Tumors of the Central Nervous System, Volume 5: Astrocytomas, Hemangioblastomas, and Gangliogliomas

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Author: M.A. Hayat

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Tumors of the Central Nervous System

Tumors of the Central Nervous System Volume 5

For other titles published in this series, go to www.springer.com/series/8812

Tumors of the Central Nervous System Volume 5

Tumors of the Central Nervous System Astrocytomas, Hemangioblastomas, and Gangliogliomas Edited by

M.A. Hayat Distinguished Professor Department of Biological Sciences, Kean University, Union, NJ, USA

123

Editor M.A. Hayat Department of Biological Sciences Kean University Union, NJ, USA [email protected]

ISBN 978-94-007-2018-3 e-ISBN 978-94-007-2019-0 DOI 10.1007/978-94-007-2019-0 Springer Dordrecht Heidelberg London New York Library of Congress Control Number: 2011936737 © Springer Science+Business Media B.V. 2012 No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

“Although touched by technology, surgical pathology always has been, and remains, an art. Surgical pathologists, like all artists, depict in their artwork (surgical pathology reports) their interactions with nature: emotions, observations, and knowledge are all integrated. The resulting artwork is a poor record of complex phenomena.” Richard J. Reed MD

Preface

It is recognized that scientific journals and books not only provide current information but also facilitate exchange of information, resulting in rapid progress in the medical field. In this endeavor, the main role of scientific books is to present current information in more detail after careful additional evaluation of the investigational results, especially those of new or relatively new therapeutic methods and their potential toxic side-effects. Although subjects of diagnosis, drug development, therapy and its assessment, and prognosis of tumors of the central nervous system, cancer recurrence, and resistance to chemotherapy are scattered in a vast number of journals and books, there is need of combining these subjects in single volumes. An attempt will be made to accomplish this goal in the projected ten-volume series of handbooks. In the era of cost-effectiveness, my opinion may be minority perspective, but it needs to be recognized that the potential for false-positive or false-negative interpretation on the basis of a single laboratory test in clinical pathology does exist. Interobservor or intraobservor variability in the interpretation of results in pathology is not uncommon. Interpretative differences often are related to the relative importance of the criteria being used. Generally, no test always performs perfectly. Although there is no perfect remedy to this problem, standardized classifications with written definitions and guidelines will help. Standardization of methods to achieve objectivity is imperative in this effort. The validity of a test should be based on the careful, objective interpretation of the tomographic images, photo-micrographs, and other tests. The interpretation of the results should be explicit rather than implicit. To achieve accurate diagnosis and correct prognosis, the use of molecular criteria and targeted medicine is important. Equally important are the translation of molecular genetics into clinical practice and evidence-based therapy. Translation of medicine from the laboratory to clinical application needs to be carefully expedited. Indeed, molecular medicine has arrived. This is the fifth volume in the series, Tumors of the Central Nervous System. As in the case of the four previously published volumes, this volume mainly contains information on the diagnosis, therapy, and prognosis of brain tumors.Various aspects of three types of brain tumors (Astrocytomas, Hemangioblastoma and Ganglioglioma) are discussed. Insights into the understanding of molecular pathways involved in tumor biology are explained, which lead to the development of effective drugs. Information on pathways facilitates targeted therapies in cancer. Tumor models are also presented, which utilize expression data, pathway sensitivity, and genetic abnormalities, representing targets in cancer. vii

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Preface

Advantages and limitations of chemotherapy (e.g., Cisplatin/carboplatin combination) for patients with pilomyxoid astrocytoma are discussed. Identification and characterization of biomarkers, including those for metastatic brain tumors, are presented. Genomic analyses for identifying clinically relevant subtypes are included. A number of imaging modalities, including time-resolved laser fluorescence spectroscopy and magnetic resonance- guided laser interstitial thermal therapy are detailed to diagnose and treat brain tumors. Introduction to new technologies and their applications to tumor diagnosis, treatment, and therapy assessment are explained. For example, nanotechnology-based therapy for malignant tumors of the CNS is explained. Molecular profiling of brain tumors to select therapy in clinical trials of brain tumors is included. Several surgical treatments, including resection, and radiosurgery, are discussed. The remaining two volumes in this series will provide additional recent information on this and other aspects of other types of CNS malignancies. By bringing together a large number of experts (oncologists, neurosurgeons, physicians, research scientists, and pathologists) in various aspects of this medical field, it is my hope that substantial progress will be made against this terrible disease. It would be difficult for a single author to discuss effectively the complexity of diagnosis, therapy, and prognosis of any type of tumor in one volume. Another advantage of involving more than one author is to present different points of view on a specific controversial aspect of the CNS cancer. I hope these goals will be fulfilled in this and other volumes of this series. This volume was written by 85 contributors representing 14 countries. I am grateful to them for their promptness in accepting my suggestions. Their practical experience highlights their writings, which should build and further the endeavors of the reader in this important area of disease. I respect and appreciate the hard work and exceptional insight into the nature of cancer provided by these contributors. The contents of the volume are divided into seven subheadings: Introduction, Diagnosis and Biomarkers, Therapy, Tumor to tumor cancer, Imaging methods, Prognosis, and Quality of life for the convenience of the reader. It is my hope that the current volume will join the preceding volumes of the series for assisting in the more complete understanding of globally relevant cancer syndromes. There exist a tremendous, urgent demand by the public and the scientific community to address to cancer, diagnosis, treatment, cure, and hopefully prevention. In the light of existing cancer calamity, government funding must give priority to eradicating this deadly malignancy over military superiority. I am thankful to Dr. Dawood Farahi and Dr. Kristie Reilly for recognizing the importance of medical research and publishing through an institution of higher education. Union, New Jersey April 2011

M.A. Hayat

Contents

Part I

Astrocytomas: Diagnosis and Biomarkers . . . . . . . . . . . .

1

1 Methylation in Malignant Astrocytomas . . . . . . . . . . . . . . . . María del Mar Inda, Juan A. Rey, Xing Fan, and Javier S. Castresana

3

2 Deciphering the Function of Doppel Protein in Astrocytomas . . . . Alberto Azzalin and Sergio Comincini

13

3 Astrocytic Tumors: Role of Antiapoptotic Proteins . . . . . . . . . . Alfredo Conti, Carlo Gulì, Giuseppe J. Sciarrone, and Chiara Tomasello

23

4 Deregulation of the Wnt/β-Catenin/Tcf Signaling Pathway in Astrocytomas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gangadhara Reddy Sareddy and Phanithi Prakash Babu

35

5 Subependymal Giant Cell Astrocytoma: Role of mTOR Pathway and Its Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . Katarzyna Kotulska and Sergiusz Jó´zwiak

45

6 Role of Progesterone Receptor Isoforms in Human Astrocytomas Growth . . . . . . . . . . . . . . . . . . . . . . . . . . Ignacio Camacho-Arroyo, Valeria Hansberg-Pastor, Edith Cabrera-Muñoz, Olivia Tania Hernández-Hernández, and Aliesha González-Arenas 7 Astrocytic Tumors: Role of Carbonic Anhydrase IX . . . . . . . . . Joonas Haapasalo, Hannu Haapasalo, and Seppo Parkkila 8 Development of Cysts in Pilocytic Astrocytomas: Role of Eosinophilic Granular Bodies (Method) . . . . . . . . . . . . . . . Jai-Nien Tung, Tang-Yi Tsao, Kun-Tu Yeh, Ching-Fong Liao, and Ming-Chung Jiang 9 Role of Synemin in Astrocytoma Cell Migration . . . . . . . . . . . . Quincy Quick, Yihang Pan, and Omar Skalli 10 Diffuse Astrocytomas: Immunohistochemistry of MGMT Expression . . . . . . . . . . . . . . . . . . . . . . . . . . David Capper

57

65

73

81

89

ix

x

11

12

13

14

15

16

Contents

Central Nervous System Germ Cell Tumors: An Epidemiology Review . . . . . . . . . . . . . . . . . . . . . . . . Daniel L. Keene and Donna Johnston

95

RAF Fusion Genes and MAPK Activation in Pilocytic Astrocytomas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sally R. Lambert and David T.W. Jones

99

Biomarker Discovery in Central Nervous System Neoplasms: Past, Present and Future . . . . . . . . . . . . . . . . . . Anne F. Buckley, Roger E. McLendon, Carol J. Wikstrand, and Darell D. Bigner Astrocytomas: Role of Taurine in Apoptosis Using Magnetic Resonance Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . Kirstie S. Opstad

121

Imaging of Hypoxia-Inducible Factor-1-Active Regions in Tumors Using a POS and 123 I-IBB Method . . . . . . . . . . . . . Masashi Ueda and Hideo Saji

129

Diffuse Low-Grade Astrocytomas: P53-Mediated Inhibition of Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Timo Gaiser and Markus D. Siegelin

135

17

Spontaneous Regression of Cerebellar Astrocytomas . . . . . . . . . Mansoor Foroughi, Shibu Pillai, and Paul Steinbok

18

Subependymal Giant Cell Astrocytoma: Gene Expression Profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Magdalena Ewa Tyburczy and Bozena Kaminska

Part II 19

20

21

22

23

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Astrocytomas: Therapy . . . . . . . . . . . . . . . . . . . . . .

Time-Resolved Laser Induced Fluorescence Spectroscopy (TRLIFS): A Tool for Intra-operative Diagnosis of Brain Tumors and Maximizing Extent of Surgical Resection . . . . . . . . Pramod Butte and Adam N. Mamelak

143

149

159

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Magnetic Resonance-Guided Laser Interstitial Thermal Therapy for Brain Tumors . . . . . . . . . . . . . . . . . . . . . . . . Kevin Beccaria, Michael S. Canney, and Alexandre C. Carpentier

173

Nanotechnology-Based Therapy for Malignant Tumors of the Central Nervous System . . . . . . . . . . . . . . . . . . . . . Abraham Boskovitz, Abdullah Kandil, and Al Charest

187

Pilocytic Astrocytoma: Pathological and Immunohistochemical Factors Affecting Surgical Treatment and Surveillance . . . . . . . . . . . . . . . . . . Devon Haydon and Jeffrey Leonard Pilomyxoid Astrocytomas: Chemotherapy . . . . . . . . . . . . . . . Hitoshi Tsugu, Shinya Oshiro, Fuminari Komatsu, Hiroshi Abe, Takeo Fukushima, Tooru Inoue, Fumio Yanai, and Yuko Nomura

195 203

Contents

xi

Part III

Astrocytomas: Prognosis . . . . . . . . . . . . . . . . . . . . .

211

24 Astrocytomas: Predicting Survival and Recurrence Using Cerebral Blood Volume Measurements . . . . . . . . . . . . . . . . . Sotirios Bisdas

213

25 Electronic Patient-Reported Outcome Monitoring (ePROM) in Brain Tumour Patients . . . . . . . . . . . . . . . . . . . . . . . . Lisa M. Wintner, Johannes M. Giesinger, Gabriele Schauer-Maurer, and Bernhard Holzner Part IV

223

Hemangioblastoma . . . . . . . . . . . . . . . . . . . . . . . .

231

26 Intra-operative ICG Use in the Management of Hemangioblastomas . . . . . . . . . . . . . . . . . . . . . . . . . . Loyola V. Gressot and Steven W. Hwang

233

27 Hemangioblastoma Cysts: Diagnosis Using Fluorescence with 5-Aminolevulinic Acid . . . . . . . . . . . . . . . . . . . . . . . Satoshi Utsuki, Hidehiro Oka, and Kiyotaka Fujii

239

28 Hemangioblastoma: Stereotactic Radiosurgery . . . . . . . . . . . . Anand Veeravagu, Bowen Jiang, and Steven D. Chang

245

Part V

Ganglioglioma . . . . . . . . . . . . . . . . . . . . . . . . . . .

251

29 Gangliogliomas: Molecular Pathogenesis and Epileptogenesis . . . . Eleonora Aronica and Pitt Niehusmann

253

30 Epilepsy-Associated Gangliogliomas: Identification of Genes with Altered Expression . . . . . . . . . . . . . . . . . . . . . . . . . Albert J. Becker

267

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

275

Contents of Volume 1

1 Introduction 2 Molecular Classification of Gliomas 3 Glioblastoma: Endosialin Marker for Pericytes 4 Glioma Grading Using Cerebral Blood Volume Heterogeneity 5 The Role of Ectonucleotidases in Glioma Cell Proliferation 6 Gliomas: Role of Monoamine Oxidase B in Diagnosis 7 Glioma: Role of Integrin in Pathogenesis and Therapy 8 Proton Magnetic Resonance Spectroscopy in Intracranial Gliomas 9 Infiltration Zone in Glioma: Proton Magnetic Resonance Spectroscopic Imaging 10 Malignant Gliomas: Role of E2F1 Transcription Factor 11 The Role of Glucose Transporter-1 (GLUT-1) in Malignant Gliomas 12 Malignant Gliomas: Role of Platelet-Derived Growth Factor Receptor A (PDGFRA) 13 Molecular Methods for Detection of Tumor Markers in Glioblastomas 14 Role of MGMT in Glioblastomas 15 Glioblastomas: Role of CXCL12 Chemokine 16 Cell Death Signaling in Glioblastoma Multiforme: Role of the Bcl2L12 Oncoprotein 17 Glioblastoma Multiforme: Role of Polycomb Group Proteins 18 Glioblastoma Multiforme: Role of Cell Cycle-Related Kinase Protein (Method) 19 Markers of Stem Cells in Gliomas 20 Efficient Derivation and Propagation of Glioblastoma Stem-Like Cells Under Serum-Free Conditions Using the Cambridge Protocol

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21

Glioma Cell Lines: Role of Cancer Stem Cells

22

Glioblastoma Cancer Stem Cells: Response to Epidermal Growth Factor Receptor Kinase Inhibitors

23

Low- and High-Grade Gliomas: Extensive Surgical Resection

24

Brainstem Gangliogliomas: Total Resection and Close Follow-Up

25

Glioblastoma: Temozolomide-Based Chemotherapy

26

Drug-Resistant Glioma: Treatment with Imatinib Mesylate and Chlorimipramine

27

Glioblastoma Multiforme: Molecular Basis of Resistance to Erlotinib

28

Enhanced Glioma Chemosensitivity

29

Malignant Glioma Patients: Anti-Vascular Endothelial Growth Factor Monoclonal Antibody, Bevacizumab

30

Aggravating Endoplasmic Reticulum Stress by Combined Application of Bortezomib and Celecoxib as a Novel Therapeutic Strategy for Glioblastoma

31

Targeted Therapy for Malignant Gliomas

32

Glioblastomas: HER1/EGFR-Targeted Therapeutics

33

Epidermal Growth Factor Receptor Inhibition as a Therapeutic Strategy for Glioblastoma Multiforme

34

Role of Acyl-CoA Synthetases in Glioma Cell Survival and Its Therapeutic Implication

35

Malignant Glioma Patients: Combined Treatment with Radiation and Fotemustine

36

Malignant Glioma Immunotherapy: A Peptide Vaccine from Bench to Bedside

37

Malignant Glioma: Chemovirotherapy

38

Intracranial Glioma: Delivery of an Oncolytic Adenovirus

39

Use of Magnetic Resonance Spectroscopy Imaging (MRSI) in the Treatment Planning of Gliomas

40

Malignant Glioma Cells: Role of Trail-Induced Apoptosis

41

Long-Term Survivors of Glioblastoma

42

Glioblastoma Patients: p15 Methylation as a Prognostic Factor


1 Introduction 2 Gliomagenesis: Advantages and Limitations of Biomarkers 3 Molecular Subtypes of Gliomas 4 Glioblastoma: Germline Mutation of TP53 5 Familial Gliomas: Role of TP53 Gene 6 The Role of IDH1 and IDH2 Mutations in Malignant Gliomas 7 Malignant Glioma: Isocitrate Dehydrogenases 1 and 2 Mutations 8 Metabolic Differences in Different Regions of Glioma Samples 9 Glioblastoma Patients: Role of Methylated MGMT 10 Brain Tumor Angiogenesis and Glioma Grading: Role of Tumor Blood Volume and Permeability Estimates Using Perfusion CT 11 Vasculogenic Mimicry in Glioma 12 Newly Diagnosed Glioma: Diagnosis Using Positron Emission Tomography with Methionine and Fluorothymidine 13 Role of Diffusion Tensor Imaging in Differentiation of Glioblastomas from Solitary Brain Metastases 14

131 I-TM-601

SPECT imaging of Human Glioma

15 Assessment of Biological Target Volume Using Positron Emission Tomography in High-Grade Glioma Patients 16 Skin Metastases of Glioblastoma 17 Diffuse Low-Grade Gliomas: What Does “Complete Resection” Mean? 18 Quantitative Approach of the Natural Course of Diffuse Low-Grade Gliomas 19 Impact of Extent of Resection on Outcomes in Patients with High-Grade Gliomas

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20

Glioma Surgery: Intraoperative Low Field Magnetic Resonance Imaging

21

Low-Grade Gliomas: Intraoperative Electrical Stimulations

22

Malignant Gliomas: Present and Future Therapeutic Drugs

23

Recurrent Malignant Glioma Patients: Treatment with Conformal Radiotherapy and Systemic Therapy

24

Glioblastoma: Boron Neutron Capture Therapy

25

Glioblastoma: Anti-tumor Action of Cyclosporin A and Functionally Related Drugs

26

Glioblastoma Patients: Chemotherapy with Cisplatin, Temozolomide and Thalidomide

27

Glioblastoma: Role of Galectin-1 in Chemoresistance

28

Glioma-Initiating Cells: Interferon Treatment

29

Glioblastoma: Anti-tumor Action of Natural and Synthetic Cannabinoids

30

Patients with Recurrent High-Grade Glioma: Therapy with Combination of Bevacizumab and Irinotecan

31

Monitoring Gliomas In Vivo Using Diffusion-Weighted MRI During Gene Therapy-Induced Apoptosis

32

High-Grade Gliomas: Dendritic Cell Therapy

33

Glioblastoma Multiforme: Use of Adenoviral Vectors

34

Fischer/F98 Glioma Model: Methodology

35

Cellular and Molecular Characterization of Anti-VEGF and IL-6 Therapy in Experimental Glioma

36

Adult Brainstem Gliomas: Diagnosis and Treatment

37

The Use of Low Molecular Weight Heparin in the Treatment and Prevention of Thromboembolic Disease in Glioma Patients

38

Brainstem Gliomas: An Overview

39

Tumor-Associated Epilepsy in Patients with Glioma

40

Brain Tumors Arising in the Setting of Chronic Epilepsy

41

Low-Grade Gliomas: Role of Relative Cerebral Blood Volume in Malignant Transformation

42

Angiocentric Glioma-Induced Seizures: Lesionectomy


1 Introduction 2 Brain Tumor Classification Using Magnetic Resonance Spectroscopy 3 Cellular Immortality in Brain Tumors: An Overview 4 Tumor-to-Tumor Metastasis: Extracranial Tumor Metastatic to Intracranial Tumors 5 Brain Metastases from Breast Cancer: Treatment and Prognosis 6 Brain Metastasis in Renal Cell Carcinoma Patients 7 Coexsistence of Inflammatory Myofibroblastic Tumor in the Lung and Brain 8 Breast Cancer and Renal Cell Cancer Metastases to the Brain 9 Breast Cancer Brain Metastases: Genetic Profiling and Neurosurgical Therapy 10 Central Nervous System Tumours in Women Who Received Capecitabine and Lapatinib Therapy for Metastatic Breast Cancer 11 Functional Role of the Novel NRP/B Tumor Suppressor Gene 12 Brain Tumors: Diagnostic Impact of PET Using Radiolabelled Amino Acids 13 Malignant Peripheral Nerve Sheath Tumors: Use of 18FDG-PET/CT 14 Brain Tumors: Evaluation of Perfusion Using 3D-FSEPseudo-Continuous Arterial Spin Labeling 15 Cerebral Cavernous Malformations: Advanced Magnetic Resonance Imaging 16 Nosologic Imaging of Brain Tumors Using MRI and MRSI 17 Brain Tumor Diagnosis Using PET with Angiogenic Vessel-Targeting Liposomes 18 Frozen Section Evaluation of Central Nervous System Lesions 19 Clinical Role of MicroRNAs in Different Brain Tumors

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20

Electrochemotherapy for Primary and Secondary Brain Tumors

21

Brain Tumors: Convection-Enhanced Delivery of Drugs (Method)

22

Brain Metastases: Clinical Outcomes for Stereotactic Radiosurgery (Method)

23

Noninvasive Treatment for Brain Tumors: Magnetic Resonance-Guided Focused Ultrasound Surgery

24

Radioguided Surgery of Brain Tumors

25

Implications of Mutant Epidermal Growth Factor Variant III in Brain Tumor Development and Novel Targeted Therapies

26

Endoscopic Port Surgery for Intraparenchymal Brain Tumors

27

Intracranial Tumor Surgery in Elderly Patients

28

Intracranial Hemangiopericytoma: Gamma Knife Surgery

29

Stereotactic Radiosurgery for Cerebral Metastases of Digestive Tract Tumors

30

Malignant Brain Tumors: Role of Radioresponsive Gene Therapy

31

Brain Tumors: Quality of Life

32

Health-Related Quality of Life in Patients with High Grade Gliomas

33

Epilepsy and Brain Tumours and Antiepileptic Drugs

34

Familial Caregivers of Patients with Brain Cancer

35

Pain Management Following Craniotomy

36

Air Transportation of Patients with Brain Tumours


1 Epidemiology of Primary Brain Tumors 2 Supratentorial Primitive Neuroectodermal Tumors 3 Epileptic Seizures and Supratentorial Brain Tumors in Children 4 Breast Cancer Metastasis to the Central Nervous System 5 Melanoma to Brain Metastasis: Photoacoustic Microscopy 6 Extraaxial Brain Tumors: The Role of Genetic Polymorphisms 7 Central Nervous System Germ Cell Tumor 8 Microvascular Gene Changes in Malignant Brain Tumors 9 Role of MicroRNA in Glioma 10 Glioblastoma Multiforme: Cryopreservation of Brain Tumor-Initiating Cells (Method) 11 Relationship Between Molecular Oncology and Radiotherapy in Malignant Gliomas (An Overview) 12 High-Grade Brain Tumours: Evaluation of New Brain Lesions by Amino Acid PET 13 Cyclic AMP Phosphodiesterase-4 in Brain Tumor Biology: Immunochemical Analysis 14 Molecular Imaging of Brain Tumours Using Single Domain Antibodies 15 Quantitative Analysis of Pyramidal Tracts in Brain Tumor Patients Using Diffusion Tensor Imaging 16 Differentiation Between Gliomatosis Cerebri and Low-Grade Glioma: Proton Magnetic Resonance Spectroscopy 17 Peripheral Nerve Sheath Tumors: Diagnosis Using Quantitative FDG-PET 18 Tumor Resection Control Using Intraoperative Magnetic Resonance Imaging

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19

Brain Tumors: Clinical Applications of Functional Magnetic Resonance Imaging and Diffusion Tensor Imaging

20

Trigeminal Neuralgia: Diagnosis Using 3-D Magnetic Resonance Multi-Fusion Imaging

21

Epilepsy-Associated Brain Tumors: Diagnosis Using Magnetic Resonance Imaging

22

Growth of Malignant Gliomas In Vivo: High-Resolution Diffusion Tensor Magnetic Resonance Imaging

23

Resection of Brain Lesions: Use of Preoperative Functional Magnetic Resonance Imaging and Diffusion Tensor Tractography

24

Paradigms in Tumor Bed Radiosurgery Following Resection of Brain Metastases

25

Rat Model of Malignant Brain Tumors: Implantation of Doxorubicin Using Drug Eluting Beads for Delivery

26

Electromagnetic Neuronavigation for CNS Tumors

27

Stereotactic Radiosurgery for Intracranial Ependymomas

28

Is Whole Brain Radiotherapy Beneficial for Patients with Brain Metastases?

29

Triggering Microglia Oncotoxicity: A Bench Utopia or a Therapeutic Approach?

30

Preoperative Motor Mapping

31

Intraoperative Monitoring for Cranial Base Tumors

32

Brain Tumours: Pre-clinical Assessment of Targeted, Site Specific Therapy Exploiting Ultrasound and Cancer Chemotherapeutic Drugs

33

Headaches in Patients with Brain Tumors

34

Headache Associated with Intracranial Tumors

35

Patients with Brain Cancer: Health Related Quality of Life

36

Emerging Role of Brain Metastases in the Prognosis of Breast Cancer Patients

Contributors

Hiroshi Abe Department of Neurosurgery, Fukuoka University Faculty of Medicine, Jonan-ku, Fukuoka 814-0180, Japan Eleonora Aronica Department of Neuropathology, Academic Medical Center, 1105 AZ, Amsterdam, The Netherlands, [email protected] Alberto Azzalin Institute of Molecular Genetics, IGM-CNR Pavia via Abbiategrasso 207 (OR via Ferrata 1), 27100 Pavia, Italy, [email protected] Phanithi Prakash Babu Department of Biotechnology, School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India, [email protected] Kevin Beccaria Department of Neurosurgery and Advanced Surgical Technologies Research Team, Hopital de la Pitie-Salpetriere, Assistance Publique Hopitaux de Paris, Université Paris VI – Pierre & Marie Curie, 75013 Paris, France Albert J. Becker Department of Neuropathology, University of Bonn Medical Center, D-53105 Bonn, Germany, [email protected] Darell D. Bigner Section(s) of Surgical Pathology, Duke University Medical Center, Durham, NC 27710, USA Sotirios Bisdas Department of Diagnostic and Interventional Neuroradiology, Karls Eberhard University, Tübingen, Germany, [email protected] Abraham Boskovitz Department of Neurosurgery, Tufts Medical Center, Tufts University, Boston, MA 02111, USA Anne F. Buckley Section(s) of Surgical Pathology, Duke University Medical Center, Durham, NC 27710, USA Pramod Butte Department of Neurosurgery, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA, [email protected] Edith Cabrera-Muñoz Facultad de Quimica, Universidad Nacional Autonoma de Mexico, Coyoacan, Mexico 04510, D.F. Mexico Ignacio Camacho-Arroyo Facultad de Quimica, Universidad Nacional Autonoma de Mexico, Coyoacan, Mexico 04510, D.F. Mexico, [email protected]

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Michael S. Canney Department of Neurosurgery and Advanced Surgical Technologies Research Team, Hopital de la Pitie-Salpetriere, Assistance Publique Hopitaux de Paris, Université Paris VI – Pierre & Marie Curie, 75013 Paris, France David Capper Department of Neuropathology, Institute of Pathology, Ruprecht-Karls-University, 69120 Heidelberg, Germany, [email protected] Alexandre C. Carpentier Department of Neurosurgery and Advanced Surgical Technologies Research Team, Hopital de la Pitie-Salpetriere, Assistance Publique Hopitaux de Paris, Université Paris VI – Pierre & Marie Curie, 75013 Paris, France, [email protected] Javier S. Castresana Unidad de Biologia de Tumores Cerebrales, Universidad de Navarra, 31008 Pamplona, Spain, [email protected] Steven D. Chang Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA 94305-5484, USA, [email protected] Al Charest Department of Neurosurgery, Tufts Medical Center, Tufts University, Boston, MA 02111, USA, [email protected] Sergio Comincini Department of Genetics and Microbiology, University of Pavia, via Abbiategrasso 207 (OR via Ferrata 1), 27100 Pavia, Italy Alfredo Conti Department of Neuroscience, University of Messina, Messina, Italy, [email protected] Xing Fan Department of Neurosurgery, University of Michigan Medical School, Ann Arbor, MI, USA Mansoor Foroughi Division of Neurosurgery, B.C.’s Children Hospital, Vancouver, BC, Canada Kiyotaka Fujii Department of Neurosurgery, Kitasato University School of Medicine, 1-15-1, Kitasato, Minami, Sagamihara, Kanagawa 252-0374, Japan Takeo Fukushima Department of Neurosurgery, Fukuoka University Faculty of Medicine, Jonan-ku, Fukuoka 814-0180, Japan Timo Gaiser University of Massachusetts, Amherst, MA LRB 460 E, USA; Pathology Mannheim, University Medical Center Mannheim, Theodor-Kutzer Ufer 1-3, 68167 Mannheim, Germany, [email protected] Johannes M. Giesinger Department of Psychiatry and Psychotherapy, Innsbruck Medical University, Anichstr.35, 6020 Innsbruck, Austria Aliesha González-Arenas Facultad de Quimica, Universidad Nacional Autonoma de Mexico, Coyoacan, Mexico 04510, D.F. Mexico Loyola V. Gressot Department of Neurosurgery, Baylor College of Medicine, Houston, TX, USA Carlo Gulì Departments of Neuroscience and Clinical Oncology, University of Messina, Messina, Italy Hannu Haapasalo Department of Pathology, Tampere University Hospital, FI-33521 Tampere, Finland, [email protected]

Contributors

Contributors

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Joonas Haapasalo Department of Pathology, Tampere University Hospital, FI-33521 Tampere, Finland Valeria Hansberg-Pastor Facultad de Quimica, Universidad Nacional Autonoma de Mexico, Coyoacan, Mexico 04510, D.F. Mexico Devon Haydon Department of Neurosurgery, Washington University School of Medicine, St. Louis, MO 63110, USA Bernhard Holzner Department of Psychiatry and Psychotherapy, Innsbruck Medical University, Anichstr.35, 6020 Innsbruck, Austria, [email protected] Steven W. Hwang Department of Neurosurgery, Tufts Medical Center, Boston, MA, USA, [email protected] María del Mar Inda Ludwig Institute for Cancer Research, San Diego, CA, USA Tooru Inoue Department of Neurosurgery, Fukuoka University Faculty of Medicine, Jonan-ku, Fukuoka 814-0180, Japan Bowen Jiang Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA 94305-5484, USA, [email protected] Ming-Chung Jiang Section of Haematology-Oncology, Department of Medicine, Taipei Medical University and Hospital, Taipei 110, Taiwan, [email protected] Donna Johnston Division of Neurology, Department of Pediatrics, Children’s Hospital of Eastern Ontario, Ottawa, ON, Canada K1H 8 David T.W. Jones Molecular Genetics of Pediatric Brain Tumors (B062), German Cancer Research Centre (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Sergiusz Jó´zwiak Klinika Neurologii I Epileptologii, Instytut Pomnik “Centrum Zdrowia Dziecka”, 04-730 Warszawa, Poland Bozena Kaminska Laboratory of Transcription Regulation, The Nencki Institute of Experimental Biology, University of Warsaw, Warsaw, Poland Abdullah Kandil Department of Neurosurgery, Tufts Medical Center, Tufts University, Boston, MA 02111, USA Daniel L. Keene Division of Neurology, Department of Pediatrics, Children’s Hospital of Eastern Ontario, Ottawa, ON, Canada K1H 8, [email protected] Fuminari Komatsu Department of Neurosurgery, Fukuoka University Faculty of Medicine, Jonan-ku, Fukuoka 814-0180, Japan Katarzyna Kotulska Klinika Neurologii I Epileptologii, Instytut Pomnik “Centrum Zdrowia Dziecka”, 04-730 Warszawa, Poland, [email protected] Sally R. Lambert Department of Pathology, University of Cambridge, Addenbrooke’s Hospital Box 231, Cambridge, CB2 0QQ, UK, [email protected] Jeffrey Leonard Department of Neurosurgery, Washington University School of Medicine, St. Louis, MO 63110, USA, [email protected]

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Ching-Fong Liao Section of Haematology-Oncology, Department of Medicine, Taipei Medical University and Hospital, Taipei 110, Taiwan Adam N. Mamelak Department of Biomedical Engineering, University of Southern California, Los Angeles, CA 90089, USA Roger E. McLendon Section(s) of Surgical Pathology, Duke University Medical Center, Durham, NC 27710, USA, [email protected] Pitt Niehusmann Department of Neuropathology, University of Bonn, Medical Center, Sigmund-Freud-Str. 25, 53105 Bonn, Germany, [email protected] Yuko Nomura Department of Neurosurgery, Fukuoka University Faculty of Medicine, Jonan-ku, Fukuoka 814-0180, Japan Hidehiro Oka Department of Neurosurgery, Kitasato University School of Medicine, 1-15-1, Kitasato, Minami, Sagamihara, Kanagawa 252-0374, Japan Kirstie S. Opstad Division of Clinical Sciences, St. George’s, University of London, Cranmer Terrace, London SW17 0RE, UK, [email protected] Shinya Oshiro Department of Neurosurgery, Fukuoka University Faculty of Medicine, Jonan-ku, Fukuoka 814-0180, Japan Yihang Pan Department of Cellular Biology and Anatomy, Louisiana State University Medical Center, Shreveport, LA 71103, USA Seppo Parkkila Department of Pathology, Tampere University Hospital, FI-33521 Tampere, Finland Shibu Pillai Division of Neurosurgery, B.C.’s Children Hospital, Vancouver, BC, Canada Quincy Quick Department of Cellular Biology and Anatomy, Louisiana State University Medical Center, Shreveport, LA 71103, USA Juan A. Rey Research Unit, La Paz University Hospital, Madrid, Spain Hideo Saji Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan, [email protected] Gangadhara Reddy Sareddy Department of Biotechnology, School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India Gabriele Schauer-Maurer Department of Psychiatry and Psychotherapy, Innsbruck Medical University, Anichstr.35, 6020 Innsbruck, Austria Giuseppe J. Sciarrone Departments of Neuroscience and Clinical Oncology, University of Messina, Messina, Italy Markus D. Siegelin Department of Pathology & Cell Biology, Columbia University College of Physicians & Surgeons, 630 W. 168th Street, New York, NY 10032, USA, [email protected] Omar Skalli Department of Cellular Biology and Anatomy, Louisiana State University Medical Center, Shreveport, LA 71103, USA, [email protected]

Contributors

Contributors

xxv

Paul Steinbok Division of Neurosurgery, B.C.’s Children Hospital, Vancouver, BC, Canada, [email protected] Olivia Tania Hernández-Hernández Facultad de Quimica, Universidad Nacional Autonoma de Mexico, Coyoacan, Mexico 04510, D.F. Mexico Chiara Tomasello Departments of Neuroscience and Clinical Oncology, University of Messina, Messina, Italy Tang-Yi Tsao Section of Haematology-Oncology, Department of Medicine, Taipei Medical University and Hospital, Taipei 110, Taiwan Hitoshi Tsugu Department of Neurosurgery, Fukuoka University Faculty of Medicine, Jonan-ku, Fukuoka 814-0180, Japan, [email protected] Jai-Nien Tung Section of Haematology-Oncology, Department of Medicine, Taipei Medical University and Hospital, Taipei 110, Taiwan Magdalena Ewa Tyburczy Translational Medicine Division, Brigham and Women’s Hospital, Boston, MA, [email protected] Masashi Ueda Radioisotopes Research Laboratory, Kyoto University Hospital, Sakyo-ku, Kyoto, 606-8507, Japan, [email protected] Satoshi Utsuki Department of Neurosurgery, Kitasato University School of Medicine, 1-15-1, Kitasato, Minami, Sagamihara, Kanagawa 252-0374, Japan, [email protected] Anand Veeravagu Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA 94305-5484, USA Carol J. Wikstrand Department of Microbiology, Saba University School of Medicine, Saba, Dutch Caribbean Lisa M. Wintner Department of Psychiatry and Psychotherapy, Innsbruck Medical University, Anichstr.35, 6020 Innsbruck, Austria Fumio Yanai Department of Neurosurgery, Fukuoka University Faculty of Medicine, Jonan-ku, Fukuoka 814-0180, Japan Kun-Tu Yeh Section of Haematology-Oncology, Department of Medicine, Taipei Medical University and Hospital, Taipei 110, Taiwan

Part I

Astrocytomas: Diagnosis and Biomarkers

Chapter 1

Methylation in Malignant Astrocytomas María del Mar Inda, Juan A. Rey, Xing Fan, and Javier S. Castresana

Abstract The term epigenetics is used to describe the study of stable and heritable alterations in gene expression potential that arise during development and cell proliferation. Two epigenetic mechanisms have been thoroughly investigated in the past few years: DNA methylation and histone modifications. The failure of the maintenance of these heritable epigenetic marks can lead to inappropriate activation or inactivation of signaling pathways and result in disease, such as cancer. Promoters of tumor suppressor genes have been assessed for hypermethylation with a variety of techniques, both at specific loci or genome wide. Methylation of the MGMT gene, which favors treatment results with temozolomide, is a clear example of the influence of methylation in a specific gene in astrocytomas. At the clinical level, the emphasis is now on combining inhibitors of DNA methyl transferases and of histone deacetylases. Keywords DNA methylation · CpG islands · DNMT · O6 -Methylguanine · MGMT methylation

and are retained throughout mitosis. They do not involve mutations of the DNA itself and are referred to as epigenetic alterations. Originally, the term epigenetics, which literally means outside conventional genetics, was defined as the casual interactions between genes and their products, which bring the phenotype into being (Waddington, 1942). Nowadays, the term epigenetics is used to describe the study of stable and heritable alterations in gene expression potential that arise during development and cell proliferation (Jaenisch and Bird, 2003). Two epigenetic mechanisms have been thoroughly investigated in the past few years: DNA methylation and histone modifications. Epigenetic mechanisms are essential for development and differentiation, but they can also arise in adults, either by random change or under the influence of the environment, allowing the organism to respond to the environment by modulating gene expression. The failure of the maintenance of these heritable epigenetic marks can lead to inappropriate activation or inactivation of signaling pathways and result in disease, such as cancer.

Understanding the Word Epigenetics DNA Methylation Even though they are genetically identical, cells from a multicellular organism present differential gene expression and are structurally and functionally heterogeneous. These differences occur during development

J.S. Castresana () Unidad de Biologia de Tumores Cerebrales, Universidad de Navarra, 31008 Pamplona, Spain e-mail: [email protected]

Methylation might be responsible for the stable maintenance of a particular gene expression pattern through mitotic cell division. Ample support to this hypothesis has been provided and now, DNA methylation is recognized as an important mechanism for establishing a silent chromatin state by collaborating with proteins that modify nucleosomes. These epigenetic modifications can be copied after DNA synthesis, resulting in heritable changes in chromatin structure. Genes

M.A. Hayat (ed.), Tumors of the Central Nervous System, Volume 5, DOI 10.1007/978-94-007-2019-0_1, © Springer Science+Business Media B.V. 2012

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can be transcribed from methylation-free promoters even though adjacent transcribed and non-transcribed regions are extensively methylated. In mammals, DNA methylation is predominantly found in cytosines of the dinucleotide sequence CpG and consists in the addition of a methyl group to the 5 -position of cytosines, altering the appearance of the major grove of DNA to which DNA binding proteins bind. CpG dinucleotides are not evenly distributed in the genome but rather are concentrated in short CpGrich DNA stretches called CpG islands, defined as regions of DNA greater than 200 bp, with a C + G content >50%, and an observed/expected presence of CpG >60%. In non-embryonic cells, methylation is found in approximately 80% of CpG dinucleotides. An exception for this global methylation of the genome are the CpG islands. The majority of CpG islands are associated with genes unmethylated in the germline and often located within promoter regions of genes. Approximately 60% of the human gene promoters contain CpG islands at the 5 end. How CpG islands in non-embryonic cells remain unmethylated is still unknown, but it is known that in cancer cells, methylation of CpG islands contributes to gene silencing of tumor suppressor genes. Methylation of certain CpG island promoters during development, resulting in long-term transcriptional silencing, has been observed.

Relevance of DNA Methylation in Normal Cells The relevance of DNA methylation in mammal development has been demonstrated by targeted mutagenesis of the different DNA methyltransferases (DNMT) genes in mice (Bestor, 2000). Genes involved in the establishment, maintenance or interpretation of genomic methylation pattern are essential for normal development. The first Dnmt to be discovered was Dnmt1 and it seems to act as a maintenance methyltransferase. Dnmt1 knock-out mice resulted in global demethylation and embryonic lethality (Li et al., 1992). In contrast, Dnmt3a and Dnmt3b are highly expressed in mouse embryo and are responsible for global de novo methylation after implantation. No obvious phenotype has been observed in mice after deletion of Dnmt2, but this gene is highly expressed

M. del Mar Inda et al.

during oogenesis and lacks biochemically detectable methyltransferase activity, but it seems to be responsible for the small amount of non-CpG methylation observed in the fly embryo (Lyko et al., 2000). CpG islands can normally be methylated in four cases: imprinted genes, X-chromosome inactivation in women, germline-specific genes, and tissue specific genes. X-chromosome inactivation in women is a well-characterized developmental phenomenon associated with DNA methylation in CpG islands assuring monoallelic gene expression (Jaenisch and Bird, 2003). The X-inactivation process and the genomic imprinting share some epigenetic mechanisms. The choice of the inactive X-chromosome and the initiation of the inactivation depends on Xist RNA, a noncoding transcript that originates at the X inactivation center (Xic) and coats the inactive X chromosome. Dnmt1 activity is needed for the maintenance of imprinting as well as for the X inactivation. Some studies suggest that Dnmt3L, which has no detectable methyltransferase activity, is required to establish maternal imprinting through the cooperation with de novo methyltransferase Dnmt3a. Some tissue-specific gene silencing through CpG island methylation has been reported in a variety of somatic tissues to silence these tissue-specific genes in tissues that should not express them, a well characterized example are the methionine adenosyltransferases 1A and 2A in rodents. A similar case are the germline-specific genes to restrict the expression of these genes to the male or the female germline and that later in the adult tissues will not be expressed, such as MAGE and LAGE gene families. Another interesting function for the normal DNA methylation is its role in repressing parasitic sequences. The methylation of the parasitic promoters inactivates them; over time, and thanks to the promutagenicity of the methylated cytosine, cytosines can be substituted by thymidine and destroy many transposons.

Epigenetics, Environment, Diet and Aging Epigenetic states are reversible and can be modified by environmental factors, diet and ageing and may contribute to the development of abnormal phenotypes. In addition, normal response to certain environmental

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Methylation in Malignant Astrocytomas

stimuli may be mediated by epigenetic mechanisms. In mammals, hypo- and hypermethylation have been associated with ageing; however, the functional significance remains to be determined. It is known that age is a major risk factor for cancer development, probably through the methylation of CpG islands and silencing of tumor suppressor genes. Some examples of genes hypermethylated in ageing individuals are estrogen receptor, IGF2 and MYOD (Jaenisch and Bird, 2003). In addition, some dietary supplements, such as folate or vitamins, can affect the activity of enzymes supplying methyl groups for the methylation processes and influence the rate of disease manifestation. A methyl-deficient diet has been shown to induce liver cancer associated with both hypomethylation and the enhanced expression of oncogenes such as c-ras, c-myc or c-fos (Dizik et al., 1991).

DNA Methylation in Human Disease and Cancer The failure of the maintenance of the DNA methylation or disruption of its machinery can be the cause of disease or cancer. Mutations in the DNMT3B gene, the human homolog of Dnmt3b, cause the ICF syndrome (immunodeficiency, centromeric region instability, and facial abnormalities), a human heritable genetic disease with deficient methylation of the pericentromeric repetitive DNA and at CpG islands of the X chromosome. Another X-linked neurological disorder, the Rett’s syndrome, is due to a failure in DNA methylation-related system; more specifically, it is due to mutations in the methyl binding protein MeCP2, responsible for recruiting histone deacetylases (HDACS) and other chromatin factors to methylated DNA. These two diseases suggest that DNA methylation is not only needed to complete embryonic development, but it is also required for development after birth. DNA methylation plays a critical role in the development and differentiation of mammalian cells, and its deregulation has been involved in oncogenesis. The alteration of the DNA methylation pattern results in global dysregulation of gene expression profiles, leading to the development and progression of cancer. Since these alterations are hereditable, cells with epigenetic alterations conferring a growth advantage

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are rapidly selected and result in uncontrolled tumor growth. Cancer can be considered to be a genetic disease at the same level as an epigenetic disease, and DNA methylation can be an excellent candidate to explain how certain environmental factors or ageing can increase the risk of cancer. In fact, DNA methylation plays an essential role in all three mechanisms by which cancer cells eliminate tumor suppressor gene function: point mutation, silencing by promoter hypermethylation and deletion by LOH due to genomic instability (Gronbaek et al., 2007). CpG sites have been considered to be mutation hotspots in the human germline and recently, it has become apparent that they are also hotspots for inactivating mutations of tumor suppressor genes such as p53 which is mutated in CpG sites in 25% of the cases. That CpG dinucleotides constitute hotspots for point mutations is due to the fact that methylated cytosines can be spontaneously deaminated to thymine and result in a C–T transition. If C–T transitions are not repaired and occur in the coding region of genes, they may activate an oncogene or suppress a tumor suppressor gene (Gronbaek et al., 2007). More than 30% of the point mutations in the germline related to disease occur at CpG dinucleotides. For example, in colorectal cancer, 44% of the mutations are C–T transitions. In addition, methylated cytosines also favor the formation of adducts on the neighboring G in the presence of some carcinogens, such as the benzo(a)pyrene present in tobacco smoke, resulting in a G–T transversion (Gronbaek et al., 2007). Commonly, cancer cells are characterized by global genomic hypomethylation and hypermethylation of CpG islands that are generally unmethylated in normal cells. DNA hypomethylation plays a critical role in tumorigenesis and may lead to the upregulation and activation of oncogenes, such as R-Ras and MAPSIN in gastric cancer, or MAGE in melanoma. The mechanisms by which DNA methylation can contribute to tumorigenesis can be summarized in three: reactivation of retrotransposons, increasing chromosomal instability, and loss of imprinting. DNA hypomethylation can allow the transcription and/or translocation of retrotransposons, increasing the genomic instability, or lead to the upregulation of oncogenic microRNAs. Loss of methylation has been observed in Alu repeats and in LINES in cancer cells, and some imprinted genes, such as H19 or IGF-2, present loss of methylation in pediatric tumors. Hypomethylation may allow

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the formation of chromosomal breaks, translocations and/or allelic loss by illegitimate mitotic recombination, and the demethylation in pericentromeric regions of chromosomes plays a role in aneuploidy. In contrast to DNA hypomethylation which can lead to the activation of proto-oncogenes or increase genomic instability, hypermethylation of CpG islands that are unmethylated in normal cells leads to inactivation of tumor suppressor genes by silencing their expression, and several reports have shown a correlation between expression and loss of DNA methylation. How these genes are targeted for hypermethylation still remains unclear, and in some tumors, silencing by promoter hypermethylation occurs at a very high frequency. Many genes of key pathways in cancer are affected by promoter hypermethylation; however, methylation of the downstream gene sequences usually has no effect on gene expression (Jones, 1999). Examples of tumor suppressor genes silenced by hypermethylation were found in cancer and include: MGMT, Rb, p16Ink4a, BRCA1, p14ARF, APC, retinoic acid receptor-β2, RASFF1, etc (Gronbaek et al., 2007). Recently, experimental data has provided support to the idea that genes can be transcriptionally activated by removing DNA methylation (Baylin et al., 1998, 2001; Lorente et al., 2009), providing an attractive target for cancer therapeutics.

Methods to Detect Methylation Aberrant methylation is the most common alteration found in cancer cells, while silencing of tumor suppressor genes by CpG island promoter hypermethylation is the change of DNA methylation most studied in neoplasms. The detection of methylation in clinical samples (Table 1.1) may be useful in the early detection of cancer screening; therefore, it has become the focus of research in many clinical and translational laboratories. The reason for this is partially due to the early occurrence of alterations in the methylation pattern (hypo or hypermethylation) in carcinogenesis. Furthermore, since they are DNA markers, they are more stable than RNA or proteins, and studies can be performed in formalin-fixed and paraffin-embedded tissues (Fan et al., 2002). It has been demonstrated that DNA methylation can be detected in blood, sputa, ductal lavage fluids, urine, saliva, mammary aspiration


fluid, stool, and biopsy specimens by using highly sensitive PCR-based methods after bisulfite modification. In addition to being a tumor-specific change, different tumor types have different DNA methylation profiles that are helpful in diagnosing difficult cases (Shames et al., 2007). In glioblastoma multiforme, the detection of methylation in the promoter of the MGMT gene (O6 -methylguanine-DNA methyltransferase) predicts a favorable outcome in patients treated with alkylating agents (Hegi et al., 2005). The initial studies of DNA methylation relied on the use of methylation-sensitive restriction enzymes that were able to distinguish between unmethylated and methylated recognition sites and Southern blot hybridization. This approach has many drawbacks: the limitation of the sites that can be analyzed, the problem of incomplete restriction cutting, the necessity of using high-molecular weight and elevated amounts of DNA to perform the Southern blot analysis, and the fact that the method is labor-intensive. In addition, only CpGs located within sequences recognized by methylation-sensitive enzymes can be analyzed. The majority of the methods used to detect DNA methylation are based on the chemical modification of DNA with sodium bisulfite followed by PCR with primers specific for methylated sequences. These methods, especially the ones that use primers designed specifically to amplify the methylated sequence, provide a very sensitive and specific analytical tool for detecting methylation at single loci. The treatment of DNA with sodium bisulfite deaminates cytosines to uracil, and because deamination of 5-methylcytosine is much slower, it is generally assumed that only unmethylated cytosines are transformed. There are three processes in the DNA modification by the bisulphite reaction: the reversible cytosine sulphonation, the irreversible hydrolytic deamination of the sulphonated cytosine, and the removal of the bisulfite adduct to give uracil by alkali treatment (Clark et al., 1994). It has been determined that the conversion rate under ideal conditions of unmethylated cytosines is about 99% (Taylor et al., 2007). Several groups have worked on optimizing the bisulfite treatment (Cottrell et al., 2004; Fan et al., 2002; Grunau et al., 2001). Once DNA is treated and modified with sodium bisulfite, different techniques can be used so as to make it possible for every laboratory and hospital to assess DNA methylation. Bisulfite sequencing provides a quantitative way to determine the methylation state of a genomic

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Table 1.1 Comparison among some of the different techniques to detect methylation Specimen Method treatment Application Sensitivity Quantitative

Advantages

Disadvantages Expensive and timeconsuming

No

Methylation status of individual CpG sites can be analyzed Easy to perform

Low

Yes

Reproducible

Genomewide

Low

Yes

Novel marker discovery

Bisulfite conversion

Specific locus

High

No

Cost-effective and needs small amounts of DNA

Q-MSP or Methylight


Specific locus

High

Yes

Easy and high throughput

Heavymethyl


Specific locus

High

Yes

MALDI-TOF MS


Genomewide/specific locus

Medium

Yes

Low false positives and high throughput Quantitative data on individual CpG sites can be obtained

Bisulfite sequencing


Specific locus

Low

Yes

Southern blot

Methylationsensitive enzyme

Genomewide

Low

RLGS

Methylationspecific restriction enzyme Immnunoprecipitation + array

Genomewide

MSP

ChIP-on-chip

region at a single-nucleotide resolution and is the gold standard of the methods based on the bisulfite DNA treatment. Unfortunately, this method is too expensive and time consuming to be used in a clinical setting. In this chapter we will discuss the methods most often used for detecting methylation at a single locus or multiple loci, as well as genome-wide (Table 1.1).

Limited sites available, needs high amounts of high quality DNA and is labor intensive Needs high quality DNA

No correlation with expression False positives and does not allow discrimination between unmethylated and partially methylated Does not allow discrimination between unmethylated and partially methylated Many oligonucleotides are used Expensive equipment required

The most widely used assay for sensitively detecting methylation is called methylation-specific PCR (MSP) (Herman et al., 1996). Before PCR amplification, genomic DNA is modified by sodium bisulfite treatment in order to convert all unmethylated cytosines to uracil which, after amplification, will be transformed into thymidine. Two sets of primers are designed for

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PCR amplification: one set is designed to hybridize with the methylated sequence (M), while the other set of primers is designed to match with the nonmethylated sequence (U). After PCR, products are visualized in agarose gels by ethidium bromide staining. The sensitivity of this technique has been estimated to be one in 1000 (detection of methylated DNA in 1000-fold excess of unmethylated DNA). This method is simple, inexpensive, highly specific and sensitive, and does not require special equipment. However, MSP also presents some drawbacks. For example, it does not allow discrimination between partial levels of methylation and complete lack of methylation, leading to false positive results if the PCR conditions and sodium bisulfite modification are not optimized. The fact that MSP is gel-based makes this method unsuitable for a clinical setting because of the need to be high-throughput and homogeneous and not be quantitative. To address some of the drawbacks of the conventional MSP, a method based on Taqman technology, known as quantitative MSP (QMSP) or Methylight, has been developed (Eads et al., 2000). Each round of PCR leads to an increase in fluorescence proportional to the amount of target in the sample, and the signal is only observed when the probe has hybridized between the primers, thus eliminating the nonspecific amplification, such as primer dimer formation. This method is more suitable for routine clinical use because it does not need a secondary electrophoresis step eliminating cross-contamination problems, and it is quantitative. Other methods derived from the MSP are also very helpful in the methylation analysis of specific loci, overcoming some of the disadvantages of the MSP. This is the case of the quantitative MSP-Sybr green based, sensitive melting analysis after real-time MSP (SMART-MSP), methylation specific amplicon generation (MS-FLAG), multiplex Q-MSP (QM-MSP), methylation specific nested PCR (MSnested-PCR), etc. (Cottrell et al., 2004; Fackler et al., 2004). MALDI-TOF mass spectrometry (MALDI-TOF MS) is a novel strategy for high-throughput DNA which is based on a base-specific cleavage reaction combined with mass spectrometric analysis (Coolen et al., 2007). Briefly, DNA is converted with sodium bisulfite and amplified using a T7 promoter tagged primer. A single strand RNA molecule is then formed and it is base-specific cleaved by RNase A. Fragments originated by cleavage are analyzed by MALDI-TOF


MS. The differences in methylation status are reflected in differences in fragment size, and quantification of abundance of each fragment represents the amount of DNA methylation in the sample. This technique is quantitatively accurate, relatively sensitive, with possible high-throughputs, but it is very complex and requires expensive equipment. Another method that uses primers which are nonspecific for the methylated or unmethylated sequence and is highly sensitive and specific is the denominated Heavy Methyl. The primers are designed to hybridize close to a region that contains a CpGrich sequence. During the reaction, there are also blockers that are designed to hybridize only with the unmethylated sequence; thus, if DNA is methylated, the blockers cannot hybridize, and amplification will occur. However, if the sequence is unmethylated, the blockers will bind to the sequence and block amplification. Amplification is detected with a probe that contains CpG sites, a quencher and a fluorophore label. Fluorophore is released from the quencher when the exonuclease activity of the polymerase cleaves the probe and light is emitted. The emitted light is proportional to the amount of PCR product, allowing an accurate quantification of methylation level. The use of the blocker increases the sensitivity and decreases the number of false positives. Other advantages of this method are that it is close tube-based, so it eliminates cross-contamination and allows highthroughputs, making it appropriate for a clinical setting. It is also possible to analyze DNA methylation globally. Global approaches to methylation analysis are high-throughput, but they are relatively expensive and labor-intensive, including microarray expression profiling, restriction landmark genomic scanning (RLGS) and immunoprecipitation of methylated DNA followed by array-based comparative genome hybridization (ChIP on chip) analysis. The RLGS has been defined as a method which provides quantitative genetic and epigenetic assessment of thousands of CPG islands in a single gel without prior knowledge of gene sequence (Costello et al., 2002). The basis of the RLGS method is the two-dimensional separation of radiolabeled DNA fragments obtained after successive digestions of genomic DNA with different restriction enzymes. DNA is first digested with an infrequently cutting enzyme sensitive to methylation, such as NotI or AscI. It is then radiolabeled, digested with a second

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enzyme and electrophoresed through a narrow tubeshaped gel. The DNA in the tube-gel is digested again with a more frequent cutting restriction enzyme and electrophoresed perpendicularly in a non-denaturing polyacrylamide gel. Approximately 2000 fragments originate, with a high probability of containing gene and/or promoter sequences, and making it possible to detect new hypermethylated sequences in the genome. The resulting gel autoradiography gives a determined RLGS profile that is highly reproducible, and comparisons between different samples can be performed. DNA quality is a critical parameter for generating high quality RLGS profiles. One of the disadvantages of this technique is that the loss of a fragment in the RLGS profile can be due to deletion or methylation. Recently, another method to detect global methylation has been developed. The ChIP-on-chip method is based on immunoprecipitation of methylated DNA with a monoclonal antibody to 5-methylcytosine after sonication or restriction digestion. The DNA is then labeled and hybridized to a DNA microarray with probes of the regions of interest. Methylated sequences are detected by comparing the fluorescent signal for each probe (Shames et al., 2007). As RLGS, ChIP-onchip is a genome-wide, high resolution and quantitative method, but both methods are very expensive and labor-intensive.

DNA Methylation in Astrocytic Tumors: Genes Frequently Methylated, Relevance for Diagnosis and Prognosis DNA methylation plays a critical role in mammalian central nervous system development and function, and global DNA methylation levels are dynamic during brain development varying among different brain regions (Nagarajan and Costello, 2009). As we previously discussed, genes involved in DNA methylation machinery such as DNA methyltransferases are important for normal development and several human neurodevelopmental disorders, such as Rett or human ICF syndromes, have been associated with alterations in genes involved in DNA methylation, such as MECP2 or DNMT3B. Attention has been focused on studying epigenetic alterations in glioblastoma multiforme because of the

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possibility of being used as potential prognostic factors and as response factors for treatment. One of the most widely studied genes is O 6 -methylguanineDNA-methyltransferase (MGMT) due to the relationship between MGMT methylation status and response to alkylating agents, such as temozolomide. MGMT is involved in DNA repair, removing alkyl groups from O6 -methylguanine nucleotides and not allowing them to pair with thymine or to chemically react with other bases (Esteller et al., 1999) and protecting the cells from carcinogens. Approximately 56–68% of GBM present MGMT promoter hypermethylation (Nakamura et al., 2001), and is negatively correlated with expression. MGMT promoter hypermethylation is considered to be a biomarker of poor prognosis in GBM due to the fact that it has a critical role in DNA repair system and MGMT silencing has been correlated with increased TP53 mutations (Nakamura et al., 2001), in particular G to A transitions. On the other hand, it has been demonstrated that patients with MGMT hypermethylation respond better to treatment with alkylating agents, such as temozolomide (Hegi et al., 2005). Moreover, methylation of the pro-apoptotic gene TMS1/ASC coincides with MGMT methylation, suggesting that different types of glioma presenting differences in survival and response to treatment might have different epigenetic marks (Martinez et al., 2007). In GBM, promoter hypermethylation occurs in genes involved in different functions related to tumorigenesis and tumor progression, including apoptosis, DNA repair, drug resistance, invasion, cell cycle regulation, or angiogenesis. Methylation of cell cycle regulatory genes, such as p14, p15 and p16, has been observed in primary GBM, although at low frequencies, being more frequently methylated in low-grade astrocytomas and secondary GBM. PTEN tumor suppressor gene is mutated in 20–40% of GBM, and inactivation of PTEN expression by promoter methylation has also been found frequently in gliomas and glioma cell lines (Wiencke et al., 2007). Several groups have studied methylation patterns of different grades of glioma and found differences in the frequency of methylation of some tumor suppressor genes between different grades of glioma, primary GBM or secondary GBM or between gliomas and their relapses (Gonzalez-Gomez et al., 2003; Kunitz et al., 2007; Martinez et al., 2007; Nakamura et al., 2001; Uhlmann et al., 2003).

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A large study of 139 tissues samples (Uhlmann et al., 2003), including 33 control tissues and 106 gliomas of different grades (33 pilocytic astrocytomas; 34 diffuse astrocytomas, 11 anaplastic astrocytomas, 7 secondary GBM and 3 primary GBM, among other oligoastrocytomas and oligodendrogliomas) assessed methylation at 15 loci including RB1, ARF, CDKN2B, APC or TIMP3. They defined an epigenotype of those tumors, showing that 7 of 15 loci analyzed presented tumor specific methylation. Notoriously, no hypermethylation was detected in the lower grade of gliomas or pilocytic astrocytomas. However, they were significantly hypomethylated at MYODI compared to control tissues. Grade II astrocytomas presented the most significant changes in methylation compared to normal controls. Some new genes were found to be methylated in gliomas such as CALCA, CDH1 or PTGS2, and the authors proposed that methylation fingerprinting for gliomas appears to be possible and could provide an additional diagnostic method for the future. In another study, 13 loci of genes involved in DNA repair, apoptosis, cell cycle regulation or tumor suppression were analyzed for DNA methylation in 32 paired tumor samples of GBM and relapses (Martinez et al., 2007). They found that the hypermethylation profile of GBM relapses was different in 62.5% of the patients, with CASP8 being the gene that was more significantly hypermethylated in GBM relapses, suggesting that a significant epigenetic silencing of this gene occurs during progression of primary to recurrent GBM. In contrast, other pro-apoptotic genes, such as CASP3 and CASP9, were unmethylated in both GBM and relapses. Characterization of diffuse astrocytomas (grade II) that underwent recurrence or progression revealed that tumors with p14ARF methylation at first biopsy were associated with shorter patient survival. Furthermore, they found that methylation of p14ARF and MGMT were frequent events in diffuse astrocytomas and were mutually exclusive. While p14ARF methylation was associated with a shorter survival, MGMT methylation was indicative of better clinical outcome after chemotherapy (Kleihues et al., 1994). No methylation of p21Waf/Cip1, p27Kip1 or p73 was observed in this study. It has been shown that primary or de novo GBM and secondary GBM present different genetic abnormalities. While primary GBM are characterized by LOH


10q (70%), EGFR amplification (36–40%), p16INK4a deletion (31%) and PTEN mutations (25–40%), the main genetic abnormalities found in secondary GBM are LOH 10q (63%) and TP53 mutations (60%), which present a higher proportion in secondary GBM (Furnari et al., 2007; Ohgaki and Kleihues, 2007). Secondary and primary GBM also differ significantly in their pattern of DNA methylation and RNA and protein expression. In general, the promoter hypermethylation frequency is higher for secondary GBM than for primary GBM, maybe due to slower progression of the disease and the accumulation of alterations. RB1, p16INK4a, p14ARF, MGMT or TIMP-3 are methylated in a higher frequency in secondary GBM (Ohgaki and Kleihues, 2007), and for example, p14ARF is already methylated in a third of low-grade astrocytomas. Loss of MGMT expression by promoter hypermethylation was found in 75% of secondary GBM versus 36% of primary GBM. The correlation found between MGMT methylation and TP53 mutation might explain the higher frequency of TP53 mutations in secondary GBM (60–65% versus 28% in primary GBM) (Nakamura et al., 2001). The epithelial membrane 3 protein gene (EMP3), whose methylation is considered to be an unfavorable prognostic marker in neuroblastoma, is frequently methylated in low grade astrocytomas as well as secondary GBM (80%), but no in primary GBM (17%) suggesting that EMP3 methylation might be an early alteration in astrocytomas (Kunitz et al., 2007). In addition, some studies have suggested that methylation does not play a critical role in primary high grade gliomas and low frequency of RB1, p14ARF, p15INK4b, p16INK4a, p21Waf/Cip1, p27Kip1 or p73 were found in grade III and grade IV gliomas. Other genes frequently found to be methylated in gliomas are RASSF1A, BLU, Death receptor 4 (DR4), estrogen receptor (ER), or RARbeta.

Relevance of Methylation in the Clinic The importance of methylation in the clinic has been increasing due to the development of sensitive techniques for detecting methylation; methylation is a tumor-specific change that occurs early in tumorigenesis and the correlation between methylation and prognosis. In many neoplasms, therapeutic advances have

1


been related to the availability of plasma biomarkers with prognostic and therapeutic significance. It is known that tumors released substantial amounts of DNA, containing the genetic and epigenetic alterations present in the primary tumor, into the systemic circulation, probably through apoptosis and cellular necrosis. It has been demonstrated that it is possible to detect promoter methylation in total plasma from glioma patients (Weaver et al., 2006): they analyzed promoter methylation in 4 gene locus in tumor tissue and total plasma from patients with different glioma grades, and demonstrated that 90% of the patients presented methylation of at least one locus in the primary brain tumor and that in 67% of these patients, methylation was also found in blood. As we mentioned previously, MGMT hypermethylation is associated with significant longer survival in patients with GBM and low-grade gliomas treated with alkylating agents such as temozolomide. Therefore, the detection of MGMT methylation can be used as a prognostic factor (Hegi et al., 2005). On the contrary, the detection of methylation of p14ARF promoter is associated with malignant progression and shorter survival; methylation of the pro-apoptotic gene Caspase-8 is frequently associated with relapsed GBM (Martinez et al., 2007).

Epigenetic Therapy The DNMT inhibitor Decitabine (5-aza-2 deoxycytidine) and the HDAC inhibitor Vorinostat (SAHA: suberoylanilide hydroxamic acid) are currently in use in multiple cancers, although only SAHA is in clinical trials in GBM. The advantage of epigenetic mutations is their reversibility compared to genetic mutations, but the principal problem of the epigenetic therapy is the target specificity. Even though the use of demethylating agents can reactivate silenced tumor suppressor genes, they can also activate oncogenes through hypomethylation. Another caveat of the use of 5-aza-2 -deoxycytidine is its toxicity. To overcome this problem, the combination of 5-aza-2 deoxycytidine and drugs that inhibit HDAC activity reduce the effective drug concentration and systemic toxicity while resulting in a more effective reactivation of tumor suppressor genes.

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Future Directions Epigenetic studies of gliomas will provide further understanding of glioma biology and might identify new therapeutic targets. Consortiums, such as The Cancer Genome Atlas (TCGA; http://cancergenome. nih.gov), are helping to unravel the genetic and epigenetic alterations in GBM using high-throughput genomic and epigenomic approaches. The causes and consequences of DNA methylation in glioma are not entirely known and why some genes or pathways are preferentially targeted for methylation still remains unclear. Acknowledgements We are grateful to Laura Stokes for helping with editing the manuscript. This research was supported in part by grants from the Departmento de Salud del Gobierno de Navarra (9/07), Caja Navarra (08/13912), and Fundación Universitaria de Navarra, Pamplona; and Fondo de Investigación Sanitaria (PI081849), Madrid, Spain.

References Baylin SB, Herman JG, Graff JR, Vertino PM, Issa JP (1998) Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv Cancer Res 72:141–196 Baylin SB, Esteller M, Rountree MR, Bachman KE, Schuebel K, Herman JG (2001) Aberrant patterns of DNA methylation, chromatin formation and gene expression in cancer. Hum Mol Genet 10:687–692 Bestor TH (2000) The DNA methyltransferases of mammals. Hum Mol Genet 9:2395–2402 Clark SJ, Harrison J, Paul CL, Frommer M (1994) High sensitivity mapping of methylated cytosines. Nucleic Acids Res 22:2990–2997 Coolen MW, Statham AL, Gardiner-Garden M, Clark SJ (2007) Genomic profiling of cpg methylation and allelic specificity using quantitative high-throughput mass spectrometry: Critical evaluation and improvements. Nucleic Acids Res 35:e119 Costello JF, Plass C, Cavenee WK (2002) Restriction landmark genome scanning. Methods Mol Biol 200:53–70 Cottrell SE, Distler J, Goodman NS, Mooney SH, Kluth A, Olek A, Schwope I, Tetzner R, Ziebarth H, Berlin K (2004) A real-time pcr assay for DNA-methylation using methylationspecific blockers. Nucleic Acids Res 32:e10 Dizik M, Christman JK, Wainfan E (1991) Alterations in expression and methylation of specific genes in livers of rats fed a cancer promoting methyl-deficient diet. Carcinogenesis 12:1307–1312

12 Eads CA, Danenberg KD, Kawakami K, Saltz LB, Blake C, Shibata D, Danenberg PV, Laird PW (2000) Methylight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res 28:E32 Esteller M, Hamilton SR, Burger PC, Baylin SB, Herman JG (1999) Inactivation of the DNA repair gene O6methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. Cancer Res 59:793–797 Fackler MJ, McVeigh M, Mehrotra J, Blum MA, Lange J, Lapides A, Garrett E, Argani P, Sukumar S (2004) Quantitative multiplex methylation-specific pcr assay for the detection of promoter hypermethylation in multiple genes in breast cancer. Cancer Res 64:4442–4452 Fan X, Inda MM, Tunon T, Castresana JS (2002) Improvement of the methylation specific pcr technical conditions for the detection of p16 promoter hypermethylation in small amounts of tumor DNA. Oncol Rep 9:181–183 Furnari FB, Fenton T, Bachoo RM, Mukasa A, Stommel JM, Stegh A, Hahn WC, Ligon KL, Louis DN, Brennan C, Chin L, DePinho RA, Cavenee WK (2007) Malignant astrocytic glioma: Genetics, biology, and paths to treatment. Genes Dev 21:2683–2710 Gonzalez-Gomez P, Bello MJ, Arjona D, Lomas J, Alonso ME, De Campos JM, Vaquero J, Isla A, Gutierrez M, Rey JA (2003) Promoter hypermethylation of multiple genes in astrocytic gliomas. Int J Oncol 22:601–608 Gronbaek K, Hother C, Jones PA (2007) Epigenetic changes in cancer. APMIS 115:1039–1059 Grunau C, Clark SJ, Rosenthal A (2001) Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res 29:E65–65 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 (2005) Mgmt gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med 352: 997–1003 Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB (1996) Methylation-specific pcr: a novel pcr assay for methylation status of cpg islands. Proc Natl Acad Sci USA 93:9821–9826 Jaenisch R, Bird A (2003) Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet 33 Suppl:245–254 Jones PA (1999) The DNA methylation paradox. Trends Genet 15:34–37 Kleihues P, Lubbe J, Watanabe K, von Ammon K, Ohgaki H (1994) Genetic alterations associated with glioma progression. Verh Dtsch Ges Pathol 78:43–47

M. del Mar Inda et al. Kunitz A, Wolter M, van den Boom J, Felsberg J, Tews B, Hahn M, Benner A, Sabel M, Lichter P, Reifenberger G, von Deimling A, Hartmann C (2007) DNA hypermethylation and aberrant expression of the emp3 gene at 19q13.3 in human gliomas. Brain Pathol 17:363–370 Li E, Bestor TH, Jaenisch R (1992) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69:915–926 Lorente A, Mueller W, Urdangarin E, Lazcoz P, Lass U, von Deimling A, Castresana JS (2009) Rassf1a, blu, nore1a, pten and mgmt expression and promoter methylation in gliomas and glioma cell lines and evidence of deregulated expression of de novo dnmts. Brain Pathol 19:279–292 Lyko F, Ramsahoye BH, Jaenisch R (2000) DNA methylation in drosophila melanogaster. Nature 408:538–540 Martinez R, Schackert G, Esteller M (2007) Hypermethylation of the proapoptotic gene tms1/asc: prognostic importance in glioblastoma multiforme. J Neurooncol 82:133–139 Nagarajan RP, Costello JF (2009) Epigenetic mechanisms in glioblastoma multiforme. Semin Cancer Biol 19:188–197 Nakamura M, Watanabe T, Yonekawa Y, Kleihues P, Ohgaki H (2001) Promoter methylation of the DNA repair gene mgmt in astrocytomas is frequently associated with g:C –> a:T mutations of the tp53 tumor suppressor gene. Carcinogenesis 22:1715–1719 Ohgaki H, Kleihues P (2007) Genetic pathways to primary and secondary glioblastoma. Am J Pathol 170:1445–1453 Shames DS, Minna JD, Gazdar AF (2007) Methods for detecting DNA methylation in tumors: From bench to bedside. Cancer Lett 251:187–198 Taylor KH, Kramer RS, Davis JW, Guo J, Duff DJ, Xu D, Caldwell CW, Shi H (2007) Ultradeep bisulfite sequencing analysis of DNA methylation patterns in multiple gene promoters by 454 sequencing. Cancer Res 67:8511–8518 Uhlmann K, Rohde K, Zeller C, Szymas J, Vogel S, Marczinek K, Thiel G, Nurnberg P, Laird PW (2003) Distinct methylation profiles of glioma subtypes. Int J Cancer 106:52–59 Waddington CH (1942) The epigenotype. Endeavour 1:18–20 Weaver KD, Grossman SA, Herman JG (2006) Methylated tumor-specific DNA as a plasma biomarker in patients with glioma. Cancer Invest 24:35–40 Wiencke JK, Zheng S, Jelluma N, Tihan T, Vandenberg S, Tamguney T, Baumber R, Parsons R, Lamborn KR, Berger MS, Wrensch MR, Haas-Kogan DA, Stokoe D (2007) Methylation of the pten promoter defines low-grade gliomas and secondary glioblastoma. Neuro Oncol 9:271–279

Chapter 2

Deciphering the Function of Doppel Protein in Astrocytomas Alberto Azzalin and Sergio Comincini

Abstract Doppel is a newly recognized prion-like protein encoded by a novel gene locus, PRND, located on the same chromosomal region of the prion coding gene (PRNP); doppel is considered a prion paralogue and together they constitute the prion-gene family, originated through an ancestral gene duplication event. Prion and doppel have different expression patterns, suggesting that the gene products exhibit different biological functions. Actually, doppel is not involved in the etiology of the transmissible spongiform encephalopathies (TSEs) or “prion diseases” and it is highly expressed only within the testicular tissue, where its important physiological role in the process of spermatogenesis and fertilization in human and mouse has been described. Importantly, doppel is toxic when ectopically overexpressed in the central nervous system (CNS), with concomitant prion protein absence: this evidence suggests deeper investigations within particular pathological contexts, such as in Parkinson and Alzheimer’s diseases as well as in cancer. In the latter scenario several studies are showing that doppel represents a novel and attractive diagnostic molecular marker, and that it might provide insights into the regulatory pathways of tumor-cell transformation. In particular, doppel protein has been recently associated with the ability of tumor cells to migrate, as one of the most important hallmarks of cancer. In conclusion, since its discovery, the intriguing spectrum of biological and pathological functions of this new

A. Azzalin () Institute of Molecular Genetics, IGM-CNR Pavia via Abbiategrasso 207 (OR via Ferrata 1), 27100 Pavia, Italy e-mail: [email protected]

prion-like protein is constantly considered for novel investigations. Keywords Doppel · Transmissible spongiform encephalopathies · Bovine spongiform encephalopathy · CNS · GPI · Prion–doppel

Introduction Historical Background of Doppel Discovery The doppel gene was identified rather incidentally during the sequence analysis of a murine cosmid clone containing the prion gene, isolated from the I/LnJ inbred strain of mice (Moore et al., 1999): the rationale of this large scale genomic sequence project was to find regulatory elements and additional genes that might influence or contribute to the abnormal expression of the pathological isoform of the prion protein, the etiological agent of TSEs or “prion diseases” (Prusiner, 1998). In particular, it was noted that, directly adjacent to the prion gene, there was an attractive open reading frame (ORF) sequence which coded for a protein with striking similarities with the prion counterpart, as shown in Fig. 2.1. However, despite the protein structure similarity, the novel identified coding sequence shared low nucleotide similarity with the prion gene. This novel gene was therefore termed “prion–doppel” or “doppel” (for downstream of prion protein-complex or doppelgänger, i.e., a ghost-like companion). Further studies reported the evolutionary conservation of doppel gene sequences in different


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Fig. 2.1 The prion-gene family and its gene products. The prion-gene family and its gene products. The figure shows the human Prn genomic locus and, in detail, the PRNP and PRND gene structures (a), both composed of two exons (red and blue boxes, respectively) containing single ORFs; numbers above the structure indicate sizes in base pairs. In the section b) of the figure, a schematic diagram of prion and doppel protein secondary structures are reported, showing major structural and biochemical features, such as disulfide bonds and threonine (Thr) and

A. Azzalin and S. Comincini

asparagines (Asn) glycosylation sites; aminoacid residues numbering is indicated. The two proteins show many features in common as revealed by NMR analysis (c): in both cases the proteins are composed of three α-elixes and two β-sheets, in blue and red respectively. Between square brackets the accession numbers to NCBI databases are reported (http://www.ncbi.nlm.nih.gov/). GPI, glycosyl-phosphatidyl-inositol; H, helix; OR, octarepeats; S, sulphate; SP-N and -C, amino- and carboxy-signal peptide; TM, trans-membrane domain

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Deciphering the Function of Doppel Protein in Astrocytomas

mammalian species, such as humans and ruminants (Comincini et al., 2001). It was then inferred that the doppel gene might have derived from a proto-prion gene duplication event, thus originating the Prn-gene family, composed of prion (PRNP) and doppel (PRND) genes, sharing a common architecture (Mastrangelo and Westaway, 2001). At present, two other genes belonging to this small gene family, PRNT and SPRN, respectively, have been described, although their biological functions are still unclear. Further studies, that mirrored the studies by Moore et al. (1999) in murine species, confirmed that doppel was nearly undetectable in the CNS, markedly different from the prion protein. Doppel expression pattern is more temporary and spatially restricted compared to prion, being particularly expressed in mammal testicular tissues (Peoc’h et al., 2002). Of note, once discovered, doppel provided important insights into the puzzling phenotypes observed in Prnp knock-out mice: in particular, when doppel is overexpressed in CNS derived cells or tissues (where it is usually not expressed), together with the absence of the prion gene expression, doppel causes severe ataxic phenotype, roughly similar to that observed in prion diseases. Strikingly, however, the reintroduction of the Prnp transgene restores the normal phenotype (Weissmann and Aguzzi, 1999). Therefore different studies in mice highlighted that doppel might represent a toxic product, particularly at the CNS level and that its expression is strictly regulated in these tissues, with the exception of a limited window of expression within a week postnatal interval. Furthermore, different molecular models have been proposed in order to decipher a possible mechanism of antagonistic interaction between the prion and doppel gene products in CNS (Sakaguchi, 2008). Doppel discovery, followed by its biochemical and structural characterization (Lührs et al., 2003), provided new hypothetical avenues on the molecular pathogenesis of the prion diseases, in particular in the aetiology of the ataxic correlated phenotype. Unfortunately, extensive DNA sequencing surveys on patients affected by Creutzfeldt–Jakob (CJ) disease, Gerstmann–Sträussler–Scheinker (GSS) disease, familiar fatal insomnia (FFI) (Mead et al., 2000), as well as on scrapie and bovine spongiform encephalopathy (BSE)-affected animals (Comincini et al., 2001) failed to reveal doppel-genetic determinants associated with these pathologies. Moreover,

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the expression of doppel protein in the CNS does not modulate TSEs transmission in mouse models, excluding a direct involvement of this protein in the disease progression (Rossi et al., 2001). The testisrestricted expression pattern of doppel, mainly identified in sperms and in Sertoli cells, confirmed in different mammalian species, prompted to investigate if this gene might have a possible function in the male reproductive tract: different studies reported that doppel gene knock-out mice gave rise to infertility in mice, because their mutated spermatozoa were unable to perform the acrosome reaction (Paisley et al., 2004). Analogously in humans, doppel DNA polymorphisms were associated with fertility defects (Peoc’h et al., 2003). Moreover, it was reported that an interaction between doppel and P34H (also knows as dicarbonyl/L -xylulose reductase), a glycosylphosphatidyl-inositol (GPI)-anchor protein with testisspecific expression, localized particularly on the acrosomal cap of spermatozoa (Azzalin et al., 2006). Interestingly, P34H protein, similarly to doppel, has been demonstrated to be involved in one of the prerequisites of human fertilization, i.e., the binding of spermatozoa to the zona pellucida. In recent years, different contributions enlarged the spectrum of interest regarding the doppel gene functions in pathological context. Because of the structural homology with the prion protein, the analysis of doppel in this field moved directly to the CNS diseases. Therefore, the examination of doppel expression and its possible role in human neurodegenerative pathologies different from prion diseases (Alzheimer’s, Pick’s, Parkinson’s and diffuse Lewy body diseases) and brain tumors was undertaken, as discussed here.

Doppel Gene Expression Analysis in Astrocytomas: A Novel Potential Tumor Marker Doppel possesses similar exon–intron architecture to that of the prion gene, as shown in Fig. 2.1a; additionally, the two genes share a chromosomal synteny in different mammalian species (Comincini et al., 2006a). As previously stated, these evidences reinforced the concept that an ancestral gene duplication event originated the prion-family gene chromosomal locus, designated Prn. Similarly to the prion gene,

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the doppel coding region is contained within a single exon. However, the two genes exhibited marked different expression patterns, with prion gene being mostly expressed within the CNS tissues, while doppel is uniquely expressed in the adult testis tissue but, notably, at very low levels in nervous system. Therefore, while prion gene expression is relevantly related to the prion disease onset and epidemiology, doppel related studies were targeted to male gametogenesis and fertilization processes. In addition to neurodegenerative and reproductive investigations, a novel doppel-related branch of research was proposed (Comincini et al., 2004). The rationale was to investigate doppel expression perturbation within CNS-related diseases such as brain cancers, where it is expected to be physiologically not expressed. These studies led to the identification and characterization of a novel expression marker within the human glial tumors and to the association with the malignant grading progression. Glial tumors are histo-pathologically divided into four grades of malignancy, according to the WHO classification (Louis et al., 2007): pilocytic (WHO I), low-grade (WHO II), anaplastic astrocytomas (WHO III) and glioblastoma multiforme (WHO IV). In contrast to the longstanding and well defined histo-pathological criteria, the underlying molecular and genetic basis emerged only recently. In particular, several genes and pathways have been identified as being associated with tumorigenesis and with the anaplastic progression (Comincini, 2001). Adopting a high sensitive Real-time PCR based approach, doppel gene expression was investigated in large cohorts of patients with glial tumors. As a result, doppel expression was directly related to the malignancy of the tumor: highest in glioblastoma multiforme, lower in anaplastic astrocytomas, and even lower in low grade astrocytoma specimens, as graphically reported in Fig. 2.2 (Comincini et al., 2004). Extensive differences in doppel gene expression were also found within each grade of malignancy, suggesting that the quantification of doppel expression might be useful to distinguish astrocytoma subtypes. In addition, it was demonstrated that its expression is helpful in disease stratification and in the identification of patient subsets with specific molecular signatures (Comincini et al., 2007). Further molecular doppel gene expression investigations were then performed within human glial tumor specimens and in derived cell lines. These studies revealed that the


upregulated doppel transcripts underwent a significant nuclear retention process within the glial tumor cells, as shown in the image of Fig. 2.2 (Comincini et al., 2006b): this alternative post-transcriptional pathway might directly regulate the excess of potentially deleterious transcripts. In addition, this aberrant nuclear retention may have a functional meaning, as other genes with this trademark were described. In fact, nuclear mRNA retention is increasingly recognized as an important mechanism to regulate the activity of transcription related proteins and to modulate cell growth and death. For this reason, the export of nuclear mRNA is constantly challenged by the opposing force of mRNA retention from one side, and its decay from the other. This balance ensures that only perfect transcripts persist and that nonfunctional and potentially deleterious transcripts are differently regulated in their biogenesis. In detail, doppel human mRNA underwent alternative maturation processes within glial tumor cells, thus originating an alternative shorter transcript of 1.9 kb, significantly different from that originated in testis tissue (Comincini et al., 2006b). Altogether, these data might suggest that glial tumor cells overproduce doppel transcripts in primis, and that these transcripts are then subjected to an initial quality/quantity control through a nuclear retention process. In a similar manner, the expression pattern and the distribution of doppel were investigated in tumors with a non-glial origin and, in a former study, high levels of doppel transcripts were detected in gastric adenocarcinoma and in anaplastic meningioma specimens (Comincini et al., 2004). Additionally, Travaglino et al. (2005) investigated doppel expression in bone marrowderived cells of patients with acute myeloid leukaemia (AML) and with myelo-dysplastic syndrome (MDS). As a result, while doppel transcripts were barely detectable in normal bone marrow samples, AML and MDS cases exhibited a marked increase in doppel expression, particularly localized in blast-like cells with a significant phenomenon of nuclear retention of its transcripts. As a consequence of the ectopically expression of doppel gene in different tumor biopsies, one may hypothesize that the corresponding gene product belongs to the group of the cancer-testis antigens (CTA), that recently captured considerable interest (Zendman et al., 2003). In fact, along with its physiological expression restricted to germ cells of the testis that exclusively reappears in neoplastictransformed cells, the doppel gene reflects other typical

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Fig. 2.2 Doppel molecular and cellular signatures in human astrocytoma. Doppel molecular and cellular signatures in human astrocytoma. As described, doppel gene and protein expression levels increase following the glioma malignancy grading; a significant nuclear retention of the doppel transcripts has been previously reported (inset a), as well as an increase of

the complexity in the glycan moiety composition (Comincini et al., 2006b). Additionally, as illustrated (inset b), the cellular localization of the doppel protein shifts from plasma membrane to the cytosol, particularly within lysosome organelles (Sbalchiero et al., 2008)

features that characterise CTA, such as its belonging to a gene family and the single-exon ORF gene structure. To date, the molecular mechanisms responsible for doppel over-expression in transformed cells remain summarily defined. Doppel altered expression could merely be an epiphenomenon because of the widespread change in gene methylation patterns observed within different tumor types (Travaglino et al., 2005). To better delve into doppel gene regulatory mechanisms, investigations in different species were performed; in general, these experimental and bioinformatics computational analysis supported the concept that doppel gene expression is tightly regulated through an interplay of positive and negative cisacting factors that specifically recognize activating and

inhibiting elements in the promoter and in its surrounding sequence (Del Vecchio et al., 2005). Furthermore, doppel expression is affected by the methylation status of its promoter sequence, differently from the prion paralogue gene (Comincini, unpublished data). It is therefore conceivable that doppel is positively regulated in testis and negatively regulated in CNS. Interestingly, as a conclusive remark, the high expression profiles of doppel gene, physiologically in the first stage of the brain development and ectopically in glial tumor specimens, may indicate the neoplastic reacquisition of tumor cells of a primitive expression behaviour (Comincini et al., 2004).

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Doppel Protein in Astrocytomas Biochemical Features and Cellular Localization Doppel is a membrane protein that was initially identified as the first prion-like protein, due to its significant sequence homology with the cellular prion protein, as previously mentioned (Moore et al., 1999). In particular, the sequence analysis revealed that doppel is a truncated version of the prion protein, lacking the prion-typical octarepeat motifs, as graphically shown in the comparison between the secondary structures of Fig. 2.1b. Besides the protein sequence similarity, the two proteins share a number of structural and biochemical similarities: they covalently link a GPI anchor molecule for location to the outer leaflet of the cell membrane and, at the C-terminus, each protein has a super-imposable three-dimensional structure, characterized by three α-helices and by two short antiparallel β-sheets (Mo et al., 2001). Furthermore, the proteins were reported to bind copper ions with different affinity, but the physiological relevance of this biochemical property has not been clearly defined yet. Because of the already stated extensive similarities, doppel was primarily investigated as an alternative element to explore the physiological and pathological functions of the prion protein. As a consequence of these investigations, it was suggested that, instead of sharing similar activities, the two evolutionary-derived proteins appear to show different and possibly opposite activities, according to the “paralogue compensation process”. In particular, unlike the prion protein, doppel is not required for prion replication, and it is most likely unable to originate a pathogenic proteaseresistant isoform (Mo et al., 2001). Other functional differences between the two proteins were related to the adverse effect on neuronal viability and the proapoptotic behaviour of doppel (Qin et al., 2006), compared to the importance of prion protein expression in neuronal protection to oxidative stress, and in cell growth and maturation (Aguzzi and Polymenidou, 2004). In the glial tumor context, doppel protein showed peculiar features, graphically summarized in Fig. 2.2, compared to the physiological conditions, as derived from comparative testicular tissue examinations. Biochemical studies primarily highlighted that the


levels of astrocytoma doppel protein seem to increase with the tumor grade, showing a similar trend to that observed for the doppel transcripts (Chiarelli, unpublished data). In addition, the examination of astrocytoma surgical samples showed that doppel was present in the soluble fraction, whereas the corresponding protein in the testis tissue was detected in the microsomial fraction, as expected for a GPI-anchored membrane molecule. These differences in doppel localization between glial tumors and normal testis might reflect a complex recycling machinery of the astrocytoma membrane molecules, where GPI-linked proteins might have remarkably different traffic within the cells. It is known that GPI-proteins contribute to the overall organization of other membrane-bound proteins and they also play a critical role in a variety of receptor mediating signal transduction pathways. Another significant biochemical feature of the doppel protein in glioma cells was the presence of different glycoforms as revealed by Comincini and collaborators (unpublished data) in astrocytoma cell lines (2006) and further confirmed in tumor specimens. In particular, an abnormal post-translational maturation process, specifically an hyper-glycosylation, with a reduced content in sialic acids, resulted in a significant increase of the doppel protein molecular mass. It is known that protein-linked oligosaccharide moieties are crucial to serve diverse functions: they stabilize the proteins against denaturation and proteolysis, enhance solubility, modulate immune responses, facilitate orientation of proteins, confer structural rigidity to proteins, and regulate their turnover. Furthermore, it has long been predicted that the carbohydrate moieties of cell surface glycoproteins play important roles in the physical function and in the structural stability of the proteins. In the case of doppel, it has been demonstrated that the protein is subjected to post-translational modifications and that the doppel membrane localization in glioma cells seems not efficient and/or not well tolerated (Sbalchiero et al., 2008). Therefore, it is possible to suppose that altered glycosylation processes, common in cancers, can cause the altered localization of doppel, resulting in a shift from the membrane to the cytosol; therefore, this highly glycosylated protein was probably targeted directly to lysosomes where it accumulates for degradation. This parsimonious explanation of the catabolism of a potentially cytotoxic protein in the lysosomal compartments would not take into account that doppel-linked oligosaccharide moieties might be

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additionally important in conferring a tumor phenotype. For example, the absence of doppel expression at membranes or its rapid turnover within glioma cells might also contribute to a complex re-assortment of plasma membrane proteins, likely plausible in such cancers.

Functional Pathways and Interaction Analysis Recently the first interactome characterization map of the prion-protein family members was reported (Watts et al., 2009). This includes a cell-based quantitative analysis of neuroblastoma-derived interactors of prion, and of the two known paralogues, i.e., doppel and shadoo proteins. The results highlighted, in particular, that all prion-family members shared similar molecular and subcellular microenvironments (i.e., endoplasmic reticulum, Golgi and raft-like membranes), due to cointeraction with similar protein candidates. In detail, a reciprocal prion–doppel interaction has been reproposed, confirming the sharing of prion and doppel in common plasma membrane microdomains and in internalization pathways in neuroblastoma cells, as suggested by Massimino et al. (2004). The functional interplay between doppel and prion proteins was originally raised in prion-deficient mice, overexpressing doppel, after the rescue of the doppel-dependent ataxic phenotype, by the reintroduction of the prion transgene (Moore et al., 1999). In fact, the demonstration of the prion and doppel proteins interaction was of primary importance, considering the relevance of the cell membrane microdomains in the pathogenesis of prion and other neurodegenerative diseases. However, in literature, contrasting results on the putative interaction between the two proteins have been reported: whereas in neuronal cells the results supported an interaction, in testis this was not revealed, probably because the association of these proteins to different sub-cellular localizations could account for their different functions in each tissue. Similarly, in astrocytoma-derived cells a direct and detectable interaction between prion and doppel proteins was not documented; furthermore, doppel protein, even abundantly and ectopically expressed at the cytoplasmic level in such tumor cells, failed to physically interact with some prion-interacting proteins, such as the glial

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fibrillary acidic (GFAP) and the growth factor receptorbound 2 (Grb2) proteins (Azzalin et al., 2005). Because of the soluble form of doppel in glioma, one could hypothesize that if the prion–doppel complex gets disrupted, and/or malfunctions, the latter does not interact with prion thus reversing the beneficial effect of a neuro-protective signal of the prion protein towards doppel; as a consequence, doppel is sorted at the lysosomes, as described. However, the doppel–prion functional interplays, whether the derived proteins might physically interact and coparticipate or antagonize in biological processes, need to be finally delineated. Many results confirmed, at least in the CNS derived cells, the potential toxicity of the doppel gene product and a plausible cellular adaptive response in activating cell-death circuits. Within the brain, in particular in neuroblastoma N2a and in primary rat astrocytes, Qin et al. (2006) demonstrated that doppel caused apoptosis through a caspase-10 mediated mechanism, in a mitochondrion-independent manner, probably through a direct interaction with death cellular receptors. However, doppel itself showed a reduced apoptotic effect in neuronal cells expressing the prion protein, but a significant increase in cell death induction rate was reported in the same cells devoid of prion protein expression. In this scenario, other studies pointed to the direct involvement of the proapoptotic Bax protein in promoting the doppel induced apoptosis in transgenic murine Purkinje cells, deficient in prion protein expression. As a counterpart, further data supported the rescuing of the neuronal survival through the involvement of the anti-apoptotic Bcl2-dependent pathways, suggesting that the Bcl2-like property of the prion protein might impair doppel-induced neurotoxic effects (Heitz et al., 2008). To additionally complicate the functional networks involving doppel, more recent studies indicated that the expression of this protein in a neuronal context, in the absence of the prion protein counterpart, might also trigger autophagic cell death processes. It was therefore speculated that, as observed in amyloid neurodegenerative diseases, the doppel-induced upregulation of autophagic markers, resulted in extensive accumulation of autophagosomes, might likely reflect a progressive dysfunction of neuronal cells that finally lead to cell death (Heitz et al., 2010). Although doppel possesses an intrinsic toxic effect if not counteracted by another effector (for example the prion protein), astrocytoma cells acquired the

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ability to protect themselves from the doppel potential toxicity, delocalizing the protein from the plasma membrane towards the cytosol and to the lysosomes. For this reason, the peculiar localization of doppel in tumor astrocytic cells, focused the research of potential interactants within the cytosol as well as into specific organelles. In this regard, RACK1 (receptor for activated C-kinase), a well-characterized cytosolic adaptor molecule, was recently documented as a doppel interacting protein (Azzalin et al., 2006). This partner was identified among a group of doppel interactor candidates isolated from a glioblastoma-derived expression library, by means of a yeast two-hybrid assay. This interaction has also been subsequently verified by coimmunoprecipitation experiments and, interestingly, bioinformatic analysis underlined that doppel protein sequence shows conserved amino acids with the sequence of the PH (Pleckstrin protein) domain, an important domain contained in many cell signaling proteins that mediates the interaction with RACK1. In addition, RACK1 is homologous to the hetero-trimeric G protein ß-subunit, that regulates cell signaling via Src- and PKC (protein kinase C)-dependent pathways. In particular, it has been described to modulate the integrin-mediated cellular adhesion and migration (Kiely et al., 2008). RACK1 has a peculiar secondary structure composed of seven WD (tryptophan-aspartic acid) domains and, due to this conformation, it can manage contemporaneously many protein partners and coordinate different cell signaling inputs. In particular, the doppel-RACK1 interaction region was mapped between the C-terminal portion of doppel and the WD(1-4) domains of RACK1, respectively. It was, therefore, suggested that doppel might participate with RACK1 in a common molecular pathway involved in migration within astrocytic tumor context, where both proteins showed altered expression patterns. In this branch of research, the involvement of doppel in cell migration, as a typical hallmark of cancer, was investigated. The movement of cells is a sophisticated mechanism that is rigidly controlled by a spectrum of different genes during the various stages of embryonic and adult development. During carcinogenesis, the motile behaviour of cells is manifested without tight cellular controls and regulations and this causes tumor cells to spread throughout the healthy tissue; consequently, cancer cells motility plays a pivotal role in tumor invasion and in metastasis formation. In particular, in astrocytomas, the infiltration ability


of tumor cells constitutes one of the main causes of the unfortunate prognosis for these malignancies, and it explains the intensive efforts in aiming the development of novel therapies. Several key proteins are described as effectors of migratory ability of astrocytoma cells and deeper analysis of the signaling pathway involved in this process are assiduously carried out to discover novel molecular-based targets (Teodorczyk and Martin-Villalba, 2010). In this context, doppel protein was demonstrated to influence the cell migration, suggesting that this ability may be an intrinsic feature of the protein, independently from the biological scenario, as it was documented in different tumor cell lines, namely glioblastoma- and uterus carcinoma-derived cells (Azzalin et al., 2008). Of note, prion and doppel seemed to differently contribute to the migratory phenotype of tumor cells: in fact, it was demonstrated that doppel expression is related to migration and, in general, to the cell morphology condition, while the expression of the prion counterpart did not affect these phenotypes. In addition, an inverted correlation between migration and growth rates was reported in this study, as first described by Giese et al. (1996) as the paradigm of the “go or grow” dichotomy; however, the molecular and cellular mechanisms involved in these mutually exclusive phenotypes, are up to now not completely clarified. In the case of human astrocytomas, it might also be hypothesized that doppel is expressed mostly in the highly migratory cells of the external edge of the tumor area. In this context, doppel might influence cell migration by means of its membrane localization, promoting cell-to-cell contacts. However, doppel localization in these tumor cells could not directly sustain this speculation because of the prevalent cytoplasmic localization of the protein. For this reason, other protein partners and pathways should be considered to clarify the contribution of doppel in the cell migration process.

Conclusion Since its discovery in 1999, important data have emerged in the biology of the first prion-like protein, doppel. As the function of the cellular prion protein is still rather unclear, with the exception of the involvement of its pathological isoform within the TSEs, the

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study of the antagonistic roles of doppel and prion proteins in the neuronal cell survival remains of pivotal interest to directly discern the functions of doppel. Although these proteins share biochemical properties, doppel is unlikely to play a major role in prion diseases. The physiological function of doppel seems to be restricted to the male reproductive apparatus, in particular regulating male fertility. The functional involvement of doppel in a novel identified pathological scenario, such as tumors, in particular those deriving from glial cell transformation, would contribute in the near future to gain further insights into the molecular biology of such dramatic pathologies, possibly with the improvement of the molecular-assisted diagnosis and treatment.

References Aguzzi A, Polymenidou M (2004) Mammalian prion biology: one century of evolving concepts. Cell 116:331–327 Azzalin A, Del Vecchio I, Chiarelli LR, Valentini G, Comincini S, Ferretti L (2005) Absence of interaction between doppel and GFAP, Grb2, PrPC proteins in human tumor astrocytic cells. Anticancer Res 25:4369–4374 Azzalin A, Del Vecchio I, Ferretti L, Comincini S (2006) The prion-like protein doppel (Dpl) interacts with the human receptor for activated C-kinase 1 (RACK1) protein. Anticancer Res 26:4539–4548 Azzalin A, Sbalchiero E, Barbieri G, Palumbo S, Muzzini C, Comincini S (2008) The doppel (Dpl) protein influences in vitro migration capability in astrocytoma-derived cells. Cell Oncol 30:491–501 Comincini S (2001) Searching for molecular markers of human gliomas. Funct Neurol 16:291–298 Comincini S, Foti MG, Tranulis MA, Hills D, Di Guardo G, Vaccari G, Williams JL, Harbitz I, Ferretti L (2001) Genomic organization, comparative analysis, and genetic polymorphism of the bovine and ovine prion Doppel genes (PRND). Mamm Genome 12:729–733 Comincini S, Facoetti A, Del Vecchio I, Peoc’h K, Laplanche JL, Magrassi L, Ceroni M, Ferretti L, Nano R (2004) Differential expression of the prion-like protein doppel gene (PRND) in astrocytomas: a new molecular marker potentially involved in tumor progression. Anticancer Res 24:1507–1517 Comincini S, Del Vecchio I, Azzalin A (2006a) The doppel gene biology: a scientific journey from brain to testis, and return. Central Eur J Biol 1:494–505 Comincini S, Chiarelli LR, Zelini P, Del Vecchio I, Azzalin A, Arias A, Ferrara V, Rognoni P, Dipoto A, Nano R, Valentini G, Ferretti L (2006b) Nuclear mRNA retention and aberrant doppel protein expression in human astrocytic tumor cells. Oncol Rep 16:1325–1332 Comincini S, Ferrara V, Arias A, Malovini A, Azzalin A, Ferretti L, Benericetti E, Cardarelli M, Gerosa M, Passarin MG,

21 Turazzi S, Bellazzi R (2007) Diagnostic value of PRND gene expression profiles in astrocytomas: relationship to tumor grades of malignancy. Oncol Rep 17:989–996 Del Vecchio I, Azzalin A, Guidi E, Amati G, Caramori T, Uboldi C, Comincini S, Ferretti L (2005) Functional mapping of the bovine doppel gene promoter region. Gene 356: 101–108 Giese A, Loo MA, Tran N, Haskett D, Coons SW, Berens ME (1996) Dichotomy of astrocytoma migration and proliferation. Int J Cancer 67:275–282 Heitz S, Gautheron V, Lutz Y, Rodeau JL, Zanjani HS, Sugihara I, Bombarde G, Richard F, Fuchs JP, Vogel MW, Mariani J, Bailly Y (2008) BCL-2 counteracts Doppel-induced apoptosis of prion-protein-deficient Purkinje cells in the Ngsk Prnp(0/0) mouse. Dev Neurobiol 68:332–348 Heitz S, Grant NJ, Leschiera R, Haeberlé AM, Demais V, Bombarde G, Bailly Y (2010) Autophagy and cell death of Purkinje cells overexpressing Doppel in Ngsk Prnp-deficient mice. Brain Pathol 20:119–132 Kiely PA, Baillie GS, Lynch MJ, Houslay MD, O‘Connor R (2008) Tyrosine 302 in RACK1 is essential for insulin-like growth factor-I-mediated competitive binding of PP2A and beta1 integrin and for tumor cell proliferation and migration. J Biol Chem 283:22952–22961 Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, Scheithauer BW, Kleihues P (2007) The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol 114:97–109; Erratum in: Acta Neuropathol 2007. 114:547 Lührs T, Riek R, Güntert P, Wüthrich K (2003) NMR structure of the human doppel protein. J Mol Biol 326:1549–1557 Massimino ML, Ballarin C, Bertoli A, Casonato S, Genovesi S, Negro A, Sorgato MC (2004) Human Doppel and prion protein share common membrane microdomains and internalization pathways. Int J Biochem Cell Biol 36:2016–2031 Mastrangelo P, Westaway D (2001) The prion gene complex encoding PrPC and Doppel: insights from mutational analysis. Gene 275:1–18 Mead S, Beck J, Dickinson A, Fisher EM, Collinge J (2000) Examination of the human prion protein-like gene Doppel for genetic susceptibility to sporadic and variant CreutzfeldtJakob disease. Neurosci Lett 290:117–120 Mo H, Moore RC, Cohen FE, Westaway D, Prusiner SB, Wright PE, Dyson HJ (2001) Two different neurodegenerative diseases caused by proteins with similar structures. Proc Natl Acad Sci USA 98:2352–2357 Moore RC, Lee IY, Silverman GL, Harrison PM, Strome R, Heinrich C, Karunaratne A, Pasternak SH, Chishti MA, Liang Y, Mastrangelo P, Wang K, Smit AF, Katamine S, Carlson GA, Cohen FE, Prusiner SB, Melton DW, Tremblay P, Hood LE, Westaway D (1999) Ataxia in prion protein (PrPC )-deficient mice is associated with up-regulation of the novel PrPC -like protein doppel. J Mol Biol 292:797–817 Paisley D, Banks S, Selfridge J, McLennan NF, Ritchie AM, McEwan C, Irvine DS, Saunders PT, Manson JC, Melton DW (2004) Male infertility and DNA damage in Doppel knockout and prion protein/Doppel double-knockout mice. Am J Pathol 164:2279–2288 Peoc’h K, Serres C, Frobert Y, Martin C, Lehmann S, Chasseigneaux S, Sazdovitch V, Grassi J, Jouannet P, Launay JM, Laplanche JL (2002) The human “prion-like” protein

22 Doppel is expressed in both Sertoli cells and spermatozoa. J Biol Chem 277:43071–43078 Peoc’h K, Volland H, De Gassart A, Beaudry P, Sazdovitch V, Sorgato MC, Creminon C, Laplanche JL, and Lehmann S (2003) Prion-like protein Doppel expression is not modified in scrapie-infected cells and in the brains of patients with Creutzfeldt-Jakob disease. FEBS Lett 11:61–65 Prusiner SB (1998) Prions. Proc Natl Acad Sci USA 95: 13363–13383 Qin K, Zhao L, Tang Y, Bhatta S, Simard JM, Zhao RY (2006) Doppel-induced apoptosis and counteraction by cellular prion protein in neuroblastoma and astrocytes. Neuroscience 141:1375–1388 Rossi D, Cozzio A, Flechsig E, Klein MA, Rülicke T, Aguzzi A, Weissmann C (2001) Onset of ataxia and Purkinje cell loss in PrP null mice inversely correlated with Dpl level in brain. EMBO J 20:694–702 Sakaguchi S (2008) Antagonistic roles of the N-terminal domain of prion protein to doppel. Prion 3:107–111 Sbalchiero E, Azzalin A, Palumbo S, Barbieri G, Arias A, Simonelli L, Ferretti L, Comincini S (2008) Altered cellular

A. Azzalin and S. Comincini distribution and sub-cellular sorting of doppel (Dpl) protein in human astrocytoma cell lines. Cell Oncol 30:337–347 Teodorczyk M, Martin-Villalba A (2010) Sensing invasion: cell surface receptors driving spreading of glioblastoma. J Cell Physiol 222:1–10 Travaglino E, Comincini S, Benatti C, Azzalin A, Nano R, Rosti V, Ferretti L, Invernizzi R (2005) Overexpression of the Doppel protein in acute myeloid leukaemias and myelodysplastic syndromes. Br J Haematol 128:877–884 Watts JC, Huo H, Bai Y, Ehsani S, Jeon AH, Shi T, Daude N, Lau A, Young R, Xu L, Carlson GA, Williams D, Westaway D, Schmitt-Ulms G (2009) Interactome analyses identify ties of PrP and its mammalian paralogs to oligomannosidic N-glycans and endoplasmic reticulum-derived chaperones. PLoS Pathog 5:e1000608 Weissmann C, Aguzzi A (1999) Perspectives: neurobiology. PrPC’s double causes trouble. Science 286: 914–915 Zendman AJ, Ruiter DJ, Van Muijen GN (2003) Cancer/testisassociated genes: identification, expression profile, and putative function. J Cell Physiol 194:272–288

Chapter 3

Astrocytic Tumors: Role of Antiapoptotic Proteins Alfredo Conti, Carlo Gulì, Giuseppe J. Sciarrone, and Chiara Tomasello

Abstract Apoptosis is a fundamental anti-neoplastic mechanism to prevent tumorigenesis. Nearly all neoplastic changes during the development of a normal cell to a cancer cell, such as DNA-damage, oncogene activation or cell cycle deregulation, are potent inducers of the programmed cell death pathway. Therefore, overcoming the apoptotic failsafe is a key mechanism in the genesis and progression of tumors. In astrocytic brain tumors, the apoptotic failure has been documented and involves both the intrinsic or mitochondrial and extrinsic or receptor pathways of apoptosis. This breakdown may be caused by an imbalance of pro- and antiapoptotic members of the Bcl-2 protein family, inhibition of the activity of caspases by specific factors, changes in the p53 system. Detailed molecular knowledge of the anti-apoptotic mechanisms of astrocytic tumor cells is essential for the improvement of conventional chemotherapies and the development of new potent targeted therapies. In this chapter, authors describe the multiple antiapoptotic signals that have been demonstrated to be active in astrocytomas. Keywords Apoptosis · Tumorigenesis · Programmed cell death · FasL · TRAIL · TNF

Introduction The term programmed cell death was introduced by Lockshin and Williams (1964) to describe the destiny of some cells to die as driven by a cell-intrinsic program. Later, Kerr et al. (1972) introduced the term “apoptosis” to describe a series of common morphological features associated with this form of cell death, including cytoplasm shrinkage, membrane blebbing, nuclear fragmentation, intranucleosomal DNA fragmentation, phosphatidylserine exposure, and fragmentation into membrane-enclosed apoptotic bodies sequestered by macrophages or other engulfing cells. Apoptosis has a widespread biological significance, being involved in development, differentiation, proliferation/homeostasis, regulation and function of the immune system, and in the removal of defect and, therefore, harmful cells. Apoptosis is a fundamental anti-neoplastic mechanism in normal cells to prevent tumorigenesis. Nearly all neoplastic changes during the development of a normal cell to a cancer cell, such as DNA-damage, oncogene activation or cell cycle deregulation, are potent inducers of the programmed cell death pathway. Therefore, overcoming the apoptotic failsafe is observed in many cancers.

Death Ligands, Receptors and Messengers

A. Conti () Department of Neuroscience, University of Messina, Messina, Italy e-mail: [email protected]

The extrinsic apoptosis signalling is mediated by the activation of so-called death receptors which are cell surface molecules that transmit apoptotic signals after ligation with specific ligands (Fig. 3.1). The death ligands are members of the TNF superfamily, including: TNFα, CD95 ligand (CD95L), also known as Fas


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Fig. 3.1 Apoptosis is activated through two major signalling pathways. The first pathway is the intrinsic or mitochondrial pathway, because the mitochondria take the key position by initiating apoptosis. Initiation by different apoptotic stimuli is still not entirely clear, but likely involves an imbalance of proand antiapoptotic members of the Bcl-2 protein family. This imbalance finally leads to the activation of the proapoptotic Bcl-2 family members BAX and/or BAK and the perturbance of the integrity of the outer mitochondrial membrane. This induces the release of cytochrome c and other apoptotic regulators, like apoptosis-inducing factor (AIF), Smac (second mitochondria-derived activator of apoptosis)/DIABLO (direct inhibitor of apoptosis protein (IAP)-binding protein with low PI), endonuclease G or Omi/HtrA2 from the intermembraneous space of mitochondria. In the cytosol, cytochrome c binds to monomeric APAF-1 which then, in a dATP-dependent conformational change, oligomerizes to assemble the apoptosome, a complex of wheel-like structure with 7-fold symmetry that triggers the activation of the initiator procaspase-9. Caspase9 provokes the cleavage of the executioner caspases, such as caspase-3. Furthermore, the potent endogenous inhibitors of caspases, the inhibitor of apoptosis proteins (IAPs), are neutralized by Smac/DIABLO or Omi/HtrA2. The second pathway is the extrinsic pathway. Extrinsic apoptosis signaling is mediated by the activation of so-called “death receptors”, which are cell surface receptors that transmit apoptotic signals after ligation with specific ligands. Death receptors belong to the tumor necrosis factor receptor (TNFR) gene superfamily, including TNFR-1, Fas/CD95, and the TRAIL receptors DR-4 and DR-5. All members of the TNFR family consist of cysteine rich extracellular

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subdomains which allow them to recognize their ligands with specificity, resulting in the trimerization and activation of the respective death receptor. Subsequent signalling is mediated by the cytoplasmic part of the death receptor which contains a conserved sequence termed death domain (DD). Adapter molecules like FADD or TRADD themselves possess their own DDs by which they are recruited to the DDs of the activated death receptor, thereby forming the so-called death inducing signaling complex (DISC). In addition to its DD, the adaptor FADD also contains a death effector domain (DED), which through homotypic DED-DED, interaction sequesters procaspase-8 to the DISC. The local concentration of several procaspase-8 molecules at the DISC leads to their autocatalytic activation and release of active caspase-8. Active caspase-8 then processes downstream effector caspases which subsequently cleave specific substrates resulting in cell death. Cells harboring the capacity to induce such direct and mainly caspase-dependent apoptosis pathways were classified to belong to the so-called type I cells. In type II cells, the signal coming from the activated receptor does not generate a caspase signalling cascade strong enough for execution of cell death on its own. In this case, the signal needs to be amplified via mitochondria-dependent apoptotic pathways. The link between the caspase signalling cascade and the mitochondria is provided by the Bcl-2 family member Bid. Bid is cleaved by caspase-8 and in its truncated form (tBID) translocates to the mitochondria where it acts in concert with the proapoptotic Bcl-2 family members BAX and BAK to induce the release of cytochrome c and other mitochondrial proapoptotic factors into the cytosol (modified from Ashkenazi, 2002)

3 Astrocytic Tumors: Role of Antiapoptotic Proteins

ligand (FasL) or Apo-1 ligand, and Apo-2 ligand/TNFrelated apoptosis-inducing ligand (Apo2L/TRAIL). The FasL and Apo2L/TRAIL act on target cells through binding their specific receptors CD95 and death receptors (DR)4/DR5, respectively. Between ligands and effector caspases, there are a number of factors that suppress apoptosis. The most proximal step to suppress a death receptor pathway is inhibition of ligand binding. This could be achieved by the lack of or mutations in death receptors or the presence of antagonistic (decoy) receptors. In glioma cells, the existence of both agonistic and decoy receptors for TNF has been demonstrated: agonistic Fas receptors are present on the glioma cells (Rieger et al., 1998), but a decoy soluble receptor (DcR3) for FasL is also released by glioma cells, likely protecting them from the FasL-induced apoptosis. The efficacy of TNF-induced apoptosis is enhanced by protein synthesis inhibition, which points to the role of expression of factors with specific antiapoptotic function. The TNF-to-TNFR ligation provokes the formation of the DISC, initiating the apoptotic cascade (Fig. 3.1). However, such activation corresponds to a concurrent and parallel activation of the transcription factor nuclear factor (NF)-κB. The nuclear factor-κB is a dimeric transcription factor controlling the expression of several regulators of immune, inflammatory, and acute phase responses (Conti et al., 2007). A role of NF-κB in the genesis and progression of cancer has also been demonstrated; in particular a constitutive NF-κB activation has been described in a variety of epithelial and lymphoid cancers. As seen in other cell systems, TNF-induced NF-κB activation in astrocytoma cells may be mediated by the TNFR-associated factor (TRAF) family, which consists of a group of six adapter proteins (TRAF1–TRAF6) that participate in the intracellular signalling activity of several members of the TNFR superfamily. Through appropriate ligand stimulation of TNFRs found on the surface of these cells, TRAF proteins can induce activation of NF-κB, resulting in both cytokine secretion and resistance to apoptosis (Conti et al., 2005). The signal transduction mechanism emanating from the TNFR is thought to be mediated by TRAF2, a signalling intermediate that has been shown to be recruited to the cytoplasmic tail of TNFR through a TNFR/TRADD/TRAF2 interaction. On the basis of this hypothesis, TNF can either induce apoptosis through FADD (FAS associating protein with death domain) and caspase recruitment or promote survival

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through TRAF2 recruitment and NF-κB induction. As the cytosolic NF-κB concentration rises, the expression of several antiapoptotic genes is amplified. Candidate antiapoptotic genes for NF-κB induction include, cIAP (inhibitor of apoptosis) 1 and 2, Bcl-2, Bcl-X L, XIAP, and survivin. Characteristics and the role of these antiapoptotic factors will discussed in specific sections in this chapter. There are other possible anti-apoptotic mechanisms related to the death ligands and receptors. The U373MG cell line appears to be resistant to death receptor mediated apoptosis due to lack of crucial signalling components. Expression of caspase-8 sensitizes this cell line to FasL and TRAIL mediated apoptosis. Furthermore, recent studies have reported methylation of the caspase-8 gene as a mechanism for decreased levels of protein expression in neuroblastomas, rendering cells resistant to apoptosis (Teitz et al., 2000). Recent progress in the understanding of the varying susceptibility of glioma cell lines to Apo2L/TRAILinduced apoptosis has revealed that resistant cell lines expressed 2-fold higher levels of the apoptosis inhibitor phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-15 kDa (PED/PEA-15). This phosphoprotein protects astrocytes from TNF-α-induced apoptosis through interruption of FADD-Caspase-8 binding (Condorelli et al., 1999). Preclinical studies have established the potential for using TNF factors as a therapy in gliomas. In the field of death ligands and receptors, inducing cancer cell apoptosis via local or systemic application of Apo2L/TRAIL is one of the most promising strategies. Local injection of TRAIL exerted strong antitumor activity on intracranial human malignant glioma xenografts in athymic mice without neurotoxicity (Roth et al., 1999). However, a significant number of glioma cell lines remain resistant to TRAIL when it is used as monotherapy. In combination with conventional DNA-damaging chemotherapy, TRAIL showed synergistic cytotoxicity for human gliomas in vivo and in vitro. Of particular concern is the fact that TRAIL and FasL administration have been shown to have profound toxicity toward normal human hepatocytes, resulting in a massive and rapid induction of cell death. The local application of adenoviral vectors expressing FasL may be a strategy to circumvent systemic side effects in gliomas. Transferring the gene encoding FADD into glioma cells also inhibits glioma growth in vitro and in vivo. Finally, even more down-stream

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effectors of death receptor-mediated apoptosis, the caspases, have been successfully employed to promote glioma cell death.

The p53 The p53 is a nuclear phosphoprotein which acts as a tumor suppressor. The gene for p53 is located on the short arm of chromosome 17 at 17p13.105-p12. The open reading frame of p53 encodes for a protein with 393 amino-acids (53 kDa). The central region of the protein contains the DNA-binding domain. The structure of p53 consists of a large beta-sandwich which encompasses three loop-based elements and is composed of two anti-parallel beta-sheets encompassing four and five beta-strands, respectively. The first loop binds to DNA within the major groove and the second loop binds to DNA within the minor groove. The function of the third loop is stabilization of the second loop. p53 has been considered “the guardian of the genome” because it plays a key role in several processes of cellular physiology including control of cell cycle, genome stability, senescence, angiogenesis, and induction of apoptosis. Those activities are promoted through pathways that are both dependent and independent by transcription regulation. Mutations involving the p53 gene are the most common among those occurring in cancer, with more than one half of human cancers expressing a mutant p53. Particularly, it is the DNA-binding domain of the protein to be frequently modified in the neoplastic cells. p53 exerts its function only after the constitution of homo-oligomeric complexes (homo-tetramers). A single mutation for each tetramer is sufficient to compromise the possibility of DNA-binding and interaction. Wild-type p53 has a shorter half-life (∼20 min) than mutated p53 (3–7 h). This means that in cancer, even in heterozygosis (a normal TP53 allele and a mutated TP53 allele) p53 homotetramers are formed often by mutant monomers. The physiological role of p53 in normal cells depends on correct expression of regulator proteins with activation and inhibition functions.

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including gliomas. MDM2 encodes for p90MDM2 , which negatively regulate p53 by promoting its degradation through translocation from cell nucleus into cytoplasm, where p53 is a target of the proteasome 26S. Proteasomes 26S are large catalytic complexes responsible for extra-lisosomial endocellular proteolysis. In senescent cells, p90MDM2 expression is downregulated, and the inhibition of p53 is also depressed. Conversely, in gliomas amplified MDM2 inhibits p53 activity. With an opposite mechanism the p53 activity is stabilized by other factors. When a DNA damage occurs due to radiation or others toxic agents, it activates DNA-dependent protein kinase (DNAPK), ataxia telangiectasia mutated (ATM), ATM- and Rad3-related (ATR) kinases. DNA-PK is a nuclear serine/threonine kinase composed of a catalytic subunit and a DNA binding subunit. It has close interaction with p53, because they form a protein complex that preferentially binds to abnormal DNA structures. Also ATM and ATR can be activated by DNA damage, although the mechanism is not exactly known. These proteins can phosphorylate p53 on a specific serine residue in 15 position avoiding its degradation by the product of MDM2, p90MDM2 . This stabilization increases p53 half-life and its intranuclear concentration, activating p21, also known as cyclin-dependent kinase inhibitor 1A (CKN1A). The p21 is a gene located on chromosome 6 (6p21.2), and encodes a potent cyclin-dependent kinase inhibitor that is able to bind to and inhibit the activity of cyclin-CDK2 or -CDK4 complexes, major regulators of cell cycle progression at G1. In this way, raised p21 activity arrests the cell cycle in G1 phase (Fig. 3.2). Stabilization of p53 also leads to activation of transcription of several genes involved in apoptosis such as GADD45, PCNA, WAF1/CIP1, MDM2, BAX, NOXA, PUMA, KILLER/DR5, PIG, Caspase-1, and inhibition of others such as Bcl-2 and survivin. Particularly, the key role in apoptosis is repression of Bcl-2 and survivin, and raising the activity of BAX in a manner discussed in other sections.

P53 and Cell Cycle Progression Regulation of p53 Activity p53 activates transcription of the Murine Double Minute 2 (MDM2) gene. This is amplified in many tumors

Blockage of cyclin-cdk2 -cdk4 modulated by increased p21 level is also involved in regulating the activity of Rb protein. This is a tumor suppressor that is

3 Astrocytic Tumors: Role of Antiapoptotic Proteins

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Fig. 3.2 Mechanism of p53 control of cell cycle progression. Amplification of MDM2 occurs in many tumors including glioma and is the most important inhibiting factors of p53. On the other hand, DNA-pk stabilizes p53 increasing its half-life and its intranuclear concentration. P53 activates p21. p21 is a potent cyclin-dependent kinase inhibitor that is able to bind to and inhibit the activity of cyclin-CDK2 or -CDK4 complexes, major regulators of cell cycle progression at G1. The complex cyclin D1-CDK4 can cause the hyper-phosphorylation of

the retinoblastoma protein (Rb) causing the progression of the cell cycle. When Rb is hypophosphorylated, it keeps locked the E2F-DP complex, and the cell remains in the G1 stage. Hyperphosphorylation of Rb determines dissociation of E2F-DP from Rb and its activation. Unbound E2F activates factors like cyclins (e.g. cyclin E and A), which push the cell through the cell cycle by activating cyclin-dependent kinases

dysfunctional in many cancer types. The name Rb derives from the retinoblastoma, the tumor in which this protein was identified. It is encoded by the RB1 gene that is located on chromosome 13 (13q14.1q14.2). Because it is a tumor suppressor gene, both alleles must be mutated for the development of cancer. Rb can be hyper-phosphorylated or hypophosphorylated. Hyper-phosphorylation, due to raised activity of cyclin-cdk4, inhibits Rb. On the other hand, hypophosphorylated Rb represents the active form, and it is able to inhibit cell cycle progression by binding and inhibiting the transcription factors of the E2F family. E2F is a transcription factor widely believed to integrate cell-cycle progression with the transcription apparatus through its cyclical interactions with important regulators of the cell cycle, such as Rb, cyclins and cyclin-dependent kinases. E2F exerts its role by binding specific DNA sequences to the promoter of the target genes. E2F family protein has a complex structure that presents some domains. A specific domain is responsible for binding with a protein called DP, that functions as an inhibitor of E2F. As long as Rb is hypophosphorylated, it keeps locked E2FDP complex, and the cell remains in the G1 stage. Hyper-phosphorylation of Rb determines dissociation of E2F-DP from Rb and its activation. Unbound E2F

activates factors such as cyclins (e.g. cyclins E and A), which push the cell through the cell cycle by activating cyclin-dependent kinases, and a molecule called proliferating cell nuclear antigen (PCNA) which favors the DNA replication.

P53 and p16/INKa4 In glioblastoma and anaplastic astrocytomas, a correlation between p53, MDM2, p16/INKa4, PTEN, and EGFR and survival rates seem to be present. Loss of p16/INKa4 is essential for maintenance of the transformed neoplastic phenotype (Shapiro et al., 1995). This property of p16/INKa4 protein suggests that it is a tumor suppressor gene product that exerts its function in association with p53, both of which inhibit cyclindependent kinases involved in the Rb pathway. It is probable that cells unable to produce p16/INKa4 protein may be vulnerable to neoplastic transformation. p16/INKa4 protein is a potent inhibitor of cdk-4 that blocks cdk4-mediated phosphorylation of the tumor suppressor Rb protein, allowing Rb-mediated growth suppression (Fig. 3.2). The cdk4/cyclin D1 complex phosphorylates the Rb protein, thereby inducing release of the E2F transcription factor that activates

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genes involved in the G1 to S transition. p16/INKa4 binds to CDK4, inhibits the cdk4/cyclin D1 complex, and thus inhibits the G1 to S transition. Thus, loss of normal RB1 function may result from altered expression of any of the RB1, p16/INKa4, or Cdk4 gene. This means that pathways of both p53 and p16/INKa4 have a convergence of activity on the pRB protein.

P53 and PTEN A broad relationship between p53 and PTEN (phosphatase and tensin homolog deleted on chromosome 10) has been described. PTEN is a tumor suppressor gene whose mutation was found in various types of sporadic tumors, and was originally described in malignant gliomas. The promoter of PTEN has a p53 binding site. In fact, wild-type p53 can promote PTEN transcription, whereas TGF-β down-regulates it. The protein encoded by PTEN is involved in the regulation of many important cellular functions, such as cellcycle progression, cell migration and spreading, cell growth, and apoptosis. As p53 modulates transcription of PTEN gene, the protein protects p53 from MDM2mediated degradation through a PI3K/Akt pathway. In fact, there is a positive feedback between p53 and PTEN which aims to control the cellular response to stress, DNA damage, and cancer. PTEN has a phosphatase activity against phosphoinositide substrates; it dephosphorylates, with high activity, the 3 -OH position of the inositol ring of phosphatidylinositol phosphates, in particular of phosphatidylinositol 3-phosphate (PIP3), thereby acting as the counterpart of phosphoinositide-3-kinase (PI3K). Actually, PI3K phosphorylates phosphatidynositol4,5-bisphosphate to the respective 3-phosphate (PIP3), which functions as a second messenger molecule that is important for the activation of protein kinase B/Akt (for reviews, see Vanhaesebroeck and Alessi, 2000). In addition, PIP3 facilitates the translocation of Akt to the plasma membrane and activates PDK1, which in turn phosphorylates Akt on threonine 308 within the kinase domain. Activated Akt in turn can phosphorylate a variety of substrates and thereby regulates important cellular processes, including cell-cycle progression, cell growth, cell survival, cell motility and adhesion, translation of mRNA into protein, glucose metabolism, and angiogenesis. PTEN may influence the activity

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of several cellular signalling pathways other than the PI3K/Akt pathway. For example, the lipid phosphatase activity of PTEN may also contribute to the inhibition of Ras and MAPK pathway activation by EGF. In gliomas, PTEN mutations are preferentially found in glioblastomas, with reported frequencies of up to 40%. PTEN mutations are frequent in primary (de novo) glioblastomas, less frequent ( 4.2 (p value = 0.0007) while the shorter progression-free survival was 6.7 times increased in patients with WHO-grades III and IV compared to WHO grades I and II (p value = 0.05). These results support the hypothesis that MR-derived perfusion parameters may be comparable or even

Fig. 24.1 T2-weighted fluid-attenuation inversion recovery (FLAIR) MR image (a) and color-coded cerebral blood volume (CBV) map (overlaid on the corresponding FLAIR image) (b) of a male patient with a supratentorial astrocytoma (WHO grade IV) in the left hemisphere shows a markedly hyperperfused area

in the tumor. Progression-free survival curves in patients with low- and high-grade astrocytomas have shown the significantly longer time to progression by dichotomizing the patients according to a rCBVmax cut-off value of 4.2, group 3 WHO-grade = III-IV and rCBVmax ≤ 4.2, and group 4 WHO-grade = III-IV and rCBVmax > 4.2. The results showed that the histopathological grading-CBV joint classification provided statistically significant results for both the one-year survival and recurrence (Bisdas et al., 2009). Using this classification system, the patients in groups 1 and 3 had a significantly higher progression-free survival (761 days) compared to the progression-free survival in patients in group 4 (135 days) (p value = 0.0001). The patients with high-grade astrocytomas and rCBVmax > 4.2 had a 20 times increased relative risk for a shorter progression-free survival than the patients with astrocytomas and low rCBV, regardless of the grade (p value < 0.0001). The rCBVmax in patients stratified in groups 1 and 3 (2.11 ± 0.99) was significantly lower than the rCBVmax in patients of group 4 (6.98 ± 2.38) (p value = 0.0005) (Bisdas et al., 2009). Thus, it seems that the combination of rCBV values and histological grading appears to provide the strongest predictive value for recurrence/progression and this issue has to be elucidated in future studies. There is also initial evidence that the rCBV measurements may not only have a predictive role as a baseline parameter but the longitudinal changes in the rCBV values obtained after combined radiation and temozolamide therapy may correlate with the overall survival (Mangla et al., 2010). Specifically, increased rCBV after treatment was shown to be a strong predictor of poor survival (median survival, 235 days versus 529 days with decreased rCBV) (p value < 0.008, log-rank test). The ROC curves for 1-year

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survival also showed a greater area under the curve (p value = 0.005) with rCBV than with tumor size. The longitudinal perfusion-weighted MR imaging has also recently received increased attention as the significant increases in rCBV up to 12 months before contrast enhancement appearance on T1-weighted images may predict the malignant transformation of low-grade astrocytomas and thus, the survival rates (Danchaivijitr et al., 2008), though other authors advocate conventional imaging criteria, like the six-month tumor growth, as more important (Brasil Caseiras et al., 2009). In conclusion, preoperative grading of gliomas based on conventional MR imaging is often unreliable, whereas histopathologic grading frequently suffers from inherent shortcomings. Independently and in combination with histopathology, rCBV measurements on pre- and postreatment MR imaging scans in patients with astrocytomas demonstrate the significance of cerebral blood volume estimates as a clinical, objective biomarker for predicting the survival and recurrence in these patients. The combined assessment of histopathologic and perfusion MR imaging findings obtained before the inception of treatment may be useful to determine optimal management strategies in patients with high-grade astrocytomas. On the other hand, the response to therapy as assessed by CBV measurements may also detect the early non-responders and thus, lead to a modification of the initial therapy regimen. Threshold values, as proposed in literature and under consideration of possible methodological flaws, may be used in a clinical setting to evaluate tumors preoperatively for histologic grade and provide a means for guiding treatment and predicting postoperative patient outcome. Ongoing data collection from longitudinal studies is crucial to determine if rCBV can, in the long run, be superior to histopathologic examination in predicting tumor behavior and patient prognosis.

References Aronen HJ, Glass J, Pardo FS, Belliveau JW, Gruber ML, Buchbinder BR, Gazit IE, Linggood RM, Fischman AJ, Rosen BR (1995) Echo-planar MR cerebral blood volume mapping of gliomas. Clinical uitility. Acta Radiol 36:520–528 Bisdas S, Kirkpatrick M, Giglio P, Welsh C, Spampinato MV, Rumboldt Z (2009) Cerebral blood volume measurements by

220 perfusion-weighted MR imaging in gliomas: ready for prime time in predicting short-term outcome and recurrent disease? AJNR Am. ANJR Am J Neuroradiol 30:681–688 Brasil Caseiras G, Ciccarelli O, Altmann DR, Benton CE, Tozer DJ, Tofts PS, Yousry TA, Rees J, Waldman AD, Jager HR (2009) Low-grade gliomas: six-month tumor growth predicts patient outcome better than admission tumor volume, relative cerebral blood volume, and apparent diffusion coefficient. Radiology 253:505–512 Brookes MJ, Morris PG, Gowland PA, Francis ST (2007) Noninvasive measurement of arterial cerebral blood volume using Look-Locker EPI and arterial spin labeling. Magn Reson Med 58:41–54 Bruening R, Kwong KK, Vevea MJ, Hochberg FH, Cher L, Harsh GRt, Niemi PT, Weisskoff RM, Rosen BR (1996) Echo-planar MR determination of relative cerebral blood volume in human brain tumors: T1 versus T2 weighting. AJNR Am J Neuroradiol 17:831–840 Caseiras GB, Chheang S, Babb J, Rees JH, Pecerrelli N, Tozer DJ, Benton C, Zagzag D, Johnson G, Waldman AD, Jager HR, Law M (2010) Relative cerebral blood volume measurements of low-grade gliomas predict patient outcome in a multi-institution setting. Eur J Radiol 73:215–220 Chaskis C, Stadnik T, Michotte A, Van Rompaey K, D’Haens J (2006) Prognostic value of perfusion-weighted imaging in brain glioma: a prospective study. Acta Neurochir (Wien) 148:277–285, discussion 285 Danchaivijitr N, Waldman AD, Tozer DJ, Benton CE, Brasil Caseiras G, Tofts PS, Rees JH, Jager HR (2008) Low-grade gliomas: do changes in rCBV measurements at longitudinal perfusion-weighted MR imaging predict malignant transformation?. Radiology 247:170–178 Dhermain F, Saliou G, Parker F, Page P, Hoang-Xuan K, Lacroix C, Tournay E, Bourhis J, Ducreux D (2010) Microvascular leakage and contrast enhancement as prognostic factors for recurrence in unfavorable low-grade gliomas. J Neurooncol 97:81–88 Emblem KE, Scheie D, Due-Tonnessen P, Nedregaard B, Nome T, Hald JK, Beiske K, Meling TR, Bjornerud A (2008) Histogram analysis of MR imaging-derived cerebral blood volume maps: combined glioma grading and identification of low-grade oligodendroglial subtypes. AJNR Am J Neuroradiol 29:1664–1670 Fuss M, Wenz F, Essig M, Muenter M, Debus J, Herman TS, Wannenmacher M (2001) Tumor angiogenesis of low-grade astrocytomas measured by dynamic susceptibility contrastenhanced MRI (DSC-MRI) is predictive of local tumor control after radiation therapy. Int J Radiat Oncol Biol Phys 51:478–482 Galban CJ, Chenevert TL, Meyer CR, Tsien C, Lawrence TS, Hamstra DA, Junck L, Sundgren PC, Johnson TD, Ross DJ, Rehemtulla A, Ross BD (2009) The parametric response map is an imaging biomarker for early cancer treatment outcome. Nat Med 15:572–576 Hacklander T, Reichenbach JR, Modder U (1997) Comparison of cerebral blood volume measurements using the T1 and T2∗ methods in normal human brains and brain tumors. J Comput Assist Tomogr 21:857–866 Hirai T, Murakami R, Nakamura H, Kitajima M, Fukuoka H, Sasao A, Akter M, Hayashida Y, Toya R, Oya N, Awai K, Iyama K, Kuratsu JI, Yamashita Y (2008) Prognostic

S. Bisdas value of perfusion MR imaging of high-grade astrocytomas: long-term follow-up study. AJNR Am J Neuroradiol 29:1505–1510 Kassner A, Annesley DJ, Zhu XP, Li KL, Kamaly-Asl ID, Watson Y, Jackson A (2000) Abnormalities of the contrast re-circulation phase in cerebral tumors demonstrated using dynamic susceptibility contrast-enhanced imaging: a possible marker of vascular tortuosity. J Magn Reson Imaging 11:103–113 Knopp EA, Cha S, Johnson G, Mazumdar A, Golfinos JG, Zagzag D, Miller DC, Kelly PJ, Kricheff II (1999) Glial neoplasms: dynamic contrast-enhanced T2∗ -weighted MR imaging. Radiology 211:791–798 Law M, Oh S, Babb JS, Wang E, Inglese M, Zagzag D, Knopp EA, Johnson G (2006) Low-grade gliomas: dynamic susceptibility-weighted contrast-enhanced perfusion MR imaging–prediction of patient clinical response. Radiology 238:658–667 Law M, Yang S, Babb JS, Knopp EA, Golfinos JG, Zagzag D, Johnson G (2004) Comparison of cerebral blood volume and vascular permeability from dynamic susceptibility contrastenhanced perfusion MR imaging with glioma grade. AJNR Am J Neuroradiol 25:746–755 Lev MH, Ozsunar Y, Henson JW, Rasheed AA, Barest GD, Harsh GRt, Fitzek MM, Chiocca EA, Rabinov JD, Csavoy AN, Rosen BR, Hochberg FH, Schaefer PW, Gonzalez RG (2004) Glial tumor grading and outcome prediction using dynamic spin-echo MR susceptibility mapping compared with conventional contrastenhanced MR: confounding effect of elevated rCBV of oligodendrogliomas [corrected]. AJNR Am J Neuroradiol 25:214–221 Li KL, Zhu XP, Checkley DR, Tessier JJ, Hillier VF, Waterton JC, Jackson A (2003) Simultaneous mapping of blood volume and endothelial permeability surface area product in gliomas using iterative analysis of first-pass dynamic contrast enhanced MRI data. Br J Radiol 76:39–50 Ludemann L, Grieger W, Wurm R, Wust P, Zimmer C (2006) Glioma assessment using quantitative blood volume maps generated by T1-weighted dynamic contrast-enhanced magnetic resonance imaging: a receiver operating characteristic study. Acta Radiol 47:303–310 Mangla R, Singh G, Ziegelitz D, Milano MT, Korones DN, Zhong J, Ekholm SE (2010) Changes in relative cerebral blood volume 1 month after radiation-temozolomide therapy can help predict overall survival in patients with glioblastoma. Radiology 256:575–584 Mills SJ, Patankar TA, Haroon HA, Baleriaux D, Swindell R, Jackson A (2006) Do cerebral blood volume and contrast transfer coefficient predict prognosis in human glioma? ANJR Am J Neuroradiol 27:853–858 Oh J, Henry RG, Pirzkall A, Lu Y, Li X, Catalaa I, Chang S, Dillon WP, Nelson SJ (2004) Survival analysis in patients with glioblastoma multiforme: predictive value of cholineto-N-acetylaspartate index, apparent diffusion coefficient, and relative cerebral blood volume. J Magn Reson Imaging 19:546–554 Paulson ES, Schmainda KM (2008) Comparison of dynamic susceptibility-weighted contrast-enhanced MR methods: recommendations for measuring relative cerebral blood volume in brain tumors. Radiology 249:601–613

24 Astrocytomas: Predicting Survival and Recurrence Using Cerebral Blood Volume Measurements Persigehl T, Wall A, Kellert J, Ring J, Remmele S, Heindel W, Dahnke H, Bremer C (2010) Tumor blood volume determination by using susceptibility-corrected DeltaR2∗ multiecho MR. Radiology 255:781–789 Pignatti F, van den Bent M, Curran D, Debruyne C, Sylvester R, Therasse P, Afra D, Cornu P, Bolla M, Vecht C, Karim AB (2002) Prognostic factors for survival in adult patients with cerebral low-grade glioma. J Clin Oncol 20:2076–2084 Saraswathy S, Crawford FW, Lamborn KR, Pirzkall A, Chang S, Cha S, Nelson SJ (2009) Evaluation of MR markers that predict survival in patients with newly diagnosed GBM prior to adjuvant therapy. J Neurooncol 91:69–81 Stupp R, Roila F (2009) Malignant glioma: ESMO clinical recommendations for diagnosis, treatment and follow-up. Ann Oncol 20(Suppl 4):126–128 Tzika AA, Astrakas LG, Zarifi MK, Zurakowski D, Poussaint TY, Goumnerova L, Tarbell NJ, Black PM (2004)

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Spectroscopic and perfusion magnetic resonance imaging predictors of progression in pediatric brain tumors. Cancer 100:1246–1256 Uematsu H, Maeda M, Sadato N, Matsuda T, Ishimori Y, Koshimoto Y, Kimura H, Yamada H, Kawamura Y, Yonekura Y, Itoh H (2001) Blood volume of gliomas determined by double-echo dynamic perfusion-weighted MR imaging: a preliminary study. AJNR Am J Neuroradiol 22:1915–1919 Wetzel SG, Cha S, Johnson G, Lee P, Law M, Kasow DL, Pierce SD, Xue X (2002) Relative cerebral blood volume measurements in intracranial mass lesions: interobserver and intraobserver reproducibility study. Radiology 224:797–803 Young R, Babb J, Law M, Pollack E, Johnson G (2007) Comparison of region-of-interest analysis with three different histogram analysis methods in the determination of perfusion metrics in patients with brain gliomas. J Magn Reson Imaging 26:1053–1063

Chapter 25

Electronic Patient-Reported Outcome Monitoring (ePROM) in Brain Tumour Patients Lisa M. Wintner, Johannes M. Giesinger, Gabriele Schauer-Maurer, and Bernhard Holzner

Abstract The diagnosis of brain tumour commonly goes along with short survival, bad outcome prognosis and strongly impaired health-related quality of life (HRQOL) due to the disease itself, anti-cancer treatment or ancillary medication. Patient-reported outcome monitoring (PROM) ensures the capture of individual problematic issues threatening patients’ HRQOL for a targeted and patient-tailored intervention. Practical barriers of PROM can be avoided by electronic data capture (ePROM), which saves time, human resources and can help to deepen patientphysician communication and to improve patients’ satisfaction with care. Furthermore, tele-monitoring incorporates usually neglected time points when patients are discharged from hospital and symptom burden is only partly communicated to physicians and nurses. For comprehensive health care of brain tumour patients the usage of ePROM and tele-monitoring could help to meet the patients’ needs. Keywords ePROM · quality of life · computerized · Brain tumours

Introduction In 2008, brain tumours and cancer of the nervous system ranged number 15 of the most often diagnosed cancer types within the European Union (Ferlay

B. Holzner () Department of Psychiatry and Psychotherapy, Innsbruck Medical University, Anichstr.35, 6020 Innsbruck, Austria e-mail: [email protected]

et al., 2010). Compared to the five most common cancer types (colorectum, breast, prostate, lung and bladder) the incidence rate of brain tumours appears to be small-sized, however, the mortality rate for brain and nervous systems cancers is in percentage terms higher than those of most high-incidencecancers. Survival expectance depends strongly on the WHO grade of the tumour and ranges from possibly no impact on life expectancy (grade I) to a survival of only 10 months (grade IV) (Reardon and Wen, 2006). Therefore, although brain cancer is only seldom diagnosed, patients experience an extremely high negative impact as survival prognosis is poor and deteriorated HRQOL is common (Taphoorn et al., 2010b). There is a large variety of brain tumours and their differentiation between primary tumours, which originate directly from brain tissue, and secondary tumours, which are brain metastasis from other malignant diseases, is often difficult to make. However, their distinction is of particular importance since the most favourable treatment modalities e.g. in glioblastoma and in brain metastasis differ strongly and inadequate intervention endangers patients’ HRQOL. The treatment possibilities of brain tumours include surgery, chemotherapy, radiotherapy and supplementary medical therapies. Not only the tumour itself, but also treatment-related side effects and adverse events caused by supportive medication (e.g. steroids and antiepileptic drugs) confront the patient with a high level of physical and psychosocial burden. Brain tumour patients are affected by a variety of tumour symptoms, which might be caused by e.g. increased intracranial pressure (e.g. headache, anorexia, nausea, fatigue, vomiting, seizures, sleeping longer at night, drowsiness, napping during the day). Other


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physical impairments may occur due to focal neurological deficits (e.g. motor deficit, aphasia, visual field defects). Even more burdensome both for patients and caregivers might be symptoms like personality changes, mood disturbances, decrease in mental capacity and concentration, cognitive dysfunction, anxiety and depression (Heimans and Taphoorn, 2002). These symptoms were found to negatively influence both patients’ and their carers’ overall QOL if compared to general population (Janda et al., 2007). As brain tumour patients cannot be cured in most cases, the treatment has to focus on maintaining HRQOL in at least the same extent as on life prolongation.

Patient-Reported Outcomes Patient-reported outcomes (PROs) allow the comprehensive evaluation of patients’ perception of aspects of functioning and well-being in regard to their health status, disease, and its treatment. The U.S. Food and Drug Administration presents a short and concise definition of PROs: “A PRO is a measurement of any aspect of a patient’s health status that comes directly from the patient (i.e., without the interpretation of the patient’s responses by a physician or anyone else).” (U.S. FOOD AND DRUG ADMINISTRATION, 2006). In this respect, PROs comprise a variety of directly reported health-related issues: disease-related symptoms, treatment-related adverse events, functioning, well-being, HRQOL, perceptions about treatment, satisfaction with care and professional communication (Rothman et al., 2007). The assessment of HRQOL issues include numerous domains like depression, anxiety, pain, fatigue, gastrointestinal symptoms, social functioning, or perceived cognitive dysfunction. PROs are therefore versatile in application possibilities, as they may be used for adverse event detection in drugsafety reports and medical product development, for HRQOL evaluation both in clinical research and routine, and for guidance in medical decision making. PRO-data may not only support patients in choosing between available treatments, but also enhance understanding of the disease and improve its treatment and the management of treatment-related symptom burden. The usage of PROs measurement provides several benefits, as patients namely favour the discussion of

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HRQOL domains with their clinician, but often do not take the initiative themselves. Concerning emotional functioning, daily activities and familial issues approximately 25–29% of patients wish their clinician to start discussion and even 37% of patients expect their clinician to address the topic of social functioning (Detmar et al., 2000). In this respect PROs can successfully contribute to satisfaction of clinicians and patients, as clinicians attain a deeper insight in patient’s needs and may properly address relevant topics without asking the patient a variety of possibly irrelevant questions during the face-to-face consultation. The usage of PROs may also sharpen clinicians’ awareness for psychological, social and spiritual functioning and diminish the domination of physical functioning in patientdoctor conversation. In clinical routine the usage of PROs already showed a number of benefits. Based on PRO-data clinicians adjusted the dosage of analgesics in a more sophisticated way than without PRO-data (Trowbridge et al., 1997). The patient-clinician communication was improved and enhanced concerning discussed domains, if clinicians took advantage of provided PRO-data. Furthermore patients felt more emotional support, clinicians became more sensitive for normally underestimated HRQOL domains (Detmar et al., 2002), patients were more satisfied with care and the building of a relationship to the clinician (Velikova et al., 2010) and reported a better QOL (Velikova et al., 2004). The literature for HRQOL in brain tumour patients grows considerably and reports a plenitude of results: they are partly consistent and matching but also puzzling and contradictory. HRQOL and its sub-domains are reported to depend on tumour location and laterality, on low- or high-grade classification, the tumour itself and its histological type. Derived from results of clinical trials the disease itself seems to have the major negative impact on HRQOL, which can be improved by treatment, whereby treatment side-effects may limit the extent of the benefits (Taphoorn et al., 2010b). Due to the seldom occurrence of brain tumour and the differentiated types of brain tumours, scientific investigations on large patient populations with a homogeneous diagnosis is very difficult to obtain. Regardless of this methodological constraint contemporary studies report brain tumour survivors to suffer from cognitive deficits and impairs HRQOL e.g. increased fatigue or depression (Taphoorn et al., 2010b). Only low baseline QOL scores are reported for brain tumour patients after

25 Electronic Patient-Reported Outcome Monitoring (ePROM) in Brain Tumour Patients

surgery and before adjuvant therapy. It is supposed that the site of disease (well operable or badly surgically accessible) influences patients’ QOL and not surgery itself (Budrukkar et al., 2009).

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Index (FBrSI) for the FACT-G) (see Table 25.1 for details).

EORTC QLQ-C30 and BN20 Cancer-Targeted and Brain Tumour-Specific PROs For HRQOL-assessment in cancer patients usually a generic PRO core-questionnaire is administered, which can be supplemented by disease-specific questionnaire-modules. Two internationally used, validated and reliable cancer-targeted instruments are the European Organization for Research and Treatment of Cancer core questionnaire (EORTC QLQ-C30) (Aaronson et al., 1993) and the Functional Assessment of Cancer Therapy general version (FACT-G) (Cella et al., 1993). Both instruments can be expanded with a brain cancer-specific module (BN20 for the EORTC QLQ-C30 and FACT-Br and the FACT-Br Symptom

The EORTC QLQ-C30 is a validated and widely used PRO instrument, which was originally developed to evaluate QOL in cancer patients participating in clinical trials (Aaronson et al., 1993). A modular approach was used for questionnaire design. The core questionnaire, which consists of thirty questions, is the basic part of the QOL-instrument and can be expanded by additional elements, which supplementary focus diagnosis-specific symptoms and impairments. The QLQ-C30 consists of five functioning scales (physical, role, social, emotional and cognitive functioning), a scale for global QOL, three symptom scales and six single item symptoms. Except for the two questions about general QOL every question has four response options: “not at all”, “a little”, “quite a bit” and “very

Table 25.1 Commonly used PRO instruments Instrument Scales

Val.

Items

Period

FACT-G Functional Assessment of Cancer Therapy-General

– – – –

Physical well-being Social/Family well-being Emotional well-being Functional well-being

x

27

1 week

FACT-Br Functional Assessment of Cancer Therapy-Brain Module

– – – – –

Cognitive functioning Neurological functioning Sensory functioning Psychological functioning Impact of changes in functioning on daily living.

x

23

1 week

FBrSI FACT-Br Symptom Index

– Symptom list

x

15

1 week

EORTC QLQ-C30 European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire Core 30

– Functioning (physical, role, social, emotional and cognitive) – Global QOL – Pain, fatigue, nausea/vomiting – Dyspnoea, sleeping disturbances, appetite loss, constipation, diarrhoea and financial impact

x

30

1 week

EORTC QLQ-BN20 European Organisation for Research and Treatment of Cancer Quality of Life Brain Module

– – – – –

x

20

1 week

Future uncertainty Visual disorder Communication deficit Motor dysfunction) Headache, seizure, drowsiness, hair loss, itching, weakness of both legs, difficulty controlling bladder function

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much”. The overall QOL scale asks the patient to rate how they experienced their general QOL and physical condition between 0 “very poor” and 6 “excellent”. Except the physical functioning scale, all questions shall be rated in regard to the week before the examination date. The EORTC Brain Cancer Module (BN20) contains 20 items, which focus on symptoms that particularly concern brain cancer patients under chemotherapy or radiotherapy. Therefore both disease symptoms and treatment toxicities are included in the item list. The items are grouped into four scales (future uncertainty, visual disorder, communication deficit, motor dysfunction) and seven single items (headache, seizure, drowsiness, hair loss, itching, weakness of both legs, difficulty controlling bladder function). The QLQ-BN20 proved adequate psychometric properties in multi-national and multi-lingual validation study (Taphoorn et al., 2010a).

FACT-G and FACT-Br/FBrSI The FACT-G is a cancer-specific PRO instruments provided by the Functional Assessment of Chronic Illness Therapy (FACIT) measurement system, which supplies questionnaires concerning a variety of chronic illnesses and their conditions (cancer, HIV/AIDS, multiple sclerosis) (Webster et al., 2003). Nowadays the fourth version of the FACT-G is widely used and comprises 27 items grouped into four primary QOL domains (physical well-being, social/family well-being, emotional well-being, and functional wellbeing). The patient has to rate each item on a five-point Likert-scale between “not at all”, “a little bit”, “somewhat”, “quite a bit” and “very much” in relation to symptom severity during the last week. The FACT-G was examined concerning its ease of administration, brevity, reliability, validity and responsiveness to clinical change and was found to fit all the stipulated requirements (Cella et al., 1993). The questionnaire’s validity was also tested for a variety of language versions (Sanchez et al., 2011). The brain cancer-specific FACT-Br module consists of 23 items and explores how patients perceive their cognitive, neurological, sensory and psychological functioning and the impact of changes in these domains on their daily living. The FACT-Br Symptom Index (FBrSI) is available for symptom

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rating of brain tumour patients. Following symptoms are included: headaches, seizures, weakness of extremities, self-caring issues, lack of energy, difficulties with expression, trouble with coordination, frustration due to limited agency, nausea, aphasia, hopelessness, social functioning, anxiety and enjoyment of life. Both the FACT-Br and FBrSI proved to be reliable, valid and responsive to change (Nickolov et al., 2005).

Computer-Based QOL Monitoring: ePROM Although many advantages of PRO assessments can be communicated, there are still popular counterarguments circulating. Even if PRO-data is available many clinicians do not pay attention to them because of lack of time, human resources and an adequate PRO instrument and the assumption that directly from patients obtained information does not add any additional value (Luckett et al., 2009). Furthermore some clinicians argue that information on HRQOL are not of same importance as treatment decisions, equality of PRO instruments is doubtful and the methodology of PRO measurement seems to be dubious (Barlesi et al., 2006). These objections can be devitalised, though. Meanwhile a broad variety of internationally validated and widely used PRO instruments is available (especially for cancer population the EORTC QLQ-C30 and the FACT-G with their supplemental modules). The use of PRO instruments does usually not or only a few minutes prolong the clinical appointment (Frost et al., 2007). Particularly the implementation of electronic patient-reported outcome monitoring (ePROM), which means the routinely and electronic collection of PRO-data on a systematic basis, solves the problem of time and resource constraints. Data are directly entered by patients, scores are calculated automatically and immediate information processing is possible. Electronic data capture was shown to need less time for instrument completion than paper-pencil versions (Velikova et al., 1999). The usage of ePROM is well accepted by patients (Mullen et al., 2004; Velikova et al., 2010), equivalent to paper-pencil versions (Coons et al., 2009) and valid (Abernethy et al., 2010). Also in brain tumour populations the usage of ePROM was feasible and useful for both patient


and clinician (Erharter et al., 2010; Holzner et al., 2011). Furthermore, the ePROM allows the use of computer-adaptive testing (CAT), which reduces the burden of PRO completion for patients, as the questionnaire is fitted to the patients’ individual needs and health condition. Depending on the answers to preceding questions appropriate following questions are chosen from a PRO-item bank. Currently a large project funded by the US-American National Institutes of Health (NIH) is developing generic computeradaptive PRO-instruments for use in the chronical ill (www.nihpromis.org). Administrative failure is reported as the most important reason for missing data (72.2%) in PRO-data collection in patients suffering from malignant glioma (Walker et al., 2003). About 21.7% of patients did not fill out the PRO-instruments due to their very bad health condition and only 6.1% of patients refused to participate in PRO-assessments. The major problem of administrative failures in this study can therefore be traced to irregular administration time points of PROs, too little explanation of instrument completion and missing specialized staff for QOL research who checks questionnaires completeness. Such barriers can easily be overcome with the routine usage of ePROM, which avoid a high percentage of missing data, as a standardized questionnaire explanation is integrated in the procedure and further questions are only displayed after all preceding questions have been answered. Additionally, the implementation of ePROM for the assessment of HRQOL or adverse events in clinical drug evaluation studies facilitates and accelerates data flow because complicated and defective data collection is cut short. Without ePROM clinicians ask patients about their symptoms and write a construed summary of these symptoms down in patients’ charts, from which research assistants collect information and consign them into research data bases (Trotti et al., 2007). This process is highly endangered to lose and/or alter information given by patients and needs much more human and time resources than the usage of ePROM. Admittedly, at the beginning of ePROM implementation some time burden may be set on clinicians and nurses, as they need to be trained in software handling and result interpretation, but after a phase of familiarization ePROM contributes in time saving. Firstly, the completion of a PRO instrument

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encourages the patient to reflect more detailed on his/her health status and symptom burden and facilitates in that way the communication between patient and clinician/nurse about relevant discussion areas. Secondly, ePROM is immediately accessible for the clinician, whose attention can be called to clinically relevant deteriorations or improvements. In fact, such an alert system helps clinicians to focus on issues important for the patient without long-winded enquiry of possible difficulties. Moreover, queue times of patients might meaningfully be padded by ePROM completion.

EPRO Software For obtaining the highest grade of feasibility and utility of PRO-assessments, the use of specialized software that fits perfectly the needs of both patients and clinicians is obligatory. In the last years there have been a few attempts on constructing and implementing a software solution for QOL-monitoring in clinical routine undertaken e.g. by Joerg Sigle (AnyQuest), Galina Velikova and Irma Verdonck (OncoQuest). These tools using touch screens have shown feasibility in daily clinical practice and implementation studies suggest some important benefits for the physicians, the patients and medical care. A further example for a specific software solution is the Computer-based Health Evaluation System (CHES). The CHES program has especially been developed for electronic PRO-data capture and offers a variety of useful features for clinical routine and research purposes. Any required paper-pencil questionnaire can be implemented into CHES to facilitate all steps from data collection to result calculation, interpretation and report generation as well. CHES provides a database (e.g. MySQL or Oracle) for supplementary medical and psychosocial data. This database is particularly useful for research purposes, as it improves study logistics, reduces the need for human resources and increased data quality. Database connection can be established via LAN or Wifi. Connection via LAN is more laborious as the study nurse needs to connect the tablet PC to the LAN with the purpose of preparing the patient list to who the questionnaire shall be administered. The tablet PC has to be disconnected, handed over to patients for bed-side assessment and again connected to the

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LAN for uploading the collected information into the database. A Wifi-connection is much more comfortable, as the database can be updated anytime without special constraints. Connection via WiFi eases work for both data collectors and clinicians, as data is transferred instantly and the immediate access to patients’ data is possible. In clinical routine the use of specialized software as CHES offers several advantages: it decreases the need of personal and time resources, diminished possible error sources and missing data, accelerates the calculation of PRO-data and immediately provides easily interpretable results by means of eyecatching and comprehensible graph charts. By means of touch screen equipped tablet PCs patients can easily complete the PRO assessment. Missing data is prevented by forwarding only to the remaining questions when all prior items were answered. The possibility to fit font and button sizes to the needs of different patients groups allow that readability and handling of the assessment is practicable for e.g. elderly people as well. As no computer literacy of patients is needed for PRO completion, in principle all patients are able to participate in ePROM. Due to the instantly performed data calculation, clinicians directly can be informed after instrument completion about clinically remarkable PRO-data that need adequate intervention. Furthermore individual PRO-data can be displayed as longitudinal as well as cross-sectional illustrations. The electronic administration of PROs by means of CHES was well accepted by brain tumour patients (Erharter et al., 2010). The on average required time of ten minutes for completion of the EORTC QLQ30 and the QLQ-BN20 even decreased with repeated instrument administration. Clinicians reported the ePRO to be beneficial, as loss of bladder controlling would not have been detected adequately without ePRO. Additionally CHES was implemented for routine HRQOL monitoring at the neuro-oncological outpatient unit of the Medical University Clinic of Neurology and reported to be feasible and profitable for patient care (Holzner et al., 2011).

Pro-Tele-Monitoring Although PROs and ePROM work hard on the routinely incorporation of the patients’ individual per-

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spective in clinical practice and cancer clinical trials, there is still a piece missing. The usage of ePROM should not be limited by the walls of hospitals and research institutions, but can also be extended into the domestic environment of patients. The step into the real world of patients is absolutely necessary to capture all possible time points, which can be of special importance for patients’ well-being and adequate medical assistance. Especially with regard to chemotherapy, side-effects and related symptom burden are known to be most severe a few days after application of cytostatic drugs (Hawkins and Grunberg, 2009); a time point at which most patients are already back at their home environment. This gap cannot be bridged by traditional ePROM, since periods without attendance of an in- or outpatient unit of the hospital are of interest. The missing knowledge about patients’ well-being at their home environment may lead to an underestimation of perceived symptom burden. This shortcoming can be overcome by a web-based assessment tool or phone-based home monitoring (so called tele-monitoring). Measuring HRQOL frequently may increase longitudinal information, and potentially enable clinicians to identify early signs of adverse events and thus intervene prospectively to minimize complications. Despite the early development-stage of telemonitoring, few studies document already its usage within oncological care. One study with patients who received hematopoietic stem cell transplantation stands out for its complex design and favourable results (Bush et al., 2005). They assessed QOL online on a daily basis by means of short and dynamic question-sets and monthly via a comprehensive questionnaire. Data show that not only the less sick patients were very compliant with online QOL, since only three patients discontinued study participation due to their bad health condition. The study reports high feasibility of telemonitoring, good patient compliance and high user satisfaction. The close-meshed assessment of patients’ health and well-being during their stays at home will require additional administrative resources. The internet, though, provides an inexpensive technology that allows a simple, user friendly and reliable data collection. Frequent HRQOL assessment is via tele-monitoring much easier and more inexpensive practicable than via traditional paper-pencil questionnaires. The use of tele-monitoring ensures that patients’ HRQOL outside the hospital setting is no


longer neglected, and that it can easily be integrated into symptom estimation, monitoring and treatment.

Usefulness of Proxy Ratings and Future Directions It is undoubtedly that ePROM must be restricted to patients who are able to report their experiences. Due to the nature of brain tumours the usage of ePROM is not possible in some patients over the whole course of disease, since cognitive impairments are next to a poor physical condition often occurring disease symptoms. Patients who suffer from a lack of concentration, thought disorder, communication deficits or visual disorders cannot successfully participate in ePROM. At this point, the usage of proxy-rating can be useful to maintain the assessment of the patients’ perspective for symptom management. Proxies like spouses, children, family members or close friends can bridge the gap in communication arising from patients’ restricted agency. Studies provide evidence, that agreement between patients and proxies are moderate and good, whereas proxies tend to underestimate patients perceived HRQOL. For patients with mental confusion, cognitive impairment, poor performance status and motor deficits only low agreement rates with significant others were found. Background characteristics like age, gender, culture, the relationship between patient and proxy or housing situation did not influence patient-proxy agreement. One study reveals a differentiation of HRQOL sub-domains in regard to patient-proxy agreement (Giesinger et al., 2009). More obvious issues and physical symptoms like physical functioning, sleeping disturbances, appetite loss, constipation, financial impact and taste alterations showed appropriate agreement. Less noticeable aspects like emotional functioning, cognitive functioning, fatigue, pain, dyspnoea and seizures had only low agreement between patients and proxies. To fully benefit from all advantages ePROM and tele-monitoring have to offer, the implementation of ePROM in clinical routine and telemonitoring in patient-home-assistance is one major step. Additionally, further research on ePROM in brain tumour patients has to focus on establishing reference scores for patients with different diagnoses undergoing

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different treatments. The steadily increasing role of ePROM in medical decision-making and the constantly ongoing software development encourages the use of these measures with neurooncological patients on a regular basis. A special focus should be put on evaluation studies to investigate in detail the impact of ePROM on medical care. Clinicians’ contribution to ePROM conduction and use of PRO-data is in the same extent necessary as patients’ compliance, and further research is needed to examine how ePROM can effectively and smoothly be integrated in clinicians’ daily routine. Although studies have already shown that routine ePROM usage is feasible, physicians and nurses in the neurooncological setting need to be directly addressed to play an active role in PRO-data collection and application in clinical practice. A sophisticated knowledge about the effect of ePROM on the patientphysician communication and on the administration of medical and psychosocial interventions would help to provide patient-tailored and satisfactory intervention and improve HRQOL.

References Aaronson NK, Ahmedzai S, Bergman B, Bullinger M, Cull A, Duez NJ, Filiberti A, Flechtner H, Fleishman SB, de Haes JC (1993) The European Organization for Research and Treatment of Cancer QLQ-C30: a quality-of-life instrument for use in international clinical trials in oncology. J Natl Cancer Inst 85(5):365–376 Abernethy AP, Zafar SY, Uronis H, Wheeler JL, Coan A, Rowe K, Shelby RA, Fowler R, Herndon JE 2nd (2010) Validation of the Patient Care Monitor (Version 2.0): a review of system assessment instrument for cancer patients. J Pain Symptom Manage 40(4):545–558 Barlesi F, Tchouhadjian C, Doddoli C, Astoul P, Thomas P, Auquier P (2006) Quality of life: attitudes and perspectives of doctors in a thoracic oncology regional care network. Sante Publique 18(3):429–442 Budrukkar A, Jalali R, Dutta D, Sarin R, Devlekar R, Parab S, Kakde A (2009) Prospective assessment of quality of life in adult patients with primary brain tumors in routine neurooncology practice. J Neurooncol 95(3):413–419 Bush N, Donaldson G, Moinpour C, Haberman M, Milliken D, Markle V, Lauson J (2005) Development, feasibility and compliance of a web-based system for very frequent QOL and symptom home self-assessment after hematopoietic stem cell transplantation. Qual Life Res 14(7):77–93 Cella DF, Tulsky DS, Gray G, Sarafian B, Linn E, Bonomi A, Silberman M, Yellen SB, Winicour P, Brannon J et al. (1993) The Functional Assessment of Cancer Therapy scale: development and validation of the general measure. J Clin Oncol 11(3):570–579

230 Coons S, Gwaltney C, Hays R, Lundy J, Sloan J, Revicki D, Lenderking W, Cella D, Basch EI. e. T. Force (2009) Recommendations on evidence needed to support measurement equivalence between electronic and paper-based patient-reported outcome (PRO) measures: ISPOR ePRO Good Research Practices Task Force report. Value Health 12(4):419–429 Detmar SB, Aaronson NK, Wever LD, Muller M, Schornagel JH (2000) How are you feeling? Who wants to know? Patients’ and oncologists’ preferences for discussing health-related quality-of-life issues. J Clin Oncol 18(18):3295–3301 Detmar SB, Muller MJ, Schornagel JH, Wever LDV, Aaronson NK (2002) Health-related quality-of-life assessments and patient-physician communication: a randomized controlled trial. JAMA 228(23):3027–3034 Erharter A, Giesinger J, Kemmler G, Schauer-Maurer G, Stockhammer G, Muigg A, Hutterer M, Rumpold G, Sperner-Unterweger B, Holzner B (2010) Implementation of computer-based quality-of-life monitoring in brain tumor outpatients in routine clinical practice. J Pain Symptom Manage 39(2):219–229 Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM (2010) GLOBOCAN 2008, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 10 [Internet]. Lyon, France: International Agency for Research on Cancer. Available from: http://globocan.iarc.fr Frost M, Bonomi A, Cappelleri J, Schunemann H, Moynihan T, Aaronson N (2007) Applying quality-of-life data formally and systematically into clinical practice. Mayo Clin Proc 82(10):1214–1228 Giesinger JM, Golser M, Erharter A, Kemmler G, SchauerMaurer G, Stockhammer G, Muigg A, Hutterer M, Rumpold G, Holzner B (2009) Do neurooncological patients and their significant others agree on quality of life ratings? Health Qual Life Outcomes 7:87 Hawkins R, Grunberg S (2009) Chemotherapy-induced nausea and vomiting: challenges and opportunities for improved patient outcomes. Clin J Oncol Nurs 13(1):54–64 Heimans J, Taphoorn MJ (2002) Impact of brain tumour treatment on quality of life. J Neurol 249(8):955–960 Holzner B, Schauer-Maurer G, Stockhammer G, Muigg A, Hutterer MGJ (2011) Computergestütztes Patient-reported Outcome Monitoring in der Neuroonkologie: Lebensqualität und Rezidiv beim Glioblastom.Wien Med Wochenschr 1611213–19 Janda M, Steginga S, Langbecker D, Dunn J, Walker D, Eakin E (2007) Quality of life among patients with a brain tumor and their carers. J Psychosom Res 63(6):617–623 Luckett T, Butow PN, King MT (2009) Improving patient outcomes through the routine use of patient-reported data in cancer clinics: future directions. Psychooncology 18(11):1129– 1138 Mullen KH, Berry DL, Zierler BK (2004) Computerized symptom and quality-of-life assessment for patients with cancer part II: acceptability and usability. Oncol Nurs Forum 31(5):E84–89 Nickolov A, Beumont J, Victorson D, Peterman A, Cella D, Liepa A, HA F (2005). Validation of functional assessment of cancer therapy: brain (FACT-Br) questionnaire and FACT-Br

L.M. Wintner et al. symptom index (FBrSI) in patients with recurrent high-grade glioma. Paper presented at: Chicago Supportive Oncology Conference, 2005, Chicago, IL Reardon DA, Wen PY (2006) Therapeutic advances in the treatment of glioblastoma: rationale and potential role of targeted agents. Oncologist 11(2):152–164 Rothman ML, Beltran P, Cappelleri JC, Lipscomb J, Teschendorf B (2007) Patient-reported outcomes: conceptual issues. Value Health 10(Suppl 2):S66–75 Sanchez R, Ballesteros M, Arnold BJ (2011) Validation of the FACT-G scale for evaluating quality of life in cancer patients in Colombia. Qual Life Res 20(1):19–29 Taphoorn MJ, Claassens L, Aaronson NK, Coens C, Mauer M, Osoba D, Stupp R, Mirimanoff RO, van den Bent MJ, Bottomley A (2010a) An international validation study of the EORTC brain cancer module (EORTC QLQBN20) for assessing health-related quality of life and symptoms in brain cancer patients. Eur J Cancer 46(6): 1033–1040 Taphoorn MJ, Sizoo EM, Bottomley A (2010b) Review on quality of life issues in patients with primary brain tumors. Oncologist 15(6):618–626 Trotti A, Colevas AD, Setser A, Basch E (2007) Patient-reported outcomes and the evolution of adverse event reporting in oncology. J Clin Oncol 25(32):5121–5127 Trowbridge R, Dugan W, Jay SJ, Littrell D, Casebeer LL, Edgerton S, Anderson J, O’Toole JB (1997) Determining the effectiveness of a clinical-practice intervention in improving the control of pain in outpatients with cancer. Acad Med 72(9):798–800 U.S. FOOD AND DRUG ADMINISTRATION (2006). Guidance for industry: patient-reported outcome measures: use in medical product development to support labeling claims Velikova G, Wright EP, Smith AB, Cull A, Gould A, Forman D, Perren T, Stead M, Brown J, Selby PJ (1999) Automated collection of quality-of-life data: a comparison of paper and computer touch-screen questionnaires. J Clin Oncol 17(3):998–1007 Velikova G, Booth L, Smith AB, Brown PM, Lynch P, Brown JM, Selby PJ (2004) Measuring quality of life in routine oncology practice improves communication and patient well-being: a randomized controlled trial. J Clin Oncol 22(4):714–724 Velikova G, Keding A, Harley C, Cocks K, Booth L, Smith AB, Wright P, Selby PJ, Brown JM (2010) Patients report improvements in continuity of care when quality of life assessments are used routinely in oncology practice: secondary outcomes of a randomised controlled trial. Eur J Cancer 46(13):2381–2388 Walker M, Brown J, Brown K, Gregor A, Whittle I, Grant R (2003) Practical problems with the collection and interpretation of serial quality of life assessment in patients with malignant glioma. J Neurooncol 63(2): 179–186 Webster K, Cella D, Yost K (2003) The functional assessment of chronic illness therapy (FACIT) measurement system: properties, applications, and interpretation. Health Qual Life Outcomes 1:79

Part IV

Hemangioblastoma

Chapter 26

Intra-operative ICG Use in the Management of Hemangioblastomas Loyola V. Gressot and Steven W. Hwang

Abstract Technology that assists in obtaining a safe, complete resection of central nervous system (CNS) tumors can have a tremendous impact on patient outcomes. Indocyanine green (ICG) is a fluorescent dye that has established utility in the intraoperative visualization of intracranial vasculature during aneurysm surgery. Recently, the use of ICG as an adjunct in tumor resection has been described. Intraoperative ICG use may help visualize vascular tumors, such as hemangioblastomas, to ascertain the tumor margins and to assure that no residual tumor remains. ICG can also assist in intraoperative localization of tumors and operative planning by visualizing the tumor in real time. Intraoperative ICG videography has the potential to become a powerful adjunct in the resection of specific CNS neoplasms. Keywords von Hippel-Lindau syndrome · Hemangioblastomas · Indocyanine green · ICG · Videography

Introduction Neoplasms of the central nervous system remain a challenging management dilemma. Although significant advances have been made over the last few decades, profound limitations still remain.

S.W. Hwang () Department of Neurosurgery, Tufts Medical Center, Boston, MA, USA e-mail: [email protected]

Improvements in treatment modalities have included the development of tools and techniques for safer surgical resection, advances in radiation therapy and improved chemotherapeutic agents. Overall, advances in technology and treatment modalities as well as systemic control have prolonged survival, but with select pathologies, the importance of gross total surgical resection (GTR) has become paramount in treatment. Achieving a gross total resection can potentially be curative for low grade tumors and has been shown to effect survival outcomes in high grade lesions including glioblastoma multiforme (Stummer et al., 2008), and anaplastic astrocytoma (Keles et al., 2006; McGirt et al., 2008). Gross total resection has also been shown to increase disease free progression in spinal intramedullary hemangioblastomas, ependymomas (Graces-Ambrossi et al., 2009) and pilocytic astrocytomas (Karikari et al., 2011). Therefore adjuvant tools that improve our ability to safely achieve a gross total resection are critical to the advancement of oncologic care. Hemangioblastomas are rare vascular tumors typically located in the infratentorial space or within the spine and often associated with von HippelLindau syndrome. Complete resection can be curative, although as hemangioblastomas are highly vascular tumors, surgical resection can be complicated and limited by catastrophic intra-operative bleeding (Cristante and Herrmann, 1999; Gläsker et al., 2010). Therefore technology permitting the visualization of tumor vessels in real time and verifying the complete resection of the lesion is an invaluable tool. Furthermore, most intramedullary spinal tumors occur in the cervical region (Karikari et al., 2011) therefore increasing the potential iatrogenic risk of devastating


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neurological injury, such as respiratory depression and quadriparesis. Thus the utilization of intraoperative technology to optimize a complete resection while minimizing trauma to neural tissue would further improve the safety of surgical intervention. Therefore, development of adjuvant tools, such as use of indocyanine green video fluoroscopy, to optimize safe and complete resection of these lesions has a significant role in oncological care.

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desired view prior to injection of the dye. The operative field must be clear of debris, cottonoids, blood product, or other obstructive material for the desired structures to be visualized. Calcification or atherosclerosis of the intracranial vasculature may obscure the field and marginalize interpretation of the imaging (Raabe et al., 2005). The anesthesiologist and surgical team should be cognizant that, as ICG absorbs light, it may cause a transient drop in pulse oximetry readings when administered intravenously.

Background Initially, ICG was designed by Kodak labs and applied to cardiac output research, but was quickly adapted to hepatologic study given its clearance attributes (Hemming et al., 1992). It has also been used extensively in ophthalmology to diagnose choroidal hemangiomas (Arvelo et al., 2000), to differentiate ocular melanoma from nevi (Kubicka-Trzaska et al., 2002), as a stain intra-operatively for cataract extraction (Yi and Sullivan, 2002), and has been proposed as an ancillary diagnostic tool for the identification of ocular tumors (Schalenbourg et al., 2000). Indocyanine green (ICG) is a water-soluble, tricarbocyanine, near infrared fluorescent dye that is not metabolized in human physiology. It is excreted into bile by the liver and does not flow through enterohepatic recirculation. The half-life is approximately 3–4 min in plasma. ICG is typically administered as an intravenous bolus after reconstitution in sterile water and the recommended dose is 0.2–0.5 mg/kg with a maximum of 5 mg/kg per day. ICG absorbs light at 805 nm and emits it at 835 nm which is in the near infrared spectrum. Most commercial operating microscopes can be fitted with a filter to provide high resolution images that can visualize the dye both in real time, permitting the evaluation of transit speed, and recorded for playback (Raabe et al., 2003). ICG administration may be repeated multiple times during a procedure after a short latency (Woitzik et al., 2005). Intraoperative ICG videography is visualized through the filter on the monitor and therefore images are only perceived in two dimensions, without depth perception. Hence, the surgeon must optimize the

Intracranial Practical Applications of ICG The use of intraoperative ICG is becoming more prevalent in vascular neurosurgery. Intraoperative ICG is used in vascular neurosurgery to evaluate clip position, to assure adequate occlusion of an aneurysm, and to visualize filling of perforating vessels as well as patency of parent vessels (Dashti et al., 2009). Though intraoperative digital subtraction angiography remains the gold standard, ICG is arguably comparable in identifying surgically relevant details and is faster while requiring less equipment and mobilization of personnel (Raabe et al., 2005). Intraoperative ICG can also be used to assess extra-cranial- intra-cranial (EC-IC) bypass patency as well as denote the site of graft stenosis or obstruction in the event of inadequate flow (Woitzik et al., 2005). Intraoperative ICG has also been used to identify the site of residual arteriovenous malformation (AVM) nidus (Takagi et al., 2007), and as an adjunct in the resection of extracranial vascular malformations including an intramuscular arteriovenous hemangioma (Nazzi et al., 2008). ICG has even been used intraoperatively to characterize unexpected findings such as an AVM encountered during a microvascular decompression for trigeminal neuralgia (Ferroli et al., 2010) and to identify a developmental venous anomaly by observing the patterns of dynamic flow (Ferroli et al., 2008). The use of ICG has also been shown to stain glioma margins in animal models which may be of potential use to demarcate normal parenchyma brain from tumor intraoperatively (Hansen et al., 1993; Britz et al., 2002). Hansen et al. (1993) using a rat glioma

26 Intra-operative ICG Use in the Management of Hemangioblastomas

model, proved that ICG staining is reliable to identify tumor margins within 1 mm of actual parenchyma. The stain persisted for up to an hour but required doses greater than the lethal level for a rat. Britz et al. (2002) showed that adjunct administration of the bradykinin analogue RMP-7 lowers the dose of ICG required to produce margin staining to non-toxic levels. More studies are needed to further delineate if this technology will be applicable or feasible in human physiology. Ferroli et al. (2011) reported their use of ICG in the resection of 100 craniotomies of mixed pathology and attempted to delineate applications of this new surgical tool. They felt that ICG use permitted identification of arterial vasculature that supplied normal parenchyma in 7 of 71 cases and accurately identified hypervascular or hypovascular areas of the tumor reliably. In 51 of 83 tumors, ICG identified high flow arterio-venous fistulas within the tumors. These intraoperative observations influenced their decisions to sacrifice selected venous outflow (Ferroli et al., 2011). Although they subjectively noted a benefit with the use of ICG, further objective evaluation documenting potential advantages of it use such as greater surgical resection, lower complications, less blood loss or even long-term clinical outcomes is still required.

Use of ICG in Resection of Intracranial Hemangioblastomas Hemangioblastomas are slow growing, benign, highly vascular tumors that typically occur in adults and are often located in the infratentorial space. These tumors are rare and often associated with Von Hippel Lindau syndrome (Gläsker et al., 2010). The incidence is low has not been clearly defined with no gender preference associated (Aldape et al., 2007). Hemangioblastomas typically present as an enhancing nodular mass with an associated cyst or syrinx and are routinely encountered infratentorially or in the spinal cord. Although the application of ICG to intracranial tumors is still being defined, its use in vascular neurosurgery has been well documented. Hence resection of highly vascular tumors may have the greatest benefit with use of ICG. However, little clinical experience has been reported with use of ICG and hemangioblastomas

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thus far (Murai et al., 2011; Ferroli et al., 2011). Murai et al. (2011) described their use of ICG in three cases resecting a hemanglioblastoma and Ferroli et al. (2011) applied it to the three hemangioblastomas of their tumor series. Both authors noted good visualization of the tumor and associated arterio-venous structures. Murai et al. (2011) even reported that it helped identify the location of one lesion underneath the parenchyma through inference of superficial abnormal vasculature. Intraoperative ICG videography is especially helpful in identifying regions of tumor that may be obscured from traditional microscopy by scar tissue and can be a useful adjunct in the pursuit of an en bloc resection for these tumors. The pursuit of an en bloc resection of hemangioblastomas is important not only for an oncological cure, but also to limit intraoperative hemorrhage as these tumors are highly vascular. Multiple authors have emphasized that careful development of a surgical plane between the tumor and neural tissue is paramount to avoiding massive tumor bleeding intraoperatively (Cristante and Herrmann, 1999; Gläsker et al., 2010). Identification of feeding arteries and draining veins via ICG videography can facilitate safer removal of these lesions (Murai et al., 2011). Post-resection ICG can also be utilized to confirm a gross total resection. However clinical experience using this tool is still early and its limitations have not yet been defined.

Use of ICG in Resection of Spinal Hemangioblastomas Spinal hemangioblastomas are much more likely to be associated with VHL than hemangioblastomas in other locations (Takai et al., 2010). Hemangioblastomas are the third most commonly encountered intramedullary spinal tumor comprising 5–15% of reported series (Baleriaux, 1999; Karikari et al., 2011; Cristante and Herrmann, 1999; Xu et al., 1996). Primary management of these lesions is surgical resection. Gross total resection is potentially curative, whereas subtotal resection has a higher risk of recurrence and disease progression (Takai et al., 2010). Intraoperative ICG videography has also been used for localization of intradural tumors to optimize the

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location of the durotomy overlying the tumor which may have shifted to some extent during patient positioning. This technology permits the confirmation of the location of the tumor while the dura is intact avoiding epidural veins. Schubert et al. (2010) demonstrated that the tumor could be identified prior to dural opening in 93% intradural spinal cord tumors. Tumors located in a circumscribed ventral location were difficult to visualize during a posterior approach. In one case, ICG imaging revealed the need to extend the laminectomy exposure which was then accomplished prior to performing the durotomy. Also, use of intraoperative ICG videography has been useful for the identification of feeding arteries and draining veins during the resection of spinal vascular malformations (Murakami et al., 2010; Hanel et al., 2010).

Intraoperative ICG videography has similarly been applied to visualize spinal hemangioblastomas (Figs. 26.1, 26.2 and 26.3). The same potential intraoperative advantages of visualizing arterio-venous anatomy, extent of tumor, confirming complete resection, and minimizing blood loss are applicable to spinal hemangioblastomas. However, only the superficial vessels and vascular lesions are visible and the field must be clear of obstructive foreign bodies and blood to optimize the interpretation of ICG videography. Various authors have reported successful imagining using ICG doses ranging from 5 to 50 mg boluses with imaging about 1–2 min post injection (Hwang et al., 2010; Murakami et al., 2010; Schubert et al., 2010).

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Fig. 26.1 (a) Sagittal T1-weighted, (b) Sagittal T1-weighted post-gadolinium pre-operative MRI demonstrating a recurrent hemangioblastoma with enhancing nodule at C1 and significant spinal cord edema

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Fig. 26.2 (a) Intraoperative images using microscopy, (b) video ICG demonstrating a hemangioblastoma with surrounding vasculature. Note the area of tumor visible on ICG but hidden by scar tissue on plain microscopic view

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26 Intra-operative ICG Use in the Management of Hemangioblastomas

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Fig. 26.3 Postresection intra-operative images demonstrating no residual tumor visible on either (a) microscopic view or (b) ICG videography or (c) post-operative MRI (sagittal post-gadolinium T1-weighted MRI)

Conclusion

References

Intraoperative ICG videography is a relatively new technology and its application in neuro-oncology is still being investigated. Intraoperative ICG videography appears to be useful for characterization and localization of vascular tumors. However, further evaluation is needed to delineate the benefits and limitations of this tool including types of tumor enhancement, depth of tumor visualization, intra-op changes with hyperemia, and clinical outcomes or patients treated in conjunction with this technology.

Aldape KD, Plate KH, Vortmeyer AO, Zagzag D, Neumann HPH (2007). Haemangioblastoma. WHO classification of tumours of the central nervous system, 4th edn. International Agency for Research on Cancer. Lyon, pp 184–186 Arevalo JF, Shields CL, Shields JA, Hykin PG, De Potter P (2000) Circumscribed choroidal hemanigoma:characteristic features with indocyanine green videoangiography. Ophthalmology 107:344–350 Baleriaux DL (1999) Spinal cord tumors. Eur Radiol 9:1252–1258 Britz GW, Ghatan S, Spence AM, Berger MS (2002) Intracarotid RMP-7 enhanced indocyanine green staining of tumors in a rat glioma model. J Neuro Oncol 56:227–232

238 Cristante L, Herrmann HD (1999) Surgical management of intramedullary hemangioblastoma of the spinal cord. Acta Neurochir (Wien) 141:333–340 Dashti R, Laakso A, Niemela M, Porras M, Hernesniemi J (2009) Mircoscope-integrated near-infrared indocyanine green videoangiography during surgery of intracranial aneurysma: the Helsinki experience. Surg Neurol 71:543–550 Ferroli P, Tringali G, Albanese E, Broggi F (2008) Developmental venous anomaly of petrous veins: intraoperative findings and indocyanine green video angiographic study. Neurosurgery 62:418–421 Ferroli P, Acerbi F, Broggi M, Broggi G (2010) Arteriovenous micromalformation of the trigeminal root: intraoperative diagnosis with indocyanine green videoangiography: case report. Neurosurgery 67:E309–310 Ferroli P, Acerbi F, Albanese E, Tringali G, Broggi M, Franzini A, Broggi G (2011) Application of intraoperative indocyanine green angiography for CNS tumors: results on the first 100 cases. Acta Neurochir Suppl 109:251–257 Gläsker S, Klingler JH, Müller K, Würtenberger C, Hader C, Zentner J, Neumann HP, Velthoven VV (2010) Essentials and pitfalls in the treatment of CNS hemangioblastomas and von Hippel-Lindau disease. Cen Eur Neurosurg 71:80–87 Graces-Ambrossi GL, McGirt MJ, Mehta VA, Sciubba DM, Witham TF, Bydon A, Wolinksy JP, Jallo GI, Gokaslan ZL (2009) Factors associated with progression-free survival and long-term neurological outcome after resection of intramedullary spinal cord tumors: analysis of 101 consecutive cases. J Neurosurg Spine 11:591–599 Hanel RA, Nakaji P, Spetzler RF (2010) Use of microscopeintegrated near-infrared indocyanine green videoangiography in the surgical treatment of spinal dural arteriovenous fistulae. Neurosurgery 66:978–985 Hansen DA, Spence AM, Carski T, Berger MS (1993) Indocyanine green (ICG) staining and demarcation of tumor margins in a rat glioma model. Surg Neurol 40:451–456 Hemming AW, Scudamore CH, Shackleton CR, Pudek M, Erb SR (1992) Indocyanine green clearance as a predictor of successful hepatic resection in cirrhotic patients. Am J Surg 163:515–518 Hwang SW, Malek AM, Schapiro R, Wu JK (2010) Intraoperative use of indocyanine green fluorescence videography for resection of a spinal cord hemangioblastoma. Neurosurgery 67:300–303 Karikari IO, Nimjee SM, Hodges TR, Cutrell E, Hughes BD, Powers CJ, Mehta AI, Hardin C, Bagley CA, Isaacs RE, Haglund MM, Friedman AH (2011) Impact of tumor histology on resectablility and neurological outcome in primary intramedullary spinal cord tumors: A single-center experience with 102 patients. Neurosurgery 86:188–197 Keles GE, Change EF, Lambron KR, Tihan T, Chang CJ, Change SM, Berger MS (2006) Volumetric extent of resection and residual contrast enhancement on initial surgery as predictors of outcome in adult patients with hemispheric anaplastic astrocytoma. J Neurosurg 105:34–40 Kubicka-Trzaska A, Starzycka M, Romanowska-Dixon B (2002) Indocyanine green angiography in the diagnosis of small choroidal tumors. Ophthalmologica 216:316–319

L.V. Gressot and S.W. Hwang McGirt MJ, Chaichana KL, Attenello FJ, Weingart JD, Than K, Burger PC, Olivi A, Brem H, Quinones-Hinojosa A (2008) Extent of surgical resection is independently associated with survival in patients with hemispheric infiltrating low-grade gliomas. Neurosurgery 63:700–707 Murai Y, Adachi K, Matano F, Tateyama K, Teramoto A (2011) Indocyanin Green Videoangiography Study of Hemangioblastomas. Can J Neurol Sci 38:41–47 Murakami T, Koyangi I, Kaneko T, Iihoshi S, Houkin K 2010. Intraoperative indocyanine green videoangiography for spinal vascular lesions. Neurosurgery [epub ahead of print] Nazzi V, Messina G, Dones I, Ferroli P, Broggi G (2008) Surgical removal of intramuscular arterovenous hemangioma of the upper left forearm compressing radial nerve branches. J Neurosurg 108:808–811 Raabe A, Beck J, Gerlach R, Zimmermann M, Seifert V (2003) Near-infrared indocyanine green video angiography: a new method for intraoperative assessment of vascular flow. Neurosurgery 52:132–139 Raabe A, Nakaji P, Beck J, Kim LJ, Hsu FP, Kamerman JD, Seifert V, Spetzler RF (2005) Prospective evaluation of surgical microscope-integrated intraoperative near-infrared indocyanine green videoangiography during aneurysm surgery. J Neurosurg 103:982–989 Schalenbourg A, Piguet B, Zografos L (2000) Indocyanine green angiographic findings in choroidal hemangiomas: a study of 75 cases. Ophthalmologica 214:246–252 Schubert GA, Barth M, Thome C (2010) The use of indocyanine green videography for intraoperative localization of intradural spinal tumors. Spine 35:E212–E217 Stummer W, Reulen HJ, Meinel T, Pichlmeier U, Schumacher W, Toon JC, Rohde V, Oppel F, Turowski B, Woiciechowsky C, Franz K, Pietsch T, ALA-Glioma Study Group. 2008. Extent of resection and survival in glioblastoma multiforme: identification of and adjustment of bias. Neurosurgery 62: 564–576 Takagi Y, Kikuta KI, Nozaki K, Sawamura K, Hashimoto N (2007) Detection of residual nidus by surgical microscopeintegrated intraoperative near-infrared indocyanine green videoangiography in a child with a cerebral arteriovenous malformation. J Neurosurg. 107:416–418 Takai K, Taniguchi M, Takahashi H, Usui M, Saito N (2010) Comparative analysis of spinal hemangioblastomas in sporadic disease and von hippel-lindau syndrome. Neurol Med Chir (Tokyo) 50:560–567 Woitzik J, Horn P, Vajkoczy P, Schmiedek P (2005) Intraoperative control of extracranial-intracranial bypass patency by near-infrared indocyanine green videoangiography. J Neurosurg. 102:692–698 Xu QW, Bao WM, Mao RL, Yang GY (1996) Aggressive surgery for intramedullary tumor of cervical spinal cord. Surg Neurol 46:322–328 Yi DH, Sullivan BR (2002) Phacoemulsification with indocyanine green versus manual expression extracapsular cataract extraction for advanced cataract. J Cataract Refract Surg 28:2165–2169

Chapter 27

Hemangioblastoma Cysts: Diagnosis Using Fluorescence with 5-Aminolevulinic Acid Satoshi Utsuki, Hidehiro Oka, and Kiyotaka Fujii

Abstract Hemangioblastoma is a benign tumor which often emerges sporadically in adult cerebellum or is associated with von Hippel-Lindau disease. Though it can be surgically removed, in rare occasions it can reappear, necessitating a second treatment to remove the tumor. Recurrences of hemangioblastoma are located either in an area removed from the initial tumor site or locally due to an incomplete excision of the tumor. The latter recurrence can be controlled by confirming that none of the tumor remains at the time of surgery. Intraoperative fluorescence diagnosis using 5-aminolevulinic acid (5-ALA) utilizes of protoporphyrin IX (PpIX) accumulation in tumor cells, resulting in PpIX accumulation in residual tumor cells which can be seen reacting under ultraviolet light. This is a simple, non-invasive technique to detect the presence of any residual tumor while still in the operating room. Although there are no tumor cells in cysts of hemangioblastoma, there are cases where a recurrence is thought originate from a residual cyst. Because rupturing these thin-walled cysts can cause damage to the surrounding healthy cerebellum, routine extirpation of hemangioblastoma cysts is not recommended unless the cysts have been invaded by tumor cells, in which case extirpation is necessary to prevent recurrence. Intraoperative fluorescence diagnosis using 5-ALA is

S. Utsuki () Department of Neurosurgery, Kitasato University School of Medicine, 1-15-1, Kitasato, Minami, Sagamihara, Kanagawa 252-0374, Japan e-mail: [email protected]

a useful way to determine the presence of tumor cells in cyst walls associated with hemangioblastoma. Keywords 5-aminolevulinic acid · Hemangioblastoma · Intraoperative fluorescence diagnosis · Protoporphyrin IX · Residual tumor

Introduction Hemangioblastoma is a benign tumor which often emerges in adult cerebellum, approximately 62% of which occur sporadically and 38% of which occur in relation to von Hippel-Lindau (VHL) disease (Singounas, 1978). Hemangioblastoma can be extirpated microsurgically, producing mainly positive surgical results. However, even in cases where complete excision of the initial tumor was successful, longterm follow-up has established a recurrence rate of 15–27% (de la Monte and Horowitz, 1989; Niemela et al., 1999) and the risk of delayed recurrence may be high. Initial diagnosis at a young age, the presence of VHL disease, and multicentricity of CNS tumors can be cited as risk factors for recurrence. The categories of recurrence are local recurrence in the area where the initial tumor existed, and new region recurrence where no tumors were detected initially. In the latter cases, an undetected tumor may already have existed at initial surgery, a predisposition to VHL-associated tumors may have facilitated a new tumor (Niemela et al., 1999; Wanebo et al., 2003), or the recurrence may be due to a condition called hemangioblastomatosis (Weil et al., 2002). Local recurrence is caused by an incomplete excision of the tumor during initial surgery. Such incomplete excisions of tumors result


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from either minute residual tumor infiltration or cases where the cyst is invaded with tumor cells. Cyst walls with hemangioblastoma are composed of collagen fiber, astrocytic gliosis and rosenthal fibers and is usually devoid of tumor cells (Hussein, 2007). It is therefore unnecessary to extirpate cyst walls with hemangioblastoma; Conversely, extirpation should be avoided due to the risk of additional neurological damage. However, there are reports indicating the presence of tumor cells in cyst walls (Utsuki et al., 2010) as well as recurrences resulting from residual tumors in cyst walls (Bishop et al., 2008). Accordingly, the cyst wall should be extirpated if tumor cells exist in the cyst wall to prevent recurrence of the tumor. This should be assessed during surgery. The intraoperative photodynamic diagnosis (PDD) using 5-aminolevulinic acid (5-ALA) is an efficient detection method to easily and objectively ascertain the presence of residual tumors in real time.

Mechanisms of Cyst Formation in Hemangioblastoma According to MRI studies, hemangioblastoma can be divided into four different types (Lee et al., 1989; Richard et al., 1998); tumors that are not associated with cysts, tumors that are associated with intratumoral cysts, tumors that are associated with peritumoral cysts and tumors that are associated with both peritumoral and intratumoral cysts. The frequency of each type is reported to be 46, 51, 27 and 7% respectively (Jagannathan et al., 2008). The mechanisms for the formation of intratumoral and peritumoral cysts differ from each other. While intratumoral cysts are formed by intratumoral necrosis, peritumoral cysts develop as a result of a tumor interstitial process that begins with the occurrence of edema (Lohle et al., 1998; Lonser et al., 2005). Increased tumor vascular permeability associated with hydrodynamic forces promotes fluid extravasation. Once this fluid extravasation from a tumor overcomes the capacity of the surrounding tissue to reabsorb excess fluid, edema and subsequent cyst formation occur (Lonser et al., 2005). Aquaporin-1 (AQP1), an envelope protein of the plasma membrane, has been shown to be involved in the balance between extravasation and reabsorption,

S. Utsuki et al.

operating as a constitutive channel for water transport (Chen et al., 2006). Protein identification of fluid in the cyst using two-dimensional proteomic profiling demonstrate that the proteomic patterns of intratumoral and peritumoral cyst fluid are the same (Glasker et al., 2006). Since both are considerably similar to the serum, the formation of hemangioblastoma cysts is indicated to be caused by an extravascular leakage of the serum. Accordingly, the mechanisms of both intratumoral necrosis and extravascular leakage of serum relate to intratumoral cysts associated with hemangioblastoma.

Regulation of Heme Synthesis Heme Synthesis ALA is generated in vivo from glycine and succinylCoA in mammals by ALA synthase (ALAS); an enzyme in the mitochondrial membrane. Subsequently, porphobilinogen (PBG) is produced from two ALA molecules by cytoplasmic ALA dehydratase. PBG can be transformed to uroporphyrinogen III by PBG deaminase (PBGD) and uroporphyrinogen III synthase, both of which are enzymes found in the cytoplasm, but PBGD is the rate-limiting step under normal conditions. Uroporphyrinogen decarboxylase converts uroporphyrinogen III to coproporphyrinogen III and coproporphyrinogen oxidase, which exists in the intermembrane space of the mitochondria and generates protoporphyrinogen in the cell nucleus. Protoporphyrinogen oxidase converts Protoporphyrinogen IX into Protoporphyrin IX (PpIX) and heme is synthesized by introducing iron to the tetrapyrrole structure using ferrochelatase, which is found in the inner mitochondrial membrane.

Regulation of the Heme Synthesis Pathway All enzymes of the heme pathway operate irreversibly and are partially adjusted by the feedback inhibition of ALAS. ALAS activity is the lowest after only PBGD; other enzymes have far higher activities. 5-ALA is

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drawn into the cells when administered, and no further reaction occurs due to PBGD, the rate-limiting step. No excessive metabolism of 5-ALA occurs and the feedback inhibition of ALAS controls intercellular 5-ALA synthesis, while exogenous 5-ALA drawn into the cells is rapidly metabolized in the tumor cells, where PBGD is not the rate-limiting step. Furthermore, the ferrochelatase activity in tumor cells is low (Itoh et al., 2000) causing excessive PpIX to accumulate.

Detection of Tumor Cells by ALA-Derived PpIX Fluorescence (Methodology) PpIX accumulates only in tumor cells when 5-ALA is administered, and not in healthy cells due to the respective difference in enzymic activities. Because PpIX produces red fluorescence when irradiated by ultraviolet light and 5-ALA alone does not produce fluorescence, tumor areas, which accumulate PpIX, can be visually identified by this red fluorescence. Patients received 1 g of orally administered 5-ALA two hours prior to the introduction of an anesthesic. Tumor masses were extirpated under a microscope and the extirpated tumor mass, the extirpated area and the remaining cysts were irradiated with 405 nm of excitation light using a semiconductor laser device (VLD-V1 version 2; M&M Co., Ltd., Tokyo, Japan). The presence of PpIX fluorescence was observed through a low-cut filter (cut, 420 nm, M & M Co., Ltd.). This method was used to perform a PDD on the cerebellum hemangioblastoma of all patients. When we classified the cysts, there were no cases with no cysts, one case associated with intratumoral cysts, five cases associated with peritumoral cysts and one case associated with both peritumoral and intratumoral cysts. PpIX fluorescence was observed on the tumor masses in all of the cases. In addition, PpIX fluorescence was also observed on the intratumoral cyst walls in all of the cases. Although the enhancing effect of the cyst was not clear in all five cases considered to be peritumoral cysts (Fig. 27.1), PpIX fluorescence was observed on the cyst walls in two out of the five cases when the PDD was performed. In both of these cases, no obvious abnormality was seen under micrographic surgery and we believed there were no residual tumors (Fig. 27.2). However, residual tumors were suspected

Fig. 27.1 Contrast-enhanced axial T1-weighted MR image showing a homogenous enhanced mass lesion with peritumoral cyst at the right cerebellum hemisphere. The cyst wall showed no enhancement

Fig. 27.2 Intraoperative photograph showing peritumoral cyst wall after resection of a nodal lesion in microscope view. A residual tumor cannot be identified

with the PDD and an additional extirpation was performed on the cyst wall. In both cases of the extirpated samples, the cyst walls contained a thin layer of tumor cells (Fig. 27.3).

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Fig. 27.3 Photomicrograph showing histological findings of the peritumoral cyst wall where a residual tumor cannot be identified in Fig. 27.2. The tumor cells and capillary vessels formed a thin line to border the cyst wall, and gliosis surrounds the circumference (hematoxylin and eosin staining, original magnification ×100)

Discussion Hemangioblastoma is a tumor with a clear boundary distinct from the brain and does not usually have a tumor capsule. Although brain tissue such as gliosis and Rosenthal fibres adjacent to the tumor form a boundary between the tumor and the brain, tumors do, in rare cases, invade brain parenchyma (Hussein, 2007). However, although hemangioblastoma is a benign tumor, long-term follow-up observation indicates a high incidence of recurrence. The cases of local recurrence may be due to the incomplete removal of the invading tumor cells from the brain parenchyma and the cyst wall (Bishop et al., 2008). Such residual tumor cells can be identified during surgery with a PDD using 5-ALA. This is mainly used for malignant glioma surgery (Stummer et al., 2000) and an improvement of prognosis can be achieved by extirpating areas where red fluorescence can be seen. This method assesses tumor invasions more objectively and promotes a decrease in residual tumors. Other than malignant glioma, this technique is also used to assess tumor invasions and assess residual tumors with meningioma (Kajimoto et al., 2007) and benign ependymoma (Shimizu et al., 2006). Jagannathan et al. examined all cyst walls, including peritumoral cysts, following the extirpation of hemangioblastoma and reported that the recurrence of tumors can be prevented

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by performing an additional extirpation on tumor-like lesions (Jagannathan et al., 2008). However, a minute traces of residual tumors may be difficult to detect during surgery. The presence of tumors in the cyst wall could not be confirmed during surgery in two of our aforementioned cases, although the presence of tumor cells was confirmed histologically. PDD using 5-ALA is an effective way to objectively detect residual tumors in hemangioblastoma. In cases such as these, intratumoral cysts develop around the tumor and may imitate peritumoral cysts with the wall becoming thinner as the cyst grows bigger (Bishop et al., 2008; Utsuki et al., 2010). Moreover, because some tumor cells were floating in the cyst fluid (Lallu et al., 2008), they could engraft to genuine peritumoral cysts and multiplied. Also, although it is rare, tumor cells not associated with either the mural nodule or with the enhancing effect of the wall may appear similar to an arachnoid cyst on the image (Vatsal et al., 2002). To prevent the recurrence of a tumor, it is necessary to extirpate the cells of such tumors completely, and PDD using 5-ALA is one of the methods which enables this.

References Bishop FS, Liu JK, Chin SS, Fults DW (2008) Recurrent cerebellar hemangioblastoma with enhancing tumor in the cyst wall: case report. Neurosurgery 62:E1378–E1379 Chen Y, Tachibana O, Oda M, Xu R, Hamada J, Yamashita J, Hashimoto N, Takahashi JA (2006) Increased expression of aquaporin 1 in human hemangioblastomas and its correlation with cyst formation. J Neurooncol 80:219–225 de la Monte SM, Horowitz SA (1989) Hemangioblastomas: clinical and histopathological factors correlated with recurrence. Neurosurgery 25:695–698 Glasker S, Vortmeyer AO, Lonser RR, Lubensky IA, Okamoto H, Xia JB, Li J, Milne E, Kowalak JA, Oldfield E H, Zhuang Z (2006) Proteomic analysis of hemangioblastoma cyst fluid. Cancer Biol Ther 5:549–553 Hussein MR (2007) Central nervous system capillary haemangioblastoma: the pathologist’s viewpoint. Int J Exp Pathol 88:311–324 Itoh Y, Henta T, Ninomiya Y, Tajima S, Ishibashi A (2000) Repeated 5-aminolevulinic acid-based photodynamic therapy following electro-curettage for pigmented basal cell carcinoma. J Dermatol 27:10–15 Jagannathan J, Lonser RR, Smith R, DeVroom HL, Oldfield EH (2008) Surgical management of cerebellar hemangioblastomas in patients with von Hippel-Lindau disease. J Neurosurg 108:210–222 Kajimoto Y, Kuroiwa T, Miyatake S, Ichioka T, Miyashita M, Tanaka H, Tsuji M (2007) Use of 5-aminolevulinic acid in

27 Hemangioblastoma Cysts: Diagnosis Using Fluorescence with 5-Aminolevulinic Acid fluorescence-guided resection of meningioma with high risk of recurrence. Case report J Neurosurg 106:1070–1074 Lallu S, Naran S, Palmer D, Bethwaite P (2008) Cyst fluid cytology of cerebellar hemangioblastoma: a case report. Diagn Cytopathol 36:341–343 Lee SR, Sanches J, Mark AS, Dillon WP, Norman D, Newton TH (1989) Posterior fossa hemangioblastomas: MR imaging. Radiology 171:463–468 Lohle PN, Wurzer HA, Seelen PJ, Kingma LM, Go KG (1998) The pathogenesis of cysts accompanying intra-axial primary and metastatic tumors of the central nervous system. J Neurooncol 40:277–285 Lonser RR, Vortmeyer AO, Butman JA, Glasker S, Finn MA, Ammerman JM, Merrill MJ, Edwards NA, Zhuang Z, Oldfield EH (2005) Edema is a precursor to central nervous system peritumoral cyst formation. Ann Neurol 58:392–399 Niemela M, Lemeta S, Summanen P, Bohling T, Sainio M, Kere J, Poussa K, Sankila R, Haapasalo H, Kaariainen H, Pukkala E, Jaaskelainen J (1999) Long-term prognosis of haemangioblastoma of the CNS: impact of von Hippel–Lindau disease. Acta Neurochir (Wien) 141:1147–1156 Richard S, Campello C, Taillandier L, Parker F, Resche F (1998) Haemangioblastoma of the central nervous system in von Hippel-Lindau disease. French VHL Study Group J Intern Med 243:547–553

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Shimizu S, Utsuki S, Sato K, Oka H, Fujii K, Mii K (2006) Photodynamic diagnosis in surgery for spinal ependymoma. Case illustration. J Neurosurg Spine 5:380 Singounas EG (1978) Haemangioblastomas of the central nervous system. Acta Neurochir (Wien) 44:107–113 Stummer W, Novotny A, Stepp H, Goetz C, Bise K, Reulen HJ (2000) Fluorescence-guided resection of glioblastoma multiforme by using 5-aminolevulinic acid-induced porphyrins: a prospective study in 52 consecutive patients. J Neurosurg 93:1003–1013 Utsuki S, Oka H, Sato K, Shimizu S, Suzuki S, Fujii K (2010) Fluorescence diagnosis of tumor cells in hemangioblastoma cysts with 5-aminolevulinic acid. J Neurosurg 112:130–132 Vatsal DK, Husain M, Husain N, Chawla S, Roy R, Gupta RK (2002) Cerebellar hemangioblastoma simulating arachnoid cyst on imaging and surgery. Neurosurg Rev 25:107–109 Wanebo JE, Lonser RR, Glenn GM, Oldfield EH (2003) The natural history of hemangioblastomas of the central nervous system in patients with von Hippel-Lindau disease. J Neurosurg 98:82–94 Weil RJ, Vortmeyer AO, Zhuang Z, Pack SD, Theodore N, Erickson RK, Oldfield EH (2002) Clinical and molecular analysis of disseminated hemangioblastomatosis of the central nervous system in patients without von Hippel-Lindau disease. J Neurosurg 96:775–787

Chapter 28

Hemangioblastoma: Stereotactic Radiosurgery Anand Veeravagu, Bowen Jiang, and Steven D. Chang

Abstract CNS hemangioblastomas are rare, vascular neoplasms that arise primarily in the posterior cranial fossa. Prognosis is generally favorable, with a recurrence rate of fewer than 25% in multiple surgical series. Although current standard of care for CNS hemangioblastomas is surgical resection, other treatment modalities including endovascular embolization and stereotactic radiosurgery (CyberKnife, LINAC, Gamma Knife) are being applied. Increasing evidence has suggested the effectiveness of stereotactic radiosurgery in managing CNS hemangioblastomas. Herewithin, we review the indications and multiinstitutional experiences in using such a treatment modality. Keywords Radiosurgery · Hemangioblastomas · Tumor

Mast

cell

·

Introduction Central nervous system (CNS) hemangioblastomas were first described in the cerebellum by Jackson in 1872. Hemangioblastomas are usually slow growing tumors which account for 1–3% of all CNS neoplasms and 7–10% of posterior fossa tumors. These highly vascular and histologically benign (WHO I) lesions consist of a small mural nodule with an

S.D. Chang () Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA 94305-5484, USA e-mail: [email protected]

associated pseudocyst in 30–80% of cases, with the remaining lesions consisting of solid tumors. The tumor tissue itself has a well defined border and has a bright red color on gross morphological examination (Fig. 28.1). Histologically, hemangioblastomas are vascular lesions containing channels lined by cuboidal epithelium and are interspersed with nests of foamy stromal cells and pericytes. Mast cells are also found and may be responsible for the production of erythropoietin which can cause erythrocytosis (Fig. 28.2). Although debate still exists, the clusters of stromal cells surrounding the vascular plexus are thought to be the neoplastic component of the lesions. These lesions typically occur in the cerebellum (63%), spinal cord (32%), and brainstem (5%), though some cases of lumbosacral nerve root and supratentorial lesions have been reported as well. There is not thought to be any sex or ethnic predominance and the mean age at diagnosis is in the late third or early fourth decade of life. CNS hemangioblastomas are most commonly treated by surgical resection, which is an effective strategy capable of achieving curative results. A number of large clinical case series have shown that when appropriately applied, surgical resection is often necessary to provide symptomatic improvement. In the case of unfavorable anatomic location or post surgical recurrence, radiosurgery is often the next line of treatment. In particular, our experience and the reported literature surrounding the use of stereotactic radiosurgery (CyberKnife, LINAC, Gamma Knife) highlight favorable outcomes in certain clinical settings. The size, morphology, location, and clinical presentation of hemangioblastomas must all be considered when choosing treatment.


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Fig. 28.1 Histologic Appearance of hemangioblastoma. (a) low power (100×; H&E): well-demarcated proliferation of vessels and stromal cells with adjacent gliosis (b) medium power

(200×; H&): capillary network and admixed stromal cells (c) higher power (400×; H&E): vacuolated stromal cells and fine capillaries

Fig. 28.2 MRI with Gadolinium enhancement at T1/T2/FLAIR weightings. (a) Axial T1-weight post contrast images showing recurrent cerebellar hemangioblastoma associated with a large, cystic cavity. (b) Coronal T1-weight post contrast images again

demonstrating focus of recurrent hemangioblastoma and associate cystic cavity. (c) Axial T1-weight post contrast images demonstrating interval resection of mural nodule and drainage of associated cerebellar cyst with decompression of posterior fossa

Conventional Radiotherapy

a high dose of radiation. One study of 24 patients noted a 10-year survival rate of 57% for patients treated with more than 36 Gy, compared with a survival rate of 27% for patients treated with less than 36 Gy. In another review of 27 patients irradiated postoperatively (19 with gross residual disease and 6 with VHL disease), local control was achieved for 33% of patients treated with less than 50 Gy, compared with 57% of those who received more than 50 Gy. Although conventionally fractionated irradiation seems to increase the probability of hemangioblastoma control, local control even with increased radiation doses is less than optimal. Furthermore, the exposure to significant volumes of normal tissue with radiotherapy remains a concern.

Radiosurgical treatment of hemangioblastomas has become increasingly popular. Current indications for radiosurgery for hemangioblastomas include: 1) Unfavorable and inaccessible region of the CNS axis, 2) Recurrence after surgical resection (particularly in VHL patients), 3) Medical co-morbidities that preclude surgery. As with other radiosurgical targets, hemangioblastomas treated with radiosurgery are typically less than 3 cm in size. Contraindications to radiosurgery for hemangioblastomas include tumors greater than 3 cm and those inducing significant neurologic symptoms due to mass effect and edema requiring urgent decompression. The rationale for radiosurgery for hemangioblastomas comes from prior use of conventionally fractionated external beam radiation for residual or unresectable hemangioblastomas. Studies suggest that control of hemangioblastomas depends upon achieving

Stereotactic Radiosurgery Because single fractions of 20–25 Gy have been estimated to achieve the biological equivalence of 50–100 Gy administered by conventional fractionated

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irradiation, stereotactic radiosurgery may provide better control than that achieved with standard radiotherapy, especially for highly vascular, benign tumors. The steep dose gradient achieved with this technique minimizes damage to eloquent structures in the posterior fossa. Because hemangioblastomas are typically small, well-defined tumors that show no histological infiltration, they are ideally suited for radiosurgical treatment. In a recent study from the NIH, 20 patients with 44 lesions were treated with Gamma Knife SRS. At a mean follow-up of 8.5 years (range 3.0–17.6 years), all 20 patients remained living. Local control was reported to be 91, 83, and 61% at 2, 5, and 10 years after Gamma Knife SRS, respectively (Asthagiri et al., 2010). This group noted that 33% of SRS treated asymptomatic, small ( 70 %) reported for GG WHO grade I (Blumcke and Wiestler, 2002; Louis et al., 2007). In GG with anaplastic features (WHO grade III) the temporal lobe appears to be less frequently affected (Blumcke and Wiestler, 2002).

Imaging The computed tomography (CT) appearance of GG consists in a low density, well circumscribed lesion.

E. Aronica and P. Niehusmann

The solid portion of the tumor can be isodense, hypodense or mixed. Calcification of the tumor can be detected on CT. Contrast enhancement is variable. On magnetic resonance imaging (MRI) GG may display a solid or cystic appearance or be cystic with a mural nodule. On T1-weighted images the signal intensity is variable, the tumor is generally hypointense (less often, iso-intense); T2-weighted sequences generally show an hyperintense circumscribed mass; variable enhancement can be observed within the solid portion of the tumor (Louis et al., 2007); (Fig. 29.1a–c).

Symptoms The clinical history of patients with GG consists of a protracted history of focal symptoms, which vary depending on the size and location of the tumor. When the tumor is located in the region of the third ventricle and hypothalamus, symptoms related to hypothalamic dysfunction can be observed. Tumors located in the neocortex or temporal lobe are frequently associated with a history of chronic seizures, varying from months to decades before diagnosis. The seizures are often represented by drug-resistant complex partial seizures, although simple partial seizures and secondary generalization of partial seizures may also occur. Tumors involving the brainstem or the spinal cord often present with a shorter duration of symptoms before diagnosis (due to the tumor mass effect).

Neurophysiological Features On preoperative electroencephalogram (EEG) examination, patients with GG show focal, multifocal or widespread epileptiform discharges. Acute intraoperative electrocorticography (ECoG) provides an unique opportunity to assess the epileptogenicity of exposed areas of the cortex during surgery and to correlate these findings with histopathology (Fig. 29.1d). Continuous

Fig. 29.1 (continued) interval between two subsequent spikes being 1 s at the most (frequency ≥ 1 Hz); bursts of spikes, sudden occurrence of spikes for at least 1 s with a frequency of 10 Hz

or more; (4) recruiting discharges, rhythmic spike activity characterised by an increased amplitude and a decreased frequency (electrocorticographic seizure; (Ferrier et al., 2006)

29 Gangliogliomas: Molecular Pathogenesis and Epileptogenesis

Fig. 29.1 Imaging and electrocorticography (ECoG) in ganglioglioma (GG). Panels (a)–(c): coronal MRI images revealing a focal lesion in the left temporal lobe. Panel (a) T1-weighted inversion recovery image; Panel (b) T1-weighted image with contrast; Panel (c) T2-weighted image (images kindly provided by Prof Urbach, Dep. of Neuroradiology; University of Bonn Medical Center, Germany). Panel (d) acute intraoperative ECoG provides an unique opportunity to assess the epileptogenicity of exposed areas of the cortex during surgery. Panel (d) shows silicon (5×4-) grid electrodes, with intercontact distances of 10 mm placed on the exposed surface of the cortex in a 2 year

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old patient with temporal GG. ECoG can guide the extent of the resection, which may include a part of the macroscopically normal appearing perilesional zone (insert in d). (e) representative ECoG showing bursts of spikes recorded from the electrodes overlying the temporal cortex (arrows; ECoG kindly provided by Dr. C. Ferrier and Dr. F.S.S. Leijten, Department of Clinical Neurophysiology, University Medical Center, Utrecht, The Netherlands). (f) representative epileptiform ECoG discharge patterns. (1) sporadic spikes, spikes occurring at irregular time intervals at several sites; (2) continuous spiking, occurring rhythmically theat regular time intervals for at least 10 s, the

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spiking, bursts, and recruiting discharges can occur in patients with GG (Fig. 29.1e and f). When continuous spiking is found, GNTs appear to be associated with a high neuronal density and with dysplastic regions surrounding the tumor (Ferrier et al., 2006). The close relationship between epileptiform discharge patterns and histopathological changes is a strong argument for electrocorticographic tailoring of the resection in GG. However, further research is required to evaluate whether tailoring resection to these discharge patterns has an impact on the surgical outcome of GG.

Pathology Macroscopy Macroscopically GG appear as a glassy, grayyellowish solid lesion with often cyst/nodular configuration; focal mineralization can be encountered on cutting the surgical specimen. Only rarely can hemorrhage and necrosis be observed (in anaplastic lesions).

Intraoperative Diagnosis It is often difficult to obtain a confident diagnosis using smear preparations, even in cases in which the diagnosis is suggest by clinical and imaging features, if the distribution of glial and neuronal elements is not preserved or the tumor smears poorly. In cases where the tumor does smear, dysplastic neurons with large nuclei and prominent nucleoli surrounded by astroglial cells


can be detected. However, when the smear preparations contain only astroglial cells the appearance can be similar to a low grade astroglial tumor (pilocytic or fibrillary astrocytomas). Frozen sections may be useful to better identify the neuronal and glial components, supporting the intraoperative diagnosis of GG.

Histopathological Features GG consist of a mixture of dysplastic neurons and glial cells (Fig. 29.2a). The glial component is represented by a large spectrum of glial cells, including cells resembling the cell type of a fibrillary or pilocytic astrocytoma, with different degrees of cellularity. The neuronal component is represented by dysplastic neurons, which vary in amount and characteristically show prominent Nissl substance, lack uniform orientation and have abnormal shapes and sizes, vesicular nuclei and prominent nucleoli (Fig. 29.2a). Bi- or multinucleate neurons may also be observed (Blumcke and Wiestler, 2002; Louis et al., 2007). The large spectrum of histopatological features observed in GG represents a diagnostic challenge to neuropathologists. In some tumors the neuronal component is relatively inconspicuous and a GG could be misdiagnosed as a low grade astrocytoma. In contrast, in cases with a predominant neuronal phenotype, cortical dysplasia and gangliocytoma should be considered in the differential diagnosis. In some GG the predominant cell type may be represented by smaller neuronal cells or cells resembling oligodendrocytes (clear cell morphology), raising the differential diagnosis of oligodendroglioma or dysembryoplastic neuroepithelial tumor (DNT). A pseudopapillary architecture can also be observed in

Fig. 29.2 Histopathological feautures of ganglioglioma (GG). (a) Hematoxylin/Eosin (HE) staining of GG showing the mixture of neuronal cells, lacking uniform orientation (arrows and insert) and glial cells. (b) NeuN staining detects the neuronal component (nuclear staining) of GG. (c) GFAP staining detects the astroglial tumor component. (d) confocal merged image showing co-expression (yellow) of two presynaptic vesicle proteins, synaptophysin (SYN; red) with synaptic vesicle protein 2A (SV2A; green) in a dysplastic neuronal cell. Note the strong perineuronal staining. Insert in (d) shows synaptophysin staining of a large binucleate cell. Panels (e) and (f): phosphorylated ribosomal S6 protein (PS6) IR in dysplastic cells (arrows in e and high magnification in f). (g) HLA-DR detects the presence of cells of

the microglia/macrophage lineage. (h) CD3 detects the presence of T-lymphocytes around a blood vessel. (i) CD34 (precursor cell marker) IR in GG; insert a in (i) shows a CD34 immunoreactive cell with intense ramification of processes; insert b in (i) shows an AMOG positive cell with a similar morphology. (j) low magnification photograph showing CD34 positive tumor aggregates infiltrated into adjacent neocortex. (k) NeuN staining showing alterations in architectural composition (cortical dislamination and layer I hypercellularity) of the neocortex adjacent the tumor, but not infiltrated by tumor cells. Scale bar: a, g–i: 80 μm; b, c: 40 μm; d, f: 15 μm e: 60 μm; d: 1.5 mm; g: 30 μm; j–h: 160 μm


Fig. 29.2 (continued)

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GG and recently, a new variant with prominent papillary appearance, designed as papillary glioneuronal tumor, has been included as a separate entity (Louis et al., 2007). Additional histopathological features commonly observed in GG include the presence of eosinophilic granular bodies, Rosenthal fibers, calcifications and a prominent capillary network. The reticulin staining can be useful to visualize the fiber network in GG. In addition, perivascular lymphocytic infiltration, as well as the presence of an abundant population of activated microglial cells represent common features of this tumor (Fig. 29.2g and h; (Louis et al., 2007; Aronica et al., 2008)). Mitotic activity is rare and the majority of cases show a low expression of proliferation markers (such as Ki67 labeling) within the glial component. The large majority of GG correspond to WHO grade I. According to the WHO classification (Louis et al., 2007) GG with anaplastic glial features are considered WHO grade III. These tumors are characterized by increased cellularity and proliferation activity within the gilal component of the tumor. The detection of microvascular proliferation and necrosis support the anaplastic transformation of the tumor. The existence of GG grade II is still a matter of debate and clear criteria for grade II are not yet established (Luyken et al., 2004; Louis et al., 2007; Majores et al., 2008).

Immunohistochemistry The panel of antibodies used for the immunohistochemical characterization of GG includes both glial and neuronal markers. A well-characterized glial marker protein, such as glial fibrillary acidic protein (GFAP) can be used to demonstrate the neoplastic gial component of the tumor (Fig. 29.2c). Several neuronal markers, such as neuronal nuclear protein (NeuN), microtubule-associated protein 2 (MAP2) and synaptophysin can be used to detect the neuronal component (Fig. 29.2b). Immunocytochemical detection of synaptophysin, as well as of the synaptic vesicle protein 2A (SV2A; both presynaptic vesicle proteins) often show a strong perikaryal and occasionally cytoplasmic immunoreactivity (IR) in dysplastic neurons within GG specimens (Fig. 29.2d). Strong perineuronal synaptophysin IR can be found in non-neocortical


areas (spinal cord, thalamus, brain stam), but is rarely observed in the human neocortex (Quinn, 1998). Thus synaptophysin- and SV2A-positive neurons represent a common feature of glioneuronal tumors. However, this staining has to be interpreted with caution, particularly in cases with extra-cortical localization or without clear evidence of neuronal differentiation (Quinn, 1998). Immunocytochemical detection of the onco-fetal protein CD34 can also be helpful in the diagnosis of GG. CD34, negative in neural cells in adult brain, has been shown to be consistently expressed in 70– 80% of GG, also detecting peritumoral satellite lesions (Fig. 29.2i and j); (Blumcke and Wiestler, 2002). Immunocytochemical detection of AMOG (adhesion molecule on glia) shows a pattern of IR similar to that observed for CD34 and AMOG colocalizes with CD34 (Fig. 29.2i(b)); (Boer et al., in press). Thus both CD34 and AMOG may help to identify a population of glioneuronal precursor cells in GG. The phosphorylated ribosomal S6 protein (PS6) represents an additional marker, which can be used to detected in the neuronal component of GG, indicating activation of the Pi3K-mTOR pathway in this tumor (Fig. 29.2e and f) (Boer et al., in press).

Differential Diagnosis Considering the broad spectrum of histopathological features displayed by GG a careful differential diagnosis with other glioneuronal and glial entities is required. Differential diagnosis is particularly difficult in stereotactic biopsy where the typical architectural features of GG are not represented and only few neurons can be detected within the specimen. Establishing the nature of these neurons, differentiating dysplastic neurons of GG from entrapped neurons of an astroglial tumor, represents a common problem in the diagnosis of GG.

Gangliocytoma Gangliocytoma is characterized by the presence of clusters of large dysplastic neurons and non-neoplastic glial cells (Louis et al., 2007). The absence of neoplastic glial cells represents the most important feature in


the differential diagnosis with GG. Whether gangliocytoma represents a clearly distinct tumor entity is still matter of discussion and requires further studies. Pilocytic Astrocytoma Many GG contain a predominant glia component, displaying a pilocytic astrocytoma pattern. The differential diagnosis is particularly difficult in tumors located in the cerebellum, where a pilocytic astrocytoma is often expected. The lack of CD34 IR, the presence of strong MAP2-positive neoplastic glial cells, as well as the usually higher proliferation activity (>1%) support the diagnosis of a pilocytic astrocytoma. Astrocytoma (WHO Grade II) As discussed above, the detection of dysplastic neurons and their differentiation from entrapped neurons within pre-existing brain parenchyma infiltrated by a diffuse astrocytoma represent a major diagnostic problem which may be addressed immunohistochemically. In this differential diagnosis, the presence of MAP2-positive neoplastic glial cells, the lack of CD34 staining, as well as the higher proliferation activity compared to GG support the diagnosis of astrocytoma. Pleomorphic Xanthoastrocytoma This tumor may have a prominent reticulin stroma, eosinophilic bodies and large pleomorphic cells that can resemble the dysplastic neurons of GG. Immunocytochemical analysis may be helpful to reveal the astroglial nature of these large cells, displaying strong GFAP IR; CD34 expression has been also observed (Blumcke and Wiestler, 2002). Thus, the lack of a neuronal dysplastic component together with the MAP2- positivity of neoplastic astrocytes and the higher proliferation activity compared to GG may help to differentiate these two entities. However, tumors with features of both pleomorphic xanthoastrocytoma and GG have been reported (Evans et al., 2000). Oligodendroglioma (WHO Grade II) A differential diagnosis with grade II oligodendroglioma could be considered in cases of GG with

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prominent clear cell elements, resembling oligodendrocytes. The absence of dysplastic neuronal cells, as well as the distinct pattern of IR observed with MAP2 in oligodendroglioma and the higher proliferation activity may be helpful in the differential diagnosis. Dysembryoplastic Neuroepithelial Tumor (DNT) The differential diagnosis with DNT may represent a problem in case of small biopsies with few typical architectural features (such as multifocal nodular appearance and “floating” neurons in DNT) or in case of a DNT with a prominent glial component.

Coexistence with Cortical Dysplasia Dysplastic disorganization of the cortex near but separate from the tumor has been reported in patients with GNTs (Blümcke et al., 2009). According to the presently used classification of cortical dysplasia (Palmini et al., 2004) the large majority of GNT cases are combined with mild malformations of cortical development (mMCD) or with focal cortical dysplasia (FCD) type I (Fig. 29.2k). Whether cortical dysplasia combined with GNTs represents a distinct type of dysplasia (different from isolated FCDs) is a matter of ongoing debate. In addition, the potential contribution of this peritumoral cortical disorganization to the mechanisms of epileptogenesis will need further research and evaluation.

Prognostic Factors and Surgical Outcome Although GG are generally benign neoplasms, tumor recurrence, malignant progression and secondary glioblastoma multiforme (GBM) have been observed in some patients. Tumor mass effects and pharmacoresistent seizures are common manifestations of GG. Early surgical resection of GG reduces longterm morbidity and mortality from seizures, making surgery the treatment of choice for most patients when compared to medical management (Morris et al., 1998; Aronica et al., 2001a). Comparability of postoperative seizure outcome between different series is

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complicated by differences in the follow up period and definition of seizure freedom, i.e. implication of patients suffering exclusively from auras or not. In large series the reported results of postoperative seizure freedom range from 63% to 90 (Morris et al., 1998; Aronica et al., 2001a; Im et al., 2002; Giulioni et al., 2006; Ogiwara et al., 2010). Some authors reported improved prognosis regarding seizure outcome in younger patients (Aronica et al., 2001a; Majores et al., 2008), whereas others found no correlation with age at the time of surgery (Giulioni et al., 2006; Ogiwara et al., 2010). Complete surgical resection indicates an optimal prognosis, even if significant reduction of symptoms including seizure freedom can be often achieved with partial resection. Particularly anaplastic features and malignant progression were associated with older age at surgery, subtotal resection of the tumor, extratemporal location and absence of epilepsy (Aronica et al., 2001a; Luyken et al., 2004; Majores et al., 2008). Additionally, tumor localisation and resection strategies affect surgical outcome. Whereas in series with tailored surgery using intraoperative electrocorticography (ECoG) best results are reported in patients with temporal GG (Ogiwara et al., 2010), resection of tumor alone (lesionectomy) has been associated with a less satisfactory outcome in temporo-mesial GG than in neocortical temporal or extratemporal GG (Giulioni et al., 2006). Those findings support an epileptogenic effect of the peritumoral zone especially in temporo-mesial GG. Furthermore, association with cortical dysplasia has been reported to determine a less effective control of seizures after surgery (Im et al., 2002). Concerning the histopathological parameters, anaplastic changes in the glial component, such as increased cellularity and mitotic activity, the presence of microvascular proliferation and necrosis, as well as high Ki-67 and TP53 indices, indicate a malignant progression and are associated with a less favorable outcome. A more recent analysis of the neuropathological features of GG with recurrence and malignant progression indicates the presence of a gemistocytic cell component and focal tumor cell associated CD34 immunolabeling as significant predictors of an adverse clinical course (Majores et al., 2008). However, malignant progression to a GBM usually is associated with loss of GG features, including loss of CD34 immunolabeling (Majores et al., 2008). Post-operative complications correspond


with tumor localisation. Most frequently neurological deficits like quadrantanopia, transitory aphasia, as well as memory decline are reported (Clusmann et al., 2002; Ogiwara et al., 2010).

Molecular Genetics and Pathogenesis In comparison to other tumor entities, little is known about the molecular pathogenesis of GG. Screening for genomic alterations by chromosomal and array-based comparative genomic hybridization (CGH) in a large series of 61 GG showed aberrations in 66% of GG (Hoischen et al., 2008). The most frequent aberration was gain of chromosome 7 (21%). Unsupervised cluster analysis of genomic profiles detected two major subgroups (group I: complete gain of 7 and additional gains of 5, 8 or 12; group II: no major recurring imbalances, mainly losses). A comparison of CGH data from GG and diffuse astrocytomas (WHO grade II) revealed gain of chromosome 5 significantly more frequent in GG. Interestingly, by unsupervised cluster analysis, all but one diffuse astrocytoma formed subclusters within group II of GG showing no concordant pattern of genomic imbalances. This finding suggests that the GG cluster defined by combined gains of chromosomes 5, 7, 8 and 12 (group I) represents a subgroup that can be genetically distinguished from diffuse astrocytomas, although cellular elements in the latter and the glial component of GG are generally not distinguishable with respect to their cytoand histopathological characteristics. With interphase FISH experiments the authors localized the imbalances on the cellular level to a subpopulation of glial cells, while no dysplastic neuronal cells carried those aberrations. In the same study, the analysis of two primary GG and their anaplastic recurrences identified genetic aberrations commonly associated with malignant gliomas (losses of CDKN2A/B and DMBT1 or a gain/amplification of CDK4), which were found in the anaplastic tumors and were already present in the respective GG (Hoischen et al., 2008). Interestingly, GG from patients with long-standing epilepsy had a significantly lower median number of imbalances per case than tumors from patients without epilepsy. There may be an association between these results and the observation that GG-patients with long-standing epilepsy have a lower recurrence rate and a better


clinical course than patients without epilepsy (Luyken et al., 2004). In the limited number of cytogenetically analyzed GG in further studies, numerical or structural aberrations of chromosome 7 were the only alterations detected in more than two cases (for review see Louis et al., 2007). Gain of chromosome 7 was also found recurrently by chromosomal CGH analysis (Squire et al., 2001; Yin et al., 2002; Pandita et al., 2007). The focal nature and differentiated glioneuronal phenotype as well as the expression of the stem cell epitope CD34 and coexistence with cortical dysplasia point toward an origin of GG from developmentally compromised or dysplastic precursor lesions (Blumcke and Wiestler, 2002; Fauser et al., 2004). In addition, histological similarities hint at an etiological relation with other primary low-grade brain tumors such as dysembryoplastic neuroepithelial tumours (DNETs) or diffuse and pilocytic astrocytomas. However, genetic studies have argued against the contribution of mutational events in genes with pathogenic relevance in other brain tumors (eg, TP53, EGFR, PTEN) to play a major role in GG (reviewed in Becker et al., 2006). Analysis of genes involved in the reelin pathway with a major role in neuronal development and cellular migration showed no evidence for mutations (e.g. CDK5, DCX, CDK5R1, DAB1) (Becker et al., 2002; Kam et al., 2004) On the other hand, GG did show lower mRNA expression of these genes compared with normal central nervous system tissue controls. Array analysis of epilepsy-associated GG revealed reduced expression, of the LIM-domain-binding 2 (LDB2) gene (Fassunke et al., 2008). Since LDB2 is known to play a critical role in brain development during embryogenesis, its reduced expression may represent an additional molecular mechanism contributing to the development of an aberrant neuronal network in GG (Fassunke et al., 2008). Further molecular genetic studies of GG suggest an activation of the PI3K-mTOR pathway, which is involved in cell size- and growth-control, cortical development and neuronal migration. Ligand binding to insulin receptors or growth factor receptors activates the cascade components phosphatidylinositol-3 kinase (Pi3K), Akt, TSC1 (hamartin), TSC2 (tuberin), mTOR (mammalian target of rapamycin), and the transcription factors p70S6kinase (S6K) and ribosomal S6 protein (S6) (Fig. 29.3a).

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Hamartin and tuberin constitute a tumor suppressor complex with a central role in the PI3K-mTOR pathway (Potter et al., 2001). Several studies examined aberrant patterns of allelic variants in the TSC1 and TSC2 genes in GG and FCD IIa/IIb (reviewed in Becker et al., 2006). In contrast to FCD IIb, that showed an accumulation of partially coding polymorphisms in TSC1, FCD IIa and GG exhibit increased incidences of polymorphisms in TSC2. Polymorphisms in intron 4 and exon 41 of TSC2 were substantially increased in GG compared with controls. A somatic mutation in TSC2 intron 32 was identified in the glial cellular elements but not in the neurons of a single GG, compatible with a clonal expansion of the glial component within this tumor. The differential patterns of allelic variants in TSC1 and TSC2 argue against FCD IIb as a dysplastic precursor lesion for GG. The ERM proteins (ezrin, radixin and moesin) interact with hamartin and regulate cell adhesion and migration. Whereas sequence analysis of ezrin and radixin showed only occasional polymorphisms in epilepsy-associated tumors and FCDs, immunohistochemical data revealed aberrant labeling of ERM proteins in a high percentage of dysplastic elements in different glioneuronal lesions including GG (reviewed in Becker et al., 2006). Recent findings of further alterations in the Pi3K-mTOR cascade strongly support a pathogenetic relevance of this pathway for GNT (Boer et al., in press); (Fig. 29.3a). In addition, increased expression of components of this signaling pathway was not detected in patients with DNT, suggesting a different pathogenetic origin for this tumor. An intriguing question for the future is to further elucidate the mechanisms that regulate the Pi3K-mTOR signaling to explain the observed differences in activation of this pathway in the different developmental lesions.

Mechanisms of Epileptogenesis The association between epilepsy and brain tumors has been observed for over one century. In 1882 Hughlings Jackson made the important observation that often epilepsy represents the initial and only clinical manifestation of glial tumors; moreover he was the first to recognize the relationship between

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Fig. 29.3 Pathogenesis and epileptogenesis. Panel (a): Schematic representation of the Pi3K-mTOR signaling pathway. Ligand binding to insulin receptors or growth factor receptors trigger phosphatidylinositol-3 kinase (Pi3K), which in turn activates the phosphoinositide-dependent protein kinase 1 (PDK1) by phosphorylation. Akt is phosphorylated and activated by phosphorylated (p)-PDK1 or by the adhesion molecule on glia (AMOG) independently of PDK1 and Pi3K. P-Akt inactivates the tumor suppressor protein tuberin (TSC2) by phosphorylation which results in the indirect activation of the mammalian target of rapamycin (mTOR). Downstream phosphorylation of the eukaryotic initiation factor 4E binding protein

1 (4E-BP1) releases the eukaryotic initiation factor 4E (eIF4E). EIF4E interacts with phosphorylated eIF4G to activate capdependent mRNA translation which enhances cell size and cell proliferation. Cell size and proliferation are also regulated by phosphorylation of the ribosomal protein S6 kinase (p70S6K) and its downstream effector ribosomal protein S6. The ERM proteins (Ezrin, radixin and moesin) interact with hamartin (TSC1) and regulate cell adhesion and migration. Components of the pathway studied in ganglioglioma are shaded in blue. Panel (b): alterations at cellular/molecular/circuit level in the lesion, in the perilesional region and in the global network potentially contributing to epileptogenesis in GG

tumor epileptogenicity and involvement of cortical gray matter in patients with brain tumors (Jackson, 1882). The advent of the neurochirurgical treatment of epilepsy associated brain lesions confirmed these initial observations and several clinical studies emphasize that pharmacologically intractable epilepsy critically affects the daily life of patients with brain tumors, even if the tumor is under control (for review see van Breemen et al., 2007; Shamji et al., 2009). The incidence of brain tumors in patients with epilepsy is about 4% and the frequency of epilepsy in patients with brain tumors is 30% or more depending on the type of the tumor (van Breemen et al., 2007). In principle, any tumor (extra-axial, intra-axial, benign or malignant, common or uncommon) can cause seizures. However, patients with supratentorial low-grade tumors are more likely to develop epilepsy (van Breemen et al., 2007). In particular GG represents the most frequent tumor in patients with focal pharmacologically intractable epilepsy (Blumcke and Wiestler, 2002; Luyken et al., 2004).

The cellular mechanism(s) underlying the epileptogenicity of brain tumors are still not clearly understood. A number of hypotheses have been put forward during the past decades that could explain increased excitability in patients with brain tumors (van Breemen et al., 2007; Shamji et al., 2009). It is likely that multiple mechanisms are involved, including both tumor related factors (tumor type, tumor location), as well as genetic and peritumoral and changes (van Breemen et al., 2007; Shamji et al., 2009). Figure 29.3b shows the mechanisms potentially contributing to epileptogenesis in GG.

Tumor Related Factors The usual localization of GG in the temporal lobe, often with cortical involvement, may only partially explain the raised likelihood of epileptic activity in this entity. The strong association of GG with chronic


seizures could also reflect the peculiar cellular composition and neurochemical profile of these tumors. Several studies have demonstrated the intrinsic epileptogenicity of GG, indicating the presence of a hyperexcitable neuronal component (for review see Blumcke and Wiestler, 2002). Analysis of electrocorticographic patterns in GG reveals that a relatively high neuronal density in the lesion is associated with highly epileptiform discharge patterns, such as continuous spiking or recruiting discharges (Ferrier et al., 2006). The immunocytochemical profile of the dysplastic neuronal component of GG indicates high expression of specific glutamate receptors (GluR) subtypes including both ionotropic and metabotropic glutamate receptors (mGluR). These findings support a central role for glutamatergic transmission in the mechanisms underlying the intrinsic, high epileptogenicity of GG (Aronica et al., 2001b). More recent studies analyzing the gene expression profile of GG support the existence of developmental alterations of the balance between excitation and inhibition within the lesion (Samadani et al., 2007; Aronica et al., 2008; Fassunke et al., 2008). Single cell analysis demonstrates a prominent expression of the mGluR5 in the neuronal component of GG (Samadani et al., 2007). In contrast, a downregulation of several GABAa receptor (GABAA R) subunits (including α1, α5, ß1, ß3 and δ subunit) was detected in GG, suggesting an impairment of GABAergic inhibition (Samadani et al., 2007; Aronica et al., 2008). Increase of the sodium-potassium chloride co-transporter (NKCC1) expression and a reduced expression of the potassium-chloride co-transporter KCC2 has been also reported (Aronica et al., 2008). The expression patterns of NKCC1 and KCC2 resemble the expression patterns observed in immature brain and support the hypothesis of a failure of developmental maturation in the pathogenesis of GG. Moreover, the reported deregulation of these transporters could actively contribute to the epileptogenicity of GG, via modulation of GABA receptors (Yamada et al., 2004). Both gene expression and immunocytochemical studies provide evidence for a prominent activation of the inflammatory response in GG. In particular, upregulation of inflammatory interleukins (such IL1β), activation of the complement cascade and the Toll-like receptor pathway are particularly consistent in GG (Aronica et al., 2008). Interestingly, several observations strongly support the proconvulsant and ictogenic properties of inflammatory molecules and

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suggest a pathogenic role for inflammation in epilepsy (reviewed in Vezzani and Granata, 2005). The prominent increased expression of the immune response with activation of the complement cascade and the production of pro-inflammatory cytokines observed in GG may induce alterations of the blood brain barrier (BBB), which could play an additional role in the tumor epileptogenicity (Shamji et al., 2009). Decreased expression of glial glutamate transporters, in GG in combination with an enhanced amount of astrocytes, may also contribute to tumor epileptogenesis, increasing the extracellular glutamate concentration (Samadani et al., 2007). An aditional potential mechanism underlying tumorassociated epilepsy is represented by an altered potassium homeostasis. The gene expression profile of GG with low expression of several potassium channel genes suggests a disturbed ion homeostasis and transport that could lead to increased excitability (Aronica et al., 2008). Alterations in Pi3K-mTOR pathway components in GG have been recently reported (Samadani et al., 2007; Schick et al., 2007; Boer et al., in press). Interestingly, recent studies have demonstrated the critical role of this pathway in epileptogenesis and the epileptogenic potential of mTOR inhibitors (Zeng et al., 2008). However, additional experimental work is needed to clarify the molecular mechanisms by which mTOR pathway activation may influence the epileptogenesis in GG. Alterations in antiepileptic drug targets (such as ion channels and neurotransmitter receptors) and overexpression of multidrug transporters, such as P-glycoprotein (P-gp;(Aronica et al., 2003)) reported in GG, may likely underlie the drug refractoriness observed in patients with GG.

Peritumoral Changes The peritumoral region may also be relevant for the generation and propagation of seizure activity (van Breemen et al., 2007). The epileptogenicity of the peritumoral zone is supported by both functional and immunocytochemical studies, showing network alterations and revealing cytoarchitectural and neurochemical changes in the cortex resected from patients with intractable epilepsy associated with different types of focal brain lesions, including glial tumors (van Breemen et al., 2007; Shamji et al., 2009).

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Abrupt tissue damage with isolation of cortical area (deafferentation) and hemosiderin deposition have been suggested to be implicated as cause of seizure activity in rapidly progressive high grade tumors. Experimental studies have shown that glioma invasion may alter the discharge properties of neighbouring neurons, converting them to bursting cells and hence providing “pacemaker” cells driving the neuronal networks surrounding the tumour (Kohling et al., 2006). Hypoxia and acidosis, ionic changes, enhanced intercellular communication through increased expression of gap junction channels and increased level in the peritumoral region have also been suggested as potential mechanisms affecting epileptogenesis in gliomas (van Breemen et al., 2007; Shamji et al., 2009). Perilesional changes, involving both excitatory and inhibitory pathways have been reported in GG (Aronica et al., 2007; for review see Blumcke, 2009). The reported perilesional changes suggest, in particular, a complex alteration of the GABAergic system in patients with GG (Aronica et al., 2007). Finally, the potential contribution of peritumoral cortical disorganization (coexistence with cortical dysplasia; as discussed above) has to be considered in the evaluation of the epileptogenicity of GG. Acknowledgments We thank Dr. Albert Becker (Dept. of Neuropathology, University of Bonn Medical Center) for his input in the final version of this chapter.

References Aronica E, Gorter JA, Jansen GH, van Veelen CW, van Rijen PC, Leenstra S, Ramkema M, Scheffer GL, Scheper RJ, Troost D (2003) Expression and cellular distribution of multidrug transporter proteins in two major causes of medically intractable epilepsy: focal cortical dysplasia and glioneuronal tumors. Neuroscience 118:417–429 Aronica E, Boer K, Becker A, Redeker S, Spliet WG, van Rijen PC, Wittink F, Breit T, Wadman WJ, Lopes da Silva FH, Troost D, Gorter JA (2008) Gene expression profile analysis of epilepsy-associated gangliogliomas. Neuroscience 151:272–292 Aronica E, Leenstra S, van Veelen CW, van Rijen PC, Hulsebos TJ, Tersmette AC, Yankaya B, Troost D (2001a) Glioneuronal tumors and medically intractable epilepsy: a clinical study with long-term follow-up of seizure outcome after surgery. Epilepsy Res 43:179–191 Aronica E, Yankaya B, Jansen GH, Leenstra S, van Veelen CW, Gorter JA, Troost D (2001b) Ionotropic and metabotropic glutamate receptor protein expression in glioneuronal tumors

E. Aronica and P. Niehusmann from patients with intractable epilepsy. Neuropathol Appl Neurobiol 27:1–16 Aronica E, Redeker S, Boer K, Spliet WG, van Rijen PC, Gorter JA, Troost D (2007) Inhibitory networks in epilepsyassociated gangliogliomas and in the perilesional epileptic cortex. Epilepsy Res 74:33–44 Becker AJ, Klein H, Baden T, Aigner L, Normann S, Elger CE, Schramm J, Wiestler OD, Blumcke I (2002) Mutational and expression analysis of the reelin pathway components CDK5 and doublecortin in gangliogliomas. Acta Neuropathol 104:403–408 Becker AJ, Blumcke I, Urbach H, Hans V, Majores M (2006) Molecular neuropathology of epilepsy-associated glioneuronal malformations. J Neuropathol Exp Neurol 65:99–108 Blumcke I (2009) Neuropathology of focal epilepsies: a critical review. Epilepsy Behav 15:34–39 Blumcke I, Wiestler OD (2002) Gangliogliomas: an intriguing tumor entity associated with focal epilepsies. J Neuropathol Exp Neurol 61:575–584 Blümcke I, Vinters HV, Armstrong D, Aronica E, Thom M, Spreafico R (2009) Malformations of Cortical Development and Epilepsies: Neuropathological findings with emphasis on Focal Cortical Dysplasia. Epileptic Disord 11:181–193 Boer K, Troost D, Timmerman W, Spliet WGM, van Rijen PC, Aronica E (2009) Pi3K-mTOR signaling and AMOG expression in epilepsy-associated glioneuronal tumors. Brain Pathol Apr 7. [Epub ahead of print] Clusmann H, Schramm J, Kral T, Helmstaedter C, Ostertun B, Fimmers R, Haun D, Elger CE (2002) Prognostic factors and outcome after different types of resection for temporal lobe epilepsy. J Neurosurg 97:1131–1141 Evans AJ, Fayaz I, Cusimano MD, Laperriere N, Bilbao. JM (2000) Combined pleomorphic xanthoastrocytomaganglioglioma of the cerebellum. Arch Pathol Lab Med 124:1707–1709 Fassunke J, Majores M, Tresch A, Niehusmann P, Grote A, Schoch S, Becker AJ (2008) Array analysis of epilepsyassociated gangliogliomas reveals expression patterns related to aberrant development of neuronal precursors. Brain 131:3034–3050 Fauser S, Becker A, Schulze-Bonhage A, Hildebrandt M, Tuxhorn I, Pannek HW, Lahl R, Schramm J, Blumcke I (2004) CD34-immunoreactive balloon cells in cortical malformations. Acta Neuropathol 108:272–278 Ferrier CH, Aronica E, Leijten FS, Spliet WGM, van Huffelen AC, van Rijen PC, Binnie CD (2006) Electrocorticographic discharge patterns in glioneuronal tumors and focal cortical dysplasia. Epilepsia 47:1477–1486 Giulioni M, Gardella E, Rubboli G, Roncaroli F, Zucchelli M, Bernardi B, Tassinari CA, Calbucci F (2006) Lesionectomy in epileptogenic gangliogliomas: seizure outcome and surgical results. J Clin Neurosci 13:529–535 Hoischen A, Ehrler M, Fassunke J, Simon M, Baudis M, Landwehr C, Radlwimmer B, Lichter P, Schramm J, Becker AJ, Weber RG (2008) Comprehensive characterization of genomic aberrations in gangliogliomas by CGH, array-based CGH and interphase FISH. Brain Pathol 18:326–337 Im SH, Chung CK, Cho BK, Wang KC, Yu IK, Song IC, Cheon GJ, Lee DS, Kim NR, Chi JG (2002) Intracranial ganglioglioma: preoperative characteristics and oncologic outcome after surgery. J Neuro Oncol 59:173–183

29 Gangliogliomas: Molecular Pathogenesis and Epileptogenesis Jackson JH (1882) Localized convulsions from tumor of the brain. Brain 5:364–374 Kam R, Chen J, Blumcke I, Normann S, Fassunke J, Elger CE, Schramm J, Wiestler OD, Becker AJ (2004) The reelin pathway components disabled-1 and p35 in gangliogliomas– a mutation and expression analysis. Neuropatho Appl Neurobiol 30:225–232 Kohling R, Senner V, Paulus W, Speckmann EJ (2006) Epileptiform activity preferentially arises outside tumor invasion zone in glioma xenotransplants. Neurobiol Dis 22:64–75 Louis, DN, Ohgaki, H, Wiestler, OD and Cavanee, WK (eds) (2007) WHO classification of tumours of the central nervous system. IARC, Lyon Luyken C, Blumcke I, Fimmers R, Urbach H, Wiestler ODand, Schramm J (2004) Supratentorial gangliogliomas: histopathologic grading and tumor recurrence in 184 patients with a median follow-up of 8 years. Cancer 101:146–155 Majores M, von Lehe M, Fassunke J, Schramm J, Becker AJ, Simon M (2008) Tumor recurrence and malignant progression of gangliogliomas. Cancer 113:3355–3363 Morris HH, Matkovic Z, Estes ML, Prayson RA, Comair YG, Turnbull J, Najm I, Kotagal Pand, Wyllie E (1998) Ganglioglioma and intractable epilepsy: clinical and neurophysiologic features and predictors of outcome after surgery. Epilepsia 39:307–313 Ogiwara H, Nordli DR, DiPatri AJ, Alden TD, Bowman RM, Tomita T (2010) Pediatric epileptogenic gangliogliomas: seizure outcome and surgical results. J Neurosur Pediatr 5:271–276 Palmini A, Najm I, Avanzini G, Babb T, Guerrini R, FoldvarySchaefer N, Jackson G, Luders HO, Prayson R, Spreafico R, Vinters HV (2004) Terminology and classification of the cortical dysplasias. Neurology 62:S2–8 Pandita A, Balasubramaniam A, Perrin R, Shannon P, Guha A (2007) Malignant and benign ganglioglioma: a pathological and molecular study. Neuro Oncol 9:124–134

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Potter CJ, Huang Hand, Xu T (2001) Drosophila Tsc1 functions with Tsc2 to antagonize insulin signaling in regulating cell growth, cell proliferation, and organ size. Cell 105:357–368 Quinn B (1998) Synaptophysin staining in normal brain: importance for diagnosis of ganglioglioma. Am J Surg Pathol 22:550–556 Samadani U, Judkins AR, Akpalu A, Aronica E, Crino PB (2007) Differential cellular gene expression in ganglioglioma. Epilepsia 48:646–653 Schick V, Majores M, Koch A, Elger CE, Schramm J, Urbach H, Becker AJ (2007) Alterations of phosphatidylinositol 3kinase pathway components in epilepsy-associated glioneuronal lesions. Epilepsia 48(Suppl 5):65–73 Shamji MF, Fric-Shamji ECand, Benoit BG (2009) Brain tumors and epilepsy: pathophysiology of peritumoral changes. Neurosurg Rev 32:275–284 Squire JA, Arab S, Marrano P, Bayani J, Karaskova J, Taylor M, Becker L, Rutka J, Zielenska M (2001) Molecular cytogenetic analysis of glial tumors using spectral karyotyping and comparative genomic hybridization. Mol Diagn 6:93–108 van Breemen MS, Wilms EB, Vecht CJ (2007) Epilepsy in patients with brain tumours: epidemiology, mechanisms, and management. Lancet Neurol 6:421–430 Vezzani A, Granata T (2005) Brain inflammation in epilepsy: experimental and clinical evidence. Epilepsia 46:1724–1743 Yamada J, Okabe A, Toyoda H, Kilb W, Luhmann HJ, Fukuda A (2004) Cl- uptake promoting depolarizing GABA actions in immature rat neocortical neurones is mediated by NKCC1. J Physiol 557:829–841 Yin XL, Hui AB, Pang JC, Poon WS, Ng HK (2002) Genomewide survey for chromosomal imbalances in ganglioglioma using comparative genomic hybridization. Cancer Genet Cytogenet 134:71–76 Zeng LH, Xu L, Gutmann DH, Wong M (2008) Rapamycin prevents epilepsy in a mouse model of tuberous sclerosis complex. Ann Neurol 63:444–453

Chapter 30

Epilepsy-Associated Gangliogliomas: Identification of Genes with Altered Expression Albert J. Becker

Abstract Gangliogliomas are the most frequent neoplasms in patients with temporal lobe epilepsy (TLE). The characteristic histopathological composition of glial and neuronal elements, the focal nature and their differentiated phenotype and benign biological behavior suggest an origin from a developmentally compromised or dysplastic precursor lesion e.g. by neoplastic transformation of the glial component. The complex cellular admixture of these glioneuronal tumors represents a particular challenge for functional genomics. Considering the large number of expressed genes already in normal neuronal and glial cell types, unraveling of comprehensive, transcriptional patterns in glioneuronal neoplasms as well as cell-specific expression profiles are critical to further understand the molecular pathology of gangliogliomas. New developments in microarray and gene expression profiling strategies allow transcriptional studies for (a) many mRNAs in parallel or even the entire number of human genes as well as (b) starting from small amounts of tissue or individual cells. In concert with mRNA amplification strategies from single cells as well as laser-microdissection approaches and quantitative reverse transcription-polymerase chain reaction (RT-PCR) technologies, comprehensive expression analyses can be carried out in bioptic tumor tissue or even cellular subpopulations. The integrated interpretation of gene expression data with comprehensive data from genomic as well as promoter analyses represents an intriguing perspective to improve the

A.J. Becker () Department of Neuropathology, University of Bonn Medical Center, D-53105, Bonn, Germany e-mail: [email protected]

understanding and develop new treatment regimens for gangliogliomas. Keywords Gangliogliomas · Temporal lobe epilepsy · Microdissection · Oligonucleotide · Reelin

Introduction A major clinical feature of glioneuronal tumors is given by their manifestation with partial seizures. Within a number of different entities, gangliogliomas represent the most frequent tumors in surgical specimens from epileptic patients. They represent approximately 5% of brain tumors in childhood, but are rare in adults (Blümcke et al., 1999; Luyken et al., 2004). Gangliogliomas WHO grade I are most frequently present within the temporal lobe (>70%) (Blümcke and Wiestler, 2002). In gangliogliomas with anaplastic features (WHO grade III) the temporal lobe appears to be less frequently affected (Blümcke and Wiestler, 2002). The dual composition of dysmorphic neuronal cell combined with glial cells represents a histopathological hallmark of gangliogliomas (Fig. 30.1). The neoplastic nature of the tumor is reflected by the proliferative but rarely mitotic activity of the glial cell component, whereas the neuronal tumor element is generally considered to be non-neoplastic. Nuclear labeling for the proliferating cell nuclear antigen Ki-67 can observed in astrocytic tumor elements (Wolf et al., 1994). The focal nature of gangliogliomas, the differentiated glioneuronal phenotype and the benign clinical character have substantiated the hypothesis that these tumors derive from developmentally compromized or dysplastic precursor lesions


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Fig. 30.1 Neuropathological hallmarks of gangliogliomas. (a) Gangliogliomas show a composition of glial and dysmorphic neuronal elements. The arrow points to a large, irregularly oriented dysmorphic neuron with aberrantly clustered Nissl-substance (hematoxylin-eosin (HE), scale bar: 50 μm). (b) Immunohistochemically, neoplastic astroglial cells are marked by strong expression of glial fibrillary acid protein

(GFAP) in delicate processes, that form a fibrillary matrix. (c) Immunohistochemistry with an antibody directed against synaptophysin underlines the irregular orientation of neurons interspersed within the glial matrix of the tumor. Perisomatic accumulation of synaptophysin is a typical finding in gangliogliomas (arrow)

(Blümcke et al., 1999). Correspondingly, the stem cell epitope CD34 has been observed abundantly expressed in gangliogliomas (Blümcke et al., 1999). These characteristics of gangliogliomas, i.e. an admixture of strikingly heterogeneous cellular populations suggest a complex etiology and pathogenesis. In order to learn more about the pathological basis of these intriguing neoplasm, refined gene expression analysis tools provide excellent technical options, which are complementary applied.

microarrays as well as to resolute gene expression on a cellular level (Fassunke et al., 2008, 2004). An important prerequisite for expression profiling experiments is given by the use of suitable controls or matched pairs of samples such as histopathologically or immunohistochemically characterized cellular elements, identified cell populations, brain regions or groups of neuropathologically characterized individuals (Crino et al., 2001; Fassunke et al., 2004). With respect to the composition of gangliogliomas the aspect of appropriate control tissue is particularly important. One approach to overcome this problem is to use ‘control’ brain tissue adjacent to ganglioglioma portions matched for grey and white matter composition of identical patients, which excludes differential expression due to differences in genetic background of different tumor and control individuals (Fassunke et al., 2008). With respect to expression array technologies largescale oligonucleotide (Lipshutz et al., 1999) and glass microscope slide DNA arrays (Brown and Botstein,

Expression Analysis: Methodology The development of expression microarray technology, i.e. cDNA and oligonucleotide arrays, allows highly parallel analysis of human tissue gene expression profiles (Bowtell, 1999; Brown and Botstein, 1999; Lipshutz et al., 1999). In addition to genome-wide studies, particularly real-time RT-PCR is a powerful approach to validate expression profiles obtained with

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Fig. 30.2 Gene expression analysis – technical approaches. (a) Oligonucleotide arrays (e.g. Affymetrix system) represent a standard approach for highly parallel gene expression analyses based on non-competitive hybridization. Initial double strand cDNA synthesis is followed by an RNA-polymerase step, i.e. moderate amplification of templates biotin-labeling resulting in cRNA, which is hybridized. Each template transcript is represented by several short oligonucleotide sequences on the array. The specificity of hybridization for perfect sequences (PM) is monitored by a mismatch (MM) sequence with minute alteration in the DNA-sequence composition. This procedure allows large-scale parallel expression analyses with high specificity. (b) In order to learn about specific gene expression patterns in individual, highly defined cellular elements, complementary

approaches are useful such as in situ-RT and immunolaser microdissection. This procedure was used to unravel the cellular nature of CD34-expressing ganglioglioma components. As initial step we applied in situ-reverse transcription on fresh frozen sections from gangliogliomas. After in situ-RT and an immunohistochemical reaction with an antibody directed against CD34 (arrow marks a CD34 expressing cellular element, scale bar: 200 μm) laser microdissection of an individual cell was carried out. PCR analysis with lineage specific primers reveals represented the final step of this experiment (lane 1: GFAP, lane 2: NFM, lane 3: MBP, lane 4: HLA-DQ, lane 5: CD34). The result suggests a neuronal nature of CD-34 expressing cellular elements of gangliogliomas

1999) hybridized with fluorescent probes and analyzed by specific detection systems are widely applied (Fig. 30.2). Large-scale expression arrays have proven to constitute useful tools for monitoring gene expression and open new avenues for the identification of pathogenesis-related molecules and mechanisms of central nervous tissue disorders including low-grade brain tumors (Aronica et al., 2008; Fassunke et al., 2008). A certain limitation of microarray systems with respect to complex tissue samples reflected by summative expression profiles is the need of sufficient amounts of region/cell type specific mRNA (Becker et al., 2003; Duggan et al., 1999). The analysis of cellular pathology particularly in complex glioneuronal lesions and tumors represents a critical issue since mRNA isolates from gangliogliomas contain combinations of different cell types (Becker et al., 2007). Therefore, expression differences may be

contaminated due to heterogeneous cellular composition within the sample of mRNA origin for expression analysis rather than to prove true disease associated alterations in gene expression. In order to analyze specific cellular populations, separation may be needed. As a particular powerful technique used in glioneuronal lesions, aRNA amplification has been applied to generate sufficient amounts of aRNA for array hybridization starting from individual cells (Crino et al., 2001). A particular advantage of this method is the option to determine differential expression of large numbers of genes starting from minute amounts of mRNA isolated of even individual cells. (Laser)-microdissection of individual cells or cellular groups represents a highly useful technology to be combined with aRNA or RT-PCR protocols to determine changes of gene expression in ganglioglioma pathogenesis (Becker et al., 2006).

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Differential Gene Expression in Gangliogliomas In addition to their biphasic composition by dysplastic neuronal cells as well as neoplastic glial components, the latter showing proliferative activity (Wolf et al., 1994), particularly the expression of the stem cell epitope CD34 in gangliogliomas has substantiated the hypothesis of an origin from developmentally compromised or dysplastic precursor malformations (Blümcke et al., 1999; Fassunke et al., 2004). However, the cellular nature of CD34 expressing elements in gangliogliomas had remained enigmatic. In order to characterize CD34 expressing ganglioglioma components, we have performed single-cell mRNA analysis in gangliogliomas (Fig. 30.2) (Fassunke et al., 2004). After in situ-RT and immunohistochemistry by an antibody against CD34 we used laser microdissection of individual ganglioglioma components. Subsequently, we used PCR systems with different lineage markers, and observed co-expression of CD34 and neurofilament (NFM) protein but not other lineage markers (Fig. 30.2). This finding is in line with neuronal elements of compromised differentiation indicated by expression of CD34 as inherent components of gangliogliomas. Complementary to this qualitative expression analysis approach starting from distinct laser microdissected cellular elements, we have addressed alterations in components of a major signal pathway for neuronal development and migration, i.e. the signaling cascade (Gilmore and Herrup, 2000). Lower mRNA transcript numbers of CDK5, DCX, p35 and dab1, coding for proteins that operate in the reelin signaling cascade, were found in gangliogliomas compared to normal brain tissue controls (Becker et al., 2001; Kam et al., 2004). Low expression of several components of the reelin signaling cascade may point towards impaired reeling cascade signaling in gangliogliomas as factor related to its dysmorphic appearance. Epigenetic modification of factors of the reelin signaling cascade (Kobow et al., 2009) should be taken into consideration in this context in the future. In order to learn about differential expression patterns with emphasis on developmental mechanisms more comprehensively, several large scale expression array studies have been carried out in gangliogliomas.

A.J. Becker

Recently, we have carried out an expression array study starting from discrete microdissected ganglioglioma and adjacent control brain tissue obtained from the neurosurgical access area to the tumor of identical patients that were carefully matched for equivalent glial and neuronal elements (Fassunke et al., 2008). A cluster analysis with all present genes based on euclidean distance as a samplewise distance measure and average linkage as a setwise distance measure showed tumors and controls to be fairly well separated (Fassunke et al., 2008). Intriguingly, even in highly differentiated tumors such as gangliogliomas, the parameter “tumor” apparently was more distinctive than “identical genetic background” with respect to comprehensive gene expression. Although gene expression in a considerable range of molecular cascades was affected in gangliogliomas, differential expression of more than individual genes related to only a certain circumscribed number of elementary cellular functions and molecular pathways, i.e., regulation of chromatin state and transcription factors, intracellular signal transduction, transduction of extracellular signals and cell adhesion, control of cell cycle and proliferation, development and differentiation (Fig. 30.3). As particularly interesting molecules, we observed two LIM domain-interacting transcripts, i.e. LIM domain only 4 (LMO4) and LIM domain binding 2 (LDB2), as strikingly lower in expression in gangliogliomas compared to controls (Fassunke et al., 2008). LIM domain-containing proteins are critical regulators determining cellular fate and differentiation in embryonal development (Dawid et al., 1998). Furthermore, LIM-HD transcription factors were observed as substantially present in neurons and essential for their development (Benveniste et al., 1998; Pfaff et al., 1996; Way and Chalfie, 1988). In order to follow further on a potential functional role of LDB2 in neuronal and ganglioglioma development, we used LDB2 silencing by specific shRNAs in cultured primary neurons. These experiments demonstrated a striking deficit of arborization in neuronal development (Fassunke et al., 2008) reflecting features of dysmorphic neuronal components of gangliogliomas. Functionally, neuronal elements with impaired connectivity by significantly contribute to impaired neuronal network function and resulting hyperexcitability.

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Fig. 30.3 Overview of differential gene expression in gangliogliomas. Intriguingly, differential expression of more than individual genes in gangliogliomas related to a rather defined number of elementary cellular functions and molecular pathways. Only a minor fraction of differentially expressed transcripts was not sorted in one of the functional groups. A detailed list of differentially expressed transcript has been presented before (Fassunke et al., 2008)

Correspondingly, alterations of the balance between excitation and inhibition were recently reported by important comprehensive expression array studies in gangliogliomas (Aronica et al., 2008; Samadani et al., 2007). A highly refined single cell expression analysis showed abundance of the metabotropic glutamate receptor (mGluR) 5 in neuronal ganglioglioma elements (Samadani et al., 2007). Conversely, reduced expression of several GABAa receptor (GABAAR) subunits was present in gangliogliomas, which may reflect insufficient GABAergic inhibition (Aronica et al., 2008; Samadani et al., 2007). Aronica and colleagues reported on aberrant expression of a variety of ion channels and ion transporters (Aronica et al., 2008) that may point to impaired ion homeostasis in gangliogliomas, which represents a general factor for epileptogenicity (Beck and Yaari, 2008; Becker et al., 2008). A recent factor that gains considerable importance in the pathogenesis of gangliogliomas and seizure development is given by the strong activation of particularly innate inflammatory reactions (Aronica et al., 2008). Increased expression of inflammatory interleukins (e.g., IL-1β), activation of the complement cascade and the Toll-like receptor pathway have been detected partially by expression array studies (Aronica

et al., 2005, 2008). As these data and considerations may have demonstrated, expression array studies have provided substantially improved insights in molecules and pathogenetic cascades operating in gangliogliomas. How are we going to optimally use and exploit this data treasure in the future?

Discussion Large-scale expression data on gangliogliomas has come up with a substantial number of differentially expressed transcripts. Which of these findings are pathogenetically important, which represent side effects? Can we learn from differential expression patterns about transcriptional command structures active in gangliogliomas? Considering these questions, expression array data can gain an overadditive power by integration with complementary technical approaches that aim to understand the functional impact of differential gene expression as well as to unravel genomic aberrations that interfere with gene expression in gangliogliomas. Complex analyses starting from expression array data to characterize concerted promoter control

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modules taking into account differential promoter activation due to slight allelic variants of gene promoter and resulting variability in transcription factor binding affinity in humans will provide completely novel insights into the pathogenesis of complex disorders such as glioneuronal neoplasms (Kasowski et al., 2010; Marsh et al., 2009). Further important insights into the pathogenesis of gangliogliomas may be expected by the comprehensive analysis of expression array and comparative genomic hybridization (CGH) data. A recent, somewhat unexpected finding was given by a high incidence of genomic aberrations observed in more than 50% of gangliogliomas under study (Hoischen et al., 2008). Considering the glial ganglioglioma component as neoplastic and cytologically resembling many features of low-grade astrocytomas, we compared the CGH patterns of gangliogliomas and diffuse astrocytomas (WHO grade II). In an unsupervised cluster analysis, diffuse astrocytomas formed subclusters within a group of gangliogliomas that demonstrated no strong similarities in the pattern of genomic imbalances. In contrast a genetically clearly distinct ganglioglioma cluster characterized by combined gains of chromosomes 5, 7, 8 and 12 (group I) was clustered distinct from diffuse astrocytomas, albeit strong histological similarities of the constituting astroglial elements. The analysis of these CGH data with comprehensive gene expression array results of respective gangliogliomas will provide novel information on putative functional consequences of genetic aberrations with respect to gene expression. This approach will comprehensively provide insight in amplification of oncogenes/loss of heterozygosity of tumor suppressor genes and corresponding alterations of gene expression. Considering the recently published fact that array-CGH detected genetic alterations such as amplification of the oncogene CDK4 in a fraction of tumor cells in gangliogliomas (WHO grade I) can predict a malignant recurrence as glioblastoma multiforme, in which tumor cells derived from this fraction represent the majority of cellular elements (Hoischen et al., 2008), expression-/CGH-array based biomarkers for also low grade brain tumors may be expected in the future. Such strategies will be a starting point for personalized therapy also of generally rather benign brain neoplasms such as gangliogliomas.

A.J. Becker

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Index

A Abdulrauf, S.I., 139 Acetazolamide, CA inhibitor, 68 Actin-associated proteins, 84 Adenomatous polyposis coli gene (APC), 41 Ageing, and cancer, 5 Agonistic Fas receptors, 25 AGT, see O6 -methylguanine-DNA methyltransferase (MGMT) Alkylating agents, 89 Allograft implantation models, 188 Alpha-carbonic anhydrase family, 65 Altinoz, M.A., 60 5-Aminolevulinic acid (5-ALA), 239 Anaplastic astrocytomas, 36–37, 135, 213 CBV measurements, 216 MGMT IHC of, 92 p16/INKa4 protein, relation with p53, 27–28 Angiogenesis, 135–136 Angiogenesis-related proteins, 137–138 Antiapoptotic proteins, see Astrocytoma(s) Antisera, 108 ANXA1 (annexin 1), 49 Aphasia, 224 Apolipoprotein Apia-I, 191 Apoptosis, 136, 146, 153 activation, and signaling pathways, 23–24 Bcl-2 controls, 29–30 biological significance, 23 caspases role in, 30 defined, 23, 121–122 FasL and TRAIL mediated, 25 in gliomas, study using MRS apoptotic cell density, 123 ca 2.8 ppm Lip/MM peak from PUFAs, 125–126

methodology, 122 taurine concentration in glioma biopsies, 124–126 ligands and effector caspases suppress, 25 pathways of, 121 proteins, inhibitors of, 30–31 regulatory function of PTEN, 28 repression of Bcl-2 and survivin, 26, 32 role in gliomas, 30, 33 TNF-induced, 25 ubiquitination role in, 31 Aquaporin-1 (AQP1), 240 Argon lasers, 174 Aronica, E., 271 Assimakopoulou, M., 61 Asthagiri, A.R., 247 Astrocytic tumors, see Astrocytoma(s) Astrocytoma(s), 57–58, 70, 135, 213–214, 259 anaplastic astrocytoma, 36 antagonist RU486 role in, 60 antiapoptotic proteins role in Bcl-2 proteins family, 29–30 death ligands, 23–26 IAPs, activation in gliomas, 30–32 p53, and cell cycle progression E2F role, 26–27 PTEN relationship with p53, 28–29 receptors and messengers role, 23–26 survivin and cell cycle progression, 32–33 TNF-induced NF-κB activation in, 25 biopsy, analysis of, 122–126 carbonic anhydrase IX diagnostic tool in grading astrocytomas, studies, 69–70 evaluation in tumors, 68 prognostic significance of, 68–69 role, 68–70

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276

Astrocytoma(s) (cont.) CBV/rCBV measurements predictive role, in survival and recurrence, see Cerebral blood volume (CBV) cell motility, IF proteins role GFAP, 85–86 nestin, 81, 85–86 synemin, 82–85 vimentin, 81, 85–86 cerebellar, spontaneous regression of, see Cerebellar astrocytomas (CA) diffuse astrocytoma, 36 doppel protein functionality biochemical features, and cellular localization, 18–20 distinguishing subtypes of, 16 functional pathways analysis, 19–20 gene expression analysis, tumor marker, 13, 15–17 glial tumors, gene expression, 16, 18 interaction analysis, 19–20 over-expression, 16–17 glioblastoma multiforme, 35 higher MVD in p53 mutated low-grade, 140 high recurrence rate of, factors, 81 hormonal therapy for, 58, 62 Human Angiogenesis Array, 137–138 IF proteins role in cell motility, 85–86 IHC for MVD, 136–137 Laser-Induced Fluorescence Spectroscopy (LIFS) for, 162 low grade, p53 involvement, 135 pilocytic astrocytoma, 36 P53 mutation, 138 association with MVD, 138–139 characterization, 137 higher number of vessels, 138 PR isoforms expression regulation, and malignancy grades, 59–60, 62 function in growth of, 57, 60–61 progesterone (P) genomic mechanism of action, 58 role in cell growth and proliferation of, 57, 60 regression, 143 SEGA, see Subependymal giant cell astrocytomas (SEGAs) statistical analyses, 138 taurine role, in apoptosis, see Apoptosis

Index

therapy, synemin and other IF proteins prospects, 86–87 treatments, depending factors, 58 See also Gliomas Autism, 149–150, 154 Axin, tumor suppressor, 40–41 B Bannykh, S.I., 199 Bcl-2 protein family, 23 controls apoptosis, action mechanism, 29–30 groups, 29 Bigner, S.H., 111 Biomarker discovery, methods cytogenetic method, 111–112 digital karyotyping technique, 114–115 EGFR amplification technique, 111–112 EGFRvIII expression, 112 genomic characterization technique, 116–117 immunologic methods, 108–109 integrated genomic analysis, 115–116 karyotypic analysis, 111 large-scale array analysis, 113 using double-minute (DM) chromosomes, 111 using gangliosides, 109–110 using gene expression arrays, 114 using GLI transcript, 112–113 using tenascin, 110 Bisdas, S., 216–218 Bisulfite DNA treatment, 6–7 Bjerkvig, R., 109 Bleier, A.R., 180 Blood brain barrier (BBB), 188 Bonéy-Montoya, J., 60 Borit, A., 143 Bourdon, M.A., 110 Bouvier, C., 199 Bovenzi, V., 60 Bovine spongiform encephalopathy (BSE), 13 Bowers, D.C., 201 Bown, S.G., 173–174 Brain Cancer, nanotechnology based therapy methods to increase targeting specificity, 191–192 nanomaterials delivery, 190–191 convection-enhanced delivery (CED), 192–193 systemic delivery, 190–191 nanoparticle formulations, 189–190 strategies to overcome BBB, 192 See also Cancer

Index

Brain thermal lesions, evolution on MRI, 181–182 Brain tumors, 57, 60, 135, 195 cell cultures, 107 computer-based QOL monitoring, ePROM, 226–227 fluorescence signature and, 170 Laser-Induced Fluorescence Spectroscopy (LIFS) for, 162 malignant, nanotechnology-based therapy for, 187–189 PROM, see electronic Patient-Reported Outcome Monitoring (ePROM) specific Pros, 225 symptoms, 223–224 TR-LIFS, tool for intra-operative diagnosis, see Time-resolved laser induced fluorescence spectroscopy (TR-LIFS) variety of, and treatment possibilities, 223 Breast cancer, CA IX as tumor biomarker, 67 Brell, M., 92 Britz, G.W., 235 Brunetaud, J.M., 177 C Cabrera-Munoz, E., 61 Cachexia, 196 CA9 gene, 65, 67 Calcifications, 196, 199 Camacho-Arroyo, I., 58–59 Camptothecin, 146 Cancer ageing and, 5 and DNA methylation failure, 5–6 ERK/MAPK pathway deregulation, 102 importance, of imaging of HIF-1-active tumors, 129 NF-κB in genesis and progression of, 25 p16/INKa4 protein role, 27–28 role of synemin in, 83 -targeted PROs, 225 types, 223 Cantharidinare, 147 Cao, V.T., 93 Capper, D., 92–93 Carbonic anhydrase (CAs), 65 Carbonic Anhydrase IX (CA IX), 65–66 diagnostic evaluation monoclonal M75 anti-human CA IX antibody use, 68

277

diagnostic tool, in grading astrocytomas, 70 expression in Barrett’s-associated adenocarcinomas, 66 and disease prognosis, 67 ectopic, 66 and hypoxia responsive element (HRE), 67 increased, 66–67 malignant gliomas, 67 in normal tissue, 66 predominant CA isozyme in tumors, 66 prognostic significance (studies), as valuable marker, 67–70 role in astrocytomas, 68–70 role in carcinogenesis, function mechanism, 67–68 Carboplatin, in treatment of PMAs, 207 Carpentier, A., 175–176, 180, 183 Carroll, R.S., 60 β-Catenin, 35, 37–39, 42–43 See also Wnt signaling pathway Cathepsin D, lysosomal proteinase, 74 CBV, see Cerebral blood volume (CBV) CD95 ligand (CD95L), 23 CD34 protein, 256 Cellular differentiation, 136 Central nervous system (CNS), 187–188 and doppel protein expression, 13, 15, 19 germ cell tumors, 95 arise from pleuropotential embryonic cells, 95 in Asian population, higher incidence, 97 benign and malignant, 95 clinical presentation, tumors in, 96–97 germinoma and non-germinoma, histological examination, 95–96 locations, mainly midline, 95–97 hemangioblastomas, see Hemangioblastomas low-grade neoplasms PAs, pediatric tumors of, see Pilocytic astrocytomas (PAs) malignant tumors, nanotechnology-based therapy for, 187–189 tumors and EGBs presence diagnosis, 78 Ceppa, E.P., 198, 207 Cerebellar astrocytomas (CA), 143 regression, 144, 147 criteria for, 144 CT scans, spontaneous regression, 144 hormonal changes and chances of, 145 imaging surveillance in, 145 incidence and time course of, 144–145 mechanism and multiple factors, 145–146

278

Cerebellar astrocytomas (CA) (cont.) residual tumours, smaller volume and higher chances of, 146 therapy for, surgical resection, 144 Cerebellar lesions, 195 Cerebral blood volume (CBV) estimation, by MRI arterial spin labeling techniques, 215 DSC-MRI, 214–215 gradient echo pulse sequence use, 215 preload approach, problem, 214 T1-based methods, 214 measurements, predictive value, 217–219 biases in prediction, 217 correlation between histopathological grade and, 218 cut-off values, 218 data collection from longitudinal studies, 219 EORTC criteria, 218 rCBV value, after combined radiation and temozolamide therapy, 219 methodological considerations, and survival rates, 215–216 CBV mapping, 215 histogram analysis, of rCBV values, 216 hot-spot ROI method, 216 limitations, 216 in predicting astrocytoma histopathologic grade, 214 Cerebrospinal fluid (CSF), 149, 203 Chang, S.D., 248 Chemotherapy, for PMA, 203–204, 206–208 Chen, Y.Y., 43 Chikai, K., 207 Chinese herbs, 146–147 ChIP-on-chip method, 9 Choi, Y.J., 47, 51 Choroid plexus, 65 Cisplatin (CDDP), in treatment of PMAs, 203, 207 Coagulation factor III (CF III), 138, 140 Coagulation factor VIIa, 140 CO2 lasers, 174 Colchicinamide, 147 Colchicine, 146 Coley’s vaccine, 145 α(II) collagen prolyl-4-hydroxylase, 139 Comincini, S., 18 Computer-based Health Evaluation System (CHES), 227

Index

Cottingham, S.L., 206–207 CpG islands, 4–5, 90 Curzerenone, 147 CyberKnife SRS, 249 Cyst, 239–240 Cystic tumors, see Pilocytic astrocytomas (PAs) Cytogenetic method, 111–112 Cytoskeletal protein, 81 D Death domain (DD), 24 Death effector domain (DED), 24 Death inducing signaling complex (DISC), 24 Death ligands, 23 Death receptors, 23–24 De Porter, J., 175, 180 Desmuslin, 82 Devaux, B.C., 176 Dhermain, F., 218 Di, C., 115 Dickkopf family proteins, 35, 39 Diencephalic syndrome, 204 Dietary supplements, anti-cancer effects, 146–147 Diffuse astrocytoma(s), 10, 36 main tumor entities, 89–90 MGMT IHC expression, 89–90 compared to MGMT promoter methylation, 91 in glioma and non-neoplastic cells, 89, 91–92 marker of patient outcome, 92–93 technical considerations, 90–91 MGMT protein and resistance alkylating agents, 89–90 Digital karyotyping technique, 114–115 Digital oscilloscope, 164 3,3-Diindolylmethane (DIM), 146 Diode laser, 176 Dirven, C.M., 200–201 Discriminant function analysis (DFA), 165 Dishevelled (Dvl), 40 D-54 MG glioma cells, 109 DNA, apoptotic DNA fragmentation, 121 DNA methylation, 3–4 analysis, methods based on DNA chemical modification, 6 ChIP-on-chip method, 9 MALDI-TOF mass spectrometry, 8 methylation-sensitive restriction enzymes usage, 6 methylation-specific PCR (MSP), 7

Index

microarray expression profiling, 8 primers usage, 8 restriction landmark genomic scanning (RLGS) method, 8–9 techniques comparison, 7 in astrocytic tumors, for diagnosis and prognosis, 9–10 failure, as cause of disease or cancer, 5–6 hypomethylation, 5–6 relevance in normal cells, 4 role in mammalian CNS development and function, 9 DNA methyltransferases, 4 DNA methyltransferases (DNMT) genes, 4 DNA repair, 136 DNMT, see DNA methyltransferases Doppel gene, discovery, 13–15 Doppel protein biochemical features, 15, 18–19 cell migration process, contribution in, 13, 20 functional pathways, 19–20 and interaction analysis, 19–20 post-translational modifications, 18 and prion proteins, comparison and similarities, 13–15, 18, 20 similar exon–intron architecture, with prion gene, 14 and structural characterization, 15 Double-minute (DM) chromosomes, 111 Doxorubicin-loaded, PEG coated PHDCA nanoparticles, 191 Dysembryoplastic neuroepithelial tumours (DNETs), 259, 261 E E2F family protein, 27 EGBs, see Eosinophilic granular bodies (EGBs) EGFR, see Epidermal growth factor receptor gene (EGFR) EGFRvIII expression, 112 electronic Patient-Reported Outcome Monitoring (ePROM) implementation of, 226–227 software, 227 tele-monitoring, 227–228 usage of proxy-rating, 228–229 Ellmann, S., 60 Endostatin, 139 EORTC Brain Cancer Module (BN20), 226

279

EORTC QLQ-BN20, 226, 228 Eosinophilic granular bodies (EGBs), 73–74 cathepsin D role, in cell apoptosis, 76 contents and morphologies, in tumor, 74 H&E (hematoxylin and eosin) staining and anti-GFAP and anti-CSE1L antibodies, 75 Mayer’s hematoxylin for staining, 75–76 observation protocol using immunohistochemistry, 77 with antibodies against LAMP-1 and LAMP-2, 73–74 origin, 75–77 PAS (periodic acid-Schiff) staining and anti-GFAP and anti-CSE1L antibodies, 75 relation to lysosomal system, 73 role in cyst development in pilocytic astrocytomas, 73, 75–77 Epidermal growth factor receptor gene (EGFR), 111–112, 192 Epigenetics defined, 3 states, modification, 4–5 Epigenetic therapy, 11 Epileptogenicity, of brain tumors cellular composition and neurochemical profile, 262–263 of peritumoral zone, 263–264 ERM proteins, 261 European Organization for Research and Treatment of Cancer core questionnaire (EORTC QLQ-C30), 225, 229 Everolimus, 45, 50–51 F FACT-Br Symptom Index (FBrSI), 225–226 FasL (Fas ligand), 25 Fernandez, C., 207 Ferroli, P., 235 Fiber optic probe, 164 Fisher, B.J., 201 Fisher, P.G., 195 FLAIR image, 218 Fluorescence measurements types, 162 Fluorescence spectroscopy, 162 Fluorophores, 162, 169 Foltz, G., 39 Franz, D.N., 52 FRAT1 (frequently arranged in advanced T-cell lymphomas-1), 40

280

Freilinger, A., 47 Functional Assessment of Cancer Therapy-Brain Module (FACT-Br), 225–226 Functional Assessment of Cancer Therapy general version (FACT-G), 225 Functional Assessment of Chronic Illness Therapy (FACIT) measurement system, 225 Furnari, F.B., 187 Fusion genes BRAF and KIAA1549, in PAs, 100–101 MAPK activation via RAF1, 101 in pilocytic astrocytoma, KIAA1549, 100–101 Fuss, M., 218 FVIII-immunostaining, 139 G Gajjar, A., 195 Gamma-glutamyl-Semethylselenocysteine (GGMSC), 146 Gamma Knife SRS, 247 Gangliocytoma, 258–259 Gangliogliomas (GG) alterations in Pi3K-mTOR pathway components, 263 association with temporal lobe epilepsy (TLE), 267 cellular pathology analysis, 269 coexistence with cortical dysplasia, 259 CT for imaging, 254 differential diagnosis, 258–259 differential gene expression in, 270–271 LIM domain-interacting transcripts, 270 reelin signaling cascade, factors modification, 270 epileptogenicity, 263 expression of specific glutamate receptors (GluR) subtypes, 263 gene expression analysis, approaches, 272 aRNA amplification method, 269 glass microscope slide DNA arrays, 268–269 microarray systems, limitations, 269 oligonucleotide arrays, 268–269 in situ-RT and immunolaser microdissection, 268–269 histopathological features, and hallmark of, 257–258, 268–269 IHC characterization of, 258 imaging, 254 incidence and age/sex distribution, 254 intraoperative diagnosis, 256

Index

localization, 254 macroscopy of, 256 mechanisms of epileptogenesis, 261–264 molecular pathogenesis of, 260–261 neurophysiological features, 254–256 origin from, 268–269 pathogenesis, 263 perilesional changes, 264 prognostic factors and surgical outcome, 259–260 symptoms, 254 Gangliosides antigens, 109–110 patterns, 109–110 GBM, see Glioblastoma multiforme (GBM) Genetically engineered mouse models (GEMMs), 188 Genomic characterization technique, 116–117 Germ cell tumors arise from pleuropotential embryonic cells, 95 in Asian population, higher incidence, 97 benign and malignant, 95 clinical presentation, tumors in basal ganglia region, 96 pineal region, symptoms, 96–97 predilection for males, during puberty time, 97 suprasellar region, 96 germinona, 95 histological examination, cell types, 96 magnetic resonance imaging of, 96 locations, mainly midline, 95 and non-germinoma, histological examination, 95 choriocarcinoma, and teratomas, 96 yolk-sac tumors and embryonal carcinoma, 96 SEER-17 registry data for study, 97 See also Central nervous system (CNS) GFAP (Glial fibrillary acidic protein), 75, 82–83, 85–87 Giannini, C., 200 Glial tumors, malignancy grades, 16 Glioblastoma multiforme (GBM), 187, 214 develop de novo, 36 doppel expression, 16 epigenetic alterations in, 9 genetic changes associated, 37 GPNMB gene role, 153 gross total surgical resection (GTR) in, 233 nanotechnology-based delivery of therapeutics, features, 188–189

Index

primary and secondary, chromosomal aberrations in, 36 TP53 mutation, 37 Glioblastomas, 36, 135–136, 161 CA IX expression in, 69–70 cell types, 37 EGFRvIII mutation, 112 GBM, see Glioblastoma multiforme (GBM) p16/INKa4 protein, relation with p53, 27–28 poor prognosis, 58 Gliomas, 57, 135, 161 animal model systems, for preclinical trials, 188 astrocytomas, 35–36, 57 cell culture studies, limitation, 188 diagnosis, in vivo measurements fluorescence signature of diffuse astrocytoma, 170 spectroscopic parameters analysis, 170 TR-LIFS characteristics, 170 ependymomas, 35–36 of grades III-IV, 187 histological characteristics for grading, 35 IAPs activation in, 31–32 malignancy grades, and survival rates, 57–58 malignant, 187–188 mixed oligoastrocytomas, 35 nuclear polymorphism, 36 oligodendrogliomas, 35–36 progress, microvascular hyperplasia, 135 spectroscopic classification, 164 surgical resection of, 161–162 survivin and cell cycle progression, 32–33 Wnt/β-catenin/Tcf signaling pathway components in, 35, 38 Axin and APC proteins, 38 Axin-APC-GSK3β, 41 β-catenin, central player, 38, 41–42 Dishevelled (Dvl), 40 DKK family, 39–40 FRAT1, 40 Frizzled family, 37–38 frizzled receptors, 40 Lef/Tcf family transcription factors, 42 LRP5 and LRP6, 37, 39 neural stem cells regulation, 38 overview, 37 pygopus 2, 43 sFRPs and Dickkopf family, extracellular inhibitors, 39

281

Wnt genes associated with, 37, 39–40 Wnt inhibitory factor-1 (WIF-1), antagonist, 39 Wnt proteins, 39–40 Wnt receptor complex, 37 in women, 146 Glioneuronal lesions, 253, 261 Glioneuronal tumors, see Gangliogliomas (GG) GLI transcript, 112–113 Glutamate decarboxylase (GAD), 169 Glycogen synthase kinase 3 (GSK 3), 41 Glycosylphosphatidyl-inositol (GPI), 14–15, 18 Gonzalez-Aguero, G., 60–61 Goodwin, T., 97 Gottfried, O.N., 207 Gotze, S., 39 GPI, see Glycosylphosphatidyl-inositol (GPI) GPNMB (glycoprotein nmb), 49 Grasbon-Frodl, E.M., 91 Groucho (Grg/TLE) family, transcriptional co-repressors, 42 GTPase-activating protein, 47 Guba, M., 49 Guerra-Araiza, C., 59 Gunny, R.S., 146 Guo, G., 41 H Haapasalo, H., 201 Harada, H., 130 Harringtonine, 146 Health-related quality of life (HRQOL) assessment, 223 Helin, K., 58 Hemangioblastomas, 233, 239–240, 242, 245, 250 associated with von Hippel-Lindau syndrome, 233 conventional radiotherapy for, 246 cysts, formation mechanisms in aquaporin-1 (AQP1) role, 240 histologic appearance of, 246 intraoperative ICG videography use in, 233–235 resection of intracranial and spinal, 235–237 photodynamic diagnosis (PDD), with 5-ALA, 239–240 radiosurgery for contraindications, 246 current indications, 246 radiosurgical complications, 249–250 rationale for use, 246 stereotactic, 246–249

282

Hemangioblastomas (cont.) radiosurgical treatment of, key studies, 247–249 rare vascular tumors, 233 recurrence rate, 239, 245 residual tumors detection, by ALA-derived PpIX fluorescence method, 241–242 treatment modalities, 250 types, as per MRI studies, 240 Heme pathway regulation, ALAS feedback inhibition role, 240–241 synthesis steps, 240 Hengstschlaeger, M., 50 Henske, E.P., 48 Hernandez-Hernandez, T., 60 HIF-1-active tumors imaging, using 123 I-IPOS based on pretargeting approach, concept, 131–132 hypoxia imaging concept, 130–131 size-exclusion analysis, 131 High mobility group (HMG), 42 High-resolution magic angle spinning (HRMAS) 1 H MRS, 121–122 lipid peaks, 126 of non-necrotic (top spectrum) and necrotic (middle spectrum) biopsy, 123, 125 taurine concentrations, 126 Higuchi, N., 178 Hilvo, M., 68 Hirai, T., 218 Histology, of LITT-induced lesions, see Laser interstitial thermotherapy (LITT) Histone modifications, 3 HMG, see High mobility group (HMG) Homoharringtonine, 146 Horbinski, C., 199 Howng, S.L., 40 HRMAS; 1H MRS, see High-resolution magic angle spinning (HRMAS) Hulleman, E., 58 Human angiogenesis array, 137–138 Human ICF syndrome, 5 Humphrey, P.A., 112 Hydrocephalus, 149–150 Hydroxycamptothecin, 146 Hyperplasia, 135 Hyperthermia, 177 Hypoxia-inducible factors (HIFs), 47, 67 Hypoxic regions, 129

Index

I ICG, see Indocyanine green (ICG) Ichikawa, T., 48 Ichimura, K., 58 IHC markers, 90 ([123 I]iodobenzoyl)norbiotinamide (123 I-IBB), 129 123 I-IPOS, probe, 129 Immunohistochemistry, 131 Indirubin, 147 Indocyanine green (ICG), 234 intracranial practical applications, 234–235 intraoperative use, in hemangioblastomas, 233–237 resection of intracranial and spinal, 235 Inhibitor of apoptosis (IAP) proteins family activation in gliomas, 31–32 classification, 30–31 eight human members, 30 survivin, and cell cycle progression, 32–33 Inoki, K., 49 Inorganic nanoparticles, 189 Integrated genomic analysis, 115–116 Intermediate filament (IF) proteins functions, 81–82 GFAP (Glial fibrillary acidic protein), 75, 82–83, 85–87 nestin, 81–82, 85–87 prospects for astrocytoma therapy, 86–87 synemin, 81–83 vimentin, 81, 83, 85–87 See also Astrocytoma(s) Interstitial hyperthermia concept, 173–174 Intracranial germ cell tumors, malignant, 95 Intracranial xenografts, 191 Intraoperative ICG videography, applications, 234 in identifying regions of tumor, 235 in the resection of 100 craniotomies of mixed pathology, 235 in the resection of hemangioblastoma, 235 in resection of spinal hemangioblastomas, 235–236 to stain glioma margins in animal models, 234–235 in vascular neurosurgery, 234 Intratumoral, 240–241 accumulation, 191 administration, 191 cysts, 240–242 hemorrhages, 150 infusion, 192 necrosis, 240 Isbert, C., 177

Index

Ischemic tumour necrosis, 145 3 6 -isoLD1, expression of, 109 Ivanov, S., 69 Ivarsson, K., 177 J Jagannathan, J., 242 Jawahar, A., 248 Johnson, M.W., 48 Jolesz, F.A., 179 Jó´zwiak, S., 48, 51–52 K Kageji, T., 207 Kahn, T., 178, 180–181, 183 Kangasniemi, M., 176, 178, 181–182 Kano, H., 247 Karabagli, H., 247, 249 Karayan-Tapon, L., 92 Karnofsky performance score, 204 Karyotypic analysis, 111 Kastner, P., 58–59 Kazuno, M., 140 Keene, D., 97 Kerr, J.F., 23 Khalid, H., 60 KIAA1549 protein, 101 Kickhefel, A., 180 Klein, R., 200 Knudson’s two-hit model, tumor development, 48 Kohler, G., 108 Komakula, S.T., 207 Komotar, R.J., 207 Kondo, I., 111 Korkolopoulou, P., 69 Korur, S., 41 Kou, L., 176 Kraus, W.L., 59 Krueger, D.A., 52 Kudo, T., 130 Kuratsu, J., 97 L Lam, C., 45, 52 LAMP-1 and 2 (Lysosomal membrane proteins), 74–78 Lange, C.A., 58–59 Large-scale array analysis, 113 Laser fibers, 176

283

Laser-induced fluorescence spectroscopy (LIFS), 162 Laser interstitial thermotherapy (LITT), 173 applied to brain tumors, clinical studies CT-guided stereotactic procedures, 182 devices, manufactured for, 184–185 Gd-DTPA-enhancing rim, 183 heating process and temperature elevation, 183 histological analysis, 183 hyperthermia treatment with Nd-YAG laser, 182 real-time magnetic resonance-guided LITT system, 183 for deepseated tumors, 174 limitations, 175 MRI imaging and, 179 brain thermal lesions on MRI, evolution, 181–182 MRI thermal imaging sequences, 179–180 real time computation, 180–181 procedures on patients, with brain metastases, 174 with real-time MRI, 174 treatments consist of, 173 Laser (Light Amplification by Stimulated Emission of Radiation), 173–174 control delivery software with real time with MRI thermometry analysis, 181 emission in active medium, 175 functioning principles, 175–176 history, 174–175 interactions with biological tissues, mechanisms, 177 histology of LITT-induced lesions in brain tissue, 178–179 immediate and secondary, 177–178 thermal dosimetry, 179 main elements, 175 physics, 175 produce high energy light, properties, 175 technology, fundamental principles, 175 transmission of beam, 176 used in LITT, 176–177 use in neurosurgery, 173 Law, M., 218 LDL receptors (LDLR), 191 Lee, N., 52 Lef/Tcf family transcription factors, 42 Leonhardt, S.A., 58 Leon, S.P., 139 Lev, M.H., 218

284

Libermann, T.A., 111–112 LIM-domain-binding 2 (LDB2) gene, 261 LINAC-based radiosurgery, 247 Lipid NPs, categories, 190 Liposomes, 190 LITT, see Laser interstitial thermotherapy (LITT) Liu, X., 42 Liu, Z.J., 60 Lockshin, R.A., 23 Loncaster, J.A., 67 LTF (lactotransferrin), 49 Lycobetaine, 147 Lysosomal protease, 73–74 Lysosomes, 73–74 M Magnetic resonance imaging (MRI), 145, 254 astrocytomas characterization, imaging method, 214 brain, case report, 204–206 contrast T1 MRI follow-up after LITT treatment, 182 coronal MRI images, 255 diffusion-weighted, 214 DSC-MRI, 214–215 for evaluating intracranial, neoplastic disease, 196 with gadolinium enhancement at T1/T2/FLAIR weighting, 246 of gangliogliomas (GG), 253 imaging and LITT, 179 perfusion-weighted, 214 for pilocytic astrocytomas, 197 thermal imaging sequences, 179–180 tumor determined by, 183 T2-weighted (FLAIR) MR image, 218 to visualize spinal hemangioblastomas, 236 Mahaley, S.M., 108 MALDI-TOF mass spectrometry, 8 Male fertility, and doppel, 15, 21 Mamelak, A.N., 207 Mammal development, and DNA methylation role, 4–5 MAPK, see Mitogen activated protein kinase (MAPK) pathway Maser (Microwaves Amplification by Stimulated Emission of Radiation), 174 Massimino, M.L., 19 Mast cells, 245 Matrix metalloproteinase-9 (MMP-9), 140

Index

Matsunaga, S., 248 Maxwell, J.A., 91 McCowage, G., 207 McKenna, N.J., 58 Menovsky, T., 179 Mental retardation, 150 Methylation, importance in clinic, 10–11 Methylation-specific PCR (MSP) method, 7–8 O6 -Methylguanine-DNA methyltransferase (MGMT), 9, 89 IHC expression in diffuse gilomas, see Diffuse astrocytoma(s) immunohistochemistry with clone MT3.1 and MT23.3, 92 double, 91–92 expression, 9, 89–90 marker of patient outcome, 93 and non-neoplastic cells, 91–92 technical considerations, 90–91 tumors identification with loss of MGMT expression, 92–93 promoter methylation assays application and limitations of, 91 MGMT, see O6 -Methylguanine-DNA methyltransferase MGMT methylation, 9–11 MIB-1 labeling index, 200 Microarray expression profiling, 8 Microdissection approach, 267, 269–270 Microvascular hyperplasia, 135 Microvessel density (MVD) astrocytoma evaluation, 136 degree of vascularization, 136 function estimatimation, of p53 mutation status, 137 IHC detected P53 protein, 138 Milstein, C., 108 Mineura, K., 91 Mi, R., 51 Missense mutant p53 proteins, 139 Mitogen activated protein kinase (MAPK) pathway activation via BRAF fusion gene, in PAs, 103 alternative activation mechanisms, 103–104 targeted therapy against AZD6244, 103 PLX4032 inhibitor, 103 Sorafenib, target, 103 Mizobuchi, Y., 40 Momparler, R.L., 60

Index

Monoclonal antibodies (MAbs), 108–110 Monocrotaline, 147 Moore R.C., 15 Mordon, S., 177 Moss, J.M., 247, 249 Motor deficit, 224 MRI imaging, 145 See also Magnetic resonance imaging (MRI) MRI thermal imaging sequences, 179–180 MSP derived methods, 8 mTOR (mammalian Target Of Rapamycin) pathway, 46 inhibitors, anti-angiogenic effects effects on cell cultures and animal models of TSC, 51–52 everolimus (RAD001), selective nature, 52 rapamycin action process, 49–50 sirolimus, mechanism involved, 51 temsirolimus (CCI-779), action mode, 49–51 in Subependymal Giant Cell Astrocytoma, 48 genes regulating activity, 48 targeting, 52–53 in Tuberous Sclerosis Complex, 46–47 autophagy inhibition, 47 genes regulated by HIFs, 46 inactivating mutations TSC1/TSC2 gene, 46 Ras-homoloque-enriched in brain (Rheb) target, 46 mTOR protein, 46 Mulac-Jericevic, B., 59, 61 Muller, W., 40 Murai, Y., 235 N Nakasu, S., 93 Nanooncology, 187 Nanotechnology, 187–189 based delivery of therapeutics to GBM, 188 for brain cancer, 189 nanomaterials delivery, 190 convection-enhanced delivery (CED), 192–193 methods to increase targeting specificity, 191–192 strategies to overcome BBB, 192 systemic delivery, 190–191 nanoparticles (NPs), 188 designed to delivery, 188–189 formulations, 189

285

inorganic, 189 lipid, 190 liposomes, 190 polymeric, 189 polymeric micelles, 190 xenograft systems models, tumor biology, 188 Natural remedies and herbs, anti-cancer effects, 146–147 Necrosis prediction, 180 Necrotic biopsies, analysis of, 122–126 Neodimium(Nd)-YAG lasers, 173–177, 179–180, 182–183 Neovascularization, 135–136, 139 Nestin, 81–82, 85–87 Neural stem cells-gliomas, 39 Neurooncology, 107 Neurosurgery interstitial hyperthermia in, 174 ultrasound-based monitoring techniques not feasible for, 179 use of intraoperative ICG, 234 use of lasers in, 173 NF1 (Neurofibromatosis 1) gene, 100, 102 Niemela, M., 249 Nikuseva Martic, T., 41 NPTX1 (neuronal pentraxin I), 49 Nuclear factor-κB, 25 Nuclear medicine imaging, 130 Nuclear polymorphism, in tumor cells, 36 O Oligodendroglioma, 259 Oligonucleotide arrays, 268–269 Olson, J.J., 60 Oridonin, 147 Oxygen-dependent degradation domain (ODD) fusion proteins, 130 P PAI-1 gene, 140 Paixão Becker, A., 199 Paralogue compensation process, 18 Park, Y.S., 248 Parsons, D.W., 115 Pastorek, J., 65 Pastoreková, S., 68 Patient-reported outcomes (PROs), 224–225 commonly used PRO instruments, 229 computer-based, 226–228

286

Patient-reported outcomes (PROs) (cont.) tele-monitoring, 227–228 See also electronic Patient-Reported Outcome Monitoring (ePROM) Pecina-Slaus, N., 41 Pediatric gliomas, 195 high grade, and for MGMT IHC, 93 See also Pilocytic astrocytomas (PAs) Petronio, J., 207 P53 gene expression, 140 immunohistochemistry, 137 -mediated regulation on angiogenesis, in low grade astrocytomas, 135, 139–140 mutations, 138 in astrocytomas, 135, 137–138 characterization of, 137 protein expressions, 138–139 tumor suppressor, 136 P-glycoprotein (P-gp), 263 Phosphatidylethanolamine (PE), 190 Phospholipid, 190–191 Photoablative effect, 177 Photochemical effect, 177 Photodynamic diagnosis (PDD), for residual tumors, 240 Photomechanical effect, 177 Photosensitizing agent, 177 Photothermal effect, 177 PI3K-mTOR pathway, 261 Pilocytic astrocytomas (PAs), 36, 73–74, 195–196, 203, 259 biphasic tumors, 76, 99 cerebellum, most frequent site, 99 classic form, Rosenthal fibers presence, 196–198 common pediatric tumors of CNS, 73, 99 EGBs presence in microcysts, 76–77 histological analyses, 198–199 histopathology, 73 imaging characteristics, 196–197 immunohistochemical analyses, 199–201 and increased intracranial pressure in, 76, 78 LAMP-1, LAMP-2, and cathepsin D involvement in EGBs formation, 76 leptomeningeal infiltration in, 199 lysosomal proteinases, in EGBs role, 76–77 management complexity, 196

Index

MAPK activation via RAF fusion genes, 99–101 BRAF:KIAA1549 fusion genes, 100–101 BRAF V600E mutation, 103 fusion variants, 100–101 mutation of KRAS and BRAF, 101–102 NF1 gene mutation, 102 targeted therapy against MAPK pathway, 102–103 MIB-1 labeling indices of, 200–201 molecular genetic changes in, 100 oligodendroglioma-like features, 199 of optic pathways, 195 role of EGBs in cyst development in, 73, 75–77 slow-growing tumors, 76 treatment strategies, 196 tumorigenesis, importance of MAPK signaling, 99–100 variants, 99, 197–198 See also Eosinophilic granular bodies (EGBs) Pilomyxoid astrocytoma (PMA), 198, 203 case reports, 204–206 chemotherapeutic regimens, 207–208 clinical characteristics, 203 different features from PA, 203, 206 drug combination cisplatin (CDDP)/carboplatin (CBDCA) and etoposide, 203 higher rate of recurrence, 203, 207 management of, 203 MIB-1 labeling index, 204 with monomorphous pilomyxoid features, 206 patients with CSF dissemination, 204 temozolomide as first line adjuvant chemotherapy, 207–208 therapeutic strategies for PMA patients, 207 WHO classification, 207 See also Pilocytic astrocytomas (PAs) Pinski, J., 60 Piroli, G., 60 Plasminogen activator (PA), 140 Pleomorphic xanthoastrocytoma, 259 P53, nuclear phosphoprotein activity regulation, 26 cell cycle progression, control mechanism, 26–27 as genome guardian, 26 p16/INKa4 protein, relation with, 27–28 relationship between PTEN and, 28–29 Pollack, I.F., 93, 195 Polymeric micelles, 190

Index

Polymeric nanoparticles, 189 POS and 123 I-IBB method for imaging HIF-1 active regions in tumors, 130–131 in vivo molecular imaging, 130 pretargeting approach, 131–133 oxygen-dependent degradable probe, development, 130 P response elements (PRE), 58 Pretargeting approach, 129, 131–133 advantages, 132 defined, 129, 131 for imaging of HIF-1-active tumors, principle, 132–133 Preusser, M., 92 Prion diseases, see transmissible spongiform encephalopathies (TSEs) Prion–doppel interaction, 13, 19–20 Prion-like protein, see Doppel protein PR isoforms, 57 expression, regulation of, 59–60, 62 estrogens effects, and interaction with ERs, 59 PR-B and PR-A, 57 regulation and function in astrocytomas, 57, 60–61 PR-A transfection effects, on U373 human astrocytomas growth, 61 regulation by phosphorylation, 58–59, 62 transcriptional activity of, 58–59 U373 and D54 cell lines, expressed in, 57 up-regulation by E, 59 See also Progesterone (P) Probe, 129–132 Proescholdt, M.A., 70 Progesterone (P), 57 genomic mechanism of action, PR interaction with, 58–59 induced cell proliferation, in cell lines, 60 interaction with intracellular receptor (PR), 57 and PR isoforms role in astrocytomas cell growth, 57, 60 Programmed cell death, see Apoptosis Proliferating cell nuclear antigen (PCNA), 27 Proptosis, 195 Proton-resonance frequency (PRF), 175 Protoporphyrin IX (PpIX), 239 PTEN, tumor suppressor gene, 28–29 P53, tumor suppressor gene, 136 Pu, P., 40–41 Pygopus 2, 42

287

Q Qin, K., 19 QOL-instrument, 225 R Radionuclides, 132 Radiosurgery, 173, 182, 245–246 contraindications, 246 current indications, 246 frame-based linear accelerator, 249 indications, 246 key studies, 247–249 radiosurgical complications, 250 rationale for use, 246 stereotactic, 245–249 See also Hemangioblastomas Radiotherapy, 145 Rapamycin, 45, 49–50 rCBV, see Relative cerebral blood volume (rCBV) Real time computation, 180–181 Real time MRI thermal imaging, 180 Reelin, 270 Relative cerebral blood volume (rCBV), 213, 215–218 Relaxivity, 214–215 Reoxygenation, 131 Restriction landmark genomic scanning (RLGS) method, 8–9 disadvantages, 9 Reticuloendothelial system (RES), 190 Rett’s syndrome, 5 Richardson, E.P. Jr, 143 Richer, J.K., 58–59, 61 RND3 (Rho family GTPase 3), 49 Rodriguez, F.J., 197 Roggendorf, W., 200 Roninson, I.B., 113 Rosenfeld, M.G., 58 Rosenthal fibers, 203, 206 Rosner, M., 50 Roth, W., 39 Rousseau, A., 57 Roux, F.X., 176–177, 179, 182 Ruby-based laser, 174–176 Rüegg, S., 49 S Saarnio, J., 66 Sager, G., 60 Sandlund, J., 67

288

Sapareto, S.A., 179 Saraswathy, S., 218 Sarcomas, 145 Sasai, K., 91 S100A11 (S100 calcium binding protein a11), 49 Sathornsumetee, S., 69 Saunders, D.E., 145 Schatz, S.W., 178 Schlosser, S., 93 Schober, R., 178 Schubert, G.A., 236 Schulze, C.P., 178–180 Schulze, P.C., 183 Schwabe, B., 181, 183 Secondary glioblastomas, 139 Seizures, 150 Serpin E1, 138, 140 sFRP (secreted Frizzled Related Protein), 39 SFRP4 (secreted frizzled-related protein4), 49 Shadoo proteins, 19 Shimizu, N., 111 Shou, J., 40 Silica nanocarriers, 189 Sirolimus, antiangiogenic effects, 49 Smith, M., 97 Smoots, D.W., 143, 145 Sorafenib, 103 Span, P.N., 67 Spontaneous regression, of tumour, 143–144 See also Cerebellar astrocytomas (CA) Spriggs, A.I., 111 Stafford, R.J., 176 Steady state fluorescence spectroscopy, 162 Stellar, S., 174 Stening, K., 60 Stereotactic radiosurgery, 246–249 Steroid hormones, 146 Steroid receptor coactivator (SRC) family, 58 Streptavidin, 137 Strong, J.A., 196 Students’t-test, 4 Stupp, R., 207 Subependymal giant cell astrocytomas (SEGAs), 47–49, 51–52 analysis of gene expression profiling in, 151 defined, 149–150 everolimus impact on, 45 gene expression profiling analysis Affymetrix microarrays, 151

Index

gene regulation, by mTOR kinase at transcriptional level, 155–156 genes with highest up-or down-regulation scores in, 152–153 mixed-lineage phenotype, 155 mTOR effector genes in, 49 mTOR pathway in, 48, 150–151 genes regulating activity, 49 targeting, everolimus and rapamycin treatment, 52–53 TSC1/TSC2 disruption, 48 restricted ability to differentiate into glial cells or neurons, 154 with Tuberous sclerosis complex (TSC), 45, 149–150 dual neuronal and glial origin, 48 epilepsy, symptom, 150 gene expression profiling, 151 mental retardation, 150 mixed cells glial/giant, 47 molecular pathophysiology of, 150–151 neurologic dysfunctions, genes down-regulation association with, 154 positive for GFAP, 48 tumorigenesis, genes up-regulation link with, 151–154 Subependymal giant cell tumor (SEGT), 48 Subependymal nodules (SENs), 149 Sugiyama, K., 174, 178–179, 182 Surgery cellular necrosis following vascular damage during, 145 treatment options for glioma, 161 treatment possibilities of brain tumours, 223 See also Neurosurgery Survivin, and cell cycle progression, 32–33 Sutton, C., 173–174 Synemin, 82–83 α- and β-synemin, expressed in astrocytoma cells, 83 characteristic, 82 contribution to astrocytoma cells, malignant behavior Boyden chamber assays, 83–84 down-regulation impact, 83–84 interaction with α-actinin, 83–84 invasion, 83 present in leading edges and ruffled membranes, 83, 85

Index

RNAi experiments, 83 siRNAs and scrape wound assays, 83–84 exhibit alternative splice variants, 82 intermediate filament (IF) protein, 82–83 positive regulator, of cell motility and proliferative capacity, 81, 83–84 prospects for astrocytoma therapy, 86–87 regulation in pathologies of CNS, 83 U-373 MG human glioblastoma cells staining, 85 See also Astrocytoma(s); Intermediate filament (IF) proteins Synthetic low-density lipoproteins (LDL), 191 T Tago, M., 248 Takei, H., 201 TAT-ODD-procaspase-3 (TOP3), 130 Temozolomide, chemotherapy for PMA, 207–208 Temporal lobe epilepsy (TLE), 267 Temsirolimus (CCI-779), 49–51 Tenascin, 110 Teratomas, 95 Tetrapyrrole, 240 Thermal dosimetry, 179 Thrombospondin-1, 138–139 Thrombospondin-2, 140 Tibbetts, K.M., 199, 201 Tihan, T., 198, 203, 206–207 Time-resolved fluorescence measurements, 162 Time-resolved laser induced fluorescence spectroscopy (TR-LIFS) advantages, 170 classification and prediction, fluorescence signal classification algorithm elements, 165–166 linear discriminant function analysis (DFA), 165 method limitation, 169 training and test phase, 165–166 TR-fluorescence characteristics, 166–168 classification results using, 168 clinical methods, 163 data analysis, 164 reduction and statistical analysis, 165 delivery catheter, 163–164 penetration depth for astrocytoma, 164

289

differentiate clearly LGG from normal tissues, 168–169 fluorescent data collection, 164 goal, 163, 170 histopathological analysis, of tumors, 164 intra-operative tool, instrumental setup, 163–164 in vivo measurements, for glioma diagnosis, see Gliomas NC measurements, 169 parameters selection, 164–165 statistical analysis, and classification, 168 time-resolved fluorescence characteristics, 166–168 TNFR associated factor (TRAF) family, 25 TNF superfamily, 23, 25, 32 TP53 mutation, 138 Tracz, R.A., 175, 178, 180 TRAIL, 24–25, 28–30 Transcriptional regulation, 149 Transfection, 136 Transmissible spongiform encephalopathies (TSEs), 13 TR-LIFS, see Time-resolved laser induced fluorescence spectroscopy (TR-LIFS) Trypsin-Giemsa banding technique, 111 TSC, see Tuberous sclerosis complex (TSC) Tsc1, 150–151, 154 TSC2-Rheb-mTOR pathway, 47 TSP-1 expression, 140 Tuberous sclerosis complex (TSC), 45–53, 149–150 Akt activation, 49 -associated lesions, 48 defined, 45 glial dysfunction and, 150 global gene expression profiling, 151 and analysis, 151 GTPase-activating protein (GAP), 46 loss of heterozygosity (LOH) in, 48 molecular pathophysiology, 150–151 role of mutations in TSC1 and TSC2, 151 mTORC1 functions, 151 mTOR inhibitors, cell cultures and animal models studies, 51–52 mTOR pathway in, 46–47 autophagy inhibition, 47 genes regulated by HIFs, 46 inactivating mutations TSC1/TSC2 gene, 46 Ras-homoloque-enriched in brain (Rheb) target, 46

290

Tuberous sclerosis complex (TSC) (cont.) SEGAs in, 51 dual neuronal and glial origin, 48 mixed cells glial/giant, 48 positive for GFAP, 48 TSC1/TSC2-mTOR signaling pathway, 151, 155–156 tumorigenesis hamatrin-tuberin complex inactivation, 49 Knudson’s two-hit model, 48 See also Subependymal giant cell astrocytomas (SEGAs) Tumorigenesis apoptosis relation with, 23 EGFRvIII role in, 112 genes up-regulated in SEGA, link to, 151, 153–154 hypomethylation role, 5 MAPK signaling to PA, 102 OTX2 gene, medulloblastoma, 115 and survivin overexpression, 32 in TSC, see Tuberous sclerosis complex (TSC) Tumorigenesis apoptosis relation with, 23 EGFRvIII role in, 112 genes up-regulated in SEGA, link to, 151, 153–154 hypomethylation role, 5 MAPK signaling to PA, 102 OTX2 gene, medulloblastoma, 115 and survivin overexpression, 32 in TSC, see Tuberous sclerosis complex (TSC) Tumours animal models for preclinical trials, 188 associated with NF1, 143 autoimmunity to, 145 of central nervous system, 203 hemangioblastoma, see Hemangioblastomas hypoxia, importance, 129–130 imaging HIF-1 active regions by 123 I-IBB and 123 IPOS method, 129, 131–133 in vivo molecular imaging, 130 pretargeting approach, 131–133 nuclear medicine imaging, 130 oxygen-dependent degradable probe, development, 130 regression, 146 slow-growing, 213 Tumstatin, 139 Turner, J.R., 66

Index

U Ubiquitin–proteasome system, 58 Ueda, M., 132 Ushio, Y., 97 V Vaporization, 177 Ventana Benchmark IHC system, 90 Videography, intraoperative ICG, 233–237 Vimentin, 81–83, 85–87 Vincristine, 207 Visual field defects, 224 von Hippel-Lindau syndrome, 233, 235 W Wang, E.M., 248 Wang, Z.X., 42 Weidner, N., 136 Wesseling, P., 139 Williams, C.M., 23 Wnt antagonists, sFRP and Dickkopf class, 39 Wnt genes, 37 Wnt signaling pathway, 35, 37–39 Axin-APC-GSK3β, 41–42 β-catenin role, 40–41 destruction complex, 38 Dishevelled (Dvl), and FRAT1 action, 40 inhibition by extracellular Wnt antagonists DKK glycoproteins family, 39 sFRPs and Dickkopf family, 39 Wnt inhibitory factor-1 (WIF-1), 39 Lef-1 and Tcf-4 factors, 42 neural stem cells-gliomas, 38 pygopus 2, 42 See also Gliomas Wong, A.J., 111 X Xenograft models, 188 Y YAG (Yttrium, Aluminum and Garnet) laser, 176 Yang, Z., 39 Yolk-sac tumors, 96 Yu, J.M., 40 Z Zhang, L.Y., 42 Zhang, Z., 40

Tumors of the Central Nervous System, Volume 5: Astrocytomas, Hemangioblastomas, and Gangliogliomas

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MR Imaging and Spectroscopy of Central Nervous System Infection

Primary Central Nervous System Tumors: Pathogenesis and Therapy (Current Clinical Oncology)

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