• Towards identifying potent new hits for glioblastoma

      Sherer, Chris; Prabhu, Saurabh; Adams, David; Hayes, Joseph; Rowther, Farzana; Tolaymat, Ibrahim; Warr, Tracy; Snape, Timothy J. (Royal Society of Chemistry, 2018-10-02)
      Glioblastoma is a devastating disease of the brain and is the most common malignant primary brain tumour in adults. The prognosis for patients is very poor with median time of survival after diagnosis measured in months, due in part to the tumours being highly aggressive and often resistant to chemotherapies. Alongside the ongoing research to identify key factors involved in tumour progression in glioblastoma, medicinal chemistry approaches must also be used in order to rapidly establish new and better treatments for brain tumour patients. Using a computational similarity search of the ZINC database, alongside traditional analogue design by medicinal chemistry intuition to improve the breadth of chemical space under consideration, six new hit compounds (14, 16, 18, 19, 20 and 22) were identified possessing low micromolar activity against both established cell lines (U87MG and U251MG) and patient-derived cell cultures (IN1472, IN1528 and IN1760). Each of these scaffolds provides a new platform for future development of a new therapy in this area, with particular promise shown against glioblastoma subtypes that are resistant to conventional chemotherapeutic agents.
    • Genomic and transcriptomic characterisation of undifferentiated pleomorphic sarcoma of bone

      Ali, Naser M.; Niada, Stefania; Brini, Anna T.; Morris, Mark R.; Kurusamy, Sathishkumar; Alholle, Abdullah; Huen, David; Antonescu, Cristina R.; Tirode, Franck; Sumathi, Vaiyapuri; Latif, Farida (Wiley, 2018-10-03)
      Undifferentiated pleomorphic sarcoma of bone (UPSb), is a rare primary bone sarcoma that lacks a specific line of differentiation. There is very little information about the genetic alterations leading to tumourigenesis or malignant transformation. Distinguishing between UPSb and other malignant bone sarcomas, including dedifferentiated chondrosarcoma and osteosarcoma, can be challenging due to overlapping features. To explore the genomic and transcriptomic landscape of UPSb tumours, whole-exome sequencing (WES) and RNA Sequencing (RNA-Seq) were performed on UPSb tumours. All tumours lacked hotspot mutations in IDH1/2 132 or 172 codons, thereby excluding the diagnosis of dedifferentiated chondrosarcoma. Recurrent somatic mutations in TP53 were identified in 4/14 samples (29%). Moreover, recurrent mutations in histone chromatin remodelling genes, including H3F3A, ATRX and DOT1L, were identified in 5/14 samples (36%), highlighting the potential role of deregulated chromatin remodelling pathways in UPSb tumourigenesis. The majority of recurrent mutations in chromatin remodelling genes identified here are reported in COSMIC, including the H3F3A G35 and K36 hotspot residues. Copy number alteration analysis identified gains and losses in genes that have been previously altered in UPSb or UPS of soft tissue. Eight somatic gene fusions were identified by RNA-Seq, two of which, CLTC-VMP1 and FARP1-STK24, were reported previously in multiple cancers. Five gene fusions were genomically characterised. Hierarchical clustering analysis, using RNA-Seq data, distinctly clustered UPSb tumours from osteosarcoma and other sarcomas, thus molecularly distinguishing UPSb from other sarcomas. RNA-Seq expression profiling analysis and quantitative RT-PCR showed an elevated expression in FGF23 which can be a potential molecular biomarker in UPSb. To our knowledge, this study represents the first comprehensive WES and RNA-Seq analysis of UPSb tumours revealing novel protein-coding recurrent gene mutations, gene fusions and identifying a potential UPSb molecular biomarker, thereby broadening the understanding of the pathogenic mechanisms and highlighting the possibility of developing novel targeted therapeutics.
    • Randomised controlled trial of a home-based physical activity intervention in breast cancer survivors

      Lahart, Ian M.; Metsios, George S.; Nevill, Alan M.; Kitas, George D.; Carmichael, Amtul R. (2016-03-17)
      Background: To improve adherence to physical activity (PA), behavioural support in the form of behavioural change counselling may be necessary. However, limited evidence of the effectiveness of home-based PA combined with counselling in breast cancer patients exists. The aim of this current randomised controlled trial with a parallel group design was to evaluate the effectiveness of a home-based PA intervention on PA levels, anthropometric measures, health-related quality of life (HRQoL), and blood biomarkers in breast cancer survivors. Methods: Eighty post-adjuvant therapy invasive breast cancer patients (age = 53.6 ± 9.4 years; height = 161.2 ± 6.8 cm; mass = 68.7 ± 10.5 kg) were randomly allocated to a 6-month home-based PA intervention or usual care. The intervention group received face-to-face and telephone PA counselling aimed at encouraging the achievement of current recommended PA guidelines. All patients were evaluated for our primary outcome, PA (International PA Questionnaire) and secondary outcomes, mass, BMI, body fat %, HRQoL (Functional assessment of Cancer Therapy-Breast), insulin resistance, triglycerides (TG) and total (TC), high-density lipoprotein (HDL-C) and low-density lipoprotein (LDL-C) cholesterol were assessed at baseline and at 6-months. Results: On the basis of linear mixed-model analyses adjusted for baseline values performed on 40 patients in each group, total, leisure and vigorous PA significantly increased from baseline to post-intervention in the intervention compared to usual care (between-group differences, 578.5 MET-min∙wk−1, p = .024, 382.2 MET-min∙wk−1, p = .010, and 264.1 MET-min∙wk−1, p = .007, respectively). Both body mass and BMI decreased significantly in the intervention compared to usual care (between-group differences, −1.6 kg, p = .040, and −.6 kg/m2, p = .020, respectively). Of the HRQoL variables, FACT-Breast, Trial Outcome Index, functional wellbeing, and breast cancer subscale improved significantly in the PA group compared to the usual care group (between-group differences, 5.1, p= .024; 5.6, p = .001; 1.9 p = .025; and 2.8, p=.007, respectively). Finally, TC and LDL-C was significantly reduced in the PA group compared to the usual care group (between-group differences, −.38 mmol∙L−1, p=.001; and −.3 mmol∙L−1, p=.023, respectively). Conclusions: We found that home-based PA resulted in significant albeit small to moderate improvements in selfreported PA, mass, BMI, breast cancer specific HRQoL, and TC and LDL-C compared with usual care.
    • Germline Mutations in the CDKN2B Tumor Suppressor Gene Predispose to Renal Cell Carcinoma.

      Jafri, Mariam; Wake, Naomi C; Ascher, David B; Pires, Douglas E V; Gentle, Dean; Morris, Mark R.; Rattenberry, Eleanor; Simpson, Michael A; Trembath, Richard C; Weber, Astrid; Woodward, Emma R; Donaldson, Alan; Blundell, Tom L; Latif, Farida; Maher, Eamonn R (American Association for Cancer Research, 2015-07)
      Familial renal cell carcinoma (RCC) is genetically heterogeneous and may be caused by mutations in multiple genes, including VHL, MET, SDHB, FH, FLCN, PTEN, and BAP1. However, most individuals with inherited RCC do not have a detectable germline mutation. To identify novel inherited RCC genes, we undertook exome resequencing studies in a familial RCC kindred and identified a CDKN2B nonsense mutation that segregated with familial RCC status. Targeted resequencing of CDKN2B in individuals (n = 82) with features of inherited RCC then revealed three candidate CDKN2B missense mutations (p.Pro40Thr, p.Ala23Glu, and p.Asp86Asn). In silico analysis of the three-dimensional structures indicated that each missense substitution was likely pathogenic through reduced stability of the mutant or reduced affinity for cyclin-dependent kinases 4 and 6, and in vitro studies demonstrated that each of the mutations impaired CDKN2B-induced suppression of proliferation in an RCC cell line. These findings identify germline CDKN2B mutations as a novel cause of familial RCC.
    • Non-Steroidal Anti-Inflammatory Drugs, DNA Repair and Cancer

      Dibra, Harpreet K.; Perry, Chris J.; Nicholl, Iain D. (InTech, 2011)
    • Eukaryote polyphosphate kinases: is the 'Kornberg' complex ubiquitous?

      Hooley, Paul; Whitehead, Michael P.; Brown, Michael R. W. (Elsevier, 2008)
      Polyphosphate (poly P) is a polymer of up to several hundred phosphate residues and is important to a variety of cell processes. The main poly P synthetic enzyme in many bacteria is poly P kinase 1 (PPK1), which until recently had been detected among eukaryotes in some protists only. There is now evidence for the presence in several other eukaryotes of PPK1 homologues and also a second bacteria-type enzyme, PPK2. The latest genome databases reveal that the 'Kornberg' enzyme complex of three actin-related proteins, termed DdPPK2 in Dictyostelium discoideum, might also be ubiquitous in eukaryotes. Owing to the intimate association of poly P synthesis with the formation of structural fibres, this ubiquity indicates a central role for this molecule in the evolution of eukaryotic cells.
    • Hydrophobins: new prospects for biotechnology

      Cox, P. W.; Hooley, Paul (2009)
      Hydrophobins are small amphipathic molecules found uniquely in fungi. They perform crucial roles in allowing filamentous species to break through interfaces during aerial hyphae formation, sporulation, fruit body production and cell penetration. Initial biotechnological applications have exploited materials coated with hydrophobins to switch hydrophobic surfaces to hydrophilic and vice versa. Recent improvements in our understanding of the biophysics of hydrophobin layer formation, including the use of mixed types of molecules, together with advances in genomics promise to extend greatly the potential for hydrophobin biotechnologies.
    • The Aspergillus nidulans stress response transcription factor StzA is ascomycete-specific and shows species-specific polymorphisms in the C-terminal region.

      Chilton, Ian J.; Delaney, C. E,; Barham-Morris, J.; Fincham, Daron A.; Hooley, Paul; Whitehead, Michael P. (Elsevier, 2008)
      Orthologues of the Aspergillus nidulans gene stzA were identified and characterised in an additional 19 fungi. These orthologues were restricted to, and found within all the Pezizomycotina subphyla of the Ascomycota, for which data are available, but not the Saccharomycotina or Taphrinomycotina subphyla. Intron analysis indicated that both intron loss and gain have occurred in this gene. The orthologous proteins demonstrate considerable size variation (between 663 and 897 amino acids); with almost all this variability accounted for by a hyper-variable region that is carboxy terminal to the zinc finger region. The Hypocrea jecorina orthologue (ACE1) has the binding site 5'AGGCA. There is evidence of competition, or interaction, between the ACE1/StzA and AreA binding sites in promoters of stzA and its orthologues, as well as genes involved in the metabolism of amino acids. The A. nidulans and A. fumigatus cpcA promoters have seven potential ACE1/StzA binding sites, six of which are highly conserved in position. Two very closely positioned sites are conserved across 14 of the 19 fungi analysed. Potential CpcA binding sites (5'TGAC/GTCA) have been identified between -50 and -170bp of the ATG start in the promoters of 16 of the stzA orthologues.
    • Aspirin and alterations in DNA repair proteins in the SW480 colorectal cancer cell line.

      Dibra, H. K.; Brown, J. E.; Hooley, Paul; Nicholl, I. D. (Spandios Publications, 2010)
      Regular aspirin intake is associated with a reduction in the incidence of colorectal cancer. Aspirin has been shown to be cytotoxic to colorectal cancer cells in vitro. The molecular basis for this cytotoxicity is controversial, with a number of competing hypotheses in circulation. One suggestion is that the protective effect is related to the induction of expression of the DNA mismatch repair (MMR) proteins hMLH1, hMSH2, hMSH6 and hPMS2 in DNA MMR proficient cells. We report that treatment of the DNA MMR competent/p53 mutant colorectal cancer cell line SW480 with 1 mM aspirin for 48 h caused changes in mRNA expression of several key genes involved in DNA damage signalling pathways, including a significant down-regulation in transcription of the genes ATR, BRCA1 and MAPK12. Increases in the transcription of XRCC3 and GADD45alpha genes are also reported. Regulation of these genes could potentially have profound effects on colorectal cancer cells and may play a role in the observed chemo-protective effect of aspirin in vivo. Although a correlation was not seen between transcript and protein levels of ATR, BRCA1 and GADD45alpha, an increase in XRCC3 encoded protein expression upon aspirin treatment in SW480 cells was observed by immunoblotting, immunofluorescence and immunohistochemical analysis. This is the first report of XRCC3 gene transcription and encoded protein expression being susceptible to exposure to the non-steroidal anti-inflammatory drug, aspirin. Furthermore, this study indicates that alterations in gene transcription seen in microarray studies must be verified at the protein level.
    • A role for human endogenous retrovirus-K (HML-2) in rheumatoid arthritis: investigating mechanisms of pathogenesis.

      Freimanis, Graham L.; Hooley, Paul; Ejtehadi, H Dava; Ali, H. A.; Veitch, A.; Rylance, P.; Alawi, A.; Axford, J.; Nevill, Alan M.; Murray, Paul G.; Nelson, Paul N. (Wiley-Blackwell, 2010)
      Human endogenous retroviruses (HERVs) are remnants of ancient retroviral infections within the human genome. These molecular fossils draw parallels with present-day exogenous retroviruses and have been linked previously with immunopathology within rheumatoid arthritis (RA). Mechanisms of pathogenesis for HERV-K in RA such as molecular mimicry were investigated. To clarify a role for HERVs in RA, potential autoantigens implicated in autoimmunity were scanned for sequence identity with retroviral epitopes. Short retroviral peptides modelling shared epitopes were synthesized, to survey anti-serum of RA patients and disease controls. A novel real-time polymerase chain reaction (PCR) assay was also developed to quantify accurately levels of HERV-K (HML-2) gag expression, relative to normalized housekeeping gene expression. Both serological and molecular assays showed significant increases in HERV-K (HML-2) gag activity in RA patients, compared to disease controls. The real-time PCR assay identified significant up-regulation in HERV-K mRNA levels in RA patients compared to inflammatory and healthy controls. Exogenous viral protein expression and proinflammatory cytokines were also shown to exert modulatory effects over HERV-K (HML-2) transcription. From our data, it can be concluded that RA patients exhibited significantly elevated levels of HERV-K (HML-2) gag activity compared to controls. Additional factors influencing HERV activity within the synovium were also identified. The significant variation in RA patients, both serologically and transcriptionally, may be an indication that RA is an umbrella term for a number of separate disease entities, of which particular HERV polymorphisms may play a role in development.
    • Molecular biology masterclasses – developing practical skills and building links with Higher Education in years 12/13

      Hooley, Paul; Cooper, Phillippa; Skidmore, Nick (Higher Education Academy, 2008)
      A one day practical course in molecular biology skills suitable for year 12/13 students is described. Colleagues from partner schools and colleges were trained by university staff in basic techniques and then collaborated in the design of a course suitable for their own students. Participants carried out a transformation of E.coli cells with a plasmid and cut lambda virus DNA with restriction enzymes for display via agarose gel electrophoresis. Practical demonstrations of the polymerase chain reaction (PCR) and fermentation technology were also given. An evaluation of year 12/13 student responses revealed considerable enthusiasm for the activities.
    • A rhegnylogic strategy for the synthesis of signal transduction modulatory, cell penetrating peptides

      Jones, Sarah; Ostlund, Pernilla; Langel, Ulo; Zorko, Matjaz; Nicholl, Iain D.; Howl, John D. (Wiley InterScience, 2006)
      INTRODUCTION: Many cell-penetrating peptides (CPP) have been utilised as biologically inert vectors. A majority of these studies employ sychnologically organised constructs in which a bioactive cargo (message) is chemically conjugated to the CPP (address). Previously, we have adopted a sychnologic strategy to modulate intracellular signal transduction. Using chimeric constructs composed of the CPP transportan 10, conjugated to partial sequences that correspond to functional domains of signal transduction proteins, we have selectively modulated a variety of cellular activities including secretion and activation of p42/p44 mitogen-activated protein kinases [1, 2]. However, a QSAR-based algorithm can now be used to predict CPP that reside within the primary sequences of proteins [3]. We have adapted this strategy to identify CPP within signal transducing proteins including functional domains that govern protein-protein interactions. Data presented herein indicate that it is now feasible to identify rhegnylogic sequences, containing vectoral-independent discontinuously organised pharmacophores, that are cell penetrant modulators of signal transduction pathways.
    • Molecular pathology of brain tumours - how will molecular and cell biology contribute to improved outcomes in patients with malignant brain tumours?

      Darling, John L. (Czech Republic: Palacky University, Olomouc: Faculty of Medicine and Dentistry, 2006)
      Although in comparison to breast, lung and colon cancer, the brain is a relatively uncommon site for the development of cancer, the brain is the tenth most common site for the development of cancer in men and about the twelfth in women. This translates to about 6,000 individuals in the UK developing a primary malignant brain tumour every year. Cancer of the brain develops in two distinct age groups, although the types of tumour that develop in these two age groups differ markedly. There is a peak of incidence in the first decade of life, and brain tumours rank with leukaemia as a leading cause of cancer death in children. These tumours tend to be indolent low-grade astrocytomas or highly malignant primitive neuroectodermal tumours like medulloblastoma. However, the vast majority of brain tumours occur with increasing frequency in the sixth, seventh and eight decade of life and they are the second fastest growing cause of cancer death among those over 65. These tumours tend to be malignant astrocytomas particularly the most malignant variety, glioblastoma multiforme (GBM). Unlike lung cancer or malignant melanoma there is no strong evidence of an environmental carcinogen associated with the development of these tumours and no change in behaviour reduces risk.
    • Spectrum of copy number changes in low grade paediatric brain tumours detected by comparative genomic hybdidisation

      Warr, Tracy; Ward, Samantha; Idowu, Michael O.; Gregory, S.; Darling, John L.; Thomas, David G. (Society for Neuro-Oncology and Duke University Press, 2001)
      Several different types of low-grade brain tumours arise predominantly in the paediatric population, including astrocytoma, craniopharygioma and choroid plexus papilloma (CPP). As yet, the genetic events that contribute to their development have not been well defined. We have used comparative genomic hybridisation (CGH) to identify regions of genetic loss and gain in a series of 30 low grade tumours comprising 7 pilocytic astrocytoma (WHO grade I), 12 grade II astrocytoma, 5 craniopharyngioma (WHO grade I) and 6 CPP (WHO grade I). In the group of astrocytoma, there were no detectable regions of genomic imbalance in all 7 grade I tumours and in 9 of 12 grade II tumours. The copy number changes in the remaining 3 cases of grade II astrocytoma were gain of 7p and 12p; loss of 1p, 12q and 22; gain of 2q, 3, 8q and 18q, respectively. Similarly, 4 of 5 craniopharyngioma appeared to have normal CGH profiles, although multiple changes including gain of 4, 6q, 8q and 18q and loss of 17, 20q and 22 were observed in the sixth case. In contrast, copy number aberrations were detected in 3 of 6 CPP. In one tumour, gain of 7 was the sole change, whereas gain of 3q and 6q and loss of 9q and 21 were detected in a second case. However, in the remaining CPP all chromosomes except X and Y were involved in copy number changes with gain of 1, 7, 9, 12, 15, 16, 18, 20 and 22 and loss of the remaining autosomes. Overall, genomic imbalance was detected in only 7 of these low grade tumours and it was not possible to identify consistent regions of loss and gain. Considerably larger numbers of each tumour type need to be analysed in order to elucidate the genetic pathways involved in tumourigenesis.
    • TP53 mutation is infrequent in aneuploid choroid plexus tumours

      Warr, Tracy; Idowu, Michael O.; Suarez-Merino, Blanca; Ward, Samantha; Harkness, William; Hayward, Richard; Thompson, Dominic; Darling, John L.; Thomas, David G. (Society for Neuro-Oncology and Duke University Press, 2003)
      Choroid plexus tumours are uncommon primary brain tumours that arise predominantly in the paediatric population. Although cytogenetic studies are limited, it appears that in both benign choroid plexus papilloma (WHO grade I) and histologically malignant choroid plexus carcinoma (WHO grade III), there is a prevalence of numerical rather than structural chromosomal aberrations. In particular, additional copies of chromosomes 7, 8, 12, 15, 18, and 20 have been reported in several tumours. In the present study, we analysed 10 choroid plexus tumours comprising 9 papillomas and 1 carcinoma for genomic loss or gain using comparative genomic hybridisation. Four cases had no detectable regions of imbalance. The remaining tumours had multiple copy number aberrations (CNAs) ranging from 2 to 22 (mean 8.2), and all of the autosomes were involved at least once. The most common changes were gains of 7, 9p, and 20, and losses of 3p and 10q, which were each seen in 3 cases. In contrast to previous reports, chromosome loss was as frequent as gain. Additionally, imbalance of single chromosome arms was observed in 3 tumours, which may be indicative of structural abnormalities. In many other tumour types, aneuploidy is often associated with loss of functional p53. To investigate the role of TP53 in the development of choroid plexus tumours, we screened all 10 cases for mutations by direct sequencing of exons 4–8 and their corresponding splice junctions. In 1 papilloma, a missense mutation was detected at codon 273 in which a G to A transition results in the replacement of an arginine residue by a histidine residue. Codon 273 is a hotspot for mutation in many types of tumour, including astrocytoma. Interestingly, this tumour was aneuploid for every autosome. There were no mutations present in the remaining 9 cases, and it is unlikely that TP53 inactivation is critical to the pathogenesis of choroid plexus tumours. Further investigations are necessary to elucidate the genetic pathways involved in tumourigenesis.
    • MGMT methylation status and expression level do not correlate with sensitivity to CCNU in short-term cultures derived from malignant astrocytoma

      Warr, Tracy; Poh, R.; Suarez-Merino, Blanca; Ward, Samantha; Warren, Phil; Darling, John L.; Thomas, David G. (Society for Neuro-Oncology and Duke University Press, 2005)
      Adjuvant chemotherapy using DNA-damaging agents has largely failed to make a significant impact on the outcome of patients with malignant astrocytoma. One of the primary mechanisms of resistance to nitrosureas such as CCNU is mediated through O6-methylguanine-DNA methyltrans-ferase (MGMT). This DNA repair enzyme removes the cytotoxic alkyl adducts from O6-guanine, and hence the level of MGMT activity in tumor cells is related to their sensitivity to nitroureas. It has been proposed that functional inactivation of MGMT through hypermethylation of the gene promotor region could be predictive of chemosensitivity. We have previously reported differential sensitivity to CCNU in a panel of 17 short-term cultures derived from malignant astrocytoma. In this study, we determined the methylation status of MGMT using methylation-specific PCR in these 17 cultures. We also assessed the amounts of MGMT mRNA and protein present in each culture using real-time quantitative PCR and immunohistochemistry with a commercial antibody against MGMT. There was good correlation between MGMT promotor methylation and presence of MGMT mRNA and protein in all but 2 cases. In both these cultures, mRNA and protein were not detected even though the MGMT promotor was unmethylated. However, there was no correlation between sensitivity to CCNU and MGMT status. In the 2 most resistant cultures, the MGMT gene was methylated and was not expressed. Similarly, in 4/5 of the most sensitive cultures, MGMT was unmethylated, and in 2 of these cases, there was commensurate MGMT expression. However, in the remaining 2 cultures, MGMT expression was not detected, indicating that an alternative mechanism to gene methylation is responsible for MGMT inactivation. This study highlights that the resistance of malignant astrocytoma to nitroureas may be more complex than simple reliance on MGMT activity and prediction of response to such agents by MGMT methylation status should be used with caution.
    • Correlation of copy number aberrations with clinico-pathological criteria in paediatric glial tumours

      Ward, Samantha; Hayward, Richard; Harkness, William; Phipps, Kim; Thompson, Dominic; Harding, Brian; Wilkins, Peter; Darling, John L.; Thomas, David G.; Warr, Tracy (Society for Neuro-Oncology and Duke University Press, 2003)
      Glial cell tumours represent the largest group of brain tumours in childhood and include astrocytoma (WHO grades I-IV) and ependymoma (WHO grade II-III). However, little is known about the pathogenesis of these tumours. We have used comparative genomic hybridisation (CGH) to investigate the genetic alterations in 128 tumours from children and young adults (< 30 years of age) comprising 52 ependymoma, including 40 samples that have previously been reported (44 grade II and 8 grade III) and 76 astrocytoma (consisting of 34 grade I, 17 grade II, 7 grade III, and 18 grade IV). Genetic alterations were compared to clinicopathological data such as histology, tumour recurrence, and survival in order to identify potential prognostic markers. In ependymoma, 39% of the tumours had no detectable copy number aberrations (CNAs). In the remaining tumours, the most common regions of gain were 4q (29%), 6q (21%), 1q (17%), and 2q (15%). The most common regions of loss were 22 (29%), 16p (17%), 17p (13%), and 20q (13%). Three regions of high copy number amplification were observed in 3 tumours at 1q24-31 (3 cases), 8q21-23 (3 cases), and 9p (1 case). There was no association between any CNA and histology, tumour recurrence, or length of survival. In contrast, in the astrocytoma group there was a clear association between histology and the presence of CNAs. The pattern of genetic alterations became increasingly complex with tumour grade, and grade IV tumours were more likely to have CNAs than lower grade tumours (p = 0.0502). Overall, the most frequent alterations observed in astrocytoma were gain of 4q (11%), loss 16p (10.5%), and loss 17p (10.5%). However, several CNAs were seen predominantly in grade IV tumours (gain 1q, 2q, 4q, and 5q). Fourteen amplicons were observed in 8 tumours of all grades, of which the most common were localised to 7q31 (4 cases), 8q21-22 (3 cases), 19p (2 cases), 2q (2 cases), and 12q15-21 (2 cases). From this study, it appears that paediatric glial tumours are much more genetically heterogeneous than their adult counterparts, and further molecular investigations are needed to define clinically useful subgroups.
    • Identification of differentially expressed genes in paediatric ependymoma

      Suarez-Merino, Blanca; Hubank, Mike; Hayward, Richard; Harkness, William; Thompson, Dominic; Phipps, Kim; Revesz, Tamas; Darling, John L.; Thomas, David G.; Warr, Tracy (Society for Neuro-Oncology and Duke University Press, 2003)
      To date, the genetic events that contribute to the pathogenesis of ependymoma are essentially unknown. Furthermore, previous cytogenetic studies have demonstrated that approximately 50% of tumors have no detectable chromosome abnormalities. In this study, we used the Affymetrix GeneChip U95Av2 microarrays to generate gene expression profiles in 11 pediatric ependymoma, comprising 6 fresh frozen samples and 5 short-term cultures. A total of 107 genes were differentially expressed in both the biopsies and cell cultures compared with normal control tissue derived from corpus callosum. The 84 genes with increased expression included many encoding adhesion and extracellular matrix proteins; genes involved in the cell cycle, such as cyclin D1, CDK2, CDK4, and Wee1; transcription factors such as Zic1; and known oncogenes such as c-myc, WNT5A, JUN, TC21, RAB36, and FOP. An interesting group also found to be overexpressed comprises the insulin-like growth factor binding proteins IGFBP2, IGFBP3, and IGFBP4, which may be responsible for the autostimulation of cell growth in these tumors. Of the 23 genes that were underexpressed by 5-fold or less, 6 were of unknown function. Others included the apoptotic control gene ATIP the DAAM1 gene associated with the Wnt/frz oncogenic pathway, and genes involved in vesicle trafficking and recycling within and across cells, such as NPC1, RAB40B, TJP2, and SH3GL3. We have identified a number of candidate genes and genetic pathways that have not previously been associated with the pathogenesis of pediatric ependymoma. Further evaluation of the expression or mutation status of these genes will elucidate their roles in ependymoma development.
    • Expression profiling in ependymoma reveals differences between benign and anaplastic ependymoma

      Suarez-Merino, Blanca; Hubank, Mike; Hayward, Richard; Harkness, William; Thompson, Dominic; Phipps, Kim; Revesz, Tamas; Darling, John L.; Thomas, David G.; Warr, Tracy (Society for Neuro-Oncology and Duke University Press, 2003)
      Ependymomas arise from the ependymal cells lining the ventricular system of the CNS and account for approximately 10% of paediatric brain tumours. Approximately 70% of ependymomas are histologically benign and correspond to WHO grade II, whilst the remainder are anaplastic (WHO grade III). The 5-year survival rates in children are 34–45%, with local recurrence being the major source of therapeutic failure. Anaplasia does not appear to be associated with worse prognosis, and at present there are no molecular or genetic markers which can be used as predictors of outcome. Indeed, the genetic events that contribute to the pathogenesis of ependymoma are essentially unknown. We have used human oligonucleotide arrays to generate gene expression profiles in 10 ependymoma samples from patients with different histopathological/clinical parameters in order to identify new prognostic markers. Our sample group is composed of 7 ependymoma (WHO grade II) and 3 anaplastic ependymoma (WHO grade III). Three patients were <3 years of age at first presentation. Five tumours have chromosomal aberrations identified by comparative genomic hybridisation (CGH). Our preliminary data show that overexpression of specific functional categories of genes is dependant on histology when compared to normal controls. Cell cycle and adhesion related genes, oncogenes, and genes involved in apoptosis were mainly overexpressed in anaplastic tumours. Benign tumours, however, overexpressed mainly growth factor related genes. Some common candidates emerged for all tumours; Wee1+, a cell cycle related gene that regulates entry into mitosis, was up to six fold overexpressed in tumours. The oncogene c-myc, which maps to an amplicon at 8q24 detected by CGH in a subset of ependymomas, was also overexpressed in some tumours and may be an interesting candidate in their development. To our knowledge, none of these genes have been associated previously with this class of brain tumours. Further analysis of differential gene expression profiles using large series of tumours will help in the identification of molecular markers. This information, when linked to clinical and pathology data, could also help in the classification of these tumours and the choice of therapy.
    • Abnormal gene expression can be linked to chromosomal gains and losses in paediatric astrocytoma

      Potter, N.; Poh, R.; Ward, Samantha; Phipps, Kim; Hayward, Richard; Harkness, William; Thompson, Dominic; Thomas, David G.; Rees, J.; Darling, John L.; Warr, Tracy (Society for Neuro-Oncology and Duke University Press, 2006)
      Brain tumors are the most frequently found solid tumor in children, 40% of which are astrocytomas. These are graded according to the WHO classification into the more common low-grade (I and II) and high-grade (III and IV) tumors. Little is known about the genetic basis underlying the development of pediatric astrocytomas. In this study, we have studied the correlation between abnormal gene expression in pediatric astrocytoma with genomic copy number changes. We used the Affymetrix HGU133A array to identify differentially expressed genes in a group of pediatric astrocytoma short-term cell cultures comprising 9 grade I, 11 grade II and 12 grade IV tumors. Data analysis was carried out using Genespring version 6.0 software. In addition, we used the Spectral Chip 2600 to generate array-comparative genomic hybridization (aCGH) profiles of each short-term cell culture. Chromosome regions of gain and loss were then compared with differential gene expression using Formatter software. Hierarchical clustering of the short-term cultures according to expression profile similarity showed that the tumors clustered into 3 clear groups that were independent of grade. Two groups were predominantly low-grade tumors, comprised of a mixture of grade I and II tumors with 3 grade IV tumors, and the third group contained predominantly high-grade tumors with 2 low-grade tumors. Genes involved in the phosphatidylinositol signaling system, the cell cycle pathway, and the regulation of the actin cytoskeleton, were significantly differentially expressed between the 3 groups. Differential disruption of these cell pathways may be associated with subtypes of pediatric astrocytoma. Most tumors in the third group (including the low-grade tumors) showed copy number changes that can be correlated with changes in gene expression. In specific tumors, the downregulation of TSB1 (thrombospondin-1) correlated with loss at 15q15. This gene has previously been found to be downregulated in astrocytoma and is involved in cell adhesion. This finding suggests that gene expression in a subset of pediatric astrocytomas is influenced by gene dosage.