Gene expression profiles of human glioblastomas are associated with both tumor cytogenetics and histopathology

Ana Lusa Vital 0 1 3 4 5 Maria Dolores Tabernero 0 1 3 4 Abel Castrillo 1 3 4 Olinda Rebelo 1 3 4 Hermnio Ta o 1 3 4 Fernando Gomes 1 3 4 Ana Belen Nieto 1 3 4 Catarina Resende Oliveira 1 3 4 Maria Celeste Lopes 1 2 3 4 Alberto Orfao 1 2 3 4 0 Both authors should be considered as first author 1 Service (H.T. , F.G.), University Hospital of Coimbra , Coimbra, Portugal 2 Both authors have equally contributed to this work and they should be both considered as last authors 3 University of Coimbra , Coimbra, Portugal ; Research Unit of University Hospital of Salamanca , Salamanca 4 Center for Neuroscience and Cell Biology (A.L.V. , C.R.O., M.C.L.), Faculty of Pharmacy (A.L.V. , M.C.L.) 5 PhD Programme on Experimental Biology and Biomedicine (PDBEB), Center for Neurosciences and Cell Biology, University of Coimbra , Coimbra, Portugal Spain (M.D.T., A.C.); Center for Cancer Research (CIC IBMCC-CSIC/USAL), Salamanca, Spain (M.D.T., A.B.N., A.O.); IECSCYL, Spain (M.D.T.); Neuropathology Laboratory, Neurology Service (O.R.), Neurosurgery and tumor histopathology; additionally, among grade IV astrocytoma, GEP are significantly associated with the cytogenetic profile of the ancestral tumor cell clone. Further studies in larger series of patients are necessary to confirm our observations. - Despite the increasing knowledge about the genetic alterations and molecular pathways involved in gliomas, few studies have investigated the association between the gene expression profiles (GEP) and both cytogenetics and histopathology of gliomas. Here, we analyzed the GEP (U133Plus2.0 chip) of 40 gliomas (35 astrocytic tumors, 3 oligodendrogliomas, and 2 mixed tumors) and their association with tumor cytogenetics and histopathology. Unsupervised and supervised analyses showed significantly different GEP in low- vs high-grade gliomas, the most discriminating genes including genes involved in the regulation of cell proliferation, apoptosis, DNA repair, and signal transduction. In turn, among glioblastoma multiforme (GBM), 3 subgroups of tumors were identified according to their GEP, which were closely associated with the cytogenetic profile of their ancestral tumor cell clones: (i) EGFR amplification, (ii) isolated trisomy 7, and (iii) more complex karyotypes. In summary, our results show a clear association between the GEP of gliomas Iinformation has accumulated about the cytogenetic n the last decades an increasingly high amount of characteristics of human gliomas.1 3 Although different and unique cytogenetic profiles have been described in low- vs high-grade gliomas, no clearly defined cytogenetic subgroups have been further defined among the most frequent tumor subtypeseg, glioblastoma multiforme (GBM). Because of this, more recent attempts have been made to search for new relevant molecular markers that could be of help for a better subclassification of low- and high-grade gliomas and the identification of putative new therapeutic targets.4 6 Until now, such efforts have mainly concentrated on genes that are specifically altered in a significant fraction of all gliomas, particularly among high-grade tumors (eg, EGFR, PTEN, TP53, CDKN2A, MDM2, and PDGFA genes) and their potential association with specific intracellular signaling pathways such as those involving STAT3, MAPK, and AKT;7 10 however, these studies have only been partially successful.11 13 In recent years, the availability of large-scale genomic approaches and new bioinformatic tools has fostered the identification of molecular profiles that could be characteristic of human gliomas and define specific subgroups of GBM. Thus, it has been shown that the patterns of gene expression of gliomas correlate with tumor histopathology14 18 and patient survival19,20 and gene expression profiles (GEP) emerge in some studies as an independent prognostic factor among high-grade gliomas.20 According to these studies, most informative genes include genes involved in cell proliferation, RNA processing, signal transduction, and the proteasome functional activity.18 However, no clear association between the GEP of gliomas and tumor cytogenetics has been clearly established so far, which could help on the understanding of the various GEP described. In a recent paper21 using multicolor interphase fluorescence in situ hybridization (iFISH), we showed the existence of highly variable and heterogeneous intratumoral patterns of cytogenetic abnormalities in human gliomas; such cytogenetic profiles are typically characterized by complex karyotypes consisting of combined gains of chromosome 7, del(9p21)/null 9p, and/or del(10q23) in association with a variable number of other more heterogeneous cytogenetic changes; interestingly, the acquisition of these cytogenetic lesions followed different clonal pathways.21 In this regard, it should be noted that different pathways of intratumoral cytogenetic evolution were found in low- vs high-grade gliomas, which further suggests that the latter tumors might not always correspond to more advanced stages of histologically low-grade gliomas. Even more interestingly, different recent pathways of intratumoral clonal evolution were defined within GBM.21 On the basis of these results, the potential impact of these unique cytogenetic pathways in tumor behavior through the expression of different GEP remains to be elucidated. The aim of the present study was to identify whether the reported cytogenetic heterogeneity of human gliomasparticularly that of high-grade tumorstranslates into the existence of different GEP, which could contribute to a better understanding of the behavior of the tumor and patient outcome. Overall, our results based on a pilot series of 40 gliomas confirm the existence of a clear association between GEP of gliomas and tumor histopathology; in addition, for the first time we show that among grade IV astrocytomas, GEP are significantly associated with the cytogenetic profile of the ancestral tumor cell clone, further supporting its potential impact on the outcome of the disease. Materials and Methods Patients and Samples A total of 40 consecutive adult patients22 males (55%) and 18 females (45%)diagnosed with primary gliomas were studied. Mean age at diagnosis was 57 + 17 years (range: 21 84 years). In all cases, resected tumor samples were obtained by conventional surgical procedures, after informed consent was given by the patient, according to the recommendations of the local Ethics Committee of the University Hospital of Coimbra (Coimbra, Portugal). For each case, the most relevant histopathological, clinical, and biological features of the disease were recorded, including patient age, gender, performance status (Karnofsky index), and tumor localization (Table 1). At the moment of closing this study, 24 patients (60%) had died and 7 had relapsed after a median follow-up of 15 and 18 months, respectively. Tumor Histopathology Histopathological studies included assessment of both the hi (...truncated)