H3F3A Mutations Research Articles

Radiological, clinical, and molecular analyses reveal distinct subtypes of butterfly glioblastomas affecting the prognosis

Abstract Background Glioblastoma (GB) is known for its highly invasive nature. Images of butterfly GB (bGB) often illustrate this characteristic, but the molecular background and origins of bGB remain unknown. Methods We analyzed a cohort of 34 bGB patients from our dataset (K-cohort) and 46 bGB patients from publicly available datasets, including TCGA-GBM, CPTAC-GBM, IvyGAP, and UPENN-GBM. Results In the K-cohort, the median age was 66 years, and molecular analyses revealed TERT promoter mutations in 55.9% of cases, with no cases exhibiting H3F3A, HIST1H3B, or BRAF mutations. Sequential radiological imaging from the K-cohort provided unique insights, showing one case originating in the corpus callosum (CC) and three cases originating in the cerebral hemisphere before developing into bGB. Multi-regional sampling supported a mutational trajectory from the hemisphere to the CC. These observations indicate the presence of two distinct radiological origins for bGB. Consequently, we classified cases into CC-type and Hemispheric-type based on the tumor volume ratio within the CC. This subgrouping was clinically meaningful; the CC-type is an independent poor prognostic factor for overall survival, with a hazard ratio of 1.8 (95% confidence interval 1.1-3.0, P = 0.033), and is molecularly distinct by a higher frequency of methylated MGMTp (P = 0.0039) compared to the Hemispheric-type. Conclusions Our results highlight that the radiological features of bGB are not homogenous and can indicate two potential subtypes based on their origins. Further studies are mandatory, but CC-type and Hemispheric-type exhibit distinct clinical backgrounds, outcomes, and molecular features.

Characterization of the Rat Osteosarcoma Cell Line UMR-106 by Long-Read Technologies Identifies a Large Block of Amplified Genes Associated with Human Disease.

The rat osteosarcoma cell line UMR-106 is widely used for the study of bone cancer biology but it has not been well characterized with modern genomic methods. To better understand the biology of UMR-106 cells we used a combination of optical genome mapping (OGM), long-read sequencing nanopore sequencing and RNA sequencing.The UMR-106 genome was compared to a strain-matched Sprague-Dawley rat for variants associated with human osteosarcoma while expression data were contrasted with a public osteoblast dataset. Using the COSMIC database to identify the most affected genes in human osteosarcomas we found somatic mutations in Tp53 and H3f3a. OGM identified a relatively small number of differences between the cell line and a strain-matched control animal but did detect a ~45 Mb block of amplification that included Myc on chromosome 7 which was confirmed by long-read sequencing. The amplified region showed several blocks of non-contiguous rearranged sequence implying complex rearrangements during their formation and included 14 genes reported as biomarkers in human osteosarcoma, many of which also showed increased transcription. A comparison of 5mC methylation from the nanopore reads of tumor and control samples identified genes with distinct differences including the OS marker Cdkn2a. This dataset illustrates the value of long DNA methods for the characterization of cell lines and how inter-species analysis can inform us about the genetic nature underlying mutations that underpin specific tumor types. The data should be a valuable resource for investigators studying osteosarcoma, in general, and specifically the UMR-106 model.

DIPG-48. PRECISION GUIDED THERAPY PROVIDES CLINICAL BENEFIT IN H3K27-ALTERED DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND The utility of precision-guided therapeutic approaches to H3K27-altered diffused midline glioma (DMG) remains uncertain. The Australian Zero Childhood Cancer (ZERO) program combines molecular profiling (whole-genome sequencing (WGS), whole-transcriptome sequencing (RNAseq), and DNA methylation profiling), with in vitro high-throughput drug screening (HTS) and patient-derived xenograft (PDX) drug-efficacy testing in patients with high-risk cancer. METHODS We report on the cohort of patients enrolled with a DMG in the ZERO clinical trial between 2017 and 2023. RESULTS 68 patients were enrolled, predominantly at diagnosis (87%), with pontine lesions (66%) at a mean age of 8.7 years. 59 (87%) patients had WGS + RNAseq successfully performed. Most tumors harbored an H3F3A mutation (n=50), followed by HIST1H3B (n=11), HIST1H3C (n=2), HIST2H3C (n=1) and EZHIP overexpression (n=4). 37 samples were suitable for cell culture, with HTS successfully performed in 25 cases, and PDXs established in 7. Precision-guided therapy (PGT) was recommended by the ZERO multidisciplinary tumor board for 53 patients (78%), including 5 recommendations guided by HTS. PI3K/mTOR inhibitors and MEK inhibitors were the most recommended PGTs. 21 patients (31%), receiving 24 PGTs, were eligible for survival analysis. The clinical benefit rate (per RAPNO) was 52% for patients receiving PGT, with 5 patients exhibiting PR and 6 patients SD >6 months. Notably, the 5 PR included: a PR in a patient with BRAFV600E mutation to dabrafenib/trametinib, a near-CR in a patient with germline BRCA2 mutation to olaparib/durvalumab, a sustained PR in a patient with FGFR1 mutation to trametinib/bevacizumab and 2 patients with PIK3CA mutations who received PGT in the context of recent radiotherapy. Median overall survival of the PGT group was 21.2 months versus 11.8 months for the rest of the cohort (hazard ratio 0.67 [95%CI 0.39-1.1]). CONCLUSIONS These results, for the first time, highlight the potential clinical benefit of PGT in H3K27-altered DMG.

Clinicopathological characteristics of gangliogliomas with anaplastic morphology

Objective: To investigate the clinical, radiological, and pathological features of anaplastic gangliogliomas (AGGs) and to determine whether these tumors represent a distinct entity. Methods: Consecutive 667 cases of ganglioglioma (GG) diagnosed at the Xuanwu Hospital, Capital Medical University, Beijing, China between January 2015 and July 2023 were screened. Among these cases, 9 pathologically confirmed AGG cases were identified. Their clinical, radiological, treatment, and outcome data were analyzed retrospectively. Most of the tumor samples were subject to next-generation sequencing, while a subset of them were subject to DNA methylation profiling. Results: Among the 9 patients, there were five males and four females, with a median age of 8 years. Epileptic seizures (5/9) were the most frequently presented symptom. Radiological examinations showed three types of radiological manifestations: four cases showed abnormal MRI signals with no significant mass effects and mild enhancement; two cases demonstrated a mixed solid-cystic density lesion with peritumoral edema, which showed significant heterogeneous enhancement and obvious mass effects, and one case displayed cystic cavity formation with nodules on MRI, which showed evident enhancements. All cases exhibited mutations that were predicted to activate the MAP kinase signaling pathway, including seven with BRAF p.V600E mutation and two with NF1 mutation. Five AGGs with mutations involving the MAP kinase signaling pathway also had concurrent mutations, including three with CDKN2A homozygous deletion, one with a TERT promoter mutation, one with a H3F3A mutation, and one with a PTEN mutation. Conclusions: AGG exhibits a distinct spectrum of pathology, genetic mutations and clinical behaviors, differing from GG. Given these characteristics suggest that AGG may be a distinct tumor type, further expansion of the case series is needed. Therefore, a comprehensive integration of clinical, histological, and molecular analyses is required to correctly diagnose AGG. It will also help guide treatments and prognostication.

Expression of Interleukin-13 Receptor Alpha 2 in Brainstem Gliomas.

The objective of this study was to investigate IL13Ra2 expression in brainstem glioma (BSG) and its correlation with key markers, functions, and prognostic implications, evaluating its therapeutic potential. A total of 80 tumor samples from BSG patients were analyzed. Multiplex immunofluorescence was used to examine six markers-IL13Ra2, H3.3K27M, CD133, Ki67, HLA-1, and CD4-establishing relationships between IL13Ra2 and these markers. Survival analysis, employing Kaplan-Meier and Cox proportional hazard regression models, encompassed 66 patients with complete follow-up. RNA-Seq data from a previously published study involving 98 patients were analyzed using the DESeq2 library to determine differential gene expression between groups. Gene Ontology (GO) enrichment and single-sample gene set enrichment analysis (ssGSEA) via the clusterProfiler library were used to delineate the gene functions of differentially expressed genes (DEGs). Nearly all the BSG patients displayed varying IL13Ra2 expression, with 45.0% (36/80) exhibiting over a 20% increase. Elevated IL13Ra2 levels were notably observed in pontine gliomas, diffuse intrinsic pontine gliomas (DIPGs), H3F3A-mutant gliomas, and WHO IV gliomas. IL13Ra2 expression was strongly correlated with H3.3K27M mutant protein, Ki67, and CD133. Patients with IL13Ra2 expression >20% showed shorter overall survival compared to those with ≤20% IL13Ra2 expression. The Cox proportional hazard regression model identified H3F3A mutations, rather than IL13Ra2 expression, as an independent prognostic factor. Analysis of RNA-Seq data from our prior cohort confirmed IL13Ra2's correlation with H3.3, CD133, and Ki67 levels. Widespread IL13Ra2 expression in BSG, particularly elevated in the H3F3A mutant group, was strongly correlated with H3F3A mutations, increased proliferation, and heightened tumor stemness. IL13Ra2 represents a promising therapeutic target for BSGs, potentially benefiting patients with H3K27M mutations, DIPGs, WHO Grade IV, and pontine location-specific BSGs, particularly those with H3K27M mutations.

10184-BT-14 CLINICAL FEATURE OF HEMISPHERIC GLIOMA WITH H3F3A, PEDIATRIC-TYPE HIGH GRADE GLIOMA, H3 K27-ALTERED, NEC AND DIFFUSE HEMISPHERIC GLIOMA, H3 G34-MUTANT, CNS WHO GRADE 4

Abstract INTRODUCTION Diffuse midline glioma (DMG), H3K27-altered, is a CNS WHO grade 4 glioma that usually located mainly in the brainstem, thalamus and spinal cord. However, DMG located in non-midline lesions are now described as pediatric-type high grade glioma, H3K27-altered, NEC (NDMG). On the other hand, diffuse hemispheric glioma, H3G34-mutant (DHG) located in the cerebral hemispheres, and is classified under the WHO 2021 classification. It is unknown that the differences in clinical characteristics of these hemispheric tumors with H3F3A mutations. In the present study, we report a comparative study of the clinical characteristics of two groups of NDMG and DHG. METHODS Among 4128 brain tumor samples collected in the Kansai Network for Molecular Diagnosis of Central Nervous System Tumors, 16 NDMG and 9 DHG cases were examined for comparison of clinical characteristics. RESULTS There were no differences in gender, tumor location, or pathology between NDMG and DHG. The median age was 47.3 years in NDMG and 26.2 years in DHG, and NDMG was significantly older than DHG (p=0.003). There was no significant difference in MGMT promoter methylation between NDMG and DHG (p=0.087). The Kaplan-Meier survival curve showed no significant difference, with a median survival of 495 days for NDMG and 587 days for DHG (p=0.765). There was no significant different survival rate between WHO grade 3 (n=15) and grade 4 (n=9) for pathological diagnosis. DISCUSSION AND CONCLUSION We compared the clinical characteristics of NDMG and DHG. There are some reports that NDMG and DHG gliomas located in non-midline lesion. Removing much tumor volume may improve the prognosis of non-midline glioma. We discuss the gliomas with H3F3A mutations located in a hemispheric lesions by literature review.

Giant cell tumor of bone-an update

In the past few years, numerous new insights have been gained in the field of giant cell tumor of bone (GCTB). On the one hand, the detection of the highly characteristic histone mutation in the H3F3A gene in GCTB is becoming increasingly important in diagnostics in differentiating GCTB from other giant cell-rich lesions of bone as well as for defining rare variants of GCTB without osteoclastic giant cells. On the other hand, the effects of the H3F3A mutation were shown to have an impact on the epigenetic profile of tumor-driving stromal cells, providing new insights into tumorigenesis of GCTB.

PATH-18. MORE UNUSUAL FEATURES IN H3 K27M-MUTANT GLIOMAS

Abstract Diffuse midline glioma, H3 K27M-altered, WHO grade 4 is an infiltrating midline glioma; however, H3 K27M mutation can also be seen in midline circumscribed, non-infiltrating gliomas and rarely in non-midline tumors. We report 2 unusual examples. A 37-year-old female developed a right posterior frontal infiltrating glioma; resection showed infiltrative tumor with brisk mitotic activity, elevated MIB-1 (20%), negative p53 expression, ATRX retention, but no necrosis or microvascular proliferation. Molecular workup showed neither IDH1/2/TERT promoter mutations or co-deletion 1p, 19q; further testing showed gain of whole chromosome 7 + loss of whole chromosome 10, meeting molecular criteria for glioblastoma. Recurrent tumor 2 years later in the same area prompted re-resection again of infiltrating tumor devoid of necrosis or microvascular proliferation; molecular testing now revealed a H3F3A (p.K28M) mutation. Both tumors showed protein expression by H3 K27M IHC+. She succumbed post-therapy at 3 ½ years. A 30-year-old woman developed a circumscribed, non-enhancing third ventricular mass; resection demonstrated circumscribed, non-infiltrating glioneuronal tumor with monotonous rounded tumor nuclei, synaptophysin IHC+, negative GFAP, H3 K27M IHC+, and low MIB-1. Molecular testing identified alterations in H3F3A p. K28M and FGFR1 p.N546K. She was treated, but lost to follow-up, until she re-presented 5 years later with hemorrhagic left frontal lobe tumor contiguous with the midline, with dorsal midbrain and 4th ventricular involvement. After resection, tumor now showed infiltrative features, necrosis and microvascular proliferation, diffuse GFAP IHC+, retention of nuclear ATRX, p53 (20%), MIB-1 (22%), and H3 K27M IHC+. New tumor had no FGFR1 alteration or PDGFRA amplification to suggest radiation-induced tumor, instead manifesting PTPRZ1-MET fusion. She survives at 5 years. These cases offer new insights into survival of a non-midline example and transformation of a circumscribed, non-infiltrating tumor to secondary infiltrating H3 K27-altered tumor with new molecular alterations, possibly representing second independent tumor development.

NIMG-61. RADIOLOGICAL, CLINICAL, AND MOLECULAR ANALYSES UNRAVEL DISTINCT SUBTYPES OF ADULT GRADE 4 BUTTERFLY GLIOMAS AFFECTING THE PROGNOSIS

Abstract Gliomas located at the corpus callosum (CC) and invading the bilateral hemispheres is known as butterfly gliomas (bG). Images of bG are frequently used as the hallmark of glioblastoma (GB); however, the molecular background and origins of bG remain unknown. We collected 37 bG patients, including 32 cases of GB and 5 cases of astrocytoma, WHO grade 4, IDH-mutant. Our dataset included extremely rare sequential radiological imaging before receiving a definitive diagnosis of bG. One case emerged from CC and subsequently became bG, whereas five cases emerged from the cerebral hemisphere and became bG, suggesting distinct radiological origins exist in bG. Reflecting this observation to further analysis, we subdivided the cohort into CC-type (n = 15) and hemispheric-type (n = 22) based on the ratio calculated by the tumor volume at CC and hemisphere. Survival analysis presented that CC-type was significantly poorer overall survival (OS) than hemispheric-type (P = 0.010), indicating that bG has clinically distinct two subtypes. Molecular analyses revealed that TERT promoter mutations and IDH1 mutations were observed in 73.3% and 0% in the CC-type, whereas 36.4% and 22.7% in the hemispheric-type, respectively. Other alterations such as MGMT promoter methylation, EGFR amplification/gain, CDKN2A hemi/homozygous deletion, and PTEN hemi/homozygous deletion remained the same between two groups. None of the patients had H3F3A, HIST1H3B, or BRAF mutations. In the hemispheric-type, the multisampling of hemispheric and CC specimens revealed accumulating alterations in the CC lesions, indicating an invasive trajectory from the hemisphere to the CC region. For the validation, we reviewed 993 MRI obtained from the public datasets (TCGA-GBM, CPTAC-GBM, IvyGap, and UPenn-GBM) and found the same trend: CC-type presented poor OS. bG is not a sole subtype with just extensive invasion from the hemisphere. Our results highlight the pathophysiology of bG and may influence its management.

BIOM-46. STANDARDIZATION OF LIQUID BIOPSY FOR PEDIATRIC DIFFUSE MIDLINE GLIOMA USING DDPCR

Abstract 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 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. 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 A >T 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 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. CONCLUSION 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.

Clinicohistoradiological and surgical outcome in diffuse midline glioma.

Diffuse Midline Glioma (DMG) with H3K27M mutation is a rare and aggressive midline high grade glioma with a predominant astrocytic differentiation and K27M mutation in either H3F3A or HIST1H3B/C. This tumor is more common in children than in adults. The current study was aimed to determine clinicohistoradiologicaland surgical outcome of patients who have undergone surgery for DMG and study disease severity of patients with DMG. This is an observational study in which 29 DMG patients were evaluated for clinicohistoradiological and surgical outcomes by assessing the pre and postoperative neurological status. Survival duration was significantly high in patients with age > 18years (p = 0.02). Patients who had undergone Radiation Therapy showed higher survival rate (p = 0.05) and the cases with low levels of Ki 67 index had improved post operative outcome (p = 0.002). DMG with H3K27M mutation in newly classified Central Nervous System tumor areWHO grade IVTumors, comprising H3K27M mutation as molecular marker for diagnosis and related with a poor prognosis.

DIPG-47. DISCOVERING A MOLECULAR BASIS FOR VULNERABILITY OF H3K27M-DMG TO HISTONE DEACETYLASE INHIBITORS

Abstract Using two H3K27M-DMG treatment-naive preclinical models (PBT22 and PBT29), we detected a 20-fold differential response to the histone deacetylase inhibitor, Quisinostat. PBT-22 harbors mutations in H3F3A, TP53, and ASXL2, while PBT-29 has mutations in H3F3A, TP53, PIK3CA and FGFR1. Acetylation and deacetylation of wtH3 alter chromatin structure as part of normal regulation of gene expression. The methionine substitution for Lysine in H3K27M removes this histone from participating in acetylation/deacetylation regulated gene expression. Concomitantly, H3K27M induces a marked reduction in global acetylation of histone tails on wtH3. In the native state of H3K27M DMG with hypoacetylated H3, we posit that the specific pattern of nucleosome integration with genes responsible for cell survival/death underlies the differential vulnerability of DMGs to HDACi. Additionally, the nucleosome integration pattern with cell survival/death genes may be based on the relative abundance of wtH3wt versus H3K27M- histones. Following Quis treatment (48 hrs) in both preclinical models, total wtH3ac protein abundance increased 3-fold, suggesting HDACi stabilizes or impedes K27 acetylated H3 histone turnover. Comparison of differentially expressed genes (DEGs) from control and Quis-treated PBT22 and PBT29 will seed gene ontology analysis focusing on chromatin remodeling, cell death, and growth arrest pathways accounting for differential vulnerability. Findings will be validated by measuring transcripts and proteins (qRT-PCR and ELISA or mass spectrometry) from analytes from a larger panel of DMG cell lines. The data depicts DMG preclinical models with large differential sensitivity to Quis. The long-term goal is to discover a molecular profile of DMGs predictive of the vulnerability to HDACi. The discovery exercise is also likely to yield insight into the development of resistance to HDAC inhibitors.

Distinct molecular features and potential therapeutic strategies for spinal cord gliomas.

2056 Background: Spinal cord gliomas (SCG) are rare primary CNS tumors with varied prognosis. Standard treatment remains poorly defined, as no clear survival advantage exists with extensive resection, radiation, or chemotherapy. Mounting evidence suggests that tumors from different regions of the CNS harbor unique molecular signatures. In a retrospective study, we comprehensively characterized molecular alterations to identify potential therapeutic strategies in the largest cohort of SCG reported to date. Methods: We performed centralized pathology review and analyzed SCG with next-generation sequencing of DNA (592 gene NextSeq or WES, Novaseq), RNA (WTS, NovaSeq) and pyrosequencing (MGMT promoter methylation, MGMTme). We estimated tumor microenvironment cell infiltration by quanTIseq & Epithelial-Mesenchymal Transition (EMT) by RNAseq. We used X2/Fisher’s-exact/Mann-Whitney U tests for comparison & determined significance (p < 0.05), adjusting for multiple comparison by the Benjamini-Hochberg method (q < 0.05). Results: We analyzed 39 surgically accessible SCG (19 high grade, 17 low grade, 3 NOS); ependymomas were excluded. The most common alterations were mutations in H3F3A (31%), TP53 (25%), ATRX (21%), NF1 (18%) & BRAF fusion (19%). H3F3A mutations occurred exclusively in high grade SCG (63%, q < 0.05) while BRAF fusions occurred exclusively in low grade SCG (60%, p < 0.05). TP53 mutations were more prevalent in high grade SCG (50% vs. 8%, p < 0.05). Compared to intracranial gliomas (n = 6732), SCG harbor significantly more frequent H3F3A (31% vs. 1.6%), KRAS mutations (10% vs. 0.7%) & BRAF fusions (19% vs. 0.7%) but less frequent TERT (4.3% vs. 66%) & PTEN (0 vs. 27%) mutations or EGFR amplification (0 vs. 28%) (all q < 0.05). SCG rarely harbor the canonical intracranial alterations IDH1 (2.6% vs. 17%), EGFRvIII (0 vs. 17%) or MGMTme (19% vs. 47%) (p < 0.05). SCG have greater penetration of immune infiltrates of DCs (median cell fraction/MCF: 13% vs. 9%), T regs (positive percent: 47% vs. 26%) & B cells (MCF: 9% vs. 7%), with less penetration of monocytes (MCF: 0 vs. 2%) (q < 0.05). EMT score associates with high grade glioma in intracranial disease (q < 0.05) but does not recapitulate in SCG (p > 0.05). Conclusions: Clinical management of SCG is currently drawn from experience with intracranial gliomas. Our results identify unique molecular features of SCG suggesting an underlying biology distinct from intracranial gliomas. In SCG, H3F3A mutations are exclusive to high grade while BRAF fusions are exclusive to low grade; SCG rarely harbor canonical intracranial alterations such as IDH1, EGFR or MGMTme; and SCG have greater penetration of DCs with less penetration of monocytes. We provide a biological explanation for limited effectiveness of current therapies in SCG with potential implications for chemotherapy, targeted and immunotherapy. Our work underscores the need for investigations dedicated uniquely to SCG.

Abstract 4740: Molecular effects of histone deacetylase inhibitor Quisinostat on diffuse midline glioma of the pons

Abstract Diffuse Midline Glioma of the pons (DMG) is a lethal, aggressive heterogeneous brain stem tumor. Median overall survival is less than a year, with radiation as the only standard treatment. Recently, mutations in DMGs have arisen as potential therapeutic targets, specifically a mutation in one of the histone H3 genes, resulting in methionine substituted for lysine at site 27 (H3K27M). H3K27M induces a marked reduction in global acetylation of histone tails, altering chromatin structure and causing aberrant gene expression. Histone deacetylase inhibitors, HDACis, are epigenetic drugs that show anticancer activity. In our studies, Quisinostat (Quis) is used to preserve histone acetylation. Using two H3K27M-DMG treatment-naive preclinical models (PBT22 and PBT29) we detected a 100-fold differential response to the histone deacetylase inhibitor, Quisinostat. PBT-22 harbors mutations in H3F3A, TP53, and ASXL2, while PBT-29 has mutations in H3F3A, TP53, PIK3CA and FGFR1. Following Quis treatment (48 hrs) in both preclinical models, total H3K27ac protein abundance increased 3-fold, suggesting HDACi stabilizes or impedes turnover of K27 acetylated H3 histone. We are pursuing studies to test whether sensitivity to HDACi in DMGs is determined by a shift in relative or total abundance of respective H3wt- and H3K27M- histones. We posit that changes in H3K27ac manifest as shifts in nucleosome integration with genes responsible for cell survival/death. This project will profile differentially expressed genes (DEGs) from Quis-treated PBT22 and PBT29. Additionally, total and relative abundance of H3 proteins (wt and mt, me, me2, me3, and ac) from treated cells will be determined. Gene ontology analysis will focus on pathways accounting for chromatin remodeling, cell death, and growth arrest. RNA (qRT-PCR) and proteins (western blot) from analytes from a larger panel of DMG cell lines and neural stem cells treated with Quis will be used to validate the findings. Overall, the data depicts DMG preclinical models with large differential sensitivity to Quis, which may be partially due to different oncoprints between the models. The markedly different sensitivity of these models enables mechanistic study of the consequences of elevated abundance of histone 3 acetylation. The long term goal is to discover a molecular profile of DMGs indicative of the vulnerability to HDACi. Citation Format: Danyelle Paine, Nanyun Tang, Yue Hao, Matt Biery, Carrie Meyers, Alyssa Noll, Nicholas Vitanza, Michael Berens. Molecular effects of histone deacetylase inhibitor Quisinostat on diffuse midline glioma of the pons. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4740.

Abstract 936: Clinicogenomic predictors for the pattern of failure in high-grade glioma

Abstract Background: High-grade gliomas, the most common primary brain tumors, are highly lethal, and treatment options remain limited. Despite advances in genomic technologies, there are few molecular biomarkers to guide precision medicine for high-grade glioma. Here, we aimed to identify the clinicogenomic features associated with its prognosis and recurrence patterns. Materials/Methods: Our single-institution retrospective analysis included 223 patients diagnosed with high-grade gliomas who underwent next-generation sequencing targeting 82 brain tumor-relevant genes. We categorized them according to the 2021 WHO classification, and their clinicopathological statuses, treatment characteristics, survival, and genomic profiles were analyzed. Results: The most common genomic variants were TERT promoter (51%), TP53 (39%), IDH1/2 (18%), NF1 (13%), ATRX (12%), PTEN (11%), PIK3CA (9%), SETD2 (7%), and H3F3A (6%) mutation. Regarding copy number variants, amplification of EGFR (21%), PDGFRA (11%), CDK4 (9%) and loss of CDKN2A/2B (55%), PTEN (29%), 19q (17%), NF2 (14%), and RB1 (10%) were the most common copy number aberrations. At a median follow-up of 18 months (range, 2-54 months), 174 patients (78%) had progression or recurrences. A total of 15.2% of our patients were re-classified under the 2021 WHO classification, and there was a clear separation of overall survival(OS) and progression-free survival(PFS) curves between grade 3 and 4 groups using the revised grading system. On multivariate cox regression analysis, MYCN amplification (HR 6.08, p = 0.002) and SETD2 mutation (HR 0.19, p =0.06) were independent predictors of OS and PFS. Conclusion: The assessment of genomic characteristics in conjunction with the pattern of failure for high-grade glioma aids in identifying patients who are likely to benefit from personalized medicine. The 2021 WHO grading system explains survival differences between grade 3 and 4 tumors better than the 2016 WHO classification dose. We identified SETD2 mutation and MYCN as prognostic biomarkers with potential therapeutic implications in patients with high-grade glioma. Citation Format: Joo Ho Lee, Byung-Hee Kang, Seungbok Lee. Clinicogenomic predictors for the pattern of failure in high-grade glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 936.

The clinicopathological features and prognosis of multifocal high-grade gliomas in adults withH3F3Amutation

To explore the clinicopathological features and prognosis of multifocal high-grade gliomas (M-HGGs) with H3F3A mutation in adults. Four adult patients with H3F3A-mutant M-HGGs who were treated at our institution from August 2020 to December 2021 were reviewed, including clinical, pathological and radiologic data. A series of 16 adult patients with M-HGGs without H3F3A mutation was used as a comparative group. Progression-free survival (PFS) and overall survival (OS) were compared between the groups using the Kaplan-Meier method. All patients were IDH wild-type and TERT wild-type, and P53 was overexpressed. A patient with the H3 G34R mutation and 1 of 3 patients with the H3 K27M mutation had MGMT promoter methylation. The lesions with the H3 G34R mutation were located in the cerebral hemisphere; the lesions with H3 K27 alterations were mainly in the midline structure, and the cerebral hemisphere could also be involved. One patient underwent subtotal resection (STR), and 3 patients underwent biopsy. All patients received radiotherapy, and the median PFS and OS were 9.5 months and 14.5 months, respectively. The clinical outcomes were similar to those of non-H3F3A-mutated M-HGGs patients (median PFS and OS were 7.0 months and 18.0 months, respectively). We describe the clinicopathological features and outcomes of 4 adult M-HGGs patients with H3F3A mutation, and found this mutation doesn't appear to have a negative outcome with the administration of current therapies.

Novel genetically engineered H3.3G34R model reveals cooperation with ATRX loss in upregulation of Hoxa cluster genes and promotion of neuronal lineage.

Pediatric high-grade gliomas (pHGGs) are aggressive pediatric CNS tumors and an important subset are characterized by mutations in H3F3A, the gene that encodes Histone H3.3 (H3.3). Substitution of Glycine at position 34 of H3.3 with either Arginine or Valine (H3.3G34R/V), was recently described and characterized in a large cohort of pHGG samples as occurring in 5-20% of pHGGs. Attempts to study the mechanism of H3.3G34R have proven difficult due to the lack of knowledge regarding the cell-of-origin and the requirement for co-occurring mutations for model development. We sought to develop a biologically relevant animal model of pHGG to probe the downstream effects of the H3.3G34R mutation in the context of vital co-occurring mutations. We developed a genetically engineered mouse model (GEMM) that incorporates PDGF-A activation, TP53 loss and the H3.3G34R mutation both in the presence and loss of Alpha thalassemia/mental retardation syndrome X-linked (ATRX), which is commonly mutated in H3.3G34 mutant pHGGs. We demonstrated that ATRX loss significantly increases tumor latency in the absence of H3.3G34R and inhibits ependymal differentiation in the presence of H3.3G34R. Transcriptomic analysis revealed that ATRX loss in the context of H3.3G34R upregulates Hoxa cluster genes. We also found that the H3.3G34R overexpression leads to enrichment of neuronal markers but only in the context of ATRX loss. This study proposes a mechanism in which ATRX loss is the major contributor to many key transcriptomic changes in H3.3G34R pHGGs. GSE197988.

Diagnostic value of H3F3A mutation and clinicopathological features of giant cell tumours in non-long bones

AimsA histone H3F3A (H3.3) mutation involving a substitution in H3.3 G34 recently has been reported in GCTB within the frequency range (from 69 % to 96 %) and is a helpful diagnostic indicator of GCTB. However, the relationship between H3F3A mutations and the clinicopathological feature of GCTB involving non-long bones (irregular bones and small bones) is unclear. Methods and resultsH3F3A mutations were observed in a cohort of specimens (230 samples of GCTB) using immunohistochemistry and Sanger sequencing. The relationship between H3F3A mutations and the clinicopathological characteristics of patients with GCTB occurring in the non-long bones of the appendicular skeleton was investigated. No significant difference between H3F3A mutations in GCTB arising in non-long bones and the classic sites was found (P = 0.483). GCTB in non-long bones occurred more common in female (31/49, 63.3 %) than in male patients (P = 0.016). GCTB with H3.3 G34L/V/R mutation occurred more often in younger patients compared with those with H3.3 G34W mutation (P = 0.009). The majority of GCTB with soft tissue extension developed in irregular bones but not in small bones (P = 0.061). The H3.3 G34L/V/R mutations rate (7/45) in the non-long bones was significantly higher than that in long bones. The recurrence rate of the GCTB in long bones and non-long bones was 23.3 % (45/193) including 43 cases with local recurrene and 2 cases with lung metastasis. No recurrence occurred in cases with G34V/L/R mutations. ConclusionsH3F3A was an effective diagnostic marker for GCTB of the non-long bones. The younger patients with GCTB of the non-long bones harboured H3.3 G34L/V/R mutations and may had a female preference and rarely recurrent.

Bone and soft tissue tumors: clinicoradiologic-pathologicmolecular-genetic correlation of novel fusion spindled, targetable-ovoid, giant-cell-rich, and round cell sarcomas.

New entities in the classification of bone and soft tissue tumors have been identified by use of advanced molecular-genetic techniques,including next-generation sequencing. Clinicoradiologic and pathologic correlation supports diagnostic classification. Tumors from four morphologically grouped areas are selected to enhance diagnosis and awareness among the multidisciplinary team. These include select round cell tumors, spindle cell tumors, targetable tyrosine kinase/RAS::MAPK pathway-ovoid (epithelioid to spindled)tumors, and giant-cell-rich tumors of bone and soft tissue. Round cell tumors of bone and soft tissue include prototypical Ewing sarcoma, newer sarcomas with BCOR genetic alteration and CIC-rearranged, as well as updates onFUS/EWSR1::NFATc2, an EWSR1 non-ETS tumor that is solid with additional amplified hybridization signal pattern of EWSR1.This FUS/EWSR1::NFATc2 fusion hasnow been observed in seemingly benign to low-grade intraosseous vascular-rich and simple (unicameral) bone cyst tumors. Select spindle cell tumors of bone and soft tissue include rhabdomyosarcoma with FUS/EWSR1::TFCP2, an intraosseous high-grade spindle cell tumor without matrix. Targetable tyrosine-kinase or RAS::MAPK pathway-tumors of bone and soft tissue include NTRK, ALK, BRAF, RAF1, RET, FGFR1, ABL1, EGFR, PDGFB,and MET with variable ovoid myopericytic to spindled pleomorphic features and reproducible clinicopathologic and radiologic clues to their diagnosis. Giant-cell-rich tumors of bone, joint, and soft tissue are now respectivelycharacterized by H3F3A mutation, CSF1 rearrangement (targetable), and HMGA2::NCOR2 fusion. This article is an update for radiologists, oncologists, surgeons, and pathologists to recognize these novel ovoid, spindled, giant-cell-rich, and round cell tumors, for optimal diagnostic classification and multidisciplinary team patient care.

PEDT-12 NATIONWIDE LANDSCAPE OF GENOMIC ANALYSIS OF PEDIATRIC CENTRAL NERVOUS SYSTEM TUMORS

Abstract Background and Purpose In Japan, cancer-gene profiling (CGP) tests, such as the OncoGuide NCC Oncopanel System, FoundationOne CDx Cancer Genome Profile (F-one), and FoundationOne Liquid CDx Cancer Genome Profile, are covered by insurance and clinically applied to promote cancer genomic medicine. Information from CGP tests is managed at the Center for Cancer Genomics and Advanced Therapeutics (C-CAT) and shared with clinicians after discussions at the Expert Panel (EP). We provide an overview of the registered pediatric CNS tumors. Methods We extracted and analyzed CNS/brain data from the C-CAT registry from June 2019 to June 2022 for ages 0–19 years. Variables such as age, gender, histopathological diagnosis, CGP test type, ECOG Performance Status (PS), registration status, genetic abnormalities, and treatments were analyzed. Results Among 1133 patients of all ages with CNS tumors, 361 (31.9%) were aged 0–19 years; Of the 918 patients aged 0-19 years with any type of cancer, those with CNS tumors accounted for 34.2%; 188 patients were aged under 10 years, 173 patients were teenagers, 174 patients were males, 187 patients were females; PS was 0 in 47.9% of patients. The most common histopathological diagnoses were medulloblastoma, diffuse midline glioma and glioblastoma. Genetic abnormalities included TP53 mutations in 85 cases, H3F3A mutations in 38 cases, STK11 mutations in 36 cases, BRAF mutations in 33 cases, BRAF-KIAA1549 fusions in 24 cases, and TMB high in 16 cases. Seventy-six patients (21.1%) were offered treatment options in the EP, and 26 patients (7.2%) received the recommended treatment, including two in physician-initiated trials, four in company trials, eleven in treatments within insurance coverage, and eleven in others. Discussion This is the first report of genetic analysis of pediatric CNS tumors on a nationwide scale. Pediatric CNS tumors have a large number of registered cases; thus, drug development for them is urgently needed.