H3F3A K27M Research Articles

Primary spinal cord gliomas: Pathologic features associated with prognosis.

Primary spinal cord gliomas are rare and are associated with high mortality. Unlike brain tumors, the clinicopathological features of spinal cord gliomas are not well defined. We analyzed clinical, histopathology, and immunohistochemical features and overall survival (OS) of 25 patients with primary spinal cord gliomas treated between 1994 and 2023 at 4 institutions. IDH1 R132H, H3K27M, and p53 were assessed by immunohistochemistry (IHC). Four (16%), 5 (20%), 2 (8%), and 13 (52%) patients were diagnosed as having grades 1, 2, 3, and 4 gliomas according to the World Health Organization (WHO) 2021 classification, respectively. One case (4%), with a circumscribed diffuse midline glioma, H3K27-altered, had a rare molecular profile and could not be graded. IHC demonstrated H3K27M positivity, indicative of H3F3A K27M or HIST1H3B K27M mutation, in 9 (36%) patients. H3K27me3-loss was evident in 13 (52%) patients. In one patient with a grade 1 tumor that showed negative staining for H3K27M and H3K27me3 loss, numbers of EZHIP-positive cells were increased, suggesting diffuse midline glioma, H3K27-altered (WHO grade 4). H3K27me3 loss, frequency of p53 positive cells (≥10%), MIB-1 index (≥10%), and high histopathological grades significantly correlated with poor OS. These results indicate the pathological and immunohistochemical characteristics of primary spinal cord gliomas that impact prognosis.

DIPG-44. DIFFUSE MIDLINE GLIOMA WITH EXTRACRANIAL METASTASIS

Abstract Diffuse midline gliomas (DMGs) are malignant infiltrative gliomas enriched in the pediatric population and characterized by loss of the H3 K27me3 epigenetic marker, most frequently via mutation of the H3F3A gene. Few cases of extracranial DMG metastasis with redemonstrated H3F3A p.K28M (K27M) mutation in metastatic tissue are reported in the literature. Here, we report two such patients, both females (ages 12 and 15), with DMG and extracranial metastasis who died 5 years after initial diagnosis. The first patient with thalamic DMG underwent biopsy, radiation, chemotherapy, and subsequent decompression of spinal metastasis. Initial tumor biopsy demonstrated mutation in BRAF p.V600E, H3F3A K27M, and TERT promoter. The epidural spinal metastasis had mutations in BRAF p.V600E, H3F3A, TERT, and TP53. The second patient with brainstem DMG, diagnosed by radiographic appearance, underwent conventional external beam radiation therapy, chemotherapy including panobinostat and direct delivery of radioimmunotherapy, intra-arterial delivery of bevacizumab, and subsequent decompression of spinal metastasis. The histology and molecular profile for the spinal metastasis was consistent with DMG H3 K27M-mutant, with mutations in BRAF p.G464V, PTPN11 p.R498L, PDGFRA, and KIT. Bone marrow biopsy demonstrated atypical extrinsic cells with strong and diffuse immunolabeling for OLIG2. Autopsy revealed solid organ and osseous metastases. Both patients significantly exceeded the median life expectancy for DMG, raising the possibility that prolonged overall survival permitted progression to a rarely observed disseminated state of disease. There was absence of TP53/p53 modulating pathway mutation seen in classic DMG in the thalamic biopsy of the first patient, as well as the metastatic disease for the second patient, which may contribute to the prolonged survival observed. Molecular analysis of metastatic disease is important, as clinically and prognostically relevant alterations that vary from the primary site of disease may be detected, which shed light on clonal evolution patterns and further our understanding of disease biology.

H3F3A K27M Mutation Promotes the Infiltrative Growth of High-Grade Glioma in Adults by Activating β-Catenin/USP1 Signaling.

Simple SummaryGliomas is a primary type of tumor in the central nervous system. High-grade glioma is a malignant cancerous disease and grows rapidly. This study reports the expression of H3.3K27M in high-grade glioma tissues and the association with malignant glioma cell behavior. Moreover, the results suggested that a high expression of H3.3K27M promotes the migration and invasion of glioma cells, leading to a poor prognosis by promoting the infiltration of glioma through aggravating aberrant activation of β-catenin signaling-driven pathway. H3F3A K27M (H3.3K27M) is a newly identified molecular pathological marker in glioma and is strongly correlated with the malignancy of diffuse intrinsic pontine glioma (DIPG). In recent years, accumulating evidence has revealed that other types of glioma also contain the H3.3K27M mutation. However, the role of H3.3K27M in high-grade adult glioma, the most malignant glioma, has not been investigated. In this study, we focused on exploring the expression and function of H3.3K27M in high-grade glioma in adults. We found that H3.3K27M was highly expressed at high levels in some high-grade glioma tissues. Then, we introduced H3.3K27M into H3.3 wild-type glioma cells, U87 cells and LN229 cells. We found that H3.3K27M did not affect the growth of glioma cells in vitro and in vivo; however, the survival of mice with transplanted tumors was significantly reduced. Further investigation revealed that H3.3K27M expression mainly promoted the migration and invasion of glioma cells. Moreover, we confirmed that H3.3K27M overexpression increased the levels of the β-catenin and p-β-catenin (Ser675) proteins, the ubiquitin-specific protease 1 (USP1) mRNA and protein levels, and the enhancer of zeste homolog 2 (EZH2) protein level. In addition, the β-catenin inhibitor XAV-939 significantly attenuated the upregulation of the aforementioned proteins and inhibited the increased migration and invasion caused by the H3.3K27M mutation. Overall, the H3.3K27M mutation in high-grade glioma is a potential biomarker for poor prognosis mainly due to the infiltration of glioma cells that is at least partially mediated by the β-catenin/USP1/EZH2 pathway.

Posterior fossa ependymoma H3 K27-mutant: an integrated radiological and histomolecular tumor analysis

Posterior fossa group A ependymomas (EPN_PFA) are characterized by a loss of H3 K27 trimethylation due to either EZHIP overexpression or H3 p.K27M mutation, similar to H3 K27-altered diffuse midline gliomas (DMG), but in reverse proportions. Very little data is available in the literature concerning H3 K27M-mutant EPN_PFA. Here, we retrospectively studied a series of nine pediatric tumors initially diagnosed as H3 K27M-mutant EPN_PFA to compare them to EZHIP-overexpressing EPN_PFA in terms of radiology, follow-up, histopathology, and molecular biology (including DNA-methylation profiling). Seven tumors clustered within EPN_PFA by DNA-methylation analysis and t-distributed stochastic neighbor embedding. Among the two remaining cases, one was reclassified as a DMG and the last was unclassified. H3 K27M-mutant EPN_PFA cases were significantly older than their counterparts with an EZHIP overexpression. Radiological and histopathological central review of our seven H3 K27M-mutant EPN_PFA cases found them to be similar to their counterparts with an EZHIP overexpression. Sequencing analyses revealed HIST1H3B (n = 2), HIST1H3C (n = 2), H3F3A (n = 1), and HIST1H3D (n = 1) K27M mutations (no sequencing analysis available for the last case which was immunopositive for H3K27M). Consequently, HIST1H3C/D mutations are more frequently observed in EPN_PFA than in classic pontine DMG, H3K27-mutant. Overall survival and event-free survival of EZHIP-overexpressing and H3 K27M-mutant EPN_PFA were similar. After surgery and radiation therapy, 5/7 patients were alive at the end of the follow-up. In summary, the diagnosis of EPN_PFA must include tumor location, growth pattern, Olig2 expression, and DNA-methylation profiling before it can be differentiated from DMG, H3 K27-altered.

Somatic structural variations in pediatric brain tumors.

Pediatrics brain tumors are the second most frequent malignancy in children, and the most common cause of cancer-related deaths in both the 0-14-year and the 15-24-year age group. Although, pediatrics high-grade glioma (pHGG) is a histologically similar tumor to that arising in adults, these are distinct biological diseases, differing in copy number profiles and driver genetic alterations. Recent sequencing initiatives have conclusively shown the existence of subgroups of HGG marked by distinct driver mutations, which are significantly enriched in young children (H3F3A K27M), teenagers and young adults (H3F3A G34R/V), and middle- aged adults (IDH1/2). Structural rearrangements resulting in novel fusion genes are strongly associated with cancer, and numerous examples exist in both adult and childhood malignancies. Although, only few have been described in pHGG. Structural variants (SV) frequently result in chimeric proteins targetable by novel therapeutic approaches, an outcome desperately needed in pHGG.

The prognostic significance of HIST1H3B/C and H3F3A K27M mutations in diffuse midline gliomas is influenced by patient age.

Diffuse midline gliomas (DMGs) are infiltrative midline gliomas harboring H3K27M mutations and are generally associated with poor outcomes. H3K27M mutations include mutations in HIST1H3B/C (H3.1), HIST2H3B/D (H3.2), or H3F3A (H3.3) genes. It is still unclear whether these mutations each portend a universally poor prognosis, or if there are any factors which modulate outcome. The main objective of this study was to study overall survival (OS) of H3.1 versus H3.3 K27M-mutant DMGs in pediatric and adult patients. PubMed and Web of Science were searched, and we included studies if they have individual patient data of DMGs with available H3K27M genotype. Kaplan-Meier analysis and Cox regression models were used to analyze the survival of H3.1 and H3.3 mutations in each subgroup. We included 26 studies with 102 and 529 H3.1 and H3.3-mutant DMGs, respectively. The H3.1 mutation was more commonly seen in younger age. In pediatric population, H3.3 mutation conferred a shorter survival (median OS of 10.1 vs 14.2months; p < 0.001) in comparison to H3.1-positive patients, which was further confirmed in the multivariate Cox analysis. Conversely, H3.3 was associated with a prolonged survival in adult patients as compared with H3.1 mutation (median OS of 14.4 vs 1.7months; p = 0.019). We demonstrated that the prognosis of H3.1 and H3.3 K27M mutation in DMG patients is modulated by patient age. Routine H3K27M mutation genotyping in newly diagnosed DMGs may further stratify patients with these difficult tumors.

GEN-7 Liquid biopsy in brain tumor patients -The present and future-

We have previously published liquid biopsy for the diagnosis of brain tumors including PCNSL (JCO Precision Oncology, 2019; Leukemia and Lymphoma, 2019) and diffuse midline gliomas (DMG) (Diagnostics, 2021). We used the Maxwell RSC cfDNA extraction kit to extract circulating tumor DNA (ctDNA from) 1 milliliter of cerebrospinal fluid (CSF), and droplet digital PCR to detect MYD88 L265P mutations in PCNSL and H3F3A K27M mutations in DMG. From our initial experience, we were able to detect a high rate of MYD88 mutations in PCNSL, but not H3F3A mutations in DMG. We also observed that higher concentrations of ctDNA were obtained when prompt centrifugation and storage were done after obtaining CSF. Application of liquid biopsy to early detection of relapse and monitoring of treatment relapse are highly anticipated. In cases of PCNSL, we perform liquid biopsy when relapse is suspected on post-contrast MRI. However interestingly, the rate of MYD88 mutations detected is lower than that of newly-diagnosed cases. We would also like to share our experience performing liquid biopsy in conjunction with CSF cytology in brain tumor patients with evidence of leptomeningeal disease. From our initial experience, we would like to discuss the present limitations and future prospects of liquid biopsy in brain tumor patients.

PATH-25. H3 K27M-MUTANT DIFFUSE NON-MIDLINE GLIOMA: REPORT OF A CASE WITH EXTENDED FOLLOW-UP

Abstract H3 K27M-mutant diffuse gliomas without midline involvement are extremely rare and their clinical behavior remains elusive due to limited reported follow-up data. We describe an H3 K27M-mutant diffuse non-midline glioma patient with extended follow-up. A 34-year-old woman presented with headache, memory loss, periods of changes in taste and smell, and confusion. Imaging studies revealed an 2.3 cm expansile T2/flair hyperintensity with patchy postcontrast enhancement centered in the left amygdala without associated restricted diffusion and no involvement of the midline structures. The tumor was debulked and consisted of a mitotically active infiltrating astrocytic glioma without tumor necrosis or microvascular proliferation, consistent with anaplastic astrocytoma. Targeted 187-gene neuro-oncology NGS testing detected an H3F3A K27M mutation along with a PTPN11 and a PPM1D mutation. FISH 1p/19q-codeletion testing was negative. MGMT promoter was unmethylated. The patient was treated with chemoradiation with temozolomide for 6 weeks followed by 12 cycles of temozolomide. Four years after initial resection, an area of increased post-contrast enhancement indicating tumor recurrence/progression was noted. Partial resection of the recurrent tumor revealed a mitotically active infiltrating astrocytic glioma with tumor necrosis consistent with glioblastoma. Molecular profiling of the recurrent tumor by the neuro-oncology NGS panel detected similar mutational profile (identical H3F3A K27M and PTPN11 mutation; different PPM1D mutation) with an additional SOS1 mutation. The patient completed 4 cycles of Lomustine and although clinically stable, imaging studies showed slight increase in residual tumor size concerning for tumor progression. Lomustine was discontinued, Bevacizumab therapy was initiated and patient was enrolled in clinical trial (NCT02525692). Despite tumor progression, this patient has had a relatively long disease course (5 years) suggesting that H3 K27M-mutant non-midline diffuse gliomas although molecularly similar, may follow a more favorable clinical course than their midline counterparts possibly due to the hemispheric location, which is more amenable for surgical resection.

OS02.4.A Molecular characterization of adult cerebellar glioblastomas identifies distinct prognosis subgroups

Abstract BACKGROUND Adult cerebellar glioblastomas (cGBM) are very rare and recent studies have shown that they constitute a heterogeneous group of gliomas. The aim of the present study was to characterize the prevalence and prognostic significance of major driver molecular alterations in a large series of cGBM. MATERIAL AND METHODS Adults with histologically proven cGBM diagnosed between 2003 and 2017 were identified from the French brain tumor database and the Club de Neuro-Oncologie of the Société Française de Neurochirurgie. Tumors were reviewed and reclassified according to WHO 2016. Targeted sequencing was performed, including determination of H3F3A, TERTp, IDH1/2, FGFR1, BRAF and EGFR status. RESULTS A total of 83 adult patients (median age 57 years) with cGBM (hemispheric n= 47, vermian n=14 or both n=22) were identified. Median overall survival was 10 months. Main molecular alterations observed were TERT promoter, H3F3A K27M, hotspot FGFR1 (N546 and K656), BRAF mutations, EGFR amplification and ATRX loss of expression in 19.2%, 18.8%, 10.9%, 2.6%, 19.5% and 22.7% of patients, respectively. cGBM could be classified into 6 mutually exclusive subgroups associated with age at diagnosis and prognosis: pTERT and/or EGFR amplified tumors (n=22, 26.5%, median age = 62 years, median OS = 4 months), H3K27M-mutant tumors (n=15, 18.1%, median age = 48 years, median OS =8 months), tumors with MAPK pathway activating mutations (FGFR1, BRAF) or occurring in NF1 patients (n=15, 18.1%, median age = 41 years, median OS = 57 months), radiation-induced tumors (n=5, 6%, median age = 47 years, median OS = 5 months), IDH-mutant tumors (n=1) and unclassified tumors (n=25, 30.1%, median age = 63 years, median OS = 17 months). In multivariate analysis, MAPK activating mutations and ATRX loss of expression were independently associated with a better outcome and pTERT/EGFR alterations with a worse outcome. CONCLUSION About 18% of tumors diagnosed as cGBM harbor actionable MAPK activating genetic alterations. Targeted sequencing enables to classify these tumors into clinically relevant subgroups.

Low Detection Rate of H3K27M Mutations in Cerebrospinal Fluid Obtained from Lumbar Puncture in Newly Diagnosed Diffuse Midline Gliomas.

Recent studies have suggested the feasibility of detecting H3K27M mutations in the cerebrospinal fluid of diffuse midline glioma (DMG) patients. However, cerebrospinal fluid from patients in these studies were collected mainly during biopsy, ventriculo-peritoneal shunt procedures or postmortem. We assessed circulating tumor DNA (ctDNA) extracted from cerebrospinal fluid (CSF) and plasma in a series of 12 radiographically suspected and/or pathologically confirmed diffuse midline glioma patients and assessed for H3F3A K27M mutation using digital droplet PCR. In 10 patients, CSF was obtained by lumbar puncture at presentation. A definitive detection of H3F3A K27M mutation was achieved in only one case (10%); H3F3A K27M mutation was suspected in three other cases (30%). H3F3A K27M mutation was detected in two patients in CSF obtained by ventricular tap during a ventriculo-peritoneal shunt for obstructive hydrocephalus. Cases in which a definitive assessment was possible (definite H3F3A K27M or definite H3F3A wildtype) tended to be younger (median 7.5 years vs. 40.5 years; p = 0.07) and have a higher concentration of CSF protein (median 123 mg/dL vs. 27.5 mg/dL; p = 0.21) compared to nondefinite cases. Low proliferation and apoptotic rates seemed to be characteristics of DMG unfavorable for liquid biopsy. More advanced lesions with necrosis and evidence of dissemination were unlikely to be candidates for lumbar puncture due to the fear of exacerbating obstructive hydrocephalus. Methods to safely sample CSF and a more sensitive detection of ctDNA are necessary for reliable liquid biopsy of DMG at presentation.

Genetic signature and treatment of pediatric high-grade glioma.

Pediatric high-grade glioma (HGG) is a type of malignancy that carries a poor prognosis. The genetic analysis of HGGs has previously identified useful mutations, the targeting of which has improved prognosis. Thus, further research into the more common mutations, such as H3 histone variants (HIST1H3B and H3F3A) and BRAF V600E, may be useful in identifying tumors with different prognoses, as the mutations are considered to drive two distinct oncogenic programs. The present study performed a retrospective analysis of pediatric HGGs. In total, 42 cases of HGG, including 32 cases (76.1%) of anaplastic astrocytoma and 10 cases (23.8%) of glioblastoma multiforme (GBM), were assessed. The median age of the patients was 7 years (range, 0-32 years). Mutations on histone H3, in particular the K27M and G34R mutations in the distinct variants HIST1H3B and H3F3A, in addition to the presence of the BRAF V600E mutation, were analyzed in 24 patients. The H3F3A K27M mutation was identified in 7 patients (29.1%), while the HIST1H3B K27M mutation was only observed in 1 patient with GBM. In addition, 5 patients harbored a BRAF V600E mutation (21%), while the H3F3A G34R mutation was not recorded in any of the patients. The overall survival of the wild-type patients at 20 months was 68% [confidence interval (CI): 38-85%] compared with 28% (CI: 0.4-60%) in patients with the H3F3A K27M mutation. These results suggested that patients with the H3F3A K27M mutation had a worse prognosis compared with wild-type patients (P=0.0045). Moreover, 3/5 patients with the BRAF V600E mutation had HGGs that were derived from a previous low-grade glioma (LGG; P=0.001). In conclusion, these results suggested that histone H3 mutations may help predict the outcome in patients with HGG. In addition, the BRAF V600E mutation was found to be associated with an increased risk of anaplastic progression. The novel data of the present study may help better define the clinical and radiological characteristics of glioma.

H3.3-K27M drives neural stem cell-specific gliomagenesis in a human iPSC-derived model.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive childhood tumor of the brainstem with currently no curative treatment available. The vast majority of DIPGs carry a histone H3 mutation leading to a lysine 27-to-methionine exchange (H3K27M). We engineered human induced pluripotent stem cells (iPSCs) to carry an inducible H3.3-K27M allele in the endogenous locus and studied the effects of the mutation in different disease-relevant neural cell types. H3.3-K27M upregulated bivalent promoter-associated developmental genes, producing diverse outcomes in different cell types. While being fatal for iPSCs, H3.3-K27M increased proliferation in neural stem cells (NSCs) and to a lesser extent in oligodendrocyte progenitor cells (OPCs). Only NSCs gave rise to tumors upon induction of H3.3-K27M and TP53 inactivation in an orthotopic xenograft model recapitulating human DIPGs. In NSCs, H3.3-K27M leads to maintained expression of stemness and proliferative genes and a premature activation of OPC programs that together may cause tumor initiation.

HGG-49. A PEDIATRIC THALAMIC HIGH-GRADE GLIOMA WITH H3F3A K27M AND BRAF V600E DOUBLE MUTATIONS

CASEA 18-month-old boy presented with approximately 2 months history of progressive left hemiparesis and left exotropia. MRI study showed a 3–4 cm T1-iso, T2-high tumor at right thalamus to midbrain with little contrast enhancement. The patient underwent endoscopic biopsy of the tumor, which showed relatively dense proliferation of small cells with round nuclei, mitosis of the tumor cell, but no necrosis. Immunohistochemical showed positive stain of GFAP and Olig2. Ki-67 was 34%. The histopathological diagnosis was compatible with high grade glioma. Chemotherapy with vincristine, cyclophosphamide, cisplatin and etoposide was initiated. Molecular testing of the tumor revealed H3F3A K27M and BRAF V600E double mutations in DNA from frozen tumor tissue.DISCUSSIONThe concurrent mutation of H3F3A K27M and BRAF V600E in pediatric glioma is very rare, but there are several cases previously reported in literature. Interestingly those cases are heterogenous in age, location, histopathological subtypes and clinical outcome.

DIPG-08. ELECTRONIC SEQUENCING PROVIDES OPTIMIZED QUANTIFICATION OF SERIAL, MULTI-GENE MOLECULAR RESPONSE IN THE CSF OF CHILDREN WITH HIGH-GRADE GLIOMA

BACKGROUNDFor pediatric high-grade glioma (pHGG), non-invasive methods for diagnosis and surveillance are needed. Tumors release DNA (tDNA) into cerebrospinal fluid (CSF), allowing for detection of tumor-associated mutations by CSF sampling. We hypothesized that direct, electronic analysis of tDNA with a novel, hand-held platform (Oxford Nanopore MinION) could quantify patient-specific CSF tDNA variant allele fraction (VAF) with improved speed and limit of detection compared to established methods.METHODSWe integrated required multi-timepoint (0, 2, and 6 months) correlate lumbar punctures (LP) in two ongoing pHGG clinical trials. Using Nanopore technology, we performed amplicon-based PCR on CSF tDNA for recurrent mutations from patient samples (n=19) and normal controls. VAF were determined via MinKNOW, Guppy, MiniMap2, and Integrated Genome Browser.RESULTSNanopore CSF tDNA demonstrated improved sensitivity (91%) when compare to NGS sequencing (50%). Nanopore analysis of serially diluted CSF sample demonstrated significantly lower limit of detection (attomolar) than typical NGS sample requirement (nanomolar). H3K27M mutation was reliably detected with 1,000x depth sequencing, which was achieved in less than 15 minutes of sequencing after amplification. Multiplexed Nanopore analysis of H3F3A and HIST1H3B was employed when H3 status was unknown. Serial CSF tDNA analysis confirmed multi-gene (H3F3A K27M, PIK3CA, and TP53) molecular remission in a 17-year-old with thalamic diffuse midline glioma that correlated with sustained clinical response to ONC201 (14 months and ongoing).CONCLUSIONSUse of a hand-held, electronic DNA analysis platform allows quantification of multi-gene molecular response with improved speed and limit of detection in the CSF of children with high-grade glioma.

DIPG-19. TARGETING ATM MUTATION IN METASTATIC DIFFUSE MIDLINE GLIOMA – A CASE OF SUSTAINED RESPONSE USING PARP INHIBITOR

Diffuse midline glioma (DMG) with H3.3K27M mutation is associated with an extremely poor prognosis, with a median survival of 10 to 12 months. Radiation remains the standard of care however there is no established curative therapy available. We describe a patient diagnosed with a diffuse intrinsic pontine glioma at 5 years of age by clinical and radiological criteria. He was treated with focal radiation 59Gy which resulted in reduction in size of the tumour, and partial improvement of T2 changes on MRI. At 18 months post diagnosis, the patient developed metastatic recurrence at the anterior fornix. This was biopsied and histopathology demonstrated a high grade glioma. Next generation sequencing revealed a H3F3A K27M mutation, and an ATM R3008H mutation. He received whole ventricular radiation 36Gy and boost to the lesion to 45Gy, followed by Olaparib 135mg/m2/day twice daily. He remains in radiological remission 20 months post metastatic relapse and has no organ toxicity to Olaparib. CONCLUSION: H3.3K27M and ATM co-segregating mutations are described in DMG. This is the first case report of targeting ATM mutation with a PARP inhibitor which resulted in prolonged remission of metastatic DMG. Olaparib was well tolerated.

Evaluating H3F3A K27M and G34R/V somatic mutations in a cohort of pediatric brain tumors of different and rare histologies.

Somatic mutations on H3 histone are currently considered a genetic hallmark for midline pediatric high-grade gliomas (HGGs). Yet, different tumor histologies have been occasionally described to carry these mutations. Since histone modifications can lead to major epigenetic changes with direct impact on prognosis and treatment, we thought to investigate the occurrence of H3F3A K27M and G34R/V mutations in a cohort of pediatric tumors which included HGGs, low-grade gliomas, ependymomas, medulloblastomas, and a series of rare brain tumor lesions of different histologies. A total of 82 fresh-frozen pediatric brain tumor samples were evaluated. PCR or RT-PCR followed by Sanger sequencing for the exon 2 of H3F3A (containing both K27 and G34 hotspots) were obtained and aligned to human genome. Loss of trimethylation mark (H3K27me3) in H3F3A/K27M-mutant samples was confirmed by immunohistochemistry. We found H3F3A/K27M mutation in 2 out of 9 cases of HGGs; no H3F3A/K27M mutations were detected in low-grade gliomas (27), ependymomas (n = 10), medulloblastomas (n = 21), or a series of rare pediatric brain tumors which included meningiomas, dysembryoplastic neuroepithelial tumors (DNETs), central nervous system (CNS) germ-cell tumors, choroid plexus tumors, cortical hamartoma, subcortical tubers, and schwannomas (n = 15). H3F3A/G34R/V mutation was not observed in any of the samples. Our investigation reinforces the low frequency of H3F3A somatic mutations outside the HGG setting. Interestingly, an atypical focal brainstem glioma carrying H3F3A K27M mutation that showed protracted clinical course with late-onset tumor progression was identified.

Prognostic role of H3K27M mutation, histone H3K27 methylation status, and EZH2 expression in diffuse spinal cord gliomas.

The objective of this study is to clarify clinical significance of the H3F3A K27M mutation (H3K27M) and analyze the correlation between H3K27M, H3K27me3 status, and EZH2 expression and prognosis in spinal cord gliomas. Patients with spinal cord diffuse glioma regardless of World Health Organization (WHO) grade underwent genetic analysis for H3F3A, HIST1H3B, TERT promoter, IDH1/2, and BRAF. H3K27me3 status and EZH2 expression were analyzed through immunohistochemistry. Thereafter, the association between H3K27M, H3K27me3 status, and EZH2 expression and prognosis was retrospectively analyzed using the log-rank test. A total of 26 cases, 5 with WHO grade 4, 9 with grade 3, and 12 with grade 2 glioma, were analyzed. Although WHO grade 2 cases tended to present favorable overall survival, the difference was not statistically significant. H3K27M, which was detected in four grade 4 cases (80%) and three grade 3 cases (33%), was not associated with prognosis among grade 3 and 4 cases. Among WHO grade 2-4 cases, the combination of retained H3K27me3 and negative EZH2 expression was correlated with favorable overall survival (p = 0.03). The combination of H3K27me3 status and EZH2 expression was considered as a potential prognostic marker in WHO grade 2-4 diffuse spinal cord gliomas.

Abstract A35: From map to model: Genetic evolution of diffuse intrinsic pontine glioma provides the framework for accurate tumor modeling

Abstract Diffuse intrinsic pontine glioma (DIPG) is a devastating pediatric high-grade glioma (HGG) that diffusely penetrates the brainstem and is the leading cause of pediatric brain tumor death. Around 80% of DIPGs harbor lysine 27-methionine (K27M) substitutions in histone H3. H3K27M inhibits the activity of Polycomb repressive complex 2 (PRC2) that deposits H3K27me3, leading to widespread H3K27me3 loss, but how this leads to cancer is unclear. Further, despite clonal analysis indicating that H3K27M is the tumor-initiating mutation in DIPG, in vivo models of H3K27M-driven cancer have failed to generate tumors without additional oncogenic drivers. Thus, the early events driven by H3K27M alone remain obscure as does the most relevant cell of origin. To address the role of H3K27M in tumor initiation we generated a transgenic murine model with a tetracycline-repressible, Cre-inducible H3F3A-K27M transgene within the ROSA26 locus. This allows for both spatial and temporal control of K27M expression, via the cre-inducible LSL cassette and tetracycline treatment, respectively. These mice were crossed with the brain specific -cre drivers Nestin-, Olig2-, or GFAP-, targeting neural stem cells, oligodendrocyte progenitor cells, and glial progenitor cells, respectively. H3F3A K27M/-cre double-positive mice were born significantly below mendelian ratios, the most severe being those crossed with Olig2-cre (Nestin- 18/100; 0.18, GFAP- 15/114; 0.132, Olig2- 9/106; 0.08). All mice displayed a degree of ataxia and a smaller overall stature compared to littermate controls, with the most severe phenotype being particularly evident in Olig2-cre mice. Additionally, both Nestin- and GFAP-cre crosses experienced decreased survival post-weaning age (p21) (Nestin- 13/18 alive, GFAP- 10/15 alive); however, this decrease was greatly exaggerated in Olig2-cre (1/9 alive). Together, these data suggest that the displayed phenotypes are dependent upon the cell population in which H3F3A K27M is expressed. We also found that in addition to spatial control, temporal control of H3F3A K27M expression is critical to survival. Doxycycline (dox) was administered to breeding pairs for each H3F3A K27M/-cre line to repress transgene expression. Dox-H3F3A K27M/-cre double-positive mice were born at higher mendelian ratios than their no-dox counterparts (Nestin- 26/79; 0.33, GFAP- 14/50; 0.28, Olig2- 17/52; 0.327). Litters remained on dox treatment until either p0 or p21. Delayed H3F3A K27M expression led to an increase in overall survival in both treatment groups across all -cre lines, particularly Olig2-cre (Nestin- p0: 11/11, p21: 14/15, GFAP- p0: 4/4, p21: 9/10, Olig2- p0: 8/8, p21: 7/9), as well as an absence of ataxia and an increase in stature. This suggests that H3F3A K27M expression during a developmental window before p0 is critical to the onset of the observed phenotypes, in addition to the progenitor population being afflicted. Overall these data give insight into the spatial-temporal effects of H3K27M in early tumor development. Citation Format: Evan Lubanszky, Scott Ryall, Robert Siddaway, Byunjin Kim, Taylor Bridge, Sanja Pajovic, Cynthia Hawkins. From map to model: Genetic evolution of diffuse intrinsic pontine glioma provides the framework for accurate tumor modeling [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A35.

Diffuse midline glioma of the cervical spinal cord with H3 K27M genotype phenotypically mimicking anaplastic ganglioglioma: a case report and review of the literature

Here, we report on a 28-year old male patient presenting with neck and shoulder pain, dysesthesia of all four limbs and hypesthesia of both hands, without motor deficits. Magnetic resonance imaging showed an intradural, intramedullary mass of the cervical spinal cord of 6.4 cm length and 1.7 cm diameter. The patient underwent surgical resection. Histological and immunohistochemical evaluation showed pleomorphic glial tumor cells, mitoses, calcifications, and atypical ganglioid cells compatible with the morphology of anaplastic ganglioglioma (WHO Grade III). Extensive molecular workup revealed H3F3A K27M, TERT C228T and PDGFRα Y849C mutations indicating poor prognosis. The H3F3A K27M mutation assigned the tumor to the molecular group of diffuse midline glioma (WHO Grade IV). Epigenome-wide methylation profiling confirmed the methylation class of diffuse midline glioma. Thus, this is a very rare case of malignant glioma with H3 K27M genotype phenotypically mimicking anaplastic ganglioglioma. This case emphasizes the importance of comprehensive morphological and molecular workup including methylome profiling for advanced patient care.

Management of incidental brain tumors in children: a systematic review.

Due to technical advancements and availability of neuroimaging, detection of incidental pediatric brain tumors (IPBT) is growing rapidly. The management of these asymptomatic lesions remains unclear; radiological, pathological, and clinical risk factors for further growth and malignant transformation (MT) are not well defined. We systematically reviewed the literature on the dilemmas and management of IPBT suggestive of a low-grade brain tumor (LGBT). Keyword searches of the PubMed and Medline (NCBI) databases identified studies on IPBT describing the prevalence, neuroimaging, management, or risk of MT through July 2019. References of the identified articles were also reviewed. A total of 2021 records were screened. Fifty-nine full-text articles were reviewed, and 34 published studies were included. IPBT are diagnosed in 0.2-5.7% of children undergoing brain imaging for various reasons. The accepted approach for management of lesions showing radiological characteristics suggestive of LGBT is radiological follow-up. The rate at which additional intervention is required during follow-up for these apparently low-grade lesions is 9.5%. Nevertheless, the dilemma of early surgical resection or biopsy vs. clinical and radiological follow-up of IPBT is still unresolved. The risk in these cases is missing a transformation to a higher grade tumor. However, MT of pediatric LGBT is very rare, occurring in less than 3% of the cases of proven low-grade gliomas in children. The risk of future MT in pediatric low-grade gliomas seems to be greater in the presence of specific molecular markers such as BRAF V-600E, CDKN2A, and H3F3A K27M. The natural history, management, and prognosis of IPBT remain ambiguous. It seems that lesions suggestive of LGBT can initially be followed, since many of these lesions remain stable over time and MT is rare. However, controversy among centers concerning the ideal approach still exists. Further observational and prospective cohort studies, focusing on potential clinical and radiological characteristics or risk factors suggestive of high-grade tumors, tumor progress, or MT of IPBT, are needed.