Amanda Saratsis

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.

Insights into pediatric diffuse intrinsic pontine glioma through proteomic analysis of cerebrospinal fluid

Diffuse intrinsic pontine glioma (DIPG) is a leading cause of brain tumor-related death in children. DIPG is not surgically resectable, resulting in a paucity of tissue available for molecular studies. As such, tumor biology is poorly understood, and, currently, there are no effective treatments. In the absence of frozen tumor specimens, body fluids--such as cerebrospinal fluid (CSF), serum, and urine--can serve as more readily accessible vehicles for detecting tumor-secreted proteins. We analyzed a total of 76 specimens, including CSF, serum, urine, and normal and tumor brainstem tissue. Protein profiling of CSF from patients with DIPG was generated by mass spectrometry using an LTQ-Orbitrap-XL and database search using the Sequest algorithm. Quantitative and statistical analyses were performed with ProteoIQ and Partek Genomics Suite. A total of 528 unique proteins were identified, 71% of which are known secreted proteins. CSF proteomic analysis revealed selective upregulation of Cyclophillin A (CypA) and dimethylarginase 1 (DDAH1) in DIPG (n = 10), compared with controls (n = 4). Protein expression was further validated with Western blot analysis and immunohistochemical assays using CSF, brain tissue, serum, and urine from DIPG and control specimens. Immunohistochemical staining showed selective upregulation of secreted but not cytosolic CypA and DDAH1 in patients with DIPG. In this study, we present the first comprehensive protein profile of CSF specimens from patients with DIPG to demonstrate selective expression of tumor proteins potentially involved in brainstem gliomagenesis. Detection of secreted CypA and DDAH1 in serum and urine has potential clinical application, with implications for assessing treatment response and detecting tumor recurrence in patients with DIPG.

Mutations in chromatin machinery and pediatric high-grade glioma

Mutations in chromatin machinery define pediatric high-grade gliomas; efforts to define and target their functions are under way. Pediatric central nervous system tumors are the most common solid tumor of childhood. Of these, approximately one-third are gliomas that exhibit diverse biological behaviors in the unique context of the developing nervous system. Although low-grade gliomas predominate and have favorable outcomes, up to 20% of pediatric gliomas are high-grade. These tumors are a major contributor to cancer-related morbidity and mortality in infants, children, and adolescents, with long-term survival rates of only 10 to 15%. The recent discovery of somatic oncogenic mutations affecting chromatin regulation in pediatric high-grade glioma has markedly improved our understanding of disease pathogenesis, and these findings have stimulated the development of novel therapeutic approaches targeting epigenetic regulators for disease treatment. We review the current perspective on pediatric high-grade glioma genetics and epigenetics, and discuss the emerging and experimental therapeutics targeting the unique molecular abnormalities present in these deadly childhood brain tumors.

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.

Granulocytic Sarcoma in a Patient With Blast Crisis Mimicking a Chronic Subdural Hematoma

Case Report A 34-year-old black man was admitted to the neurosurgical intensive care unit with a 1-day history of progressive weakness and decreased mental status. He was known to the neurosurgical service, having presented 1 week prior for evaluation of persistent headaches for 1 month. At that time, he was diagnosed with bilateral chronic subdural hematomas and observed in the neurosurgical unit for 1 week, after which he left against medical advice. He had no history of head trauma, anticoagulation, or thrombocytopenia. His medical history was notable for chronic myelogenous leukemia (CML) diagnosed at our institution 10 months prior, for which he was receiving imatinib 600 mg daily. The patient had a known history of poor adherence to his leukemia management. On admission, the patient was intubated. He opened his eyes to voice. The right pupil was 4 mm and minimally reactive, and the left pupil was 3 mm and sluggishly reactive. He followed simple commands, moved all extremities purposefully against gravity, and had no additional focal neurologic deficits. The remainder of the physical examination was notable only for splenomegaly. A noncontrast head computed tomography (CT) scan revealed a hypodense right frontotemporal subdural collection measuring 1 cm, a small hypodense left subdural collection measuring 5 mm, and 7 mm of right-to-left midline shift (Fig 1A). Both lesions were interpreted as chronic subdural hematomas. Complete blood count revealed leukocytosis (247.9 K/CCM) consisting of 30% metamyelocytes, 25% myelocytes, 16% blastocytes, and 3% promyelocytes; anemia (hemoglobin of 10.3 g/dL); and normal platelets (138 K/CMM). The patient’s international normalized ratio was slightly elevated (1.43), and a blood toxicology screen was negative. The patient was taken emergently to the operating room for a right frontotemporal craniotomy and evacuation of the subdural collection. Durotomy revealed a thick green membranous substance diffusely covering the right cerebral cortex. Small pockets of chronic subdural blood were also identified. All visible membranes were dissected off the pia during the procedure and immediately sent to the pathology department for analysis. Bacterial and fungal cultures collected intraoperatively showed no growth. The subdural space was thoroughly explored, revealing no other evidence of abnormal tissue. The hemisphere was found to have diminished pulsatility and did not re-expand after a satisfactory decompression. The calvarium was inspected and found to be normal in appearance. Several hours after surgery, the patient developed fixed and dilated pupils, extensor posturing, and loss of brainstem reflexes. A noncontrast head CT revealed no significant residual subdural collection and unchanged midline shift. The patient was not brought back to surgery because it was felt that his decline was not the result of reaccumulation of the subdural lesion. The patient continued to deteriorate clinically despite aggressive CSF drainage and medical intervention to reduce intracranial pressure and cerebral edema. Laboratory values reflected progressive multisystem organ failure and persistent leukocytosis despite urgent leukophoresis. CSF examination was notable for elevated total protein (106 mg/dL) and pleocytosis (40 cells/uL) with 17% bands, 15% metamyelocytes, and 4% myelocytes. CSF cultures were negative. A noncontrast head CT on postoperative day 2 revealed an acute right-sided subdural hematoma, 1 cm right-to-left midline shift, and diffuse cerebral edema and infarction in the distribution of the posterior cerebral arteries suggesting tentorial herniation (Fig 1B). At this point, the patient’s physical examination was

Putting residents in the office: an effective method to teach the systems-based practice competency.

OBJECTIVES Systems-based practice (SBP) was 1 of 6 core competencies established by the Accreditation Council for Graduate Medical Education and has proven to be one of the most difficult to effectively implement. This pilot study presents an immersion workshop as an effective tool to teach the SBP competency in a way that could easily be integrated into a residency curriculum. DESIGN In 2006, 16 surgical residents rotated through 3 stations for 30 minutes each: coding and billing, scheduling operations and return appointments, and patient check-in. Participants were administered a pretest and posttest questionnaire evaluating their knowledge of SBP, and were asked to evaluate the workshop. SETTING Outpatient clinic at MedStar Georgetown University Hospital, Washington, DC. PARTICIPANTS Residents in the general surgery residency training program at MedStar Georgetown University Hospital. RESULTS Most residents (62.5%) improved their score after the workshop, whereas 31.25% showed no change and 6.25% demonstrated a decrease in score. Overall within their training levels, all groups demonstrated an increase in mean test score. Postgraduate year-2 residents demonstrated the greatest change in mean score (20%), whereas postgraduate year-4 residents demonstrated the smallest change in mean score (3.3%). CONCLUSIONS An immersion workshop where general surgery residents gained direct exposure to SBP concepts in situ was an effective and practical method of integrating this core competency into the residency curriculum. Such a workshop could complement more formal didactic teaching and be easily incorporated into the curriculum. For example, this workshop could be integrated into the ambulatory care requirement that each resident must fulfill as part of their clinical training.

Abstract C30: Four dimensional molecular analysis of pediatric diffuse intrinsic pontine glioma

Introduction: Diffuse Intrinsic Pontine Glioma (DIPG) is a highly morbid form of pediatric brainstem glioma. Molecular characterization is limited due to lack of tissue. Recent investigations suggest possible molecular subtypes may account for the historical poor response to therapy. We previously generated protein profiles of CSF and formalin fixed DIPG tumor specimens to characterize patterns of protein expression. Here, we present the first comprehensive tissue proteome of fresh frozen DIPG tumor specimens (n=16) and normal brain tissue (n=10). We characterize differential protein expression in DIPG tumor specimens, and compare these to gene expression and DNA methylation profiles of the same tissue. Methods: Normal brain and tumor tissue was collected intraoperatively or post-mortem. Extracted total tissue protein was quantified by mass spectrometry (MS/MS) via LTQ-Orbitrap-XL and database search using the Sequest algorithm. Gene expression profiles were detected using whole-genome Human HT-4 v12 Gene Expression Bead Chips. DNA methylation profiles were characterized after bisulphite conversion using Infinium HumanMethylation 450K BeadChip arrays. Quantitative and statistical analysis was performed with Genome Studio, ProteoIQ, and Partek Genomics Suite. Functional analysis was performed using Ingenuity Pathways Analysis. Gene and protein expression was validated via western blot and immunohistochemical staining of tumor and normal brain tissue. Results: 1,918 differentially expressed genes were detected in DIPG tumor tissue (ANOVA, p 2 or 2), with differential ShH pathway (GLI1 z-score -0.626 vs. 2.254) and protein expression COL1A2, LMNA, MAP4, NES, NRCAM, STMN1, and TNC between subgroups (ANOVA, p 2 or 2). Conclusions: We present the first comprehensive protein profile of DIPG fresh frozen tumor tissue and correlate to tissue gene expression profiles, which suggest differential activity of SHh pathway. This may in part be explained by differential methylation patterns of key genes. Proteomic analysis of DIPG tumor tissue reveals protein expression profiles reflective of this differential pathway activation, and hence may be useful tool for elucidating mechanisms of brainstem gliomagenesis in what likely is a heterogeneous tumor population. Biomarkers identified through proteomic analysis may in turn serve to more accurately diagnose patients with DIPG and measure response to therapy. Citation Format: Amanda Saratsis, Sridevi Yadavilli, Madhuri Kambhampati, Eric Raabe, Suresh Magge, Javad Nazarian. Four dimensional molecular analysis of pediatric diffuse intrinsic pontine glioma. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C30.