Neuroblastoma: Molecular Mechanisms and Therapeutic Interventions

Neuroblastoma: Molecular Mechanisms and Therapeutic Interventions comprehensively reviews current concepts in molecular and histopathological mechanisms that influence the growth of human malignant neuroblastoma, along with exciting therapeutic interventions. This book features a broad collection of contributions from leading investigators in histopathology, molecular mechanisms, genetics, epigenetics, microRNAs, proteomics, and metabolism in controlling growth and death in neuroblastoma. Recent developments in therapeutic interventions for neuroblastoma are also covered extensively, including chapters on surgery, chemotherapy, targeted therapy and immunotherapy. This book is ideal for advanced undergraduate students, graduate students, medical students, postdoctoral fellows, and investigators with an interest in current molecular concepts and therapeutic interventions.

• Comprehensively covers the histopathological characterization, molecular mechanisms, and most recent therapeutic interventions in neuroblastoma
• Includes recent developments and therapeutic interventions for neuroblastoma, including chapters on surgery, chemotherapy, targeted therapy and immunotherapy
• Presents a broad scope that provides basic researchers, practitioners and students with the most current overview of recent advances

• Language: English
• Publisher: Academic Press
• Release date: Jan 11, 2019
• ISBN: 9780128120040

Book preview

Neuroblastoma

Molecular Mechanisms and Therapeutic Interventions

Editor

Swapan K. Ray

Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine, Columbia, SC, United States

Table of Contents

Cover image

Title page

Copyright

List of Contributors

Chapter 1. Neuroblastoma Pathology and Classification for Precision Prognosis and Therapy Stratification

Introduction

Pathology Diagnosis

International Neuroblastoma Pathology Classification (INPC)

Searching for Actionable/Druggable Targets Associated with Therapy Resistance

Proposed Subgroups of Unfavorable Histology Neuroblastoma for Precision Medicine

Molecular Targeting Therapies for Unfavorable Histology Neuroblastoma Resistant to the Current Therapy

Conclusion

Chapter 2. Role of Genetic and Epigenetic Alterations in Pathogenesis of Neuroblastoma

Introduction

Genetic Alterations in Neuroblastoma Pathogenesis

Epigenetic Alterations

Conclusions

Chapter 3. Neuroblastoma: Molecular Mechanisms and Therapeutic Interventions

Introduction

Growth Factor Signaling and Oncogenes

Immunotherapy

Norepinephrine-Targeted Therapy

Conclusions

Chapter 4. GD2-Targeted Immunotherapy of Neuroblastoma

Introduction

Structure, Biosynthesis, and Distribution of GD2

Functions of GD2

Cancer Immunotherapeutics

GD2-Specific Antibodies

Anti-GD2 Monoclonal Antibodies

Chimeric Anti-GD2 Monoclonal Antibody

Humanized Anti-GD2 Antibody

GD2-Specific Antibodies and Cytokines

GD2-Specific Antibody in Combination With Chemotherapy

Bispecific Antibody

GD2 Chimeric Antigen Receptor

GD2 Specific Vaccines

GD2 Peptide Mimotope

Anti-GD2 Idiotype Monoclonal Antibody

O-Acetyl GD2-Specific Antibody

Conclusions

Chapter 5. Targeting Angiogenesis in Neuroblastoma

Biological and Clinical Aspects of Neuroblastoma

Angiogenesis in Neuroblastoma

Antiangiogenesis in Neuroblastoma

Concluding Remarks

Chapter 6. Autophagy and Novel Therapeutic Strategies in Neuroblastoma

Introduction

The Autophagy-Lysosome System

Autophagy in Neuroblastoma

The Cross-talk Between Autophagy and Apoptosis

Molecular Mechanisms of Autophagy Activation in Neuroblastoma Cells

Conclusions

Chapter 7. Energy Metabolism and Metabolic Targeting of Neuroblastoma

Introduction

Alterations in the OXPHOS System in NB

Glucose Metabolism

Amino Acid Metabolism

Lipid Metabolism

The Nexus Between Common Genetic Abnormalities and Energy Metabolism in NB

Metabolic Adaption to Hypoxia in NB

Therapeutic Opportunities

Conclusion

Chapter 8. Molecular Imaging in Neuroblastoma

Metaiodobenzylguanidine (MIBG) Scintigraphy

Skeletal Scintigraphy

Fluorodeoxyglucose (FDG) PET

Somatostatin Receptor Scintigraphy

131I-MIBG Therapy

Conclusion

Chapter 9. Immunotherapy for Neuroblastoma

Introduction

Targets for NB Immunotherapy

Effectors of Immunotherapy for NB

Cytokines

Monoclonal Antibodies

Chemoimmunotherapy

Immunocytokines

Radioimmunotherapy

Adoptive Cell Therapy

Vaccines

Limitations of Immunotherapy for NB

Future Directions

Conclusions

Chapter 10. Advances in the Surgical Treatment of Neuroblastoma

Introduction

Chapter 11. Role of Stemness Factors in Neuroblastoma: Neuroblastoma Stem Cells, Tumor Microenvironment, and Chemoresistance

Cancer Stem Cells

Heterogeneity and CSCs in Neuroblastoma

Identification of CSCs in Neuroblastoma

Microenvironment

Tumor-Associated Macrophages (TAMs) and Cancer-Associated Fibroblasts (CAFs)

Immune Cells of the Microenvironment

Mesenchymal Stromal Cells and the Microenvironment

The Extracellular Matrix

Cell Adhesion Molecules (CAMs)

Angiogenesis and the Microenvironment

Hypoxia and the Microenvironment

Chemoresistance

Conclusions

Chapter 12. Current Pharmacotherapy for Neuroblastoma

Introduction

Stage-Guided Chemotherapy

Chemotherapy for Adults With Neuroblastoma

Late Effects of Chemotherapy for Neuroblastoma

Future Directions

Conclusions

Chapter 13. Current Challenges in the Management of Neuroblastoma: Noncoding RNA Influences

Introduction

Current Status of Neuroblastoma Theranostics

Noncoding RNAs

Clinical Importance of ncRNAs in NB Management Challenges

Conclusion and Perspectives

Chapter 14. Novel Therapeutic Targets in Neuroblastoma

Introduction

Clinical and Molecular Features of Neuroblastoma

Current Approach to Treatment

Clinical Trials of Targeted Therapies

Precision Medicine

Future Directions

Conclusion

Chapter 15. Current and Future Strategies for Treatment of Relapsed Neuroblastoma

Introduction

Standard Chemotherapy Approaches

Novel Chemotherapy Combinations

Targeted Molecular Radiotherapy

Precision Medicine Approaches

Genetic and Molecular Targeted Agents

Novel Immunotherapy Strategies

Challenges, Controversies, and Conclusions

Chapter 16. Emerging Evidence for Krüppel-Like Factor 4 (KLF4) as a Tumor Suppressor in Neuroblastoma

Introduction

Neuroblastoma in Need for New and More Reliable Prognostic Biomarkers

Members of KLF Family and Subfamilies

Molecular Structure of KLF4 to Account for Its Transcription Regulatory Roles

Regulation of Expression and Activity of KLF4

Involvement of KLF4 in Mechanism of Transactivation of Its Target Genes

Involvement of KLF4 in Mechanism of Transrepression of Its Target Genes

Neuroblastoma Shows Rare Genetic Complexion and Paradoxical Outcomes

Evidence for KLF4 as a Tumor Suppressor in Neuroblastoma

Conclusions

Chapter 17. Modulation of Expression of miRNAs for Therapeutic Effects in Human Malignant Neuroblastoma

Introduction

Biogenesis of miRNAs and Their Roles in Cancers

Deregulation of miRNAs in Human Malignant Neuroblastoma

Suppression of Expression of Oncogenic miRNAs in Malignant Neuroblastoma in Preclinical Models

Promotion of Expression of Tumor Suppressor miRNAs in Malignant Neuroblastoma in Preclinical Models

Transcriptional Activation of the Epigenetically Silenced Tumor Suppressor miRNA Genes in Malignant Neuroblastoma in Preclinical Models and Patients

Inhibition of Oncogenic miRNA or Induction of Tumor Suppressor miRNA and Pharmacotherapy to Enhance Therapeutic Effects in Malignant Neuroblastoma in Preclinical Models

Conclusions

Index

Copyright

Academic Press is an imprint of Elsevier

125 London Wall, London EC2Y 5AS, United Kingdom

525 B Street, Suite 1650, San Diego, CA 92101, United States

50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States

The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom

Copyright © 2019 Elsevier Inc. All rights reserved.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions.

This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).

Notices

Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary.

Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility.

To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein.

Library of Congress Cataloging-in-Publication Data

A catalog record for this book is available from the Library of Congress

British Library Cataloguing in Publication Data

A catalogue record for this book is available from the British Library

ISBN: 978-0-12-812005-7

For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Nikki Levy

Senior Acquisition Editor: Melanie Tucker

Editorial Project Manager: Gabriela D. Capille

Production Project Manager: Bharatwaj Varatharajan

Designer: Mark Rogers

Typeset by TNQ Technologies

List of Contributors

Sepideh Aminzadeh-Gohari,     Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, University Hospital of the Paracelsus Medical University, Salzburg, Austria

Sanja Aveic,     Neuroblastoma Laboratory, Pediatric Research Institute-Città della Speranza, Padua, Italy

J. Aye,     University of Alabama, Birmingham, AL, United States

Duncan Ayers

Centre for Molecular Medicine and Biobanking, University of Malta, Msida, Malta

Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom

E.A. Beierle,     University of Alabama, Birmingham, AL, United States

Nicole J. Croteau,     Pediatric Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, United States

Michael A. Dyer,     Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN, United States

René Günther Feichtinger,     Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, University Hospital of the Paracelsus Medical University, Salzburg, Austria

Paolo Grumati,     Institute of Biochemistry II, Goethe-Universität Frankfurt am Main, Frankfurt am Main, Germany

Ravi Kant Gupta,     Department of Nuclear Medicine, All India Institute of Medical Sciences, New Delhi, India

William Clay Gustafson,     University of California San Francisco, San Francisco, CA, United States

Jung-Tung Hung,     Institute of Stem Cell and Translational Cancer Research, Chang Gung Memorial Hospital at Linkou & Chang Gung University, Taoyuan, Taiwan

Naohiko Ikegaki,     Department of Anatomy and Cell Biology, College of Medicine, University of Illinois at Chicago, Chicago, IL, United States

Mariia Inomistova

National Cancer Institute of MPH of Ukraine, Kyiv, Ukraine

Educational and Scientific Center Institute of Biology and Medicine, Taras Shevchenko National University of Kyiv, Kyiv, Ukraine

Meredith S. Irwin,     Division of Haematology/Oncology, Hospital for Sick Children, Toronto and Department of Pediatrics, University of Toronto, Canada

Natalia Khranovska,     National Cancer Institute of MPH of Ukraine, Kyiv, Ukraine

Barbara Kofler,     Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, University Hospital of the Paracelsus Medical University, Salzburg, Austria

Anupa Kudva,     Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, United States

Prerna Kumar,     University of Illinois College of Medicine at Peoria, Peoria, IL, United States

Rakesh Kumar,     Department of Nuclear Medicine, All India Institute of Medical Sciences, New Delhi, India

Michael P. La Quaglia

Pediatric Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, United States

Department of Surgery, Weill Cornell Medical College, New York, NY, United States

Katherine K. Matthay,     University of California San Francisco, San Francisco, CA, United States

Shakeel Modak,     Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, United States

Daniel A. Morgenstern,     Division of Haematology/Oncology, Hospital for Sick Children, Toronto and Department of Pediatrics, University of Toronto, Canada

Rosa Nguyen,     Department of Oncology, St. Jude Children's Research Hospital, Memphis, TN, United States

Swapan K. Ray,     Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine, Columbia, SC, United States

Domenico Ribatti,     Department of Basic Medical Sciences, Neurosciences and Sensory Organs, University of Bari Medical School, Bari, Italy

James A. Saltsman,     Pediatric Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, United States

Nina F. Schor

Departments of Pediatrics, Neurology, and Neuroscience, University of Rochester, Rochester, NY, United States

National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, United States

Hiroyuki Shimada,     Department of Pathology and Laboratory Medicine, Children's Hospital Los Angeles, University of Southern California Keck School of Medicine, Los Angeles, CA, United States

Oksana Skachkova,     National Cancer Institute of MPH of Ukraine, Kyiv, Ukraine

L.L. Stafman,     University of Alabama, Birmingham, AL, United States

Gian Paolo Tonini,     Neuroblastoma Laboratory, Pediatric Research Institute-Città della Speranza, Padua, Italy

Alice L. Yu

Institute of Stem Cell and Translational Cancer Research, Chang Gung Memorial Hospital at Linkou & Chang Gung University, Taoyuan, Taiwan

Department of Pediatrics, University of California in San Diego, San Diego, CA, United States

Chapter 1

Neuroblastoma Pathology and Classification for Precision Prognosis and Therapy Stratification

Hiroyuki Shimada ¹ , and Naohiko Ikegaki ²       ¹ Department of Pathology and Laboratory Medicine, Children's Hospital Los Angeles, University of Southern California Keck School of Medicine, Los Angeles, CA, United States      ² Department of Anatomy and Cell Biology, College of Medicine, University of Illinois at Chicago, Chicago, IL, United States

Abstract

Tumors in neuroblastoma group offer one of the best models for investigating the biologically significant relationship between their genetic/molecular properties and morphologic manifestations. Over the past decades, histopathological analysis according to the International Neuroblastoma Pathology Classification (INPC) has provided invaluable information for determining the prognosis and therapy stratification for the patients with this disease. Now we are facing a great challenge for developing more efficient and less toxic treatment modalities for the patients with aggressive neuroblastomas. For this purpose, neuroblastoma pathology research needs to shift its gear to the direction of identifying prognostic factors to defining actionable/druggable targets in close collaboration with molecular biology, based on the concept of precision medicine.

Keywords

Actionable/druggable targets; ALK mutation/overexpression; Alternative lengthening of telomere; ATRX; Ganglioneuroblastoma; Ganglioneuroma; Intermixed; International neuroblastoma pathology classification; MYC family driven neuroblastoma; MYC protein; MYCN protein; Neuroblastoma; Nodular; Telomere maintenance and elongation; TERT

Introduction

Neuroblastoma is often used as a collective term for all types of peripheral neuroblastic tumors of neural crest origin and includes Neuroblastoma, Ganglioneuroblastoma, and Ganglioneuroma. Historically tumors in this group were described as enigmatic because of their unique and often unpredictable clinical behaviors, such as spontaneous regression, tumor maturation, and aggressive progression refractory to therapy. These clinical features are now considered to be closely associated with their genetic/molecular characteristics [1–3]. It is also noted that tumors in this group offer one of the best models for investigating the biologically significant relationship between their genetic/molecular properties and morphologic manifestations [4].

Pathology Diagnosis

Whenever feasible, it is recommended to obtain biopsied/surgically resected samples before starting chemotherapy/irradiation therapy for pathology evaluation. Determining the histologic/biologic characteristics of the tumors is critical for patient stratification and appropriate protocol assignment at the time of diagnosis. International Neuroblastoma Pathology Classification (INPC) is applied only to the tumor specimen obtained before starting chemotherapy/irradiation therapy [4,5]. After chemotherapy, tumor samples, especially of biologically/clinically unfavorable cases, show acute chemotherapy effects including a large area of necrosis and extensive hemosiderin (intracellular iron storage complex) deposition. Cytologic/morphologic changes of the tumors after chemotherapy, which could mainly represent an epigenetic phenomenon, are often not reliable for predicting clinical behaviors of the individual tumors. We should conduct further studies on recurrent tumors, as they could demonstrate different genetic/molecular properties of the tumors at the time of diagnosis.

At the surgical pathology gross bench, the priority should be the securing of enough samples for histological examination. For determining biological characteristics of the given tumor, it is critical for saving the snap-frozen material for molecular tests. Diagnosis by touch preparations is recommended for MYCN and other analyses by the FISH (fluorescence-based in-situ hybridization) test.

Immunohistochemical stainings often used for confirmation of the neuroblastoma diagnosis include neuronal markers (NSE, PGP9.5, Synaptophysin, Chromogranin, CD56, etc.) and so-called neuroblastoma marker (NB84). However, those markers are not specific for neuroblastoma; for example, those neuronal markers are usually positive for Ewing's/PNET. Markers for the neural crest tumors with neuronal and neuroendocrine differentiation, which include TH (tyrosine hydroxylase) and Phox2b [6–8], are more specific for neuroblastoma diagnosis. Between these two neural crest markers, Phox2b is more stable and can be used for the staining of the bone and bone marrow samples after decalcification [9]. In our experience, Phox2b is more sensitive than TH and positive for all neuroblastomas including undifferentiated subtype [9,10]. In contrast, TH is positive only for sporadic cells or even becomes negative in some of the tumors in the undifferentiated subtype. It should be noted that both Phox2b and TH are positive for pheochromocytomas and paragangliomas [10].

International Neuroblastoma Pathology Classification (INPC)

Histologic Categories and Subtypes

The International Neuroblastoma Pathology Committee defines four categories in this group of tumors: each is characterized by the grade of neuroblastic differentiation and the degree of Schwannian stromal development [4]. They are Neuroblastoma (Schwannian stroma-poor), Ganglioneuroblastoma, intermixed (Schwannian stroma-rich), Ganglioneuroma (Schwannian stroma-dominant), and Ganglioneuroblastoma, nodular (composite, Schwannian stroma-rich/stroma-dominant and stroma-poor). It is believed that all Ganglioneuromas are once Neuroblastomas in their early stage of tumor development. The maturation sequences from Neuroblastoma to Ganglioneuroma are prompted by the cross talk between neuroblasts and Schwannian cells, comparable to the embryologically well-defined relationship in neural crest development toward ganglion structure of the autonomic nervous system [11]. The cross talk seems to be supported by various signaling pathways including trkA/nerve growth factor (NGF) signaling and Nrg1/ErbB signaling, etc. [12,13].

Neuroblastoma (Schwannian stroma-poor) - NB: Tumors in this category include three subtypes: They are: undifferentiated, poorly differentiated, and differentiating. Those tumors are characterized by the typical growth pattern of neuroblastic cells forming groups or nests demarcated by thin fibrovascular stromal septa where limited or no Schwannian cell proliferation is observed.

1. Neuroblastoma, undifferentiated subtype—NB-UD is rare and supplementary procedures, such as immunohistochemistry and/or molecular tests, are required to establish the diagnosis. The proliferating cells are uniformly primitive without clearly recognizable neurite production (Fig. 1.1A). Neuroblasts in this subtype usually do not express higher levels of a favorable marker trkA (high-affinity NGF receptor) and do not have a potential for differentiation. Accordingly, tumors in this subtype are considered as biologically unfavorable. The nuclear morphology of NB-UD cells often exhibits a vesicular euchromatic (transcriptionally active chromatin) appearance.

2. Neuroblastoma, poorly differentiated subtype—NB-PD is composed of neuroblasts having varying amounts of neurite production with or without HomerWright rosette formation. This is the most common histological form among the peripheral neuroblastic tumors. Less than 5% of tumor cells have cytomorphologic features of differentiating neuroblasts (see blow c. NB-D). Nuclear morphology of the NB-PD neuroblasts is often described as salt-and-pepper (sprinklings of heterochromatin and a few inconspicuous nucleoli) (Fig. 1.1B). It is interesting to note that some tumors in this subtype, and more in the undifferentiated subtype, show the presence of some prominent nucleoli (nucleolar hypertrophy), especially when MYCN oncogene is amplified (Fig. 1.1C). Tumors in this subtype are either biologically favorable or biologically unfavorable. Biologically favorable tumors show spontaneous regression or cellular differentiation/tumor maturation. As mentioned above, the latter seems to be supported by the cross talk between neuroblastic cells and Schwannian stromal cells; those neuroblasts express higher levels of trkA and actively recruit Schwannian stromal cells. In contrast, biologically unfavorable tumors seem to have lower levels of trkA expression, do not recruit Schwannian stromal cells, and do not have a potential of differentiation/maturation. Amplified MYCN is known to downregulate trkA expression. However, many MYCN nonamplified tumors can also express lower trkA levels, and considered to be biologically unfavorable as well.

3. Neuroblastoma, differentiating subtype—NB-D, is a tumor usually characterized by abundant neurite production. More than 5% of tumor cells show cellular differentiation and have an appearance of differentiating neuroblasts (Fig. 1.1D). Those differentiating neuroblasts are defined by synchronous differentiation of both the nucleus (enlarged, eccentrically located with a vesicular chromatin pattern and usually a single prominent nucleolus) and the cytoplasm (eosinophilic/amphophilic with a diameter or twice or more of the nucleus). A Nissl substance can be seen in the periphery of the cytoplasm. Majority of the tumors in this subtype are biologically favorable. However, some of the patients with differentiating subtype of neuroblastoma still have a poor clinical outcome (please see Prognostic Grouping section below).

Ganglioneuroblastoma, Intermixed (Schwannian stroma-rich)—GNB-I: Tumor in this category contains well-defined microscopic nests of neuroblastic cells in a background of naked neurites that are intermixed or randomly distributed in the ganglioneuromatous tissue. Those microscopic nests represent the areas where neuritic processes produced by the neuroblasts are not incorporated in the cytoplasm of Schwannian stromal cells. By definition, more than 50% of tumor tissue in this category should have a ganglioneuromatous appearance where ganglion cells are individually embedded in abundant Schwannian stromal cells. These microscopic nests are composed of a mixture of neuroblastic cells in various stages of differentiation, often dominated by differentiating neuroblasts (Fig. 1.1E). Some apoptotic cells may be seen in the nests as well. Presence of these microscopic nests is considered as a sign of the lagging behind of tumor maturation toward ganglioneuroma and the tumors are biologically favorable, leading to an excellent prognosis of the patients.

Figure 1.1 Categories and Subtypes of Peripheral Neuroblastic Tumors: (A) Neuroblastoma, Undifferentiated subtype (NB-UD); (B) Neuroblastoma, Poorly differentiated subtype (NB-PD); (C) MYCN amplified tumor showing the appearance of NB-PD with a high MKI (Mitosis-Karyorrhexis Index), (inset: neuroblastic cells having prominent nucleolar formation); (D) Neuroblastoma, Differentiating subtype (NB-D) (inset: typical differentiating neuroblasts with both cytoplasmic and nuclear enlargement); (E) Ganglioneuroblastoma, Intermixed (GNB-I); (F) Ganglioneuroma (GN) [inset: completely mature ganglion cell covered with satellite cell (arrow)]; (G) Ganglioneuroblastoma, Nodular (GNB-N) composed of two distinct histologies (clones)—Ganglioneurmatous tissue (left) and neuroblastomatous nodule (right).

Ganglioneuroma (Schwannian stroma-dominant) - GN: Tumors in this category are characterized by the presence of individually distributed ganglion cells in the Schwannian stroma (Fig. 1.1F). Neuritic processes produce by the ganglion cells are immediately enveloped by the cytoplasm of Schwann cells. Accordingly, there are no recognizable microscopic foci of naked neurites without Schwannian coverage. This category includes two subtypes: maturing and mature. The maturing subtype contains both maturing and mature ganglion cells, whereas the mature subtype contains only mature ganglion cells. The mature ganglion cells are surrounded by the satellite cells. The stromal tissue is usually well organized and shows the fascicular profile of Schwann cells bundled with perineurial cells. Ganglioneuroma is a biologically/clinically benign tumor. However, there are markedly rare cases where malignant Schwannoma develops in ganglioneuroma with or without irradiation therapy [14].

Ganglioneuroblastoma, Nodular (composite, Schwannian stroma-rich/stroma-dominant and stroma-poor)—GNB-N: Tumors in this category are characterized by the presence of grossly visible, often hemorrhagic and/or necrotic, NB nodule(s) (stroma-poor component), coexisting with GNB-I (stroma-rich component) or with GN (stroma-dominant component) (Fig. 1.1G). The term composite implies that the tumor is composed of biologically different clones.

Prognostic Grouping (Favorable Histology Vs. Unfavorable Histology)

INPC distinguishes two prognostic groups, Favorable Histology Group and Unfavorable Histology Group (Fig. 1.2) [5,15]. Tumors in the Favorable Histology Group are within a framework of age-appropriate tumor differentiation/maturation and age-appropriate mitotic and karyorrhectic activities. As for the morphologic indicators of tumor differentiating/maturation, the categories and subtypes described above are utilized. In other words, tumors in the Favorable Histology Group can demonstrate age-dependent differentiation/maturation from NB-PD to NB-D, then to GNB-I and finally to GN, based on the cross talk between tumor cells and Schwannian stromal cells. However, to observe tumor differentiation/maturation, it seems to take a certain amount of time; i.e., in vivo latent period. It is expected to take up to 18   months for those tumors of NB-PD subtype to become NB-D subtype, and up to 60   months to become GNB-I or GN. In contrast, tumors of NB-UD subtype in any age group, tumors of NB-PD subtype over 18   months of age, and tumors of NB-D subtype over 60   months of age are considered as having limited or no differentiating potential, and they are classified into the Unfavorable Histology Group.

Another morphologic indicator for predicting clinical behavior in this disease is mitotic and karyorrhectic activities of neuroblastic cells, and that is applied to tumors in the NB category [16]. One of three MKI (Mitosis-Karyorrhexis Index) classes based on the activities is assigned to the given NB tumors: They are Low (<100/5000   cells), Intermediate (100–200/5000   cells), and High (>200/5000   cells) and their prognostic effects are also age-dependent. Low MKI tumors in the patients <5   years of age at diagnosis, and Intermediate MKI tumors in the patients <18   months of age at diagnosis are classified into the Favorable Histology Group. High MKI NB tumors in any age group, Intermediate MKI tumors >18   months of age at diagnosis, and Low MKI tumors >60   months of age at diagnosis are classified into the Unfavorable Histology Group. MYCN amplified tumors are typically associated with high MKI (please see Fig. 1.1C) [17,18].

Figure 1.2 International Neuroblastoma Pathology Classification ∗: Mitosis-Karyorrhexis Index is not assigned for Ganglioneuroblastoma, Intermixed and Ganglioneuroma. ∗∗: Ganglioneuroblastoma, Intermixed, Ganglioneuroma, and Ganglioneuroblastoma, Nodular are diagnosed in older children. ∗∗∗: Prognostic distinction of Ganglioneuroblastoma, Nodular is determined by the age-linked evaluation of histologic markers (grade of neuroblastic differentiation and mitosis-karyorrhexis index) of the neuroblastomatous nodule (see text).

GNB-I and GN are always classified into the Favorable Histology Group [19], while tumors in the GNB-N category are classified into the Favorable Histology Group or Unfavorable Histology Group based on the characteristics of NB nodule(s) [15]. For this purpose, the same criteria of age-linked evaluation for the grading of neuroblastic differentiation and the MKI class utilized for the prognostic distinction of NB tumors are applied to the NB nodule(s). It should be noted that making the correct diagnosis of GNB-N is often difficult by biopsy or partial tumor resection, since NB nodule could be hidden and not sampled for pathology examination. In that situation, it is recommended to add a disclaimer based on the review of limited material in the diagnosis line after GN or GNB-I, Favorable Histology in the surgical pathology report. It is critically important since the clinical behavior of the given tumor would depend on the characteristics of NB nodule(s), if present [14].

In neuroblastoma, the patient's age at diagnosis is one of the prognostic indicators. Historically, 1   year has been used as the cutoff mark. The prognostic contribution of age to the clinical outcome seems to be naturally continuous, and the survival rates of younger patients are always better than older patients in any age cutoff. Based on the Children's Oncology Group (COG) Neuroblastoma study, London et al. reported the statistical evidence of an age cutoff greater than 1   year for risk stratification [20], and the COG is now in the process of moving the cutoff from 1   year (365   days) to 18   months (548   days). The age factor should be considered as a surrogate for other genetic/biologic risk markers. Although the INPC has an already built-in age cutoff point of 18   months, Sano et al. demonstrated that the INPC was able to add independent prognostic information beyond the prognostic contribution of age [21]. In other words, the INPC clearly distinguishes two prognostic groups (Favorable Histology identifying a significantly better prognosis group than Unfavorable Histology) in different age groups, such as < versus >12   months; < versus >18   months, and < versus >24   months of age at diagnosis (Fig. 1.3).

Importantly, the survival rate of Favorable Histology Group is estimated to be around or over 90%, whereas that of Unfavorable Histology Group has remained at 50%–40% or less [5,21,22]. It indicates that at least one in two of the Unfavorable Histology Group patients dies from the disease despite the high-intensity multimodal therapy. Clearly, new innovative therapeutic approaches are required for those in the Unfavorable Histology Group, who are resistant to the current treatment protocols. To address this problem, we have attempted to identify the expression of potentially drug-targetable proteins that appear to lay the foundation for the aggressive behavior of certain neuroblastomas existing in the Unfavorable Histology Group. Examples of those proteins are summarized in the following section. At this stage, we are planning to refine the current INPC by incorporating immunohistochemical detection/evaluation of the target proteins and developing a more precise system of pathology classification for future patient stratification and protocol assignment.

Figure 1.3 International Neuroblastoma Pathology Classification significantly distinguishes event-free survivals for FH (Favorable Histology) patients from UH (Unfavorable Histology) patients within different age groups: (A) <12   months versus >12   months; (B) <18   months versus >18   months; and, (C) <24   months versus >24   months at the time of diagnosis. 

The figure reproduced from Figure 3 in the published article by Sano H, Bonadio J, Gerbing RB, London WB, Matthay KK, Lukens JN, et al. International neuroblastoma pathology classification adds independent prognostic information beyond the prognostic contribution of age. Eur J Cancer 2006;42:1113–1119.

Searching for Actionable/Druggable Targets Associated with Therapy Resistance

In this section, some examples of our recent effort are described using immunohistochemistry in searching for actionable/druggable targets associated likely with refractory and resistance to the current intensive multimodal therapy in this disease.

MYC Driven Neuroblastoma

MYCN oncogene is considered as a significant oncogenic driver of neuroblastoma. MYCN amplification is seen in approximately 20% of neuroblastomas [23,24]. The vast majority of MYCN amplified tumors overexpress the MYCN protein, and MYC-MAX protein heterodimer formation is critical in activating downstream molecular targets through E-box gene sequences and leading to aggressive tumor growth [25]. There are rare MYCN amplified neuroblastomas without protein expression [26], and also extremely rare MYCN nonamplified neuroblastomas with MYCN protein overexpression [unpublished data].

Besides MYCN oncogene amplification and subsequent MYCN protein overexpression, MYC (aka C-Myc) protein overexpression, observed in ∼10% of undifferentiated and poorly differentiated neuroblastomas, is reported as a new prognostic factor [27]. It seems extremely rare to observe overexpression of both MYCN protein and MYC protein in the same tumor. Recently we have defined MYC driven neuroblastoma that shows augmented expression of either MYCN protein or MYC protein detectable by immunohistochemistry [27]. These neuroblastomas are highly aggressive and associated with similarly low survival rates (3-year event-free survival: 46.2   ±   12.0% for MYCN overexpressing neuroblastoma and 43.4   ±   23.1% for MYC overexpressing NB, respectively) (Fig. 1.4A).

Of note, neuroblastic cells in MYC driven neuroblastoma are often associated with the prominent nucleolar formation (nucleolar hypertrophy) [27,28], indicative of increased rRNA synthesis leading to high levels of protein expression (Fig. 1.4B-i and ii). Because of the presence of one to a few prominent nucleoli, MYC driven neuroblastoma can be cytologically distinguished from the conventional small-round-blue-cell neuroblastoma with salt-and-pepper nuclei. In addition, MYCN protein overexpressing neuroblastoma (Fig. 1.4C-i) is significantly associated with MYCN oncogene amplification, and present with High MKI. In contrast, MYC protein overexpressing neuroblastoma (Fig. 1.4C-ii) is not associated with MYCN oncogene amplification and often present with low or intermediate MKI [27]. Currently, the precise molecular mechanism(s) for MYC protein overexpression in neuroblastoma is not known, but in some rare cases, MYC gene amplification is involved [29].

MYC driven neuroblastoma, comprising more than 50% of high-risk neuroblastomas, includes rare and unique tumors named large-cell neuroblastoma that is often characterized by the bull's eye appearance of enlarged and uniquely open euchromatin-rich nuclei containing highly conspicuous nucleoli (Fig. 1.4D) [30]. Most large-cell neuroblastomas overexpress higher levels of MYCN protein or MYC protein than other MYC driven neuroblastomas. Their euchromatin-rich open nuclei also suggest the stem-cell like nature of the tumor cells [31].

Neuroblastoma With ALK Mutation/Overexpression and Amplification

ALK (Anaplastic Lymphoma Kinase) is a receptor tyrosine kinase and expressed in the developing sympathoadrenal lineage of the neural crest. However, ligand(s) of ALK have not been well established. In neuroblastoma, mutations in the ALK gene account for the majority of familial neuroblastoma cases [32]. ALK mutations/overexpression and amplification are also found in around 10% of sporadic neuroblastoma cases [33,34]. It was also found that ALK gene amplification and F1174 mutations, which are among the most active forms of the mutations in in vitro assay, are associated with MYCN amplification [35]. Interestingly, overexpression of ALK due to ALK amplification as well as gene mutations appears to be responsible for its oncogenic function [36]. In addition, a feed forward activation loop between MYCN and ALK expressions has been reported [37–39]. ALK abnormalities seem to cause dysregulation of multiple pathways, including PI3K, AKT, MEKK3, and MEK5 signal transduction pathways, to bring an uncontrolled proliferation of neuroblasts [38].

Figure 1.4 A new concept of MYC driven neuroblastoma: (A) Event-free survivals by International Neuroblastoma Pathology Classification and MYCN/MYC protein expression in Neuroblastoma, Undifferentiated and Poorly differentiated subtype. Patients with MYC protein overexpressing tumor and those with MYCN protein overexpressing tumor have a similar and significantly low survival rates. (B) Different nuclear morphologies; salt-and-pepper nuclei in non-MYC driven neuroblastoma (B–I) and prominent nucleolar formation (nucleolar hypertrophy) in MYC driven neuroblastoma (B-II). (C) MYC driven neuroblastoma with MYCN protein overexpression (C–I) and MYC protein overexpression (C-II). (D) Large-cell neuroblastoma, a member of MYC driven neuroblastoma characterized by enlarged and uniquely open nuclei containing highly conspicuous nucleoli. 

(A) The figure adapted by permission from Macmillan Publications Ltd.: Wang LL, et al. Augmented expression of MYC and/or MYCN protein defines highly aggressive MYC driven neuroblastoma: a Children's Oncology Group Study. Br J Cancer 2015;113:57–63.

However, the prognostic significance of ALK mutations/overexpression has been controversial, as some reports suggest an association of ALK mutations/overexpression with fatal outcome of the disease, whereas others suggest otherwise. Initially, De Brouwer et al. showed no significant survival difference in neuroblastomas with or without ALK mutations or amplification and reported no significant difference in the frequency of ALK mutations between low- and high-stage tumors [35]. Subsequently, however, Schulte et al. reported that high levels of mutated and wild-type ALK expression were associated with reduced survival of neuroblastoma [36]. This difference appeared because the cohort of De Brouwer et al. had included more unfavorable neuroblastomas than that of Schulte et al. Thus, if we only considered unfavorable neuroblastomas, there would be no difference in survival of neuroblastomas with or without ALK mutations or amplification. This idea is supported by the data shown in Fig. 1.5, where there is little difference in survival between ALK high- and low-expressing tumors in the high-risk subset as well as in the fatal cases of neuroblastoma patients. Passoni et al. first reported the immunohistochemical detection of ALK protein in neuroblastoma and its prognostic significance in 2009 [40], in that, high-level expression of ALK was associated with adverse outcome of the disease. However, Regairaz et al. later showed immunohistochemically that ALK and its active form pALK expression were observed in many neuroblastomas independent from ALK mutation/amplification [41]. Based on these observations, it is less likely that future INPC will incorporate the immunohistochemical detection of ALK and/or pALK expression in the pathology classification.

Telomere Maintenance and Elongation in Neuroblastoma

Telomere maintenance and even elongation could prevent neuroblasts from replication senescence/cellular death due to telomere erosion. Accordingly, neuroblasts could acquire infinite proliferating capability.

Telomere elongation by increased TERT activity (Fig. 1.6A and B): Immunohistochemically, TERT (telomere reverse transcriptase) overexpression can be observed in both MYC driven and non-MYC driven NBs in any age groups of the neuroblastoma patients [42]. In other words, there seem to be multiple mechanisms for upregulation of TERT expression: It can be associated with MYCN/MYC protein overexpression, TERT rearrangement, or promoter hypermethylation, etc. [43–47].

Alternative lengthening of telomere by ATRX loss (Fig. 1.6C and D): ATRX (alpha-thalassemia/mental retardation syndrome X-linked) mutations are reported in older children with neuroblastoma and indicate a poor prognosis [48]. Most of the tumors with ATRX mutations show alternative lengthening of telomere (ALT). ATRX mutations causing loss of ATRX expression can easily be detected by immunohistochemistry. Neuroblastomas with loss of ATRX are diagnosed in older children (>5   years of age at diagnosis), and almost exclusively found in the non-MYC driven neuroblastomas with Unfavorable Histology [49]. Also, reported are rare NBs with DAXX (Death Domain Associated Protein) gene mutations that are also known to cause ALT [50]. ATRX and DAXX are components of the molecular complex that functions to replace Histone 3.1/2 with Histone 3.3. ATRX recognizes DNA segments containing H3K4me0 and H3K9me3, and DAXX removes Histone 3.1/2 and inserts Histone 3.3 in a replication-independent fashion [51]. DAXX interacts with KAP1 and SETDB1 to catalyze H3K9me3 modification on newly deposited H3.3. ATRX/DAXX/H3.3 are able to act continuously through the cell cycle to ensure constant maintenance of H3K9me3 heterochromatin (transcriptionally inactive chromatin) at telomeres [52]. Therefore, ATRX or DAXX loss will lead to recombinogenic telomeres leading to the ALT phenotype (see below).

Figure 1.5 Prognostic significance of ALK expression in neuroblastoma: Possible prognostic significance of ALK expression was assessed using the whole cohort, High-Risk subset or Fatal cases. Analysis was done by R2 (http://r2.amc.nl), using Tumor Neuroblastoma - SEQC - 498 - RPM - seqcnb1 Dataset [88]. Note: ALK mutations and amplification result in high levels of ALK expression.

Figure 1.6 Immunostainings for ATRX (alpha-thalassemia/mental retardation syndrome X-linked) and TERT (telomerase reverse transcriptase) Expression: (A) Neuroblastoma with ATRX loss (Note: endothelial cells retain ATRX); (B) Neuroblastoma with positive ATRX; (C) Neuroblastoma with TERT overexpression; (D) Neuroblastoma without TERT overexpression.

Proposed Subgroups of Unfavorable Histology Neuroblastoma for Precision Medicine

As we have gained additional knowledge about the potential molecular targets that underlay the therapy-resistant phenotype associated with Unfavorable Histology neuroblastomas (indicated above), we may incorporate such information into the INPC toward precision prognosis and therapy stratification.

The first example of this approach was to examine the expression of MYC family proteins. Consequently, this study has led to the concept of MYC family protein driven neuroblastoma or in short MYC driven neuroblastoma, having a high-level expression of MYC family proteins with a dismal survival rate [27]. Thus, this study was able to retrospectively identify a group of high-risk patients who would likely fail the current high-risk therapy. Those patients are who would likely benefit from an alternative high-risk protocol that includes an MYC-targeted regimen (see below). For this study, we have used anti-MYCN (NCM II 100) and anti-MYC (Y69) antibodies separately to detect MYCN and MYC proteins. In future studies, we are planning to use an anti-pan-MYC antibody (NCM II 143) that can simultaneously detect all three members of the MYC family proteins, MYC, MYCN, and MYCL (though we still have not identified MYCL overexpressing neuroblastomas in our file), to streamline the process (Fig. 1.7). Recently, we have been extending the immunohistochemical approach to TERT and ATRX expression, and the preliminary results appear promising [42,49].

Figure 1.7 Neuroblastomas overexpressing MYCN protein (left column), MYC protein (middle column), and no MYCN/MYC protein (right column) shown by H&E staining, MYCN FISH (Fluorescence in situ hybridization), MYCN protein immunostaining, MYC protein immunostaining, and Pan-MYC protein immunostaining (Note: MYCN overexpressing neuroblastoma is MYCN amplified): In contrast, MYC overexpressing neuroblastoma and both protein negative neuroblastoma are MYCN nonamplified. Pan-MYC immunostaining yields positive signals in both MYCN and MYC overexpressing neuroblastomas.

Based on these observations, we could refine the Unfavorable Histology Neuroblastoma stratification using immunohistopathological analysis shown in Table 1.1. We propose to use three (or four) immunohistochemical stainings with anti-pan-MYC antibody (or anti-MYCN and anti-MYC), anti-TERT antibody, and anti-ATRX antibody to classify Unfavorable Histology Neuroblastomas into four subgroups.

MYC subgroup: As augmented MYC family protein expression stimulates rRNA synthesis and protein translation, this subgroup should exhibit prominent nucleolar formation (nucleolar hypertrophy) and hypertrophic cell morphology. In addition, MYC family protein overexpression would also lead to open chromatin as MYCN/MYC proteins recruit histone acetyltransferases on to chromatin globally. Thus, the overall histologic appearance of MYC driven tumors would ultimately become the large-cell neuroblastoma. It is also expected that a considerable number of MYC driven neuroblastomas are associated with TERT overexpression, and this is because TERT is a direct gene target of MYC family proteins, whereas TERT can stabilize MYC protein via its non-canonical enzymatic function, creating a positive feedback loop between MYCN/MYC and TERT expression [46,53].

Table 1.1

a  LCNB   =   Large-cell neuroblastoma.

b  S&P   =   Conventional neuroblastoma with salt-and-pepper nuclei.

TERT subgroup: Higher levels of TERT expression are also observed in neuroblastomas without MYCN/MYC protein overexpression. The activation of TERT expression, in this case, is likely due to long-range genomic rearrangements, but not to promoter mutations in neuroblastoma [54]. TERT promoter hypermethylation might also be its activating mechanism [45,55].

ALT subgroup: ATRX loss results in the ALT (alternative lengthening of telomere) phenotype. ATRX is rarely mutated in the MYC subgroup and TERT subgroup, and loss of ATRX is exclusively seen in the older children over 5   years of age. Because one of the normal ATRX functions is to insert the variant histone H3, namely H3.3, in concert with DAXX into chromatin to maintain transcriptionally active euchromatin [56], the absence of ATRX may result into a more transcriptionally inactive heterochromatic state. Thus, the histological/cytological appearance of the ALT subgroup could be the conventional type with salt-and-pepper nuclei rather than that with nucleolar hypertrophy or bull's eye type.

Null subgroup: There are still Unfavorable Histology Neuroblastomas without MYCN/MYC overexpression, TERT overexpression, and ATRX loss. Unless otherwise any other aggravating factors are found, the patients in the Null subgroup would hopefully respond to the current high-risk treatment regimens.

Molecular Targeting Therapies for Unfavorable Histology Neuroblastoma Resistant to the Current Therapy

Along with the well-documented ALK inhibitors for ALK overexpression [57], potential agents targeting MYC family protein overexpression and telomere maintenance/elongation are discussed in this section. Those are candidates to be included in our future protocols of molecular targeting therapy in neuroblastoma (Table 1.2).

Targeting of MYC Driven Neuroblastoma

In order to develop treatment strategies for the MYC driven neuroblastomas, new targets and potential therapeutic agents should be clearly defined. However, the direct targeting of MYC family proteins with small molecules has turned out to be a highly formidable task [58]. Hence, many have sought indirect approaches to downregulate MYC family protein expression in cancer cells. The strategies under current consideration include transcriptional repression of MYC/MYCN genes by BET (Bromo- and Extra-Terminal)-inhibitors [59,60] and CDK (Cyclin-dependent kinase) inhibitors [61,62] or destabilization of MYCN by Aurora kinase A inhibitors [63].

Table 1.2

We have been seeking other indirect strategies to down-regulate MYC family protein expression in neuroblastoma. Since MYC driven neuroblastomas are characterized by prominent nucleolar formation, inhibition of rRNA gene transcription and protein translation by small molecule inhibitors could be a potential therapeutic approach for this subset of biologically unfavorable neuroblastomas [64]. In fact, those small molecules, such as CX-5461 and Halofuginone, are currently examined for their efficacy in various human diseases in clinical trials [65,66]. CX-5461 disrupts the binding of the SL1 transcription factor to the rDNA promoter and prevents the initiation of rRNA synthesis by RNA Pol I [67]. Consequently, the ribosomal assembly will be halted, leading to the accumulation of unassembled ribosomal proteins. Free ribosomal proteins will then promote cancer-specific activation of p53 [64]. Interestingly, a similar set of freed ribosomal proteins could also down-regulate MYC protein by distinct mechanisms [68,69]. Halofuginone, on the other hand, specifically inhibits glutamyl-prolyl tRNA synthetase (EPRS) [70], resulting in the accumulation of uncharged prolyl tRNAs, which then leads to suppression in protein translation via the induction of amino acid starvation response [71]. In addition, inhibition of EPRS may also liberate one of the cofactors from the human multi-tRNA-synthetase complex (i.e., AIMP2 - Aminoacyl TRNA Synthetase Complex Interacting Multifunctional Protein 2). Freed AIMP2 could then migrate into the nucleus and inhibit FBP (FUBP1), which otherwise functions as the transcriptional activator of MYC [72,73]. Consequently, Halofuginone could induce downregulation of MYC family proteins through both global translational suppression and transcriptional inhibition.

Targeting ALK in Neuroblastoma

Approximately 60% anaplastic large-cell lymphomas (ALCLs) show activation