Section 4 -

Into the 21ST Century: Genomics and the New Mouse Models Synergistically Advancing Glioma Research Gregory N. Fuller Department of Pathology The University of Texas M. D. Anderson Cancer Center Houston , Texas

Introduction Brain tumor pathology is a rapidly evolving field, with new clinicopathologic entities (Table 1), new diagnostic antibodies (Table 2), and new molecular diagnostic tests (Table 3) continually emerging; frequent continuing medical education in this area is essential. Gliomas constitute the most common type of primary brain tumor. As with other types of brain tumors, our knowledge of the gliomas continues to expand, including newly characterized subtypes, improvements in glioma grading and attendant patient stratification for prognostic and treatment purposes, and also in our understanding of basic glioma pathobiology. Table 1. 10 New Types of Brain Tumor Described Over the Past 25 Years* Tumor Type Year Described Pleomorphic xanthoastrocytoma 1979 Central neurocytoma 1982 Dysembryoplastic neuroepithelial tumor 1988 Chordoid glioma 1998 Papillary glioneuronal tumor 1998-99 Rosetted glioneuronal tumor 1998-99 Pilomyxoid astrocytoma 1999 Rosette-forming glioneuronal tumor of the 4th ventricle 2002 Papillary tumor of the pineal region 2003 Monomorphous angiocentric bipolar glioma 2005 Table 2. Recently Introduced Diagnostic Antibodies for Brain Tumors Antibody Neoplasm(s) Inhibin alpha Hemangioblastoma INI1/SMARC5/BAF47 Rhabdoid and Atypical Teratoid/Rhabdoid Tumor OCT4 Germinoma and Embryonal Carcinoma Phospho-histone H3 Proliferation Marker; Diffuse Gliomas, Meningiomas Table 3. Potentially Useful Glioma Molecular Markers Currently Under Study* Chromosome 1p and 19q deletion status O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status Chromosome 7 Chromosome 9p Chromosome 10q EGFR gene amplification/overexpression EGFRvIII PTEN TP53 P16INK4a *Fuller GN, Aldape KD, Molecular genetics and neuropathology of intracranial tumors, In: M D Anderson Cancer Care Series: Tumors of the Brain and Spinal Cord. New York : Springer-Verlag, 2006. Molecular Classification of the Diffuse Gliomas The field of glioma classification is currently entering a new era with the introduction of new paradigms based on molecular information. Rather than supplanting traditional morphology-based classification schemes, it is anticipated that emerging molecular biologic, genomic, transcriptomic and proteomic data will complement and augment existing morphologic and immunophenotypic data, providing for a more accurate and refined stratification of glioma patients for directed therapies and for the resolution of several problematic issues inherent in histological classifications. Two different approaches are contributing to the improvement of glioma stratification. The first is the analysis of alterations of a small number of genes or gene products of recently demonstrated impact on patient survival and response to therapy, such as the deletion status of chromosomes 1p and 19q in oligodendroglial tumors, and O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation status in glioblastoma. The second paradigm is a more comprehensive analysis of the tumor genome, transcriptome or proteome, which may itself provide refined subclassification, or may identify specific relevant biomolecules for use in the single gene substratification approach. Both paradigms have already exerted a tangible and growing impact on glioma classification, and it is likely that we have only just begun to exploit their potential contributions. New Mouse Models of Diffuse Gliomas Historically, the most extensively studied glioma animal model has been the xenograft, in which human glioma tumor cells are injected into immunocompromised mice. The resultant tumors constitute good genotypic models because the tumor cells are human in origin. However, many human glioma cell lines in xenograft form exhibit only limited infiltration of the surrounding animal host brain parenchyma. Diffuse infiltration of brain tissue, which is a critical phenotypic feature of the diffuse gliomas that severely limits the effectiveness of many treatment modalities, can now be studied in new transgenic/somatic gene transfer mouse models in which different subtypes of diffuse glioma can be generated through the introduction of specific combinations of oncogenes. Using the RCAS/tva model, investigators have generated glioblastomas, low-grade astrocytomas, oligodendrogliomas, anaplastic oligodendrogliomas, and mixed gliomas. All of these gliomas are virtually identical to their human counterparts in terms of morphologic features, differentiation marker immunophenotype, and biologic behavior (see References for additional information about the RCAS/tva model). Synergy Between Genomics and Mouse Modeling in Advancing Glioma Research: The IGFBP2 Story Increasingly, investigators are combining the power of genomics technology with the new mouse models. One illustration of this approach is provided by the discovery and elucidation of the role of insulin-like growth factor binding protein 2 (IGFBP2) in glioma biology. IGFBP2 was initially shown to be overexpressed in 80% of glioblastomas by cDNA microarray expression profiling followed by tissue microarray confirmation. IGFBP2 overexpression was subsequently found to correlate with poor survival in diffuse gliomas. In vitro functional studies combined with transcriptome profiling provided evidence that IGFBP2 increases glioma cell migration and invasion by up-regulating the pathways responsible for those pathways. Finally, using the RCAS-tva mouse model system, IGFBP2 and several known oncogenes, including platelet-derived growth factor (PDGF), K-Ras, and Akt, were delivered separately and in combination into the developing mouse brain. The experimental results showed that chronic PDGF signaling leads exclusively to the formation of low-grade oligodendrogliomas; in contrast, PDGF delivered in combination with IGFBP2 results in anaplastic oligodendrogliomas formation, and combined activated K-Ras and Akt lead to the formation of astrocytomas. A similar paradigm is being exploited by investigators world-wide to advance our knowledge and ability to treat many different forms of cancer. References General Resources for Neuropathology and Genomics of Brain Tumors Burger PC and Scheithauer BW. Tumors of the Central Nervous System. AFIP Atlas of Tumor Pathology, 4th Series. Washington, DC: American Registry of Pathology, 2006. Fuller GN, Aldape KD, Molecular genetics and neuropathology of intracranial tumors, In: M D Anderson Cancer Care Series: Tumors of the Brain and Spinal Cord. New York : Springer-Verlag, 2006. Fuller GN, Burger PC: Central nervous system. In: Mills SE, ed. Histology for Pathologists. 3rd ed. New York : Raven Press; 2006. Kleihues P, Cavenee WK, eds. Pathology and Genetics of Tumours of the Nervous System. New York : Oxford University Press, 2000. (Revised WHO Classification scheduled for 2007) McLendon R, Rosenblum M, Bigner DD, eds. Russell & Rubinstein's Pathology of Tumors of the Nervous System, 7th ed. London: Arnold, 2006. Prayson RA. Neuropathology. Philadelphia : Elsevier, 2005. Zhang W and Fuller GN. Genomic and Molecular Neuro-Oncology. Boston : Jones and Bartlett Publishers, 2004. Molecular Classification of Brain Tumors Freije WA, Castro-Vargas FE, Fang Z, Horvath S, Cloughesy T, Liau LM, Mischel PS, Nelson SF. Gene expression profiling of gliomas strongly predicts survival. Cancer Res. 2004;64:6503-6510. Cancer Res 2003;63:6613-6625. Fuller GN: Molecular classifications. In: Barnett GH, ed. Current Clinical Oncology: High-Grade Gliomas: Diagnosis and Treatment. Totawa: Humana Press; 2006. Fuller GN, Hess, KR, Rhee, CH, Yung WKA, Sawaya RA, Bruner JM, Zhang W. Molecular classification of human diffuse gliomas by multidimensional scaling analysis of gene expression profiles parallels mophology-based classification, correlates with survival, and reveals clinically-relevant novel glioma subsets. Brain Pathology 2002;12:108-116. Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M, Kros JM, Hainfellner JA, Mason W, Mariani L, Bromberg JE, Hau P, Mirimanoff RO,Cairncross JG, Janzer RC, Stupp R. MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med 2005;352:997-1003. Iwadate Y, et al. Molecular classification and survival prediction in human gliomas based on proteome analysis. Cancer Res 2004;64:2496-2501. Jeuken JW, von Deimling A, Wesseling P. Molecular pathogenesis of oligodendroglial tumors. J Neurooncol 2004;70:161-81. Jeuken JW, Sprenger SH, Boerman RH, von Deimling A, Teepen HL, van Overbeeke JJ,Wesseling P. Subtyping of oligo-astrocytic tumours by comparative genomic hybridization. J Pathol 2001;194:81-87. McDonald JM, Colmam H, Perry A, Aldape K. Molecular and clinical aspects of 1p/19q loss in oligodendroglioma. In: Zhang W, Fuller GN, eds. Genomic and Molecular Neuro-Oncology. Boston:Jones and Bartlett, 2004:17-30. Mischel PS, Cloughesy TF, Nelson SF. DNA-microarray analysis of brain cancer: molecular classification for therapy. Nat Rev Neurosci. 2004;5:782-792. Nigro JM, Misra A, Zhang L, Smirnov I, Colman H, Griffin C, Ozburn N, Chen M,Pan E, Koul D, Yung WK, Feuerstein BG, Aldape KD. Integrated array-comparative genomic hybridization and expression array profiles identify clinically relevant molecular subtypes of glioblastoma. Cancer Res 2005;65:1678-1686. Nutt CL, et al. Gene expression-based classification of malignant gliomas correlates better with survival than histological classification. Cancer Res 2003;63:1602-1607. Reifenberger G, Louis DN. Oligodendroglioma: toward molecular definitions in neuro-oncology. J Neuropathol Exp Neurol 2003;62:111-126. Roerig P, Nessling M, Radlwimmer B, Joos S, Wrobel G, Schwaenen C, Reifenberger G, Lichter P. Molecular classification of human gliomas using matrix-based comparative genomic hybridization. Int J Cancer 2005;117:95-103. Recently Introduced Diagnostic Antibodies Colman H, Giannini C, Huang L, Gonzalez J, Hess K, Bruner J, Fuller G, Langford L, Pelloski C, Aaron J, Burger P, Aldape K. Assessment and prognostic significance of mitotic index using the mitosis marker phospho-histone H3 in low-intermediate grade infiltrating astrocytomas. Amer J Surg Pathol 2006 30:657-64 Ribalta T, McCutcheon IE, Aldape KD, Bruner JM, Fuller GN. The mitosis-specific antibody anti-phosphohistone-H3 (PHH3) facilitates rapid reliable grading of meningiomas according to WHO 2000 criteria. Am J Surg Pathol. 2004;28:1532-1536. Sung M-T, et al. OCT4 is superior to CD30 in the diagnosis of metastatic embryonal carcinomas after chemotherapy. Human Pathology 2006;37:662-667. Hattab EM, Tu PH, Wilson JD, Cheng L. OCT4 immunohistochemistry is superior to placental alkaline phosphatase (PLAP) in the diagnosis of central nervous system germinoma. Am J Surg Pathol. 2005;29:368-71. Jones TD, Ulbright TM, Eble JN, Cheng L. OCT4: A sensitive and specific biomarker for intratubular germ cell neoplasia of the testis. Clin Cancer Res. 2004; 10:8544-7. Jung SM, Kuo TT. Immunoreactivity of CD10 and inhibin alpha in differentiating hemangioblastoma of central nervous system from metastatic clear cell renal cell carcinoma. Mod Pathol. 2005;18:788-94. Hoang MP, Amirkhan RH. Inhibin alpha distinguishes hemangioblastoma from clear cell renal cell carcinoma. Am J Surg Pathol. 2003;27:1152-6. Perry A, Fuller CE, Judkins AR, Dehner LP, Biegel JA. INI1 expression is retained in composite rhabdoid tumors, including rhabdoid meningiomas. Mod Pathol. 2005;18:951-8. Sigauke E, Rakheja D, Maddox DL, Hladik CL, White CL, Timmons CF, Raisanen J. Absence of expression of SMARCB1/INI1 in malignant rhabdoid tumors of the central nervous system, kidneys and soft tissue: an immunohistochemical study with implications for diagnosis. Mod Pathol. 2006;19:717-25. RCAS/tva Mouse Model for Diffuse Gliomas Websites: http://www.retrovirus.info/RCAS/mice.html http://rex.nci.nih.gov/RESEARCH/basic/varmus/tva-web/tva2.html Dai C, Lyustikman Y, Shih A, Hu X, Fuller GN, Rosenblum M, Holland EC. The characteristics of astrocytomas and oligodendrogliomas are caused by two distinct and interchangeable signaling formats. Neoplasia. 2005;7(4):397-406. Begemann M, Fuller GN, Holland EC. Genetic modeling of glioma formation in mice. Brain Pathol. 2002;12:117-32. Holland EC, Celestino J, Dai C, Schaefer L, Sawaya RA, Fuller GN. Combined activation of Ras and Akt in neural progenitors induces glioblastoma formation in mice. Nature Genetics 25:55-57, 2000.