Pawel Buczkowicz

Genomic analysis of diffuse intrinsic pontine gliomas identifies three molecular subgroups and recurrent activating ACVR1 mutations

Diffuse intrinsic pontine glioma (DIPG) is a fatal brain cancer that arises in the brainstem of children, with no effective treatment and near 100% fatality. The failure of most therapies can be attributed to the delicate location of these tumors and to the selection of therapies on the basis of assumptions that DIPGs are molecularly similar to adult disease. Recent studies have unraveled the unique genetic makeup of this brain cancer, with nearly 80% found to harbor a p.Lys27Met histone H3.3 or p.Lys27Met histone H3.1 alteration. However, DIPGs are still thought of as one disease, with limited understanding of the genetic drivers of these tumors. To understand what drives DIPGs, we integrated whole-genome sequencing with methylation, expression and copy number profiling, discovering that DIPGs comprise three molecularly distinct subgroups (H3-K27M, silent and MYCN) and uncovering a new recurrent activating mutation affecting the activin receptor gene ACVR1 in 20% of DIPGs. Mutations in ACVR1 were constitutively activating, leading to SMAD phosphorylation and increased expression of the downstream activin signaling targets ID1 and ID2. Our results highlight distinct molecular subgroups and novel therapeutic targets for this incurable pediatric cancer.

Whole-Genome Profiling of Pediatric Diffuse Intrinsic Pontine Gliomas Highlights Platelet-Derived Growth Factor Receptor α and Poly (ADP-ribose) Polymerase As Potential Therapeutic Targets

PURPOSE Diffuse intrinsic pontine glioma (DIPG) is one of the most devastating of pediatric malignancies and one for which no effective therapy exists. A major contributor to the failure of therapeutic trials is the assumption that biologic properties of brainstem tumors in children are identical to cerebral high-grade gliomas of adults. A better understanding of the biology of DIPG itself is needed in order to develop agents targeted more specifically to these childrens disease. Herein, we address this lack of knowledge by performing the first high-resolution single nucleotide polymorphism (SNP) -based DNA microarray analysis of a series of DIPGs. PATIENTS AND METHODS Eleven samples (nine postmortem and two pretreatment surgical samples), the largest series thus far examined, were hybridized to SNP arrays (250 k or 6.0). The study was approved by the research ethics board at our institution. All array findings were validated using quantitative polymerase chain reaction, fluorescence in situ hybridization, immunohistochemistry, and/or microsatellite analysis. RESULTS Analysis of DIPG copy number alterations showed recurrent changes distinct from those of pediatric supratentorial high-grade astrocytomas. Thirty-six percent of DIPGs had gains in platelet-derived growth factor receptor alpha (PDGFRA; 4 to 18 copies) and all showed PDGFR-alpha expression. Low-level gains in poly (ADP-ribose) polymerase (PARP) -1 were identified in three cases. Pathway analysis revealed genes with loss of heterozygosity were enriched for DNA repair pathways. CONCLUSION To our knowledge, our data provides the first, comprehensive high-resolution genomic analysis of pediatric DIPG. Our findings of recurrent involvement of the PDGFR pathway as well as defects in DNA repair pathways coupled with gain of PARP-1 highlight two potential, biologically based, therapeutic targets directed specifically at this devastating disease.

Histopathological spectrum of paediatric diffuse intrinsic pontine glioma: diagnostic and therapeutic implications

Diffuse intrinsic pontine glioma (DIPG) is the main cause of brain tumour-related death in children. In the majority of cases diagnosis is based on clinical and MRI findings, resulting in the scarcity of pre-treatment specimens available to study. Our group has developed an autopsy-based protocol to investigate the histologic and biologic spectrum of DIPG. This has also allowed us to investigate the terminal pattern of disease and gain a better understanding of what challenges we are facing in treating DIPG. Here, we review 72 DIPG cases with well documented clinical history and molecular data and describe the pathological features of this disease in relation to clinical and genetic features. Fifty-three of the samples were autopsy material (7 pre-treatment) and 19 were pre-treatment biopsy/surgical specimens. Upon histological review, 62 patients had high-grade astrocytomas (18 WHO grade III and 44 WHO grade IV patients), 8 had WHO grade II astrocytomas, and 2 had features of primitive neuroectodermal tumour (PNET). K27M-H3 mutations were exclusively found in tumours with WHO grade II–IV astrocytoma histology. K27M-H3.1 and ACVR1 mutations as well as ALT phenotype were only found in WHO grade III–IV astrocytomas, while PIK3CA mutations and PDGFRA gains/amplifications were found in WHO grade II–IV astrocytomas. Approximately 1/3 of DIPG patients had leptomeningeal spread of their tumour. Further, diffuse invasion of the brainstem, spinal cord and thalamus was common with some cases showing spread as distant as the frontal lobes. These findings suggest that focal radiation may be inadequate for some of these patients. Importantly, we show that clinically classic DIPGs represent a diverse histologic spectrum, including multiple cases which would fit WHO criteria of grade II astrocytoma which nevertheless behave clinically as high-grade astrocytomas and harbour the histone K27M-H3.3 mutation. This suggests that the current WHO astrocytoma grading scheme may not appropriately predict outcome for paediatric brainstem gliomas.

BRAF Mutation and CDKN2A Deletion Define a Clinically Distinct Subgroup of Childhood Secondary High-Grade Glioma

PURPOSE To uncover the genetic events leading to transformation of pediatric low-grade glioma (PLGG) to secondary high-grade glioma (sHGG). PATIENTS AND METHODS We retrospectively identified patients with sHGG from a population-based cohort of 886 patients with PLGG with long clinical follow-up. Exome sequencing and array CGH were performed on available samples followed by detailed genetic analysis of the entire sHGG cohort. Clinical and outcome data of genetically distinct subgroups were obtained. RESULTS sHGG was observed in 2.9% of PLGGs (26 of 886 patients). Patients with sHGG had a high frequency of nonsilent somatic mutations compared with patients with primary pediatric high-grade glioma (HGG; median, 25 mutations per exome; P = .0042). Alterations in chromatin-modifying genes and telomere-maintenance pathways were commonly observed, whereas no sHGG harbored the BRAF-KIAA1549 fusion. The most recurrent alterations were BRAF V600E and CDKN2A deletion in 39% and 57% of sHGGs, respectively. Importantly, all BRAF V600E and 80% of CDKN2A alterations could be traced back to their PLGG counterparts. BRAF V600E distinguished sHGG from primary HGG (P = .0023), whereas BRAF and CDKN2A alterations were less commonly observed in PLGG that did not transform (P < .001 and P < .001 respectively). PLGGs with BRAF mutations had longer latency to transformation than wild-type PLGG (median, 6.65 years [range, 3.5 to 20.3 years] v 1.59 years [range, 0.32 to 15.9 years], respectively; P = .0389). Furthermore, 5-year overall survival was 75% ± 15% and 29% ± 12% for children with BRAF mutant and wild-type tumors, respectively (P = .024). CONCLUSION BRAF V600E mutations and CDKN2A deletions constitute a clinically distinct subtype of sHGG. The prolonged course to transformation for BRAF V600E PLGGs provides an opportunity for surgical interventions, surveillance, and targeted therapies to mitigate the outcome of sHGG.

Pathology, Molecular Genetics, and Epigenetics of Diffuse Intrinsic Pontine Glioma

Diffuse intrinsic pontine glioma (DIPG) is a devastating pediatric brain cancer with no effective therapy. Histological similarity of DIPG to supratentorial high-grade astrocytomas of adults has led to assumptions that these entities possess similar underlying molecular properties and therefore similar therapeutic responses to standard therapies. The failure of all clinical trials in the last 30 years to improve DIPG patient outcome has suggested otherwise. Recent studies employing next-generation sequencing and microarray technologies have provided a breadth of evidence highlighting the unique molecular genetics and epigenetics of this cancer, distinguishing it from both adult and pediatric cerebral high-grade astrocytomas. This review describes the most common molecular genetic and epigenetic signatures of DIPG in the context of molecular subgroups and histopathological diagnosis, including this tumor entity’s unique mutational landscape, copy number alterations, and structural variants, as well as epigenetic changes on the global DNA and histone levels. The increased knowledge of DIPG biology and histopathology has opened doors to new diagnostic and therapeutic avenues.

ATM Regulates 3-Methylpurine-DNA Glycosylase and Promotes Therapeutic Resistance to Alkylating Agents

UNLABELLED Alkylating agents are a first-line therapy for the treatment of several aggressive cancers, including pediatric glioblastoma, a lethal tumor in children. Unfortunately, many tumors are resistant to this therapy. We sought to identify ways of sensitizing tumor cells to alkylating agents while leaving normal cells unharmed, increasing therapeutic response while minimizing toxicity. Using an siRNA screen targeting over 240 DNA damage response genes, we identified novel sensitizers to alkylating agents. In particular, the base excision repair (BER) pathway, including 3-methylpurine-DNA glycosylase (MPG), as well as ataxia telangiectasia mutated (ATM), were identified in our screen. Interestingly, we identified MPG as a direct novel substrate of ATM. ATM-mediated phosphorylation of MPG was required for enhanced MPG function. Importantly, combined inhibition or loss of MPG and ATM resulted in increased alkylating agent-induced cytotoxicity in vitro and prolonged survival in vivo. The discovery of the ATM-MPG axis will lead to improved treatment of alkylating agent-resistant tumors. SIGNIFICANCE Inhibition of ATM and MPG-mediated BER cooperate to sensitize tumor cells to alkylating agents, impairing tumor growth in vitro and in vivo with no toxicity to normal cells, providing an ideal therapeutic window.

Spatial heterogeneity in medulloblastoma

Spatial heterogeneity of transcriptional and genetic markers between physically isolated biopsies of a single tumor poses major barriers to the identification of biomarkers and the development of targeted therapies that will be effective against the entire tumor. We analyzed the spatial heterogeneity of multiregional biopsies from 35 patients, using a combination of transcriptomic and genomic profiles. Medulloblastomas (MBs), but not high-grade gliomas (HGGs), demonstrated spatially homogeneous transcriptomes, which allowed for accurate subgrouping of tumors from a single biopsy. Conversely, somatic mutations that affect genes suitable for targeted therapeutics demonstrated high levels of spatial heterogeneity in MB, malignant glioma, and renal cell carcinoma (RCC). Actionable targets found in a single MB biopsy were seldom clonal across the entire tumor, which brings the efficacy of monotherapies against a single target into question. Clinical trials of targeted therapies for MB should first ensure the spatially ubiquitous nature of the target mutation.

Aurora kinase B is a potential therapeutic target in pediatric diffuse intrinsic pontine glioma.

Pediatric high‐grade astrocytomas (HGAs) account for 15–20% of all pediatric central nervous system tumors. These neoplasms predominantly involve the supratentorial hemispheres or the pons—diffuse intrinsic pontine gliomas (DIPG). Assumptions that pediatric HGAs are biologically similar to adult HGAs have recently been challenged, and the development of effective therapeutic modalities for DIPG and supratentorial HGA hinges on a better understanding of their biological properties. Here, 20 pediatric HGAs (9 DIPGs and 11 supratentorial HGAs) were subject to gene expression profiling following approval by the research ethics board at our institution. Many of these tumors showed expression signatures composed of genes that promote G1/S and G2/M cell cycle progression. In particular, Aurora kinase B (AURKB) was consistently and highly overexpressed in 6/9 DIPGs and 8/11 HGAs. Array data were validated using quantitative real‐time PCR and immunohistochemistry, as well as cross‐validation of our data set with previously published series. Inhibition of Aurora B activity in DIPG and in pediatric HGA cell lines resulted in growth arrest accompanied by morphological changes, cell cycle aberrations, nuclear fractionation and polyploidy as well as a reduction in colony formation. Our data highlight Aurora B as a potential therapeutic target in DIPG.

GLI2 Is a Potential Therapeutic Target in Pediatric Medulloblastoma

To determine whether the zinc finger transcription factors GLI1 to GLI3 and suppressor of fused (SUFU) components of the Sonic hedgehog signaling pathway may be prognostic markers and potential therapeutic targets in pediatric medulloblastoma (MB), we investigated the relationship of the expression of these proteins to prognosis in the MB of 124 patients who had undergone surgery atthe Hospital for Sick Children (Toronto, Ontario, Canada). The expressions of GLI1 (p = 0.011) and GLI2 (p = 0.003), but not of GLI3 (p = 0.774) or SUFU (p = 0.137), in the MB were associated with a worse overall survival by Kaplan-Meier analysis. Overall survival of patients positive for GLI1 and GLI2 was 6.01 ± 0.85 years and 5.27 ± 1.44 years, respectively, versus 10.11 ± 1.52 years and 10.18 ± 0.22 years for patients negative for GLI1 and GLI2, respectively. Knockdown of GLI2 in 3 MB cell lines resulted in decreased cell number and viability, as determined by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay; knockdown of GLI1 had no effect. The decrease in cell number with GLI2 knockdown was causedby G0 cell cycle arrest; there was no induction of apoptosis. These results suggest that targeting the Sonic hedgehog pathway in positive patients may be a useful adjuvant therapeutic strategy for MB.

Abstract B12: H3-K27M is a negative prognostic marker in high- and low-grade pediatric thalamic glioma

Pediatric gliomas are the most commonly diagnosed brain cancer in children, accounting for approximately 50% of all cases. Work by our group and others has revealed recurrent histone mutations in diffuse intrinsic pontine glioma (DIPG), astrocytomas that arise in the brainstem of children and boast a dismal median survival of less than one year. In ~70% of DIPG, a mutation in histone variant H3.3 or H3.1 at position 27 leads to substitution of a lysine by a methionine (K27M). When correlated to clinical data, patients harbouring H3-K27M mutations showed a worse prognosis in comparison to patients wild type for H3 (H3-WT), regardless of their histologic grade. Investigation into H3-K27M prevalence in pediatric gliomas revealed that they were not exclusively observed in the brainstem, but rather, in additional midline structures such as the thalamus. However, the clinical relevance of H3-K27M in other midline tumours has not yet been investigated. We hypothesized that H3-K27M would represent a negative prognostic marker in pediatric thalamic gliomas. We searched the Hospital for Sick Children9s tumour archives and obtained 86 radiologically confirmed cases of pre-treatment thalamic glioma. The cohort contained 46 male and 40 female patients with a combined median age of diagnosis and survival of 9.04 and 3.88 years, respectively. Of the 86 samples analyzed, 53 were histologically reviewed as low grade malignancies while 33 appeared high grade. Median survival for patients with high grade thalamic gliomas was 1.02 years vs 8.71 years for low grade thalamic glioma patients. Six (11.3%) low grade and 17 (51.5%) high grade gliomas tested positive for the H3-K27M mutation. Patients were clinically annotated for treatment, extent of resection, age of diagnosis and outcome (median follow-up of 10.3 years) and were used to determine the prognostic implications of H3-K27M. Kaplan-Meier survival analysis revealed significantly worse overall survival of thalamic glioma patients harbouring the H3-K27M mutation versus wild type samples (log rank p These findings provide the first evidence that H3-K27M is a negative prognostic indicator for pediatric thalamic glioma. Further, the survival difference between H3-K27M mutant low grade and high grade astrocytoma suggests differing biology and a role for “second hits” in dictating the aggressiveness of these tumours. Importantly, it supports prognostic subgrouping based on mutation status and histologic grade. Citation Format: Scott Ryall, Pawel Buczkowicz, Anthony Arnoldo, Rahul Krishnatry, Matthew Mistry, Joshua Rubin, Paul Steinbok, Uri Tabori, Cynthia Hawkins. H3-K27M is a negative prognostic marker in high- and low-grade pediatric thalamic glioma. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B12.