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1.  Evaluation of Clinically Translatable Magnetic Resonance Imaging Biomarkers of Therapeutic Response in the TH-MYCN Transgenic Mouse Model of Neuroblastoma 
Radiology  2012;266(1):130-140.
To evaluate noninvasive and clinically-translatable magnetic resonance imaging (MRI) biomarkers of therapeutic response in the TH-MYCN transgenic mouse model of aggressive, MYCN-amplified neuroblastoma.
Materials and methods
All experiments were performed in accordance with the local ethical review panel, the UK Home Office Animals Scientific Procedures Act 1986 and with the United Kingdom National Cancer Research Institute guidelines for the welfare of animals in cancer research. Multiparametric MRI was performed on abdominal tumors found in the TH-MYCN model. T2-weighted MRI, quantitation of native relaxation times T1 and T2, the relaxation rate R2*, and dynamic contrast-enhanced (DCE) MRI were used to monitor tumor response to cyclophosphamide (25mg/kg), the vascular disrupting agent ZD6126 (200mg/kg), or the anti-angiogenic agent cediranib (6mg/kg, daily). Any significant changes in the measured parameters, and in the magnitude of the changes upon treatment between treated and control cohorts, were identified using Student’s 2-tailed paired and unpaired t-test respectively, with a 5% level of significance.
Treatment with cyclophosphamide or cediranib induced a 54% and 20% reduction in tumour volume at 48 hours respectively (p<0.005 and p<0.005, p<0.005 and p<0.005 versus control). Treatment with ZD6126 induced a 45% reduction in mean tumor volume 24 hours after treatment (p<0.005, p<0.005 versus control). The anti-tumor activity of cyclophosphamide, cediranib and ZD6126 was consistently associated with a decrease in tumor T1 (p<0.005, p<0.005 and p<0.005, respectively, p<0.005, p<0.005 and p<0.005 versus control respectively) and with a correlation between therapy-induced changes in native T1 and changes in tumor volume (r = 0.56, p < 0.005). Tumor response to cediranib was also associated with a decrease in the DCE MRI derived volume transfer constant Ktrans (p=0.07, p<0.05 versus control) and enhancing fraction (p<0.05, p<0.01 versus control), and an increase in R2* (p<0.005, p<0.05 versus control).
The T1 relaxation time is a robust noninvasive imaging biomarker of response to therapy in tumors in TH-MYCN mice, which emulate high-risk neuroblastoma in children. T1 measurements can be readily implemented on clinical MR systems, and should be investigated in translational clinical trials of new targeted therapies for pediatric neuroblastoma.
PMCID: PMC4298658  PMID: 23169794
T1; MRI; Biomarker; Neuroblastoma; Chemotherapy
2.  Primary glioblastoma with oligodendroglial differentiation has better clinical outcome but no difference in common biological markers compared with other types of glioblastoma 
Neuro-Oncology  2013;15(12):1635-1643.
Glioblastoma multiforme with an oligodendroglial component (GBMO) has been recognized in the World Health Organization classification—however, the diagnostic criteria, molecular biology, and clinical outcome of primary GBMO remain unclear. Our aim was to investigate whether primary GBMO is a distinct clinicopathological subgroup of GBM and to determine the relative frequency of prognostic markers such as loss of heterozygosity (LOH) on 1p and/or 19q, O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation, and isocitrate dehydrogenase 1 (IDH1) mutation.
We examined 288 cases of primary GBM and assessed the molecular markers in 57 GBMO and 50 cases of other primary GBM, correlating the data with clinical parameters and outcome.
GBMO comprised 21.5% of our GBM specimens and showed significantly longer survival compared with our other GBM (12 mo vs 5.8 mo, P = .006); there was also a strong correlation with younger age at diagnosis (56.4 y vs 60.6 y, P = .005). Singular LOH of 19q (P = .04) conferred a 1.9-fold increased hazard of shorter survival. There was no difference in the frequencies of 1p or 19q deletion, MGMT promoter methylation, or IDH1 mutation (P = .8, P = 1.0, P = 1.0, respectively).
Primary GBMO is a subgroup of GBM associated with longer survival and a younger age group but shows no difference in the frequency of LOH of 1p/19q, MGMT, and IDH1 mutation compared with other primary GBM.
PMCID: PMC3829593  PMID: 24158110
glioblastoma with an oligodendroglial component; histopathology; 1p/19q; IDH1; MGMT
4.  Genomic analysis of diffuse intrinsic pontine gliomas identifies three molecular subgroups and recurrent activating ACVR1 mutations 
Nature genetics  2014;46(5):451-456.
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 choosing therapies based on assumptions that DIPGs are molecularly similar to adult disease. Recent studies have unraveled the unique genetic make-up of this brain cancer with nearly 80% harboring a K27M-H3.3 or K27M-H3.1 mutation. 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 are three molecularly distinct subgroups (H3-K27M, Silent, MYCN) and uncovering a novel recurrent activating mutation in the activin receptor ACVR1, in 20% of DIPGs. Mutations in ACVR1 were constitutively activating, leading to SMAD phosphorylation and increased expression of downstream activin signaling targets ID1 and ID2. Our results highlight distinct molecular subgroups and novel therapeutic targets for this incurable pediatric cancer.
PMCID: PMC3997489  PMID: 24705254 CAMSID: cams4215
5.  The genomic landscape of diffuse intrinsic pontine glioma and pediatric non-brainstem high-grade glioma 
Nature genetics  2014;46(5):444-450.
Pediatric high-grade glioma (HGG) is a devastating disease with a two-year survival of less than 20%1. We analyzed 127 pediatric HGGs, including diffuse intrinsic pontine gliomas (DIPGs) and non-brainstem HGGs (NBS-HGGs) by whole genome, whole exome, and/or transcriptome sequencing. We identified recurrent somatic mutations in ACVR1 exclusively in DIPG (32%), in addition to the previously reported frequent somatic mutations in histone H3, TP53 and ATRX in both DIPG and NBS-HGGs2-5. Structural variants generating fusion genes were found in 47% of DIPGs and NBS-HGGs, with recurrent fusions involving the neurotrophin receptor genes NTRK1, 2, or 3 in 40% of NBS-HGGs in infants. Mutations targeting receptor tyrosine kinase/RAS/PI3K signaling, histone modification or chromatin remodeling, and cell cycle regulation were found in 68%, 73% and 59%, respectively, of pediatric HGGs, including DIPGs and NBS-HGGs. This comprehensive analysis provides insights into the unique and shared pathways driving pediatric HGG within and outside the brainstem.
PMCID: PMC4056452  PMID: 24705251
6.  Novel Oncogenic PDGFRA Mutations in Pediatric High-Grade Gliomas 
Cancer research  2013;73(20):6219-6229.
The outcome for children with high-grade gliomas (HGG) remains dismal, with a two-year survival rate of only 10–30%. Diffuse intrinsic pontine glioma (DIPG) comprise a subset of HGG that arise in brainstem almost exclusively in children. Genome-wide analyses of copy number imbalances previously showed that platelet derived growth factor receptor alpha (PDGFRA) is the most frequent target of focal amplification in pediatric HGGs, including DIPGs. To determine whether PDGFRA is also targeted by more subtle mutations missed by copy number analysis, we sequenced all PDGFRA coding exons from a cohort of pediatric HGGs. Somatic activating mutations were identified in 14.4% (13/90) of non-brainstem pediatric HGGs and 4.7% (2/43) of DIPGs, including missense mutations and in-frame deletions and insertions not previously described. 40% of tumors with mutation showed concurrent amplification, while 60% carried heterozygous mutations. Six different mutations impacting different domains all resulted in ligand-independent receptor activation that was blocked by small molecule inhibitors of PDGFR. Expression of mutants in p53-null primary mouse astrocytes conferred a proliferative advantage in vitro, and generated HGGs in vivo with complete penetrance when implanted into brain. The gene expression signatures of these murine HGGs reflected the spectrum of human diffuse HGGs. PDGFRA intragenic deletion of exons 8 and 9 were previously shown in adult HGG, but were not detected in 83 non-brainstem pediatric HGG and 57 DIPGs. Thus, a distinct spectrum of mutations confers constitutive receptor activation and oncogenic activity to PDGFRα in childhood HGG.
PMCID: PMC3800209  PMID: 23970477
Glioma; Glioblastoma; HGG; PDGFR; Pediatric
7.  Histone H3.3 mutations drive paediatric glioblastoma through upregulation of MYCN 
Cancer discovery  2013;10.1158/2159-8290.CD-12-0426.
Glioblastomas of children and young adults have a median survival of only 12-15months and are clinically and biologically distinct from histologically similar cancers in older adults1. They are defined by highly specific mutations in the gene encoding the histone H3.3 variant H3F3A2, occurring either at or close to key residues marked by methylation for regulation of transcription – K27 and G34. Here we show that the cerebral hemispheric-specific G34 mutation drives a distinct expression signature through differential genomic binding of the K36 trimethylation mark (H3K36me3). The transcriptional program induced recapitulates that of the developing forebrain, and involves numerous markers of stem cell maintenance, cell fate decisions and self-renewal. Critically, H3F3A G34 mutations cause profound upregulation of MYCN, a potent oncogene which is causative of glioblastomas when expressed in the correct developmental context. This driving aberration is selectively targetable in this patient population by inhibiting kinases responsible for stabilisation of the protein.
PMCID: PMC3763966  PMID: 23539269
glioma; ChIP-Seq; forebrain; H3F3A; G34R/V
8.  Plant vigour at establishment and following defoliation are both associated with responses to drought in perennial ryegrass (Lolium perenne L.) 
Journal of Experimental Botany  2014;65(20):5823-5834.
Highlight text
We have developed a new methodology to assess individual perennial ryegrass plant performance under moisture stress and identified QTLs associated with improved performance during drought in this important forage species.
Periodic drought events present a significant and, with climate change, increasing constraint on temperate forage plants’ production. Consequently, improving plants’ adaptive response to abiotic stress is a key goal to ensure agricultural productivity in these regions. In this study we developed a new methodology, using both area-based comparison and soil water content measurements of individual non-irrigated and irrigated clones, to assess performance of perennial ryegrass (Lolium perenne L.) genotypes subjected to moisture stress in a simulated competitive environment. We applied this method to the evaluation of a full-sibling population from a pair cross between genotypes from a New Zealand cultivar and a Moroccan ecotype. Our hypothesis was that: (i) both leaf lamina regrowth after defoliation (LR) and plant vigour affect plant performance during drought and rehydration; and (ii) quantitative trait loci (QTLs) associated with plant performance under moisture stress could be identified. Differences amongst genotypes in dry matter (DM) production, early vigour at establishment, leaf elongation rate and LR were measured. LR explained most of the variation in DM production during exposure to moisture deficit and rehydration followed by plant vigour, indicated by initial DM production in both treatments and subsequent measures of DM production of irrigated clones. We identified two main QTL regions associated with DM production and LR, both during drought exposure and rehydration. Further research focused on these regions should improve our understanding of the genetic control of drought response in this forage crop and potentially other grass species with significant synteny, and support improvement in performance through molecular breeding approaches.
PMCID: PMC4203121  PMID: 25104762
Drought; grass; Lolium perenne; moisture stress; pasture production; perennial ryegrass; quantitative trait locus (QTL); soil water content.
9.  Lactate and Choline Metabolites Detected In Vitro by Nuclear Magnetic Resonance Spectroscopy Are Potential Metabolic Biomarkers for PI3K Inhibition in Pediatric Glioblastoma 
PLoS ONE  2014;9(8):e103835.
The phosphoinositide 3-kinase (PI3K) pathway is believed to be of key importance in pediatric glioblastoma. Novel inhibitors of the PI3K pathway are being developed and are entering clinical trials. Our aim is to identify potential non-invasive biomarkers of PI3K signaling pathway inhibition in pediatric glioblastoma using in vitro nuclear magnetic resonance (NMR) spectroscopy, to aid identification of target inhibition and therapeutic response in early phase clinical trials of PI3K inhibitors in childhood cancer. Treatment of SF188 and KNS42 human pediatric glioblastoma cell lines with the dual pan-Class I PI3K/mTOR inhibitor PI-103, inhibited the PI3K signaling pathway and resulted in a decrease in phosphocholine (PC), total choline (tCho) and lactate levels (p<0.02) as detected by phosphorus (31P)- and proton (1H)-NMR. Similar changes were also detected using the pan–Class I PI3K inhibitor GDC-0941 which lacks significant mTOR activity and is entering Phase II clinical trials. In contrast, the DNA damaging agent temozolomide (TMZ), which is used as current frontline therapy in the treatment of glioblastoma postoperatively (in combination with radiotherapy), increased PC, glycerophosphocholine (GPC) and tCho levels (p<0.04). PI-103-induced NMR changes were associated with alterations in protein expression levels of regulatory enzymes involved in glucose and choline metabolism including GLUT1, HK2, LDHA and CHKA. Our results show that by using NMR we can detect distinct biomarkers following PI3K pathway inhibition compared to treatment with the DNA-damaging anti-cancer agent TMZ. This is the first study reporting that lactate and choline metabolites are potential non-invasive biomarkers for monitoring response to PI3K pathway inhibitors in pediatric glioblastoma.
PMCID: PMC4118961  PMID: 25084455
10.  A case study of the Secure Anonymous Information Linkage (SAIL) Gateway: A privacy-protecting remote access system for health-related research and evaluation☆ 
Journal of Biomedical Informatics  2014;50(100):196-204.
Graphical abstract
•SAIL Gateway is a privacy-protecting safe haven and secure remote access system.•It provides secure data access to approved users.•It is a powerful platform for data analysis activities.•The system is able to accommodate a growing data user base.•This is a challenging field with further improvements in progress.
With the current expansion of data linkage research, the challenge is to find the balance between preserving the privacy of person-level data whilst making these data accessible for use to their full potential. We describe a privacy-protecting safe haven and secure remote access system, referred to as the Secure Anonymised Information Linkage (SAIL) Gateway. The Gateway provides data users with a familiar Windows interface and their usual toolsets to access approved anonymously-linked datasets for research and evaluation. We outline the principles and operating model of the Gateway, the features provided to users within the secure environment, and how we are approaching the challenges of making data safely accessible to increasing numbers of research users. The Gateway represents a powerful analytical environment and has been designed to be scalable and adaptable to meet the needs of the rapidly growing data linkage community.
PMCID: PMC4139270  PMID: 24440148
AIX, Advanced Interactive eXecutive; ALF, Anonymous Linking Field; CIPHER, Centre for the Improvement of Population Health through E-records Research; DB2, a family of database server products developed by International Business Machines (IBM); DP, Data Provider; HTTPS, HyperText Transfer Protocol Secure; IGRP, Information Governance Review Panel; LSOA, Lower Super Output Area; NHS, National Health Service; NWIS, NHS Wales Informatics Service; RALF, Residential Anonymous Linking Field; SAIL, Secure Anonymised Information Linkage; SQL, Structured Query Language; UKSeRP, UK Secure Research Platform; VPN, Virtual Private Network; Data linkage; Remote access system; Privacy-protection; e-Records research
11.  Discovery of Novel GPVI Receptor Antagonists by Structure-Based Repurposing 
PLoS ONE  2014;9(6):e101209.
Inappropriate platelet aggregation creates a cardiovascular risk that is largely managed with thienopyridines and aspirin. Although effective, these drugs carry risks of increased bleeding and drug ‘resistance’, underpinning a drive for new antiplatelet agents. To discover such drugs, one strategy is to identify a suitable druggable target and then find small molecules that modulate it. A good and unexploited target is the platelet collagen receptor, GPVI, which promotes thrombus formation. To identify inhibitors of GPVI that are safe and bioavailable, we docked a FDA-approved drug library into the GPVI collagen-binding site in silico. We now report that losartan and cinanserin inhibit GPVI-mediated platelet activation in a selective, competitive and dose-dependent manner. This mechanism of action likely underpins the cardioprotective effects of losartan that could not be ascribed to its antihypertensive effects. We have, therefore, identified small molecule inhibitors of GPVI-mediated platelet activation, and also demonstrated the utility of structure-based repurposing.
PMCID: PMC4074120  PMID: 24971515
12.  CNS-PNETs with C19MC amplification and/or LIN28 expression comprise a distinct histogenetic diagnostic and therapeutic entity 
Acta Neuropathologica  2014;128(2):291-303.
Amplification of the C19MC oncogenic miRNA cluster and high LIN28 expression has been linked to a distinctly aggressive group of cerebral CNS-PNETs (group 1 CNS-PNETs) arising in young children. In this study, we sought to evaluate the diagnostic specificity of C19MC and LIN28, and the clinical and biological spectra of C19MC amplified and/or LIN28+ CNS-PNETs. We interrogated 450 pediatric brain tumors using FISH and IHC analyses and demonstrate that C19MC alteration is restricted to a sub-group of CNS-PNETs with high LIN28 expression; however, LIN28 immunopositivity was not exclusive to CNS-PNETs but was also detected in a proportion of other malignant pediatric brain tumors including rhabdoid brain tumors and malignant gliomas. C19MC amplified/LIN28+ group 1 CNS-PNETs arose predominantly in children <4 years old; a majority arose in the cerebrum but 24 % (13/54) of tumors had extra-cerebral origins. Notably, group 1 CNS-PNETs encompassed several histologic classes including embryonal tumor with abundant neuropil and true rosettes (ETANTR), medulloepithelioma, ependymoblastoma and CNS-PNETs with variable differentiation. Strikingly, gene expression and methylation profiling analyses revealed a common molecular signature enriched for primitive neural features, high LIN28/LIN28B and DNMT3B expression for all group 1 CNS-PNETs regardless of location or tumor histology. Our collective findings suggest that current known histologic categories of CNS-PNETs which include ETANTRs, medulloepitheliomas, ependymoblastomas in various CNS locations, comprise a common molecular and diagnostic entity and identify inhibitors of the LIN28/let7/PI3K/mTOR axis and DNMT3B as promising therapeutics for this distinct histogenetic entity.
Electronic supplementary material
The online version of this article (doi:10.1007/s00401-014-1291-1) contains supplementary material, which is available to authorized users.
PMCID: PMC4159569  PMID: 24839957
13.  Population Genomic Analysis of Ancient and Modern Genomes Yields New Insights into the Genetic Ancestry of the Tyrolean Iceman and the Genetic Structure of Europe 
PLoS Genetics  2014;10(5):e1004353.
Genome sequencing of the 5,300-year-old mummy of the Tyrolean Iceman, found in 1991 on a glacier near the border of Italy and Austria, has yielded new insights into his origin and relationship to modern European populations. A key finding of that study was an apparent recent common ancestry with individuals from Sardinia, based largely on the Y chromosome haplogroup and common autosomal SNP variation. Here, we compiled and analyzed genomic datasets from both modern and ancient Europeans, including genome sequence data from over 400 Sardinians and two ancient Thracians from Bulgaria, to investigate this result in greater detail and determine its implications for the genetic structure of Neolithic Europe. Using whole-genome sequencing data, we confirm that the Iceman is, indeed, most closely related to Sardinians. Furthermore, we show that this relationship extends to other individuals from cultural contexts associated with the spread of agriculture during the Neolithic transition, in contrast to individuals from a hunter-gatherer context. We hypothesize that this genetic affinity of ancient samples from different parts of Europe with Sardinians represents a common genetic component that was geographically widespread across Europe during the Neolithic, likely related to migrations and population expansions associated with the spread of agriculture.
Author Summary
The analysis of the genome of the Tyrolean Iceman, a 5,300 year old mummy from Central Europe, revealed a surprising recent common ancestry with modern Sardinians for this ancient genome. However, this study was limited both by the availability of data from Sardinians and by a lack of genomic data from other ancient European samples. Here, we use genomic data from modern Sardinians and from ancient European individuals from different geographic regions and cultural contexts, to demonstrate that this ancestry component is shared among individuals associated with the onset of agriculture in Europe. Our results thus suggest that the Iceman's Sardinian ancestry actually reflects a more widespread genetic component related to the migration of people during the Neolithic transition in Central Europe.
PMCID: PMC4014435  PMID: 24809476
14.  Paediatric and adult glioblastoma: multiform (epi)genomic culprits emerge 
Nature reviews. Cancer  2014;14(2):92-107.
We have extended our understanding of the molecular biology underlying adult glioblastoma over many years. In contrast, high-grade gliomas in children and adolescents have remained a relatively under-investigated disease. The latest large-scale genomic and epigenomic profiling studies have yielded an unprecedented abundance of novel data and revealed deeper insights into gliomagenesis across all age groups, highlighting key distinctions, but also some commonalities. As we are on the verge of dissecting glioblastomas into meaningful biological subgroups, this Review summarizes the hallmark genetic alterations associated with distinct epigenetic features and patient characteristics in both paediatric and adult disease, and examines the complex interplay between the glioblastoma genome and epigenome.
PMCID: PMC4003223  PMID: 24457416
15.  A multi-disciplinary consensus statement concerning surgical approaches to low-grade, high-grade astrocytomas and diffuse intrinsic pontine gliomas in childhood (CPN Paris 2011) using the Delphi method 
Neuro-Oncology  2013;15(4):462-468.
Astrocytic tumors account for 42% of childhood brain tumors, arising in all anatomical regions and associated with neurofibromatosis type 1 (NF1) in 15%. Anatomical site determines the degree and risk of resectability; the more complete resection, the better the survival rates. New biological markers and modern radiotherapy techniques are altering the risk assessments of clinical decisions for tumor resection and biopsy. The increasingly distinct pediatric neuro-oncology multidisciplinary team (PNMDT) is developing a distinct evidence base.
A multidisciplinary consensus conference on pediatric neurosurgery was held in February 2011, where 92 invited participants reviewed evidence for clinical management of hypothalamic chiasmatic glioma (HCLGG), diffuse intrinsic pontine glioma (DIPG), and high-grade glioma (HGG). Twenty-seven statements were drafted and subjected to online Delphi consensus voting by participants, seeking >70% agreement from >60% of respondents; where <70% consensus occurred, the statement was modified and resubmitted for voting.
Twenty-seven statements meeting consensus criteria are reported. For HCLGG, statements describing overall therapeutic purpose and indications for biopsy, observation, or treatment aimed at limiting the risk of visual damage and the need for on-going clinical trials were made. Primary surgical resection was not recommended. For DIPG, biopsy was recommended to ascertain biological characteristics to enhance understanding and targeting of treatments, especially in clinical trials. For HGG, biopsy is essential, the World Health Organization classification was recommended; selection of surgical strategy to achieve gross total resection in a single or multistep process should be discussed with the PNMDT and integrated with trials based drug strategies for adjuvant therapies.
PMCID: PMC3607269  PMID: 23502427
low-grade astrocytoma
16.  Adaptive latitudinal variation in Common Blackbird Turdus merula nest characteristics 
Ecology and Evolution  2014;4(6):841-851.
Nest construction is taxonomically widespread, yet our understanding of adaptive intraspecific variation in nest design remains poor. Nest characteristics are expected to vary adaptively in response to predictable variation in spring temperatures over large spatial scales, yet such variation in nest design remains largely overlooked, particularly amongst open-cup-nesting birds. Here, we systematically examined the effects of latitudinal variation in spring temperatures and precipitation on the morphology, volume, composition, and insulatory properties of open-cup-nesting Common Blackbirds’ Turdus merula nests to test the hypothesis that birds living in cooler environments at more northerly latitudes would build better insulated nests than conspecifics living in warmer environments at more southerly latitudes. As spring temperatures increased with decreasing latitude, the external diameter of nests decreased. However, as nest wall thickness also decreased, there was no variation in the diameter of the internal nest cups. Only the mass of dry grasses within nests decreased with warmer temperatures at lower latitudes. The insulatory properties of nests declined with warmer temperatures at lower latitudes and nests containing greater amounts of dry grasses had higher insulatory properties. The insulatory properties of nests decreased with warmer temperatures at lower latitudes, via changes in morphology (wall thickness) and composition (dry grasses). Meanwhile, spring precipitation did not vary with latitude, and none of the nest characteristics varied with spring precipitation. This suggests that Common Blackbirds nesting at higher latitudes were building nests with thicker walls in order to counteract the cooler temperatures. We have provided evidence that the nest construction behavior of open-cup-nesting birds systematically varies in response to large-scale spatial variation in spring temperatures.
PMCID: PMC3967909  PMID: 24683466
Insulation quality; latitude; nest composition; nest size; spring temperature; Turdus merula
Cancer research  2010;70(22):9243-9252.
Sensitivity to temozolomide (TMZ) is restricted to a subset of glioblastoma patients, with the major determinant of resistance being a lack of promoter methylation of the gene encoding the repair protein DNA methyltransferase MGMT, although other mechanisms are thought to be active. There are, however, limited preclinical data in model systems derived from paediatric glioma patients. We have screened a series of cell lines for TMZ efficacy in vitro, and have investigated the differential mechanisms of resistance involved. In the majority of cell lines, a lack of MGMT promoter methylation, and subsequent protein overexpression, was linked to TMZ resistance. An exception was the paediatric glioblastoma line KNS42. Expression profiling data revealed a co-ordinated upregulation of HOX gene expression in resistant lines, especially KNS42, which was reversed by PI3-kinase pathway inhibition. High levels of HOXA9/HOXA10 gene expression were associated with a shorter survival in paediatric high grade glioma patient samples. Combination treatment in vitro of pathway inhibition and TMZ resulted in a highly synergistic interaction in KNS42 cells. The resistance gene signature further included contiguous genes within the 12q13-q14 amplicon including the Akt enhancer PIKE, significantly overexpressed in the KNS42 line. These cells were also highly enriched for CD133 and other stem cell markers. We have thus demonstrated an in vitro link between PI3-kinase-mediated HOXA9/HOXA10 expression, and a drug-resistant, progenitor cell phenotype in MGMT-independent paediatric glioblastoma.
PMCID: PMC3935452  PMID: 20935218
glioma; temozolomide; paediatric; MGMT; PI3-kinase; HOX; CD133
18.  The prognostic role of intragenic copy number breakpoints and identification of novel fusion genes in paediatric high grade glioma 
Paediatric high grade glioma (pHGG) is a distinct biological entity to histologically similar tumours arising in older adults, and has differing copy number profiles and driver genetic alterations. As functionally important intragenic copy number aberrations (iCNA) and fusion genes begin to be identified in adult HGG, the same has not yet been done in the childhood setting. We applied an iCNA algorithm to our previously published dataset of DNA copy number profiling in pHGG with a view to identify novel intragenic breakpoints.
We report a series of 288 iCNA events in pHGG, with the presence of intragenic breakpoints itself a negative prognostic factor. We identified an increased number of iCNA in older children compared to infants, and increased iCNA in H3F3A K27M mutant tumours compared to G34R/V and wild-type. We observed numerous gene disruptions by iCNA due to both deletions and amplifications, targeting known HGG-associated genes such as RB1 and NF1, putative tumour suppressors such as FAF1 and KIDINS220, and novel candidates such as PTPRE and KCND2. We further identified two novel fusion genes in pHGG – CSGALNACT2:RET and the complex fusion DHX57:TMEM178:MAP4K3. The latter was sequence-validated and appears to be an activating event in pHGG.
These data expand upon our understanding of the genomic events driving these tumours and represent novel targets for therapeutic intervention in these poor prognosis cancers of childhood.
Electronic supplementary material
The online version of this article (doi:10.1186/2051-5960-2-23) contains supplementary material, which is available to authorized users.
PMCID: PMC3938307  PMID: 24548782
Fusion; Paediatric; Glioblastoma; Copy number; Intragenic
19.  Distinct Phenotypic Differences Associated with Differential Amplification of Receptor Tyrosine Kinase Genes at 4q12 in Glioblastoma 
PLoS ONE  2013;8(8):e71777.
Gene amplification at chromosome 4q12 is a common alteration in human high grade gliomas including glioblastoma, a CNS tumour with consistently poor prognosis. This locus harbours the known oncogenes encoding the receptor tyrosine kinases PDGFRA, KIT, and VEGFR2. These receptors are potential targets for novel therapeutic intervention in these diseases, with expression noted in tumour cells and/or associated vasculature. Despite this, a detailed assessment of their relative contributions to different high grade glioma histologies and the underlying heterogeneity within glioblastoma has been lacking. We studied 342 primary high grade gliomas for individual gene amplification using specific FISH probes, as well as receptor expression in the tumour and endothelial cells by immunohistochemistry, and correlated our findings with specific tumour cell morphological types and patterns of vasculature. We identified amplicons which encompassed PDGFRA only, PDGFRA/KIT, and PDGFRA/KIT/VEGFR2, with distinct phenotypic correlates. Within glioblastoma specimens, PDGFRA amplification alone was linked to oligodendroglial, small cell and sarcomatous tumour cell morphologies, and rare MGMT promoter methylation. A younger age at diagnosis and better clinical outcome in glioblastoma patients is only seen when PDGFRA and KIT are co-amplified. IDH1 mutation was only found when all three genes are amplified; this is a subgroup which also harbours extensive MGMT promoter methylation. Whilst PDGFRA amplification was tightly linked to tumour expression of the receptor, this was not the case for KIT or VEGFR2. Thus we have identified differential patterns of gene amplification and expression of RTKs at the 4q12 locus to be associated with specific phenotypes which may reflect their distinct underlying mechanisms.
PMCID: PMC3749194  PMID: 23990986
20.  Genetic transformation of western clover (Trifolium occidentale D. E. Coombe.) as a model for functional genomics and transgene introgression in clonal pasture legume species 
Plant Methods  2013;9:25.
Western clover (Trifolium occidentale) is a perennial herb with characteristics compatible for its development as an attractive model species for genomics studies relating to the forage legume, white clover (Trifolium repens). Its characteristics such as a small diploid genome, self-fertility and ancestral contribution of one of the genomes of T. repens, facilitates its use as a model for genetic analysis of plants transformed with legume or novel genes.
In this study, a reproducible transformation protocol was established following screening of T. occidentale accessions originating from England, Ireland, France, Spain and Portugal. The protocol is based upon infection of cotyledonary explants dissected from mature seed with the Agrobacterium tumefaciens strain GV3101 carrying vectors which contain the bar selection marker gene. Transformation frequencies of up to 7.5% were achieved in 9 of the 17 accessions tested. Transformed plants were verified by PCR and expression of the gusA reporter gene, while integration of the T-DNA was confirmed by Southern blot hybridisation and segregation of progeny in the T1 generation.
Development of this protocol provides a valuable contribution toward establishing T. occidentale as a model species for white clover. This presents opportunities for further improvement in white clover through the application of biotechnology.
PMCID: PMC3716983  PMID: 23841995
Agrobacterium-mediated transformation; Trifolium occidentale; Genetic transformation; Organogenic regeneration; Accession
21.  An integrated genetic linkage map for white clover (Trifolium repens L.) with alignment to Medicago 
BMC Genomics  2013;14:388.
White clover (Trifolium repens L.) is a temperate forage legume with an allotetraploid genome (2n=4×=32) estimated at 1093 Mb. Several linkage maps of various sizes, marker sources and completeness are available, however, no integrated map and marker set has explored consistency of linkage analysis among unrelated mapping populations. Such integrative analysis requires tools for homoeologue matching among populations. Development of these tools provides for a consistent framework map of the white clover genome, and facilitates in silico alignment with the model forage legume, Medicago truncatula.
This is the first report of integration of independent linkage maps in white clover, and adds to the literature on methyl filtered GeneThresher®-derived microsatellite (simple sequence repeat; SSR) markers for linkage mapping. Gene-targeted SSR markers were discovered in a GeneThresher® (TrGT) methyl-filtered database of 364,539 sequences, which yielded 15,647 SSR arrays. Primers were designed for 4,038 arrays and of these, 465 TrGT-SSR markers were used for parental consensus genetic linkage analysis in an F1 mapping population (MP2). This was merged with an EST-SSR consensus genetic map of an independent population (MP1), using markers to match homoeologues and develop a multi-population integrated map of the white clover genome. This integrated map (IM) includes 1109 loci based on 804 SSRs over 1274 cM, covering 97% of the genome at a moderate density of one locus per 1.2 cM. Eighteen candidate genes and one morphological marker were also placed on the IM. Despite being derived from disparate populations and marker sources, the component maps and the derived IM had consistent representations of the white clover genome for marker order and genetic length. In silico analysis at an E-value threshold of 1e-20 revealed substantial co-linearity with the Medicago truncatula genome, and indicates a translocation between T. repens groups 2 and 6 relative to M. truncatula.
This integrated genetic linkage analysis provides a consistent and comprehensive linkage analysis of the white clover genome, with alignment to a model forage legume. Associated marker locus information, particularly the homoeologue-specific markers, offers a new resource for forage legume research to enable genetic analysis and improvement of this forage and grassland species.
PMCID: PMC3693905  PMID: 23758831
Legume; Trifolium repens; Linkage; Microsatellite; SSR; GeneThresher®; Allotetraploid; Homoeologue; Medicago truncatula; Synteny
22.  Glioblastoma blood flow measured with stable xenon CT indicates tumor necrosis, vascularity, and brain invasion 
Neuro-Oncology  2012;14(5):641-648.
Tumor vasculature is a promising therapeutic target in glioblastoma. Imaging tumor blood flow may help assess the efficacy of anti-angiogenic treatments. We determined the clinical usefulness of stable xenon CT performed preoperatively in patients with glioblastoma. This is a prospective cohort study. We determined absolute tumor blood flow before surgery in 38 patients with glioblastoma using stable xenon CT. We also histologically examined tumor specimens obtained from surgery and quantified their vascularity (by CD31 and CD105 immunostain), necrosis (by hematoxylin and eosin stain), and the presence of neuronal processes (by neurofilament immunostain). According to the xenon CT blood flow map, there are 3 types of glioblastoma. Type I glioblastomas have unimodal high blood flow histograms; histologically there is little necrosis and vascular proliferation. Type II glioblastomas have unimodal low blood flow histograms; histologically there is prominent necrosis and vascular proliferation. We propose that in type II glioblastoma, the abnormal vessels induced by hypoxia are inefficient at promoting blood flow. Type III glioblastomas have multimodal blood flow histograms. Histologically there is significant neuronal tissue within the tumor. Patients with type III glioblastomas were more likely to develop a post-surgical deficit, consistent with the inclusion of normal tissue within the tumor. Preoperative measurement of absolute blood flow with stable xenon CT in patients with glioblastoma predicts key biological features of the tumor and may aid surgical planning.
PMCID: PMC3337310  PMID: 22492958
cerebral blood flow; glioblastoma; microvessel density; necrosis; xenon CT
23.  Intraperitoneal Vancomycin Concentrations During Peritoneal Dialysis–Associated Peritonitis: Correlation with Serum Levels 
♦ Background: For the treatment of peritoneal dialysis–associated peritonitis (PDP), it has been suggested that serum concentrations of vancomycin be kept above 12 mg/L – 15 mg/L. However, studies correlating vancomycin concentrations in serum and peritoneal dialysate effluent (PDE) during active infection are sparse. We undertook the present study to investigate this issue and to determine whether achieving the recommended serum level of vancomycin results in therapeutic levels intraperitoneally.
♦ Methods: We studied patients treated with intraperitoneal (IP) vancomycin for non-gram-negative PDP. We gave a single dose (approximately 30 mg/kg) at presentation, and we subsequently measured vancomycin levels in PDE on day 5; we wanted to determine if efflux of vancomycin from serum to PDE during a 4-hour dwell was consistent and resulted in therapeutic levels.
♦ Results: Of the 48 episodes of PDP studied, serum vancomycin concentrations exceeding 12 mg/L were achieved in 98% of patients, but in 11 patients (23%), a PDE vancomycin level below 4 mg/L—the minimal inhibitory concentration (MIC) of many gram-positive organisms—was observed at the end of a 4-hour dwell on day 5. The correlation between the concentrations of vancomycin in serum and PDE (from efflux of antibiotic over 4 hours) was statistically significant, but poor (R2 = 0.18).
♦ Conclusions: Our data support the International Society for Peritoneal Dialysis statement that adequate serum vancomycin concentrations can be achieved with intermittent dosing (single dose every 5 days), but cannot guarantee therapeutic PDE levels in the treatment of PDP. Intermittent dosing of vancomycin may not consistently result in PDE concentrations markedly greater than MIC of many important pathogens. Although the clinical significance of this finding remains to be determined, it may be preferable to give smaller but more frequent doses of PDE vancomycin (continuous dosing) for adults with PDP (as is currently recommended for children).
PMCID: PMC3525447  PMID: 22045102
Peritonitis; pharmacokinetics; vancomycin
24.  Integrative genomic analyses identify LIN28 and OLIG2 as markers of survival and metastatic potential in childhood central nervous system primitive neuro-ectodermal brain tumours 
The lancet oncology  2012;13(8):838-848.
Childhood Central Nervous System Primitive Neuro-Ectodermal brain Tumours (CNS-PNETs) are highly aggressive brain tumours for which molecular features and best therapeutic strategy remains unknown. We interrogated a large cohort of these rare tumours in order to identify molecular markers that will enhance clinical management of CNS-PNET.
Transcriptional and copy number profiles from primary hemispheric CNS-PNETs were examined using clustering, gene and pathways enrichment analyses to discover tumour sub-groups and group-specific molecular markers. Immuno-histochemical and/or gene expression analyses were used to validate and examine the clinical significance of novel sub-group markers in 123 primary CNS-PNETs.
Three molecular sub-groups of CNS-PNETs distinguished by primitive neural (Group 1), oligo-neural (Group 2) and mesenchymal lineage (Group 3) gene expression signature were identified. Tumour sub-groups exhibited differential expression of cell lineage markers, LIN28 and OLIG2, and correlated with distinct demographics, survival and metastatic incidence. Group 1 tumours affected primarily younger females; male: female ratios were respectively 0.61 (median age 2.9 years; 95% CI: 2.4–5.2; p≤ 0.005), 1.25 (median age 7.9 years; 95% CI: 6–9.7) and 1.63 (median age 5.9 years; 95% CI: 4.9–7.8) for group 1, 2 and 3 patients. Overall outcome was poorest in group 1 patients which had a median survival of 0.8 years (95% CI: 0.47–1.2; p=0.019) as compared to 1.8 years (95% CI: 1.4–2.3) and 4.3 years; (95% CI: 0.82–7.8) respectively for group 2 and 3 patients. Group 3 tumours had the highest incidence of metastases at diagnosis; M0: M+ ratio were respectively 0.9 and 3.9 for group 3, versus group 1 and 2 tumours combined (p=0.037).
LIN28 and OLIG2 represent highly promising, novel diagnostic and prognostic molecular markers for CNS PNET that warrants further evaluation in prospective clinical trials.
PMCID: PMC3615440  PMID: 22691720
25.  IDH1-Associated Primary Glioblastoma in Young Adults Displays Differential Patterns of Tumour and Vascular Morphology 
PLoS ONE  2013;8(2):e56328.
Glioblastoma is a highly aggressive tumour with marked heterogeneity at the morphological level in both the tumour cells and the associated highly prominent vasculature. As we begin to develop an increased biological insight into the underlying processes driving the disease, fewer attempts have thus far been made to understand these phenotypic differences. We sought to address this by carefully assessing the morphological characteristics of both the tumour cells and the associated vasculature, relating these observations to the IDH1/MGMT status, with a particular focus on the early onset population of young adults who develop primary glioblastoma. 276 primary glioblastoma specimens were classified into their predominant cell morphological type (fibrillary, gemistocytic, giant cell, small cell, oligodendroglial, sarcomatous), and assessed for specific tumour (cellularity, necrosis, palisades) and vascular features (glomeruloid structures, arcades, pericyte proliferation). IDH1 positive glioblastomas were associated with a younger age at diagnosis, better clinical outcome, prominent oligodendroglial and small cell tumour cell morphology, pallisading necrosis and glomeruloid vascular proliferation in the absence of arcade-like structures. These features widen the phenotype of IDH1 mutation-positive primary glioblastoma in young adults and provide correlative evidence for a functional role of mutant IDH1 in the differential nature of neo-angiogenesis in different subtypes of glioblastoma.
PMCID: PMC3579823  PMID: 23451042

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