Publications by authors named "Wendy J Ingram"

10 Publications

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Functional diversity and cooperativity between subclonal populations of pediatric glioblastoma and diffuse intrinsic pontine glioma cells.

Nat Med 2018 08 2;24(8):1204-1215. Epub 2018 Jul 2.

Centre for Evolution and Cancer, The Institute of Cancer Research, London, UK.

The failure to develop effective therapies for pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG) is in part due to their intrinsic heterogeneity. We aimed to quantitatively assess the extent to which this was present in these tumors through subclonal genomic analyses and to determine whether distinct tumor subpopulations may interact to promote tumorigenesis by generating subclonal patient-derived models in vitro and in vivo. Analysis of 142 sequenced tumors revealed multiple tumor subclones, spatially and temporally coexisting in a stable manner as observed by multiple sampling strategies. We isolated genotypically and phenotypically distinct subpopulations that we propose cooperate to enhance tumorigenicity and resistance to therapy. Inactivating mutations in the H4K20 histone methyltransferase KMT5B (SUV420H1), present in <1% of cells, abrogate DNA repair and confer increased invasion and migration on neighboring cells, in vitro and in vivo, through chemokine signaling and modulation of integrins. These data indicate that even rare tumor subpopulations may exert profound effects on tumorigenesis as a whole and may represent a new avenue for therapeutic development. Unraveling the mechanisms of subclonal diversity and communication in pGBM and DIPG will be an important step toward overcoming barriers to effective treatments.
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http://dx.doi.org/10.1038/s41591-018-0086-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6086334PMC
August 2018

The ten-year evolutionary trajectory of a highly recurrent paediatric high grade neuroepithelial tumour with MN1:BEND2 fusion.

Sci Rep 2018 01 18;8(1):1032. Epub 2018 Jan 18.

Centre for Evolution and Cancer, Institute of Cancer Research, London, UK.

Astroblastomas are rare brain tumours which predominate in children and young adults, and have a controversial claim as a distinct entity, with no established WHO grade. Reports suggest a better outcome than high grade gliomas, though they frequently recur. Recently, they have been described to overlap with a newly-discovered group of tumours described as'high grade neuroepithelial tumour with MN1 alteration' (CNS HGNET-MN1), defined by global methylation patterns and strongly associated with gene fusions targeting MN1. We have studied a unique case of astroblastoma arising in a 6 year-old girl, with multiple recurrences over a period of 10 years, with the pathognomonic MN1:BEND2 fusion. Exome sequencing allowed for a phylogenetic reconstruction of tumour evolution, which when integrated with clinical, pathological and radiological data provide for a detailed understanding of disease progression, with initial treatment driving tumour dissemination along four distinct trajectories. Infiltration of distant sites was associated with a later genome doubling, whilst there was evidence of convergent evolution of different lesions acquiring distinct alterations targeting NF-κB. These data represent an unusual opportunity to understand the evolutionary history of a highly recurrent childhood brain tumour, and provide novel therapeutic targets for astroblastoma/CNS HGNET-MN1.
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http://dx.doi.org/10.1038/s41598-018-19389-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773598PMC
January 2018

Spatial and temporal homogeneity of driver mutations in diffuse intrinsic pontine glioma.

Nat Commun 2016 Apr 6;7:11185. Epub 2016 Apr 6.

Research Center for Genetic Medicine, Children's National Health System, Washington, District Of Columbia 20010, USA.

Diffuse Intrinsic Pontine Gliomas (DIPGs) are deadly paediatric brain tumours where needle biopsies help guide diagnosis and targeted therapies. To address spatial heterogeneity, here we analyse 134 specimens from various neuroanatomical structures of whole autopsy brains from nine DIPG patients. Evolutionary reconstruction indicates histone 3 (H3) K27M--including H3.2K27M--mutations potentially arise first and are invariably associated with specific, high-fidelity obligate partners throughout the tumour and its spread, from diagnosis to end-stage disease, suggesting mutual need for tumorigenesis. These H3K27M ubiquitously-associated mutations involve alterations in TP53 cell-cycle (TP53/PPM1D) or specific growth factor pathways (ACVR1/PIK3R1). Later oncogenic alterations arise in sub-clones and often affect the PI3K pathway. Our findings are consistent with early tumour spread outside the brainstem including the cerebrum. The spatial and temporal homogeneity of main driver mutations in DIPG implies they will be captured by limited biopsies and emphasizes the need to develop therapies specifically targeting obligate oncohistone partnerships.
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http://dx.doi.org/10.1038/ncomms11185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4823825PMC
April 2016

Prognostic value of medulloblastoma extent of resection after accounting for molecular subgroup: a retrospective integrated clinical and molecular analysis.

Authors:
Eric M Thompson Thomas Hielscher Eric Bouffet Marc Remke Betty Luu Sridharan Gururangan Roger E McLendon Darell D Bigner Eric S Lipp Sebastien Perreault Yoon-Jae Cho Gerald Grant Seung-Ki Kim Ji Yeoun Lee Amulya A Nageswara Rao Caterina Giannini Kay Ka Wai Li Ho-Keung Ng Yu Yao Toshihiro Kumabe Teiji Tominaga Wieslawa A Grajkowska Marta Perek-Polnik David C Y Low Wan Tew Seow Kenneth T E Chang Jaume Mora Ian F Pollack Ronald L Hamilton Sarah Leary Andrew S Moore Wendy J Ingram Andrew R Hallahan Anne Jouvet Michelle Fèvre-Montange Alexandre Vasiljevic Cecile Faure-Conter Tomoko Shofuda Naoki Kagawa Naoya Hashimoto Nada Jabado Alexander G Weil Tenzin Gayden Takafumi Wataya Tarek Shalaby Michael Grotzer Karel Zitterbart Jaroslav Sterba Leos Kren Tibor Hortobágyi Almos Klekner Bognár László Tímea Pócza Peter Hauser Ulrich Schüller Shin Jung Woo-Youl Jang Pim J French Johan M Kros Marie-Lise C van Veelen Luca Massimi Jeffrey R Leonard Joshua B Rubin Rajeev Vibhakar Lola B Chambless Michael K Cooper Reid C Thompson Claudia C Faria Alice Carvalho Sofia Nunes José Pimentel Xing Fan Karin M Muraszko Enrique López-Aguilar David Lyden Livia Garzia David J H Shih Noriyuki Kijima Christian Schneider Jennifer Adamski Paul A Northcott Marcel Kool David T W Jones Jennifer A Chan Ana Nikolic Maria Luisa Garre Erwin G Van Meir Satoru Osuka Jeffrey J Olson Arman Jahangiri Brandyn A Castro Nalin Gupta William A Weiss Iska Moxon-Emre Donald J Mabbott Alvaro Lassaletta Cynthia E Hawkins Uri Tabori James Drake Abhaya Kulkarni Peter Dirks James T Rutka Andrey Korshunov Stefan M Pfister Roger J Packer Vijay Ramaswamy Michael D Taylor

Lancet Oncol 2016 04 12;17(4):484-495. Epub 2016 Mar 12.

Division of Neurosurgery, The Hospital for Sick Children, Toronto, ON, Canada; The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children, Toronto, ON, Canada; Developmental & Stem Cell Biology Program, The Hospital for Sick Children, Toronto, ON, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada. Electronic address:

Background: Patients with incomplete surgical resection of medulloblastoma are controversially regarded as having a marker of high-risk disease, which leads to patients undergoing aggressive surgical resections, so-called second-look surgeries, and intensified chemoradiotherapy. All previous studies assessing the clinical importance of extent of resection have not accounted for molecular subgroup. We analysed the prognostic value of extent of resection in a subgroup-specific manner.

Methods: We retrospectively identified patients who had a histological diagnosis of medulloblastoma and complete data about extent of resection and survival from centres participating in the Medulloblastoma Advanced Genomics International Consortium. We collected from resections done between April, 1997, and February, 2013, at 35 international institutions. We established medulloblastoma subgroup affiliation by gene expression profiling on frozen or formalin-fixed paraffin-embedded tissues. We classified extent of resection on the basis of postoperative imaging as gross total resection (no residual tumour), near-total resection (<1·5 cm(2) tumour remaining), or sub-total resection (≥1·5 cm(2) tumour remaining). We did multivariable analyses of overall survival and progression-free survival using the variables molecular subgroup (WNT, SHH, group 4, and group 3), age (<3 vs ≥3 years old), metastatic status (metastases vs no metastases), geographical location of therapy (North America/Australia vs rest of the world), receipt of chemotherapy (yes vs no) and receipt of craniospinal irradiation (<30 Gy or >30 Gy vs no craniospinal irradiation). The primary analysis outcome was the effect of extent of resection by molecular subgroup and the effects of other clinical variables on overall and progression-free survival.

Findings: We included 787 patients with medulloblastoma (86 with WNT tumours, 242 with SHH tumours, 163 with group 3 tumours, and 296 with group 4 tumours) in our multivariable Cox models of progression-free and overall survival. We found that the prognostic benefit of increased extent of resection for patients with medulloblastoma is attenuated after molecular subgroup affiliation is taken into account. We identified a progression-free survival benefit for gross total resection over sub-total resection (hazard ratio [HR] 1·45, 95% CI 1·07-1·96, p=0·16) but no overall survival benefit (HR 1·23, 0·87-1·72, p=0·24). We saw no progression-free survival or overall survival benefit for gross total resection compared with near-total resection (HR 1·05, 0·71-1·53, p=0·8158 for progression-free survival and HR 1·14, 0·75-1·72, p=0·55 for overall survival). No significant survival benefit existed for greater extent of resection for patients with WNT, SHH, or group 3 tumours (HR 1·03, 0·67-1·58, p=0·89 for sub-total resection vs gross total resection). For patients with group 4 tumours, gross total resection conferred a benefit to progression-free survival compared with sub-total resection (HR 1·97, 1·22-3·17, p=0·0056), especially for those with metastatic disease (HR 2·22, 1·00-4·93, p=0·050). However, gross total resection had no effect on overall survival compared with sub-total resection in patients with group 4 tumours (HR 1·67, 0·93-2·99, p=0·084).

Interpretation: The prognostic benefit of increased extent of resection for patients with medulloblastoma is attenuated after molecular subgroup affiliation is taken into account. Although maximum safe surgical resection should remain the standard of care, surgical removal of small residual portions of medulloblastoma is not recommended when the likelihood of neurological morbidity is high because there is no definitive benefit to gross total resection compared with near-total resection.

Funding: Canadian Cancer Society Research Institute, Terry Fox Research Institute, Canadian Institutes of Health Research, National Institutes of Health, Pediatric Brain Tumor Foundation, and the Garron Family Chair in Childhood Cancer Research.
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http://dx.doi.org/10.1016/S1470-2045(15)00581-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4907853PMC
April 2016

Proteomic profiling of high risk medulloblastoma reveals functional biology.

Oncotarget 2015 Jun;6(16):14584-95

Center for Cancer and Immunology Research, Children's National Medical Center, Washington DC, USA.

Genomic characterization of medulloblastoma has improved molecular risk classification but struggles to define functional biological processes, particularly for the most aggressive subgroups. We present here a novel proteomic approach to this problem using a reference library of stable isotope labeled medulloblastoma-specific proteins as a spike-in standard for accurate quantification of the tumor proteome. Utilizing high-resolution mass spectrometry, we quantified the tumor proteome of group 3 medulloblastoma cells and demonstrate that high-risk MYC amplified tumors can be segregated based on protein expression patterns. We cross-validated the differentially expressed protein candidates using an independent transcriptomic data set and further confirmed them in a separate cohort of medulloblastoma tissue samples to identify the most robust proteogenomic differences. Interestingly, highly expressed proteins associated with MYC-amplified tumors were significantly related to glycolytic metabolic pathways via alternative splicing of pyruvate kinase (PKM) by heterogeneous ribonucleoproteins (HNRNPs). Furthermore, when maintained under hypoxic conditions, these MYC-amplified tumors demonstrated increased viability compared to non-amplified tumors within the same subgroup. Taken together, these findings highlight the power of proteomics as an integrative platform to help prioritize genetic and molecular drivers of cancer biology and behavior.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4546489PMC
http://dx.doi.org/10.18632/oncotarget.3927DOI Listing
June 2015

Recurrent activating ACVR1 mutations in diffuse intrinsic pontine glioma.

Nat Genet 2014 May 6;46(5):457-461. Epub 2014 Apr 6.

Institut Gustav Roussy, Villejuif, France.

Diffuse intrinsic pontine gliomas (DIPGs) are highly infiltrative malignant glial neoplasms of the ventral pons that, due to their location within the brain, are unsuitable for surgical resection and consequently have a universally dismal clinical outcome. The median survival time is 9-12 months, with neither chemotherapeutic nor targeted agents showing substantial survival benefit in clinical trials in children with these tumors. We report the identification of recurrent activating mutations in the ACVR1 gene, which encodes a type I activin receptor serine/threonine kinase, in 21% of DIPG samples. Strikingly, these somatic mutations (encoding p.Arg206His, p.Arg258Gly, p.Gly328Glu, p.Gly328Val, p.Gly328Trp and p.Gly356Asp substitutions) have not been reported previously in cancer but are identical to mutations found in the germ line of individuals with the congenital childhood developmental disorder fibrodysplasia ossificans progressiva (FOP) and have been shown to constitutively activate the BMP-TGF-β signaling pathway. These mutations represent new targets for therapeutic intervention in this otherwise incurable disease.
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http://dx.doi.org/10.1038/ng.2925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4018681PMC
May 2014

G-protein coupled receptor expression patterns delineate medulloblastoma subgroups.

Acta Neuropathol Commun 2013 Oct 10;1:66. Epub 2013 Oct 10.

Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, IA 2524 JCP, USA.

Background: Medulloblastoma is the most common malignant brain tumor in children. Genetic profiling has identified four principle tumor subgroups; each subgroup is characterized by different initiating mutations, genetic and clinical profiles, and prognoses. The two most well-defined subgroups are caused by overactive signaling in the WNT and SHH mitogenic pathways; less is understood about Groups 3 and 4 medulloblastoma. Identification of tumor subgroup using molecular classification is set to become an important component of medulloblastoma diagnosis and staging, and will likely guide therapeutic options. However, thus far, few druggable targets have emerged. G-protein coupled receptors (GPCRs) possess characteristics that make them ideal targets for molecular imaging and therapeutics; drugs targeting GPCRs account for 30-40% of all current pharmaceuticals. While expression patterns of many proteins in human medulloblastoma subgroups have been discerned, the expression pattern of GPCRs in medulloblastoma has not been investigated. We hypothesized that analysis of GPCR expression would identify clear subsets of medulloblastoma and suggest distinct GPCRs that might serve as molecular targets for both imaging and therapy.

Results: Our study found that medulloblastoma tumors fall into distinct clusters based solely on GPCR expression patterns. Normal cerebellum clustered separately from the tumor samples. Further, two of the tumor clusters correspond with high fidelity to the WNT and SHH subgroups of medulloblastoma. Distinct over-expressed GPCRs emerge; for example, LGR5 and GPR64 are significantly and uniquely over-expressed in the WNT subgroup of tumors, while PTGER4 is over-expressed in the SHH subgroup. Uniquely under-expressed GPCRs were also observed. Our key findings were independently validated using a large international dataset.

Conclusions: Our results identify GPCRs with potential to act as imaging and therapeutic targets. Elucidating tumorigenic pathways is a secondary benefit to identifying differential GPCR expression patterns in medulloblastoma tumors.
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http://dx.doi.org/10.1186/2051-5960-1-66DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3893540PMC
October 2013

ABC transporter activity linked to radiation resistance and molecular subtype in pediatric medulloblastoma.

Exp Hematol Oncol 2013 Oct 4;2(1):26. Epub 2013 Oct 4.

Background: Resistance to radiation treatment remains a major clinical problem for patients with brain cancer. Medulloblastoma is the most common malignant brain tumor of childhood, and occurs in the cerebellum. Though radiation treatment has been critical in increasing survival rates in recent decades, the presence of resistant cells in a substantial number of medulloblastoma patients leads to relapse and death.

Methods: Using the established medulloblastoma cell lines UW228 and Daoy, we developed a novel model system to enrich for and study radiation tolerant cells early after radiation exposure. Using fluorescence-activated cell sorting, dead cells and cells that had initiated apoptosis were removed, allowing surviving cells to be investigated before extensive proliferation took place.

Results: Isolated surviving cells were tumorigenic in vivo and displayed elevated levels of ABCG2, an ABC transporter linked to stem cell behavior and drug resistance. Further investigation showed another family member, ABCA1, was also elevated in surviving cells in these lines, as well as in early passage cultures from pediatric medulloblastoma patients. We discovered that the multi-ABC transporter inhibitors verapamil and reserpine sensitized cells from particular patients to radiation, suggesting that ABC transporters have a functional role in cellular radiation protection. Additionally, verapamil had an intrinsic anti-proliferative effect, with transient exposure in vitro slowing subsequent in vivo tumor formation. When expression of key ABC transporter genes was assessed in medulloblastoma tissue from 34 patients, levels were frequently elevated compared with normal cerebellum. Analysis of microarray data from independent cohorts (n = 428 patients) showed expression of a number of ABC transporters to be strongly correlated with certain medulloblastoma subtypes, which in turn are associated with clinical outcome.

Conclusions: ABC transporter inhibitors are already being trialed clinically, with the aim of decreasing chemotherapy resistance. Our findings suggest that the inhibition of ABC transporters could also increase the efficacy of radiation treatment for medulloblastoma patients. Additionally, the finding that certain family members are associated with particular molecular subtypes (most notably high ABCA8 and ABCB4 expression in Sonic Hedgehog pathway driven tumors), along with cell membrane location, suggests ABC transporters are worthy of consideration for the diagnostic classification of medulloblastoma.
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http://dx.doi.org/10.1186/2162-3619-2-26DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3851566PMC
October 2013

Targeting the inhibitor of apoptosis proteins as a novel therapeutic strategy in medulloblastoma.

Mol Cancer Ther 2012 Dec 25;11(12):2654-63. Epub 2012 Sep 25.

Children's Cancer Institute Australia, Lowy Cancer Research Centre, University ofNewSouth Wales, Australia.

Medulloblastoma is the most common malignant brain tumor of childhood. Novel therapeutic strategies are urgently needed to overcome cytotoxic resistance. We hypothesized that antiapoptotic signals contribute to resistance and that treatment with proapoptotic agents could increase the efficacy of conventional therapies. A PCR array was used to assess the status of the apoptotic signaling pathway in medulloblastoma cells after treatment with cytotoxic chemotherapy. Treatment with cisplatin led to the upregulation of antiapoptotic signals, including inhibitor of apoptosis proteins (IAP), in medulloblastoma cells. We subsequently investigated the synergistic effect of a small-molecule IAP inhibitor, LBW242, in combination with cisplatin and/or radiotherapy in three human medulloblastoma cell lines and 5 short term primary patient medulloblastoma cultures. The addition of LBW242 to chemotherapy resulted in significantly increased antitumor activity with a similar effect observed in combination with radiotherapy. Measurement of caspase-8 and -9 activity indicated that the synergy resulted from induction of both the intrinsic and extrinsic apoptotic pathways. Apoptosis was confirmed by Annexin V staining and activation of caspases 3/7. Xenograft models were used to evaluate the mechanism of action and efficacy in vivo. The combination therapy significantly reduced the tumor burden in a medulloblastoma xenograft model and TUNEL analysis in a medulloblastoma orthograft confirmed in vivo induction of apoptosis. These findings support the strategy of targeting IAPs in combination with cytotoxic therapy as a novel treatment strategy for patients with medulloblastoma.
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http://dx.doi.org/10.1158/1535-7163.MCT-12-0352DOI Listing
December 2012

Novel genes regulated by Sonic Hedgehog in pluripotent mesenchymal cells.

Oncogene 2002 Nov;21(53):8196-205

Institute for Molecular Bioscience and Department of Biochemistry and Molecular Biology, The University of Queensland, Queensland 4072, Australia.

Sonic Hedgehog is a secreted morphogen involved in patterning a wide range of structures in the developing embryo. Disruption of the Hedgehog signalling cascade leads to a number of developmental disorders and plays a key role in the formation of a range of human cancers. The identification of genes regulated by Hedgehog is crucial to understanding how disruption of this pathway leads to neoplastic transformation. We have used a Sonic Hedgehog (Shh) responsive mouse cell line, C3H/10T1/2, to provide a model system for hedgehog target gene discovery. Following activation of cell cultures with Shh, RNA was used to interrogate microarrays to investigate downstream transcriptional consequences of hedgehog stimulation. As a result 11 target genes have been identified, seven of which are induced (Thrombomodulin, GILZ, BF-2, Nr4a1, IGF2, PMP22, LASP1) and four of which are repressed (SFRP-1, SFRP-2, Mip1-gamma, Amh) by Shh. These targets have a diverse range of putative functions and include transcriptional regulators and molecules known to be involved in regulating cell growth or apoptosis. The corroboration of genes previously implicated in hedgehog signalling, along with the finding of novel targets, demonstrates both the validity and power of the C3H/10T1/2 system for Shh target gene discovery.
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http://dx.doi.org/10.1038/sj.onc.1205975DOI Listing
November 2002