility to ONC201 or ONC206, HIF-1a, HIF-2a, and HIF-3a were knocked down showing altered ISR protein expression after treating with the imipridones under hypoxic and normoxic conditions. Citation Format: Tyler J. Roady, Nolan Stubbs, Josephine Chen, Yutong Xia, Ashley Sanchez Sevilla Uruchurtu, Xiaobing Tian, Lanlan Zhou, Wafik S. El-Deiry. Impact of hypoxia on the integrated stress response activated by imipridones ONC201 and ONC206 in pediatric diffuse midline glioma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3173.

Bone Morphogenic Proteins in Pediatric Diffuse Midline Gliomas: How to Make New Out of Old?

The BMP pathway is one of the major signaling pathways in embryonic development, ontogeny and homeostasis, identified many years ago by pioneers in developmental biology. Evidence of the deregulation of its activity has also emerged in many cancers, with complex and sometimes opposing effects. Recently, its role has been suspected in Diffuse Midline Gliomas (DMG), among which Diffuse Intrinsic Pontine Gliomas (DIPG) are one of the most complex challenges in pediatric oncology. Genomic sequencing has led to understanding part of their molecular etiology, with the identification of histone H3 mutations in a large proportion of patients. The epigenetic remodeling associated with these genetic alterations has also been precisely described, creating a permissive context for oncogenic transcriptional program activation. This review aims to describe the new findings about the involvement of BMP pathway activation in these tumors, placing their appearance in a developmental context. Targeting the oncogenic synergy resulting from this pathway activation in an H3K27M context could offer new therapeutic perspectives based on targeting treatment-resistant cell states.

Abstract B047: Inhibition of the polyamine pathway: a novel therapeutic approach to treat aggressive pediatric medulloblastoma

Abstract Medulloblastoma is the most common pediatric malignant brain tumor. Polyamines, critical intracellular polycations governing key cell processes such as DNA replication, translation, and cell proliferation, are frequently upregulated in cancer. We previously showed that the polyamine pathway is critical to the growth and survival of pediatric diffuse midline gliomas and MYC amplified neuroblastoma. Given the activity of polyamine inhibition in DMG, and given the critical role of MYC in medulloblastoma, we sought to investigate the potential of polyamine pathway inhibition as a therapeutic approach for medulloblastoma. We compared the expression of regulators of the polyamine pathway in a cohort of pediatric medulloblastoma tumors from patients enrolled in the Zero Childhood Cancer (ZERO) Personalized Medicine Program and found significant over-expression compared with normal fetal brain samples. Analysis of Cavalli dataset of 763 medulloblastoma samples, found that high expression of polyamine biosynthetic genes predicted poor overall survival, and low expression of catabolic flux genes significantly correlated with better outcomes. We next treated medulloblastoma cells treated with a polyamine synthesis inhibitor, DFMO, and found that led to a compensatory increase in polyamine transporter protein expression. Conversely, treatment with the polyamine transport inhibitor AMXT 1501 led to compensatory increase in ODC1, the key polyamine synthetic enzyme. Dual targeting of the polyamine pathway, (DFMO and AMXT 1501) showed a potent and synergistic inhibition of cell proliferation and reduced the clonogenic potential of medulloblastoma cells in soft agar clonogenic assays. Flow cytometric analysis of Annexin V-stained cells showed that treatment with DFMO and AMXT 1501 in combination led to induction of apoptosis. Furthermore, we found that dual polyamine inhibition potently sensitized medulloblastoma cells, particularly Group 3, to the chemotherapeutic agent SN-38, leading to complete eradication of medulloblastoma cells in both cytotoxicity assays and clonogenic assays at nanomolar chemotherapeutic concentrations. We are currently validating this treatment strategy in aggressive orthotopic medulloblastoma models. Our findings suggest that inhibiting both polyamine synthesis and uptake holds promise for sensitizing highly aggressive pediatric medulloblastoma tumors to chemotherapy. Currently, AMXT 1501 combined with DFMO is undergoing evaluation in a Phase I clinical trial for adults (NCT05500508). Building on these promising results, we are developing a Phase I/II clinical trial for children with aggressive brain tumors through the international CONNECT consortium. Citation Format: Aaminah Khan, Anjana Gopalakrishnan, Jie Liu, Mark R. Burns, Maria Tsoli, David S. Ziegler. Inhibition of the polyamine pathway: a novel therapeutic approach to treat aggressive pediatric medulloblastoma [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B047.

Full-length isoform concatenation sequencing to resolve cancer transcriptome complexity

BackgroundCancers exhibit complex transcriptomes with aberrant splicing that induces isoform-level differential expression compared to non-diseased tissues. Transcriptomic profiling using short-read sequencing has utility in providing a cost-effective approach for evaluating isoform expression, although short-read assembly displays limitations in the accurate inference of full-length transcripts. Long-read RNA sequencing (Iso-Seq), using the Pacific Biosciences (PacBio) platform, can overcome such limitations by providing full-length isoform sequence resolution which requires no read assembly and represents native expressed transcripts. A constraint of the Iso-Seq protocol is due to fewer reads output per instrument run, which, as an example, can consequently affect the detection of lowly expressed transcripts. To address these deficiencies, we developed a concatenation workflow, PacBio Full-Length Isoform Concatemer Sequencing (PB_FLIC-Seq), designed to increase the number of unique, sequenced PacBio long-reads thereby improving overall detection of unique isoforms. In addition, we anticipate that the increase in read depth will help improve the detection of moderate to low-level expressed isoforms.ResultsIn sequencing a commercial reference (Spike-In RNA Variants; SIRV) with known isoform complexity we demonstrated a 3.4-fold increase in read output per run and improved SIRV recall when using the PB_FLIC-Seq method compared to the same samples processed with the Iso-Seq protocol. We applied this protocol to a translational cancer case, also demonstrating the utility of the PB_FLIC-Seq method for identifying differential full-length isoform expression in a pediatric diffuse midline glioma compared to its adjacent non-malignant tissue. Our data analysis revealed increased expression of extracellular matrix (ECM) genes within the tumor sample, including an isoform of the Secreted Protein Acidic and Cysteine Rich (SPARC) gene that was expressed 11,676-fold higher than in the adjacent non-malignant tissue. Finally, by using the PB_FLIC-Seq method, we detected several cancer-specific novel isoforms.ConclusionThis work describes a concatenation-based methodology for increasing the number of sequenced full-length isoform reads on the PacBio platform, yielding improved discovery of expressed isoforms. We applied this workflow to profile the transcriptome of a pediatric diffuse midline glioma and adjacent non-malignant tissue. Our findings of cancer-specific novel isoform expression further highlight the importance of long-read sequencing for characterization of complex tumor transcriptomes.

Early prognostication of overall survival for pediatric diffuse midline gliomas using MRI radiomics and machine learning: A two-center study.

Diffuse midline gliomas (DMG) are aggressive pediatric brain tumors that are diagnosed and monitored through MRI. We developed an automatic pipeline to segment subregions of DMG and select radiomic features that predict patient overall survival (OS). We acquired diagnostic and post-radiation therapy (RT) multisequence MRI (T1, T1ce, T2, and T2 FLAIR) and manual segmentations from 2 centers: 53 from 1 center formed the internal cohort and 16 from the other center formed the external cohort. We pretrained a deep learning model on a public adult brain tumor data set (BraTS 2021), and finetuned it to automatically segment tumor core (TC) and whole tumor (WT) volumes. PyRadiomics and sequential feature selection were used for feature extraction and selection based on the segmented volumes. Two machine learning models were trained on our internal cohort to predict patient 12-month survival from diagnosis. One model used only data obtained at diagnosis prior to any therapy (baseline study) and the other used data at both diagnosis and post-RT (post-RT study). Overall survival prediction accuracy was 77% and 81% for the baseline study, and 85% and 78% for the post-RT study, for internal and external cohorts, respectively. Homogeneous WT intensity in baseline T2 FLAIR and larger post-RT TC/WT volume ratio indicate shorter OS. Machine learning analysis of MRI radiomics has potential to accurately and noninvasively predict which pediatric patients with DMG will survive less than 12 months from the time of diagnosis to provide patient stratification and guide therapy.

An ERK5-PFKFB3 axis regulates glycolysis and represents a therapeutic vulnerability in pediatric diffuse midline glioma

Metabolic reprogramming in pediatric diffuse midline glioma is driven by gene expression changes induced by the hallmark histone mutation H3K27M, which results in aberrantly permissive activation of oncogenic signaling pathways. Previous studies of diffuse midline glioma with altered H3K27 (DMG-H3K27a) have shown that the RAS pathway, specifically through its downstream kinase, extracellular-signal-related kinase 5 (ERK5), is critical for tumor growth. Further downstream effectors of ERK5 and their role in DMG-H3K27a metabolic reprogramming have not been explored. We establish that ERK5 is a critical regulator of cell proliferation and glycolysis in DMG-H3K27a. We demonstrate that ERK5 mediates glycolysis through activation of transcription factor MEF2A, which subsequently modulates expression of glycolytic enzyme PFKFB3. We show that invitro and mouse models of DMG-H3K27a are sensitive to the loss of PFKFB3. Multi-targeted drug therapy against the ERK5-PFKFB3 axis, such as with small-molecule inhibitors, may represent a promising therapeutic approach in patients with pediatric diffuse midline glioma.

A road map for the treatment of pediatric diffuse midline glioma

Recent clinical trials for H3K27-altered diffuse midline gliomas (DMGs) have shown much promise. We present a consensus roadmap and identify three major barriers: (1) refinement of experimental models to include immune and brain-specific components; (2) collaboration among researchers, clinicians, and industry to integrate patient-derived data through sharing, transparency, and regulatory considerations; and (3) streamlining clinical efforts including biopsy, CNS-drug delivery, endpoint determination, and response monitoring. We highlight the importance of comprehensive collaboration to advance the understanding, diagnostics, and therapeutics for DMGs.

Chat GPT in Diagnostic Human Pathology: Will It Be Useful to Pathologists? A Preliminary Review with ‘Query Session’ and Future Perspectives

The advent of Artificial Intelligence (AI) has in just a few years supplied multiple areas of knowledge, including in the medical and scientific fields. An increasing number of AI-based applications have been developed, among which conversational AI has emerged. Regarding the latter, ChatGPT has risen to the headlines, scientific and otherwise, for its distinct propensity to simulate a ‘real’ discussion with its interlocutor, based on appropriate prompts. Although several clinical studies using ChatGPT have already been published in the literature, very little has yet been written about its potential application in human pathology. We conduct a systematic review following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, using PubMed, Scopus and the Web of Science (WoS) as databases, with the following keywords: ChatGPT OR Chat GPT, in combination with each of the following: pathology, diagnostic pathology, anatomic pathology, before 31 July 2023. A total of 103 records were initially identified in the literature search, of which 19 were duplicates. After screening for eligibility and inclusion criteria, only five publications were ultimately included. The majority of publications were original articles (n = 2), followed by a case report (n = 1), letter to the editor (n = 1) and review (n = 1). Furthermore, we performed a ‘query session’ with ChatGPT regarding pathologies such as pigmented skin lesions, malignant melanoma and variants, Gleason’s score of prostate adenocarcinoma, differential diagnosis between germ cell tumors and high grade serous carcinoma of the ovary, pleural mesothelioma and pediatric diffuse midline glioma. Although the premises are exciting and ChatGPT is able to co-advise the pathologist in providing large amounts of scientific data for use in routine microscopic diagnostic practice, there are many limitations (such as data of training, amount of data available, ‘hallucination’ phenomena) that need to be addressed and resolved, with the caveat that an AI-driven system should always provide support and never a decision-making motive during the histopathological diagnostic process.

Biopsy of diffuse midline glioma is safe and impacts targeted therapy: a systematic review and meta-analysis.

To quantify the safety and utility of biopsy of pediatric diffuse midline glioma (DMG). This study was conducted in accordance with PRISMA guidelines. PubMed, Embase, Scopus, and Web of Science were queried for relevant articles from inception until June 2023. Two reviewers identified all articles that included diagnostic yield, morbidity, and mortality rates for pediatric DMG patients. Studies that did not present original data or were not in English or peer-reviewed were excluded. Meta-analysis was conducted in R using Freeman-Tukey or logit transformation and DerSimonian-Laird random-effects models. The risk of bias was assessed using the Newcastle-Ottawa Scale. A protocol for this review was not registered. We identified 381 patients from ten studies that met all criteria. DMG biopsy is safe overall (0% mortality, 95% CI: 0-0.6%; 11.0% morbidity, 95% CI: 4.8-18.9%) and has a high diagnostic yield (99.9%, 95% CI: 98.5-100%). The use of stereotactic biopsy is a significant moderator of morbidity (p = 0.0238). Molecular targets can be identified in approximately 53.4% of tumors (95% CI: 37.0-69.0%), although targeted therapies are only delivered in about 33.5% of all cases (95% CI: 24.4-44.1%). Heterogeneity was high for morbidity and identification of targets. The risk of bias was low for all studies. We conducted the first meta-analysis of DMG biopsy to show that it is safe, effective, and able to identify relevant molecular targets that impact targeted therapy.