H3 K27M-altered glioma and diffuse intrinsic pontine glioma: Semi-systematic review of treatment landscape and future directions.

H3 K27M-mutant diffuse glioma is a recently identified brain tumor associated with poor prognosis. As of 2016, it is classified by the World Health Organization as a distinct form of grade IV glioma. Despite recognition as an important prognostic and diagnostic feature in diffuse glioma, radiation remains the sole standard of care and no effective systemic therapies are available for H3K27M mutant tumors. This review will detail treatment interventions applied to diffuse midline glioma and diffuse intrinsic pontine glioma (DIPG) prior to the identification of the H3 K27M mutation, the current standard-of-care for H3 K27M-mutant diffuse glioma treatment, and ongoing clinical trials listed on www.clinicaltrials.gov evaluating novel therapeutics in this population. Current clinical trials were identified using clinicaltrials.gov, and studies qualifying for this analysis were active or ongoing interventional trials that evaluated a therapy in at least 1 treatment arm or cohort comprised exclusively of patients with DIPG and H3 K27M-mutant glioma. Forty-one studies met these criteria, including trials evaluating H3 K27M vaccination, chimeric antigen receptor T-cell therapy, and small molecule inhibitors. Ongoing evaluation of novel therapeutics is necessary to identify safe and effective interventions in this underserved patient population.

H3K27M Mutant Glioma: Disease Definition and Biological Underpinnings.

High-grade glioma (HGG) is the most common cause of cancer death in children, and the most common primary central nervous system (CNS) tumor in adults. While pediatric HGG was once thought to be biologically similar to the adult form of disease, research has shown these malignancies to be significantly molecularly distinct, necessitating distinct approaches to their clinical management. However, emerging data have shown shared molecular events in pediatric and adult HGG including the histone H3K27M mutation. This somatic missense mutation occurs in genes encoding one of two isoforms of the Histone H3 protein, H3F3A (H3.3) or HIST1H3B (H3.1), and is detected in up to 80% of pediatric diffuse midline gliomas and in up to 60% of adult diffuse gliomas. Importantly, the H3K27M mutation is associated with poorer overall survival and response to therapy compared to patients with H3 wild-type tumors. Here, we review the clinical features and biological underpinnings of pediatric and adult H3K27M mutant glioma, offering a groundwork for understanding current research and clinical approaches for the care of patients suffering with this challenging disease.

LIN28B and Let-7 in Diffuse Midline Glioma: A Review.

Diffuse midline glioma (DMG) is the most lethal of all childhood cancers. DMGs are driven by histone-tail-mutation-mediated epigenetic dysregulation and partner mutations in genes controlling proliferation and migration. One result of this epigenetic and genetic landscape is the overexpression of LIN28B RNA binding protein. In other systems, LIN28B has been shown to prevent let-7 microRNA biogenesis; however, let-7, when available, faithfully suppresses tumorigenic pathways and induces cellular maturation by preventing the translation of numerous oncogenes. Here, we review the current literature on LIN28A/B and the let-7 family and describe their role in gliomagenesis. Future research is then recommended, with a focus on the mechanisms of LIN28B overexpression and localization in DMG.

Gorham Stout disease of the temporal bone with cerebrospinal fluid leak.

Gorham Stout disease (GSD) is a rare disease characterized by the proliferation of endothelial lined vessels and replacement of bone by fibrous tissue. The main imaging features are progressive osteolysis and cortical resorption. Temporal bone involvement is rare but presents as a destructive bone lesion that may be misinterpreted as more common lytic processes in the pediatric population, such as infection or Langerhans cell histiocytosis. GSD of the temporal bone is associated with cerebrospinal fluid (CSF) leaks, may present with otorrhea, and can mimic other causes of ear drainage. Here, we report the clinical course, imaging features, and outcomes of a 3-year-old girl with GSD of the temporal bone presenting with CSF leak initially attributed to infection.

Standardization of the liquid biopsy for pediatric diffuse midline glioma using ddPCR.

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. 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). 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. 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.

Therapeutic targeting of transcriptional elongation in diffuse intrinsic pontine glioma.

BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is associated with transcriptional dysregulation driven by H3K27 mutation. The super elongation complex (SEC) is required for transcriptional elongation through release of RNA polymerase II (Pol II). Inhibition of transcription elongation by SEC disruption can be an effective therapeutic strategy of H3K27M-mutant DIPG. Here, we tested the effect of pharmacological disruption of the SEC in H3K27M-mutant DIPG to advance understanding of the molecular mechanism and as a new therapeutic strategy for DIPG. METHODS: Short hairpin RNAs (shRNAs) were used to suppress the expression of AF4/FMR2 4 (AFF4), a central SEC component, in H3K27M-mutant DIPG cells. A peptidomimetic lead compound KL-1 was used to disrupt a functional component of SEC. Cell viability assay, colony formation assay, and apoptosis assay were utilized to analyze the effects of KL-1 treatment. RNA- and ChIP-sequencing were used to determine the effects of KL-1 on gene expression and chromatin occupancy. We treated mice bearing H3K27M-mutant DIPG patient-derived xenografts (PDXs) with KL-1. Intracranial tumor growth was monitored by bioluminescence image and therapeutic response was evaluated by animal survival. RESULTS: Depletion of AFF4 significantly reduced the cell growth of H3K27M-mutant DIPG. KL-1 increased genome-wide Pol II occupancy and suppressed transcription involving multiple cellular processes that promote cell proliferation and differentiation of DIPG. KL-1 treatment suppressed DIPG cell growth, increased apoptosis, and prolonged animal survival with H3K27M-mutant DIPG PDXs. CONCLUSIONS: SEC disruption by KL-1 increased therapeutic benefit in vitro and in vivo, supporting a potential therapeutic activity of KL-1 in H3K27M-mutant DIPG.

Mesenchymal Stem Cells Successfully Deliver Oncolytic Virotherapy to Diffuse Intrinsic Pontine Glioma.

PURPOSE: Diffuse intrinsic pontine glioma (DIPG) is among the deadliest of pediatric brain tumors. Radiotherapy is the standard-of-care treatment for DIPG, but offers only transient relief of symptoms for patients with DIPG without providing significant survival benefit. Oncolytic virotherapy is an anticancer treatment that has been investigated for treating various types of brain tumors. EXPERIMENTAL DESIGN: Here, we have explored the use of mesenchymal stem cells (MSC) for oncolytic virus (OV) delivery and evaluated treatment efficacy using preclinical models of DIPG. The survivin promoter drives the conditional replication of OV used in our studies. The efficiency of OV entry into the cells is mediated by fiber modification with seven lysine residues (CRAd.S.pK7). Patients' samples and cell lines were analyzed for the expression of viral entry proteins and survivin. The ability of MSCs to deliver OV to DIPG was studied in the context of a low dose of irradiation. RESULTS: Our results show that DIPG cells and tumors exhibit robust expression of cell surface proteins and survivin that enable efficient OV entry and replication in DIPG cells. MSCs loaded with OV disseminate within a tumor and release OV throughout the DIPG brainstem xenografts in mice. Administration of OV-loaded MSCs with radiotherapy to mice bearing brainstem DIPG xenografts results in more prolonged survival relative to that conferred by either therapy alone (P < 0.01). CONCLUSIONS: Our study supports OV, CRAd.S.pK7, encapsulated within MSCs as a therapeutic strategy that merits further investigation and potential translation for DIPG treatment.

Effects of H3.3G34V mutation on genomic H3K36 and H3K27 methylation patterns in isogenic pediatric glioma cells.

Histone H3.3 mutation (H3F3A) occurs in 50% of cortical pediatric high-grade gliomas. This mutation replaces glycine 34 with arginine or valine (G34R/V), impairing SETD2 activity (H3K36-specific trimethyltransferase). Consequently, reduced H3K36me3 is observed on H3.3G34V nucleosomes relative to wild-type, contributing to genomic instability and driving a distinct gene expression signature associated with tumorigenesis. However, it is not known if this differential H3K36me3 enrichment is due to H3.3G34V mutant protein alone. Therefore, we set to elucidate the effect of H3.3G34V mutant protein in pediatric glioma on H3K36me3, H3K27me3 and H3.3 enrichment in vitro. We found that the doxycycline-inducible shRNA knockdown of mutant H3F3A encoding the H3.3G34V protein resulted in loss of H3.3G34V enrichment and increased H3K36me3 enrichment throughout the genome. After knockdown, H3.3G34V enrichment was preserved at loci observed to have the greatest H3.3G34V and H3K36me3 enrichment prior to knockdown. Induced expression of mutant H3.3G34V protein in vitro was insufficient to induce genomic H3K36me3 enrichment patterns observed in H3.3G34V mutant glioma cells. We also observed strong co-enrichment of H3.3G34V and wild-type H3.3 protein, as well as greater H3K27me3 enrichment, in cells expressing H3.3G34V. Taken together, our study demonstrates the effects of H3.3G34V mutant protein on genomic H3K36me3, H3K27me3 and H3.3 enrichment patterns in isogenic cell lines.

Ventricular Cerebrospinal Fluid Sampling in Pediatric Diffuse Midline Glioma Patients: Institutional Experience and Review of the Literature.

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

Histone tail analysis reveals H3K36me2 and H4K16ac as epigenetic signatures of diffuse intrinsic pontine glioma.

BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor. Most DIPGs harbor a histone H3 mutation, which alters histone post-translational modification (PTM) states and transcription. Here, we employed quantitative proteomic analysis to elucidate the impact of the H3.3K27M mutation, as well as radiation and bromodomain inhibition (BRDi) with JQ1, on DIPG PTM profiles. METHODS: We performed targeted mass spectrometry on H3.3K27M mutant and wild-type tissues (n = 12) and cell lines (n = 7). RESULTS: We found 29.2 and 26.4% of total H3.3K27 peptides were H3.3K27M in mutant DIPG tumor cell lines and tissue specimens, respectively. Significant differences in modification states were observed in H3.3K27M specimens, including at H3K27, H3K36, and H4K16. In addition, H3.3K27me1 and H4K16ac were the most significantly distinct modifications in H3.3K27M mutant tumors, relative to wild-type. Further, H3.3K36me2 was the most abundant co-occurring modification on the H3.3K27M mutant peptide in DIPG tissue, while H4K16ac was the most acetylated residue. Radiation treatment caused changes in PTM abundance in vitro, including increased H3K9me3. JQ1 treatment resulted in increased mono- and di-methylation of H3.1K27, H3.3K27, H3.3K36 and H4K20 in vitro. CONCLUSION: Taken together, our findings provide insight into the effects of the H3K27M mutation on histone modification states and response to treatment, and suggest that H3K36me2 and H4K16ac may represent unique tumor epigenetic signatures for targeted DIPG therapy.

Correction to: Comparative multidimensional molecular analyses of pediatric diffuse intrinsic pontine glioma reveals distinct molecular subtypes.

The original version of this article unfortunately contained a mistake.

Correction: Detection of histone H3 K27M mutation and post-translational modifications in pediatric diffuse midline glioma via tissue immunohistochemistry informs diagnosis and clinical outcomes.

[This corrects the article DOI: 10.18632/oncotarget.26430.].

Detection of histone H3 K27M mutation and post-translational modifications in pediatric diffuse midline glioma via tissue immunohistochemistry informs diagnosis and clinical outcomes.

Pediatric diffuse midline glioma is a highly morbid glial neoplasm that may arise in the thalamus or brainstem (also known as diffuse intrinsic pontine glioma or DIPG). Because tumor anatomic location precludes surgical resection, diagnosis and treatment is based on MR imaging and analysis of biopsy specimens. Up to 80% of pediatric diffuse midline gliomas harbor a histone H3 mutation resulting in the replacement of lysine 27 with methionine (K27M) in genes encoding histone H3 variant H3.3 (H3F3A) or H3.1 (HIST1H3B). H3K27M mutant glioma responds more poorly to treatment and is associated with worse clinical outcome than wild-type tumors, so mutation detection is now diagnostic for a new clinical entity, diffuse midline glioma H3K27M mutant, as defined in the most recent WHO classification system. We previously reported patterns of histone H3 trimethylation (H3K27me3) and acetylation (H3K27Ac) associated with H3K27M mutation that impact transcription regulation and contribute to tumorigenesis. Given the clinical implications of the H3K27M mutation and these associated H3 post-translational modifications (PTMs), we set to determine whether they can be characterized via immunohistochemistry (IHC) in a cohort of pediatric glioma (n = 69) and normal brain tissue (n = 4) specimens. We observed 100% concordance between tissue IHC and molecular sequencing for detecting H3K27M mutation. In turn, H3K37M and H3K27me3 results, but not H3K27Ac staining patterns, were predictive of clinical outcomes. Our results demonstrate H3K27M and H3K27me3 staining of pediatric glioma tissue may be useful for diagnosis, stratification to epigenetic targeted therapies, and longitudinal monitoring of treatment response.

Immunohistochemical analysis of H3K27me3 demonstrates global reduction in group-A childhood posterior fossa ependymoma and is a powerful predictor of outcome.

Posterior fossa ependymomas (EPN_PF) in children comprise two morphologically identical, but biologically distinct tumor entities. Group-A (EPN_PFA) tumors have a poor prognosis and require intensive therapy. In contrast, group-B tumors (EPN_PFB) exhibit excellent prognosis and the current consensus opinion recommends future clinical trials to test the possibility of treatment de-escalation in these patients. Therefore, distinguishing these two tumor subtypes is critical. EPN_PFA and EPN_PFB can be distinguished based on DNA methylation signatures, but these assays are not routinely available. We have previously shown that a subset of poorly prognostic childhood EPN_PF exhibits global reduction in H3K27me3. Therefore, we set out to determine whether a simple immunohistochemical assay for H3K27me3 could be used to segregate EPN_PFA from EPN_PFB tumors. We assembled a cohort of 230 childhood ependymomas and H3K27me3 immunohistochemistry was assessed as positive or negative in a blinded manner. H3K27me3 staining results were compared with DNA methylation-based subgroup information available in 112 samples [EPN_PFA (n = 72) and EPN_PFB tumors (n = 40)]. H3K27me3 staining was globally reduced in EPN_PFA tumors and immunohistochemistry showed 99% sensitivity and 100% specificity in segregating EPN_PFA from EPN_PFB tumors. Moreover, H3K27me3 immunostaining was sufficient to delineate patients with worse prognosis in two independent, non-overlapping cohorts (n = 133 and n = 97). In conclusion, immunohistochemical evaluation of H3K27me3 global reduction is an economic, easily available and readily adaptable method for defining high-risk EPN_PFA from low-risk posterior fossa EPN_PFB tumors to inform prognosis and to enable the design of future clinical trials.

Detection of Histone H3 mutations in cerebrospinal fluid-derived tumor DNA from children with diffuse midline glioma.

Diffuse midline gliomas (including diffuse intrinsic pontine glioma, DIPG) are highly morbid glial neoplasms of the thalamus or brainstem that typically arise in young children and are not surgically resectable. These tumors are characterized by a high rate of histone H3 mutation, resulting in replacement of lysine 27 with methionine (K27M) in genes encoding H3 variants H3.3 (H3F3A) and H3.1 (HIST1H3B). Detection of these gain-of-function mutations has clinical utility, as they are associated with distinct tumor biology and clinical outcomes. Given the paucity of tumor tissue available for molecular analysis and relative morbidity of midline tumor biopsy, CSF-derived tumor DNA from patients with diffuse midline glioma may serve as a viable alternative for clinical detection of histone H3 mutation. We demonstrate the feasibility of two strategies to detect H3 mutations in CSF-derived tumor DNA from children with brain tumors (n = 11) via either targeted Sanger sequencing of H3F3A and HIST1H3B, or H3F3A c.83 A > T detection via nested PCR with mutation-specific primers. Of the six CSF specimens from children with diffuse midline glioma in our cohort, tumor DNA sufficient in quantity and quality for analysis was isolated from five (83%), with H3.3K27M detected in four (66.7%). In addition, H3.3G34V was identified in tumor DNA from a patient with supratentorial glioblastoma. Test sensitivity (87.5%) and specificity (100%) was validated via immunohistochemical staining and Sanger sequencing in available matched tumor tissue specimens (n = 8). Our results indicate that histone H3 gene mutation is detectable in CSF-derived tumor DNA from children with brain tumors, including diffuse midline glioma, and suggest the feasibility of "liquid biopsy" in lieu of, or to complement, tissue diagnosis, which may prove valuable for stratification to targeted therapies and monitoring treatment response.

Therapeutic targeting of polycomb and BET bromodomain proteins in diffuse intrinsic pontine gliomas.

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brainstem tumor characterized by rapid and uniform patient demise. A heterozygous point mutation of histone H3 occurs in more than 80% of these tumors and results in a lysine-to-methionine substitution (H3K27M). Expression of this histone mutant is accompanied by a reduction in the levels of polycomb repressive complex 2 (PRC2)-mediated H3K27 trimethylation (H3K27me3), and this is hypothesized to be a driving event of DIPG oncogenesis. Despite a major loss of H3K27me3, PRC2 activity is still detected in DIPG cells positive for H3K27M. To investigate the functional roles of H3K27M and PRC2 in DIPG pathogenesis, we profiled the epigenome of H3K27M-mutant DIPG cells and found that H3K27M associates with increased H3K27 acetylation (H3K27ac). In accordance with previous biochemical data, the majority of the heterotypic H3K27M-K27ac nucleosomes colocalize with bromodomain proteins at the loci of actively transcribed genes, whereas PRC2 is excluded from these regions; this suggests that H3K27M does not sequester PRC2 on chromatin. Residual PRC2 activity is required to maintain DIPG proliferative potential, by repressing neuronal differentiation and function. Finally, to examine the therapeutic potential of blocking the recruitment of bromodomain proteins by heterotypic H3K27M-K27ac nucleosomes in DIPG cells, we performed treatments in vivo with BET bromodomain inhibitors and demonstrate that they efficiently inhibit tumor progression, thus identifying this class of compounds as potential therapeutics in DIPG.

Inhibition of DNA damage repair by the CDK4/6 inhibitor palbociclib delays irradiated intracranial atypical teratoid rhabdoid tumor and glioblastoma xenograft regrowth.

BACKGROUND: Radiation therapy is the most commonly used postsurgical treatment for primary malignant brain tumors. Consequently, investigating the efficacy of chemotherapeutics combined with radiation for treating malignant brain tumors is of high clinical relevance. In this study, we examined the cyclin-dependent kinase 4/6 inhibitor palbociclib, when used in combination with radiation for treating human atypical teratoid rhabdoid tumor (ATRT) as well as glioblastoma (GBM). METHODS: Evaluation of treatment antitumor activity in vitro was based upon results from cell proliferation assays, clonogenicity assays, flow cytometry, and immunocytochemistry for DNA double-strand break repair. Interpretation of treatment antitumor activity in vivo was based upon bioluminescence imaging, animal subject survival analysis, and staining of tumor sections for markers of proliferation and apoptosis. RESULTS: For each of the retinoblastoma protein (RB)-proficient tumor models examined (2 ATRTs and 2 GBMs), one or more of the combination therapy regimens significantly (P < .05) outperformed both monotherapies with respect to animal subject survival benefit. Among the combination therapy regimens, concurrent palbociclib and radiation treatment and palbociclib treatment following radiation consistently outperformed the sequence in which radiation followed palbociclib treatment. In vitro investigation revealed that the concurrent use of palbociclib with radiation, as well as palbociclib following radiation, inhibited DNA double-strand break repair and promoted increased tumor cell apoptosis. CONCLUSIONS: Our results support further investigation and possible clinical translation of palbociclib as an adjuvant to radiation therapy for patients with malignant brain tumors that retain RB expression.

The emerging role of NG2 in pediatric diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine gliomas (DIPGs) have a dismal prognosis and are poorly understood brain cancers. Receptor tyrosine kinases stabilized by neuron-glial antigen 2 (NG2) protein are known to induce gliomagenesis. Here, we investigated NG2 expression in a cohort of DIPG specimens (n= 50). We demonstrate NG2 expression in the majority of DIPG specimens tested and determine that tumors harboring histone 3.3 mutation express the highest NG2 levels. We further demonstrate that microRNA 129-2 (miR129-2) is downregulated and hypermethylated in human DIPGs, resulting in the increased expression of NG2. Treatment with 5-Azacytidine, a methyltransferase inhibitor, results in NG2 downregulation in DIPG primary tumor cells in vitro. NG2 expression is altered (symmetric segregation) in mitotic human DIPG and mouse tumor cells. These mitotic cells co-express oligodendrocyte (Olig2) and astrocyte (glial fibrillary acidic protein, GFAP) markers, indicating lack of terminal differentiation. NG2 knockdown retards cellular migration in vitro, while NG2 expressing neurospheres are highly tumorigenic in vivo, resulting in rapid growth of pontine tumors. NG2 expression is targetable in vivo using miR129-2 indicating a potential avenue for therapeutic interventions. This data implicates NG2 as a molecule of interest in DIPGs especially those with H3.3 mutation.

A standardized autopsy procurement allows for the comprehensive study of DIPG biology.

Diffuse intrinsic pontine glioma (DIPG) is one of the least understood and most deadly childhood cancers. Historically, there has been a paucity of DIPG specimens for molecular analysis. However, due to the generous participation of DIPG families in programs for postmortem specimen donation, there has been a recent surge in molecular analysis of newly available tumor specimens. Collaborative efforts to share data and tumor specimens have resulted in rapid discoveries in other pediatric brain tumors, such as medulloblastoma, and therefore have the potential to shed light on the biology of DIPG. Given the generous gift of postmortem tissue donation from DIPG patients, there is a need for standardized postmortem specimen accrual to facilitate rapid and effective multi-institutional molecular studies.We developed and implemented an autopsy protocol for rapid procurement, documenting and storing these specimens. Sixteen autopsies were performed throughout the United States and Canada and processed using a standard protocol and inventory method, including specimen imaging, fixation, snap freezing, orthotopic injection, or preservation. This allowed for comparative clinical and biological studies of rare postmortem DIPG tissue specimens, generation of in vivo and in vitro models of DIPG, and detailed records to facilitate collaborative analysis.

Comparative multidimensional molecular analyses of pediatric diffuse intrinsic pontine glioma reveals distinct molecular subtypes.

Diffuse intrinsic pontine glioma (DIPG) is a highly morbid form of pediatric brainstem glioma. Here, we present the first comprehensive protein, mRNA, and methylation profiles of fresh-frozen DIPG specimens (n = 14), normal brain tissue (n = 10), and other pediatric brain tumors (n = 17). Protein profiling identified 2,305 unique proteins indicating distinct DIPG protein expression patterns compared to other pediatric brain tumors. Western blot and immunohistochemistry validated upregulation of Clusterin (CLU), Elongation Factor 2 (EF2), and Talin-1 (TLN1) in DIPGs studied. Comparisons to mRNA expression profiles generated from tumor and adjacent normal brain tissue indicated two DIPG subgroups, characterized by upregulation of Myc (N-Myc) or Hedgehog (Hh) signaling. We validated upregulation of PTCH, a membrane receptor in the Hh signaling pathway, in a subgroup of DIPG specimens. DNA methylation analysis indicated global hypomethylation of DIPG compared to adjacent normal tissue specimens, with differential methylation of 24 genes involved in Hh and Myc pathways, correlating with protein and mRNA expression patterns. Sequencing analysis showed c.83A>T mutations in the H3F3A or HIST1H3B gene in 77 % of our DIPG cohort. Supervised analysis revealed a unique methylation pattern in mutated specimens compared to the wild-type DIPG samples. This study presents the first comprehensive multidimensional protein, mRNA, and methylation profiling of pediatric brain tumor specimens, detecting the presence of two subgroups within our DIPG cohort. This multidimensional analysis of DIPG provides increased analytical power to more fully explore molecular signatures of DIPGs, with implications for evaluating potential molecular subtypes and biomarker discovery for assessing response to therapy.