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

Upfront Biology-Guided Therapy in Diffuse Intrinsic Pontine Glioma: Therapeutic, Molecular, and Biomarker Outcomes from PNOC003

PURPOSE PNOC003 is a multicenter precision medicine trial for children and young adults with newly diagnosed diffuse intrinsic pontine glioma (DIPG). PATIENTS AND METHODS Patients (3-25 years) were enrolled on the basis of imaging consistent with DIPG. Biopsy tissue was collected for whole-exome and mRNA sequencing. After radiotherapy (RT), patients were assigned up to four FDA-approved drugs based on molecular tumor board recommendations. H3K27M-mutant circulating tumor DNA (ctDNA) was longitudinally measured. Tumor tissue and matched primary cell lines were characterized using whole-genome sequencing and DNA methylation profiling. When applicable, results were verified in an independent cohort from the Children's Brain Tumor Network (CBTN). RESULTS Of 38 patients enrolled, 28 patients (median 6 years, 10 females) were reviewed by the molecular tumor board. Of those, 19 followed treatment recommendations. Median overall survival (OS) was 13.1 months [95% confidence interval (CI), 11.2-18.4] with no difference between patients who followed recommendations and those who did not. H3K27M-mutant ctDNA was detected at baseline in 60% of cases tested and associated with response to RT and survival. Eleven cell lines were established, showing 100% fidelity of key somatic driver gene alterations in the primary tumor. In H3K27-altered DIPGs, TP53 mutations were associated with worse OS (TP53mut 11.1 mo; 95% CI, 8.7-14; TP53wt 13.3 mo; 95% CI, 11.8-NA; P = 3.4e-2), genome instability (P = 3.1e-3), and RT resistance (P = 6.4e-4). The CBTN cohort confirmed an association between TP53 mutation status, genome instability, and clinical outcome. CONCLUSIONS Upfront treatment-naïve biopsy provides insight into clinically relevant molecular alterations and prognostic biomarkers for H3K27-altered DIPGs

Identification and Characterization of Pediatric Midline Glioma Specific Antigens: Wilms\u27 Tumor in Diffuse Intrinsic Pontine Glioma

Pediatric high grade gliomas (HGGs), especially those associated with the pons, known as diffuse intrinsic pontine gliomas (DIPGs) are deadly pediatric brain cancer that makes up 10-15% of all central nervous system (CNS) tumors in children. Its anatomical location and infiltrative nature makes it one of the most challenging tumors to treat. Immunotherapy is a technique that is gaining more interest in CNS tumors. Identification of tumor associated antigens is one of many requirements in developing an effective immunotherapy. Wilms’ tumor protein (WT1) has been ranked number 1 cancer immunotherapy target by National Cancer Institute. Many types of solid tumors have been shown to express WT1 and it is being examined as one of potential immunotherapeutic targets. Here we validated WT1 as a potential tumor associated antigen in pediatric diffuse midline gliomas using formalin fixed paraffin embedded (FFPE) tumor and intra-patient healthy specimens, fresh frozen post-mortem tissues and patient-derived DIPG primary cell lines. Our immunohistochemistry (IHC) staining of patient FFPE specimens showed strong WT1 immunoreactivity in tumor compared to adjacent normal tissue. Western blot of tumor tissues and cell lines were performed to further validate WT1 levels in the tumors versus adjacent normal tissues. Tumors showed cytoplasmic expression of WT1, confirmed by immunofluorescent (IF) staining. In addition, H3.1K27M subtype gliomas showed weak to absent WT1 immunoreactivity compared to strong to moderate in H3.3K27M subtypes. Western blots also validated the differential expression of the protein. Our study suggests that WT1 is a potential target protein for pediatric midline glioma immunotherapy

Liquid biopsy for detection and monitoring of driver mutations in Diffuse Intrinsic Pontine Gliomas (DIPGs)

Background: Diffuse Intrinsic Pontine Glioma (DIPG) is a deadly pediatric brain cancer that makes up 10-15% of all central nervous system (CNS) tumors in children. Surgery is not an option due to its anatomical location and infiltrative nature. It is amongst the most challenging tumors to treat. Combination of chemotherapy and focal radiation therapy is the primary therapy for DIPG, but the benefits of the radiation therapy is temporary. Immunotherapy is a technique that is gaining more interest in CNS tumors. Identification of tumor associated antigens is one of many requirements in developing an effective immunotherapy. Objective: To validate Wilms\u27 tumor protein (WT1) as a potential tumor associated antigen in DIPGs using patient derived cell lines and patient formalin fixed paraffin embedded (FFPE) specimens. Methods/Design: DIPG patient FFPE specimens were immunohistochemically stained for WT1 using mouse monoclonal anti-WT1 antibody. FFPE specimens and patient derived cell lines were also co-stained for K27M histone H3 protein mutation (p.Lys27Met) and WT1 using immunofluorescent (IF) staining methods. The fluorescence intensity was measured to compare WT1 level in the two histone mutation subgroups of DIPG (H3.3K27M and H3.1K27M). In addition, frozen tumor tissues and patient derived cell lines were used to validate WT1 protein levels in western blot. Results/Discussion: Immunohistochemistry (IHC) staining of patient FFPE specimens showed strong WT1 immunoreactivity in tumor compared to adjacent normal tissue. In addition, our IHC staining showed weak to absent WT1 immunoreactivity in H3.1K27M subtype tumor specimens compared to strong to moderate in H3.3K27M subtype tumor specimens. IF staining of cell lines confirm this differential WT1 levels in the subtypes. Western blot of tumor tissues and cell lines were performed to further validate WT1 levels. These results suggest that WT1 is a potential DIPG tumor associated antigen which can be utilized for immunotherapy

Tumor surveillance using liquid biome in pediatric high grade gliomas

Immunotherapy is currently being used to treat pediatric brain cancer, though its efficacy in treating patients with diffuse intrinsic pontine glioma (DIPG), the deadliest pediatric brain tumor, has not been evaluated. MRI is the gold standard for monitoring tumor response to therapy, but is limited by pseudoresponse and psuedoprogression: post-treatment, immune cells infiltrate the primary tumor causing transient tumor enlargement, which falsely resembles tumor progression on MRI. Thus, it is critical to develop more accurate approaches to monitor tumor response to immunotherapy. Here, we use a liquid biopsy platform we have already established to monitor tumor response to therapy, to assess whether this platform is sufficient to monitor response to immunotherapy. The most frequent driver mutation in DIPG is the conversion of lysine 27 to methionine in histone H3, encoded by H3 variants H3.3 (H3F3A) and H3.1 (HIST1H3B/C). Here, we report detection of H3F3A mutation allelic frequency (MAF) in circulating tumor DNA in plasma obtained from DIPG patients undergoing immunotherapy with Newcastle disease virus (NDV) vaccine. NDV selectively targets tumor cells and induces immunogenic cell death; we hypothesize that this treatment will decrease tumor burden, corresponding to lower levels of mutant H3F3A and HIST1H3B in plasma. We used our digital droplet PCR liquid biopsy platform to determine histone mutation status (H3.3, H3.1, H3 wild type) at time of diagnosis, and to monitor MAF through the course of treatment, in order to correlate MAF to tumor response. This platform allows for more sensitive, accurate monitoring of tumor response than conventional MRI

Histological and molecular analysis of a progressive diffuse intrinsic pontine glioma and synchronous metastatic lesions: a case report.

There is no curative treatment for patients with diffuse intrinsic pontine glioma (DIPG). However, with the recent availability of biopsy and autopsy tissue, new data regarding the biologic behavior of this tumor have emerged, allowing greater molecular characterization and leading to investigations which may result in improved therapeutic options. Treatment strategies must address both primary disease sites as well as any metastatic deposits, which may be variably sensitive to a particular approach.In this case report, we present a patient with DIPG treated with irradiation and serial investigational agents. The clinical, pathological and molecular phenotypes of both the progressive primary tumor as well as concomitant metastatic deposits obtained at autopsy are discussed. While some mRNA differences were demonstrated, all analyzed sites of disease shared similar mutational arrangements, suggesting that targeting the mutations of the primary tumor may be effective for all sites of disease

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