Immune landscape of oncohistone-mutant gliomas reveals diverse myeloid populations and tumor-promoting function.

Histone H3-mutant gliomas are deadly brain tumors characterized by a dysregulated epigenome and stalled differentiation. In contrast to the extensive datasets available on tumor cells, limited information exists on their tumor microenvironment (TME), particularly the immune infiltrate. Here, we characterize the immune TME of H3.3K27M and G34R/V-mutant gliomas, and multiple H3.3K27M mouse models, using transcriptomic, proteomic and spatial single-cell approaches. Resolution of immune lineages indicates high infiltration of H3-mutant gliomas with diverse myeloid populations, high-level expression of immune checkpoint markers, and scarce lymphoid cells, findings uniformly reproduced in all H3.3K27M mouse models tested. We show these myeloid populations communicate with H3-mutant cells, mediating immunosuppression and sustaining tumor formation and maintenance. Dual inhibition of myeloid cells and immune checkpoint pathways show significant therapeutic benefits in pre-clinical syngeneic mouse models. Our findings provide a valuable characterization of the TME of oncohistone-mutant gliomas, and insight into the means for modulating the myeloid infiltrate for the benefit of patients.

Whole-exome sequencing and copy number alterations analysis in a case of expansive florid cemento-osseous dysplasia.

Whole-exome sequencing (WES) analysis of an expansive case florid cemento-osseus dysplasia were reported for the first time. Also, the new potential candidate genes were reported to expand our knowledge about their molecular pathogenesis. Abstract: We report a case of expansive florid cemento-osseus dysplasia in a 32-year-old female patient who presented an expansive tumoral lesion in the anterior mandible. As florid cemento-osseus dysplasia have only been molecularly investigated using targeted-sequencing, fragments of the lesion were collected and subjected to molecular investigation using WES to assess somatic mutations as well as copy number alterations. No gains and losses of chromosomal arms or segments longer than 1 Mb were detected. Our findings revealed a pathogenic stopgain variant at the KIF5C gene, a stoploss variant at MAPK10, and missense SNV at COL6A2 at DCDC1, suggesting potential candidate genes associated with florid cemento-osseus dysplasia.

Comments and Controversies in Oncology: The Tribulations of Trials Developing ONC201.

Our international team highlights issues with efficacy reports in several studies on DMG with the new drug ONC201.

Canadian Consensus for Treatment of BRAF V600E Mutated Pediatric and AYA Gliomas.

Background: The treatment of BRAF V600E gliomas with BRAF inhibitors (BRAFis) and MEK inhibitors (MEKis) has been increasingly integrated into clinical practice for pediatric low-grade gliomas (PLGGs) and pediatric high-grade gliomas (HGGs). However, some questions remain unanswered, such as the best time to start targeted therapy, duration of treatment, and discontinuation of therapy. Given that no clinical trial has been able to address these critical questions, we developed a Canadian Consensus statement for the treatment of BRAF V600E mutated pediatric as well as adolescent and young adult (AYA) gliomas. Methods: Canadian neuro-oncologists were invited to participate in the development of this consensus. The consensus was discussed during monthly web-based national meetings, and the algorithms were revised until a consensus was achieved. Results: A total of 26 participants were involved in the development of the algorithms. Two treatment algorithms are proposed, one for the initiation of treatment and one for the discontinuation of treatment. We suggest that most patients with BRAF V600E gliomas should be treated with BRAFis ± MEKis upfront. Discontinuation of treatment can be considered in certain circumstances, and we suggest a slow wean. Conclusions: Based on expert consensus in Canada, we developed algorithms for treatment initiation of children and AYA with BRAF V600E gliomas as well as a discontinuation algorithm.

TULIPs decorate the three-dimensional genome of PFA ependymoma.

Posterior fossa group A (PFA) ependymoma is a lethal brain cancer diagnosed in infants and young children. The lack of driver events in the PFA linear genome led us to search its 3D genome for characteristic features. Here, we reconstructed 3D genomes from diverse childhood tumor types and uncovered a global topology in PFA that is highly reminiscent of stem and progenitor cells in a variety of human tissues. A remarkable feature exclusively present in PFA are type B ultra long-range interactions in PFAs (TULIPs), regions separated by great distances along the linear genome that interact with each other in the 3D nuclear space with surprising strength. TULIPs occur in all PFA samples and recur at predictable genomic coordinates, and their formation is induced by expression of EZHIP. The universality of TULIPs across PFA samples suggests a conservation of molecular principles that could be exploited therapeutically.

Epidemiology of Pediatric Tumors in Quebec: A 17-Year Report of Cancer in Young People in the Canada Registry.

BACKGROUND: Cancer is the leading cause of disease-related death among children of more than 1 year of age. However, childhood cancer risk factors and etiology are yet to be fully understood. The goal of this study is to identify geographic variation among children and adolescents diagnosed with pediatric tumors between 2001 and 2018 in the province of Quebec. METHODS: We analyzed pediatric patients less than 15 years of age from the Cancer in Young People in Canada (CYP-C) surveillance system who were diagnosed between 2001 and 2018 with cancer in the province of Quebec. The age-standardized age-adjusted incidence rates (AAIR) per 100,000 person years were calculated for all childhood cancers by cancer subgroups, Quebec Health regions, and age groups. RESULTS: Overall, 3904 pediatric patients less than 15 years old were diagnosed with cancer in the province of Quebec in 2001-2018. The overall incidence rate (IR) in the province of Quebec was 16.14 (95%CL [15.56-16.73]) per 100,000 person years. For childhood cancers, regions that presented a higher AAIR were Chaudière-Appalaches and Capitale-Nationale with 18.2 and 17.5 per 100,000 person years, respectively. The incidence rates (IRs) in Chaudière-Appalaches (95% CI 1.0439-1.3532) and in Capitale-Nationale (95% CI 1.0124-1.2942) were statistically higher than the incidence in the province of Quebec (p = 0.0090 and p = 0.0310, respectively). When comparing the AAIR of the CNS tumor subgroup in Chaudière-Appalaches and in Capitale-Nationale, with the provincial average, we noticed a statistically higher incidence in Chaudière-Appalaches and a trend for Capitale-Nationale (p < 0.0001 and p = 0.0602, respectively). CONCLUSION: There is evidence of spatial clusters in Chaudière-Appalaches and Capitale-Nationale as areas for all childhood cancers. Further studies should be performed to investigate potential risk factors in these regions.

Purine salvage promotes treatment resistance in H3K27M-mutant diffuse midline glioma.

BACKGROUND: Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPGs), are a fatal form of brain cancer. These tumors often carry a driver mutation on histone H3 converting lysine 27 to methionine (H3K27M). DMG-H3K27M are characterized by altered metabolism and resistance to standard of care radiation (RT) but how the H3K27M mediates the metabolic response to radiation and consequent treatment resistance is uncertain. METHODS: We performed metabolomics on irradiated and untreated H3K27M isogenic DMG cell lines and observed an H3K27M-specific enrichment for purine synthesis pathways. We profiled the expression of purine synthesis enzymes in publicly available patient data and our models, quantified purine synthesis using stable isotope tracing, and characterized the in vitro and in vivo response to de novo and salvage purine synthesis inhibition in combination with RT. RESULTS: DMG-H3K27M cells activate purine metabolism in an H3K27M-specific fashion. In the absence of genotoxic treatment, H3K27M-expressing cells have higher relative activity of de novo synthesis and apparent lower activity of purine salvage demonstrated via stable isotope tracing of key metabolites in purine synthesis and by lower expression of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), the rate-limiting enzyme of purine salvage into IMP and GMP. Inhibition of de novo guanylate synthesis radiosensitized DMG-H3K27M cells in vitro and in vivo. Irradiated H3K27M cells upregulated HGPRT expression and hypoxanthine-derived guanylate salvage but maintained high levels of guanine-derived salvage. Exogenous guanine supplementation decreased radiosensitization in cells treated with combination RT and de novo purine synthesis inhibition. Silencing HGPRT combined with RT markedly suppressed DMG-H3K27M tumor growth in vivo. CONCLUSIONS: Our results indicate that DMG-H3K27M cells rely on highly active purine synthesis, both from the de novo and salvage synthesis pathways. However, highly active salvage of free purine bases into mature guanylates can bypass inhibition of the de novo synthetic pathway. We conclude that inhibiting purine salvage may be a promising strategy to overcome treatment resistance in DMG-H3K27M tumors.

Advances in pediatric gliomas: from molecular characterization to personalized treatments.

Pediatric gliomas, consisting of both pediatric low-grade (pLGG) and high-grade gliomas (pHGG), are the most frequently occurring brain tumors in children. Over the last decade, several milestone advancements in treatments have been achieved as a result of stronger understanding of the molecular biology behind these tumors. This review provides an overview of pLGG and pHGG highlighting their clinical presentation, molecular characteristics, and latest advancements in therapeutic treatments.  Conclusion: The increasing understanding of the molecular biology characterizing pediatric low and high grade gliomas has revolutionized treatment options for these patients, especially in pLGG. The implementation of next generation sequencing techniques for these tumors is crucial in obtaining less toxic and more efficacious treatments. What is Known: • Pediatric Gliomas are the most common brain tumour in children. They are responsible for significant morbidity and mortality in this population. What is New: • Over the last two decades, there has been a significant increase in our global understanding of the molecular background of pediatric low and high grade gliomas. • The implementation of next generation sequencing techniques for these tumors is crucial in obtaining less toxic and more efficacious treatments, with the ultimate goal of improving both the survival and the quality of life of these patients.

Very Long-term Survivorship in Pediatric DIPG: Case Report and Review of the Literature.

Diffuse intrinsic pontine gliomas are lethal tumors with a prognosis generally less than 1 year. Few cases of survivors of 5 years or more have been reported. This case report highlights the journey of a 9.5-year survivor who underwent 3 rounds of focal radiotherapy; she experienced 6 years of progression-free survival following the first round but ultimately succumbed to her disease. An autopsy revealed a favorable IDH1 mutation and the absence of H3K27M. This case reiterates the importance of extensive molecular analyses in diffuse intrinsic pontine gliomas and explores the potential benefit of re-irradiation in patients with positive responses and long periods of remission.

The Virtual Child.

SUMMARY: We are building the world's first Virtual Child-a computer model of normal and cancerous human development at the level of each individual cell. The Virtual Child will "develop cancer" that we will subject to unlimited virtual clinical trials that pinpoint, predict, and prioritize potential new treatments, bringing forward the day when no child dies of cancer, giving each one the opportunity to lead a full and healthy life.

Recurrent primary intracranial sarcoma, DICER1-mutant in a pediatric patient with DICER1 syndrome: the importance of molecular testing.

Pediatric intracranial sarcomas are rare, aggressive tumors with a poor prognosis in general. Here we report the case of a child who was initially diagnosed with a primary intracranial sarcoma, DICER1-mutant; subsequent genetic analyses confirmed a pathogenic germline DICER1 mutation. She received multimodal standard treatments consisting of surgery, radiotherapy and chemotherapy. The tumor recurred 2.5 years later within the surgical cavity. Following the gross tumor resection of this new lesion, the same multimodal standard approach was used. From a molecular perspective, evidence of hyperactivation of the MAPK-kinase pathway with a pathogenic KRAS mutation at both diagnosis and recurrence was present. The patient is currently in remission, 18 months post-end of treatment.

Impact of trametinib on the neuropsychological profile of NF1 patients.

PURPOSE: The use of trametinib in the treatment of pediatric low-grade gliomas (PLGG) and plexiform neurofibroma (PN) is being investigated in an ongoing multicenter phase II trial (NCT03363217). Preliminary data shows potential benefits with significant response in the majority of PLGG and PN and an overall good tolerance. Moreover, possible benefits of MEK inhibitor therapy on cognitive functioning in neurofibromatosis type 1 (NF1) were recently shown which supports the need for further evaluation. METHODS: Thirty-six patients with NF1 (age range 3-19 years) enrolled in the phase II study of trametinib underwent a neurocognitive assessment at inclusion and at completion of the 72-week treatment. Age-appropriate Wechsler Intelligence Scales and the Trail Making Test (for children over 8 years old) were administered at each assessment. Paired t-tests and Reliable Change Index (RCI) analyses were performed to investigate change in neurocognitive outcomes. Regression analyses were used to investigate the contribution of age and baseline score in the prediction of change. RESULTS: Stable performance on neurocognitive tests was revealed at a group-level using paired t-tests. Clinically significant improvements were however found on specific indexes of the Wechsler intelligence scales and Trail Making Test, using RCI analyses. No significant impact of age on cognitive change was evidenced. However, lower initial cognitive performance was associated with increased odds of presenting clinically significant improvements on neurocognitive outcomes. CONCLUSION: These preliminary results show a potential positive effect of trametinib on cognition in patients with NF1. We observed significant improvements in processing speed, visuo-motor and verbal abilities. This study demonstrates the importance of including neuropsychological evaluations into clinical trial when using MEK inhibitors for patients with NF1.

Comprehensive Genomic Analysis of Cemento-Ossifying Fibroma.

Cemento-ossifying fibroma (COF) of the jaws is currently classified as a benign mesenchymal odontogenic tumor, and only targeted approaches have been used to assess its genetic alterations. A minimal proportion of COFs harbor CDC73 somatic mutations, and copy number alterations (CNAs) involving chromosomes 7 and 12 have recently been reported in a small proportion of cases. However, the genetic background of COFs remains obscure. We used a combination of whole-exome sequencing and RNA sequencing to assess somatic mutations, fusion transcripts, and CNAs in a cohort of 12 freshly collected COFs. No recurrent fusions have been identified among the 5 cases successfully analyzed by RNA sequencing, with in-frame fusions being detected in 2 cases (MARS1::GOLT1B and PARG::BMS1 in one case and NCLN::FZR1 and NFIC::SAMD1 in the other case) and no candidate fusions identified for the remaining 3 cases. No recurrent pathogenic mutations were detected in the 11 cases that had undergone whole-exome sequencing. A KRAS p.L19F missense variant was detected in one case, and 2 CDC73 deletions were detected in another case. The other variants were of uncertain significance and included variants in PC, ACTB, DOK6, HACE1, and COL1A2 and previously unreported variants in PTPN14, ATP5F1C, APOBEC1, HDAC5, ATF7IP, PARP2, and ACTR3B. The affected genes do not clearly converge on any signaling pathway. CNAs were detected in 5/11 cases (45%), with copy gains involving chromosome 12 occurring in 3/11 cases (27%). In conclusion, no recurrent fusions or pathogenic variants have been detected in the present COF cohort, with copy gains involving chromosome 12 occurring in 27% of cases.

The type II RAF inhibitor tovorafenib in relapsed/refractory pediatric low-grade glioma: the phase 2 FIREFLY-1 trial.

BRAF genomic alterations are the most common oncogenic drivers in pediatric low-grade glioma (pLGG). Arm 1 (n = 77) of the ongoing phase 2 FIREFLY-1 (PNOC026) trial investigated the efficacy of the oral, selective, central nervous system-penetrant, type II RAF inhibitor tovorafenib (420 mg m-2 once weekly; 600 mg maximum) in patients with BRAF-altered, relapsed/refractory pLGG. Arm 2 (n = 60) is an extension cohort, which provided treatment access for patients with RAF-altered pLGG after arm 1 closure. Based on independent review, according to Response Assessment in Neuro-Oncology High-Grade Glioma (RANO-HGG) criteria, the overall response rate (ORR) of 67% met the arm 1 prespecified primary endpoint; median duration of response (DOR) was 16.6 months; and median time to response (TTR) was 3.0 months (secondary endpoints). Other select arm 1 secondary endpoints included ORR, DOR and TTR as assessed by Response Assessment in Pediatric Neuro-Oncology Low-Grade Glioma (RAPNO) criteria and safety (assessed in all treated patients and the primary endpoint for arm 2, n = 137). The ORR according to RAPNO criteria (including minor responses) was 51%; median DOR was 13.8 months; and median TTR was 5.3 months. The most common treatment-related adverse events (TRAEs) were hair color changes (76%), elevated creatine phosphokinase (56%) and anemia (49%). Grade ≥3 TRAEs occurred in 42% of patients. Nine (7%) patients had TRAEs leading to discontinuation of tovorafenib. These data indicate that tovorafenib could be an effective therapy for BRAF-altered, relapsed/refractory pLGG. ClinicalTrials.gov registration: NCT04775485 .

Bevacizumab in the Treatment of Refractory Brain Edema in High-grade Glioma.

We report the case of a 14-year-old boy with a steroid-dependent refractory tumor whose longstanding dexamethasone treatment was successfully discontinued after a course of bevacizumab. The use of bevacizumab despite the absence of clear evidence of radionecrosis allowed a significant decrease in the amount of the brain edema.

Pediatric glioma histone H3.3 K27M/G34R mutations drive abnormalities in PML nuclear bodies.

BACKGROUND: Point mutations in histone variant H3.3 (H3.3K27M, H3.3G34R) and the H3.3-specific ATRX/DAXX chaperone complex are frequent events in pediatric gliomas. These H3.3 point mutations affect many chromatin modifications but the exact oncogenic mechanisms are currently unclear. Histone H3.3 is known to localize to nuclear compartments known as promyelocytic leukemia (PML) nuclear bodies, which are frequently mutated and confirmed as oncogenic drivers in acute promyelocytic leukemia. RESULTS: We find that the pediatric glioma-associated H3.3 point mutations disrupt the formation of PML nuclear bodies and this prevents differentiation down glial lineages. Similar to leukemias driven by PML mutations, H3.3-mutated glioma cells are sensitive to drugs that target PML bodies. We also find that point mutations in IDH1/2-which are common events in adult gliomas and myeloid leukemias-also disrupt the formation of PML bodies. CONCLUSIONS: We identify PML as a contributor to oncogenesis in a subset of gliomas and show that targeting PML bodies is effective in treating these H3.3-mutated pediatric gliomas.

H3K27me3 spreading organizes canonical PRC1 chromatin architecture to regulate developmental programs.

Polycomb Repressive Complex 2 (PRC2)-mediated histone H3K27 tri-methylation (H3K27me3) recruits canonical PRC1 (cPRC1) to maintain heterochromatin. In early development, polycomb-regulated genes are connected through long-range 3D interactions which resolve upon differentiation. Here, we report that polycomb looping is controlled by H3K27me3 spreading and regulates target gene silencing and cell fate specification. Using glioma-derived H3 Lys-27-Met (H3K27M) mutations as tools to restrict H3K27me3 deposition, we show that H3K27me3 confinement concentrates the chromatin pool of cPRC1, resulting in heightened 3D interactions mirroring chromatin architecture of pluripotency, and stringent gene repression that maintains cells in progenitor states to facilitate tumor development. Conversely, H3K27me3 spread in pluripotent stem cells, following neural differentiation or loss of the H3K36 methyltransferase NSD1, dilutes cPRC1 concentration and dissolves polycomb loops. These results identify the regulatory principles and disease implications of polycomb looping and nominate histone modification-guided distribution of reader complexes as an important mechanism for nuclear compartment organization. Highlights: The confinement of H3K27me3 at PRC2 nucleation sites without its spreading correlates with increased 3D chromatin interactions.The H3K27M oncohistone concentrates canonical PRC1 that anchors chromatin loop interactions in gliomas, silencing developmental programs.Stem and progenitor cells require factors promoting H3K27me3 confinement, including H3K36me2, to maintain cPRC1 loop architecture.The cPRC1-H3K27me3 interaction is a targetable driver of aberrant self-renewal in tumor cells.

Adaptive rewiring of purine metabolism promotes treatment resistance in H3K27M-mutant diffuse midline glioma.

Background: Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPGs), are a fatal form of brain cancer. These tumors often carry a driver mutation on histone H3 converting lysine 27 to methionine (H3K27M). DMG-H3K27M are characterized by altered metabolism and resistance to standard of care radiation (RT), but how the H3K27M mediates the metabolic response to radiation and consequent treatment resistance is uncertain. Methods: We performed metabolomics on irradiated and untreated H3K27M isogenic DMG cell lines and observed an H3K27M-specific enrichment for purine synthesis pathways. We profiled the expression of purine synthesis enzymes in publicly available patient data and in our models, quantified purine synthetic flux using stable isotope tracing, and characterized the in vitro and in vivo response to de novo and salvage purine synthesis inhibition in combination with RT. Results: DMG-H3K27M cells activate purine metabolism in an H3K27M-specific fashion. In the absence of genotoxic treatment, H3K27M-expressing cells have higher relative activity of de novosynthesis and lower activity of purine salvage due to decreased expression of the purine salvage enzymes. Inhibition of de novo synthesis radiosensitized DMG-H3K27M cells in vitro and in vivo. Irradiated H3K27M cells adaptively upregulate purine salvage enzyme expression and pathway activity. Silencing the rate limiting enzyme in purine salvage, hypoxanthine guanine phosphoribosyl transferase (HGPRT) when combined with radiation markedly suppressed DMG-H3K27M tumor growth in vivo. Conclusions: H3K27M expressing cells rely on de novo purine synthesis but adaptively upregulate purine salvage in response to RT. Inhibiting purine salvage may help overcome treatment resistance in DMG-H3K27M tumors.

A Review of the Clinical Features and Management of Systemic Congenital Mastocytosis through the Presentation of An Unusual Prenatal-Onset Case.

Mastocytosis is a heterogeneous group of rare hematological disorders that can occur in infancy. We report a 16-year-old girl who presented with an aggressive form of systemic congenital mastocytosis, associated with a significant global developmental delay, deafness, and multiple anomalies. At 4 years of age, she developed a germinoma presenting as an invasive spinal mass. Extensive cytogenetic, metabolic, and molecular genetic studies that included whole-exome sequencing studies revealed a KIT alteration (NM_000222.3(KIT):c2447A > 7 pAsp816Val) and likely pathogenic variant in the DNA from peripheral blood and skin lesions. C-kit was also found to be overexpressed in the spinal tumor cells. We compared the features of this child to those of six previously reported pediatric patients with cutaneous mastocytosis, microcephaly, microtia, and/or hearing loss reported in OMIM as mastocytosis, conductive hearing loss, and microtia (MIM 248910), for which the etiology has not yet been determined. This report extends the currently recognized spectrum of KIT-related disorders and provides clues as to the potential etiology of a syndromic form of congenital mastocytosis. International efforts to understand the benefits of long-term targeted therapy with tyrosine kinase inhibitors for this KIT-altered rare disease should continue to be evaluated in clinical trials.