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9781841846149-FM 8/24/07 4:28 PM Page i

Advances in
Nuclear Oncology
9781841846149-FM 8/24/07 4:28 PM Page ii
9781841846149-FM 8/24/07 4:28 PM Page iii

Advances in
Nuclear Oncology
Diagnosis and
Therapy
Edited by

Emilio Bombardieri
Director, Department of Diagnostic Imaging and Therapy, Nuclear Medicine Division,
IRCCS Foundation, National Cancer Institute, Milan, Italy
John Buscombe
Consultant, Department of Nuclear Medicine, Royal Free Hospital, London, UK

Giovanni Lucignani
Director, Unit of Nuclear Medicine, Hospital San Paolo,
Institute of Radiological Sciences, University of Milan, Milan, Italy
Otmar Schober
Director, Department of Nuclear Medicine, University of Münster, Münster, Germany
9781841846149-FM 8/24/07 4:28 PM Page iv

© 2007 Informa UK Ltd

First published in the United Kingdom in 2007 by Informa Healthcare,

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9781841846149-FM 8/24/07 4:28 PM Page v

Contents

List of contributors vii 9. Ovarian cancer 133

Sandro Sironi, Maria Picchio, Luca
Preface xi
Guerra, Cristina Messa, and
Acknowledgments xii Ferruccio Fazio

I: Biology of cancer 10. Breast cancer 143

Emilio Bombardieri, Marco Maccauro,
1. What is cancer? 1 Orazio Schillaci, and Flavio Crippa
Uwe Haberkorn
11. Thyroid cancer 161
2. Targets 17 Peter Lind and Susanne Kohlfürst
Christoph Bremer and Michael Schäfers
12. Adrenal tumors 177
II: Diagnostic applications Christiane Franzius and
Otmar Schober
3. Primary brain tumors 23
Eva Orunesu, Andreas Jacobs, 13. Neuroendocrine 189
Andrea Falini, Angelo Del Sole, and gastroenteropancreatic tumors
Giovanni Lucignani Emilio Bombardieri, Angela Coliva,
Marco Maccauro, Ettore Seregni,
4. Head and neck cancer 51 and Irene Virgolini
Flavio Crippa, Alberto Gerali, and
Alessandra Alessi 14. Lymphoma 203
Simona Ben-Haim and Ora Israel
5. Lung cancer 61
Richard P Baum, Vikas Prasad, and 15. Bone and soft tissue tumors 223
Wolfgang A Weber Gopinath Gnanasegaran, Gary Cook,
Sanjay Vijayanathan, and Ignac
6. Esophageal cancer 89 Fogelman
Cristina Nanni, Sara Taddayon,
Guido Biasco, Stefano Fanti, and 16. Malignant melanoma 253
Arturo Chiti Ehab M Kamel and Angelika Bischof
Delaloye
7. Colorectal cancer 99
Ken Herrmann and 17. Cancer of unknown primary 265
Bernd Joachim Krause Emilio Bombardieri, Stefano Fanti,
Arturo Chiti, Cristina Nanni, Angela
8. Prostate cancer 109 Coliva, Enrico Pelosi, and Gianni Bisi
Werner Langsteger, Mohsen Beheshti,
Reza Vali, Avi Beri, Michael Nader,
and Guenter Janetschek
9781841846149-FM 8/24/07 4:28 PM Page vi

vi Contents

III: Therapeutic applications IV: Methods and technology

18. Therapy of differentiated thyroid 27. New instrumentation 397
cancer 275 Jörg Eckardt, Klaus Schäfers,
Martin Biermann, and and Lars Stegger
Otmar Schober
28. New radiopharmaceuticals 413
19. Therapy of medullary thyroid cancer 293 for cancer diagnosis
Aurore Oudoux, Catherine Ansquer, Jane K Sosabowski, Matthias Glaser,
Jacques Barbet, David M Goldenberg, and Stephen J Mather
Jean-François Chatal, and Françoise
29. Radiopharmaceuticals for therapy 433
Kraeber-Bodéré
Margaret S Cooper and
20. Therapy of lymphoma 299 John Buscombe
Tim Illidge and Yong Du
30. New concepts in dosimetry 443
21. Therapy of neuroendocrine tumors 315 and radiation protection
Irene Virgolini, Ulrich Andergassen, Michael Lassmann, Manuel Bardies,
Tatjana Traub-Weidinger, Dirk Heute, Carlo Chiesa, Wolfgang Eschner,
Elisabeth von Guggenberg, Dorota Glenn Flux, Myriam Monsieurs, Sauli
Kendler, Margarida Rodrigues, Savolainen, and Sven-Erik Strand
Georg Dobrozemsky, Boris Warwitz,
31. Advances in nuclear medicine 467
Daniel Putzer, Roy Moncayo, Clemens
imaging in oncology
Decristoforo, and Michael Gabriel
Carlo Chiesa, Roberta Matheoud,
22. Therapy of bone metastases 341 Marco Brambilla, and Gian Luca Poli
Holger Palmedo
32. Nuclear medicine-guided 499
23. Therapy of liver tumors 351 radiotherapy
John Buscombe Diana Salvo, Annibale Versari,
Cinzia Iotti, and Marta Paiusco
24. Therapy of colorectal cancer 359
John Buscombe Index 521
25. Therapy of brain tumors 367
Chiara Maria Grana, Mirco
Bartolomei, Mahila Ferrari, and
Giovanni Paganelli
26. Therapy of ovarian cancer 383
Angela Coliva, Ettore Seregni,
Mariangela Figini, Alberto Zacchetti,
Rosanna Fontanelli, Francesco
Raspagliesi, Silvana Canevari, and
Emilio Bombardieri
9781841846149-FM 8/24/07 4:28 PM Page vii

Contributors

Ulrich Andergassen Emilio Bombardieri

Medical University Innsbruck, Innsbruck, Austria Nuclear Medicine Division, National Cancer Institute,
Milan, Italy
Catherine Ansquer
Service de Gynecologie-Obstetrique, Hopital de la Marco Brambilla
Mere-et-de-l’Enfant, France. Ospedale Maggiore della Carità, Novara, Italy
Alessandra Alessi Christoph Bremer
PET Unit, Division of Nuclear Medicine, National Cancer Department of Clinical Radiology, University of Münster,
Institute, Milan, Italy Münster, Münster, Germany
Jacques Barbet John Buscombe
Institut National de la Sante et de la Recherche Medicale, Department of Nuclear Medicine, Royal Free Hospital,
Institut de Biologie, Nantes France London, UK
Manuel Bardies Silvana Canevari
INSERM UMR, Nantes, France Experimental Oncology, National Cancer Institute,
Milan, Italy
Mirco Bartolomei
European Institute of Oncology, Milan, Italy Jean-François Chatal
Centre de Reserche sur le Cancer, Universite de Nantes,
Richard P Baum
Nantes, France
Department of Nuclear Medicine /Centre for PET,
Zentralklinik Bad Berka, Bad Berka, Germany Carlo Chiesa
Division of Nuclear Medicine, National Cancer Institute,
Mohsen Beheshti
Milan, Italy
PET/CT Center Linz, St Vincent’s Hospital, Linz, Austria
Arturo Chiti
Simona Ben-Haim
Istituto Clinico Humanitas, Rozzano – Milano, Italy
Institute of Nuclear Medicine, University College London,
London, UK Angela Coliva
Division of Nuclear Medicine, National Cancer Institute,
Avi Beri
Milan, Italy
Endourology and Laparoscopic Surgery,
Elisabethinen Hospital, Linz, Austria Gary Cook
Royal Marsden Hospital, London, UK
Guido Biasco
Institute Haematology and Oncology, Universita di Bologna, Margaret S Cooper
Policlinico S. Orsola-Maplpighi, Bologna, Italy St Bartholomew’s Hospital, London, UK
Martin Biermann Flavio Crippa
University of Bergen, Haukeland University Hospital, PET Unit, Division of Nuclear Medicine, National Cancer
Bergen, Norway Institute, Milan, Italy
Angelika Bischof Delaloye Clemens Decristoforo
Nuclear Medicine, Lausanne Medical University Innsbruck, Innsbruck, Austria
University Hospital, Lausanne, Switzerland
Angelo Del Sole
Gianni Bisi Institute of Radiological Sciences, University of Milan, Italy
Nuclear Medicine Division, University of Turin,
Turin, Italy
9781841846149-FM 8/24/07 4:28 PM Page viii

viii List of contributors

Georg Dobrozemsky David M Goldenberg

Medical University Innsbruck, Innsbruck, Austria Center for Molecular Medicine and Immunology, Garden
State Cancer Center Belleville, New Jersey, USA
Yong Du
Institute of Nuclear Medicine, University College Hospital, Chiara Maria Grana
London, UK European Institute of Oncology, Milan, Italy
Jörg Eckardt Luca Guerra
University Hospital, Westfalische Wilhelms-University San Gerardo Hospital, Monza, Italy
Münster, Münster, Germany
Uwe Haberkorn
Wolfgang Eschner University of Heidelberg Clinical Cooperation Unit Nuclear
Klinik und Poliklinik für Nuklearmedizin der Universität zu Medicine German Cancer Research Center, Heidelberg,
Köln, Köln, Germany Germany
Andrea Falini Ken Herrmann
Department of Neuroradiology, Scientific Institute and Clinic and Polyclinic of Nuclear Medicine, Klinikum Rechts
University Ospedale San Raffaele, Milan, Italy der Isar, Technical University of Munich, Munich, Germany
Stefano Fanti Dirk Heute
University of Bologna, Policlinico S Orsola-Malpighi, Medical University Innsbruck, Innsbruck, Austria
Bologna, Italy
Tim Illidge
Ferruccio Fazio Cancer Studies Division, Christie Hospital, University of
University of Milano-Bicocca, Milan, and Scientific Institute Manchester, UK
San Raffaele, Milan, and IBFM-CNR, Milan, Italy
Cinzia Iotti
Mahila Ferrari Hospital ‘Santa Mario Nuova’, Reggio Emillia, Italy
European Institute of Oncology, Milan, Italy
Ora Israel
Mariangela Figini Rambem Medical Center, Haifa, Israel, & The Bruce
National Cancer Institute, Experimental Oncology, Rappaport Faculty of Medicine, Technion, Israel Institute of
Milan, Italy Technology, Haifa,
Glenn Flux Andreas Jacobs
Royal Marsden Hospital, London, UK Laboratory for Gene Therapy and Molecular Imaging,
Max Planck Institute for Neurological Research, University
Ignac Fogelman
of Cologne, Cologne, Germany
Guys and St Thomas’ Hospital NHS Trust, London, UK
Guenter Janetschek
Rosanna Fontanelli
Elisabethinen Hospital, Linz, Austria
Division of Gynecological Oncology, National Cancer
Institute, Milan, Italy Ehab M Kamel
Department of Nuclear Medicine, Lausanne University
Christiane Franzius
Hospital, Lausanne, Switzerland
University Hospital, Westfalische Wilhelms-University
Münster, Münster, Germany Dorota Kendler
Medical University Innsbruck, Innsbruck, Austria
Michael Gabriel
Medical University Innsbruck, Innsbruck, Austria Susanne Kohlfürst
PET/CT Center Klagenfurt, Klagenfurt, Austria
Alberto Gerali
PET Unit, Division of Nuclear Medicine, Françoise Kraeber-Bodéré
National Cancer Institute, Milan, Italy Réné Gauducheau Cancer Center, Nantes, France
Matthias Glaser Bernd Joachim Krause
Hammersmith Imanet Ltd, Hammersmith Hospital, Clinic and Polyclinic of Nuclear Medicine, Klinikum Rechts
London, UK der Isar, Technical University of Munich, Munich, Germany
Gopinath Gnanasegaran Werner Langsteger
Guys and St Thomas’ Hospital NHS Trust, London, UK PET/CT Center Linz, St Vincent’ s Hospital,
Linz, Austria
9781841846149-FM 8/24/07 4:28 PM Page ix

List of contributors ix

Michael Lassmann Maria Picchio

Clinic for Nuclear Medicine, University Clinic, Scientific Institute San Raffaele, Milan, Italy
University of Würzburg, Würzburg, Germany
Gian Luca Poli
Peter Lind Ospedali Riuniti, Bergamo, Italy
PET/CT Center Klagenfurt, Klagenfurt, Austria
Vikas Prasad
Giovanni Lucignani Zentralklinik Bad Berka, Bad Berka, Germany
Unit of Nuclear Medicine, Hospital San Paolo,
Daniel Putzer
Institute of Radiological Sciences, University of Milan,
Medical University Innsbruck, Innsbruck, Austria
Milan, Italy
Francesco Raspagliesi
Marco Maccauro
Division of Gynecological Oncology, National Cancer
Division of Nuclear Medicine, National Cancer Institute,
Institute, Milan, Italy
Milan, Italy
Margarida Rodrigues
Roberta Matheoud
Medical University Innsbruck, Innsbruck, Austria
Ospedale Maggiore della Carita Novara, Italy
Diana Salvo
Stephen J Mather
Hospital ‘Santa Maria Nuova’, Reggio Emilia, Italy
Centre for Cancer Imaging, Barts and the London, Queen
Mary’s School of Medicine and Dentistry, University of Sauli Savolainen
London, UK HUS, Medical Imaging Centre and Department of Physical
Sciences, University of Helsinki, Helsinki, Finland
Cristina Messa
University of Milano-Bicocca, Milan, Italy, San Gerardo Klaus Schäfers
Hospital, Monza, Italy and IBFM-CNR, Milan, Italy University Hospital, Westfalische Wilhelms-University
Münster, Münster, Germany
Roy Moncayo
Medical University Innsbruck, Innsbruck, Austria Michael Schäfers
University Hospital, Westfalische Wilhelms-University
Myriam Monsieurs
Münster, Münster, Germany
Ghent University, Ghent, Belgium
Orazio Schillaci
Michael Nader
Department of Biopathology and Diagnostic Imaging,
ARGOS Zyklotron Ltd, Member of the IASON Group, Linz,
University ‘Tor Vergata’, Rome, Italy
Austria
Otmar Schober
Cristina Nanni
Department of Nuclear Medicine, University of Münster,
University of Bologna Policlinico S Orsola-Malpighi,
Münster, Germany
Bologna, Italy
Ettore Seregni
Eva Orunesu
Division of Nuclear Medicine, National Cancer Institute,
Institute of Radiological Sciences, University of Milan,
Milan, Italy
Milan, Italy
Sandro Sironi
Aurore Oudoux
University of Milano-Bicocca, Milan, Italy and San Gerardo
Réné Gauducheau Cancer Center, Nantes, France
Hospital, Monza, Italy
Giovanni Paganelli
Jane K Sosabowski
European Institute of Oncology, Milan, Italy
Centre for Cancer Imaging, Barts and the London, Queen
Marta Paiusco Mary’s School of Medicine and Dentistry, University of
Hospital ‘Santa Maria Nuova’, Reggio Emilia, Italy London, UK
Enrico Pelosi Lars Stegger
Nuclear Medicine Division, University of Turin, Turin, Italy University Hospital, Westfalische Wilhelms-University
Munster, Munster, Germany
Holger Palmedo
Nuclear Medicine Department, University Hospital, Sven-Erik Strand
Bonn, Germany Medical Radiation Physics, Lund University, Lund, Sweden
9781841846149-FM 8/24/07 4:28 PM Page x

x List of contributors

Sara Taddayon Elisabeth von Guggenberg

Istituto Clinico Humanitas, Rozzano, Milano, Italy Medical University Innsbruck, Innsbruck,
Austria
Tatjana Traub-Weidinger
Medical University Innsbruck, Innsbruck, Austria Boris Warwitz
Reza Vali Medical University Innsbruck, Innsbruck,
PET/CT Center Linz, St Vincent’ s Hospital, Linz, Austria
Austria Wolfgang A Weber
Annibale Versari Zentralklinik Bad Berka, Bad Berka, Germany
Hospital ‘Santa Maria Nuova’, Reggio Emilia, Italy Alberto Zacchetti
Sanjay Vijayanathan Experimental Oncology, National Cancer Institute,
Musculoskeletal Radiology, Guys and St Thomas’ Hospital Milan, Italy
NHS Trust, London, UK
Irene Virgolini
Medical University Innsbruck, Innsbruck,
Austria
9781841846149-FM 8/24/07 4:28 PM Page xi

Preface
There has been enormous progress in nuclear medicine in Radiopharmaceuticals developed specifically to target and
recent years, and the impact of this progress has been par- visualize malignant tumors can also be used, at high doses,
ticularly noticeable in oncology. Research into molecular for therapeutic purposes. Nuclear medicine therapeutics
imaging has led to the development of radiopharmaceuti- thus takes advantage of selective radiopharmaceuticals that
cals that can explore the cellular metabolism, and visualize have demonstrated marked anticancer efficacy in many
at molecular and subcellular levels the pathological types of tumors. For example, in recent years, these tech-
processes specific to cancer. Equipment development has niques have been used and shown greatest efficacy in the
produced high-technology instruments such as those used treatment of lymphomas and neuroendocrine tumors.
in positron emission tomography (PET), able to produce The diagnostic and therapeutic achievements in nuclear
high-quality images that have become indispensable in the medicine are the result of the interdisciplinary research
diagnostic work-up of cancer patients, because they often efforts of cell biologists, chemists, pharmacologists, physi-
reveal alterations and lesions not demonstrated by conven- cists, computer scientists, engineers, nuclear medicine
tional morphologically oriented techniques such as X-ray physicians, and oncologists. The clinical implications of
imaging, ultrasound, computed tomography (CT), or mag- these achievements have made nuclear medicine indispen-
netic resonance imaging (MRI). Research in the area of sable in the management of cancer.
image fusion techniques has led to the design of software
programs able to merge in a single image the molecular, This textbook on modern nuclear medicine applications in
functional, and metabolic information of nuclear medicine the diagnosis and treatment of cancer describes the state of
with the morphological information provided by radiol- the art and the current position of nuclear medicine in the
ogy. Hybrid instruments (PET/CT, SPECT/CT) are now light of these recent developments. It is intended as a valu-
available which allow the fusion of images of a patient in able update also for non-nuclear medicine specialists work-
just one diagnostic session. Intensive research is ongoing to ing in oncology. Nuclear medicine as part of molecular
obtain detectors, hardware, and software able to perform imaging and therapy has changed radically in the past
whole-body scans faster and with increasing spatial resolu- decade. The growing importance and clinical impact of
tion, so that it may become possible to detect lesions on a these changes for the near future has impelled the authors
submillimeter level. Nuclear medicine has made the step to record them in this book.
from bench to bedside, to a significant extent. Emilio Bombardieri
All of these achievements have had a great impact on not only
John Buscombe
the diagnosis but also the treatment of cancer. Improved, Giovanni Lucignani
individually tailored therapy is now on the horizon. Otmar Schober
9781841846149-FM 8/24/07 4:28 PM Page xii

Acknowledgments
The Editors are grateful to Ms Anna Luisa De Simone Sorrentino for her secretarial help in organizing this book. We also
appreciate the initial approach from our publisher at Informa Healthcare, Alan Burgess and members of his team: Lindsay
Campbell and Kathryn Dunn.
9781841846149-Ch01 8/18/07 2:18 PM Page 1

1
What is cancer?
Uwe Haberkorn

At first sight cancer is a disease induced by the failure of whole chromosomes may disturb the balances that regulate
control mechanisms. The cancer cell does not respond to normal growth control. On the other hand, many normal
control signals because of damage to its DNA, the presence cells, both in vitro and in vivo, may become cancerous after
of oncogene products, or because the homeostatic control the right combination of oncogenes is introduced.4
mechanisms themselves are disturbed. Biologically this However, only a fraction of these cells will give rise to
corresponds to uncontrolled proliferation occurring at the cancer, implying that other yet unknown factors might also
wrong place and time driven by oncogenic signals, be involved in tumor initiation. Therefore, both mutations
impaired differentiation, and invasion of other tissues lead- and chromosomal derangements are important in the
ing to metastases. initial stages of tumor development, and both mechanisms
In a recent review, Hanahan and Weinberg mentioned might be involved in establishment of the cancer stem cell.
six essential alterations in cell physiology which are seen as Many of the oncogenes act by mimicking normal growth
the hallmarks of cancer: self-sufficiency in growth signals, signaling. This can be accomplished by alteration of
insensitivity to growth-inhibitory signals, evasion of extracellular growth signals, or alterations of transcellular
programmed cell death (apoptosis), limitless replicative transducers of those signals or of intracellular circuits that
potential, sustained angiogenesis, and tissue invasion and translate those signals into cellular response. Many cancer
metastasis. These alterations are interpreted as results of cells acquire the ability to synthesize growth factors to
genetic changes in the cancer cell. However, mutations in which they are responsive, creating a positive feedback
the tumor genome may not be the only cause.1 signaling loop. Examples of this autocrine stimulation are
platelet derived growth factor (PDGF) and transforming
tumor growth factor β (TGFβ). Furthermore, there is over-
expression of growth factor receptors, which often carry
tyrosine kinase activities in their cytoplasmic domains.
Genetic and epigenetic This results in cells becoming hyperresponsive even to
low growth factor levels that normally would not trigger
background proliferation. As an example, members of the epidermal
Cancer has been viewed as a multistep process of genetic growth factor receptor family such as EGFR/erbB are
alterations that result in the transformation of benign cells upregulated in non-small-cell lung cancer (NSCLC) and
into malignant ones. These genetic abnormalities include head and neck, renal cell, brain, and breast tumors,
mutations in tumor-suppressor genes and oncogenes, and and HER2/neu receptors are overexpressed in stomach and
chromosomal abnormalities such as chromosomal gain, mammary tumors. Ligand independent signaling can also
loss, and/or rearrangement (Table 1.1).1,2 Such events are be achieved through structural alteration of receptors:
thought to be followed by a clonal selection of variant cells truncated versions of the EGFR lacking parts of the
that show increasingly aggressive behavior.3 cytoplasmic domain act constitutively. Finally, there are
Although it is still commonly thought that aneuploidy alterations of the downstream cytoplasmic signaling
occurs as a late-stage effect rather than as a cause of cancer pathways, which receive and process the signals emitted by
development, this might not always be true, as carcinogens ligand-activated growth factor receptors and integrins.
such as asbestos and arsenic initially do not cause gene In that respect, the Ras–Raf–MAPK (mitogen-activated
mutations, but rather lead to aneuploid lesions. Further- protein kinase) cascade plays a central role. In about 25% of
more, normal cells exposed to chemical carcinogens can human tumors, Ras proteins are present in structurally
become aneuploid long before they show signs of being altered forms. This has been exemplified in human colon
cancerous. Therefore, it is possible that gains and losses of carcinomas where about 50% of the tumors bear mutant
9781841846149-Ch01 8/18/07 2:18 PM Page 2

2 Advances in Nuclear Oncology

Table 1.1 Regulatory proteins for tumorigenesis, apoptosis, and drug resistance

Protein Role in tumorigenesis, apoptosis, and drug resistance

Suppressor proteins
p53 Mutated or altered expression in many cancers. Initiates the intrinsic apoptotic pathway. p53-negative
cells are resistant to drug-induced apoptosis
ATM Mutated in ataxia–telangiectasia syndrome. Senses DNA double strand breaks and stabilizes p53.
Increased risk for hematological malignancies and breast cancer
CHK2 Mutated in Li–Fraumeni syndrome. Senses DNA double strand breaks and phosphorylates and stabilizes p53
Rb Mutated in some cancers, functionally disrupted in many cancers. Inhibits E2F-mediated transcription.
Loss of Rb function induces p53-dependent and -independent apoptosis
Bax Mutated or decreased expression in some tumors. Mediates mitochondrial membrane damage
Bak Mutated or decreased expression in some tumors. Mediates mitochondrial membrane damage
PTEN Mutated or altered expression in cancers. Regulates Akt activation and subsequent phosphorylation
of Bad. Loss of PTEN results in resistance to many apoptotic stimuli
APAF1 Mutated and transcriptionally silenced in melanoma and leukemia cell lines. Necessary for activation
of caspase-9 following cytochrome c release. Chemoresistance
CD95/Fas Mutated and downregulated in lymphoid and solid tumors. Initiates the extrinsic apoptotic pathway.
Resistance to drug-induced cell death
TRAIL-R Mutated in metastatic breast cancers. Initiates the extrinsic apoptotic pathway. Mutations lead to
suppression of death receptor-mediated apoptosis
Caspase-8 Silenced in neuroblastomas. Activates both extrinsic and intrinsic apoptotic pathways. Resistance
to drug-induced apoptosis
Oncogenes
Bcl-2 Frequently overexpressed in many tumors. Antagonizes Bax and/or Bak and inhibits mitochondrial
membrane disruption. Inhibits drug-induced apoptosis
MDM2 Overexpressed in some tumors. Negative regulator of p53. Inhibits drug-induced p53 activation
IAPs Frequently overexpressed in cancer. Downregulation of XIAP induces apoptosis in chemoresistant tumors
NFκB Deregulated activity in many cancers. Transcriptionally activates expression of antiapoptotic members of
the Bcl-2 and IAP families. Can inhibit both the extrinsic and intrinsic death pathways and induce
drug resistance
Myc Deregulated expression in many cancers. Induces proliferation in the presence of survival factors, such
as Bcl-2, and apoptosis in the absence of survival factors. Can sensitize cells to drug-induced apoptosis
Akt Frequently amplified in solid tumors. Phosphorylates Bad. Hyperactivation induces resistance to a range
of apoptotic stimuli
PI3K Overexpressed or deregulated in some cancers. Responsible for activation of Akt and downstream
phosphorylation of Bad. Inhibition of PI3K enhances chemotherapeutic drug-induced apoptosis
Ras Mutated or deregulated in many cancers. Activates PI3K and downstream pathways.
Induces proliferation and inhibits c-myc and drug-induced apoptosis
ATM, ataxia telangiectasia mutated; Chk2, checkpoint kinase 2; PTEN, phosphatase and tensin homolog; Apaf-1, apopotic protease activating
factor 1; TRAIL-R, TNF-related apoptosis-inducing ligand receptor; MDM2, transformed 3T3 cell double minute 2; IAPs, inhibitor of apoptosis
proteins; NFκB, nuclear factor κB; Akt, protein kinase B; PI3K, phosphatidylinositol 3-kinase.
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What is cancer? 3

ras oncogenes. It is suggested that the remaining colonic Epigenetic changes are realized by three different
tumors carry defects in other components of the growth mechanisms: DNA methylation, RNA-associated silencing,
signaling pathways, with similar functional results to those and histone modification, which are known to initiate
obtained after ras oncogene activation.1 and sustain epigenetic silencing, and to interact with each
Besides response to growth signals, resistance to antigrowth other.11–13
signals is an equally important feature of cancer. These Methylation of the C5 position of cytosine residues in
antiproliferative signals are coordinated mainly by the DNA is maintained by a number of DNA methyltransferases
retinoblastoma protein (pRb) and its two relatives p107 and has multiple roles for the silencing of transposable
and p130. Hypophosphorylated pRB blocks proliferation by elements, for defense against viral sequences, and for the
sequestering and altering the function of E2F transcription transcriptional repression of genes. The resulting metabolite,
factors that control the expression of several genes which 5-methylcytosine, is highly mutagenic, causing C:G to T:A
are essential for the progression from G1 into S phase.5 transitions, and leads to a suppression of the methylated
Disruption of the pRb pathway liberates E2F and allows site in the human genome. The predominant sites, CpG
cell proliferation, rendering cells insensitive to antigrowth islands, are regions of more than 500 base pairs in size and
factors. In this respect, transforming growth factor β (TGFβ) with a GC content greater than 55%,14 and have been
is an important regulator of pRb modification by preventing conserved during evolution because they are normally
the inactivating phosphorylation of the protein. Response kept free of methylation. They are located within the
to TGFβ can be lost after downregulation of TGFβ recep- promoter regions of about 40% of mammalian genes and
tors, or mutant, dysfunctional receptors.6 In addition, can be transcriptionally silenced by methylation. Extensive
changes in the signaling pathway may occur: the function de novo methylation of CpG islands is a common feature of
of proteins such as Smad4, which transduces signals many cancers.15
from ligand-activated TGFβ receptors to downstream Histone modifications such as acetylation, phosphorylation,
targets, or p15INK4B may be changed by mutation of the and methylation of conserved lysine residues on the amino-
corresponding genes.7,8 terminal tail domains have also been defined as epigenetic
Differentiation is is also a condition which results in the modifiers. Acetylation of histones causes transcriptionally
inhibition of proliferation and is disturbed in a variety of active DNA regions, whereas hypoacetylated histones are
tumor cells. One of the target genes in this respect is the c-myc associated with transcriptionally inactive DNA regions.
oncogene, which encodes a transcription factor. During Since there is a considerable variation of all possible
normal development, the growth-stimulating action of Myc, histone modifications, and also interactions between
in association with another factor, Max, can be inhibited by histone deacetylases, histone methyltransferases, and
the formation of complexes of Max with a group of Mad methylcytosine-binding proteins occur, this is seen as a his-
transcription factors. These Mad–Max complexes result in tone code which is used by a variety of cellular factors.16,17
differentiation-inducing signals.9 Overexpression of the The role of epigenetic changes in cancer has been shown
c-Myc oncoprotein occurs in many tumors and shifts the for the MLH1 gene, where methylation and silencing of
balance to Myc–Max complexes, which impairs differentia- the gene may lead to a variety of cancers.18,19 Chromatin-
tion and thereby promotes tumor growth. A further modifying enzymes have also been associated with human
example is inactivation of the APC/β-catenin pathway leukemias, with histone acetyltransferases and histone
in colon carcinoma, which results in a block of the methyltransferases engaged in the modification of fusion
differentiation of enterocytes in the colonic crypts.2 protein activity, such as the oncogenic PML–RARα
The characteristics mentioned above are subsumed (promyelocytic leukemia–retinoic acid receptor α) in acute
under the term ‘somatic mutation theory of carcinogene- promyelocytic leukemia, which recruits a histone deacety-
sis’, which has been the dominant force driving cancer lase to repress genes essential for the differentiation of
research during the 20th century. In brief, it proposes that hematopoietic cells, or AML1-ETO (AML refers to acute
successive DNA mutations in a single cell cause cancer. This myeloid leukaemia) fusions, which recruit a histone
theory places carcinogenesis at the cellular and subcellular deacetylase 1 complex to inhibit myeloid development.20,21
hierarchical levels of biological complexity. However, Furthermore, loss or mutations of adenosine triphosphate
increasing evidence has been obtained that epigenetic (ATP)-dependent chromatin remodeling complexes
changes and also changes in surrounding or tumor infil- such as SWI–SNF, BRM, and BRG1 have been found to be
trating non-tumor cells such as fibroblasts and endothelial associated with pediatric cancer as well as a variety of
cells are important. These may interact with tumor cells by cancer cell lines and tumor tissues.22
secretion of a variety of signaling factors such as diffusible A simple way to induce a carcinogenic phenotype is the
growth factors, extracellular matrix components, or cell- transcriptional repression of tumor suppressor genes,
to-cell adhesion/interaction molecules. Evidence of a pro- which may represent an alternative mechanism to genetic
motion of cancer cells by inflammatory cells infiltrating the mutation. In addition, cancer-cell genomes simultaneously
tumor site has also been found.10 show global hypomethylation and gene promoter-specific
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4 Advances in Nuclear Oncology

hypermethylation. This might contribute to genomic insta- Epigenetic silencing may occur during the early stages of
bility, structural changes in chromosomes, and increases in tumor progression, possibly during the abnormal expansion
gene expression.15,23–25 These alterations may occur at early of stem and progenitor cells. This silencing predisposes the
stages of carcinogenesis and may determine the subsequent stem cells to abnormal clonal exposition. Changes that are
genetic changes and progression of these mutated clones. known to contribute to tumor formation such as chronic
Many genes are epigenetically silenced in cancer cells, inflammation, which leads to the production of reactive
and many epigenetically silenced genes have not been oxygen species, induce cell renewal dedicated to repair of
found to contain any genetic mutations at all, even though tissue damage. This is associated with epigenetic events
they are transcriptionally repressed in many different which lead to heritable transcriptional repression and
cancer-cell types. These facts underscore the potential value activation of stem-cell and progenitor-cell expansion at the
of screening for all epigenetic modifications, as well as expense of normal cell differentiation and maturation. The
genetic changes, that are associated with human tumor subsequent progression to malignancy would then depend
types. Examples of a combination for genetic and not only on gene mutations but also on the collection of
epigenetic changes were found in the colon cancer cell line epigenetic alterations. These epigenetic changes might
HCT116, which contains several mutations, including the occur continuously not only in epithelial cells but also in
DNA mismatch-repair protein, MLH1, and p16, which surrounding stromal cells.40
contribute to the mismatch-repair phenotype and to The reductionist approach, which sees cancer solely as a
disruption of the cyclin D-RB1 (retinoblastoma 1) cell- disease reducible to mutations, has been challenged not
cycle-control pathway, the transforming growth factor-β2 only by the finding of epigenetic changes but also by the
receptor, which causes a loss of control of a cell differentia- number of mutations occurring in cancers. These range from
tion pathway, and an activating mutation in the gene that three to anywhere from 11 000 to 100 000 mutations.41–43
encodes β-catenin, resulting in constitutive Wnt (wingless In principle these data, obtained from high-throughput
type) signaling and cell proliferation.26–30 In addition to technologies such as gene arrays, may be used to introduce
these mutations, there are at least 14 epigenetically silenced new classifications.44 However, these new technologies face
genes in these cells, all of which can be reactivated by either problems associated with bias, reproducibility, overfitting,
treating the cells with DNA-demethylating agents or and data interpretation.45
disrupting the genes that encode DNA methyltransferases, This has led to the proposal of an alternative theory,
which catalyze DNA methylation.31–35 Reactivating expres- the tissue organization field theory of carcinogenesis and
sion of these growth-control genes results in phenotypic neoplasia. Its assumptions are that proliferation is the
changes that range from reducing proliferation to inducing default state of all cells and that carcinogenesis and neoplasia
senescence or apoptosis.26,27,33 Epigenetic alterations of are defects of tissue architecture. Carcinogens would act
these genes seem to complement mutations in determining initially by disrupting the normal interactions between
the phenotype of these cells. Examples of a collaboration parenchymal cells and the stroma of the organ. In this
between epigenetic and genetic abnormalities are MLH1 model the stroma appears as the primary target of carcino-
and CDKN2A (the gene that encodes p16) in HCT116 cells: gens, stating that carcinogenesis and neoplasia occur
while one allele of each of these genes is mutated in these exclusively through supracellular phenomena. This implies
cells, the wild-type allele becomes silenced by hypermethy- that neoplastic cells may be reprogrammed to behave as
lation. Therefore, genetic and epigenetic changes can col- non-tumor cells when placed in the context of normal
laborate to prevent expression of a functional gene product tissues.46 Evidence is derived from early studies with terato-
in cancer cells. carcinomas, where the stem cells generated not only more
Another epigenetic–genetic collaboration in HCT116 stem cells but also more differentiated cells that gave rise to
cells is found in the Wnt pathway. Four members of the non-tumorigenic tissue. The teratocarcinoma cells were
secreted frizzled-related gene family (SFRP1, SFRP2, generated from tumors resulting from the implantation of
SFRP4, and SFRP5) that encode Wnt antagonists are epige- normal embryos into ectopic locations. Teratocarcinoma
netically silenced in these cells. This contributes to the cells injected into normal blastocysts were shown to generate
abnormal activation of Wnt signaling, even in cells that normal tissues in viable mosaic individuals resulting from
already carry activating mutations in β-catenin.33 In addi- this manipulation.47,48 In subsequent generations, normal
tion, silencing of the genes that encode the transcription offspring resulted from the genome of a cell that was once a
factors GATA4 and GATA5, as well as their downstream cancer cell. If cancer indeed results from the accumulation
activation targets trefoil factor 1 (TFF1), TFF2, TFF3, and of DNA mutations in a previously normal cell, it becomes
inhibin-α,32 could impair maturation of endoderm-derived problematic to explain these data. More recently, ectopic
epithelial cells.36–39 Finally, TIMP3 (tissue inhibitor of met- expression of stromelysin-1 by mammary gland epithelial
alloproteinase 3) is silenced in HCT116 cells, and loss of cells in transgenic animals resulted in mammary gland
function of its product might increase the invasive ability of carcinoma. In this case, expression of this enzyme induces
these cells.35 stromal changes that in turn would lead to carcinoma.
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What is cancer? 5

Treatment with specific protease inhibitors blocked

carcinogenesis in this model.49 Also, irradiation of the
Table 1.2 Morphological and biochemical
stroma of epithelium-free mammary glands results in
differences between apoptosis and necrosis
carcinogenesis in non-irradiated mammary epithelial cells
Features Apoptosis Necrosis
inoculated into the irradiated stroma.50 In all these experi-
ments, the parenchyma–stroma interaction seems to be an Tissue Single cells Multiple cells
important factor for cancer progression.51,52 From the distribution
evidence mentioned above it follows that cancer is more Tissue reaction Phagocytosis Cellular exudate
than a disease of specific genes. Rather, instability of the
Cell morphology Shrinkage Swelling
genome as a whole must be seen as a hallmark of malignant
tumors.53–56 Furthermore, the concept that epigenetic Organelles Intact Damaged
abnormalities could be as important as genetic ones in Chromatin Marginated, Fragmented
determining the course of tumor development, and also be condensed
involved in tumor-specific signaling pathway abnormalities,
Membrane Intact Damaged
is relevant to the future design of both preventive and
therapeutic approaches to cancer.13 Biochemistry Activated Defective ion pump
endonucleases
DNA cleavage Activated lysosomal
Activated enzymes
caspases
Cell death: Apopotosis and more
To control abnormal proliferation a cell can either enter a
quiescence-like growth arrest phase, or undergo apoptosis
or senescence. These antiproliferative programs are
induced by tumor suppressors such as p53 and pRb in
response to potential oncogenic signals.
The most common and well-defined form of programmed
cell death is apoptosis, which is a physiological cell-suicide
Fas ligand
program that is essential for embryonic development,
function of the immune system, and the maintenance of
tissue homeostasis in multicellular organisms. Apoptosis in
mammalian cells is mediated by a family of cysteine
proteases known as the caspases. To keep the apoptotic
program under control, caspases are initially expressed Fas
in cells as inactive procaspase precursors. When initiator
caspases such as caspase-8 and caspase-9 are activated by FADD ARC
DISC
oligomerization, they cleave the precursor forms of effector
caspases, such as caspase-3, caspase-6, and caspase-7.57,58 Caspase-8 FLIP
Activated effector caspases cleave a specific set of cellular (FLICE)
substrates, resulting in specific biochemical and morpho-
logical changes (Table 1.2) that are associated with the
Caspase-3
apoptotic phenotype. Dysregulation of apoptosis has been
implicated in numerous pathological conditions, including
neurodegenerative diseases, autoimmunity, and cancer.59
There are two pathways by which caspase activation is †
triggered, the extrinsic and intrinsic apoptotic pathways.
The extrinsic pathway is induced by activation of death Figure 1.1
receptors on the cell surface. Binding of ligands such as Simplified scheme of the death receptor-mediated or extrinsic
apoptosis pathway. Binding of the Fas ligand is followed by
FasL and tumor necrosis factor (TNF) to Fas and the TNF
formation of the death inducing signaling complex (DISC): the
receptor (TNFR), respectively, leads to formation of the adaptor molecule FADD binds to the death domain of Fas and
death induced signaling complex (DISC). DISC recruits recruits procaspase-8 via its death effector domain.
caspase-8 and promotes the cascade of procaspase activa- Autocatalytic activation of procaspase-8 leads to activation of
tion (Figure 1.1).60 The intrinsic pathway is triggered by the downstream effector caspase-3. The cascade may be
various extracellular and intracellular stresses, such as inhibited by FLICE inhibitory protein (FLIP) or by the apoptosis
growth-factor withdrawal, hypoxia, DNA damage, and repressor with a CARD domain (ARC).
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6 Advances in Nuclear Oncology

oncogene induction. Signals that are transduced in response different modes of inactivating proapoptotic signaling
to these stresses converge mainly on the mitochondria. pathways underlie tumorigenesis.
A series of biochemical events is induced that results in the Several non-apoptotic cell death mechanisms have been
permeabilization of the outer mitochondrial membrane, identified, including necrosis, autophagy, mitotic catastrophe,
the release of cytochrome c and other proapoptotic mole- and senescence. In contrast to apoptotic cell death, necrosis
cules, the formation of the apoptosome, a large protein is an unregulated process with membrane distortion,
complex that contains cytochrome c, apoptotic protease organelle degradation, and cellular swelling, resulting in cell
activating factor 1 (APAF1), and caspase-9, and caspase destruction and the release of intracellular components.
activation. Once cytochrome c is released, the downstream Necrosis is usually a consequence of a pathophysiological
cascade of caspase activation is irreversible. Cell death is condition, such as infection, inflammation, or ischemia,
also modified by other proteins such as endonuclease G21 which leads to the failure of normal physiological pathways
and apoptosis-inducing factor (AIF), which may induce that are essential for maintaining cellular homeostasis, such
cell death independently of caspase activation. as regulation of ion transport, energy production, and pH
In order to survive, tumor cells need to avoid apoptosis balance.
that can be induced by unregulated oncogene expression, Primary cells in culture initially proliferate rapidly, with a
and a limited supply of growth factors, oxygen, or nutrients. significant shortening of the telomeres of their chromo-
Protection from apoptosis may be achieved by modification somes. This may lead to a form of permanent cell-cycle
of the activities of antiapoptotic genes such as Bcl-2 or arrest that has been described as replicative senescence. A
suppressor genes such as p53. In human B-cell follicular senescent cell is characterized by flattened cytoplasm,
lymphomas, a chromosomal translocation linking the increased granularity, changes in metabolism, and the
Bcl-2 gene to an immunoglobulin locus was identified in induction of senescence-associated β-galactosidase activity.
the transformed cells, resulting in constitutively active Furthermore, alterations in chromatin structure and gene-
Bcl-2 and survival of the lymphoma cells.61 expression patterns are seen. The phenomenon is inducible
The tumor suppressor p53 is an important regulator of by various cellular stresses, DNA damage, and oncogene
apoptosis and is the most commonly mutated gene in activity.69 The senescence program then induces the activation
cancer.62 Normal function of p53 induces apoptosis in the of various cell-cycle inhibitors and requires the functions of
presence of genotoxic stress, causing DNA damage that p53, the CDKN1A gene product WAF1/p21, the CDKN2A
cannot be repaired during cell-cycle arrest, growth-factor gene product INK4A/p16, and the retinoblastoma protein
withdrawal, hypoxia, and dysregulated expression of mito- (pRb). The involvement of these tumor suppressors implies
genic oncogenes. In addition to the fact that most human that one of the main functions of the senescence program is
cancers have either mutations in p53 or defects in the path- to suppress tumorigenesis, a hypothesis that has been con-
way, p53-null mice are highly prone to developing cancers. firmed in mutant mice.70,71
As the Fas pathway regulates the immune system In normal cells, unwanted proteins or proteins that are
through its proapoptotic function, disruption of this no longer required are degraded by two independent mech-
pathway may lead to lymphoproliferative disorders and anisms: ubiquitin mediated proteolysis in proteasomes, and
hematopoietic cancers. Somatic mutations of the Fas gene autophagy, a mechanism by which long-lived proteins and
or its downstream effectors have been found in patients organelle components are directed to and degraded within
with multiple myeloma, non-Hodgkin’s lymphoma, and lysosomes.72 Autophagy is conserved in various species, and
other cancers.63,64 Alterations to cell-survival pathways may is activated in response to growth-factor withdrawal, differ-
also be involved in the suppression of apoptosis. entiation, starvation, and stress. After the induction of
The PI3K–Akt (phosphatidylinositol 3-kinase–protein autophagy, autophagic vesicles (autophagosomes) are
kinase B) survival signaling pathway is activated by various formed by the assembly and expansion of membrane-
intracellular and extracellular stimuli and modulates bound structures, probably originating in the endoplasmic
apoptotic pathways, resulting in the resistance of tumor reticulum around organelles and isolated proteins. The
cells to death signals. Akt signaling induces expression of the autophagosome encapsulates the cytosolic materials and
antiapoptotic molecule Bcl-XL, inhibits the proapoptotic fuses with lysosomes or other vacuoles, causing degradation
activity of FKHRL1 (FOXO3A), and leads to negative of its content. The signaling pathway that leads to autophagy
regulation of p53 mediated apoptosis.65 Akt activation also involves at least the activities of phosphatidylinositol 3-
provides cells with a survival advantage through its promotion kinase (PI3K) and the kinase target of rapamycin (TOR).73
of glucose metabolism.66,67 Another survival factor that is The TOR pathway coordinates signaling pathways that are
relevant to human tumorigenesis is nuclear factor κB initiated by nutritional and mitogenic factors, and also
(NFκB), a transcription factor that is activated by numerous controls both protein synthesis and degradation. Although
cytokines and oncogenes. De novo gene transcription that the components of the autophagic machinery are highly
is induced by NFκB prevents apoptosis that is induced conserved in a wide range of organisms, the physiological
by the engagement of death receptors.68 Therefore, many role of the process varies. There is evidence that lysosomal
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What is cancer? 7

degradation of organelles is required for cellular remodeling mitosis when a cell has been hit by DNA damage. This
due to differentiation, stress, or damage following exposure activates a number of molecules that promote cellular
to cytotoxins, and that dysregulation of autophagy can result activities such as cell-cycle arrest, DNA repair, or apoptosis,
in pathological states such as neurodegenerative diseases, if the damage cannot be repaired. However, if the G2
cardiomyopathy, and cancer.74 checkpoint is defective, a cell can enter mitosis prematurely,
Uncontrolled protein degradation by the proteasomal before DNA replication is complete or DNA damage has
pathway can contribute to tumorigenesis. Examples are the been repaired. This aberrant mitosis causes the cell to
Wnt and hedgehog (HH) signaling pathways, which are undergo death by mitotic catastrophe. Activation of the G2
regulated by the turnover of β-catenin and cubitus inter- checkpoint begins with the detection of DNA damage by
ruptus (CI). Mutations in the corresponding genes that the sensory molecules ataxia telangiectasia mutated (ATM)
lead to constitutive activation of Wnt and HH pathways and ATM- and Rad3-related (ATR). The further process
are common in human colon cancer and basal-cell skin involves activation of checkpoint kinase 2 (CHK2) and
carcinomas.75 Defects in the autophagic pathway of protein checkpoint kinase 1 (CHK1), and phosphorylation of
degradation might also be connected to cancer via some CDC25C and several G2 checkpoint genes. The inhibition
oncogenes and tumor-suppressor genes. Autophagy is partly or inactivation of any of these G2-checkpoint genes results
controlled by the PI3K pathway, and constitutive activation in the death of cells that have sustained DNA damage by
of PI3K signaling is common in human cancer cells.76 PI3K mitotic catastrophe. Recent evidence suggests an important
and its downstream effectors, Akt and TOR, might normally role of the cytoprotective protein survivin in checkpoint
contribute to the suppression of autophagy, whereas PTEN regulation.79
(phosphatase and tensin homolog), a tumor suppressor Defects in genes such as the polo-like kinase (PLK)
that negatively regulates PI3K signaling, might normally family, the NIMA (for never in mitosis, gene A) family, the
promote autophagy. Furthermore, beclin 1 (BECN1) that aurora kinase family, and a regulator of the spindle check-
interacts with PI3K and participates in the induction of point called BUB that are required for mitotic catastrophe
autophagy in response to starvation is monoallelically can also contribute to tumorigenesis.80 Overexpression of
deleted in a high percentage of human ovarian, breast, aurora-A, which occurs in a wide range of human cancers,
and prostate cancers.77,78 Transfection of BECN1 into a increases genetic instability, aneuploidy, and centrosomal
transformed cell line can decrease its tumorigenic poten- aberrations. Furthermore, the overexpression of other
tial, and studies of mice that are deficient for this protein mitotic kinases results in multinucleation and an increase
have shown that BECN1-mediated regulation of autophagy in centrosome number.81,82
is required for normal mammalian development, and that Cancer cells often have a defect in a particular cell-death
animals with heterozygous deletions in Becn1 show a pathway, but the cells can still die because of the redundancy
marked increase in the incidence of lymphomas and carci- of cell-death mechanisms. However, the nature of the
nomas of the lung and liver. Without autophagy, the natu- cell-death defect ultimately affects the clinical outcome of
ral turnover of a protein that acts as a positive regulator of treatment, depending on which mechanism is missing. At
cell growth might be blocked, promoting proliferation. present it is not clear whether apoptosis, senescence, necro-
Autophagy is also involved in removing damaged organelles sis, autophagy, and mitotic catastrophe are entirely inde-
and, therefore, in maintaining cellular homeostasis. pendent programs, whether these mechanisms overlap to
Damage to mitochondria or sections of the endoplasmic some degree, or whether one mechanism may compensate
reticulum might result in the production of endogenous for another that is inactivated by a tumorigenic mutation.
cellular oxidants that increase the basal mutation rate.
Removal of these damaged internal cellular structures by
autophagy might therefore limit the genotoxic damage that
is caused by oxidants. Conversely, reduced autophagy might
increase oxidant stress and promote the accumulation of Factors influencing
tumorigenic mutations.
Mitotic catastrophe is caused by aberrant mitosis and is
tumor growth
associated with the formation of multinucleate, giant cells Since Virchow postulated that inflammation stimulates the
that contain uncondensed chromosomes. In normal progression of cancer, evidence has been found that
somatic cells, the M phase of the cell cycle encompasses two immune-cell infiltration is a characteristic of malignant
processes: mitosis, in which sister chromatids are aligned tumors. Tumor cells produce various cytokines and
and segregated into two daughter cells; and cytokinesis, in chemokines that attract macrophages, dendritic cells, mast
which the cytoplasm and its contents are partitioned into cells, T cells, and hematopoietic progenitors. These stromal
those cells. Mitosis is further subdivided into prophase, cells sometimes even outnumber cancer cells. Besides
prometaphase, metaphase, anaphase, and telophase. The releasing mitogenic and survival factors, stimulating DNA
G2 checkpoint of the cell cycle is responsible for blocking damage, facilitating invasion by remodeling the extracellular
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8 Advances in Nuclear Oncology

matrix, and evading the host defense, inflammatory cells

also stimulate angiogenesis in tumors.83 For instance,
Table 1.3 Human cancers with overexpression
tumor-associated macrophages accumulate in hypoxic
of the epidermal growth factor receptor
tumor regions and produce angiogenic factors such as vas-
Tumor type Expression (%) Prognostic significance
cular endothelial growth factor (VEGF), VEGF-C, and
VEGF-D. Tie2-expressing monocytes, mast cells, and NSCLC 40–80 Shorter overall survival,
platelets also release proangiogenic factors.84 Bone metastases
marrow-derived hematopoietic cells become recruited to Head and neck 80–100 Shorter disease-free and
tumors in response to VEGF and placenta growth factor overall survival
(PGF) and extravasate around nascent vessels, where they
Renal cell 50–90 Poor prognosis, shorter
stimulate growth of resident vessels by releasing angiogenic survival
factors such as VEGF, PlGF and angiopoietin-2. In addition,
these cells function as hemangioblasts, producing both Bladder 31–48 More prevalent in
hematopoietic and endothelial progenitors that result in recurrent invasive
tumors
new blood vessels.85 Furthermore, leukocyte-attracting
chemokines such as interleukin-8 (IL-8) directly stimulate Gastric 40 Shorter survival in
endothelial cell growth. Moreover, in response to PlGF tumors positive
released by tumor cells, VEGFR-1 positive hematopoietic for TGFβ and EGFR
bone-marrow progenitors home to tumor-specific Pancreatic 30–50 Reduced survival
premetastatic sites, where they recruit tumor cells and
Colon 25–77 Poor patient outcome
endothelial progenitor cells.86
As mentioned above, growth factor peptides and their Breast 14–91 Worse overall survival,
receptors are often overexpressed in human cancer and are steroid receptor-
involved in cell proliferation, differentiation, and survival. negative tumors
One of the best known systems is the HER/erbB family which Ovary 35–70 Reduced disease-free
consists of four structurally similar, transmembrane tyrosine survival, overall
kinase (TK) receptors: endothelial growth factor receptor survival, drug
(EGFR or HER1/erbB-1), HER2/erbB-2, HER3/erbB-3, and resistance
HER4/erbB-4.87 Multiple ligands bind to the EGFR: endothe-
NSCLC, non-small-cell lung cancer; TGF, transforming growth
lial growth factor (EGF), transforming growth factor
factor; EGFR, epidermal growth factor.
β (TGFβ), heparin-binding EGF, amphiregulin, betacellulin,
epiregulin, and neuregulin G2b. After binding, the receptors
dimerize and trigger intracellular signaling pathways.88 HER2
heterodimerizes with either EGFR or HER3, and binds to
their ligands for receptor activation. The downstream path- cytokines (IL-2, IL-3, GM-CSF (granulocyte macrophage-
ways of activated EGFR/HER2 involve at least two major colony stimulating factor)), and hormones (insulin,
pathways: the Ras/mitogen activated protein kinase insulin-like growth factor (IGF)).92 Ras activity is regulated
(MAPK)-dependent pathway and the phosphatidylinositol by cycling between the inactive guanosine diphosphate
3-kinase (PI3K)-dependent pathway. (GDP)-bound and the active guanosine triphosphate
The HER receptors and their ligands are frequently over- (GTP)-bound forms. Active ras triggers several down-
expressed in many tumor types and are often associated stream effector pathways that mediate cell proliferation and
with advanced disease and poor prognosis (Table 1.3).89 suppression of apoptosis. The hydrolysis of GTP by Ras is
Therefore, EGFR and HER2 have been defined as promising facilitated by GTPase-activating proteins (GAPs) such as
targets of anticancer therapy, which has been realized p120GAP and NF1.92 Point mutations in the ras gene
mostly using monoclonal antibodies such as trastuzumab, render Ras insensitive to GAP stimulation and result in
to prevent ligand binding, and tyrosine kinase inhibitors, to a permanent activation of the downstream pathways.
interact with receptor autophosphorylation and signaling. Furthermore, continuous activation of Ras also occurs in
In addition to overexpression mutations may occur. the absence of any mutation in the ras gene, as a result of
Mutations in exons 18–21 lead to increased EGFR activity continuous upstream signals, for example by activation of
through overactivation of PI3K/Akt signaling and confer HER family receptors. Overexpression of Ras isoforms is
sensitivity towards TK inhibitors, such as gefitinib or reported in many solid and hematologic malignancies and
erlotinib.90 On the other hand, there are mutations in exon is associated with a worse outcome.93 Ras protein function
20 that confer resistance to TK inhibitors.91 depends on its physical association with the inner surface
Another important system is the ras signal transduction of the membrane and transmission of the signal to Raf-1,
pathway. It is activated by growth factor receptors, which involves complex post-translational modifications
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What is cancer? 9

of the protein such as farnesylation, which is catalyzed by

the enzyme farnesyl-transferase.94 Mutations may also
Table 1.4 Most important pro- and
occur downstream of the ras pathway. The Raf kinase
antiangiogenic factors
proteins include three isoforms, ARAF, BRAF, and RAF-1,
Molecule Function
which differ in their expression profile, regulation, and
ability to function in the context of the Ras–Raf–MAPK Proangiogenic
pathway. Activating mutations in the BRAF gene have been VEGF Angiogenesis, permeability,
demonstrated in 70% of human malignant melanomas and leukocyte adhesion
15% of human colon cancers.95
VEGFR Integration of angiogenic signals
Akt/protein kinase B (PKB) is a serine/threonine protein
kinase that interacts with the membrane lipid phos- Ang-1/Tie-2 Stabilization of vessels
phatidylinositol-trisphosphate (PIP3), which is produced PDGF Recruitment of smooth
by PI3K after activation by growth factor receptors. Activation muscle cells
of Akt is prevented by PTEN, a phosphatase that removes
TGFβ, TGFβR Extracellular matrix stimulation
from PIP3 the phosphate attached by PI3K.96 Akt is a positive
regulator of the mammalian target of rapamycin (mTOR), FGF, HGF, MCP-1 Angiogenesis
a central control element for cell growth and proliferation, Integrins Receptors for matrix proteinases
by phosphorylation and inactivation of mTOR inhibitors,
such as tuberin (TSC2).97 Furthermore, Akt activation VE-cadherin Endothelial junction
leads to phosphorylation and thereby inactivation of Plasminogen Matrix remodeling, release of
proapoptotic proteins, such as BAD and caspase-9, and activators growth factors
several transcriptional factors. There are multiple lines of Chemokines Pleiotropic role
evidence relating Akt to carcinogenesis: Akt is overex-
pressed in a variety of human cancer types; PTEN, a major Antiangiogenic
antagonist of Akt activity, is frequently lost or inactivated Soluble VEGFR-1 Secreted molecule, binds VEGF
by mutation in human cancer; and the Akt positive regulator,
Ang-2 Antagonist of Ang-1, function
PI3K, or one of its downstream effectors, mTOR, is depends on other factors
commonly upregulated in cancer.96,98
Angiostatin Suppresses angiogenesis
Endostatin Inhibition of endothelial survival
and migration

Angiogenesis Vasostatin Inhibition of endothelial growth

The dependence of tumor growth on the development of a Platelet factor 4 Inhibition of bFGF and VEGF
neovasculature is now a well-established aspect of cancer binding
biology.99,100 In general, increased tumor vascularization MMP inhibitors Suppression of pathological
and tumor expression of proangiogenic factors has been angiogenesis
associated with advanced tumor stage and poor prognosis
in a variety of human cancers.101–104 VEGF, vascular endothelial growth factor; VEGFR, VEGF
receptor; PDGF, platelet derived growth factor; FGF, fibroblast
Tumor angiogenesis is regulated by a balance of pro- and
growth factor; HGF, hepatocyte growth factor; MCP-1, monocyte
antiangiogenic factors (Table 1.4). A shift in this balance in chemotactic protein 1; VE-cadherin, vascular endothelial
favor of proangiogenesis molecules activates the normally cadherin; MMP, matrix metalloproteinase.
quiescent vasculature to develop new blood vessels, often
together with enlargement of the pre-existing vasculature.
This process involves the recruitment of sprouting vessels
from existing blood vessels and the incorporation of reproductive organs, organs that are undergoing physiolog-
endothelial progenitors into the growing vascular bed. ical growth, or injured tissue. Under normal physiological
This relies on the proliferation, migration, and invasion angiogenesis, new vessels rapidly mature and become
of endothelial cells, organization of endothelial cells into stable. One characteristic feature of tumor blood vessels is
functional tubular structures, maturation of vessels, and that they fail to become quiescent, resulting in a constant
vessel regression.105 angiogenesis. The tumor vasculature presents unique char-
The normal vasculature is usually quiescent in the adult, acteristics being different in architecture compared to their
and endothelial cells are among the longest-lived cells normal counterparts. Tumor vessels are usually irregularly
outside the nervous system. There are only a few adult shaped, dilated, tortuous, and sometimes with dead ends, and
tissues that require ongoing angiogenesis, including female they lack organization into definitive venules, arterioles,
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10 Advances in Nuclear Oncology

and capillaries. In consequence, the vascular network that glycoprotein secreted by most epithelial cells in the extra-
forms in tumors is often chaotic, leaky, and hemorrhagic. cellular matrix, and fragments of larger proteins including
Blood flows irregularly in tumor vessels, moving slowly, endostatin, tumstatin, vasostatin, and vasohibin.120 The
and sometimes even shows oscillations. Tumors can be precise mechanism of action of these proteins is not fully
heterogeneous in their vascular patterns, and are able to defined, although several hypotheses have been proposed,
overproduce their capillary networks. In normal tissues, by including that they bind to specific integrins.120
contrast, vessel density is dynamically controlled by the
metabolic need for nutrients and oxygen.106–108
The molecular basis of angiogenesis has been studied
extensively and has identified a number of growth factor
receptor pathways that promote tumor angiogenesis. One Hypoxia
of the major pathways involved in this process is the vascular Due to uncontrolled growth and a misbalance between
endothelial growth factor (VEGF) family of proteins and tumor mass and vascularization, oxygen limitation is a
receptors.109,110 Activation of the VEGF/VEGF-receptor common feature of malignant tumors. Oxygen concentra-
(VEGFR) axis triggers multiple signaling networks that tion inside solid tumors is reduced, which contributes to
result in endothelial cell survival, mitogenesis, migration, the tumor aggressiveness and poor prognosis of patients.121
and differentiation, and vascular permeability and mobi- Genomes of tumor cells become unstable under hypoxic
lization of endothelial progenitor cells from the bone conditions, and hypoxia can be the selective pressure for
marrow into the peripheral circulation. In addition, VEGF the expansion of clones with antiapoptotic, treatment
mediates vessel permeability, and leads to deposition of resistant, or highly metastatic potential.122,123 Resistance to
proteins in the interstitium that facilitate angiogenesis. chemotherapy and radiation therapy can be attributed, at
Especially VEGF-A has been found to be expressed in many least in part, to the hypoxic condition of tumor cells.124
human tumors and to be associated with a broad spectrum Hypoxia confers these aggressive properties on the tumors
of oncogenes such as mutant ras, erbB-2/Her2, activated through either the remodeling of tumor vasculature or the
EGFR, and bcr-abl21.111,112 VEGF-A binds to two receptor direct phenotypic changes of tumor cells themselves.
tyrosine kinases (RTK), VEGFR-1 (Flt-1) and VEGFR-2 Tumor cells under hypoxia can acquire antiapoptotic and
(KDR, Flk-1).111 VEGFR-2 is the major mediator of the chemoresistant properties through changes in the expres-
mitogenic, angiogenic, and permeability-enhancing effects sion of apoptosis-related molecules. Furthermore, the
of VEGF-A. In contrast, VEGFR-1 may function as a decoy involvement of HIF-1α in tumor progression to an anti-
receptor that sequesters VEGF and prevents its interaction apoptotic phenotype was reported.125
with VEGFR-2.111 Furthermore, VEGFR-1 has significant Oxygen deprivation is encountered by the induction of
roles in hematopoiesis, the recruitment of monocytes and various genes. Hypoxia inducible factor 1 (HIF-1) plays a
other bone marrow-derived cells that may home to tumor central role in this regulatory system (Figure 1.2). HIF-1
vasculature and promote angiogenesis.113,114 In addition, can induce the production of a variety of gene products
VEGFR-1 is involved in the induction of matrix metallo- relevant for metabolism, vascularization, survival, pH, and
proteinases (MMPs) and in the paracrine release of growth cell migration. Active HIF-1 is a heterodimer composed of
factors from endothelial cells.115 two subunits, HIF-1α and HIF-1β. HIF-1β is constitutively
Other signaling molecules involved in angiogenesis are expressed independent of environmental oxygen concen-
platelet derived growth factor β and its receptor (PDGF-B/ tration, while the expression of HIF-1α is negligible under
PDGFR-β) and the angiopoietins (Ang), the ligands of the normoxia and induced under hypoxia. Up to now, HIF-1α,
Tie-2 receptor.110,116 PDGF-B is required for the recruit- HIF-2α, and HIF-3α have been identified and cloned as
ment of pericytes and maturation of the microvasculature. the members of the HIFα family that can dimerize with
Inhibition of PDGFR-β signaling has been reported to HIF-1α and bind to hypoxia responsible elements (HRE)
result in a tumor microvascular tree that is dependent on in the genes of hypoxia responsive molecules.
VEGF-mediated survival signals. Furthermore, tumor- Among HIFα family members, HIF-1α is thought to be
derived PDGF-A and PDGFR-β signaling has been shown the key molecule regulating the cellular response to physio-
to be involved in the recruitment of an angiogenic stroma logical and pathological hypoxia. Mechanisms of hypoxia-
that produces VEGF-A and other angiogenic factors.117 induced expression of HIF-1α have been intensively
Ang-1 is required for the remodeling and maturation of the studied, and the intracellular level of HIF-1α protein under
immature vasculature. Ang-1 is the major agonist for Tie-2, reduced oxygen concentration was found to be increased
whereas Ang-2 may act as an antagonist or a partial mainly through stabilization of the protein. Turnover of
agonist.118 However, recent studies present evidence that the HIF-1α protein is regulated by the ubiquitin–protea-
Ang-2 may also play a positive role in tumor angiogenesis.119 some system, in which target proteins are degraded by pro-
Several endogenous inhibitors of angiogenesis have been teasome depending on their ubiquitylation.126 Under
identified such as thrombospondin, a multifunctional normoxia, the level of the HIF-1α protein is kept low
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What is cancer? 11

Figure 1.2 Hypoxia

Stabilization of HIF-1
HIF-mediated reactions to hypoxia
leading to disease progression. Since
HIF-1 upregulates:
hypoxia inducible factor (HIF) induces VEGF
the expression of a variety of genes, VEGFR
multiple reactions at the physiological Glycolysis
and biochemical level result. Tyrosine hydroxylase
VEGF, vascular endothelial growth etc.
factor; VEGFR, VEGF receptor. Angiogenesis
Metabolic changes
Loss of tissue-specific
properties
Antiapoptosis
Disease
progression

through rapid ubiquitylation and subsequent proteasomal mature cells of a particular tissue through differentiation
degradation. HIF-1α becomes susceptible to rapid ubiqui- (Figure 1.3). The term ‘cancer stem cell’ is an operational
tylation through hydroxylation of proline residues at Pro- term defined as a cancer cell that has the ability to
402 and Pro-564 by prolyl hydroxylase 2 (PHD2), which self-renew, dividing to give rise to another malignant stem
requires oxygen for its enzyme activity.127 In cells under cell and a cell that gives rise to the phenotypically diverse
hypoxia, the ubiquitylation and subsequent degradation of tumor cell population. Stem cells in different tissues vary
HIF-1α are suppressed due to the decrease in PHD2 activ- with respect to their intrinsic abilities to self-renew and to
ity, and therefore the level of HIF-1α protein increases. In
addition, the activity of HIF-1 as a transcription factor is
also controlled by hydroxylation of HIF-1α protein.
Hydroxylation of the asparagine residue at Asn-803 Self-renewal
inhibits the interaction between HIF-1α and p300, which is
essential for the transcriptional activity of HIF-1.128
Because the factor inhibiting HIF (FIH) that hydroxylates
Asn-803 is also an oxygen-dependent enzyme, the tran-
scriptional activity of HIF-1 increases under hypoxia due to

Proliferation
Stem
the suppressed hydroxylation at Asn-803.129,130 Cells can cell
control the transcription of HIF-1-regulated genes by sens-
ing the oxygen concentration through the activities of
oxygen dependent enzymes PHD2 and FIH, and conse-
quently regulating the intracellular level as well as the tran-
scriptional activity of HIF-1.131
Although HIF-1 can be activated by non-hypoxic path-
Clonal expansion
ways, hypoxia inside the growing tumor mass is the most Transit amplifying cells
probable candidate for the activation of HIF-1α cascade in
tumor cells. This is supported by the fact that both HIF-1α
and VEGF expression are upregulated predominantly in
Differentiation

tumor cells around the necrotic areas of highly vascularized

tumor mass in glioblastoma.132 Therefore, angiogenesis
triggered by the hypoxia–HIF-1α–VEGF cascade seems to
play an important role in tumor progression to the more Differentiated cells
aggressive phenotypes.
Figure 1.3
Stem cell paradigm. It is postulated that within every tissue a
small fraction of cells exist which have the following
Cancer stem cells properties: self-renewal, pluripotency giving rise to other cells
of the lineage, and longevity. The transit amplifying cells
Stem cells are defined as cells that have the ability to perpet- show rapid division and end up in terminally differentiated
uate themselves through self-renewal and to generate cells which lose their ability to proliferate.
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12 Advances in Nuclear Oncology

differentiate into particular cell types. Most cancers consist

of a heterogeneous population of cells with differences in
Table 1.5 Signaling pathways in stem cells
their proliferative potential as well as their ability to reconsti-
and cancer
tute the tumor on transplantation. Evidence for cancer stem
Pathway Stem cell Cancer
cells was first documented in hematological malignancies
where only a small subset of cancer cells were capable of Wnt Hematopoietic stem Lymphoblastic
forming new tumors.133–137 Because the differences in clono- cells leukemia
genicity among the leukemia cells mirrored the differences in Intestinal epithelial Colorectal cancer
stem cells
clonogenicity among normal hematopoietic cells, the
Keratinocyte stem Pilomatricoma
clonogenic leukemic cells were described as leukemic stem
cells
cells. It has also been shown for solid cancers that the cells Cerebellar granule-cell Medulloblastoma
are phenotypically heterogeneous and that only a small progenitors
proportion of cells are clonogenic in culture and in CNS stem cells Gliomas
vivo.138–142 As in the context of leukemic stem cells, these Shh Hair-follicle progenitors Basal-cell carcinoma
observations led to the hypothesis that only a few cancer Cerebellar granule-cell Medulloblastoma
cells are actually tumorigenic and that these tumorigenic progenitors
cells could be considered as cancer stem cells. However, two CNS stem cells Gliomas
possible explanations remain: either all solid cancer cells BMI1 Hematopoietic stem B-cell lymphomas,
have a low probability of proliferating extensively and cells AML
behaving in clonogenic assays as cancer stem cells, or most Notch Hematopoietic stem Lymphoblastic
cancer cells have only a limited proliferative potential and cells leukemia
cannot behave as cancer stem cells, but a small, definable Mammary epithelial Breast cancer
subset of cells is enriched for the ability to proliferate exten- stem cells
sively and form tumors. In both cases, some of the cancer PTEN Neural stem cells Gliomas
cell heterogeneity would arise as a result of environmental Wnt, wingless type; Shh, Sonic hedgehog; BMI1, B lymphoma
differences within the tumor and continuing mutagenesis. MO-MLV insertion region; AML, acute myeloid leukemia.
On transplantation, cancer stem cells give rise to tumors
consisting of both new cancer stem cells and heterogeneous
populations of non-tumorigenic cells, reminiscent of the
developmental hierarchy in the tissues from which the If the signaling pathways that normally regulate stem cell
tumors arose. To date, cancer stem cell-like cells have been self-renewal lead to tumorigenesis when dysregulated, then
identified in breast and central nervous system tumors.143–148 the stem cells themselves may represent the target of trans-
It is still not clear whether the cancer stem cells are derived formation in certain types of cancer.153,154 There are two
from true tissue-derived stem cells, bone marrow stem cells lines of evidence in support of this hypothesis. First,
or mature cells that have undergone dedifferentiation or a because stem cells have the machinery for self-renewal
transdifferentiation process. It is also not entirely clear already activated, maintaining this activation may be
whether the cancer stem cell represents one or multiple simpler than turning it on de novo in a more differentiated
phenotypes.149,150 cell: fewer mutations may be required to maintain
Further evidence for cancer stem cells comes from the self-renewal than to activate it ectopically. Second, by
study of signaling pathways. Since normal stem cells and self-renewing, stem cells often persist for long periods of
cancer cells share the ability to self-renew, it seems reason- time, instead of dying after short periods of time like many
able to propose that newly arising cancer cells apply the mature cells in highly proliferative tissues. Therefore, there
machinery for self-renewal that is normally expressed in is a much greater opportunity for mutations to accumulate
stem cells. Evidence shows that many pathways that are in individual stem cells than in most mature cell types.
classically associated with cancer may also regulate normal If cancer stem cells are responsible for tumor growth,
stem cell development. For example, the prevention of apop- this would have profound implications for cancer therapy.
tosis by enforced expression of the oncogene bcl-2 results in One possible reason for the failure of current cancer ther-
increased numbers of hematopoietic stem cells (HSCs) in apy is the acquisition of drug resistance by cancer cells;
vivo, suggesting that cell death has a role in regulating the another possibility is that existing therapies fail to kill
homeostasis of HSCs.151 Other signaling pathways associated cancer stem cells effectively. Normal stem cells from vari-
with oncogenesis, such as the Notch, Sonic hedgehog (Shh) ous tissues tend to be more resistant to chemotherapeutics
and Wnt signaling pathways, may also regulate stem cell than mature cell types from the same tissues. This may be
self-renewal (Table 1.5).75 In vivo data from transgenic mice due to high levels of expression of antiapoptotic proteins or
suggest that activation of the Wnt signaling pathway in ABC (ATP-binding cassette) transporters such as the mul-
epidermal stem cells leads to epithelial cancers.152 tidrug resistance gene.151,155,156 If the same were true of cancer
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What is cancer? 13

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2
Targets
Christoph Bremer and Michael Schäfers

(2) abundantly overexpressed (or significantly downregu-

Genomics–proteomics lated) as compared to healthy tissue (warranting appropriate
The human genome consists of approximately 34 000 genes imaging contrast), and (3) easily accessible by intravenously
encoding numerous protein structures in the body. applied molecular tracers (e.g. extracellular). Typically,
Disease-specific aberrations of gene expression profiles specific proteins (e.g. cell receptors) are chosen as molecular
are being discovered with ever-increasing speed, and are targets, since the ‘natural amplification’ mechanism from
therefore unraveling the underlying ‘molecular pathology’ gene to protein (i.e. 1 gene = several copies of proteins) can
of cancer. Thus, innumerable molecular structures can be exploited and – depending on the site of expression –
potentially serve as novel tissue specific markers for onco- proteins may be more easily accessible in general.
logical imaging in vivo. High-throughput anaylsis tools While targeting extracellular proteins seems relatively
in genomics and proteomics help to identify new target easily achievable using a huge variety of compounds
molecules which are specifically regulated (up- and/or (peptidyl vs. non-peptidyl structures) coupled to an
downregulation) in cancer. The term functional genomics isotope or fluorescent dye (tracer), intracellular targets can
refers to the systematic generation and analysis of informa- only be reached with a more complex chemical approach,
tion on what specific genes do. Moreover, the genome-wide since pharmacological barriers are substantial (see below).
analysis of gene expression (analysis of the transcriptome), In a ‘classical’ approach, tracers are constructed to bind in a
the analysis of protein expression levels (proteomics), and 1:1 fashion to the molecular target. A typical example is
their phenotypic effects are fundamental research advances given by the binding of a receptor antagonist to a cellular
which will help to discover ever new relevant targets for surface receptor2 or an inhibitor of an enzyme binding
oncology in general, and imaging in oncological disorders selectively to the activated enzyme.3 As a distinct group of
specifically. molecular targets, enzymes can be exceptionally interesting
The investigation of how genes and proteins interact to when combined, for example, with radiolabeled substrates
finally carry out cellular functions, which is also referred to which are trapped intracellularly after enzymatic conver-
as ‘systems biology’, will moreover improve our under- sion, since one target structure (e.g. hexokinase) can
standing of the ‘molecular anatomy’ as well as ‘molecular process several hundreds of reporter probes and thus vastly
pathology’. amplify the signal in vivo. Optical imaging approaches
Whereas many activities in basic research on molecular have taken the advantages of fluorescence resonance energy
pathology in cancer are pursued by analytical methods transfer (FRET) techniques to design enzymatically activat-
applied to blood or tissue samples, molecular and func- able fluorophores (so-called ‘smart’ probes), which can be
tional imaging is uniquely able to discover molecular target exploited to visualize a broad spectrum of oncologically
expression and function on a protein level non-invasively relevant enzyme systems (e.g. matrix metalloproteinases,
in vivo. Thus, profiling-mediated discovery of novel disease- cathepsins, etc.).4,5
specific targets will also spur the development of new and However, theoretically even intracellular structures
more specific imaging agents for molecular imaging.1 (e.g. caspases, RNA, DNA) can be targeted by different
Based on this knowledge, new ligands with high affinity molecular imaging approaches. For this purpose, membrane
towards newly discovered molecular targets in cancer will translocation signals can facilitate entry of the probe into
form the basis for new imaging as well as therapeutic agents the intracellular space. Different approaches have been
(see below). described to achieve improved transmembrane migration
Targets for oncological molecular imaging in vivo of reporter probes, including the human immunodeficiency
should generally be (1) clinically relevant (i.e. linked to a virus (HIV) derived tat-peptide, dendrimers, and polyamine
certain type of disease or be predictive of disease outcome), residues.1
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18 Advances in Nuclear Oncology

system, the gastrointestinal tract, and the endocrine and

Targets exocrine pancreas. A large number of human tumors
To date, numerous molecular targets have been explored to express the somatostatin receptor subtype SSTR2. SSTR
serve as targets for new molecular imaging tools. Therefore, has a very short biological half-life; however, somatostatin
we here refer to only a few classes of currently applied analogs such as octreotide have much longer residence
molecular imaging targets in cancer. Generally speaking, times. Octreotide labeled with 111In using DTPA (diethylene-
among the huge spectrum of targets being evaluated for triaminepentaacetic acid) is approved for human use in
preclinical purposes using different imaging modalities the USA and Europe as a diagnostic imaging agent for neu-
such as scintigraphy and fluorescence imaging, there are roendocrine tumors. There are numerous other ocreotide
only a few molecular imaging targets which have been based imaging agents which have been extensively validated
validated and established for clinical applications, in the for oncological molecular imaging. Recently, new SSTR
vast majority using scintigraphic approaches such as ligands for PET imaging of SSTR expression in cancer
positron emission tomography (PET) and single photon patients have been described.7,8
emission computed tomography (SPECT). Recent advances, Metaiodobenzylguanidine (MIBG) was developed in the
especially in the field of molecular imaging, have prompted early 1980s to visualize tumors arising from the adrenal
the development of new tracers being specific for molecular medulla. Today, MIBG scintigraphy using MIBG labeled
imaging of molecular targets in oncology.6 A review of new with 123I in combination with SPECT represents the imaging
molecular imaging tracers for diagnostic purposes is addi- modality of choice for pheochromocytomas, paragan-
tionally given in Chapter 31. gliomas, and neuroblastomas. It has a complementary
function in other neuroendocrine tumors such as carcinoids,
medullary thyroid carcinomas, and non-functioning
paragangliomas.9 Future developments might translate the
SPECT technology to PET, e.g. labeling MIBG with the
Clinically established targets positron emitter 124I.

for oncological imaging

Glucose transporters and hexokinase Sodium iodide symporter
Positron emission tomography with [18F]fluoro-2-deoxy- Sodium iodide symporter (NIS) is a membrane glycopro-
D-glucose (FDG) is one of the first molecular imaging
tein that couples the transport of iodine and sodium
techniques which has been extensively validated experi- into cells using a sodium gradient maintained by the
mentally and has now provided a substantial clinical sodium–potassium adenosine triphosphatase (ATPase).
benefit for oncological imaging. In a first step, FDG (as the Secondary active transport of iodine occurs in the thyroid
original molecule glucose) is taken up intracellularly gland, lactating breast, salivary glands, and gastric mucosa.
via glucose transporters (e.g. GLUT1 and GLUT4) and Radioactive isotopes of iodine (e.g. 123I and 131I) and
subsequently phosphorylated by hexokinase. In contrast to [99mTc] pertechnetate are well established tracers in nuclear
glucose-6-phosphate, FDG-6-phosphate cannot enter the medicine for the diagnosis and targeted therapy of thyroid
citrate cycle, and thus is trapped and increasingly accumu- gland pathology. NIS transport is maintained in thyroid malig-
lated intracellularly. While the target enzyme, hexokinase, nancies at lower levels compared to normal thyroid tissue,
is not specific for malignancy (being activated in inflam- which can help to detect recurrent thyroid cancer and its
mation, remodeling, etc.) the specificity of the FDG metastasis as hot spots.
imaging approach is limited per se. However, a vast number
of malignant tumors indeed show an increased rate of
glycolysis, so that FDG-PET is becoming an increasingly
important mainstay of molecular imaging in clincal oncology
Preclinical imaging targets
(see Chapter 28). Over recent years substantial progress has been made in the
development of target specific anticancer drugs. Successful
molecular targeting in cancer by treatment with target-avid
drugs include, for example: the anti-HER2/neu antibody
Neuroendocrine tumors (trastuzumab) for erbB2-expressing breast cancers,10 and
Some of the earliest reported peptide based receptor imaging the kinase inhibitor imatinib for chronic myelogenous
agents developed and investigated in cancer patients were leukemia and gastrointestinal stroma tumors. Moreover,
radiolabeled analogs of the hormone somatostatin. molecularly targeted drugs have shown efficacy in lung
Somatostatin receptors (SSTRs) are found on the cell surface cancer (e.g. the epidermal growth factor receptor (EGFR)
in a number of organs, including the central nervous inhibitor erlotinib11), multiple myeloma (e.g. the proteasome
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Targets 19

inhibitor bortezomib12), advanced colorectal cancer (e.g. the and tumor cell invasion. Migration and invasion of cells
EGFR antibody cetuximab13), and other breast cancer enti- requires the degradation of components of the extracellu-
ties (e.g. the aromatase inhibitor letrozole14). lar matrix, which is triggered by a cascade of enzymes
However, the response to target specific treatment (e.g. matrix metalloproteinases and cathepsins), and finally
regimens can be difficult to predict, which spurs the the establishment of new cell adhesion contacts mediated
development of new target specific imaging probes that by integrins.
may help to enhance the clinical risk stratification due to
improved diagnostic capabilities, optimize therapy based
on molecular target characterization, and improve efficacy
assessments.
Imaging of proliferation
Generally speaking, fundamental biological properties The nucleoside thymidine (e.g. labeled with 11C) as well
of oncological processes that are desirable to measure by as [124I] iododeoxyuridine has been successfully applied
means of molecular imaging include angiogenesis, prolifera- to monitor tumor cell proliferation and response to
tion, apoptosis, and hypoxia; these will be briefly reviewed tumor treatment.26–28 18F-labeled thymidines include
hereafter. 3′-deoxy-3′-fluorothymidine (FLT). FLT is trapped intra-
cellularly in a manner analogous to FDG, since following
initial phosphorylation by thymidine kinase the resulting
monophosphate cannot be incorporated into DNA due
Imaging of angiogenesis to lack of a hydroxyl group. In general, FLT provides
Angiogensis is a key oncological feature that is essential for complementary information to FDG imaging, showing a
tumor growth and for the formation of tumor metastases.15 good correlation with histological markers for tumor
Different key features are important hallmarks of angio- proliferation.29 FLT may substantially improve the sensitivity
genesis in solid tumors. of assessing treatment response in cancers, since responding
Cell surface tyrosine kinase receptors are closely linked to tumor cells may continue to metabolize FDG but stop
cancer progression. Angiogenic endothelial cells depend on synthesizing DNA (and thus stop accumulating FLT).
activation of the vascular endothelial growth factor Moreover, molecules that are involved in cell cycle
(VEGF) receptor VEGFR2, and undergo apoptosis upon regulation and thus trigger tumor growth can be imaged.
withdrawal of VEGF.16 The estrogen receptor is one prominent example that can
A large array of adhesion molecules is important for the be monitored, e.g. by 18F-labeled estrogens (FES).30 FES
interaction of endothelial with other endothelial cells17 and estrogen receptor (ER) imaging has been validated and
with perivascular smooth muscle cells and pericytes tested in preliminary patient studies as a tool for measuring
respectively.18 The αvβ3 integrin is one prominent example ER expression and a predictive assay for hormonal therapy.10
of an adhesion molecule that can be targeted for both Likewise, the androgen receptor can be successfully
therapeutic and diagnostic purposes. Finally, enzymes targeted by [18F] fluoro-5-dihydrotestosterone (FDHT).
degrading the extracellular matrix are an integral part of FDHT-PET is feasible in patients with advanced prostate
tumor angiogenesis, including matrix metalloproteinases, cancer with testosterone levels that are in the castrate
cathepsins, or hyaluronidase. range. Recent data suggest that FDHT may be a valuable
Different non-specific, parametric imaging modalities tracer to study prostate cancer biology and to determine
have been developed to quantify tumor perfusion, vascular the treatment efficacy of receptor blockage in these
permeability, and intravascular and interstitial volume patients.31
fractions.19–23 For direct imaging of angiogenesis related Tyrosine kinases (TKs) represent another highly relevant
molecules, various endothelial targets can be successfully class of onocological targets that regulate most of the signal
visualized, including VEGF and the αvβ3 integrin transduction in eukaryotic cells and control many
(see above). The αvβ3 integrin receptor, for example, is processes in cancer cells, including metabolism, transcrip-
upregulated in angiogenic endothelium, and has been imaged tion, cell-cycle progression, cytoskeletal rearrangement and
using Arg-Gly-Asp containing peptidic linkers which exhibit cell movement, apoptosis, and differentiation.32,33 There
a high affinity for integrins.24,25 E-selectin is another are more than 90 known protein kinase genes in the human
potential angiogensis-related target that can be imaged by genome: 58 encode transmembrane receptor TKs distrib-
appropriate affinity ligands. Other factors that are related uted into 20 subfamilies, and 32 encode cytoplasmatic,
to or trigger angiogenesis include the hypoxia inducible non-receptor TKs in ten subfamilies. The epidermal
factor 1 (HIF-1; see below) that induces the expression growth factor receptor (EGFR) is a transmembrane recep-
of VEGF. VEGF in turn causes an increase in vessel tor of TK of the erbB (also known as HER) family that is
leakiness, extravasation of plasma proteins, and activation abnormally activated in many epithelial tumors33 and has
of the coagulation cascade, which leads to an alteration of been targeted both therapeutically and diagnostically
the extracellular matrix, making it supportive of endothelial with good success.33,34
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20 Advances in Nuclear Oncology

Imaging of apoptosis 2. Wagner S, Kopka K, Law M et al. Synthesis and first in vivo
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5. Weissleder R, Tung CH, Mahmood U, Bogdanov A Jr. In vivo
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that normally maintains phosphatidylserine on the interior fluorescent probes. Nat Biotechnol 1999; 17: 375–8.
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Lys([18F]FP)-TOCA PET in patients with SSTR-positive
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Position of nuclear medicine techniques in the diagnostic
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inversely proportional to the intracellular O2 concentra-
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tion, have successfully been developed for PET imaging of
16. Abramovitch R, Dafni H, Smouha E, Benjamin LE, Neeman M.
tumor hypoxia.40 Because hypoxia is associated with poor In vivo prediction of vascular susceptibility to vascular
response to both radiation and chemotherapy, identifying susceptibility endothelial growth factor withdrawal: mag-
hypoxia should have prognostic value.10 netic resonance imaging of C6 rat glioma in nude mice.
Cancer Res 1999; 59: 5012–16.
17. Cavallaro U, Liebner S, Dejana E. Endothelial cadherins and
tumor angiogenesis. Exp Cell Res 2006; 312: 659–67.
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ties of blood vessels as targets in cancer. Curr Opin Genet
1. Feindegen LE, Shreeve WW, Eckelman WC, Wagner HNJ. Dev 2005; 15: 102–11.
Molecular Nuclear Medicine–The Challenge of Genomics 19. Brasch RC, Li KC, Husband JE et al. In vivo monitoring of
and Proteomics to Clinical Practice. Berlin: Springer-Verlag, tumor angiogenesis with MR imaging. Acad Radiol 2000; 7:
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20. Bremer C, Mustafa M, Bogdanov A Jr et al. Steady-state image analysis: associations with other prognostic factors
blood volume measurements in experimental tumors with and survival. Br J Cancer 1995; 71: 146–9.
different angiogenic burdens – a study in mice. Radiology 30. Katzenellenbogen JA, Welch MJ, Dehdashti F. The develop-
2003; 226: 214–20. ment of estrogen and progestin radiopharmaceuticals for
21. Turetschek K, Roberts TP, Floyd E et al. Tumor microvascu- imaging breast cancer. Anticancer Res 1997; 17: 1573–6.
lar characterization using ultrasmall superparamagnetic iron 31. Dehdashti F, Picus J, Michalski JM et al. Positron tomo-
oxide particles (USPIO) in an experimental breast cancer graphic assessment of androgen receptors in prostatic
model. J Magn Reson Imaging 2001; 13: 882–8. carcinoma. Eur J Nucl Med Mol Imaging 2005; 32:
22. Boellaard R, Knaapen P, Rijbroek A, Luurtsema GJ, 344–50.
Lammertsma AA. Evaluation of basis function and linear 32. Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S.
least squares methods for generating parametric blood flow The protein kinase complement of the human genome.
images using 15O-water and Positron Emission Tomogra- Science 2002; 298: 1912–34.
phy. Mol Imaging Biol 2005; 7: 273–85. 33. Baselga J, Arteaga CL. Critical update and emerging trends in
23. Bruehlmeier M, Roelcke U, Schubiger PA, Ametamey SM. epidermal growth factor receptor targeting in cancer. J Clin
Assessment of hypoxia and perfusion in human brain Oncol 2005; 23: 2445–59.
tumors using PET with 18F-fluoromisonidazole and 15O- 34. Ke S, Wen X, Gurfinkel M et al. Near-infrared optical imag-
H2O. J Nucl Med 2004; 45: 1851–9. ing of epidermal growth factor receptor in breast cancer
24. Chen X, Park R, Shahinian AH et al. 18F-labeled RGD xenografts. Cancer Res 2003; 63: 7870–5.
peptide: initial evaluation for imaging brain tumor angio- 35. Hajra KM, Liu JR. Apoptosome dysfunction in human
genesis. Nucl Med Biol 2004; 31: 179–89. cancer. Apoptosis 2004; 9: 691–704.
25. Pasqualini R, Koivunen E, Ruoslahti E. Alpha v integrins as 36. Kelloff GJ, Hoffman JM, Johnson B et al. Progress and
receptors for tumor targeting by circulating ligands. Nat promise of FDG-PET imaging for cancer patient manage-
Biotechnol 1997; 15: 542–6. ment and oncologic drug development. Clin Cancer Res
26. Blasberg RG, Roelcke U, Weinreich R et al. Imaging brain 2005; 11: 2785–808.
tumor proliferative activity with [124I]iododeoxyuridine. 37. Schlegel RA, Williamson P. Phosphatidylserine, a death knell.
Cancer Res 2000; 60: 624–35. Cell Death Differ 2001; 8: 551–63.
27. Carnochan P, Brooks R. Radiolabelled 5′-iodo-2′-deoxyuri- 38. Lahorte CM, Vanderheyden JL, Steinmetz N et al. Apoptosis-
dine: a promising alternative to [18F]-2-fluoro-2-deoxy-D- detecting radioligands: current state of the art and future
glucose for PET studies of early response to anticancer perspectives. Eur J Nucl Med Mol Imaging 2004; 31:
treatment. Nucl Med Biol 1999; 26: 667–72. 887–919.
28. Wells P, Aboagye E, Gunn RN et al. 2-[11C]thymidine 39. Reed JC, Pellecchia M. Apoptosis-based therapies for hema-
positron emission tomography as an indicator of thymidy- tologic malignancies. Blood 2005; 106: 408–18.
late synthase inhibition in patients treated with AG337. 40. Rasey JS, Hofstrand PD, Chin LK, Tewson TJ. Characteriza-
J Natl Cancer Inst 2003; 95: 675–82. tion of [18F]fluoroetanidazole, a new radiopharmac-
29. Pinder SE, Wencyk P, Sibbering DM et al. Assessment of the eutical for detecting tumor hypoxia. J Nucl Med 1999; 40:
new proliferation marker MIB1 in breast carcinoma using 1072–9.
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3
Primary brain tumors
Eva Orunesu, Andreas Jacobs, Andrea Falini, Angelo Del Sole,
and Giovanni Lucignani

genitourinary tumors, and melanomas frequently give rise

Background to a metastatic tumor in the central nervous system (CNS).
Cancerous transformation entails major biochemical The incidence of primary brain tumors varies between
changes including modifications of the energy metabolism subtypes. Incidence rates of all primary non-malignant and
of the cell, e.g. utilization of glucose and other substrates, malignant brain and central nervous system tumors
protein synthesis, and expression of receptors and antigens. published by the Central Brain Tumor Registry of the
Tumor growth also leads to heterogeneity in blood flow United States (CBTRUS) for the year 2000 are 14.8 cases
owing to focal necrosis, angiogenesis, and metabolic per 100 000 person-years (7.4 per 100 000 person-years
demands, as well as disruption of transport mechanisms of for benign and borderline tumors and 7.4 per 100 000
substrates across cell membranes and other physiological person-years for malignant tumors).2 These incidence rates
boundaries such as the blood–brain barrier. These bio- are substantially similar to those from other institutions,
chemical and histological changes, along with their macro- including the International Agency for Research on Cancer
scopic anatomical effects, can be assessed with different (IARC) (http://www-dep.iarc.fr). Considering all tumors
non-invasive imaging tools, such as X-ray computed (benign and malignant), the rate is higher in females,
tomography (CT), magnetic resonance imaging (MRI), but malignant tumors show slight predominance in males.
magnetic resonance spectroscopy (MRS), positron emis- In other words males have a 0.65% and females a 0.50%
sion tomography (PET), and single photon emission com- lifetime risk of being diagnosed with a primary malignant
puted tomography (SPECT). Whereas anatomical imaging brain/central nervous system tumor. Pediatric incidence
(i.e. CT and MRI) is aimed at defining the size and exact (ages 0–19) is 4.3 cases per 100 000 person-years.
localization of brain tumors, along with the assessment of Despite aggressive multimodal treatment strategies
invasion of surrounding structures, functional imaging (surgery, radiation, chemotherapy), median survival of
(MRS, PET, SPECT) is better suited for characterization of patients with gliomas is limited, depending on the grade
the biological properties. The identification of a tumoral and age at diagnosis, varying from 1 year for glioblastoma,
mass and the assessment of its size and vascularization to 2–3 years for grade 3 and to 5–10 years for a grade 2
are best achieved with CT and MRI, while functional glioma. Overall, the 5-year relative survival rate following
imaging can provide additional information that is crucial diagnosis of a primary malignant brain and central
for tumor classification, differential diagnosis, and nervous system tumor decreases with age, from 64.8%
follow-up.1 (ages 0–19) to 23.1% (ages 45–54) and 4.8% (over 75).
In adults, about two-thirds of brain tumors arise from
supratentorial structures, while in children about two-
thirds of brain tumors are infratentorial. Over one-half of
brain neoplasms are metastases from tumors outside the
Classification and epidemiology CNS, and involve the skull or any intracranial structure.
Intracranial brain neoplasms can be classified into primary The most frequent primary brain tumors in adults are
brain neoplasms and metastatic tumors. Primary brain gliomas, accounting for over 90% of primary brain tumors
tumors may arise from various cell types of the brain, in patients older than 20 years and for more than 60% of all
including glial cells, neurons, neuroglial precursor cells, intracranial brain tumors in all patients.1 Their incidence is
pinealocytes, the meninges, choroid plexus, pericytes of the 5–10 per 100 000 in the general population. Gliomas are
vessels, cells of the hypophysis, and lymphocytes. Brain divided histologically into astrocytomas, oligodendrogliomas,
metastases are more common than primary brain tumors, mixed gliomas, ependymal tumors, and tumors of the
since lung neoplasms, as well as breast, gastrointestinal and choroid plexus. Grading is performed according to World
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24 Advances in Nuclear Oncology

Health Organization (WHO) criteria, taking into account Astrocytomas can then be graded according to their histo-
the presence of nuclear changes, mitotic activity, endothelial logical malignancy grade into the four WHO grades, while
proliferation, and necrosis. The most frequent (65%) and oligodendrogliomas and mixed oligoastrocytomas are
most malignant histological subtype of glioma in adults is graded only into two grades, although a reassessment of this
the glioblastoma, with a peak incidence in the seventh classification is in progress, concerning the grading criteria
decade and survival of less than 2 years for most patients. with the possible addition of a grade 4 oligodendroglioma, a
Even in low-grade astrocytomas, 5-year survival is only subclass of glioblastoma with an oligodendroglial compo-
30% due to malignant progression. If we consider glioma nent.7,8 Even low-grade tumors are infiltrative, with a marked
subtypes as distinct entities, the single most frequent brain potential to increase the malignancy grade over time.
tumor in adults is not glioma but meningioma. Decreasing The only WHO grade 1 tumor is the pilocytic astrocy-
frequencies are reported for pituitary adenomas, schwan- toma, which occurs mainly in children and can be located
nomas, congenital tumors, and others. in the posterior fossa (cerebellum and brainstem), hypo-
In children, the most common brain tumors are low-grade thalamus, basal ganglia, or optic tracts (often associated
gliomas, mostly the pilocytic astrocytoma and the benign with neurofibromatosis type 1). It is characterized by a slow
cerebellar astrocytoma. Other frequent brain tumors are growth rate with good prognosis and a long-term survival.
the medulloblastoma (the most common pediatric malig- Grade 2 gliomas include the diffuse astrocytoma, the
nant brain tumor), the ependymoma (which grows into the oligodendroglioma and the oligoastrocytoma. Most low-grade
brain ventricles), and brainstem glioma. They occur more gliomas affect young adults, the incidence slowly increasing
often between the 4th and 11th years of life, rarely below from birth up to 40 years of age, with a subsequent
the first year. decrease.
Metastatic brain tumors are more frequent than primary Most oligodendrogliomas are low-grade; the malignant
ones. Up to 15% of cancer patients may have metastatic form represents only 20% of cases. Approximately one-
spread into the brain, and the incidence of brain metastasis third of patients survive for 5 years after diagnosis. Like
increases as cancer patients live longer. The most common most slowly growing brain tumors, low-grade gliomas
primary cancers metastasizing to the brain are lung cancer usually present with partial or generalized seizures. Usually
(50%), breast cancer (15–20%), unknown primary cancer they develop over a year-long silent period with slow
(10–15%), melanoma (10%), and colon cancer (5%).4 subclinical progression, followed either by diffuse spread of
Eighty percent of brain metastases are localized in the cere- the tumor throughout one or both hemispheres or by
bral hemispheres, 15% occur in the cerebellum, and 5% in malignant transformation into grade 3 or 4 gliomas.7
the brainstem.3 For diagnostic purposes it is important to The most frequent grade 3 and 4 gliomas are the anaplas-
consider that in over 70% of cases there are multiple brain tic astrocytoma (WHO grade 3) and the glioblastoma
metastases at the time of diagnosis, but solitary metastases (WHO grade 4), which account for more than half of all
may also occur.4 It is important to avoid the mistake of con- astrocytomas after the age of 60, with survival less than 2
sidering an intracranial tumor a metastasis just because the years for most patients. The peak age of incidence is the fifth
patient has had previous cancer; in this case a correct diag- decade for the anaplastic astrocytoma and the sixth decade
nosis and appropriate treatment could be missed. Brain for the glioblastoma. Typically these tumors, although highly
involvement can occur with cancers of the nasopharyngeal malignant, do not spread beyond the CNS. Despite most
region by direct extension along the cranial nerves or modern therapy approaches, these tumors virtually always
through the foramina at the base of the skull. recur, usually arising within 2 cm of surgical resection mar-
Brain tumors are named after the brain cell type they his- gins. Treatment of these tumors is palliative, and must be
tologically appear most similar to. This does not mean that individually tailored through a multidisciplinary approach
the tumor originates from the type of cell it is named after. to preserve or restore the highest possible quality of life for as
Certain tumors are composed of cells that are so undifferen- long as possible. There are no strict guidelines for the timing
tiated that no similarity to a histological category can be of treatment options. External radiation therapy seems to
determined, such as glioblastoma cells.5 In general, the slightly improve survival in these patients. A recent large ran-
malignancy grade of the tumor is based on nuclear atypia, domized phase-3 study, including 573 glioblastoma patients,
number of mitoses, vascular proliferation, and foci of necro- has shown that the addition of the alkylating drug temozolo-
sis, but specific differences between the different tumor types mide to radiotherapy as initial treatment prolongs the 2-year
must be considered. The commonly applied terminology survival rate from 10.4% to 26.5% in patients with glioblas-
follows the rules of the WHO classification published in toma.9 Large efforts are aimed at identifying molecular
2002,6 consisting of classification of the malignancy grade of markers predictive of treatment response. This may, in the
brain tumors into four different grades, from the least future, permit the selection of a particular treatment based
aggressive (grade 1) to the most malignant (grade 4). on specific molecular features of the tumor. Molecular
The three main categories of gliomas are astrocy- tumor profiles may also allow identification of novel and
tomas, oligodendrogliomas, and mixed oligoastrocytomas. promising treatment targets for malignant gliomas.10
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Primary brain tumors 25

The anaplastic oligodendroglioma which arises either The incidence rate is low in children and rises steadily with
from a lower grade oligodendroglioma or de novo is well age, reaching its maximum in the seventh and eighth
known to be chemosensitive, in contrast to malignant decades. In the immunocompetent subject the tumor is
astrocytomas.11 Several subgroups of patients with differ- often localized in the subcortical region and is multifocal in
ent response rates for chemotherapy can be differentiated 40% of cases.19 The tumor is characterized by the tendency
through genetic analysis.7,12,13 to spread diffusely throughout the CNS and to the eye;
Meningiomas are not strictly brain tumors, as they occur therefore, surgical excision is not considered useful as in
outside the brain, in the meninges. Nevertheless they grow other brain tumors. Nevertheless, diagnostic surgery or
inside the cranial cavity and present some of the features of stereotactic biopsy are necessary to establish a histological
brain tumors. Meningiomas arise especially in areas of diagnosis, showing in almost all cases a malignant large-cell
arachnoid villi, mostly along the sagittal sinus, over the diffuse B-cell lymphoma that expresses pan-B cell markers
cerebral convexity, at the base of the skull, and in the such as CD20. These tumors are sometimes called ‘ghost
parasellar regions. They are mostly asymptomatic and tumors’ because of spontaneous or steroid-induced regression
discovered accidentally, but constitute approximately 30% of the mass.7 As a consequence, administration of steroids
of all brain neoplasms. The median age at diagnosis is prior to diagnostic procedures such as imaging procedures
64 years, and the female to male ratio is almost 2:1. Slow, or biopsy with histological evaluation should be avoided.
progressive enlargement of the tumor leads to focal or Primary brain tumors are the most frequent neoplastic
generalized seizure disorders or neurological deficits disease in children after leukemia. In children, up to 50% of
caused by compression of adjacent neural tissue.14 Most of brain tumors are located in the infratentorial region; this
these tumors present with histological well-differentiated region is rarely affected by brain neoplasms in adults. In
tissue, low proliferative capacity, limited invasiveness, and general, tumors in infants and children tend to show high
good long-term prognosis after surgical treatment. Ninety variability and low specificity of clinical symptoms. General
percent of meningiomas are classified as benign, 6% atypical, symptoms of rising intracranial pressure such as irritability,
and 2–4% malignant. There is increased incidence of reduced alertness, vomiting, failure to grow, and progressive
meningiomas in patients with breast cancer, for which a macrocephaly can be observed in infants; older children can
hormonal relationship has been proposed.15 All menin- sometimes show focal symptoms, but it is not the rule.20
giomas show loss of chromosome 22q, a feature common The three most common brain tumors in children are the
to neurofibromatosis type 2. In fact, neurofibromatosis pilocytic astrocytoma (21%), the medulloblastoma (17%),
type 2 is often associated with multiple meningiomas.16 and the diffuse low-grade cerebellar astrocytoma (9%).
Adenomas of the pituitary gland are often composed of The medulloblastoma is an infratentorial primitive
well differentiated cells that produce anterior pituitary neuroectodermal tumor that shows local invasiveness,
hormones, which can cause syndromes of hormone excess subarachnoid dissemination, and extraneural metastases.
as in acromegaly and gigantism for growth hormone-, Its frequency decreases from the first decade by approxi-
galactorrhea for prolactin-, and Cushing’s syndrome for mately halving every decade. The 5-year disease-free sur-
adrenocorticotropic hormone-producing tumors. They vival for medulloblastoma is variable, with an average of
can also be non-secreting, and all can cause hormonal approximately 50%. Increased risk of recurrence is mainly
misbalances or other symptoms due to the compression of determined by the presence of metastases, age below 4
surrounding structures, such as hormonal deficiencies and years, and extensive residual disease after surgery. In addition
visual problems due to compression of the optic chiasm. to surgical resection, treatment is based on craniospinal
Aids to diagnosis are plasma hormone measurements in radiotherapy to prevent leptomeningeal dissemination
addition to MRI of the sella turcica. Pituitary adenomas are through cerebrospinal fluid. Chemotherapy is added to
typically divided on the basis of their size into ‘microade- radiotherapy in selected cases.20
nomas’ when their size is below 10 mm in diameter and Ependymomas are commonly diagnosed in children
‘macroadenomas’ when they are bigger. Nearly all of these under 4 years of age, but they occur also in adults at any
tumors are benign. Life expectancy is normal if complete age. They usually arise in the ependymal lining of the
surgical removal is achieved and the subsequent hormone ventricles and may occur anywhere within the CNS. Most
deficiency is corrected by drugs. frequently they lie within the posterior fossa arising from
Primary central nervous system lymphoma is a rare the lining of the fourth ventricle or cerebellopontine angle,
form of extranodal non-Hodgkin’s lymphoma that is con- but they may also arise in the filum terminale and the cen-
fined to the CNS and the eye.17 In immunocompromised tral spinal canal. Advances in neuroimaging, neurosurgery,
patients, the incidence is several thousand-fold higher than and radiation therapy have improved disease control and
the baseline rate in the general population, namely about 5 functional outcomes for children with ependymoma,
per 1000 person-years among patients with acquired including children under the age of 3 years.21 Supratentorial
immunodeficiency sybdrom (AIDS) and 0.3 per 100 000 ependymomas are often larger and have a cystic component.
person-years in the immunocompetent population.7,18 The 5-year survival rate depends on the histopathological
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26 Advances in Nuclear Oncology

grading, being 87% for low-grade tumors and 27% for astrocytomas, can be distinguished from primary de novo
high-grade tumors, and localization: 97% for tumors of the glioblastomas on the basis of molecular genetic findings,28
spine, 68% for infratentorial tumors, and 62% for supra- with amplification and/or overexpression of the EGFR, p16
tentorial tumors.22 deletion, PTEN mutation, pRb alteration, and LOH 10p and
Other primary brain tumors in children include choroid 10q associated with primary glioblastoma. Most important,
plexus papilloma and carcinoma. They are rare tumors molecular alterations have been identified which indicate
which arise from the cerebral spinal fluid (CSF) producing the therapeutic response of patients and, thus, are prognos-
neuroepithelial tissue. Almost half of these tumors occur tically relevant: anaplastic oligodendrogliomas with LOH
under 12 years of age, and represent 2–4% of all pediatric 1p and/or LOH 19q are characteristically sensitive to PCV
brain tumors. The choroid plexus papilloma is the most (procarbazine, lomustine, vincristine) chemotherapy, and
common brain tumor in children less than 1 year of age. patients’ survival is significantly prolonged.12,29–31
Most of these tumors are located in the lateral ventricles,
less often in the fourth and rarely the third ventricle.
Because papillomas tend to grow slowly within ventricles,
they expand to fill the ventricle and block CSF flow. When
a complete surgical resection can be achieved, the prognosis
Diagnostic modalities
of choroid plexus papilloma is good, in contrast to the The goals of diagnostic procedures in brain oncology
choroid plexus carcinoma which has an extremely poor include: primary diagnosis, ideally obviating the need for
prognosis.23 stereotactic biopsy; planning of stereotactic biopsy and
surgical resection; radiation therapy planning for target
volume definition; evaluation for chemotherapy in selected
cases as well as experimental therapies; and re-evaluation
after treatment.
Molecular basis of brain When clinical signs and symptoms lead to the suspicion of
a brain tumor, the differential diagnosis includes a number
tumorigenesis of non-oncologic causes of expanding intracranial lesions
Understanding glial tumorigenesis is crucial for the devel- such as granulomas, paracytic cysts, abscesses, hemorrhages
opment of molecular therapeutic targets to overcome current (intracerebral, extradural, or subdural), or aneurysms.
therapeutic limitations. Molecular changes are the basis of The first diagnostic evaluation is based on neuroradio-
dysregulation of the cell cycle, alterations of apoptosis and logic imaging, and when the diagnosis of brain tumor is
cell differentiation, neovascularization, and tumor cell highly suspicious, it is necessary to characterize the type of
migration and invasion into the brain parenchyma. tumor and its grade of malignancy, as the morphologic
Genetic alterations which play an important role in glioma imaging alone is not specific enough to give full compre-
development include a loss, mutation, or hypermethylation hension of brain lesions. The gold standard for diagnosis is
of the tumor suppressor gene TP53 or other genes involved histopathological evaluation, which requires the use of
in the regulation of the cell cycle, such as cyclin-dependent tissue sampling, either surgically or by stereotactic biopsy,
kinase N2A/p16, p14ARF, and primitive neuroectodermal both of which are invasive procedures. However, because of
tumor (PTEN), as well as activation or amplification of tumor heterogeneity, even biopsy does not always hit the
oncogenes and growth factors and/or their receptors, such site of highest malignancy, which should best define the
as MDM2, cyclin-dependent kinase 4, cyclin D1 and D3, grade of the tumor. Hence the histological evaluation yields
epidermal growth factor receptor (EGFR), vascular a ‘minimum grading’ which is not always the real grade of
endothelial growth factor (VEGF), PDGFR (platelet- the tumor.5 Non-invasive functional imaging techniques
derived growth factor (PDGF) receptor), and transforming can then be applied with the aim of characterizing more
growth factor β.24–26 During progression from low-grade subtle tissue differences and enable successful targeting of
astrocytoma (WHO grade 2) to anaplastic astrocytoma the biopsy to the site of highest malignancy.
(WHO grade 3) and to glioblastoma multiforme (WHO Most clinical manifestations of tumors located in the
grade 4), a step-wise accumulation of genetic alterations brain parenchyma are due to the ‘mass effect’ of the grow-
occurs. Whereas TP53 mutation and PDGF and PDGFR-α ing tumor and the consequence of non-specific events such
overexpression represent early changes during low-grade as increased intracranial pressure, edema, and shift and
glioma development, progression to anaplastic astrocy- destruction of surrounding brain tissue. There is a ten-
toma is associated with pRb (retinoblastoma protein) alter- dency for slowly growing tumors to cause partial or gener-
ation and loss of heterozygosity (LOH) of 19q, further alized seizures, especially if they are located in the cortex (as
malignant progression to glioblastoma including LOH 10q, often observed in low-grade gliomas). Rapidly growing
and mutations of the PTEN gene.27 These secondary tumors tend to cause symptoms of progressively rising
glioblastomas, which develop from better differentiated intracranial pressure, which results from the growing
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Primary brain tumors 27

tumor mass itself, from cerebral edema, or from obstructed brain structures, to assess perilesional edema, and to define
cerebrospinal fluid flow. These symptoms are headache, the presence of multiple brain lesions. Most tumors are
nausea, vomiting, drowsiness, and visual abnormalities. characterized by contrast enhancement; the increased
Other symptoms are focal neurological deficits, which can accumulation of contrast results primarily from the leakage
show the localization of the tumor. Mental status changes, of contrast into the tumor’s interstitium because of the
such as memory loss and decreased alertness, can be associ- absence of a blood–brain barrier in the blood vessels
ated with tumors of the frontal lobe, diffuse meningeal derived from tumor neoangiogenesis. The region of
spread, or diffuse brain infiltration.7 contrast-enhancement corresponds to the main tumor
Unfortunately, as mentioned above, tumor misclassifica- mass; however, malignant tumor cells are commonly found
tion can occur with morphologic imaging techniques. The beyond the contrast-enhanced region, particularly in
first crucial step to achieving correct diagnosis through gliomas. X-ray CT is superior to MRI for detecting calcifi-
histological evaluation is to target the biopsy to the most cation, skull lesions, and hyperacute hemorrhage (bleeding
malignant region within the tumor. Especially gliomas can less than 24 hours old), and helps in direct differential
be regionally extremely heterogeneous, so biopsy-targeting diagnosis as well as immediate management. On the other
is of crucial importance for a correct grading of the tumor hand, it is less sensitive than MRI in some brain regions,
and for subsequent management planning, otherwise namely those near bony areas of irregular shape, such as the
sampling errors may lead to malignancy-grade underesti- posterior fossa and the floor of the middle fossa, due to
mation. Furthermore, low-grade gliomas have the tendency the presence of beam-hardening artifacts. Nevertheless, the
to infiltrate throughout the brain parenchyma and to X-ray CT depiction of bone structures is valuable for defin-
increase malignancy grade over time, so again exact ing bone destruction or sclerosis associated with metastatic
sampling of the site of highest malignancy is crucial.7 For tumors, pituitary adenomas, meningiomas or carcinomas
correct biopsy-targeting, the ideal imaging procedure from the sinuses or pharynx, and for studying lesions with
would design a precise map of regional malignancy within calcific components or localized close to bone structures.
the tumor. Thus, if such a technique was sufficiently The use of multislice high speed X-ray CT is desirable for
sensitive and specific, it could allow a non-invasive diagno- evaluating clinically unstable patients in whom motion
sis obviating the need for biopsic sampling prior to surgical would result in degraded MR images due to artifacts.
intervention. Nevertheless, mostly for gliomas, in addition Contrast-enhanced X-ray CT allows us to detect any
to the problem of correct sampling there is another frequent blood–brain barrier disruption which occurs in most
source of error, namely the poor within- and between- brain tumors, but also in many neurological disorders
observer reproducibility in the morphological classification including acute stroke, inflammatory, infectious cerebral
of glioma subclasses, especially for differentiating astrocytic diseases, and multiple sclerosis. X-ray CT can also miss
from oligodendroglial tumors or identification of anaplastic non-enhancing tumors such as low-grade gliomas.33
astrocytomas.31,32 Continuous developments in MRI provide new insights
Detection of a brain lesion is the first diagnostic step in into the diagnosis, classification, and understanding of the
patients with symptoms and signs suggesting the presence biology of brain tumors. MRI offers several advantages as
of a brain tumor. Imaging is primarily done to prove or rule compared to X-ray CT. MRI studies are characterized by
out the presence of such a lesion. To this end, both direct higher contrast resolution associated with multiplanarity.
and indirect signs are sought. A direct sign of a brain tumor MRI is characterized by high sensitivity for structural
is an area with a density (CT) or signal (MRI) different alterations caused by tumoral growth, also in brain regions
from that of normal cerebral tissue, including changes sec- that are poorly assessed by X-ray CT, such as the infratentorial,
ondary to contrast media infusion. Such changes in density sellar, temporal, and meningeal regions. The already high
or signal occur secondary to the structural features of sensitivity for structural alterations can be further
neoplasms. enhanced by the use of paramagnetic contrast agents.
For these reasons, MRI is the procedure of choice for the
primary diagnosis of all types of brain tumor.33 Standard
T1- and T2-weighted MRI acquisitions display high
X-ray computed tomography, sensitivity for brain tumors and give information on the
size and localization of the tumor.34 MRI is characterized
magnetic resonance imaging and by high soft-tissue resolution, and it allows the detection
with high definition of lesions that are isodense on X-ray CT,
magnetic resonance spectroscopy tumor enhancement, and secondary concurrent alterations
For many years, X-ray CT with contrast enhancement has such as edema, mass effects, signs of intracranial pressure, all
been the gold standard for the diagnosis of brain tumors phases of hemorrhagic states (except hyperacute), and necro-
due to its ability to ascertain the presence of a brain lesion, sis. MRI is also the procedure of choice in the evaluation
to define its dimension and relation with surrounding of intramedullary and extramedullary spinal cord lesions.
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28 Advances in Nuclear Oncology

A normal contrast-enhanced MRI scan essentially rules out the fast expansion leads to mass effect and peritumoral edema.
the possibility of a brain tumor. Lower grade gliomas can have large cystic components; they
There are several available MRI acquisition parameters: are usually more homogeneous without central necrosis.
T1-, T-2, proton density-, diffusion-, and perfusion- Three main variables differentiate tumors from normal
weighted images, as well as fluid attenuated inversion tissue: water content, regressive events, and vascular
recovery (FLAIR). They display characteristic patterns architecture.
depending on tumor type and grade. Usually most brain Most types of brain tumor typically exhibit an increased
tumors are hypointense on T1-weighted images and hyper- water content due to the high cellularity. Changes are more
intense on FLAIR and T2-weighted images (unless fat or pronounced in those lesions having a low nucleus/cyto-
hemorrhage is present (Figure 3.1)). In highly proliferative plasm ratio (e.g. astrocytoma) than in lesions with a high
brain tumors, such as the glioblastoma, the blood–brain nucleus/cytoplasm ratio (e.g. medulloblastoma). Another
barrier is often disrupted, resulting in leakage of contrast factor responsible for the increased water content in neoplasms
media, visible in gadolinium-enhanced T1 images and is the high quantity of interstitial fluids, that is, the cyto-
contrast-enhanced X-ray CT. On plain images, without toxic intratumoral edema. Increase of water content is the
contrast agent, the margins of gliomas are ill defined. explanation of CT hypodensity and the increase in T1 and
Gliomas tend to spread to the opposite hemisphere along T2 relaxation times on MRI (T1 hypointensity and T2
the white matter through the corpus callosum. Due to their hyperintensity). Regressive events include the presence of
fast proliferation, high-grade gliomas such as the glioblastoma cysts, necrotic and hemorrhagic areas, calcifications, and
often display central necrotic areas, fluid, and hemorrhage; fatty degenerative areas.

Figure 3.1
Left hypothalamic astrocytoma typically
showing a bright hyperintensity on T2
images (a, b), hypointensity on T1 images
(c), and a hypointense signal on
diffusion-weighted images (d), suggesting
increased water molecule movement.

a b

c d
9781841846149-Ch03 8/21/07 4:34 PM Page 29

Primary brain tumors 29

The cystic appearance of some neoplasms has long been of a solid tumor. Necrotic areas are due to ischemic cell
utilized as an aid for differential diagnosis (Figure 3.2). damage or intralesional hemorrhagic events that result in
Preoperative delineation of cysts is also helpful to the the formation of pseudocystic areas. Both the discrepancy
neurosurgeon when planning the surgical approach. Intra- between tumoral growth and blood supply and microvessel
tumoral cysts are secondary to focal mucoid degeneration, thrombosis due to wall infiltration or hyalinosis can cause
and further enlargement of the cavity occurs due to fluid intralesional ischemia. On CT and MRI, the density and
transudation from cyst walls. Large cysts are commonly signal characteristics can be similar to those of true cysts.
found in low-grade lesions (pilocytic astrocytoma, heman- Morphological criteria, including an irregular shape of cav-
gioblastoma, ganglioglioma, pleomorphic xantho-astrocy- ities, with indented borders and heterogeneous content,
toma, ependymomas, craniopharyngiomas, pituitary may suggest the necrotic origin of the cyst. Generally,
adenoma, acoustic neurinoma, meningioma). Cysts can be lesions containing areas of necrosis are more likely to be
filled with pure water, or contain considerable amounts of malignant. A large hemorrhage originating from a tumor is
protein or other debris deriving from prior hemorrhage. a relatively uncommon event (1% in neuroepithelial
A homogeneous content is a common finding in large lesions), and when it occurs it is usually very difficult to
cysts; in some cases, cysts and solid tumors display similar immediately identify a tumor as the cause. However, small
imaging characteristics and cannot be differentiated. The hemorrhages are frequently seen within tumors. Certain
characteristics of the fluid contained in the cysts influence primary intracranial neoplasms (e.g. glioblastoma, ependy-
the MR signal characteristics. If the water is pure and does moma, and oligodendroglioma) and metastases from
not contain proteins, the fluid in the cyst will have the various tumors (e.g. melanoma, lung carcinoma, renal cell
same signal as cerebrospinal fluid (CSF). When the protein carcinoma, choriocarcinoma) demonstrate a characteristic
content increases, protons become bound in a hydration tendency to bleed, and this behavior can be useful for the
layer adjacent to the protein, significantly decreasing the T1 diagnosis. This implies that it is necessary to use methods
relaxation time of the water solution, with a final increase that are sensitive and specific for the detection of hemor-
in the signal intensity on both T1 and T2 images. The most rhage. Both CT and MRI are useful for depicting the
benign tumors commonly show cystic fluid with an intensity presence of hemorrhage, but the ability of CT to define its
near to that of CSF. On CT scans, cysts are characterized by etiology is very poor. On MR images it is possible to distin-
a low density, similar to that of CSF. Higher protein density guish between the signal intensity pattern of intratumoral
is reflected in greater CT density and may lead to simulation hemorrhage and that of benign intracranial hematomas.35
The signal intensity is heterogeneous in the former, with
concurrent areas of edema and hemorrhage.35 Further-
more, the evolution of blood within tumor tissue may be
slow, as compared to the evolution of benign hematomas.
These delayed changes are probably related to the hypoxic
state typical of human neoplasms,36 or to repeated episodes
of bleeding.37 Another finding that helps to differentiate
intratumoral bleeding from benign hematomas is the
5
reduction or irregularity of the hemosiderin halo that can
5
usually be found in the periphery of chronic benign intracra-
nial haematomas.35 The presence of non-hemorrhagic tumor
c c 5
tissue inside the lesion represents a clear sign of neoplasm.
w 10
w
w11 Lastly, the presence on long TR (repetition time) images of
high-intensity signal in the parenchyma surrounding tumoral
hemorrhage35 requires follow-up by MRI or a biopsy.38 In the
presence of any of these signs the hemorrhagic event can be
due to a benign cause, such as an occult cerebrovascular
2 malformation; however, a work-up must be performed to
exclude a tumor.38 The presence of calcifications in brain
5 5 tumors is common and it has a diagnostic relevance.
Tumors that commonly undergo some calcification include
meningioma, craniopharyngioma, oligodendroglioma, astro-
c 9
P w 14
c 6
w 10 cytoma, ependymoma, choroid plexus papilloma, gan-
glioneuroma, dysgerminoma, chordoma, and all tumors after
Figure 3.2 irradiation. Calcification patterns vary from punctate to
Cerebellar pilocytic astrocytoma, characterized by a cystic diffuse, and calcifications are best seen on CT as high-
mass with enhancing mural nodule (arrows). density areas. Calcium produces a void signal on MRI,
9781841846149-Ch03 8/21/07 4:34 PM Page 30

30 Advances in Nuclear Oncology

and calcifications are thus difficult to identify by this amplitude.39 Spins with higher diffusion rates generally
technique. Areas of fatty degeneration occur secondary produce a greater loss of phase coherence and a lower MR
to macrophagic phagocytosis in necrotic areas (most common signal than those with slower diffusion rates.39 The apparent
in glioblastomas). They are seen as hypodense lipidic areas diffusion coefficient (ADC) reflects physical factors, such as
on CT and with a marked reduction in relaxation time, temperature and viscosity, in addition to the restricted
especially T1, on MRI. An abnormal vascular architecture is motion of the molecules resulting from the presence of semi-
a feature of most tumors. Tumors stimulate neoangiogene- permeable tissues and membranes. Differences in ADC are
sis within the tumoral tissue and sometimes in the adjacent expected to reflect changes in cellularity, cell membrane
areas, so the detection of vascularity is not specific enough permeability, intracellular and extracellular diffusion, and
to characterize morphologic images. Thus, several efforts tissue structure.39 Preliminary studies in brain tumors have
have focused on the development of further MR-based typically shown low anisotropy in abnormal regions, which
techniques including diffusion and perfusion imaging, and reflects the loss of normal tissue structure, with an
spectroscopy studies. The implementation of echo planar increased ADC in necrosis, edema, and cysts relative to
imaging sequences on clinical MR scanners has allowed the normal-appearing white matter. There have been reports that
rapid acquisition of images with new types of contrast the ADC of regions of tumor is correlated with cellularity,
mechanisms.39 One possibility that is beginning to be with a tendency towards lower ADC values in high-grade as
investigated in association with brain tumors is the use of compared with low-grade gliomas.40 The minimum ADC
diffusion-weighted MRI (Figure 3.3). With this technique, value in patients with tumors tends to be higher in regions
magnetic field gradients are applied before and after the of low anisotropy than in regions of normal tissue
180i pulse in spin echo imaging sequences. The reversal of anisotropy. This parameter may be important in the effort
spins by the 180i pulse means that these gradients do not to distinguish regions of edema from non-enhancing
contribute any net phase shift for static spins, whereas tumor.39 Moreover, diffusion-weighted MRI can be suc-
protons that are diffusing in the medium undergo a loss of cessfully used to differentiate between extra-axial cysts and
phase coherence that is detected as a loss of MR signal epidermoid tumors.41 Another application of echo planar
imaging is in the estimation of parameters that reflect
tissue vascularization.39 This is achieved by acquiring mul-
tiple repeated images during the first pass of a bolus of MR
contrast agent. Changes in signal intensity of such dynamic
data may be used to calculate an image of regional cerebral
blood volume (rCBV).39 Although the detailed mechanisms
underlying perfusion and vascular contrast MRI are under
investigation, the potential of the technique to provide
L L
useful information in patients with brain tumors is clear.39
5 5 Neovascularization has been shown to be an important
factor in the regulation and malignant potential of many
tumors,42 and as an increasing number of new therapies that
a b specifically target angiogenesis are available, it is desirable to
FH 46 he FH 46 he
have an imaging technique able to depict angiogenesis.39
With such a methodology, necrosis may be differentiated
from tumor by virtue of its low rCBV relative to normal-
appearing white matter in the contralateral hemisphere.
The differentiation between viable tumor and adjacent gray
L L matter is more difficult because these tissues may appear
isointense on rCBV images.39 A study of glioma patients
5 5 suggested a positive correlation between rCBV and tumor
grade.43 The potential for use of rCBV imaging to define
the radiation target and monitor therapeutic success was
c d demonstrated in a study of eight glioma patients investi-
gated three to four times serially.44 This study showed
Figure 3.3 similar results for rCBV data and positron emission tomog-
Deep right basal ganglia primary central nervous system raphy (PET) scans obtained at the same time points. In a
lymphoma characterized by a very low signal on T2 images (a), more recent study the authors state that dynamic suscepti-
a sharp enhancement after gadolinium (b), and a clear bility contrast MRI is more useful for grading glioma
restriction of apparent diffusion coefficient on diffusion- than conventional MRI, and that it can also provide
weighted images (c) and maps (d). complementary information that facilitates differentiation
9781841846149-Ch03 8/21/07 4:34 PM Page 31

Primary brain tumors 31

between malignant lymphoma and glioma. The absence of The MRI finding of most intracranial tumors without
tumor neovascularization in malignant lymphoma leads to contrast enhancement consists of iso- or hypointensity on
a low rCBV, which is in contrast to findings in malignant T1-weighted, and hyperintensity on T2-weighted images, to
gliomas. Moreover, this technique can be used to differentiate a variable extent of mass effect on other structures inside the
between extra-axial tumors, e.g. between meningioma and cranial cavity. These features alone are not reliable enough
neurinoma.45 to differentiate between the different tumors. To discrimi-
Two new MRI modalities attempt to highlight functional nate between the different tumors before histological evalu-
or metabolic properties of the tumor: perfusion-weighted ation, other features can be helpful, such as morphology,
and diffusion-weighted MRI. contrast enhancement, and the presence of a cystic compo-
Perfusion-weighted MRI is able to assess the tumor nent, of calcifications, of necrosis, hemorrhage, and edema.
blood volume, which can be useful in defining the tumor MR spectroscopy (MRS) shares with MRI the principle
extent. With an intact blood–brain barrier, this measure is that makes the production of images feasible, namely that
reliable, but in most brain tumors, where the blood–brain of nuclear magnetic resonance. In MRS the signal obtained
barrier is disrupted or absent, there is a variable underesti- from a single element is further separated into its different
mation of the tumor extent.46 New techniques have been chemical forms. This is possible because the effect of a
developed to overcome this problem, but there is yet no magnetic field on nuclei is not direct: it is shielded by the
application in clinical practice. distribution of the bonding electrons around the nuclei
The apparent diffusion coefficient in diffusion-weighted being detected. Therefore, nuclei in different chemical
MRI sequences does not seem to be helpful for distinguishing environments give rise to signals at different frequencies.
tumor tissue from peritumoral brain tissue in gliomas.47 In This separation of nuclear resonance frequencies is
vivo white matter tractography by diffusion tensor imaging termed chemical shift, depends on the magnetic field
(DTI) has become a popular tool for investigation of white strength, and is expressed in dimensionless units termed
matter architecture in the normal brain (Figure 3.4). DTI parts per million (ppm). It is thus possible to define a
studies for delineating white matter organization in the spectrum of nuclear magnetic resonance signal, in which
vicinity of brain tumors have demonstrated that edema, the several chemical forms of an element (where ‘element’
tissue compression, and degeneration may cause significant indicates hydrogen, carbon, etc.) give peaks in specific
interpretation problems, but also that the combination of positions.
functional MRI (fMRI) and DTI may represent a useful With 1H-MRS, a non-invasive in vivo approach for the
tool to define the seed region of interest for DTI-based determination of some cerebral metabolites of clinical
tractography and to provide more comprehensive, func- relevance is possible. Unfortunately, the in vivo resolution
tionally related, white matter mapping in presurgical of this method is poor; only molecules small and mobile
assessment.48 enough to tumble freely in solution can be detected. There is
no way to detect signals from proteins, from most membrane
components, or from small molecules bound to larger ones.
Moreover, only compounds present in concentrations of
about 1 mmol can be measured directly. The rationale for
considering that signals originating from certain metabo-
CST CST
lites are of use in defining the viability of, or the damage to,
any cellular structure is based on in vitro histochemical and
cellular studies. These studies demonstrated that individual
CST
CST
metabolites are localized in specific cells or cerebral struc-
tures; on the basis of this evidence, signal modifications of
GBM
a particular metabolite could reflect death or injury to a cer-
tain cell population. In a brain proton MRS study the main
marker resonances include: the generally defined choline
a b peak at 3.2 ppm, which encompasses choline-containing
compounds, such as membrane phospholipids (phospho-
Figure 3.4 choline and glycerophosphocholine), and their respective
Diffusion tensor imaging (DTI) tractography combined with
degradation products; creatine and phosphocreatine,
functional magnetic resonance imaging (fMRI) in a patient
which are elements of cellular energetic metabolism;
with left rolandic glioblastoma multiforme (GBM). In (a),
three-dimensional reconstruction of the corticospinal tract mobile lipids and triglycerides; N-acetyl-aspartate (NAA)
(CST) depicts the destructive effect of the lesion on the white and N-acetylate groups, considered to be the markers of the
matter tract (white arrow). In (b), the anterior displacement of neuronal population; and the lactic acid peak, which can be
part of the left CST and of the hand motor area, demostrated measured when the glycolysis terminal metabolite concen-
with fMRI (blue spot), is shown. tration exceeds the normal value, as in hypoxic conditions.
9781841846149-Ch03 8/21/07 4:34 PM Page 32

32 Advances in Nuclear Oncology

In addition to these metabolites, it is possible to detect progression of cerebral gliomas and that serial MRS imag-
other compounds utilizing short echo time (TE) sequences ing effectively and accurately differentiates between stable
(10–50 ms). Short TEs allow the recognition of substances and progressive disease.53
with a short T2, or that are strongly spin-coupled, such as Spectroscopy studies are also very useful in the assess-
glutamate, glutamine, taurine, glycine, myoinositol, and ment of response to therapy. The sensitivity of this tech-
γ-aminobutyric acid (GABA), which is very informative nique in fact exceeds that of conventional MRI, with useful
from the clinical point of view, but very difficult to quanti- information being provided in lesions treated with
tate with the 1.5-T magnetic field that is normally used for chemotherapy or radiation therapy. There is general agree-
clinical studies. ment that within high-dose regions that correspond to the
The major clinical application of MRS considered for radiation target, treatment response is reflected in reduc-
brain tumor patients has been its potential for non-invasive tion in the levels of choline, creatine, and NAA 2–3 months
tumor grading.49–51 These studies have predominantly used after treatment. In regions that are not responsive to the
MRS techniques that detect a signal spectrum from a small radiation treatment, levels of choline may increase, corre-
region of interest (single-voxel MRS). Trends such as sponding to residual or recurrent tumor. This different
higher mean choline and lower mean NAA levels in higher- behavior is of paramount importance in helping to differ-
grade tumors have been reported (Figures 3.5 and 3.6). entiate between radionecrosis and recurrence, one of the
However, most of the studies have found large standard most difficult topics in oncological neuroradiology. The
deviations in metabolite ratios and substantial overlap in possibility of monitoring the efficacy of new antitumoral
individual values, which may restrict the accuracy of the compounds explains why MRS is included as the main tool
technique. Studies using sophisticated data analysis tech- in most new experimental protocols.
niques have shown a higher degree of accuracy for in vitro The high sensitivity of MRS is not paralleled by its speci-
and in vivo spectroscopy studies.52 A significant improve- ficity. Although several studies have reported that MRS
ment in accuracy was obtained using a two-dimensional permits the differentiation of diverse histological tumor
MRS imaging technique.51 Multivoxel techniques (chemical types or of abscesses or cystic lesions from neoplasms, the
shift imaging or spectroscopic imaging) provide spatially experience of routine daily practice has drawn attention to
encoded chemical information from large tissue slices the risks related to the technique, and warrants caution
composed of several voxels. The combination of improved when considering differential diagnosis. This is true not
spatial resolution and increased number of voxels provides only for differentiation of neoplastic lesions, but even more
many more data about tumor heterogeneity and assists in so when distinction between tumoral and non-neoplastic
exploration of the tumor margin (Figure 3.7). As a result lesions is addressed.
it is possible to measure the metabolite content of different The absolute quantitation of metabolites and short TE
areas of neoplasms and surrounding normal tissue. spectra may provide additional data to improve specificity.
This is very useful for better characterization of glial However, absolute quantitation is complex and time con-
tumors, in which areas with different grading very often suming, and short TE spectra have a poor signal to noise
coexist, and for more accurate monitoring of possible ratio. Although the problems of absolute quantitation and
malignant degeneration of benign tumors. In a serial proton reduction of TE without impairment of quality may soon
MRS imaging study it was clearly demonstrated that an be resolved, it is still debatable whether different metabolic
increased choline signal is associated with malignant patterns correspond to different types of tumors.

Figure 3.5 10 NAA Cho

Single-voxel spectroscopy (echo time (TE) 135 ms) 7
NAA
6 Cr
of a subcortical low-grade glioma. Compared to the 8 Cho
5
normal spectral profile (top left), the tumoral 6 Cr 4

spectrum (top right) shows a slight increase of 3

4 2
choline (Cho) and N-acetyl-aspartate (NAA) 1
reduction. 2
0

-1
0
4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
Other documents randomly have
different content
But here a difficulty presented itself. Was she sure of recognizing this
nurse, whom she had never seen since she was ten years old? She
had Justine's voice and accent in her memory far more clearly than
her face. She followed the ups and downs of the road as far as the
last house behind the rock, and there she saw written on the door
"Peyraque Lanion." A horseshoe nailed over this sign indicated his
occupation of farrier.
Justine had risen first, as was her custom, while the closed calico
curtains of the bed shaded the last nap of M. Peyraque. The
principal apartment on this ground-floor showed the comfort of a
well-to-do household, and the mark of this easy competence
consisted particularly in the garniture of the ceiling; which was
trellised with racks of monumental supplies of vegetables and divers
rural commodities; but the strict cleanliness, a rare deviation from
the customs of the country, removed everything which might offend
the eye or the sense of smell.
Justine was lighting her fire, and preparing to make the soup her
husband was to find smoking hot on his awakening, when she saw
Mlle de Saint-Geneix come in with her hood on, carrying her bundle.
She cast a look of perplexity upon the stranger, and said at last,
"What have you to sell?"
Caroline, hearing Peyraque snore behind his curtain, put her finger
to her lips and threw her hood back on her shoulders. Justine stood
still an instant, suppressed a cry of joy, and opened her stout arms
with rapture. She had recognized her child. "Come, come!" said she,
leading her toward a little break-neck staircase at the farther end of
the entry, "your room is all ready. We have been hoping for you
every day this year." And she called to her husband, "Get up,
Peyraque, at once, and shut the door. Here is news, O, such good
news!"
The little chamber, whitewashed and furnished in rustic fashion, was,
like the lower room, of irreproachable neatness. The view was
magnificent; and blossoming fruit-trees came up to the level of the
window. "It is a paradise!" exclaimed Caroline to the good woman.
"It only needs a little fire, which you are going to make for me. I am
cold and hungry, but happy to see you and be with you. I must tell
you something, first of all. I don't want it known here who I am. My
reasons are good ones, and you shall know them; they will meet
your approval. Let us begin by agreeing on our facts; you have lived
at Brioude?"
"Yes; I was in service there before I was married."
"Brioude is a long way from here. Is there any one from that country
in Lantriac?"
"No one; and strangers never come. There is no road except for ox-
carts."
"I saw that myself. Then you can pass me off for some one you
knew at Brioude?"
"Very easily,—the daughter of my old mistress."
"No; I'm not to be a young lady."
"But she was not a young lady; she was a little tradeswoman."
"That's it; but I must have an occupation."
"Wait a minute!—that's easy enough. Be a pedler of small wares, like
the one I am speaking of."
"But then I shall have to sell something."
"I'll see to that. Besides, you are supposed to have made your
rounds, and I shall have detained you here as a matter of friendship;
for you are going to stay?"
"A month, at least."
"You must stay always. We will find you something to do, never fear.
But, let's see; what shall be your name?"
"Charlette; you called me that when I was a little thing; so it will not
give you any trouble. I am supposed to be a widow, and you must
say 'thou' to me."
"Just as I used to. Good! it is agreed. But how will you dress, my
dear Charlette?"
"Like this. You see it's not luxurious."
"It's not very rich, to be sure; though it will pass; but this lovely
blond hair of yours will attract the eye; and a city bonnet will be a
wonder."
"I thought of that; so I bought at Brioude one of the head-dresses
worn there. I have it in my travelling-bag, and I'm going to don my
costume at once for fear of a surprise."
"Then I'll go at once and get you some breakfast. You will eat with
Peyraque, I take it?"
"And with you, I hope. To-morrow I mean to help you about the
house and in the kitchen."
"O, you may pretend to do that! I don't want you to spoil those little
hands I used to take such care of. Now I'm going to see if Peyraque
is up, and let him know what has been agreed upon; then you must
tell us why there is need of all this mystery."
While talking, Justine had kindled the wood already in the fireplace.
She had filled the pitchers with pure cold water, which had trickled
from the rock, coming through an earthen pipe to the toilet-table of
her little chamber, and then down into the kitchen sink. This was an
invention of Peyraque's, who prided himself oh having ideas of his
own.
Half an hour afterward Caroline, whose simple attire marked no
particular station, put up her fine hair under the little head-dress
from Brioude, less scantily contrived, and more prettily curved than
the round dish-cover—which, like it, is of black felt trimmed with
velvet—worn by the women of Velay. It was all in vain; she was still
charming in spite of the weariness that dimmed the large eyes
"green like the sea," formerly so bepraised by the Marchioness.
The soup of rice and potatoes was quickly served in a small room
where Peyraque at odd moments did a little carpenter-work. The
good man thought this an unsuitable reception, and wanted to
sweep away the shavings. "On the contrary," said his wife, spreading
the chips and sawdust over the floor, "you don't understand at all!
She will think it a pretty carpet. O you don't know her yet! She is a
daughter of the good Providence, this one is!"
Caroline made acquaintance with Peyraque by embracing him. He
was a man of about sixty years, still very robust though thin, of
medium height, and plain-featured, like most of the mountaineers in
this region; but that his austere and even stern countenance bore
the stamp of integrity was evident at the first glance. His rare smile
was remarkably genial. You saw in it real affection and sincerity,
which were all the more unmistakable from the fact that they were
never lavished demonstratively.
Justine also had rigid features, and a blunt way of speaking. She
was a strong generous character. An earnest Roman Catholic, she
respected the silence of her husband who was of Protestant descent,
nominally converted indeed, but a free-thinker if there ever was one.
Caroline knew these circumstances and was touched to see the
delicate respect which this superior woman knew how to weave into
her love for her husband. It must be remembered that Mlle de Saint-
Geneix, the daughter of a very weak man, and the sister of an
inefficient woman, owed the great courage she possessed first to her
mother, who was of Cévenol parentage, and afterward to the ideas
Justine had given her in early life. She perceived this very clearly
when she found herself seated between this old couple whose
precise language and notions caused her neither fear nor surprise. It
seemed as if the milk of her mountain nurse had passed into her
whole being, and as if she were there in the presence of types with
which she had already been made familiar in some previous
existence.
"My friends," said she, when Justine had brought her the cream of
the dessert, while Peyraque washed down his soup with a draught of
hot wine, followed up before long with a draught of black coffee, "I
promised to tell you my story and here it is in few words. One of the
sons of my old lady had some idea of marrying me."
"Ah, indeed! that might well be," said Justine.
"You are right, because our characters and ideas are alike. Any one
ought to have foreseen that, and I myself first of all."
"And the mother, too!" said Peyraque.
"Well, no one seems to have thought of it; and the son surprised
and even angered the mother when he told her he loved me."
"And you?" asked Justine.
"I—I—why he never told me of it at all; and, as I knew I was not
noble enough or wealthy enough for him, I should never have
allowed him to think of it."
"Yes, that's right!" returned Peyraque.
"And it's true!" added Justine.
"Then I saw I could not stay a day longer, and at the first angry
word from the mother I went away without seeing the son again;
but the son would have hurried after me if I had remained with my
sister. The Marchioness wanted me to stay a little to have an
explanation with him, to tell him I did not love him—"
"That is what ought to have been done, perhaps," said Peyraque.
Caroline was forcibly impressed by the austere logic of the peasant.
"Yes, unquestionably," thought she, "my courage ought to have been
pushed thus far."
And, as she still kept silence, the nurse, enlightened by the
penetration of a loving heart, said to her husband, sharply, "Stop
talking there, you! How you run on! How do you know she did n't
love him, this poor child?"
"Ah! that, that is another thing," replied Peyraque, bowing his
serious, thoughtful head, which now looked nobler for the sense of
delicate pity expressed upon his face.
Caroline was touched in an unspeakable degree by the
straightforwardness of this simple friendship, which with one word
touched the sorest spot in her wound. What she had not had
strength or confidence to tell her sister, she was impelled not to
disguise from these hearts, so thoroughly true and so able to read
her own. "Well, my friends, you are right," said she, taking their
hands. "I should not perhaps have been able to lie to you, for, in
spite of myself, I—I do love him!"
Hardly had she spoken the words, when she was seized with terror,
and looked around as if Urbain might have been there to hear them;
then she burst into tears at the thought that he never would hear
them.
"Courage, my daughter, the Lord will aid you," exclaimed Peyraque,
rising.
"And we will aid you, too," said Justine, embracing her. "We will hide
you, we will love you, we will pray for you!"
She led her back to her room, undressed her, and made her lie
down, with motherly care that she should be warm and not see the
sun shining in too early on her bed. Then she went down to apprise
her neighbors of the arrival from Brioude of a person named
Charlette, to answer all their questions, mentioning her paleness and
her beauty that these might not strike them too forcibly. She took
pains to tell them also that the speech of Brioude was not at all like
that of the mountains, so Charlette would be unable to talk with
them. "Ah! the poor creature," replied the gossips. "She will find it
very dull and tiresome with us!"
A week later, after having informed her sister, in the proper time and
place, of her safe arrival, Caroline gave her some detailed account of
her new mode of life. It must not be forgotten that, hiding her actual
sorrow, she was trying to reassure her sister, and to divert her own
thoughts by affecting an independence far from being so complete
or so real as it seemed.
"You can form no idea of the care they take of me, these Peyraques.
Justine is always the same noble woman, with a heart like an
angel's, whom you know, and whom our father could not bear to see
going away from us. So it is saying more than a little to declare that
her husband is worthy of her. He has even more intelligence,
although he is slower of comprehension; but what he does
understand is as if engraved on marble without spot or blemish. I
assure you I am not weary a single moment with them. I could be
alone much more than I am, for my little room is free from all
intrusion of servants, and I can dream without being disturbed; but I
rarely feel the need of this: I am contented among these worthy
people, I am conscious of being loved.
"They have, besides, something of intellectual life, like most of the
people here. They inquire about things in the world without; and it is
astonishing to find in a kind of blind alley, among such wild
mountains, a peasantry with so many notions foreign to their own
necessities and habits. Their children, their neighbors, and their
friends impress me as active, intelligent, and honest, while Peyraque
tells me it is the same in villages farther still from all civilization.
"As an offset to this, the dwellers in the little groups of cottages
scattered over the mountain, those who are only peasants,
shepherds, or laborers, live in an apathy beyond all comprehension.
The other day I asked a woman the name of a river which formed a
magnificent cascade not more than a hundred paces from her
house. 'That is water,' she replied. 'But the water has a name, has
n't it?' 'I will ask my husband; I don't know myself; we women
always call all the rivers water.'
"The husband knew enough to tell me the names of the torrent and
the cascade; but when I asked for those of the mountains on the
horizon, he said he knew nothing about them, he had never been
there. 'But you must have heard that those are the Cévennes?'
"'Perhaps so! The Mézenc and the Gerbier de Joncs [sheaf or stack
of reeds] are over there, but I don't know which they are.'
"I pointed them out to him; they are easily recognized,—Mézenc, the
loftiest of the peaks, and the Gerbier, an elegant cone, which holds
in its crater reeds and swamp-grasses. Only, the good man would
not even look. It was all precisely the same to him. He showed me
the 'grottos of the ancient savages,' that is, a kind of Gallic or Celtic
village hollowed out of the rock, with the same precautions that
beasts of the wilderness use to conceal their dens; for you can
examine this rock and follow it without discovering anything unusual
unless you know the path which penetrates this labyrinth and its
habitations. Ah, my dear Camille, am I not here a little like those
'ancient savages,' who, for fear of intrusion, hid themselves in caves
and sought their peace in forgetfulness of the whole world?
"At all events, the inhabitants of La Roche impress me as being the
direct descendants of those poor Celts, hidden in their rock, and, as
it were, bound to it. I looked at the woman, with bare legs and dull
eyes, who conducted us into the grottos, and asked myself whether
three or four thousand years had really passed away since her
ancestors took root in these stones.
"You see I go out, for prudence does not require the in-door life,
which you feared for me. On the contrary, having nothing to read
here, I feel the need of strolling about, and my movements surprise
the good people of Lantriac much less than a mysterious retreat
would do. I run no risk of meeting strangers. You saw me set out in
clothing that would not attract attention in the least. Besides, I have
a black felt hat, larger than those worn here, which shades my face
quite nicely. In case of need, too, I can conceal it entirely under the
brown hood I brought with me, which the capricious weather gives
me an excuse for wearing in my walks. I am not just like the women
of the country; but there is nothing in my appearance to create a
sensation in the places where I go.
"Then, too, I have a pretext for going out, which accounts for
everything. Justine has a little trade in small wares and gives me
charge of a box whose contents I offer for sale, while Peyraque, who
is a farrier, busies himself with visiting sick animals. This enables me
to go into the houses and observe the manners and customs of the
country. I sell but little, for the women are so absorbed in their lace-
making that they never mend for their husbands, their children, or
themselves. Here is the triumph of rags worn with pride. Their
devotion to their one occupation is so passionate as to exclude all
material well-being and all cleanliness even, as a profane superfluity.
Avarice finds its account in this, and vanity also, for if Justine gave
me jewelry to sell I should soon have customers more eager for that
than for linen and shoes.
"They produce all those marvellous black and white laces, which you
have seen Justine make at our house. It is wonderful to see, here
among the mountains, this fairy-like work coming from the hands of
these poor creatures, and the trifling sum they realize shocks the
traveller. They would cheerfully give you for twenty sous what they
ask twenty francs for in Paris, if they were allowed to trade with the
consumer; but this is strictly forbidden. Under the pretext of having
furnished silk, thread, and patterns, the dealer monopolizes and sets
a price on their work. In vain you offer to supply the peasant-woman
with materials and pay her well. The poor woman sighs, looks at the
money, shakes her head, and replies that she will not risk losing the
patronage of 'her master' in order to profit by the liberality of a
person who will not employ her permanently, and whom she may
possibly never see again. And then all these women are pious, or
pretend to be so. Those who are sincere have sworn by the Virgin
and the saints not to sell to individuals, and one is forced to honor
their respect for a promise given. Those who make religion a regular
profession (and I see there are more such than one would suppose)
are conscious of being always under the hand and beneath the eye
of the priests, nuns, monks, and seminarists, with whom this country
is literally sown and covered even in the most uninhabitable places.
The convents have the work done; and here, as elsewhere, under
conditions of trade still more lucrative than those of the dealers. You
can see, in the vestibules of the churches even, the women from the
village in a sort of community, sitting in a circle, making their
bobbins fly as they murmur litanies or chant offices in Latin; which
does not, however, prevent them from gazing curiously at the
passers-by and exchanging remarks, while they reply ora pro nobis
to the gray, black, or blue sister who oversees the work and the
psalmody.
"These women are generally kind and hospitable. Their children
interest me, and when I find those who are ill, I am glad to be able
to point out the more simple attentions that should be given them.
There is either great ignorance or great indifference on this point.
Maternity here is rather passionate than tender. It is as if they told
you that children are created for the single purpose of learning how
to suffer.
"Peyraque's business, as his services are much in demand, leads us
into some almost inaccessible places on the mountain, giving me a
chance to see the finest landscapes in the world, for this wonderful
country is like a dream,—and my own life is a strange dream also, is
it not?
"Our fashion of going in search of adventures is quite primitive.
Peyraque has a little cart, which he is pleased to denominate a
carriage, because it has an awning of canvas, which somewhat
ambitiously pretends to shelter us. He harnesses to this vehicle now
an intrepid little mule, and now a pony, spirited but gentle, all skin
and bone like its owner, but like him, too, never flinching at
anything. So, while Justine's eldest son, just returned from the
regiment, where he has been shoeing artillery horses, continues his
trade under the paternal roof, his father and I wander over hill and
vale without regard to the weather. Justine pretends this does me so
much good that I must stay with her 'always,' and vows she will find
some way for me to earn our livelihood without humiliating myself to
serve any great lady.
"Alas! I never felt humiliated so long as I knew I was loved; and
then I loved so sincerely in return! Do you know it saddens me no
longer to receive a blessing every morning from that poor old
Marchioness, and not only so, but I am quite uneasy, alarmed about
her even, as if I felt she could not live without me? God grant she
may soon forget me, that my place may already have been filled by
one less fatal than I to her peace. But will she be cared for, morally
speaking, as I cared for her? Will her fanciful whims be understood,
the dulness of her leisure hours charmed away, or her children
spoken of as she loves to hear them spoken of? On my arrival here,
I drank in the free air with long breaths; I gazed at this grand,
rugged scenery which I had felt so strong a wish to know. I said to
myself, 'Here I am then free! I shall go where I please; I will talk as
little as I please; I shall no longer write the same letter ten times a
day to ten different people; I shall not live in a hot-house; I shall not
breathe the sharp perfumes of flowers distilled by chemical
processes, or of plants half dead on the windowsills; I shall drink
from the breeze hawthorn and wild thyme in their real fragrance.'
Yes, I said all this to myself, and I could not rejoice. I saw my poor
friend sad and lonely, perhaps weeping for having made me weep so
much!
"But she chose this, and to all appearance, it was necessary. I have
no right to blame her for a moment of unjust anger. The mother
thought only of her son, and such a son well deserves all a mother's
sacrifice. Perhaps she calls me hard and ungrateful for not falling in
with her plans, and I often ask myself if I ought not to have fallen in
with them; but I always answer that the end would not have been
attained. The Marquis de V—— is not one of those men who can be
sent off with a few commonplaces of cool disdain. Besides, you have
no right to act thus toward one who, far from declaring his passion,
has surrounded you with respect and delicate affection. In vain I
seek some language, half cold, half tender, which I might have used
in telling him that I hold his mother's happiness and his own equally
sacred: I do not find in myself the requisite tact or skill. Either the
real friendship I have for him would have deceived him as to my
feelings, leading him to think I was sacrificing myself to a sense of
duty, or my firmness would have offended him, as if I were parading
a virtue whose aid he has never given me occasion to invoke. No,
no! it could not be, it ought not to be.
"I have an impression that the Marchioness hinted that I might tell
him I had an engagement, another love. For Heaven's sake, let her
invent all she will now! Let her sacrifice my life and that which I hold
still more sacred, if need be. I have left the field clear: but, for my
own part, I could never have improvised a romance for the occasion.
And would he have been duped by it?
"Camille, you will see him, you have doubtless already seen him
again since that first visit, when you admitted it was hard for you to
play your part. You say it made you very unhappy to see him; he
was almost distracted—He is certainly calm now. He has so much
moral strength, he will understand so well that I must never see him
again? However, be on your guard! He is very keen. Tell him my
nature is a cold one—no, not that; he would n't believe it. But speak
of my invincible pride. That is true; yes, I am proud, I feel it! And if I
were not, should I deserve his affection?
"Perhaps it would have been liked if I had become really unworthy of
his regard,—not the mother; not she! no, never! She is too upright,
too pious, too pure in heart; but the Duke, I mean. Now, I can recall
a number of things which I did not understand, and they appear in a
new light. The Duke is excellent; he worships his brother. I believe
his wife, who is an angel, will purify his life and thoughts; but at
Séval, when he told me to save his brother at any cost,—I think of it
now, and I blush to think of it!
"Ah, that I might be allowed to disappear, that I might be allowed to
forget all! For a year I believed myself calm, worthy, happy. One day,
one hour has spoiled the whole. With one word, Madame de Villemer
has poisoned all the memories I had hoped to carry away unsoiled,
—memories which now I dare not dwell upon. In truth, Camille, you
were right in saying, as you sometimes did, that one should not be
too ingenuous, that I ventured out into life too quixotically. This will
serve me as a lesson, and I will renounce friendship as well as love.
I ask myself why I should not from this time onward break off all
relations with a world so full of dangers and snares, why I should
not accept my misery more bravely indeed than I have done. I could
create some resources in this province even, remote as it is in point
of civilization. I could not be a school-mistress, as Justine imagined
last year; the clergy have usurped everything here, and the good
sisters would not let me teach, even in Lantriac; but in a city I could
find pupils, or I could become a book-keeper in some mercantile
house.
"First of all, I must make sure of being forgotten there; but when
this oblivion is complete, I must indeed take thought for our
children, and I dwell upon this a little in advance. After all, be at
ease. I will find something. I shall manage to conquer the malicious
fates. I do not sleep, I cannot falter; you know this perfectly. You
have enough to live on for two months more, and I need absolutely
nothing here. Do not worry, let us always trust the good God, as
you, for your part, must trust the sister who loves you."

XXII

Caroline had reason to be alarmed by the inquiries M. de Villemer

was making at her sister's. He had already returned twice to
Étampes, and, fully aware that delicacy forbade anything like a
system of cross-questioning, he confined himself to watching the
demeanor of Camille, and drawing his own inferences from her silent
evasions. Thenceforth he might take it for granted that Madame
Heudebert knew her sister's hiding-place and that Caroline's
disappearance gave her no real uneasiness. Camille held in reserve
the letter which said Caroline had found employment away from
France, and did not produce it. She saw such anguish and distress in
the features of the Marquis, which were already much changed, that
she dared not inflict this last blow on the benefactor, the protector of
her children. Besides Madame Heudebert did not share all Caroline's
scruples or comprehend all her pride. She had not ventured to blame
her, in this regard; but she herself would not have held it so great a
crime to brave the displeasure of the Marchioness a little, and
become her daughter-in-law notwithstanding. "Since the intentions
of the Marquis were so serious," thought she, "and his mother loves
him so that she dares not oppose him openly, and, finally, since he is
of age and master of his own fortune, I don't see why Caroline could
not have used her influence over the old lady, her powers of
persuasion, and the evidence of her own worth, and so led her
gently to admit the propriety of the marriage.—There! poor Caroline,
with all her valiant devotedness, is too romantic, and will go away
and kill herself in order to support us; while, with a little patient tact,
she might be happy and make us all happy too."
Here is another common-sense opinion which may be set over
against that of Peyraque and Justine. Of these two lines of reasoning
the reader is free to adopt the one that he prefers; but the narrator
must, of necessity, hold an opinion also, and he avows a little
partiality for that of Caroline.
The Marquis perceived that Madame Heudebert made, now and
then, some timid allusions to the state of things, and felt sure she
knew the whole. He threw himself on her mercy a little more than he
had done hitherto; and Camille, encouraged, asked him, with a
sufficient want of tact, whether, in case the Marchioness proved
inexorable, he was fully resolved to make Caroline an offer of his
hand. She seemed on the point of betraying her sister's secret, if the
Marquis would pledge his word of honor.
The Marquis replied without hesitation: "If I was sure of being loved,
if the happiness of Mlle de Saint-Geneix depended on my courage, I
would contrive to do away with my mother's prejudices, at any cost;
but you give me no encouragement. Only give me that, and you will
see!"
"I give you encouragement!" exclaimed Camille, amazed and
confused. She hesitated to reply. She had indeed divined Caroline's
secret; but the latter had always guarded it proudly, not by
falsehood, but by never allowing herself to be questioned, and
Madame Heudebert had not the daring to inflict a severe wound on
her sister's dignity, by taking it upon herself to compromise her.
"That is something I am no wiser about than you," said she.
"Caroline has a strong character,—one which I cannot always
fathom."
"And this strength of hers is so great," said the Marquis, "that she
would never accept my name without my mother's sincere
benediction. This I know better even than you do. So tell me
nothing; it is for me alone to act. I ask of you only one thing more,
and that is to let me watch over you and your children until
something new shall occur, and even—yes, I will venture to say it—I
am haunted by the fear that Mlle da Saint-Geneix may find herself
without resources, exposed to privations which it makes me shudder
to think of. Spare me this dread. Let me leave you a sum which you
can return, if there is no use for it, but which, in case of need, you
will remit to her as coming from yourself.'
"O, that is quite impossible," replied Camille: "she would divine the
source, and never forgive me for having taken it!"
"I see you are really afraid of her."
"Just as I am of all that commands respect."
"Then we feel alike," replied the Marquis as he took leave. "I am so
thoroughly afraid of her that I dare not seek her any farther, and yet
I must find her again or die."
Shortly afterward the Marquis drew an explanation from his mother,
which was painful enough to both of them. Although he saw her
suffering, sad, regretting Caroline a hundred times more than she
admitted, and although he had resolved to await a more propitious
moment for his inquiries, the explanation came, in his own despite
and in despite of the Marchioness, through the fatality of
circumstances. The anxiety of the situation was too intense; it could
not be prolonged. Madame de Villemer confessed that she had
conceived a sudden prejudice against the character of Mlle de Saint-
Geneix, and that at the very moment of fulfilling her promise she
had let Caroline feel the exceeding pain it caused her. Gradually,
under the eager questioning of the Marquis, the conversation grew
more animated, and Madame de Villemer, pushed to extremity,
allowed the accusation against Caroline to escape her. The
unfortunate girl had committed a fault pardonable in the eyes of the
Marchioness when acting as her friend and guardian, but one which
made it quite out of the question even to think of receiving her as a
daughter.
Before this result of calumny the Marquis did not flinch one instant.
"It is an infamous lie," he cried, beside himself,—"a base lie! And
you could believe it? Then it must have been very artful and very
audacious. Mother, you must tell me all, for I am not disposed to be
taken in so myself."
"No, my son, I shall tell you no more," replied Madame de Villemer
firmly; "and every word you add to those you have just uttered, I
shall consider a breach of filial affection and respect."
So the Marchioness remained impenetrable; she had promised not to
betray Léonie; and, besides, nothing in the world would tempt her to
sow the seeds of discord between her two sons. The Duke had so
often told her, in Urbain's presence, that he had never sought or
obtained a single kind look from Caroline! This, in the opinion of the
Marchioness, was a falsehood the Marquis would never pardon. She
knew, now, that he had taken the Duke into his confidence, and that
Gaëtan, touched by his grief, had persuaded his wife into taking
measures for seeking Caroline in all the Parisian convents. "He does
not speak," said the Marchioness to herself; "he will not dissuade his
wife and brother from this folly, when he ought, at the very least, to
have confessed the past to the Marquis, in order to cure him of it. It
is too late now to risk such avowals. I cannot do it without leading
my two sons to kill each other after having loved so warmly."
Meanwhile Caroline wrote her sister as follows:—
"You feel alarmed because I am in so uneven and rocky a region,
and ask what can be fine enough to make one run the risk of being
killed at every step. First of all, there is really no danger here for me
under the guidance of this good Peyraque. The roads, that would be
actually frightful, and, as I think, impassable for carriages like those
with which we are familiar, are just large enough for the little carts
of this region. Then, too, Peyraque is very prudent. When he cannot
measure with his eye just precisely the space he needs, he has a
method of ascertaining it, which made me laugh heartily the first
time I saw him put it in practice. He trusts me with the reins, jumps
to the ground himself, takes his whip, which has the exact size of his
cart marked with a little notch on its stock, and, advancing a few
paces on the road, he proceeds to measure the width of the passage
between the rock and the precipice,—sometimes between one
precipice on the right and another on the left. If the road has a
centimetre more than is needful he comes back triumphant, and we
go quickly by. If we have no such centimetre in which to disport
ourselves, he makes me alight, while he leads the horse by the
bridle, dragging on the carriage. When we find two little walls
hemming in a foot-path, we place one wheel on either wall and the
horse in the pathway. I assure you one soon becomes accustomed
to all this, and already I think no more about it. The horses here
have no vicious tricks, and are not inclined to shy; they know the
danger as well as we, and accidents are no more frequent in this
country than they are on the plains. I certainly exaggerated the
danger of these jaunts in my first letters; it was from vanity, or a
lingering fear, of which I am wholly cured now that I feel it was
groundless.
"As to the beauty of Velay, I could never describe it for you. I did not
dream there could be, here in the heart of France, a country so
strange and so imposing. It is far more lovely than Auvergne,
through which I passed on my way hither. The city of Le Puy is
probably unique in point of location; it is perched upon masses of
lava that seem to spring up from its very heart and form a part of its
architecture. These lava pyramids are indeed the edifices of giants;
but those which man has placed on their sides, and often on their
summits, have certainly been inspired by the grandeur and wildness
of the spot.
"The cathedral is admirable, in the Romanesque style, of the same
color as the rocks, but slightly enlivened by the blue and white
mosaics on the pediments of its façade. It is placed so as to seem
colossal, for, to reach it, you must climb a mountain of dizzy steps.
The interior is sublime in its elegant strength and solemn dimness. I
never understood the terrors of the Middle Ages, or felt them, so to
speak, as I did under these bare, black pillars, beneath these storm-
laden domes. There was a furious tempest while I was there. The
flashes sent their infernal lights across the splendid windows that
strew the walls and pavements with jewels. The thunders seemed
rolling forth from the sanctuary itself. It was Jehovah in all his wrath;
but it gave me no alarm. The true God, whom we love to-day, has
no menaces for the weak. I prayed there with a perfect faith, and
felt it had done me good. As for these beautiful temples of the faith
in ages both rude and stern, it is clear they are the expression of the
one grand word, 'mystery,' whose veil it was forbidden to lift. If M.
de Villemer had been there he would have said—
"But a course of history and religious philosophy is not to the point
now. The ideas of M. de Villemer are no longer the book from which
I may study the past or learn to anticipate the future.
"You see, thanks to good Peyraque and his desire to show me the
marvels of Velay, thanks also to my impenetrable hood, I have
ventured into the city and its suburbs. The city is everywhere
picturesque; it is still a mediæval town, closely studded with
churches and convents. The cathedral is flanked by a whole world of
ancient structures, where, under mysterious arcades, and in the
turns and twists of the rock they stand on, you can see cloisters,
gardens, staircases, and mute shadows gliding by, hidden beneath
veil and cassock. A strange silence reigns there, and a certain odor
of the past, I know not what, which makes one shiver with fear, not
of our God, the source of all confidence and spiritual freedom, but of
everything that, in the name of God, breaks up forever the ties and
duties of our common humanity. In our convent, I remember a
religious life seemed cheerful; here, it is sombre enough to make
one tremble.
"From the cathedral you must keep going down hill for an hour to
reach the Faubourg d'Aiguilhe, where another monument rears its
head, which is natural and historic, at one and the same time, and,
indeed, the most curious thing in the world. It is a volcanic sugar-
loaf three hundred feet in height, which you mount by a spiral
stairway until you reach a Byzantine chapel, necessarily quite small,
but charming, and built, it is said, on the site and from the
fragments of a temple to Diana.
"A legend is current here, which struck me forcibly. A young girl, a
Christian virgin, pursued by some miscreant, flung herself to escape
him down from the top of the terrace; she arose at once; she was
unharmed. The miracle was noised abroad. She was declared a
saint. Pride grew strong in her heart; she promised to hurl herself
down again, to show she was under the protection of angels; but
this time Heaven deserted her, and she was crushed like a vain silly
creature as she was.
"Pride! yes, God leaves the proud to themselves, and without him
what can they do? But do not tell me that I am proud. No, it is not
pride. I have no desire to prove anything to any one. I ask to be
forgotten, and that there should be no suffering on my account.
"There is near Le Puy, forming a part of its magnificent landscape, a
village that also crowns one of those singular, isolated rocks, which
break through the soil here at every step. It is called Espaly, and this
rock also bears up the ruins of a feudal castle and of Celtic grottos.
One of these caves is inhabited by two persons, aged and poor,
whose squalid misery is heart-rending. This couple live here in the
solid rock, with a single hole for chimney and window. At night they
block up the door, in winter with straw; in summer, with the old
woman's petticoat. A small, rude bed without coverlids or mattress,
two stools, a little iron lamp, a spinning-wheel, and two or three
earthen pots,—these are all the furniture.
"Nevertheless, only a few paces from them there is a vast and
splendid house belonging to the Jesuits and named the Paradise. At
the foot of the rock flows a brook which brings down precious stones
in its sand. The old woman sold me for twenty sous a handful of
garnets, sapphires, and jacinths, which I am keeping for Lili. The
stones are too small to have any actual value, but there must be a
precious deposit somewhere among these rocks. The Jesuit fathers
will find it, perhaps; I don't expect to make the discovery myself,
however; so I must think about procuring some work. Peyraque has
an idea which he has enlarged upon for the last few days, and which
was suggested to him by this very rock of Espaly; I will tell you how.
"While strolling about over this rock, I was taken with one of my
sudden fancies for a little child, playing in the lap of a pretty woman
from the village, who was strong and cheerful. This child, you see, I
can compare with no one but our Charley, for inspiring affection. He
does not look like Charley, but has the same demure playfulness,
and the shy caresses which make one his willing slave. When I
called upon Peyraque to admire him, remarking how clean he was
kept, and that his mother made no lace, but seemed wholly taken up
with him, as if she knew she had a treasure there, Peyraque at once
replied, 'You have come nearer the truth than you thought. This
child is a treasure for Dame Roqueberte. If you ask who he is, she
will tell you it is the child of a sister she has in Clermont; but this is
not true: the little one has been placed in her charge by a gentleman
whom no one knows, who pays her for rearing it, who pays her,
besides, for taking great care of it, as if it were the son of a prince.
So you see this woman is well dressed and does not work. She was
in easy circumstances before. Her husband has charge of the castle
of Polignac, whose great tower, and in fact all the ruined portion,
you can see over yonder, on a rock larger and loftier than that of
Espaly; that is where she lives, and, if you meet her here, it is
because now she has such fine chances for pleasure strolls. The real
mother of the little one must be dead, for she has never been heard
of; but the father comes to see it, leaves money, and stipulates that
it shall not be allowed to want for anything.'
"You see, dear sister, this is a romance. That is partly what attracted
me perhaps, since, according to your ideas, I am quite romantic.
Certainly this little boy has something about him which captivates
the imagination. He is not strong; they say when he first came here
he had hardly life enough to breathe; but now he is quite blooming,
and the mountain air agrees with him so well that his father, who
came here at about this time last year to take him away, decided to
leave him a year longer, in order to have him regain his strength
completely. The little creature has an angelic face, dreamy eyes, with
a far-off look in them, strange in a child of his age, and there is a
wondrous grace in all his ways.
"Peyraque, seeing me so bewitched, scratched his head with an air
of profundity and continued, 'Well, tell me, then, since you are fond
of little children, why, instead of making it your occupation to read
aloud, which must be wearisome, do you not find a little pupil like
that, whom you could educate at your sister's with the other
children? This would leave you in your own home and to your own
ways.'
"'You forget, my good Peyraque, that perhaps it will be long before I
can go to my sister.'
"'Well, then, your sister might come and live here, or else you could
stay with us for a year or two; my wife would aid you in taking care
of the child, and you would only have the trouble of watching over
him and teaching him.—Stop! I have an idea of my own about this
child, since he pleases you so that you are doting on him already.
His father will come after him one of these days. Suppose I should
tell him about you?'
"'Then you are acquainted with him!'
"'I acted as driver for him once, and carried him to the mountain in
my carriage. He seems a fine man, but too young to take upon
himself the bringing up of a child of three years. He will have to
place it in charge of some woman, and he cannot leave it any longer
with the Roqueberts, for they are not capable of teaching what a
young gentleman like him ought to know. This would be your own
task, especially, and the father would never find so good a mother
for his child. Hope, hope! (which signifies wait!) I will keep watch at
Polignac, and as soon as this father arrives, I will manage to talk
with him in the proper way.'
"I let good Peyraque cultivate this project, and Justine also, but I
have no faith in it myself, for the mysterious personage expected will
ask questions I am unwilling to have answered, unless I am quite
sure he knows none of the people, either intimately or remotely,
from whom my place of retreat must be concealed. And how could I
make sure of that? Peyraque's idea is, nevertheless, in itself a good
one. To educate some child at home for a few years would please
me infinitely better than going into a strange family again. I would
rather take a girl than a boy, as she would be left with me a longer
time; but there will be little room for choice, for these children
hidden away by their parents are not easy to find. And there must
needs be the most perfect confidence in me. I must be well
recommended. Madame d'Arglade, who knows all the secrets of
fashionable life, could find for me a chance like this; but I would
rather not apply to her: without intending to do so, she might bring
upon me some fresh misfortune."

XXIII

A few days later Caroline wrote again to her sister.

"POLIGNAC, May 15.

"Here I have been for five days past, in one of the most imposing
ruined castles left from feudal times, on the summit of a great, black
lava boulder, like those I told you about in connection with Le Puy
and Espaly. You will think my position has changed, and my dream
has become reality. No: I am certainly near little Didier, but I have
taken it upon myself to watch over him, for his father or protector
has not yet appeared. Now see what has happened.
"I felt a wish to see the child again, besides a slight wish to learn
more about him; and lastly I had a desire to examine closely this
castle of Polignac, which looks from afar like a city of giants, on a
rock from the infernal depths. It is the strongest mediæval fortress
in the country; it was the nest of that terrible race of vultures under
whose ravages Velay, Forez, and Auvergne have trembled. The
ancient lords of Polignac have left everywhere throughout these
provinces mementos and traditions worthy of the legends about the
ogre and Blue-Beard. These feudal tyrants robbed travellers, pillaged
churches, murdered the monks, carried off women, set fire to
villages, and this, too, from father to son, through long centuries.
The Marquis de Villemer worked out of these facts one of the most
remarkable chapters of his book; drawing the conclusion that the
descendants of this family though innocent, assuredly, of the crimes
of their ancestors, seem, by their misfortunes, to have been
expiating the triumphs of barbarism.
"Their citadel was impregnable. The rock is sliced down
perpendicularly on all sides. The village forms a group below on the
little hill which supports the block of lava. It is some distance from
Lantriac. The insuperable ravines here make all distances great.
Having started early, however, we arrived last Tuesday toward noon,
and our little horse carried us to the foot of the postern. Peyraque
left me there, in order to take care of our animal, and to look at
some others, for he has quite a reputation in veterinary science, and
wherever he goes, practice of this kind always comes to him.
"I found a little girl ten years of age to open the door for me; but
when I asked to see Dame Roqueberte, the child told me with tears
that her mother was dying. I hurried to where she lives,—a part of
the castle still standing, in good repair,—and I found her the victim
of a brain-fever. Little Didier was playing about the room with
another of this poor woman's children; the latter child was quite
happy, comprehending nothing, although the elder; while Didier,
between smiles and tears, was looking toward the bedside with as
much anxiety as a little creature of three years could be expected to
show. When he caught sight of me, he came to me at once, and
without coquetting before embracing me, as he did the first time, he
clung to my dress, pulling me with his little hands, and saying
'mamma,' in a voice so plaintive and gentle that my whole heart was
won by it. He was certainly telling me about the strange condition of
his adopted mother. I drew near the bed. Dame Roqueberte could
not speak; she knew no one. Her husband came in after a moment
and began to be alarmed, for she had been in this state only a few
hours. I told him it was time to send for a physician and a woman to
take care of his wife, which he did at once; and as I could not be
sure that it was not typhoid fever, I sent the children out of the
room, warning the husband that it might be dangerous to leave
them there.
"When the physician came at the expiration of two hours, he
approved what I had done, observing that the disease had not yet
defined itself and that the children must be placed in some other
house. This change I undertook to make with the help of Peyraque,
for the husband had quite lost his senses, and thought of nothing
but having candles burnt in the village church and prayers mumbled
in Latin which he could not understand, but which seemed to him of
more efficacy than the doctor's prescriptions.
"When he had calmed down a little it was already four o'clock; and it
was necessary for Peyraque to set out again with me, that the night
might not overtake us in the ravine of the Gâgne. There was no
moon for the moment, and a storm was impending. Then poor
Roquebert began to lament, saying that he was ruined unless some
one would take care of the children, and especially of 'the child,'
meaning by that Didier,—the hen with the golden eggs for his
household. Special care was needful for him; he was not strong like
the children of the country, and besides he was 'curious,' he wanted
to go everywhere, and these ruins are a labyrinth of precipices,
where a young gentleman of this adventurous temper must not be
lost sight of a single moment. He dared not trust him with any one.
The money this little one had brought into his house had made
others envious, he had enemies; what did I know about it? In short,
Peyraque said to me in a low voice, 'Come, your good heart and my
own bright ideas are at one in this matter. Remain here; I see they
have the wherewith to lodge you comfortably; I will come back to-
morrow to see how the case stands, and take you home if there is
no further need of you.'
"I confess I desired this decision; it seemed as if it were a duty as
well as a privilege to watch over the child. Peyraque returned the
next day, and as I saw that Dame Roqueberte, though out of danger,
would not be able to sit up for some days, I consented to remain,
telling Peyraque not to come after me till the end of the week.
"I am very comfortable here, in a vast room, which is, I believe, an
old hall for the guards, that has been divided into several portions
for the use of the farmers. The beds, though very rustic, are clean,
and the housekeeping I attend to myself. I have the three children at
my side all the time. The little girl does the cooking while I
superintend; I see to the attendance which must be given the
mother; I wash and dress Didier myself. He is clothed like the
others, in a little blue blouse, but with more care, especially since I
have made it my concern,—and I am so fond of him that I dread the
moment when I shall have to leave him. You know my passion for
children,—that is, for some children; this one is certainly well born.
Charley would be as jealous of him as a tiger. Because, you see, this
Didier is surely the son of a superior man or woman. He is of high,
fine descent, morally speaking; his face is of a somewhat dull
whiteness with little flushes of color like those on standard roses. He
has brown eyes of admirable shape and expression, and a forest of
black hair, half inclined to curl, which is fine and soft as silk. His little
hands are perfect, and he never soils them. He does not dig in the
earth, and never touches anything: he passes his life in looking at
things. I am sure he has thoughts beyond his years which he cannot
express, or rather, a series of dreams, charming and divine, that
cannot be translated into human language; yet he talks very fluently
for one of his age, both in French and patois. He has caught the
accent of the country, but makes it very sweet by his infantile lisp.
He has the prettiest reasons in the world for doing as he pleases,
and what he pleases is to be out of doors, climbing over the ruins, or
crawling into their crevices; once there, he sits down, gazing at the
tiny flowers, and especially at the insects, without touching them,
but following all their motions, apparently interested in these living
marvels, while the other children think only of crushing and
destroying them.
"I have tried to give him his first notions in reading, being persuaded
(contrary to the father's opinion perhaps) that the earlier you begin
with children the more you spare them the heavy strain on the
attention, so painful when their strength and activity have found
greater development. I have tested his intelligence and curiosity;
they are unusual, and with our wonderful method, which succeeded
so well with your children, I am sure I could teach him to read in a
month.
"And then this child is all soul, and his self-will melts into boundless
affection. Our fondness is growing too fast really, and I ask myself
how we are ever going to part.
"Besides, although I miss my Justine and Peyraque, I enjoy myself
exceedingly among these magnificent ruins, commanding as they do
one of the loveliest spots on earth. The air is so pure that the white
stones, mixed with rough fragments of lava, are as bright as if just
from a quarry. And then the interior of this immense castle is stored
with very curious things.
"You must know that the Polignac family pretend to a descent from
Apollo or his priests in a direct line; and that tradition consecrates
the existence here of a temple to this god,—a temple of which some
fragments yet remain. As for myself, I think there is no doubt of it,
and that just to see these fragments is enough. The question to
decide is whether the inscriptions and carvings were brought here to
decorate the castle according to Renaissance usage, or whether the
castle was built upon these vestiges. Dame Roqueberte tells me the
scientific men of the country have been disputing over it for fifty
years, and for my own part I agree with those who think the
curbstone of the well was the mouthpiece of the god's oracles. The
orifice of this immense well, with which another and a smaller well
grotesquely communicates, was closed by a colossal head of noble
outline, whose perforated mouth gave forth the subterranean voice
of the priestess. Why not? Those who say it was only the mask of a
fountain are no surer. The head has been preserved from destruction
in the lower story of a little tower, along with a pile of stone bullets
found in the well. I have amused myself by taking a sketch of it,
which I send you in this letter, with a portrait of my little Didier at its
foot, lying sound asleep at full length upon the temple of the god. It
does not look like him, to be sure; but it will give you an idea of the
fantastic and charming picture which I have had before my eyes for
the last fifteen minutes.
"As for other matters, I do not read at all here. I have not
Peyraque's eight or ten stray volumes and his big old Protestant
Bible. I no longer try to improve myself; I hardly think of it even. I
mend the clothing of my Didier, following him step by step; I dream,
I am sad, but not rebellious, and not given to wondering any further
about a state of things to which I ought to submit,—and I am in
good health, which is the most important thing.
"Good old Peyraque comes in, bringing your letter. Ah! my sister, do
not give up weakly, or I shall be in despair. You say he is pale,
already ill; and this gave you so much pain that you came near
betraying me. Camille, if you have not strength enough to see a
courageous man suffer, and if you do not understand that my
courage alone can support his, I will set out again; I will go farther
away still, and you shall not know where I am. Consider yourself
notified, that the day I see the mark of a strange foot upon the sand
of my island, I shall disappear so entirely that—"

Caroline left the sentence unfinished; Peyraque, who had just given
her Madame Heudebert's letter, came back saying, "Here is the
gentleman coming."
"Who? what?" cried Caroline, rising and evidently quite troubled.
"What gentleman?"
"The father of the unknown child,—M. Bernyer he calls himself."
"Then you know his name? No one here knew it or would tell it."
"On my word, I am not very curious; but he threw his valise on a
bench at Roquebert's door, and my eye happened to fall upon it, so I
read."
"Bernyer! I don't know any such person; perhaps I might show
myself without getting into difficulty."
"Why, certainly you must see him, to tell him about the little one;
now is the time."
Roquebert came in, however, and defeated Peyraque's design. M.
Bernyer was asking for his son; but, according to his custom, he had
gone into a room, reserved for him especially, and did not wish, just
then, to see any one not of the family.
"It is all the same," added Roquebert. "I will tell him how you took
care of my wife and the little boy, and he will certainly give me
something good to repay you with. Otherwise I will do it myself, out
of my own pocket. Be easy about that."
He took the child in his arms and went out, closing the door behind
him, as if to shut out even a curious look from following him into the
passage leading to the stranger's room.
"Well, let us set out," said Caroline, whose eyes were full of tears at
the thought that she would probably never see Didier again.
"No," replied Peyraque, "let us wait a little and see what the
gentleman will think, when he knows you have stayed here five days
to take care of his child."
"But don't you see, my friend, that Roquebert will take care not to
tell him? He will never dare to own that, during his wife's illness, he
knew of nothing better than trusting the child to a stranger. And
beside, is he not anxious to keep Didier a year longer, which would
be very feasible? Will he let us give the father a hint that the child
would not only be better cared for, with us, but also educated as he
needs to be at his age? No, no. Dame Roqueberte herself, in spite of
the care I have given her, will say that no one knows me, that
perhaps I am only an adventuress; and while seeking gratitude and
confidence, we shall look as if we were intriguing to get the few
sous which have been offered us already."
"But when we refuse them it will be seen who we are. I am known
myself; it is understood that Samuel Peyraque has never lied or held
out his hand for money."
"This stranger knows nothing of all that, and he will inquire of the
Roqueberts only because he knows nobody else. Let me set out
quickly, my dear friend; I suffer every minute I stay here."
"Just as you like," said Peyraque. "I have not unharnessed, and we
can let the horse rest at Le Puy; but nevertheless, if you would trust
me, we should remain here one or two hours. Going thither from
here, we would naturally meet on the way; the child would come to
you and ask for you himself, he is so fond of you already. Look here
now! If the gentleman should see you only one minute, I am sure he
would say, 'Here is a person who is like no one else: I must speak to
her.' And when he had talked with you—"
Arguing in this way, Peyraque followed Caroline, who had gathered
up her clothing and was turning her steps toward the castle gate,
quite determined to start. Passing before the bench where the
stranger's valise was still lying beside his travelling-cloak, she read
the name which Peyraque had reported faithfully; but at the same
time she made a gesture of surprise and hurried along with unusual
agitation.
"What is it now? asked the good man, taking the reins.
"Nothing,—a fancy!" replied Caroline, when they were out of the
enclosure. "I imagined I recognized the hand of the person who
wrote the name of Bernyer on that valise."
"Bah! it was written just like print."
"That is true; I am silly! Never mind; let us go on, my good
Peyraque."
Caroline was absorbed in thought all the way. She accounted for the
singular emotion which the sight of this disguised handwriting had
caused her by what she had just experienced in reading her sister's
letter; but she had a new anxiety. M. de Villemer had never told her
that he had seen the castle of Polignac with his own eyes, but he
had given a fine description of it, and an accurate one, in his book;
he had taken it as an example of the strength of feudal restorations
in the Middle Ages, and Caroline knew he often travelled into the
provinces, in order to get a distinct impression of historic places. She
searched all the recesses of her memory to find what could not
possibly be there, to see if the Marquis had not accidentally chanced
to tell her that he had visited Polignac. "No," replied she to herself,
"if he had said so, I should have been impressed by it on account of
the names Lantriac and Le Puy, which Justine had mentioned." Then
she tried to remember whether, in connection with Polignac, she had
not spoken of Lantriac and Justine; but she had never mentioned
either of them to him, she was quite sure; so she grew calmer.
Yet she was agitated and thoughtful. Why had she taken such a
fancy to this unknown child? What was the peculiarity in his eyes,
his attitude, and his smile? Was it that he looked like the Marquis? In
the idea which had so suddenly presented itself, of educating a little
child and wishing for this one, might there not have been a vague
instinct more powerful than chance or Peyraque's instigations?
With all this uneasiness there came, too, in Caroline's despite, the
secret torment of a confused jealousy. "He has a son, then, a child
of love?" said she to herself. "He must, then, have loved some
woman passionately before he knew me, for frivolous adventures are
incompatible with his exclusive nature, and there has been an
important mystery in his past life! The mother is still living perhaps.
Why is she supposed to be dead?"
Advancing among these feverish speculations, she recalled the
words of the Marquis under the cedar in the Jardin des Plantes, and
the struggle she had caught a glimpse of between his filial duty and
some other duty, some other love, of which she herself might not be
the object after all. Who knew whether the old Marchioness had not
been equally at fault, whether the Marquis had told his mother the
name of the person he wanted to marry; in short, whether she
herself and Madame de Villemer had not both missed the truth?
Thus working herself into an involuntary excitement, Caroline strove
in vain to feel reconciled to her fate. She loved, and for her the
stronger feeling now was the fear rather than the hope of not being
loved in return.
"What is the trouble?" asked Peyraque, who had learned to read her
anxieties in her face.
She replied by overwhelming him with questions about this M.
Bernyer whom he had seen once. Peyraque had a keen eye and a
memory; but, habitually thoughtful and reserved, he bestowed his
attention only on people who especially interested him. He drew,
then, a picture of this pretended Bernyer so vague and incomplete
that Caroline made no progress. She slept poorly that night, but
toward morning she grew calm, and awoke saying to herself that
there had been no common sense in her excitement of the day
before.
Peyraque, having to go his rounds, could not linger till her
awakening. He came in at nightfall. His air was triumphant.
"Our affair is working well," said he. "M. Bernyer will come here to-
morrow, and you may rest easy; he is an Englishman, a sailor. You
don't know any such person, do you?"
"No, not at all," replied Caroline. "You saw him again, then?"
"No, he had just gone out; but I saw Dame Roqueberte, who is
better and begins to have her senses. She told me the little one
cried last night, and before he fell asleep asked over and again for
his Charlette. The father inquired who she was. It seems that
Roquebert had no great wish to speak of you; but his wife, who is a
good Christian, and the little girl, who is fond of you too, said you
were an angel from heaven, and the gentleman replied he would like
to thank you, and make you some recompense. He asked where you
lived; he has never been at our house, but remembered me
perfectly, and said he would come and see us soon. He promised the
child this, and even that he would bring you back, in order to make
him go to sleep."
"In all this," said Caroline, "I see only one thing, and that is, this
stranger is coming to offer me money."
"Well, let him do it; so much the better! It will be an opportunity to
show him you are not what he thinks. You will see one another, you
will converse; he will find you are an educated young lady, above
what he supposes you are, and I will tell him your history, because
this history of yours does you credit."
"No, no," replied Caroline, quickly. "What! shall I intrust my secret to
a stranger, after so many precautions to conceal my name and
position?"
"But since you do not know him?" said Justine. "If you are agreed on
the matter of the child, he should be intrusted with the whole.
Having his secret, we can afford to give him ours. He would have no
inducement to betray it."
"Justine!" cried Mlle de Saint-Geneix, who was near a window that
faced the street. "Listen! Heaven! not another word. There he is,
certainly, this M. Bernyer. He is coming here, and it is—yes, I was
sure—it is he! It is M. de Villemer! O my friends, hide me! Tell him I
am gone, that I am not coming back!—If he sees me, if he speaks to
me,—can't you feel that I am lost?"

XXIV

Justine followed Caroline, who had escaped to her own room, and
made signs to Peyraque that he should receive the Marquis and be
self-possessed.
Peyraque was equal to the emergency. He received M. de Villemer
with the calm dignity of a man who has the most rigid ideas of duty.
It was no longer a question of putting him in communication with
the pretended Charlette; it was necessary to get him away before
any suspicions arose in his mind, or, in case they had already arisen,
to dispel them at once. From the first words of the Marquis,
Peyraque saw that he suspected nothing. Desirous to set out again
in a few days with his son, whom he intended to keep nearer to
himself in future, he had made the most of a fine morning to come
on foot and repay this debt of gratitude to some generous stranger.
He had not supposed the distance so great, and was, therefore, a
little late in arriving. He confessed he was somewhat tired, and, in
point of fact, his face betrayed both weariness and suffering.
Peyraque hastened to offer him food and drink, the duties of
hospitality preceding everything else. He called Justine, who had, by
this time, regained her composure; and they waited upon M. de
Villemer, who, catching at this opportunity of rewarding his
entertainers generously, accepted their services with a good grace.
He learned with regret that Charlette had gone away; but there was
no reason why he should ask many questions about her. He thought
of leaving a present for her, which Justine, in a low tone, advised her
husband to accept, that he might not be surprised at anything.
Caroline would readily find a chance to send it back. Peyraque did
not see the necessity; his pride revolted at the idea of seeming to
accept money on her account.
Caroline, in her little chamber, overheard this strife on a point of
delicacy. The voice of the Marquis sent shudders through her. She
dared not stir. It seemed as if M. de Villemer would recognize her
footfall through the flooring. He, for his part, hoping to find a way of
discharging his obligations under some different form, pretended
and really tried to eat a little; and after this inquired whether he
could hire a horse to return with. The night was dark and the rain
came on again. Peyraque agreed to carry him back and went out to
get his wagon ready; but first, he climbed up softly to Caroline's
room. "This poor gentleman makes me uneasy," said he in a low
voice. "He is very ill, that I am sure of. You can see drops of sweat
on his forehead, and yet he creeps up to the fire like a man with a
fever-chill. He could not swallow two morsels, and when he breathes
hard it seems to affect his heart like a spasm, for he puts his hand
there, smiling bravely all the while, but afterwards carrying it to his
head, as one does in severe pain.
"Heavens!" exclaimed Caroline, in alarm, "when he is ill it is so
dangerous! You must not carry him back to-night; your wagon is not
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