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CANCER ETIOLOGY, DIAGNOSIS AND TREATMENTS SERIES

SMALL CELL CARCINOMAS:


CAUSES, DIAGNOSIS AND TREATMENT

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CANCER ETIOLOGY, DIAGNOSIS
AND TREATMENTS SERIES
Cell Apoptosis and Cancer Clinical, Genetic and Molecular
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Drug Resisant Neoplasms Small Cell Carcinomas: Causes,


Ethan G. Verrite (Editor) Diagnosis and Treatment
2009. ISBN: 978-1-60741-255-7 Jonathon G. Maldonado and Mikayla K.
Cervantes (Editors)
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CANCER ETIOLOGY, DIAGNOSIS AND TREATMENTS SERIES

SMALL CELL CARCINOMAS:


CAUSES, DIAGNOSIS AND TREATMENT

JONATHON G. MALDONADO
AND
MIKAYLA K. CERVANTES
EDITORS

Nova Biomedical Books


New York
Copyright 2009 by Nova Science Publishers, Inc.

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Library of Congress Cataloging-in-Publication Data

Small cell carcinomas: causes, diagnosis and treatment / editors, Jonathon G. Maldonado and
Mikayla K. Cervantes.
p. ; cm.
Includes bibliographical references and index.
ISBN 978-1-61728-580-6 (E-Book)
1. Small cell lung cancer. 2. Bladder--Cancer. 3. Neuroendocrine tumors. I. Maldonado, Jonathon
G. II. Cervantes, Mikayla K.
[DNLM: 1. Carcinoma, Small Cell--etiology. 2. Carcinoma, Small Cell--diagnosis. 3. Carcinoma,
Small Cell--therapy. QZ 365 S635 2009]
RC280.L8.S617 2009 616.99'424--dc22
2009029739
Published by Nova Science Publishers, Inc. 
New York
Contents

Preface ix
Chapter 1 Gene Silencing Therapy in Small Cell Lung Cancer
Which Agents, Targets, and Delivery Systems? 1
J. N. Moreira, A. O. Santos, L. M. Bimbo, L. C. Gomes da Silva,
M. C. Pedroso de Lima and S. Simes
Chapter 2 Small Cell Carcinomas: Contribution of Cytologic
Tools to Diagnosis and Management 51
Dilip K. Das
Chapter 3 Radiotherapy for Small Cell Lung Cancer 79
Don Yee
Chapter 4 Small Cell Carcinoma: Distinction
from Large Cell Neuroendocrine Carcinoma 101
Kenzo Hiroshima
Chapter 5 CT/MR-Based Movement Analysis of Lung Tumors: Impact
of Tumor Motion in the 3D-Based Radiotherapy of Lung Cancer 123
Arpad Kovacs
Chapter 6 Small Cell Lung Cancer: Clinical
Presentation and Diagnostic Modalities 149
Anant Mohan, Manisha Bhutani, Subhash Budania,
Samir Naik and Randeep Guleria
Chapter 7 Biotechnology and Cancer: Uses of Biotechnology
for Prevention, Diagnosis and Treatment of Cancer 167
Matias E Valsecchi
viii Contents

Chapter 8 Small Cell Carcinoma of the Urinary Bladder: Morphology,


Histogenesis, Diagnosis, Immunohistochemical Markers
and Therapeutic Strategies; Case Report and Review
of the Literature 179
Amelia Petrescu, Gabriela Berdan, Daniel Damian,
Viorel Jinga, Valentin Ambert, Narcisa Manea,
Liviu Niculescu and Florin Andrei
Chapter 9 Is the Curative Thoracic Radiotherapy Benefit
and Cost-Effective in Extensive Stage Small Cell Lung Cancer? 193
Gulden Diniz
Chapter 10 Optimal Treatment of Small Cell Lung Cancer 199
Bernard Lebeau
Chapter 11 Primary and Pure Neuroendocrine Carcinoma of the Bladder:
Anatomoclinical Report Cases, Review of the Literature
and Discussion of the Therapeutic Strategy 205
S. Ketata, H. FakhFakh, H. Ketata, A. Bahloul and M. N. Mhiri
Index 213
Preface

Small cell carcinoma is a type of cancer that almost always affects the lungs. Small cell
carcinoma is almost always caused by smoking, but exposure to large amounts of asbestos is
also a risk factor. Small cell carcinoma usually effects men more than women and while not a
common type of lung cancer, is considered very deadly. Unlike other types of cancer, small
cell carcinoma is not staged on a numerical scale but rather as simply limited or extensive.
Limited stage refers to cancer that is contained within the lungs or bronchial tubes only.
Extensive stage indicates the cancer has spread to areas outside of the chest. Limited stage
small cell carcinoma is rare because it is usually not diagnosed until it has become extensive.
Symptoms of small cell carcinoma lung cancer are similar to other types of lung cancer and
may include chronic coughing, wheezing, shortness of breath, sputum production, and
possibly weight loss. This new book gathers the latest research from around the globe on this
disease.
Chapter 1 - Gene silencing is a strategy already available to perform functional studies
that has also a great potential for therapeutic purposes. Upon confirmation, in a clinical
setting, of some of the successes achieved in a pre-clinical stage, such molecular targeted
strategy can ultimately give rise to a novel class of drugs.
Although the pharmacological modulation of oncogenes through gene silencing is not a
recent concept, it is still hampered by several issues. In this respect, the major findings
regarding the functional relevance of the molecular target, the potency and specificity of
different classes of gene silencing molecules, as well as some of the directions being taken in
other cancers, will be addressed. Focus will be given to BCL2, a gene with functions beyond
apoptosis, highly relevant in terms of anticancer intervention, including in small cell lung
cancer (SCLC). In addition, highlights will be devoted to in vivo delivery of gene silencing
molecules to the target cells. This has been one of the main obstacles for translating gene
silencing from an effective research tool into a feasible therapeutic strategy.
The evolution in the field of gene silencing has been accompanied by both the
clarification of previously addressed issues and the generation of new concepts such as
multitargeting, off-target effects, microRNA gene regulation, cancer stem cells, among
others. Gene silencing therefore represents an opportunity to change the treatment paradigm
of wide range of diseases. In this chapter, these topics will be discussed within the scope of
SCLC.
x Jonathon G. Maldonado and Mikayla K. Cervantes

Chapter 2 - Small cell carcinoma (SCC) is a malignancy with aggressive growth pattern,
high recurrence rate, and tendency to metastatize that mainly occurs in the lung, with primary
lesions in other sites very rare. The sites affected by extrapulmonary small-cell carcinoma
(EPSCC) are parotid, minor salivary glands of the tongue, sinonasal region, breast, thymus,
pleura, esophagus, extrahepatic bile duct, kidney and renal pelvis, urinary bladder, ovary,
endometrium, uterine cervix, and prostate. EPSCC has been recognized as a
clinicopathological entity distinct from the small cell carcinoma (SCC) of the lung. EPSCC
differs from small-cell lung carcinoma (SCLC) in respect of etiology, clinical course, and
survival. Both SCLC and EPSCC can occur as part of multiple primary neoplasms. Various
cytodiagnostic tools utilized for detection of small cell carcinoma are palpation-guided fine
needle aspiration (FNA) cytology, ultrasonographic (US) or CT-guided transthoracic FNA
cytology, transbronchial needle aspiration (TBNA), endoscopic US-guided fine needle
aspiration (EUS-FNA) biopsy, ultrasonographic (US)-guided FNA cytology of abdomen or
pelvic organs, and exfoliative cytology. The tumor cells of SCC are arranged mostly in
clusters of varying sizes and have minimal cytoplasm, finely stippled (salt and pepper)
chromatin, inconspicuous nucleoli, prominent nuclear molding and smearing effect. The
lesions considered in differential diagnosis of SCC are non-Hodgkin lymphoma, squamous
cell carcinoma of small cell type, or other malignant small round cell neoplasms. In case of
diagnostic difficulties, various ancillary studies may be of help in arriving at a diagnosis.
Besides cytokeratin, the neuroenocrine markers like chromogranin, neurone-specific enolase
(NSE), synaptophysin, CD56, and CAM5.2 show varying degrees of positive reaction in
SCC. Electron microscopy demonstrates dense-core granules in neoplastic cells of SCC.
When the diagnosis of SCC is reached in a patient with a lung mass based on biopsy report
including cytodiagnosis, a surgical treatment approach is no longer considered and
chemotherapy becomes the treatment of choice. However, surgical approach with or without
radiation therapy and chemotherapy has been resorted to in EPSCC in most locations
depending upon the extent of the disease.
Chapter 3 - Small cell lung carcinoma (SCLC) comprises approximately 20% of all
primary lung cancers. Biologically, SCLC is characterized by rapid proliferation, widespread
dissemination, and despite good response rates to initial treatments, poor overall survival
rates. The most widely-used staging system for this cancer features a dichotomy between
what is termed "limited-stage" or "extensive-stage" disease. Traditionally, chemotherapy has
been the mainstay of therapy for SCLC, but in the past 10-15 years, radiotherapy
interventions have provided the largest improvements in survival outcomes for SCLC
patients. Patients with limited stage disease derive local control and overall survival benefits
from the addition of concurrent thoracic radiation treatments given with their chemotherapy.
Prophylactic cranial radiation treatments given to limited stage disease patients whose disease
responds completely to initial induction treatments provides improved freedom from CNS
disease relapse and further overall survival benefits. The benefits of concurrent thoracic
radiotherapy and prophylactic cranial radiotherapy for limited stage disease patients have
been confirmed in several published meta-analyses. Patients with extensive stage disease
have what is considered incurable disease. Usually, these patients are treated with
chemotherapy alone, with radiotherapy reserved to palliate bothersome local symptoms
attributable to disease as they arise. A recently-published randomized trial has demonstrated
Preface xi

the CNS control and overall survival benefits of giving prophylactic cranial radiation
treatments to extensive stage patients who respond to their initial chemotherapy treatments.
From a radiotherapy perspective, the weight of the current evidence available from published
clinical trials indicates a beneficial role of: 1) thoracic radiotherapy given concurrently with
chemotherapy followed by prophylactic cranial irradiation in complete responders for
patients with limited stage disease and 2) prophylactic cranial irradiation for extensive stage
disease patients who respond to chemotherapy. This chapter will outline results from seminal
clinical trials that have established the important role of radiotherapy in the management of
SCLC patients. Ongoing areas of controversy with regards to SCLC radiotherapy including
ideal radiotherapy target volume and dose/ fractionation regimen for both thoracic
radiotherapy and prophylactic cranial irradiation and the potential role of consolidation chest
radiotherapy for patients with extensive stage disease will also be discussed.
Chapter 4 - Neuroendocrine carcinomas of the lung include the three grades of low-grade
typical carcinoid, intermediate-grade atypical carcinoid, high-grade large cell neuroendocrine
carcinoma (LCNEC), and small cell lung carcinoma (SCLC). SCLC is a malignant epithelial
tumor consisting of small cells with scant cytoplasm, ill-defined cell borders, finely granular
nuclear chromatin and absent or inconspicuous nucleoli. The cells are round, oval or spindle-
shaped and nuclear molding is prominent. The mitotic count is high. SCLC requires a light
microscopic diagnosis and does not require a demonstration of neuroendocrine differentiation
by electron microscopy or immunohistochemistry. Because the distinction between SCLC
and LCNEC is difficult in some cases, some propose that these carcinomas should be
classified as one high-grade neuroendocrine carcinoma (HGNC).
The authors reviewed the histological findings of HGNC and found that there was a
definite LCNEC and a tumor with characteristics of both LCNEC and SCLC. The latter
tumor cells had polygonal shape, a small amount of cytoplasm, and relatively large nuclei.
The nuclear chromatin was finely granular. The nucleoli were usually observed but were
small or inconspicuous. The tumors had organoid, trabecular, and palisading patterns, and a
rosette-arrangement was frequently observed. They temporarily defined this subtype as
intermediate cell neuroendocrine carcinoma (ICNEC), and examined biological features of
each group of HGNC using morphometry, immunohistochemical staining, loss of
heterozygosity (LOH), and methylation status of the p16 gene. Tumor cells of ICNEC were
positive for CD56, but negative for chromogranin A and synaptophysin. The frequency of
expression of NeuroD and p63 was higher in LCNEC than in SCLC, and that of mASH1,
p16, and TTF-1 was higher in SCLC than in LCNEC. The nuclear size of ICNEC cells was
between those of LCNEC and SCLC. The expression of CD56, mASH1, and p16 was high,
and that of NeuroD and p63 was low in ICNEC. The LOH analysis suggested that ICNEC
was close to SCLC. Our data suggest that ICNEC is closer to SCLC morphologically,
phenotypically, and genetically. Although further studies are needed to analyze the biological
behavior of LCNEC, ICNEC, and SCLC including sensitivity to chemotherapy, the tentative
recognition and inclusion of ICNEC into SCLC may reduce the interobserver discrepancy in
discriminating HGNC.
Chapter 5 - Lung cancer is the leading cause of death in tumor-related morbidity, both in
the male and female populations. The complex therapy of this malignant disease is based on
surgery, chemo-, and radiotherapy. Radiotherapy is often used in the treatment of lung cancer
xii Jonathon G. Maldonado and Mikayla K. Cervantes

either postoperatively or in a definitive setting depending on the tumor stage and general
condition of the patient. Local tumor control is a crucial question in the treatment process,
since the possibility of lung cancer recurrence is very high, even in early stages.
In 3D-based conformal radiotherapy of lung cancer, accurate delineation of PTV
(planning target volume) is critical. Many factors have to be taken in account during the
definition of PTV (e.g., microscopic spread of the tumor cells, daily setup errors, tumor
motion). In common practice, standard safety margins are added to clinical target volumes
(CTV), which are derived from a spiral CT scan. These safety margins are estimated
arbitrarily, potentially resulting in either excessive exposure of normal tissues (especially in
the case of combined chemo-radio therapy) or insufficient target volume coverage.
Overestimation of the PTV can result in a higher side effect profile, especially in combined
treatment settings. With the use of inappropriate PTV volume delineation, the delivery of an
ideal tumor-destroying dose becomes doubtful.
According to the International Commission on Radiation Units and Measurements
(ICRU) Report, Recommendation Nos. 50 and 62, planning target volume includes the gross
tumor volume (GTVvisible tumor mass), the clinical tumor volume (CTV, GTV plus
microscopic tumor spread), the internal margin (IM, uncertainties resulting from intra- and
interfractional tumor and organ motions) and the set-up margin (SM, uncertainties resulting
from daily setup and positioning errors). Uncertainty resulting from tumor movement must be
considered in 3D therapy planning, especially in case of IMRT or stereotactic therapy.
Moreover, normal tissue toxicity must be considered in the definiton process, too. This factor
has high importance in the treatment of lung cancer patients (lung toxicity, heart toxicity,
spinal cord).
The characterization of the internal margins needed for the radiotherapy of upper and mid
lobe lung cancers was developed following a complex study at our institution. The main goals
were to detect tumor movements, to analyze uncertainties in treatment planning arising from
tumor motions and to study the effectiveness of the fixation system used in our department for
lung cancer radiotherapy. This chapter aims to demonstrate the results of this study.
Chapter 6 - Lung cancer is second most common non-cutaneous cancer and leading
cause of cancer deaths in the United States. According to the SEER statistics, the total
number of new cases and deaths in 2008 were estimated to be 215,020 (accounting for
approximately 15% of all cancer diagnoses) and 166,280 (accounting for around 29% of all
cancer deaths), respectively. Mortality among men decreased by 1.3% per year from 1990
1994 and by 2.0% per year from 19942004. Death rates are approaching a plateau after
continuously increasing for several decades in women. These trends in lung cancer mortality
reflect the decrease in smoking rates over the past 30 years. Lung cancer is classified into two
broad varieties for treatment purposes: Non Small Cell Lung Cancer (NSCLC) and Small
Cell Lung Cancer (SCLC). The proportion of SCLC among all lung cancers has fallen from
2025% in the past to approximately 13% currently. However, it assumes significance due to
its relatively aggressive course and differences in clinical presentation from NSCLC.
Chapter 7 Introduction: This chapter will focus in the current uses of biotechnological
tools, including functional genomics, microarrays, proteomics, pharmacogenomics, gene
therapy, nanotechnology and bioinformatics, for the design and development of revolutionary
Preface xiii

diagnostic and therapeutics modalities that will represent the new standards of care in the
near future.
Methods: Using Pubmed, Ovid and EBSCO a MEDLINE database search was perfomed
from January 2002 to June 2008. Keywords used include: cancer, biotechnology, functional
genomics, nanotechnology or nanoparticles, proteomic, gene expression signature and
pharmacogenomics. Multiple combinations were used to enhance the search.
Results: There are at least three areas where the advances in biotechnology will most
likely produce innovative changes. The first area is related to the improvement in diagnostic
and prognostic assessment. Gene-expression signature, using microarrays analysis, can
predict relapse-free and overall survival, while functional genomics can be used to interrogate
a vast number of informative cancer-relevant phenotypes. Seldi-Tof and Maldi-Ms
techniques allow the study of proteomic patterns in serum in order to identify biomarkers that
can be used for screening and diagnosis purposes, including early detection of recurrences.
The proper interpretation of these data, generate by high-throughput modalities, in a reliable
and reproducible manner requires the help of a new discipline known as bioinformatics.
However, the most spectacular progress can be attributed to the domain of the
nanotechnology, especially through the production of multifunctional nanoparticles. Both
organic and inorganic nanoparticles not only can enhance significantly most of the previously
mentioned in-vitro techniques, but also represent the cornerstone for the progress of the other
two identifiable areas, tumor imaging and new therapeutic options. Improvements in
radiological techniques, using supermagnetic metal core nanoparticles or quantum dots, will
increase the sensitivity of the MRI allowing a non-invasive tumor labeling in-vivo; tumor
cells or tumor blood vessels could be tracked through the whole body. Lastly, all efforts are
directed towards the conquest of the precious personalized medicine. In this regard, three
approaches are fundamental: 1) exact prediction of toxicity, which is possible through the
pharmacogenomics studies 2) precise drug delivery (homing), obtainable through
nanoparticles coupled with multiple cancer-specific targeting ligands 3) highly specific and
accurate selection of molecular targets, made possible through the use of monoclonal
antibodies, small inhibitors particles and siRNA.
Conclusions: The exponential progress of the biotechnological techniques and the
increasing investment in new fields such as nanotechnology, make us foresee the rapid
advent of a new kind of therapeutic agents that will make real the dream of personalized
medicine.
Chapter 8 - Small cell carcinoma of the urinary bladder is a rare malignancy, comprising
less than 1% of urinary bladder carcinomas. It is highly aggressive, with a dismal prognosis,
usually presenting with advanced-stage disease. There is a male prevalence and the most
common symptom is hematuria. Morphologically, this tumor resembles its pulmonary
counterpart. There are many theories about the histogenesis of this tumor: urothelial,
neuroendocrine and from stem cells.
The aim of this chapter is to present the authors experience in the case of a 44-year-old
man with a history of smoking (10 cigarettes/day), hospitalized for one month with
intermittent hematuria in January 2007 at the Department of Urology, Prof. Dr. Th. Burghele
Hospital, Bucharest, Romania. Ultrasonography and cistoscopy revealed a sessile mass, sized
37/30 mm. The tumor was removed by transurethral resection and the fragments were
xiv Jonathon G. Maldonado and Mikayla K. Cervantes

processed by standard histopathological methods: fixed in 10% formaldehyde, paraffin


embedded and stained with HE and VG. They also performed immunohistochemical tests
including Cromo, EMA, NSE, CD56, NK1, P53 and HCG. Follow up studies of the patient
were conducted for 20 months. Echographic, CT, cistoscopic examination and a new TUR
revealed no tumor relapse and no metastases.
Histopathological examination showed a tumor proliferation composed mainly of sheets
of small cells, uniform and rounded, mitotically active and a component of a typical low-
grade urothelial carcinoma. Immunohistochemical markers emphasized that a diffuse positive
staining of the small cell component for Cromo, EMA, NSE, CD56, NK1 and P53 was
strongly positive (80%) and the urothelial carcinoma component was focally positive for
HCG.
The microscopical diagnosis was small cell carcinoma of urinary bladder coexisting with
a low-grade papillary urothelial carcinoma, invading the lamina propria (pT1).
The aggressive behaviour of this entity is due to the presence of the small cell component
and possibly to the association with HCG positive immunoreactivity. Their patient was
treated by local instillation with farmarubicine and six series of intravenous cisplatin. The
authors mention that until now there is no agreement about a standard therapy management.
Some authors recommend only cistectomy, while others, a combined therapy: cistectomy,
chemotherapy and radiotherapy. Based on this case and data from the literature, the authors
consider that immunohistochemical profile is helpful in diagnosis, and this type of cancer
must be known not only by the pathologist, but also by the urologist and oncologist because
of its aggressive behavior and its different therapeutic strategies.
Chapter 9 - Lung cancer is the most common cause of cancer- related death and small
cell lung cancer (SCLC) comprises about 15- 20 % of all lung cancer cases. Majority of
patients present with metastatic disease at diagnosis. Therapy regimens of SCLC include
systemic chemotherapy, irradiation of primary tumor and/ or metastases, adjuvant and
prophylactic cranial radiotherapy, as well as medication of pain or other symptoms.
Treatment is primarily dependent on the stage of SCLC and presence of metastases.
Chemotherapy still is the cornerstone in treatment. The standard chemotherapy is the
combination of etoposide and cisplatin. Despite initial sensitivity to therapy, >80% of the
patients die from recurrent disease within 2 years.
In limited stage disease, curative radiation therapy is delivered with the intention of
eradication all tumor cells and thereby achieving cure. Minimal tumor doses in the range of
40- 45 Gy or more by conventional fractionation are necessary to effectively control tumors
in thorax. There is a common perception that radiotherapy for extensive stage SCLC is
essentially palliative. Unfortunately all treatment regimens continue to show only modest
improvement in outcome and their effects remain small for metastatic disease.
The therapy management of SCLC has been focus of extensive investigation over the
past two decades and several new drugs are currently been developed to improve the survival
in SCLC. It was reported to provide an additional survival benefit in advanced disease and to
be less toxic. In the most of these studies, the cost- effectiveness of these drugs was not
calculated. Moreover, in these reports, therapy benefits for some population such as elderly
patients and patients with poor performance status underrepresented. If it is emphasized that
the half of all lung cancers occurs in persons aged over 60 years, it is easily estimated how
Preface xv

difficult to decide therapeutic approach and to make the right balance between expected
benefits of treatment and potential toxicity. The elderly patients are more likely to be given
only supportive care or no therapy. Therefore intensive, high dose polychemotherapy could
not be planned for therapy of most patients.
Contrary radiation therapy may be safely delivered to elderly patients with poor
performance status. In a recent study, the efficacy of curative and palliative radiotherapy in
the treatment of E- SCLC was evaluated and compared of therapy effect on survival in with
or without metastatic disease. According to the statistical findings; the gains in duration of
median survival with the curative thoracic irradiation are 151.97 days in all 128 patients. The
gains in duration of median survival with the curative thoracic irradiation are 125.75 days in
metastatic patients and 190.6 days in others. This result raises the question of whether
treating with radical thoracic radiotherapy with concomitant chemotherapy, consisting of
first-line drugs might be more beneficial and cost-effective as well as less toxic treatment of
extensive stage SCLC.
Chapter 10 - Thirty years of clinical research activity in the field of small cell lung cancer
(SCLC) treatment leads the author to conclude that standard treatment for this disease has not
yet been precisely defined. Some recent editorial assertions are untrue, due to rapid evolution
of knowledge or to simplification risks. The author will try here to highlight some ideas
whose application presently can optimise treatments of patients affected by this cancer.
Chapter 11 Objective: Neuroendocrine carcinoma arising in the bladder has been
described in many case series. However, primary and pure small cell carcinomas (PSCC) of
the bladder are very rare, and patients commonly present with metastatic disease. No
prospective studies evaluating the most efficient treatment have been published. The authors
reviewed our experience with treating these tumors to evaluate their histopathological
characteristics and clinical outcome.
Patients and Methods: This chapter presents the authors experience in 5 patients with
PSCC of the bladder during a 7-year period. The patients tumor characteristics, therapy,
follow-up and survival status were documented.
Results: All patients were male with a mean age of 67 years. The main clinical
presentation was macroscopic hematuria. All tumors were invasive at the time of diagnosis.
Systemic chemotherapy was given in 4 patients, and one patient was treated by radical
cystectomy. The overall median survival was 17 months.
Conclusion: PSCC of the bladder should be considered a systemic disease, because most
patients present with metastases. Prospective studies are needed to determine the optimal
treatment.
In: Small Cell Carcinomas: Causes, Diagnosis and Treatment ISBN: 978-1-60741-787-3
Editors: J. G. Maldonado and M. K. Cervantes 2009 Nova Science Publishers, Inc.

Chapter 1

Gene Silencing Therapy in Small Cell


Lung Cancer Which Agents, Targets,
and Delivery Systems?

J. N. Moreiraa,b,*, A. O. Santosa,b, L. M. Bimboa,b,


L. C. Gomes da Silvaa,b, M. C. Pedroso de Limab,c and S. Simesa,b
a
Laboratory of Pharmaceutical Technology, Faculty of Pharmacy,
b
Center for Neuroscience and Cell Biology, University of Coimbra, Portugal,
c
Department of Biochemistry, Faculty of Sciences and Technology, University of
Coimbra, Portugal.

Abstract
Gene silencing is a strategy already available to perform functional studies that has
also a great potential for therapeutic purposes. Upon confirmation, in a clinical setting, of
some of the successes achieved in a pre-clinical stage, such molecular targeted strategy
can ultimately give rise to a novel class of drugs.
Although the pharmacological modulation of oncogenes through gene silencing is
not a recent concept, it is still hampered by several issues. In this respect, the major
findings regarding the functional relevance of the molecular target, the potency and
specificity of different classes of gene silencing molecules, as well as some of the
directions being taken in other cancers, will be addressed. Focus will be given to BCL2, a
gene with functions beyond apoptosis, highly relevant in terms of anticancer
intervention, including in small cell lung cancer (SCLC). In addition, highlights will be
devoted to in vivo delivery of gene silencing molecules to the target cells. This has been
one of the main obstacles for translating gene silencing from an effective research tool
into a feasible therapeutic strategy.
The evolution in the field of gene silencing has been accompanied by both the
clarification of previously addressed issues and the generation of new concepts such as
multitargeting, off-target effects, microRNA gene regulation, cancer stem cells, among
others. Gene silencing therefore represents an opportunity to change the treatment
2 J. N. Moreira, A. O. Santos, L. M. Bimbo et al.

paradigm of wide range of diseases. In the present review, these topics will be discussed
within the scope of SCLC.

Abbreviations list
Akt, v-akt murine thymoma viral oncogene homolog
Bcl-2, B-cell lymphoma/leukemia 2
CCR, chemokine (C-C motif) receptor
CXCR4, chemokine (C-X-C motif) receptor 4
DISC, death-inducing signaling complex
DSB, double strand breaks
dsRNA, double-stranded RNA
ECM, extracellular matrix
FAK, focal adhesion kinase
FGF, fibroblast growth factor
FGF-R fibroblast growth factor receptor
GPCR, G-protein coupled receptor
HGF, hepatocyte growth factor
HH, hedgehog
hTERT, human telomerase reverse transcriptase
IGF-1R, insulin-like growth factor 1 receptor
LNA, locked nucleic acid
MBO, mixed backbone
miRNA, microRNA
MOE, 2-O-(2-methoxy)ethyl
nt, nucleotides
ODNs, oligodeoxynucleotides
OMe, 2-O-methyl RNA
PDK, PI-dependent protein kinase
PTEN, phosphatase and tensin homolog
PI, phosphatidylinositol
PI3K, phosphoinositide 3-kinase
PLC, phospholypase C
PS, phosphorothioate
PTK, protein tyrosine kinase
RISC, RNA-induced silencing complex
RNAi, RNA interference
RTK, receptor tyrosine kinases
SCF, stem cell factor
SCLC, small cell lung cancer
SDF-1/CXCL12, stromal-cell-derived factor-1
shRNA, short-hairpin RNA
siRNA, short interfering RNA
Gene Silencing Therapy in Small Cell Lung Cancer 3

Skp2, S-phase kinase associated protein 2


uPAR, urokinase plasminogen activator receptor
VEGF-R, vascular endothelial growth factor receptor

Introduction
Small cell lung cancer (SCLC) is an aggressive and highly metastatic neuroendocrine
histological subtype of lung cancer, that corresponds to about 15% of the cases of lung cancer
[1]. Lung cancer, along with prostate, breast, and colon cancer, is among those with the
highest incidence in developed countries such as the USA [2], and is the one with the worst
prognosis. Tobacco smoke is responsible for a 10- to 20-fold increased risk of lung cancer in
smokers compared with never smokers (reviewed in [3]). Lung cancer comprises SCLC and
non-small cell lung cancer, which includes mainly squamous cell carcinoma,
adenocarcinoma and large cell carcinoma. Despite initially responding to chemotherapy, with
response rates of up to 80%, most SCLC patients relapse and die from chemotherapy resistant
disease [4]. The 5-year period survival rate for patients diagnosed with invasive SCLC and
bronchus cancer in the USA, from 1996-2004, was 5.9% [5]. This clearly shows the urgent
need for the development of novel therapeutic approaches.

1.1. Cancer Genes as Potential Targets of Therapeutic Gene Silencing

Cancer is a genetic disease, arising from the cellular accumulation of genetic alterations,
which enable the cells to evade their usual growth control [6]. These cells have the ability to
spread and grow in distant sites or to propagate indefinitely and can be fatal for the individual
organism in which they occur. There are several sequential steps which are necessary for the
development of a tumorigenic phenotype, where selective proliferative advantages are
gained, transforming normal cells into cancer cells [7]. The types of genetic alterations that
can occur are described below.

1.1.1. Oncogenes and tumor suppressor genes


Oncogenes are genes present in the human genome (as proto-oncogenes), that promote
the development of cancer once activated. The activation can happen either via association
with retrovirus or via mutational events that depend on non-viral mechanisms, such as point
mutation, chromosomal fusion, gene amplification, or deregulated expression [8]. Those
genes are usually involved in cellular proliferation or survival. Importantly, it is not the
simple accumulation of mutations, but rather the order in which they occur that determines
progression towards neoplasia [7]. Examples of oncogenes that are activated by gene
amplification in various tumors include MYC, ABL, MYB, ERBB, K-RAS, and MDM-2.
Conversely to oncogenes, tumor suppressor genes display the function of repressing
cellular proliferation and/or maintaining normal cellular differentiation, facilitating the
normal mechanisms of cell adhesion, stopping the progression of cell cycle (G1, S-phase,
mitosis or G2) so that the normal DNA-repair mechanisms take place, and maintaining
4 J. N. Moreira, A. O. Santos, L. M. Bimbo et al.

normal cellular shape and cell contact inhibition mechanisms [9]. Tumor suppressor genes
could be further divided in gatekeepers, that prevent unwanted cell growth by eliminating
potential cancer cells and caretakers which protect the genome from accumulating
oncogenic mutations [10]. Examples of tumor suppressor genes that are inactivated in various
tumors include phosphatase and tensin homolog (PTEN), TP53 and retinoblastoma (RB).
It has been shown that suppression of one oncogene [11, 12], or restoration of function of
tumor suppressor genes [13], can regress the malignant phenotype or cause the death of the
malignant cell.

1.1.2. MicroRNAs
MicroRNAs (miRNAs) have emerged as a large class of short endogenous non-protein
coding small RNAs. Studies have shown that numerous miRNA genes occur in chromosomal
regions that undergo rearrangements, deletions, and amplifications in cancer cells [14]. The
over- or underexpression of miRNAs is expected to result in down- or upregulation,
respectively, of the protein product of the target mRNAs. The therapeutic value of miRNA in
cancer is specially relevant in case its target is an oncogene or a tumor suppressor gene [15, 16].
Given the emerging evidence that miRNAs are important players in oncogenic or tumor
suppressor activities, it is presently considered important to pursue strategies that interfere
with miRNAs and to develop them as novel cancer therapies (reviewed in [17]).

1.1.3. The hallmarks of tumorigenic phenotype


The tumorigenic phenotype of a cell is nothing more than the manifestation of six
fundamental alterations in its physiology, which lead to neoplasic growth [18]. This model,
which states that cancer progression can be a process analogous to Darwinian evolution, has
since been adopted as canonical for tumor development. Oncogenes and tumor suppressor
genes were found to be implicated in a great extent in such process, and therefore are key
players in each of the six alterations. These hallmarks can be briefly described as: (I) Self-
sufficiency in growth signals, in which the cells override the requirement of exogenous
growth factors for proliferation; (II) Insensitivity to growth inhibitory signals, in which
cellular quiescence and terminal differentiation responses are disrupted by inactivation of
certain genes (like the tumor supressor Rb) or are circumvented by the expression or
activation of others (as the c-Myc oncogene); (III) Evasion of programmed cell death, with
several mechanisms involved, which include: 1) mutation of the tumor suppressor p53 [19];
2) elevated NF-B activity [20, 21]; 3) activated PI-3K pathway [22, 23]; 4) overexpression
of B-cell lymphoma/leukemia 2 (Bcl-2), an anti-apoptotic member of the Bcl-2 family of
proteins [24-27]. (III) Self-sufficiency in growth signals, in which the cells override the
requirements of growth factors for proliferation, achieving growth factor autonomy; IV)
Limitless replicative potential, in which cells, usually with a limited life-span related to the
number of cell divisions they undergo due to telomeres shortening [28, 29], overcome both
senescence (proliferation arrest at a certain number of doublings) and crisis (death associated
to end-to-end fusion of chromosomes) in a process termed immortalization, usually
associated with upregulation of telomerase enzyme; (V) Sustained angiogenesis, when in
order for the tumor to grow, it must recruit a network of new blood vessels, required for
proper nourishment and removal of metabolic waste [30, 31]; (VI) Tissue invasion and
Gene Silencing Therapy in Small Cell Lung Cancer 5

metastasis, which can be briefly described as the spread of cells from the primary neoplasm
to distant organs and the formation of new tumors in an organ-specific pattern [32, 33].

1.1.4. Opportunities for therapeutic intervention


It is interesting to note that a number of genes that are frequently overexpressed, belong
to more than one of the six essential groups described above (e.g. VEGF, RAS, RAF, PKC-,
MYB, BCL-2) and therefore may represent particularly promising targets for therapeutic
intervention. Suppression of their functions, namely through post-transcriptional gene
silencing, could lead to restoring normal phenotype.
In addition to the relevance of the gene to the tumor cells, the targeted mRNA of the
gene of interest should ideally have a low turnover (so that the destroyed message is not
rapidly replaced), whereas the protein should have a high turnover (so that it decays
quickly when the mRNA level is reduced) [34]. Furthermore, to maximize the silencing
effect, the proteins intracellular role should be acutely sensitive to changes in protein
concentration [34]. In the case of miRNAs, it could also be of interest to specifically
knockdown the expression of oncogenic miRNAs, or inversely to deliver artificially the
candidate tumor suppressor miRNA [35]. However, it cannot be forgotten that other
approaches, besides gene silencing, can be considered in the therapeutic inhibition of
oncogenes function, like monoclonal antibodies or small molecule inhibitors. This has been
particularly explored in receptors with tyrosine kinase activity and in intracellular enzymes,
being trastuzumab (Herceptin) and imatinib (Gleevec) successful examples, against HER-
2 receptor and Bcr-Abl fusion protein, respectively. Notably, large chemical libraries, high-
throughput screening and virtual screening by molecular modeling techniques, have eased the
process of lead compound identification and rational drug design [36, 37]. Nevertheless, once
there is a good carrier system available, gene silencing will offer the direct possibility to
interfere with any desired gene.

1.2. Gene Silencing Agents: A Novel Class of Drug Molecules

Several gene silencing molecules have been developed in the last three decades after the
discovery, in 1978 by Zamecnik et al. [38], that short DNA molecules are able to inhibit gene
expression through degradation of the complementary mRNA. The scientific community
early realized the potential of this approach to study gene function and to treat diseases with
an aberrant gene expression, such as cancer disorders. In fact, antisense approaches against
several of those targets have already reached clinical evaluation (Table 1).

1.2.1. Antisense oligodeoxynucleotides


Antisense oligodeoxynucleotides (ODNs) are chemically synthesized single-stranded
DNA, generally 13 to 25 nucleotides long [93], which hybridize to the complementary sense
mRNA or pre-mRNA by Watson-Crick base pairing. Binding to the target mRNA prevents
gene expression by one of two main mechanisms: degradation of the mRNA by the
endonuclease RNase H, which recognizes RNA-DNA hybrids ; or by blocking translation
through sterical disruption of ribosomal movement along the transcript [94, 95] (Figure 1).
6 J. N. Moreira, A. O. Santos, L. M. Bimbo et al.

Table 1. Molecular targets and current status of gene silencing approaches


in clinical evaluation in oncology.

Molecular target Drug; Size chemistry Trial References & ClinicalTrials.


phase gov identifiers
B-Cell Genasense (Oblimersen I - III [39-56]; 44 entries
Lymphoma/Leukemia 2 Sodium or G3139 ODN); 18 nt inClinicalTrials.gov,
(BCL2) PS (NCT00017251,
NCT00042978, NCT00005032
in SCLC)
SPC2996; LNA I - II NCT00285103
Survivin LY2181308 sodium; 2'-O- I-II [57]; NCT00620321;
methoxymethyl NCT00642018
X-linked inhibitor of AEG35156 (GEM 640); OMe I - II [58]; 6 entries in
apoptosis (XIAP) ClinicalTrials.gov
Raf-1 ISIS 5132 (CGP 69846A); 20 I - II [59-64]; NCT00003892
nt PS
LErafAON; 15 nt PS, in I [65, 66]; NCT00100672;
cationic liposomes NCT00024661;
NCT00024648
Protein kinase C- Affinitak (ISIS 3521 or I - III [60, 64, 67-73];
(PKC-) LY900003); 20 nt PS NCT00042679;
NCT00034268;
NCT00017407;
NCT00003989
PKC- + Raf-1 Affinitak + Isis 5132; 20 nt II [22, 26]; NCT00003236
PS
Protein Kinase A (PKA) GEM 231 (HYBO-165); 18 nt I [74, 75]; NCT00004863;
MBO NCT00004864
H-RAS ISIS 2503; 20 nt PS I - II [76-78]; NCT00005594;
NCT00004193;
NCT00006467
MYB G4460 (LR-3001); 24 nt PS I - II [79]; NCT00780052;
NCT00002592
Clusterin OGX-011 ; MBO (MOE + PS) I - II [80]; 7 entries in
ClinicalTrials.gov
Heat shock protein 27 OGX-427; MBO (MOE + PS) I NCT00487786
(HSP27)
Methyltransferase 1 MG98; 20 nt MBO (OMe + I - II [81-83]; NCT00003890
(DNMT1) PS)
M1 subunit of GTI-2501; 20 nt PS I/II [84]
ribonucleotide
reductase (RRM1)
M2 subunit of GTI-2040; 20 nt PS I - II [85-88]; 10 entries in
ribonucleotide ClinicalTrials.gov
reductase (RRM2) CALAA-01; siRNA I NCT00689065
encapsulated in a targeted
nanoparticle
Gene Silencing Therapy in Small Cell Lung Cancer 7

Table 1. (Continued)

Molecular target Drug; Size chemistry Trial References & ClinicalTrials.


phase gov identifiers
TP53 Aezea (EL625 or Cenersen I - II [89]; NCT00636155;
sodium); 20 nt PS NCT00074737
Molecular target Drug; Size chemistry Trial References & ClinicalTrials.
phase gov identifiers
Vascular endothelial Veglin (VEGF-AS); 21 nt - PS I - II [90]; NCT00668499
Growth Factor (VEGF)
VEGF receptor-1 Angiozyme (RPI.4610) II [91]; NCT00021021
(VEGFR-1) Ribozyme; Hammerhead
Hypoxia inducible EZN-2968; LNA I NCT00466583
factor-1 (HIF-1)
Bcr-Abl siRNA (single [92]
case)
nt, nucleotides; PS, phosphorothioate; LNA, locked nucleic acid; OMe, 2-O-methyl RNA; MBO,
mixed backbone; MOE, 2-O-(2-methoxy)ethyl RNA.

Figure 1. Main steps of the principal mechanism of action of antisense oligdeoxinucleotides (ODN)
(Reprinted with permission from [95]).

The mechanism that is present, largely depends on the structure and chemistry of the
oligonucleotides [96].
The use of unmodified ODNs (phosphodiester ODNs) has been limited, as they are
rapidly degraded by nucleases and are inefficiently internalized by cells. Therefore, a variety
of chemically modifications have been developed in order to increase the in vivo stability,
specificity to the target mRNA, cellular uptake, to improve biodistribution, and to decrease
toxicity [93-98]. Phosphorothioate (PS) ODNs are an example of ODNs that result from a
chemical modification in the phosphodiester backbone, in which one of the nonbridging
oxygens is replaced by sulphur. It is the most widely used ODN because of its increased
8 J. N. Moreira, A. O. Santos, L. M. Bimbo et al.

nuclease resistance, increased solubility and efficient activation of RNase H. However, the
PS backbone increases the probability of interaction with other molecules of mRNA with
reduced complementarity (off-target effects) and has high affinity for various proteins, which
could lead to several side effects [93, 94]. Other chemical modifications have been
intensively developed in the last years, and are reviewed elsewhere [94, 95].
Targeting directly the gene (antigene approach) instead of mRNA (antisense approach)
could be advantageous, as the downregulation of mRNA through inhibition of transcription is
more efficient and has a longer duration. In the antigene approach, the blocking of gene
expression is achieved by chemically synthesized single-strand or double-strand ODNs, of 10
to 30 nucleotides long, that acts upon forming DNA triple helices by reverse Hoogsteen
hydrogen bonds in a sequence-specific manner [94]. The triplex-forming
oligodeoxynucleotides bind to the purine-rich strand of the duplex at physiological pH and in
the presence of magnesium [99], preventing the access of proteins required for transcription
or blocking the initiation or elongation of the transcript complex [94].

1.2.2. Ribozymes and DNAzymes


Ribozymes and DNAzymes are, respectively, catalytic single-stranded RNA and DNA,
which bind and catalyze the cleavage of mRNA in a sequence-specific manner [98]. One of
the most studied ribozymes for cancer treatment is the hammerhead ribozyme, which consists
of two substrate-binding arms that are complementary to the target RNA, and a conserved
catalytic domain that is most efficient at triplets of AUC and GUC [98, 100]. Similarly,
DNAzymes are composed by two substrate-binding arms of 7-8 nucleotides and a catalytic
domain of 15 nucleotides. It is most efficient at AU and GU duplets. Several studies have
suggested that DNAzymes are catalytically more efficient than ribozymes [97, 98, 101].

1.2.3. RNA interference


RNA interference (RNAi) was initially described in 1998 by Fire et al. [102], who have
found that long double-stranded RNA (dsRNA) added to the worm produced a strong
silencing of complementary mRNA. The application of this long dsRNA to mammalian cells
is not as advantageous, as it activates an undesirable interferon response. However, this major
drawback was solved in 2001 by Elbashir et al. [103, 104] who demonstrated that RNAi in
mammalian cells could be mediated by dsRNA of 21 to 23 nucleotides long, with a
characteristic 3overhang of 2 nucleotides, known as small-interfering RNA (siRNA),
without activation of the same interferon response.
In mammalian cells, siRNA are obtained from exogenous long dsRNA, which are
cleaved by the ribonuclease III enzyme Dicer. The duplex siRNA is unwound and the strand
with lower internal stability at the 5 end (antisense strand) is incorporated into the RNA-
induced silencing complex (RISC) [105]. The antisense strand guides the RISC to the
complementary target mRNA (Figure 2), which is cleaved by an endonuclease of the
complex, the Argonaute 2. The cleavage takes place between nucleotides 10 and 11 relative
to the 5-end of the siRNA guide strand. The siRNA is then recycled by RISC and other
mRNA molecules are cleaved, leading to an efficient silencing of gene expression [97, 106,
107].
Gene Silencing Therapy in Small Cell Lung Cancer 9

The most common strategy to induce RNAi in mammalian cells is to transfect chemically
synthesized siRNAs, which mimic the Dicer products. Nevertheless, another strategy has
been used, namely the nuclear synthesis of short-hairpin RNA (shRNA), structures
characterized by a stem and a loop region, which are processed by Dicer (as long dsRNA and
pre-miRNA) into duplexes like siRNA. The expression of shRNA in cells is carried out by
plasmids or viral vectors (Figure 2). Upon integration, they lead to a stable and prolonged
knockdown in contrast with episomal forms or siRNAs, which are diluted over successive
cellular divisions or degraded [108]. Moreover, the generation of siRNA from shRNA by
Dicer, seems to result in higher silencing potency than direct siRNA delivery to cells [109].

Figure 2. Cellular mechanism of RNA interference (Reprinted with permission from [95]).

It is widely accepted that a siRNA has multiple effects beyond those caused by the
degradation of its target mRNA. This is due to interference with the mRNA to which the
siRNA sequence has partial complementarity, by a miRNA similar mechanism. Thus,
different siRNAs against the same mRNA target will have different gene regulation profiles
[110], and could result in a toxic phenotype that is independent of the target mRNA [111].
Interestingly, even a siRNA against an exogenous protein, such as GFP, leads to a wide
range of upregulated and downregulated genes in human cells [112]. In order to increase the
specificity and potency, several rules should be followed when a siRNA is designed, such as
low content of poly-U and GU-rich regions, avoidance of immunostimulatory motifs [111,
113, 114], low G/C content, low hybridization energy at the antisense 5, lack of inverted
repeats, among others [115]. Moreover, using all the knowledge obtained from ODNs,
several chemical modifications have been proposed not only to improve the specificity, while
reducing the off-target effects [116, 117], but also to increase the potency and stability [118,
119].
10 J. N. Moreira, A. O. Santos, L. M. Bimbo et al.

2. The Challenge of Target Validation in SCLC


The initial proposal of a gene as an oncogene and the validation of the first set of data, is
a stepwise work. Candidate genes for therapeutic intervention are in general rationally
selected based on previous knowledge in tumor biology, built by a multiplicity of studies on
singular proteins, or via high-throughput experiments, such as screenings of gene
amplification, gene expression, or siRNA libraries. The validation process is usually the
summation of independent contributions, and classically comprises evaluation of the
frequency of protein or gene overexpression and/or the frequency of gene amplification in
tumors, determination of the prognostic value of the expression levels in patients, artificial
overexpression of the gene, knockout or mutant murine models, gene knockdown by
antisense or RNAi, and the use of inhibitors of the target protein or of the target protein-
activators.
Accumulated experience with gene silencing molecules has shown that their use in target
validation requires caution. It became clear that ODN and siRNA do not present the
specificity initially expected. Ascribing a function to a certain gene, by downregulation of the
mRNA, and consequently of the protein levels, with antisense ODN or RNAi, can only be
truly trusted if different sequences and/or classes of molecules render the same outcome
(reviewed elsewhere [120]). Off-target effects are a reality, although possibly helpful if not
ignored. Off-target downregulation due to complementarities in the seed region of several
siRNA within a siRNA library, served to first set the evidence of the role of Mcl-1 in the
resistance of some SCLC cell lines to the Bcl-2/Bcl-xL inhibitor ABT-737 [121].
In respect to SCLC, target validation faces several challenges. First, tumor specimens for
evaluation are limited, since patients are usually diagnosed in an advanced stage, where there
is no longer place for tumor resection. Biopsy specimens available might not be of the
required quality, although biopsies are considered to be representative of the tumor [122].
Cell lines have been the necessary object of the majority of the studies, although in vitro cell
culture might select clones with a particular phenotype, reflecting characteristics that make
them adapted to those specific conditions [123], and ignore completely the complex signaling
network within the tumor microenvironment. Artificial overexpression of a gene might
produce artifacts, and SCLC cell lines are difficult to transfect [124, 125]. Genetic instability
allows an increased propensity for the insertion of new mutations in a reduced time frame,
and selection of stable transfectants could easily co-select resistant clones independently of
the expression of the resistance gene. Murine models of SCLC also comprise several
limitations. A commonly used model is the subcutaneous implantation of human SCLC in
immunocompromised mice, which is very different of the natural behavior of SCLC. A
model of murine SCLC that closely mimics the human disease was only developed in 2003,
based on concomitant interruption of RB and TP53 loci [126].
It is unlikely that the downregulation or inhibition of a single target will ever kill all
tumor cells, the ultimate goal of any cancer therapy. There is presently the notion that
multitargeting is a necessary approach to achieve stronger responses, and to reduce the
development of drug resistance. This is perhaps not contrary to gene silencing, conversely to
what could be first thought. By learning how to take advantage of the network of
simultaneously silenced genes produced by gene silencing molecules, mimicking what the
Gene Silencing Therapy in Small Cell Lung Cancer 11

nature achieved with miRNA could be envisaged as a way to accomplish molecular


multitargeting. Alternatively, novel synergistic drug combinations comprising gene silencing
could be pursued.

Table 2. Poor prognostic predictors in SCLC whose potential as targets of therapeutic


intervention remains to be evaluated.
Gene Comment Ref.
NCAM Correlated with poor patient survival, reduced cell-cell [127-
(Neuronal-cell adhesion adherence, high clonogenic ability and high incidence of 129]
molecule) intracutaneous metastasis in nude mice.
ERCC1 Predictor of poor outcome in SCLC patients treated with [130]
(excision repair cross- chemotherapy with or without radiotherapy, especially
complementation group in limited stage SCLC.
1 protein)
hASH1 Correlated with a significant shortened patients survival [131]
(The human achaete time.
scute homolog 1)
MRP Negatively correlated with response rate to [132]
(P-glycoprotein and chemotherapy, being increasingly expressed in
multidrug resistance metastatic cells.
protein)
MMPs Increased tumoral expression of MMP-3, -11, and -14 [133]
(Matrix were independent negative prognostic factors for
metalloproteinases) survival.
BCL2L10 (Bcl-2-like Correlation with shorter survival. [134]
10 or Bcl-B)

3. Potential Targets for Therapeutic Gene


Silencing in SCLC
Within the scope of SCLC, a limited number of potential targets susceptible of
therapeutic gene silencing have emerged. This section focuses on genes that have been object
of gene silencing in SCLC. Nonetheless, other targets considered relevant to the discussion
were included. The genes whose expression was directly correlated to poor prognosis are
promising targets, though some have not been further studied for their mechanistic
contribution or assessed for their potential as targets of therapeutic intervention in SCLC
(Table 2).

3.1. Anchorage-Independency and Anchorage Signaling

While untransformed epithelial cells require attachment to a basement membrane (and


cell-to-cell contact) to survive, SCLC cells grow in vitro in the absence of extracellular
matrix (ECM), as floating aggregates. Integrins are a classical example of a group of ECM
12 J. N. Moreira, A. O. Santos, L. M. Bimbo et al.

receptors that mediate adhesion, and transmit growth and survival signals. In the absence of
adherence, some other stimuli, constitutive activation, or gain-of-function mutation, must
provide the alternative signals to active integrins. The proteins involved in such processes
could therefore represent targets of therapeutic intervention. In SCLC, secreted growth
factors that act in autocrine or paracrine loops, are one important source of growth stimuli
[125]. Also constitutive activation of phosphoinositide 3-kinase/v-akt murine thymoma viral
oncogene homolog (PI3K/Akt) pathway or focal adhesion kinase (FAK), have been reported
[125, 135, 136] as important mediators of survival signals.
Despite anchorage independency, SCLC cells have not lost the ability to bind to ECM
components, or to ligands at the surface of stroma cells. Several reports have implicated
binding to specific ligands at the local environment of SCLC, either at the primary site or
metastatic niche, as a source of signals that protect cells from cytotoxic drugs, possibly
causing the high recurrence frequency of the disease [137].

3.1.1. Integrins and chemokine receptors


Integrins are a family of heterodimer cell adhesion receptors that mediate the binding and
signaling cascades in response to ECM. 21, 41, and 51 integrins are frequently
expressed in SCLC [138]. Although some specific associations of ECM component and
integrin receptor have been implicated in differentiation and growth inhibition of SCLC cells
[139], most reports demonstrate that integrin activation promotes resistance to serum
starvation and several cytotoxic stimuli in SCLC cells [136, 137, 140-142]. Notably, overall
survival of patients with high expression of 1-integrin was significantly worse than those
patients with low expression, being more important as a poor prognostic factor than the
clinical stage of SCLC patients [122]. In this respect, it is hypothesized that ECM overrides
treatment-induced cell-cycle arrest and apoptosis, via 1-integrin and PI3K/Akt activation
(Figure 3), allowing the survival of SCLC with persistent DNA damage, and the acquisition
of novel mutations that confer drug resistance [141].

Figure 3. Schematic representation of activated PI3K/Akt signaling cascade by lipid raft-located RTKs
or by 1-integrins, with the possible cooperation of activated CXCR4. The most promising targets of
therapeutic gene silencing are represented by a target symbol.
Gene Silencing Therapy in Small Cell Lung Cancer 13

Figure 4. Schematic representation of the signaling cascades activated by cell membrane G-protein
coupled receptors (GPCRs) in SCLC. The most promising targets of therapeutic gene silencing are
represented by a target symbol.

Chemokines expressed at the surface of stroma cells, also play a role in the drug
resistance mechanism described above, as well as in the metastization process to specific
organs. The chemokine (C-X-C motif) receptor 4 (CXCR4) for the chemokine stromal-cell-
derived factor-1 (SDF-1/CXCL12), was found to be the major chemokine receptor in 10
SCLC cell lines, and to mediate proliferation, increased cell motility, and adhesion [143]. The
CXCL12 chemokine induces adhesion to immobilized VCAM-1, fibronectin and collagen,
cooperating with integrins (Figure 3) at promoting resistance to cytotoxics [136].
Remarkably, the chemokine (C-C motif) receptors 4, 5 and 9 (CCR4, CCR5, CCR9) were
found to be expressed in all metastatic lesions of a murine human SCLC (metastatic) model,
which is in agreement with the expression of the correspondent ligands in the metastasized
organs [144].

3.1.2. Autocrine loops 1 G-protein coupled receptors and downstream


effectors
SCLC frequently expresses autocrine growth signaling systems involving
neurotransmitters such as acetylcholine [145, 146], neuropeptides, and their respective
receptors [147, 148] that belong to the large family of G-protein coupled receptors (GPCR).
The binding of a ligand increases the levels of intracellular Ca2+ through phospholypases C
(PLCs), which activate Pyk2, ultimately leading to the activation of Ras/Raf/MEK/ERK
pathway through Src kinases (Figure 4), increased proliferation and colony formation [125,
149, 150]. Paradoxically, overexpression of constitutively active Raf-1 leads to apoptosis of
SCLC cells [151], and increased expression of cytoplasmic MEK (MAPK) was shown to be
of good prognostic value for survival [152].
In addition to PLC activation, GPCRs can activate RhoGTPases, which are involved in
actin cytoskeleton reorganization [153]. This seems to be the case with SCLC cells, where
there is a trend towards higher RhoA activation states than in any other type of lung cancer.
14 J. N. Moreira, A. O. Santos, L. M. Bimbo et al.

This was hypothesized to contribute to their high metastatic potential and characteristic cell
morphology of suspension cells [154, 155]. The importance of GPCR in SCLC has been
explored by the development of several inhibitors of GPCR, which is probably why silencing
approaches are not very common. In the only report available, the in vitro targeting of the
gastrin-releasing peptide receptor with free antisense ODN, resulted in partial cell growth
inhibition in SCLC cell lines that present this autocrine signaling system [156].

3.1.3. Autocrine loops 2 - receptor tyrosine kinases and downstream


effectors
Receptor tyrosine kinases (RTKs) are transmembrane receptors with kinase activity,
whose potential as therapeutic targets in SCLC has been recently reviewed [157]. In SCLC,
the relevant RTKs implicated in autocrine growth loops are c-Met, insulin-like growth factor
1 receptor (IGF-1R), c-Kit, and fibroblast growth factor receptor (FGF-R). The vascular
endothelial growth factor receptor (VEGF-R) was detected in different SCLC cell lines [158],
although the prognostic value of VEGF serum levels or tumor expression remains doubtful
[159-165].
C-Met receptor is frequently expressed in SCLC, in contrast with its natural ligand, the
hepatocyte growth factor (HGF) [166]. In addition, some SCLC cell lines and tumors present
alternative splicing isoforms or gain-of-function mutations of this receptor [167]. In SCLC, c-
Met receptor stimulation changes cell motility and induces the production of reactive oxygen
species [168, 169]. It was found in the active state in the tumor invasive front, in good
correlation to phosphorylated (p-) FAK [Y861] and p-Akt [S473], which was in accordance
with the established activated pathway (Figure 3), but without correlation with the
proliferation marker Ki-67 [170]. Silencing of the c-Met receptor with siRNA was performed
in the H69 SCLC cell line, resulting in downregulation of the activation of Akt and FAK,
although no direct measurement of cytotoxicity or other cell behavior was reported [170].
IGF-1R is another relevant RTK expressed in SCLC that mediates mitogenic and cellular
differentiation signals. In addition, SCLC cell lines also secrete the ligands IGF-1 and IGF-2
[171, 172], being the former currently associated with lung cancer risk [173, 174]. The
therapeutic relevance of IGF-1R has been emphasized by the development of an IGF-1R-
blocking antibody and an IGF-1R inhibitor, that inhibit the growth of SCLC cell lines, unless
they have constitutively high Akt activation [175, 176].
C-Kit is a RTK involved in cell growth, survival and chemoattraction. In SCLC cell lines
c-Kit expression varies according to the report [166, 177], although a significant expression
has been detected in SCLC tumors. Nevertheless, its prognostic significance remains
controversial [152, 178]. C-Kit downregulation achieved by recombinant adenovirus vectors,
expressing antisense fragments of c-Kit transcripts, led to partial growth inhibition of cell
lines in vitro [179]. The absence of a therapeutic advantage from the use of imatinib
(Gleevec), both in murine models of c-Kit-expressing SCLC [177] and in a phase II trial with
patients with SCLC [180], suggests that c-Kit signaling is not critical in vivo. Autocrine and
paracrine mechanisms are considered more important in c-Kit-regulated SCLC growth than
activating c-Kit mutations [178], and this may influence the efficacy of c-Kit inhibitors. At
this level, the current strategy includes targeting different receptors and or/cellular pathways
with mutitargeted inhibitors [181] as well as combining different inhibitors. In this respect,
Gene Silencing Therapy in Small Cell Lung Cancer 15

combined treatments of imatinib and, an agent causing either direct inhibition of PI3K, Akt
or mTOR [142], or with c-Kit and IGF-1R inhibitors [182, 183], led to synergistic induction
of apoptosis.
Finally, the fibroblast growth factor 2 and 10 (FGF-2 and FGF-10) receptors (FGF-2R
and FGF-10R) stimulation increases chemoresistance in SCLC cells through the formation of
a specific multiprotein complex comprising B-Raf, PKC and S6K2. FGF-2R has not been
targeted itself, but RNAi-mediated downregulation of B-Raf, PKC or S6K2 abolishes FGF-
2-mediated survival [184-186]. Moreover, elevated levels of FGF-2 in serum of SCLC
patients has been correlated with poor prognosis and active angiogenesis [187].

3.2. Key Proteins in Important Cellular Signaling Pathways in SCLC

3.2.1. Protein kinase C family


PKC family is a large family of isoenzymes with serine-threonine kinase activity. The
isoforms expressed in different cells, as well as their preferential sub-cellular localization or
their preferential substrates, differ substantially. The pattern of PKCs expression and their
altered regulation have been proposed to play a role in the malignant behavior of SCLC cells
[149], namely in cell survival [188], resistance acquisition [189, 190], and at mediating
signals that promote adhesion to some ECM substrates [188].
There are not many reports of PKC downregulation. PKC silencing with siRNA
produced partial growth inhibition in SCLC cell lines, although it seems more determinant in
growth inhibition and survival in NSCLC [191].

3.2.2. PI3K/Akt pathway


PI3K complex is a critical component of signaling pathways that can be activated by a
variety of growth factors, chemokines, and ECM components (Figure 3 and 4). When
activated, it phosphorylates phosphatidylinositols (PI) (e.g. PIP2 to PIP3), which interact with
Akt, exposing its main phosphorylation sites. PI-dependent protein kinases (PDK1 and
PDK2) are then involved in Akt activation. PIP3 synthesis is reverted by the phosphatase
PTEN, which is a tumor suppressor, sometimes inactivated in SCLC [192, 193]. Constitutive
activation of this pathway promotes the growth and anchorage independence of SCLC in
vitro. Five SCLC cell lines tested (H69, H345, H510, LS274, and DMS79) presented high
basal constitutive PI3K activity [135]. Inhibition of PI3K activity markedly inhibited SCLC
cell proliferation in culture, as a result of stimulating apoptosis and promoting cell cycle
delay in G1, as well as reduced basal SCLC cell colony formation [135]. PI3K/Akt pathway,
but not the MEK/ERK pathway, was also implicated in laminin-mediated survival and
resistance to cisplatin or etoposide. These results suggest that inhibition of the PI3K/Akt
pathway might be a useful strategy to overcome SCLC resistance mediated by ECM [142].
Different SCLC cell lines overexpress distinct subsets of class IA and II PI3Ks [194].
Class IA and II have been linked to RTKs, while class IB to GPCR [194, 195]. A frequently
studied catalytic subunit of class IA PI3K, is the PIK3CA gene, encoding for p110, however
mutations in this gene were not detected in 48 SCLC cell lines analyzed. PIK3CA copy
number gains were also more frequent in squamous cell carcinoma (33.1%) than in
16 J. N. Moreira, A. O. Santos, L. M. Bimbo et al.

adenocarcinoma (6.2%) or SCLC cell lines (4.7%). RNAi-mediated knockdown inhibited


colony formation of cell lines with mutated or amplified PIK3CA but was not effective in
PIK3CA wild-type cells [196]. In contrast, the PIK3CB (p110) gene was expressed by all 8
SCLC cell lines evaluated [194], and has been proposed to be a promising target in cancer
therapy, at least in prostate tumors with PTEN loss [197].
Activated Akt, also commonly called protein kinase B (PKB), translocates from inner
membrane surface to the nucleus, where it modulates the function of numerous substrates
related to the regulation of cell proliferation (e.g. beta-catenin pathway), protein synthesis
(e.g. mTOR pathway), apoptosis (e.g. NFkappaB pathway and cyclic AMP-response element-
binding protein activation) [195, 198] and cell motility [199]. Phosphorylated Akt was
detected in 62% of the SCLC tumors analyzed, although Akt expression was not predictive of
survival [152].

3.2.3. Focal adhesion kinase family


The FAK family is a subfamily of cytoplasmic proteins belonging to the protein tyrosine
kinase (PTK) family. Either 1-integrin signaling, GPCR, or RTK signaling lead to
phosphorylation and activation of the FAK protein (also known as FAK1, among other
designations) (Figure 3), which makes it an interesting point of convergence [140, 170, 200].
In some SCLC cell lines, hyluronan-activated FAK resulted in activation of matrix
metalloproteinase-2 secretion, an event that was counteracted by anti-FAK ODN [201].
Accordingly, reduction of FAK phosphorylation or FAK downregulation by siRNA, resulted
in cell death [202]. In SCLC cell lines such as H69, H510, H82 and N592, FAK is
constitutively activated [125, 136] and in other cell lines (H446 and H345) FAK activation
can transduce fibronectin or bombesin stimulation through the PI3K pathway [140, 203]. The
therapeutic potential of silencing the expression of this class of proteins has been pointed out,
in combination with docetaxel, both in vitro and in vivo murine models of ovarian cancer
[204-206].
Another member of the FAK family of PTKs is the proline-rich tyrosine kinase 2 (Pyk2),
also known as PTK2B. In SCLC, it mediates the signaling of neuropeptides such as galanin
and bradykinin through their GPCRs, responding directly and exclusively to increases in
[Ca2+]i. Once activated, it binds and activates Src kinases, resulting in GTP-loading of Ras
and Erk activation (Figure 4). Lentiviral-mediated silencing of Pyk2 in H69 and H510 cells,
reduced in vitro both basal and galanin-stimulated cell growth, and colony formation
(survival), without showing any effect on apoptosis [125]. As stated by Roelle et al, Pyk2 has
been linked to migration and invasion in other tumors, namely by transducing CXCL12
signaling, which has been recognized as a resistance-promoting stimulus in SCLC cells
[136].

3.2.4. Scr family


Scr family also belongs to PTK family, and includes proteins such as Src, Fyn, Yes and
Lck. Src kinase basal activity in H69 SCLC cell line is very high, compared to CHO cells
[125], which is in good association with the high basal PI3K activity in those cells [135].
Several reports have described the inhibitory effect of Src kinase inhibitors on either
PI3K/Akt or Ras/Raf/MEK/ERK activation by growth factors in SCLC cell lines, thus
Gene Silencing Therapy in Small Cell Lung Cancer 17

implicating Src in important cell signaling cascades (Figure 3 and 4). Those inhibitors led to
profound consequences on cell growth, although, apparently, without implication in
apoptosis [125, 207]. At SCLC lipid rafts RTK activation, such as stem cell factor (SCF)
stimulation of c-Kit, is connected to the PI3K/Akt pathway and lipid raft localization of Src
kinase (Figure 3). A combination of siRNAs targeting SRC and LCK effectively reduced SCF-
stimulated Akt activation in H69 SCLC cells, but no further effects were reported [207].

3.2.5. Ras family


Ras genes (e.g. K-RAS, H-RAS, N-RAS) encode plasma membrane proteins, which are
small GTPases belonging to the small G protein family [153]. They transduce signals with
origin either in GPCRs or RTKs to downstream pathways (PI3K/Akt or MEK/Erk pathways,
Figure 3 and 4). Apparently, SCLC cell lines have similar mRNA levels of N-RAS, K-RAS
and H-RAS, and DNA amplifications have not been yet reported [208]. The expression of Ras
proteins, although variable among different cell lines, is frequently elevated in SCLC in
comparison to type II lung epithelial cells. In SCLC, while all three isoforms lead to
activation of MAPK pathway, N-Ras was proposed as the principal isoform at mediating
PI3K pathway activation (triggered by growth factors). Retroviral stable expression of RNAi
constructs against N-RAS inhibited the induction of Akt activation and completely inhibited
cell growth of H510 SCLC cell line. It has also inhibited the induction of both Akt- and
Erk1/2 activation in the SW2 cell line. Interestingly, Ras signaling inhibition by simvastatin
has also been reported [209]. This and other statins are drugs commonly used by
hypercholesterolemic patients and are currently in phase II and III clinical trials, in
combination to standard regimens, in SCLC.

3.3. Limitless Replicative Potential

Within a tumor, a few cancer cells or a colony of tumor cells, share with stem cells the
property of unlimited self-renewal. Therefore, one rational way to fight cancer would be to
target genes responsible for the self-renewal ability. It has not been always clear if this is an
inherited feature from adult stem cells-derived cancer, or if it is acquired by cells during
transformation.
It has not been completely clear whether, all or a minority of SCLC cells have stem or
progenitor-like properties, such as self-renewal, and are indeed responsible for the long term
maintenance and recurrence of tumors. Evidence exists that only a fraction of cells is able to
form colonies in clonogenic assays, and that the small sub-set of CD133-expressing cells has
increased tumorigenicity in mice [210]. However, this property has been related to the
capacity to form xenografts in mice and not to the intrinsic tumor maintenance property
[211]. The genes that have been described to confer unlimited proliferative potential or to be
involved in the regulation of the stem-like properties of SCLC, that therefore constitute
themselves as potential targets of gene silencing, will be addressed in the following secions.
18 J. N. Moreira, A. O. Santos, L. M. Bimbo et al.

3.3.1. Telomerase
Telomerase is a ribonucloprotein complex responsible for the maintenance of
chromosomal telomeres and the unlimited proliferation ability of cancer cells, as in stem
cells. About 98% of SCLC tumors express the telomerase RNA template [212]. Furthermore,
SCLC cells exhibit high levels of telomerase activity and the highest expression levels of the
catalytic subunit of telomerase, the human telomerase reverse transcriptase (hTERT), among
lung cancer subtypes [213, 214]. Rather than silencing telomerase, the approach in SCLC has
been to use the promoter of hTERT gene to control the replication of oncolytic viruses or
suicide gene expression [215, 216]. There are some studies involving inhibitors of telomerase
activity, with only one reported so far in SCLC [217].

3.3.2. Hedgehog signaling pathway


The Hedgehog (HH) pathway has been implicated in embryonic development,
morphogenesis, and regulation of stem cell fate. Members of the HH signaling pathway may
have variable expression in SCLC cell lines [218, 219]. However, SCLC tumor expression of
Gli1, an important effector of the HH pathway, is frequent [219]. The in vitro effects of
cyclopamine (an upstream inhibitor of the pathway) or an anti-GLI1 siRNA in the growth of
SCLC cell lines, varied according to the report. Apparently, these treatments only reduced the
cell growth of cell lines co-expressing Shh (a ligand that positively stimulates the pathway)
and Gli1 [218, 219]. It is possible, though, that Gli1 is activated non-canonically by other
means within the tumor cells, therefore turning this protein a preferential target [220].

3.3.3. Urokinase plasminogen activator


The urokinase plasminogen activator (uPA) and its receptor (uPAR/CD87) are the major
regulators of extracellular matrix degradation and are involved in cell migration and invasion
under physiological and pathological conditions. uPAR-positive cells in all SCLC lines
showed multidrug resistance, high clonogenic activity and coexpression of CD44 and MDR1,
which are putative cancer stem cell markers [221].

3.4. DNA Stability, Repair and Replication

DNA double strand breaks (DSB) promote genomic rearrangements, and contribute
dramatically to cancer development. DNA repair mechanisms respond either to a DSB or to a
region of single stranded DNA, and can activate cell cycle checkpoint arrest and/or apoptosis.
Cell cycle checkpoints monitor the structural integrity of chromosomes before progression
into S phase (the G1/S checkpoint), into mitosis (the G2/M checkpoint), as well as during
replication. The damage response signaling pathways facilitate communication between
damage recognition proteins and the checkpoint machinery, arrest of cell cycle progression
and increase the opportunity for repair, before undertaking important events, such as
replication or mitosis [222].

3.4.1. RAD51
RAD51 is a protein implicated in the repair of double-strand breaks via homologous
recombination. Its expression level was positively correlated with etoposide resistance in
Gene Silencing Therapy in Small Cell Lung Cancer 19

SCLC cell lines not expressing a multidrug-resistant phenotype. In addition, downregulation


or upregulation of RAD51 modulated etoposide sensitivity [223].

3.4.2. E2F1
E2F1 (Figure 5) is a transcription factor best known for its role in driving cell cycle
progression through the G1/S checkpoint, although it also regulates G2/M transition [224]. Its
activity on proliferation and apoptosis is unregulated in SCLC due to most common
abnormalities in the tumor suppressor genes Rb and TP53 [225-228]. E2F1 protein has been
found overexpressed in 92% (24/26) of SCLC cases, with no underlining gene amplification,
and it was associated with a high Ki-67 index, as well as a Bcl-2:Bax ratio > 1 [229].
Surprisingly, silencing carried out with anti-E2F1 siRNA, did not affect neither viability nor
proliferation [124].

3.4.3. S-phase kinase associated protein 2


The S-phase kinase associated protein 2 (Skp2) is one of the F-box proteins of the E3-
ubiquitin ligase complexes under the control of E2F1, that positively regulates the G1/S
transition (Figure 5). SKP2 was found amplified in 44% and overexpressed in 83% of
primary SCLC tumors [230]. Skp2 protein accumulated preferentially in high-grade
neuroendocrine lung carcinomas, and its overexpression was associated with advanced stages
and nodal metastasis [124]. Silencing of SKP2 with antisense ODN inhibited the growth of
SCLC cells in culture [230] and induced spontaneous apoptosis [231]. Similar results were
obtained with lentiviral vectors [232].

Figure 5. Effects and outcomes in proliferation and apoptosis of E2F1 transcription factor promoted by
SKP2. SKP2 is a promising target of therapeutic gene silencing in this pathway and is represented by a
target symbol.
20 J. N. Moreira, A. O. Santos, L. M. Bimbo et al.

4. Lessons to Take from the Validation of Classical


Oncogenes as Therapeutic Targets in SCLC
As mentioned before, target validation is a stepwise work. In this process, problems and
contradictions are often encountered, as illustrated with the course of action taken with the MYC
family and Bcl-2 in SCLC. There is already extensive data published on these targets, namely
comprising the frequency of protein or gene overexpression, the frequency of gene amplification
in tumors, determination of their prognostic value in patients, artificial overexpression of the
gene, gene knockdown by antisense ODN or RNAi, or the use of Bcl-2-family inhibitors.
Nonetheless, the specific role of these oncogenes in SCLC remains to be clarified.

4.1. Myc Family

MYC family genes are among the classical oncogenes implicated in SCLC [233]. They
encode transcription factors that heterodimerise with MAX, leading to either expression
activation or repression [234]. Gene amplification of either MYC, MYCN or MYCL is an
event with low incidence, and is usually present in SCLC patients who have been treated with
chemotherapy, in correlation with the tumor end stage [235-237]. Although the frequency of
MYC-family gene amplification is increased in SCLC cell lines (33% - 50%), it is often
considered an artifact of cell line selection. However, even in the absence of a MYC family
gene amplification, increased protein expression is more frequent (45% and 80% in tumors
and cell lines, respectively), which could be explained by a slow mRNA decay [234, 237].
Moreover, c-Myc activity can be increased by interaction with Bcl-2, a protein frequently
overexpressed in SCLC [238].
Anti-MYCL and -MYC ODNs were evaluated in vitro, producing inhibition of cell growth
and/or thymidine uptake [236, 239-241]. In contrast, anti-MYC ODN strongly inhibited
apoptosis induced by genotoxic stimuli [242]. MYC amplification in SCLC was associated
with inactivation of most pro-apoptotic components of the death-inducing signaling complex
(DISC), expression of c-FLIP (inhibitor of the extracellular apoptosis pathway), and TRAIL
resistance [243]. In addition, MYC amplification was also associated with high-level
amplification or homozygous deletion of other genes involved in apoptosis in such a way that
could, theoretically, favor tumor survival [234].
In contrast to in vitro data, in vivo, c-Myc expression in stable transfected SCLC cells
suppresses the formation of tumors and downregulates VEGF. The same study also reports a
negative correlation between the probability of tumor formation and the relative expression
of c-Myc [244]. However, such conclusion should be taken cautiously, since the natural
occurring MYC amplification is accompanied of abnormalities in the apoptotic pathway,
which the aforementioned model does not seem to clearly reproduce.
Gene Silencing Therapy in Small Cell Lung Cancer 21

4.2. Bcl-2 Family

The BCL2 gene was first cloned in B cell leukemia, that showed the chromosomal
translocation 14:18, in 1984 [245]. After the discovery of its anti-apoptotic role [24], it
became an eligible target for therapeutic intervention in different type of tumors, including
SCLC [157]. Its discovery set the birth of a novel and large family of anti- and pro-apoptotic
proteins. Anti-apoptotic Bcl-2 family members may be divided into two subclasses, one
comprising proteins such as Bcl-2, Bcl-xL and Bcl-w and the other Mcl-1 and Bcl2A1, that
interact, respectively, with Bad and Noxa [246]. Overexpression of some of these proteins is
frequent in SCLC, although rarely due to gene amplifications [234]. The main anti-apoptotic
proteins that have been object of gene silencing in SCLC are Bcl-2 and Bcl-xL.

4.2.1. Bcl-2 in SCLC prognosis


Bcl-2 is frequently expressed in SCLC cell lines [247] and tumors [248-251], suggesting
that it plays a role in the pathogenesis of the disease. Nevertheless, there is no correlation
between Bcl-2 expression and resistance to therapy and/or survival [252-256]. The relation to
tumor stage is less clear, ranging from no relation to positive correlation, depending on the
author [255, 257]. A correlation between Bcl-2 expression and decreased incidence of
metastases, indicating attenuation or resistance to apoptosis, has also been reported [258].
Recently, in a series of 205 carcinomas, including lung cancer, Bcl-2 expression was
associated with better overall survival. The risk of mortality was 2.3-fold higher in patients
without Bcl-2 expression. Bcl-2 appeared to be the key biological factor influencing clinical
behavior in the most common epithelial cancers [259]. Furthermore, despite higher Bcl-2
expression, SCLC cell lines present higher spontaneous apoptotic index than other lung
cancer cell types or other cancers in general [260-262]. The apparent contradiction between
the currently attributed anti-apoptotic function of Bcl-2 and the available clinical data is an
issue that remains to be clarified.

4.2.2. In vitro modulation of Bcl-2


The idea that Bcl-2 directly regulates survival in SCLC probably came from previous
reports either mentioning the development of chemoresistance upon overexpression of Bcl-2
[238, 263] or of cytotoxic effects or chemosensitization upon Bcl-2 downregulation by 2009
ODN [264-266], together with the strong evidence of its anti-apoptotic role in hematological
diseases. However, the therapeutic value of BCL2 silencing in SCLC is still ambiguous. We
have recently observed that downregulation of Bcl-2 with G3139 ODN or siRNA, has neither
resulted in a sequence-specific decrease of viability or inhibition of SCLC cell growth, nor in
a chemosensitization to cisplatin treatments (unpublished observations). Intriguingly, the
most effective conditions at promoting Bcl-2 downregulation resulted in higher cell viability
than the correspondent treatment with nontargeting siRNA, although the effect on cell growth
was similar (unpublished observations). Nevertheless, it is not possible to draw definite
conclusions about the role of a certain protein, exclusively based on in vitro studies and the
contribution of the in vivo environment should also be considered. This was the case with
melanoma cancer, where despite the absence of in vitro cytotoxicity upon (stable) BCL2
silencing, it further reduced the in vivo tumorigenic potential of cells [267]. Therefore, the in
22 J. N. Moreira, A. O. Santos, L. M. Bimbo et al.

vivo validation of the therapeutic potential of silencing BCL2 in SCLC is likely to be of


interest. However, the absence of implication of Bcl-2 as an anti-apoptotic protein in SCLC,
at least in vitro, might be not so surprising. In fact, it has been demonstrated that Bcl-2 has
other distinct roles of Bcl-2, in different cell models, which can be regulated by different
mechanisms [238, 268-282]. In SCLC Bcl-2 aparently cooperates with c-Myc enhacing its
half-life [238], and interacts with MSH6 attenuating the mismatch repair response (refs).
Overall, Bcl-2 should not be seen only as an anti-apoptotic protein, but rather as a
multifunctional protein.

4.2.3. Oblimersen: an anti-Bcl-2 antisense ODN in clinical evaluation


A validated molecular target by itself is of no direct therapeutic use, without an effective
and safe drug interfering with that target. In addition, the lack of specificity of a drug towards
a single target can be misleading regarding the functional role of a protein within a cell.
There were always some manifested concerns about the Bcl-2unrelated effects elicited
by G3139 ODN. In vitro, G3139 ODN cytotoxicity in PC3 prostate cancer cells is
independent of Bcl-2 downregulation and correlates with the expression of stress inducible
genes [283, 284]. In melanoma cells, it triggers cytochrome c release and therefore apoptosis,
presumably by interacting directly with a mitochondrial channel (VDAC) [285]. Moreover,
the potent antitumoral effect in murine models of human cancer, including SCLC, is in part
due to stimulation of innate immune responses [286].
The success of the initial in vivo mice experiments with G3139 ODN (Oblimersen
sodium) contributed decisively to build the confidence around BCL2 as an important target
for therapeutic intervention. After the favorable results in phase III trials against melanoma
and chronic lymphocytic leukemia [48, 287], a further phase III trial (AGENDA trial) was
recently initiated in melanoma patients. However, the phase II clinical trial of G3139 ODN,
in combination with carboplatin and etoposide in SCLC patients with extensive disease, did
not result in an improved therapeutic outcome [56]. Nonetheless, it is important to point out
that such result might be due to inefficient delivery to tumor cells, and therefore these results
do not rule out per se a possible therapeutic advantage under improved delivery conditions.

4.2.4. Bcl-xL and multitargeting of anti-apoptotic proteins


There is a report on in vitro treatment of SCLC cell lines with ODN that effectively
downregulated Bcl-xL protein levels, but did not induce apoptosis, in contradiction with
NSCLC where there was apoptosis induction [288]. The same authors, using a bi-specific
anti-BCL2 and -BCLXL ODN, reported cytotoxicity against the SW2 SCLC cell line [289]. It
is now recognized that inhibition of both subclasses of anti-apoptotic proteins is required for
apoptosis induction [246]. In SCLC, Mcl-1 expression was shown to induce resistance to the
Bcl-2 family inhibitor ABT-737 [290], that acts by simultaneously inhibiting three anti-
apoptotic proteins (Bcl-2, Bcl-xL and Bcl-w) [121, 291]. Furthermore, silencing of Mcl-1
with siRNA, although with minimal effects on cell growth by itself, was able to sensitize
resistant cells to ABT-737 [121, 290].
Gene Silencing Therapy in Small Cell Lung Cancer 23

5. Delivery of Gene Silencing Agents in SCLC


The therapeutic potential of gene silencing agents is being intensively explored in cancer
and other human diseases, due its ability to silence any gene whose deregulation is causing
disease. We are probably facing the emergence of a new class of pharmaceutical drugs that
could be part of the solution for diseases with limited treatment options [292].
Despite several clinical trials undertaken and presently ongoing (Table 1), further studies
are required to solve problems related with the sequence design and the in vivo delivery of
silencing agents to the target cells. Regarding sequence design and selection of silencing
agents, both potency and specificity should be taken into account, in order to achieve a strong
silencing with minimal off-target effects, which can be further improved by chemical
modifications, as mentioned above. In respect to the in vivo delivery to the target cells, it is
well known that unmodified or modified molecules, to less extent, have a rapid clearance
when administered in the naked form, as they are prompted to nuclease attack. In addition,
due to their negative charge, they have high affinity to serum proteins and are poorly
internalized by cells. Therefore, it is difficult to achieve the drug concentration in the target
cells that will produce a therapeutic effect. A major effort is being done in order to develop
adequate carrier systems, which can be classified into viral and non-viral delivery. Although
viral delivery could provide a durable target mRNA downregulation, as a consequence of
long term expression of the silencing agent, some have associated a major risk of
immunostimulation and cell inespecificity. These issues have been discussed with great detail
elsewhere [107, 293, 294].
Non-viral strategies such as liposomes, conjugates with cholesterol, polymers (e.g.
chitosan, cyclodextrin, atelocollagen), antibodies and proteins (e.g. protamine), are
alternative strategies generally taken as safer [107, 293, 294]. Moreover, some of these non-
viral strategies resulted in significant improvements in terms of pharmacokinetic and
biodistribution, upon systemical administration in mice [295] and in non-human primates
[296]. The cell-specific delivery that is possible to obtain with monoclonal antibodies and
natural ligands such as transferrin and folate, that are directly coupled to the silencing agent
or to the surface of the carrier system, is advantageous as lower doses are needed to achieve
an efficient drug concentration in the target tissue and the potential of side effects is
significantly reduced [297].
In Figure 6 are schematically represented the main components along with the critical
parameters involved in the design of a non-viral lipid-based strategy for targeted cancer
systemic gene silencing [95].
24 J. N. Moreira, A. O. Santos, L. M. Bimbo et al.

Figure 6. Diagram representing the main components (in black) along with the critical parameters (in
blue) involved in the design of a non-viral lipid-based strategy for targeted cancer systemic gene
silencing (Reprinted with permission from [95]).

The development of carrier systems that envisage targeting of internalizing receptors


overexpressed on the surface of tumor cells, is a promising strategy aiming at improving cell
transfection. There is extensive work on this area, but only a few reports concern SCLC.
The potential of ligand-mediated targeting of liposomes for the treatment of SCLC has
been assessed before for the first time by Moreira et al [298]. It has been shown that a
hexapeptide known as antagonist G, a growth factor antagonist, could be used as a targeting
agent for sterically stabilized liposomes in human SCLC. Antagonist G-coupled liposomes
containing doxorubicin have shown to be specifically internalized by SCLC cell lines,
through a receptor-mediated endocytosis, which led to intracellular drug accumulation and
release to intracellular sites of action, resulting in improved cytotoxicity, as compared to non-
targeted liposomes [299-301]. Moreover, targeted liposomes have shown to be long-
circulating in blood [300]. Using a poorly vascularized subcutaneous murine model of SCLC,
it was observed that the therapeutic efficacy of doxorubicin-containing targeted or non-
targeted liposomes was significantly improved over free drug, but targeted liposomes did not
improve antitumor efficacy relative to the non-targeted counterpart [302]. It is important to
emphasize that the therapeutic potential of the developed peptide-targeted liposomes might
have been underestimated due to the use of a subcutaneous tumor model that does not mimic,
by any means, the human disease, where the tumors cells are much more accessible.
Gene Silencing Therapy in Small Cell Lung Cancer 25

A second independent targeted-liposomal formulation of doxorubicin, directed to


EpCAM-overexpressing SCLC SW2 cells, through coupling of an humanized single-chain Fv
antibody fragment (4D5MOCB), led to increased tumor localization and higher antitumor
activity than the non-targeted liposomes, at low treatment doses [303]. The authors discuss
that the increased tumor localization of the targeted liposomes can be due to the high affinity
of the ligand to the antigen, its rapid internalization, and its stability in human serum [303].
Furthermore, the density of the receptor expression at the tumor cell surface might be an
important factor to consider. The knowledge gained from these approaches may contribute to
the successful development of effective delivery systems containing gene silencing molecules
towards SCLC.
The potential of Ep-CAM-targeted immunoliposomes for the delivery of a bispecific
ODN (against Bcl-2 and Bcl-xL) has been also evaluated in the cellular model mentioned in
the previous paragraph. Cellular association studies confirmed the higher specific cellular
uptake of targeted coated cationic liposomes relative to the non-targeted counterpart.
Efficient internalization by receptor-mediated endocytosis, led to downregulation of both bcl-
2 and bcl-xL expression on both the mRNA and protein level, resulting in enhanced tumor
cell apoptosis. In combination experiments, the use of targeted liposomes containing the
bispecific antisense, led to a 2- to 5-fold sensitization of EpCAM-positive tumor cells of
diverse origin to death induction by doxorubicin. Overall, these results emphasize the
potential of ligand-mediated liposomal targeting as a delivery strategy of nucleic acids.

6. Conclusion
There are presently several unanswered questions and challenges to be met in SCLC. So
far, there is still a lack of knowledge about the in vivo relevance of most of the candidate
target genes described. Therefore, it is difficult to understand which could be the most
interesting molecular targets of therapeutic gene silencing in SCLC. Perhaps anti-apoptotic
genes (in a multitargeting approach), or other genes supposed to mediate the survival of cells
in vivo, such as Integrins and Chemokine receptors, could be promising targets. Particularly
interesting are some key proteins in important cellular signaling pathways, which have
already been object of gene silencing strategies, such as FAK or PYK2, classical oncogenes
such as RAS genes, namely N-Ras, and genes involved in cell cycle control such as SKP2.
One cannot forget that other genes, whose therapeutic potential in vitro has not been relevant,
could reveal to be important in the in vivo context, as it has happened with Bcl-2 in
melanoma [267, 304]. In the meantime, novel molecular targets are being identified by gene
expression or amplification profiling techniques [305-307].
Another important aspect is the possibility to consider genes of non-coding RNA as
therapeutic targets, namely overexpressed miRNAs that function as oncogenes, such as the
cluster mir-17-99, overexpressed in SCLC [308]. The silencing of some of the miRNA from
these cluster was reported in NSCLC [309]. In addition, tumor suppressor miRNAs that are
found to be deleted, or epigenetically silenced in tumors, can be substituted by artificial
expression or upon delivery of synthetic miRNAs. This approach has already been attempted
also in NSCLC [310, 311].
26 J. N. Moreira, A. O. Santos, L. M. Bimbo et al.

A different challenge is the open question regarding the possibility of existence of a rare
subpopulation of SCLC stem cells within tumors. The burst of interest in the cancer stem
cell theory in 2003 caused by a paper of Al-Hajj et al. [312], illustrates how this theory has
opened new insights and perspectives in cancer research. However, the theory of cancer
stem cells is not a clear subject [211], and given the postulated relevance of cancer stem
cells and their distinct phenotype, it will be important to determine if they are indeed a small
minority within the tumor cell population (within CD133 positive cells), or instead, if the
majority or all of SCLC cells are able to give continuity to the tumors and metastasize.
Considering that cancer stem cells existed as a minority, it is still unclear how do they relate
with some of the discussed populational responses to downregulation of the presented
candidate targets.
It might take more than silencing a single gene to achieve the needed therapeutic effect.
In this respect, molecular multitargeting constitutes a significant challenge. Furthermore, it is
important not to forget that the downregulation of the selected targets as well as the delivery
of the nucleic acid has to be organ-specific, thus avoiding any harmful effect to healthy
organs. This new opportunity, which hopefully can translate in the future in benefits to cancer
patients, still depends on the development of carriers with the capacity to promote
intracellular targeted accumulation of nucleic acids. One of the major challenges regarding
gene silencing in SCLC is the apparent low transfectability of these cells. To achieve
improved nucleic acid delivery, we still lack important information about the internalization
pathways present in SCLC cells, and what would be the best treatment scheme to achieve an
efficient transfection. One aspect that might be relevant to SCLC cells is the absence of
caveolae-mediated endocytosis [313]. This cell entry pathway is thought to be a more
effective internalization one in terms of transfection, as compared with the clathrin-mediated
endocytic pathway, since it seems less prone to endosomal degradation of nucleic acids
[314].
It is widely accepted that gene silencing represents an opportunity to change the
treatment paradigm of wide range of diseases, including SCLC. It has become evident that
the transformation of this opportunity into clinical reality is dependent on an adequate
selection of the silencing agent, a correct target validation, and an efficient intracellular
targeted delivery.

Acknowledgments
A. Santos and L.C. Gomes da Silva are students of the international PhD program on
Biomedicine and Experimental Biology, from the Center for Neuroscience and Cell Biology,
and recipient of fellowships from the Portuguese Foundation for Science and Technology
(FCT) (ref.: SFRH/BD/11817/2003 and SFRH/BD/33184/2007, respectively). The work in
the authors laboratory was supported by a Portuguese grant from FCT, POCTI and FEDER
(ref.: POCTI/FCB/48487/2002) and by the Portugal-Spain capacitation program in
nanoscience and nanotechnology (ref.: NANO/NMed-AT/0042/2007).
Figures 1, 2 and 6 were reprinted from reference [95]. Copyright@ American Scientific
Publishers, http://www.aspbs.com.
Gene Silencing Therapy in Small Cell Lung Cancer 27

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In: Small Cell Carcinomas: Causes, Diagnosis and Treatment ISBN: 978-1-60741-787-3
Editors: J. G. Maldonado and M. K. Cervantes 2009 Nova Science Publishers, Inc.

Chapter 2

Small Cell Carcinomas: Contribution


of Cytologic Tools to Diagnosis
and Management

Dilip K. Das*
Department of Pathology, Faculty of Medicine, Kuwait University

Abstract
Small cell carcinoma (SCC) is a malignancy with aggressive growth pattern, high
recurrence rate, and tendency to metastatize that mainly occurs in the lung, with primary
lesions in other sites very rare. The sites affected by extrapulmonary small-cell
carcinoma (EPSCC) are parotid, minor salivary glands of the tongue, sinonasal region,
breast, thymus, pleura, esophagus, extrahepatic bile duct, kidney and renal pelvis, urinary
bladder, ovary, endometrium, uterine cervix, and prostate. EPSCC has been recognized
as a clinicopathological entity distinct from the small cell carcinoma (SCC) of the lung.
EPSCC differs from small-cell lung carcinoma (SCLC) in respect of etiology, clinical
course, and survival. Both SCLC and EPSCC can occur as part of multiple primary
neoplasms. Various cytodiagnostic tools utilized for detection of small cell carcinoma are
palpation-guided fine needle aspiration (FNA) cytology, ultrasonographic (US) or CT-
guided transthoracic FNA cytology, transbronchial needle aspiration (TBNA),
endoscopic US-guided fine needle aspiration (EUS-FNA) biopsy, ultrasonographic (US)-
guided FNA cytology of abdomen or pelvic organs, and exfoliative cytology. The tumor
cells of SCC are arranged mostly in clusters of varying sizes and have minimal
cytoplasm, finely stippled (salt and pepper) chromatin, inconspicuous nucleoli,
prominent nuclear molding and smearing effect. The lesions considered in differential
diagnosis of SCC are non-Hodgkin lymphoma, squamous cell carcinoma of small cell
type, or other malignant small round cell neoplasms. In case of diagnostic difficulties,
various ancillary studies may be of help in arriving at a diagnosis. Besides cytokeratin,

*
Corresponding author: Dr. Dilip K. Das, MBBS, MD, PhD, DSc, FRCPath, Associate Professor, Department of
Pathology, Faculty of Medicine, Kuwait University, P.O.Box: 24923, Safat 13110, Kuwait, Tel: 00965-2498-
6019, Fax: 00965-2533-8905, E-mail: dilip76@hotmail.com
52 Dilip K. Das

the neuroenocrine markers like chromogranin, neurone-specific enolase (NSE),


synaptophysin, CD56, and CAM5.2 show varying degrees of positive reaction in SCC.
Electron microscopy demonstrates dense-core granules in neoplastic cells of SCC. When
the diagnosis of SCC is reached in a patient with a lung mass based on biopsy report
including cytodiagnosis, a surgical treatment approach is no longer considered and
chemotherapy becomes the treatment of choice. However, surgical approach with or
without radiation therapy and chemotherapy has been resorted to in EPSCC in most
locations depending upon the extent of the disease.

Keywords: Small cell lung carcinoma, extrapulmonary small cell carcinoma, Fine needle
aspiration cytology, immunocytochemistry, small round cell tumors.

Introduction
Small cell carcinoma (SCC) is a malignancy with aggressive growth pattern, high
recurrence rate, and tendency to metastatize to other sites via lymphatics and blood stream
that mainly occurs in the lung, with primary lesions in other sites being very rare [1]. The
extrapulmonary locations are minor salivary glands, parotid gland, nasal cavity, paranasal
sinuses, (hypo) pharynx, larynx, esophagus, stomach, intestine, pancreas, gallbladder, vulva,
uterine cervix, endometrium, urinary bladder, kidney, prostate, skin, thymus and other sites
[2]. Various diagnostic tools used for diagnosis of primary lung cancers include sputum
cytology, transbronchial biopsy and lavage, thoracocentesis and pleural biopsy, transthoracic
fine needle aspiration, and open lung biopsy [3]. For the diagnosis of extrapulmonary small
cell carcinomas both exfoliative cytology and fine needle aspiration, either imaging-guided or
non-guided depending upon the sites of the lesions, have been utilized. The histologic and
cytologic features of small cell carcinoma of the lung and extrapulmonary sites are well
described. Nevertheless, some small cell carcinomas may be difficult to reproducibly
distinguish from non-small cell carcinomas, especially poorly differentiated non-small cell
cancers, in both histology and cytology and this distinction carries significant clinical
importance [4, 5]. In case of diagnostic difficulties, various ancillary studies can be used for
distinguishing small cell carcinomas from other neoplasms.

Small Cell Lung Cancer (SCLC)


Small cell lung carcinoma (SCLC) accounts for 20% to 25% of all bronchogenic
carcinomas and is associated with the poorest survival of all histologic types [6]. SCLC is
most often a lesion of the central portion of the lung but is occasionally found in the
peripheral portions [2, 7]. In contrast to other major types of lung cancer, SCLC is highly
sensitive to both chemotherapy and radiation therapy, which results in initial significant
improvements in the survival of patients with this disease but the overall results remain
unchanged in the following years due to development of drug resistance and death of the
patients [8].
Small Cell Carcinomas: Contribution of Cytologic Tools... 53

Yun et al [9] observed that the age-adjusted incidence per 100,000 person-years
(standard Poisson regression analysis) for small cell carcinoma was 0.5 (95% CI: 0.2-1.4) for
never smokers, 3.5 (95% CI: 0.4-27.3) for former smokers, and 11.1 (95% CI: 1.5-82.9) for
current smokers. In this study [9] 83.6%, 14.5%, and 1.9% patients were current smoker,
former smoker, and never smoker, respectively. The age-adjusted incidence rates of small cell
lung cancer per 100,000 person-years ranged from 6.0 to 11.1 for men and 1.0 to 2.5 for
women in different periods during 1975 to 2003 in a report from Osaka, Japan [10]. Intensity
of smoking, duration, age at starting, and dose are all directly associated with all histologic
types of lung cancer, although the odds ratio (OR) is lower for adenocarcinoma than other
cell types [11]. Yun et al [9], based on the multivariate-adjusted relative risk for current
smokers observed the strongest association with smoking for small cell lung cancer (relative
risk, 21.7; 95% CI, 8.0-58.5) followed by squamous cell carcinoma (relative risk, 11.7; 95%
CI, 7.1-19.4) and then adenocarcinoma (relative risk, 2.1; 95% CI, 1.6-2.7). Sobue et al [12]
observed that for current smokers, the relative risk for combined small cell carcinoma and
squamous cell carcinoma was 12.7 (95% CI: 4.7-34.7) and 17.5 (95% CI: 4.9-62.1), while for
adenocarcinoma it was 2.8 (95% CI: 1.6-4.9) and 2.0 (95% CI: 0.8-5.0), for men and women,
respectively. According to Baldini and Strauss [13], women who smoke appear to be at
higher risk of developing small cell lung cancer than squamous cell lung cancer, whereas
men who smoke have a similar risk for the two histologic conditions. Yun et al [9] observed
that 88.7% small cell lung cancer cases had 20 years history of cigarette smoking and
90.7% smoked 10 cigarettes per day. Khuder et al [14] reported that early age at initiation of
smoking significantly increased the risk of small cell carcinoma (odds ratio = 3.0; 9% CI, 1.1-
8.4). Whereas quitting smoking reduced the risk of squamous cell carcinoma and
adenocarcinoma, it did not affect the risk of small cell lung cancer.
The frequency of SCLC among all lung cancer cases in histopathologic samples ranges
from 12.2% to 15.8% in various studies (Table 1) [9, 10, 12, 15-17]. In men the frequency
was higher (range, 13.0 to 15.7%) than that in women (5.1 to 12.4%) [10, 12, 17] and in an
autopsy study in men [11] the frequency was very high (28.9%).

Cytodiagnostic Tools for SCLC

Besides exfoliative cytology, transthoracic fine needle aspiration cytology [18, 19],
transbronchial needle aspiration [20], endoscopic ultrasonography-guided fine needle
aspiration biopsy [21] have been utilized for the diagnosis of lung cancer. In a study of 393
primary lung cancers, the diagnostic methods used by Okutan et al [3] were sputum cytology
(27.3%), transbronchial biopsy and lavage (38.6%), thoracocentesis and pleural biopsy
(15.8%), transthoracic fine needle aspiration (13.6%) and open lung biopsy (4.7%).
Transbronchial needle aspiration (TBNA) is a safe, easy to perform, and useful tool for the
diagnosis and staging of pulmonary neoplasm, with a minimum of complications [22].
However, the frequency of satisfactory specimen by this tool varies widely, which is as low
as 46% [22] and as high as 93.4% [18]. Khoo et al [23] observed that combining TBNA with
the option for EUS-FNA immediately after unrevealing TBNA gave a yield approaching that
54 Dilip K. Das

of mediastinoscopy (90%, with no complications) and, therefore, may reduce the need for
invasive mediastinal sampling.

Figure 1.
A. Small cell carcinoma of lung (SCLC): Ultrasound-guided FNA from a right lung mass in a 57-year-
old man: Smear shows a cluster of small round tumor cells with scanty cytoplasm. Nuclear molding is
evident (MGG x 400).
B. Smear shows small round tumor cells with fine to coarsely granular chromatin pattern, accompanied
by apoptotic cells and karyorrhetic debris in the background: Same case as shown in figure 1A
(Papanicolaou x 400).
C. Metastatic SCLC: FNA smear from the left supraclavicular lymph node in a 65-year-old man. Small
round tumor cells with occasional variation in nuclear size, show nuclear molding, paranuclear blue
inclusions, and occasional rosette formation (MGG x 400).
D. Tumor cells show nuclear molding, finely granular chromatin, and karyorrhectic debris: Same case
as shown in figure 1C (Papanicolaou x 400).

Frequency of Small Cell Lung Carcinoma in Cytologic Material

The frequency of SCLC among cytologically classified malignancies of the lung shows a
wide variation (Table 2) [3, 19, 22, 24-28], ranging from 3.5% to 33.3% (median, 14.3%).
The central nature of SCLC and very frequent involvement of the mediastinal lymph nodes
by this neoplasm can explain the highest frequency observed in one of the studies from this
review (33.3%), which was based on transbronchial FNA [22]. In some other studies which
did not specify the classification of non-small cell carcinoma (NSCC) into subtypes, the ratio
of SCLC to NSCC ranged from 2: 12 to 15: 50 [23, 29, 30].

Cytomorphology of SCLC

The FNA smears in SCLC are cellular with loose to tight clusters of small cells with high
nuclear to cytoplasmic ratio and practically no visible cytoplasm [18]. The chromatin pattern
is finely granular and diffuse with inconspicuous nucleoli. Nuclear molding, necrotic
Small Cell Carcinomas: Contribution of Cytologic Tools... 55

background, and nuclear smearing are observed (Figure 1 A and B). As compared to
conventional smears (CS), the ThinPrep (TP) slides of small-cell carcinoma, however, show a
cleaner background (P<0.005), decreased nuclear molding (P<0.025), decreased nuclear
smearing (P<0.05) and greater amount of discernible cytoplasm (P< 0.005) [31].

Extrapulmonary Small Cell Carcinoma (EPSCC)


Extrapulmonary small-cell carcinoma (EPSCC) has been recognized as a
clinicopathological entity, distinct from the small cell carcinoma of the lung [32, 33]. Some
of the histopathologically proved reports on extrapulmonary sites of involvement by SCC are
skin [34-36], sinonasal region [37, 38], parotid [39, 40], minor salivary glands of the tongue
[41], larynx [42]; breast [43, 44], thymus [45], pleura [45], esophagus [46-48], stomach [49,
50], extrahepatic bile duct [4], gallbladder [51, 52], ovary [53-55], endometrium [56], cervix
[57], vulva [58], kidney and renal pelvis [45, 59], urinary bladder [60-62], and prostate [63-
65].

Table 1. Frequency of Major Primary Lung Cancers in Histologic Material.


[9- 12, 15-17].

Series No of lung Histological type of cancer and frequency (%)


cancer cases Sq cell ca Adeno ca SCLC Others
Toyoda et al 18684 6029 (32.3) 8509 (45.5) 2760 (14.8) 1386 (7.4)
(2008)10
Men 13309 5031 (37.8) 5119 (38.5) 2096 (15.7) 1063 (8.0)
Women 5375 998 (18.6) 3390 (63.1) 664 (12.4) 323 (6.0)
Santos-Martinez et 678 225 (33.2) 202 (29.8) 89 (13.1) 162 (23.9)
al (2005) 17
Men 605 219 (36.2) 161 (26.6) 82 (13.6) 143 (23.6)
Women 73 6 (8.2) 41 (56.2) 7 (9.6) 19 (26.0)
Yun et al (2005) 9 1357 469 (34.6) 465 (34.3) 214 (15.8) 209 (15.4)
Sobue et al (2002) 422 97 (23.0) 181 (42.9) 47 (11.1) 97 (23.0)
12

Men 324 91 (28.1) 119 (36.7) 42 (13.0) 72 (22.2)


Women 98 6 (6.1) 62 (63.3) 5 (5.1) 25 (25.5)
Barbone et al 755 267 (35.4) 158 (20.9) 218 (28.9) 112 (14.8)
(1997)11*
Perng et al (1996) 10910 4048 (37.1) 4179 (38.3) 1331 (12.2) 1352 (12.4)
16

Lam et al (1993) 15 2385 797 (33.4) 890 (37.3) 327 (13.7) 371 (15.6)
* Autopsy study in men., Sq cell ca = squamous cell carcinoma, Adenoca = adenocarcinoma, SCLC
= small cell lung cancer
56 Dilip K. Das

In a study of 61 patients with EPSCC [33], it was observed that the most common
primary sites were the gastrointestinal (GI) tract (56%) and uterine cervix (18%). According
to Kim et al [32], uterine cervix was the most common site (7/24 cases, 29.2%) of EPSCC,
followed by urinary bladder (20.8%), colon or rectum (12.5%), kidney (8.3%), and stomach,
esophagus, pancreas, common bile duct, larynx, parotid gland, thymus (4.2% each). The
primary disease sites in a report of 101 cases by Haider et al [66] were as follows:
gastrointestinal (20%), genitourinary (18%), gynecologic (11%), head and neck (10%), breast
(9%), thymus (2%) and unknown primary sites (31%). Kim et al [67] in a study of 34 EPSCC
found that the primary sites were esophagus and thymus in 6 patients each (17.6%), pancreas
and stomach in 5 patients each (14.7%); other sites included were the cervix, abdominal
lymph nodes, abdominal wall, bladder, colon, maxillary sinus, nasal cavity, ovary, parotid
gland and liver.

Table 2. Frequency of Major Primary Lung Cancers in Cytologic Material.


[3, 19, 22, 24-28].

Series Diagnostic tools No of Cytologic types of lung cancer


cases Sq cell ca Adeno ca SCLC Others
Shah et al Tr Th FNA 100 45 (45.0) 22 (22.0) 16 (16.0) 17(17.0)
(2007) 19
Halloush et al Fluoroscopy and 94 32 (34.0) 36 (38.3) 9 (9.6) 17(18.1)
(2007) 27 CT-guided
Lannes et al TBNA 30 7 (23.3) 7 (23.3) 10 (33.3) 6 (20.0)
(2007) 22
Okutan et al Tr. Th. FNA +* 393 116 (29.5) 178 (45.3) 81 (20.6) 18 (4.6)
(2004) 3
Huq et al Fluoroscopy and 35 20 (57.1) 1 (2.9) 6 (17.1) 8 (22.9)
(2004) 26 CT-guided
Kck et al CT-guided Tr Th 141 74 (52.5) 30 (21.3) 5 (3.5) 32(22.7)
(2004) 25 FNA
Yasufuku et al EBUS-guided 40 14 (35.0) 15 (37.5) 5 (12.5) 6 (15.0)
(2004) 24
Das (2007) 28 CT and US-guided 49 16 (32.6) 26 (53.1) 4 (8.2) 3(6.1)
Tr Th FNA
Tr. Th. FNA = transthoracic FNA. *various other tools,

Skin

Merkel cell carcinoma (MCC) of the skin is a rare, primary small cell malignancy which
occurs in elderly patients, often presenting as a nodule in the head and neck region or
extremities [68, 69]. The tumor is located in the dermis, and spares the epidermis [69]. Skoog
et al [70] described the FNA smears from 4 cases of MCC and cytologic features of 29
aspirates from 19 patients were reported by Collins et al [68]. The aspirates in Merkel cell
carcinoma (MCC) of the skin typically produce highly cellular smears and show loosely
clustered and individual tumor cells, which are small and relatively monomorphic with scanty
Small Cell Carcinomas: Contribution of Cytologic Tools... 57

or absent cytoplasm and round to oval, regularly contoured, hyperchromatic nuclei with
coarse granular chromatin (salt and pepper) and inconspicuous nucleoli. The other features
are foci of rosette formation, nuclear molding, and prominent mitotic activity [36, 68-70].

Cervix

Small cell carcinoma of the cervix is a rare and an aggressive tumor with propensity for
rapid metastasis and high mortality [57, 71, 72]. Small cell carcinoma of the uterine cervix is
histologically and cytologically identical to pulmonary small cell undifferentiated carcinoma
[57]. In small cell carcinoma of the cervix, the smears display moderate to high cellularity in
most cases [73] and may contain malignant cells in singly dispersed form or arranged as
loosely cohesive sheets or gland-like aggregates [74] or with no typical architectural pattern
[73]. Tumor cells show nuclear molding and smearing, and range in size from small to large,
having minimal cytoplasm, extremely pleomorphic, hyperchromatic and angulated nuclei
with finely stippled (salt and pepper) chromatin [73, 74]. Mitotic figures are common and
karyorrhetic debris identified in all cases [74].

Urinary bladder

Small cell carcinoma of the urinary bladder accounts for 0.35-0.75% of all bladder
tumors [62]. It is an aggressive neoplasm that often demonstrates multidirectional
differentiation, including frequent but not invariable expression of neuroendocrine features
[60]. Whereas 27 (61.4%) of the 44 cases described by Choong et al [62] were pure SCC, 12
cases reported by Mills et al [60] resembled the intermediate and oat cell variants of SCLC.
Cytologic features of SCC urinary bladder have been described in a few reports [75-77].
In cytologic urinary specimen small cell carcinoma was characterized by numerous small,
lymphocyte-like round cells with high nuclear/cytoplasmic ratios, scant cytoplasm, and
hyperchromatic nuclei with fine to coarsely granular chromatin and small nucleoli [75, 76].

Gastrointestinal Tract

Small cell carcinoma of the esophagus is a rare but highly aggressive malignant tumor
[78]. Among 335 esophageal carcinomas, Koide et al [47] found 10 cases of SCC. SCC of
stomach is also a very rare disease and the prognosis is very poor compared with the common
type of gastric carcinoma [49, 79].
In a SCC of stomach, the peritoneal washing cytology show the presence of many
undifferentiated, small malignant cells with naked, hyperchromatic nuclei and finely granular
chromatin, in a necrotic background [79]. Some tumor cells contain paranuclear blue
inclusions (PBIs) in the cytoplasm.
58 Dilip K. Das

Breast

Sebenik et al [2] described FNA smears of small cell anaplastic carcinoma of breast as
highly cellular specimen consisting of loosely arranged clusters of small cells, slightly larger
than lymphocytes, showing prominent nuclear molding and having high nuclear-cytoplasmic
ratio; uniform, round nuclei with finely dispersed chromatin and inconspicuous nucleoli.
These neoplastic cells in breast aspirates resemble the small cell carcinoma of the lung [80].

Head and Neck Region

Nine primary neuoendocrine carcinomas of the salivary gland studied by Klijanienko et


al [81] displayed consistent cytological findings that included dispersed or loose clusters of
poorly differentiated small-to medium-sized cells, and occasional smudged nuclei; mild to
moderate nuclear pleomorphism, scant or absent cytoplasm, and nuclear molding. Rosette-
like patterns and multinucleated cells were occasionally observed. The cytologic features of
sinonasal undifferentiated carcinomas described by Bellizzi et al [38] included hypocellular
smears with a single-cell-predominant pattern. The cells were intermediate-sized with
irregular nuclear contours and small nucleoli accompanied by nuclear crush and mitotic
figures. Perez-Guillermo et al [82] described the FNA cytologic features of a SCC of the
eyelid which included round to ovoid and very uniform cells, either isolated or in loose
cohesive sheets with finely granular chromatin and small faintly stained juxtanuclear caps.
Rosette-like structures and frequent mitotic figures were noted.

Metastatic Lesions of SCC


SCLC represents a group of highly malignant tumors giving rise to early and widespread
metastasis at the time of diagnosis to sites such as lymph nodes, adrenal glands, liver, lung,
brain, and bones [83]. The common sites for lymph node metastasis are cervical (Figure 1 C
and D) and mediastinal (Figure 2 A and B) lymph nodes. Even if the external
lymphadenopathy is not clinically evident, in a case of suspected lung cancer, the external
lymph nodes should be assessed by imaging studies. In study of 101 suspected lung cancer
patients with enlarged mediastinal lymph nodes but no palpable supraclavicular
lymphadenopathy, US-guided FNA of supraclavicular lymph nodes yielded 49 metastatic
lesions including 33 non-small cell lung cancers, 8 SCLC, and 8 other malignancies [84].
Whether it the primary SCLC or its metastatic deposits in lymph nodes, the smears reveal
single and loose aggregates of small to medium-sized cells with a high nuclear-cytoplasmic
(N/C) ratio, round to angulated nuclei, and fine salt-andpepper chromatin, showing nuclear
molding. Cytologic features such as a significant crush artifact, streaking cytoplasm, and
cytoplasmic blobs can be appreciated in FNA smears. Among the uncommon and rare sites of
metastasis from SCLC, we observed a case of metastasis in the pancreas (Figure 3 A and B).
Chhieng et al [85] analyzed the records of 256 patients with SCLC. Of these, 8 (2.7%)
developed pleural effusion, indicating that SCLC rarely presents as pleural effusion. But
Small Cell Carcinomas: Contribution of Cytologic Tools... 59

whenever, a SCLC presents with pleural effusion one should carefully look for the malignant
cells. Of the 9 samples examined by Chhieng et al [85], 4 were positive and 2 were
suspicious of involvement by SCLC.
Like its pulmonary counterpart, EPSCC is an aggressive tumor with a high rate of
metastasis [87]. 49 FNA biopsies performed in 36 patients with various primary sites for SCC
other than lung and diagnosed as metastatic SCC were reviewed by Shin and Caraway [87].
The site distribution of FNAs in 36 extrapulmonary SCC (including MCC), were lymph
nodes (20), liver (7), bone (2), breast (1), pancreas (1), and skin/soft tissue (18). The primary
tumor sites included the prostate (14), skin (11; MCC), cervix (5), urinary bladder 93),
urethra (1), ovary (1), and parotid (1). Cheng et al [88] reported bilateral breast metastasis
diagnosed by FNA cytology from small cell carcinoma of the ovary detected following
oophorectomy in a 14-year-old girl. The cytologic features of metastatic MCC in inguinal
lymph node (Figure 4 A and B) [89, 90] and parotid [91] have also been described. Kuwatani
et al [92] described the cytologic features of a small cell carcinoma of the uterine cervix and
its metastasis in pancreas. In cytologic studies of metastatic SCC from various
extrapulmonary sites, the aspirates are found to be highly cellular and the smears reveal
predominantly dispersed single tumor cells with occasional clustering. Tumor cells are small
to medium with round to oval nuclei, scant cytoplasm, delicate nuclear membrane, fine
powdery chromatin, and inconspicuous nucleoli. Nuclear molding, mitotic figures, and
apoptotic bodies are frequently observed. Paranuclear pinkish, intermediate filament
buttons may be abundant in Romanowsky stained FNA smears; but rosette formation is
rare. Frequent mitotic figures and individual cell necrosis are observed [87, 89-92].

Figure 2.
A. Metastatic SCLC in mediastinum: US-guided FNA smear from an anterior mediastinal mass in a 47-
year-old man. Small tumor cells with round to oval nuclei show prominent nuclear molding and nuclear
smearing (MGG x 1000).
B. Tumor cells show fine to coarsely granular, salt and pepper chromatin pattern (Papanicolaou x
1000).
C. Neoplastic cells are strongly positive for cytokeratin x 1000).
D. Tumor cells are positive for neurone specific enolase (x 1000).
60 Dilip K. Das

Figure 3.
A. Metastatic SCLC in pancreas: Ultrasound-guided FNA from the pancreatic mass in a 66-year-old
man who presented with superior venacaval syndrome. Highly cellular smear shows small round tumor
cells, nuclear molding and paranuclear blue inclusions. Histology of transbronchial needle biopsy also
showed a small cell carcinoma (MGG x 400).
B. Nuclei show fine to coarsely stippled chromatin (Papanicolaou x 400).
C. Tumor cells show cap like positive reaction for cytokeratin. A small group of pancreatic epithelial or
mesothelial cells also show strong diffuse positive reaction for Ck (x 400).
D. A few tumor cells are positive for chromogranin (x 1000).
E. A group of tumor cells show positive reaction for neurone specific enolase (x 1000).

Figure 4.
A. Metastatic Merkel cell carcinoma (MCC): FNA from the 3 x 2 cm. right inguinal swelling in a 61-
year-old woman. Smear show cohesive groups of tumor cells showing nuclear molding, acinar
formation, pinkish paranuclear stained areas and paranuclear blue inclusions corresponding to
intermediate filament buttons (MGG x 400).
B. Fine chromatin pattern in tumor cells (Papanicolaou x 400).
C. Strong focal cap-like positive reaction for cytokeratin, corresponding to intermediate filament
buttons, in almost all tumor cells (x 1000).
D. Diffuse positive reaction for chromogranin in tumor cells (x 1000).
Small Cell Carcinomas: Contribution of Cytologic Tools... 61

Efficacy of Cytology in Diagnosis


of SCLC and EPSCC
Transthoracic FNAC of the lung is an accurate diagnostic tool for the diagnosis of lung
malignancies and is an excellent technique for distinguishing small cell carcinoma from other
malignant neoplasms. The efficacy of transthoracic FNAC in the diagnosis of small cell
carcinoma and other lung malignancies was 96% versus 88%, respectively, with an equal
specificity of 100% and sensitivity of 67% versus 81% [18]. Mitchell et al [93] observed that
the predictive values of FNA cytology for specific morphologic variants were 70% for
squamous cell carcinoma, 86% for adenocarcinoma, and 95% for small cell lung carcinoma.
Weisbrod et al [94] found that an unequivocal cytological diagnosis of SCLC had a positive
predictive value of 0.90 at their institution. According to Caya et al [95] none of the 31
patients with autopsy-and/or biopsy proven disease had false positive results for SCLC.
Typing accuracy for SCLC versus NSCC was 97% in their material. A review by Degaldo et
al [18], showed that the sensitivity of FNAC for diagnosis of small cell carcinoma ranged
from 87 to 100% (median, 100%), specificity ranged from 99 to 100% (median, 100%), and
efficacy ranged from 98 to 100% (median, 100%). By addition of transbronchial needle
aspiration to conventional diagnostic methods (CMD), diagnostic sensitivity increased from
60% to 82% (p= 0.001); diagnostic success increased from 52% to 76% in the non-small cell
cancer group (p= 0.001) and 81% to 95% in the small cell lung cancer group (p= 0.250) [20].
Kim et al [73] could diagnose or suggest the presence of small cell carcinoma in cervical
smears in 79% cases before histologic confirmation. Comparing cervical smears of 13 small
cell carcinoma uterine cervix with four cases of malignant lymphoma, 20 cases of squamous
cell carcinoma in situ, and five cases of chronic lymphocytic cervicitis, Kim et al [72]
observed that the cytologic differential diagnostic points of small cell carcinoma were nuclear
molding and smearing (100%), salt and pepper chromatin (100%), exudative and necrotic
background (91.7%), various architectures including individual cells (83.3%), tight clusters
(75%), feathering and strip (50%), and inconspicuous nucleoli (75%).

Limitations of Cytomorphology and


Differential Diagnostic Problems
Experiences of cytopathologists on the role of cytology in diagnosing small cell
carcinoma differ widely. According to Nicholson and Ryan [96], small cell carcinomas,
especially in bronchial brush and wash preparations, may be difficult to classify beyond
malignant. Kim et al [73] feel that cytologic features of small cell carcinoma of the uterine
cervix are characteristic enough for specific diagnoses or at least an early detection of it.
However, according to Zhou et al [74], the routine cervical smear is a relatively insensitive
and nonspecific method of detecting small cell carcinoma of the cervix. Although malignant
cells are detected in nearly 50% cases, the specific diagnosis of small cell carcinoma is
difficult, since the tumor cells can mimic inflammatory cells, follicular cervicitis, endometrial
cells, endocervical adenocarcinoma, squmous cell carcinoma of small cell type, non-Hodgkin
62 Dilip K. Das

lymphoma, or other malignant neoplasms. In practice of cytology, the diagnostic problem for
SCLC starts with its intermediate cell variant itself and extends to carcinoid tumor, EPSCC,
small cell variant of NSCC, and small round cell tumors (SRCT).

Morphologic Variants of SCC

The intermediate (polygonal cell, spindle cell) cell variant of SCLC incorporated in the
1981 World Health Organization (WHO) classification of lung tumors [97] continues create
diagnostic difficulty. 17 high-grade neuroendocrine carcinomas of the lung were classified into
5 small cell, 9 large cell and 3 mixed small/large cell types by Nicholson and Ryan [96].
According to these authors [96], review of mixed small and large cell NEC reveals the difficulty
in discerning and interpreting both small and large cell components in cytologic specimens.
Mixed small and large NEC may demonstrate both a range in cell size from small to large as
well as a rather abrupt transition between the two. The potential diagnostic pitfall is the
misinterpretation of SCC as a more poorly differentiated component of NSCC or spindle cell
SCC as tumoral desmoplastic response. Mugler and Sun [98] described a dimorphic variant of
small cell carcinoma mimicking lymphoma.

SCLC vs. Carcinoid Tumor

In the College of American Pathologists Non-Gyneclogic Cytology Program, Renshaw et


al [99] found that frequent misclassification of carcinoid tumor as small cell carcinoma in
lung fine-needle aspiration specimens correlated strongly with specific cytologic features,
some of which are common in small cell carcinoma (fine chromatin, molding, smudgy
chromatin) and others that are not (spindle-shaped cells). According to these authors [99]
awareness of these patterns including the commonly described carcinoid feature of streaming
vascularity (prominent streaming vascular pattern with tumor cells attached to the endothelial
cell core) may aid in avoiding misdiagnosis.

SCLC vs. EPSCC

The distinction between SCLC and SCC from other sites may be difficult by routine
cytology [100]. According to Shin and Caraway [87], cytologically diagnosed SCC from
different sites can not be differentiated, and its distinction requires clinical and radiographic
correlation. Due to difficulty in differentiating metastatic small cell undifferentiated
carcinoma from Merkel cell carcinoma (MCC), exclusion of metastatic neuroendocrine
tumors from other sites, especially small cell undifferentiated carcinoma of the lung, is a must
before the diagnosis of MCC is justified [89].
Small Cell Carcinomas: Contribution of Cytologic Tools... 63

SCLC vs. NSCC of Lung

Nuclear molding, cell size and scant basophilic cytoplasm were highly sensitive and
specific by univariate analysis for distinguishing SCLC from NSCC. Other features, such as
salt-and-pepper chromatin, crush artifact and apoptotic bodies, also had significantly high
specificity; however, their low sensitivity precluded their usefulness in separating SCLC from
NSCC [30]. In the College of American Pathologists Non-Gyneclogic Cytology Program,
Renshaw et al [4] found hat frequent misclassification of small cell carcinoma as non-small
cell carcinoma in the lung fine needle aspiration specimens strongly correlated with the
presence of cytoplasmic features that may suggest non-small cell carcinoma (increased
cytoplasm and apparent intracytoplasmic lumina) or with the presence of paranuclear blue
bodies.

Association with Other Neoplasms with SCLC and EPSCC

SCLC can occur as part of multiple primary neoplasms and EPSCC can also occur
simultaneously with other neoplasms (Table 3) [49, 56, 57, 60, 65, 101, 102]. The SCC of
urinary bladder may be associated with transitional carcinoma in situ [76], transitional cell
carcinoma [75, 77] or transitional cell as well as squamous cell carcinoma [77]. Presence of
more than one tumor in the same organ is likely to create diagnostic difficulty and confusion,
especially in cytologic samples

Table 3. Association of Other Neoplasms with Small Cell Carcinomas of the Lung
and Extrapulmonary Sites. [49, 56, 57, 60, 65, 101, 102].

Series SCC Associated neoplasms (when the site is


Site No of cases different, it is specified).
Murate et al Lung 1 Moderately differentiated adenocarcinoma of
(1989) 101 stomach and olfactory groove meningioma.
Matsui et al Stomach 1 Adenocarcinoma.
(1997) 49
Mebis et al Ovary 1 Endometroid adenocarcinoma of the
(2004) 102 ipsilateral ovary and Brenner tumor of the
contralateral ovary.
Huntsman et Endometrium 13 Endometroid adenocarcinoma in 8.
al (2004) 56
Gersell et al Cervix 15 Epidermoid carcinoma and adenocarcinoma
(1988) 57 (in situ and invasive forms) in 3 cases each.
Mills et al Urinary bladder 12 8 cases were associated with in situ or
(1987) 60 invasive transitional carcinoma (7 cases),
adenocarcinoma (3 cases), squamous cell
carcinoma (3 cases), spindle cell carcinoma
(1 case), and atypical carcinoid (1 case).
Sakuma et al Prostate 2 Adenocarcinoma.
(2007) 65
64 Dilip K. Das

Place of SCLC and EPSCC in the Spectrum of SRCT

Small cell carcinoma is a member of a group of neoplasms called small round cell tumors
(SRCT) which includes nonHodgkin lymphoma, neuroblastoma, retinoblastoma,
hepatoblastoma, nephroblastoma, embryonal rhabdomyosarcoma, small cell anaplastic
carcinoma, Ewing sarcoma/primitive neuroectodermal tumor, and desmoplastic small round
cell tumor [103]. Neuroblastoma, retinoblastoma, heptoblastoma, nephroblastoma, and
embryonal rhabdomyosarcoma, which are childhood tumors, should not come in the
differential diagnosis of small cell carcinoma. Among the remaining SRCTs, NHL is possibly
the main differential diagnostic entity. Certain characteristics of SCC such as nuclear
molding (100% cases), acinar formation (93.3%) and paranuclear blue inclusions (60%), are
conspicuously absent in NHL. On the other hand, exclusive noncohesive cell (95.7%) and
lymphoglandular bodies (87%), which are important features of NHL, are not seen or very
rarely observed in SCC [104]. According to De Las Casas et al [105], the identification of
distinct cytologic findings such as evenly dispersed fine-granular chromatin, paranuclear blue
inclusions, and nuclear fragments (significantly higher in small cell carcinoma, p<0.01) can
be a valuable aid to accurately diagnose and differentiate metastatic small cell carcinoma
from NHL in FNAB preparations from lymph nodes.
Bavikatty and Michael [31] felt that the presence of nuclear molding was the single most
useful feature in differentiating small-cell carcinoma from other small round-cell tumors.
However, nuclear molding to an extent has been observed in SRCTs other than SCAC [104,
106-108]. Although lymphoglandular bodies (LGB) in the background of a cytologic smear
from a malignant tumor are useful in alerting pathologists to the possibility of lymphoma,
there are exceptions, since LGB can be observed in a very small percentage of non-lymphoid
malignancies including small cell anaplastic carcinoma [104, 109, 110]. Under these
circumstances, there is a need for ancillary studies to solve the problem.

Contribution of Ancillary Studies


Carcinomas of Lung

TTF-1 is a nuclear transcription protein selectively expressed in the thyroid, the


diencephalons, and respiratory epithelium. It is positive in carcinomas of the thyroid. It is
expressed in 90% of pulmonary small cell carcinomas (SCLCs) and in almost 75% of
pulmonary non-small cell carcinomas (NSCLCs), especially in adenocarcinomas but is absent
in typical pulmonary carcinoids (TCs) [111]. The role of TTF-1 or TTF-1 and p63 in the
diagnosis of SCLC and its differentiation from NSCC and pulmonary carcinoids, have been
investigated by a number of investigators (Table 4) [5, 111-114]. Wu et al [114] observed
that p63 and TTF-1 were useful markers for differentiating SCLC from PDSqCC of lung in
formalin-fixed and alcohol-fixed, formalin-post fixed material. All 23 SCLC were negative
or, rarely, equivocal for p63 and 20/23 (87%) were TTF-1 positive. All 13 PDSqCCs were
TTF-1 -/p63+ with intense staining of 50% to 100% of tumor cells. TTF-1 immunostaining
showed equal or increased sensitivity in alcohol-fixed cytologic cell block samples compared
Small Cell Carcinomas: Contribution of Cytologic Tools... 65

with formalin-fixed biopsy material. Subsequently, Wu et al [5] found that the panel of p63
and TTF-1 were useful in the diagnostic evaluation cytologic cell block samples. 15/16
(93.8%) SCLC samples were p63-/TTF-1+; 4 primary lung adenocarcinomas were p63-/TTF-
1+; 4 nonpulmonary adenocarcinomas were p63-/TTF-1-; and 4 PDSqCC were p63+/TTF-1-.
Di Loreto et al [112] observed positive reaction for TTF-1 in 38 (92.7%) of 41 cases of
SCLCs, 5 (62.5%) of 8 adenocarcinomas, and one (11%) of 9 cases of squamous cell
carcinoma (SqCC) in cytological specimen and confirmed it in subsequent bronchial biopsy
specimens. According to these authors [112], TTF-1 is strictly associated with SCLC, but is
weakly expressed in various types of NSCC. Strum et al [113] observed positive
immunostaining for TTF-1 in 47/55 (85.5%) pure SCLC, 31/64 (48.4%) of large cell
neuroendocrine carcinomas (LCNECs), but in none of the 15 neuroendocrine hyperplasias
(NEH), 23 tumorlets, and 50 carcinoid tumors. In 19/20 (95.0%) combined SCLC and
LCNECs, TTF-1 expression was identical in both NE and non-NE components. These
observations challenge the concept of a spectrum of NE proliferations and tumors and lends
credence to the alternative hypothesis of a common derivation of SCLC and non-SCLC
including LCNEC. Using formalin-fixed material and heat-induced epitope retrieval, Folpe et
al [111] observed TTF-1 expression in 18 (35.3%) of 51 TCs, all of 9 (100%)
adenocarcinomas, 6 (75%) of the 8 LCNECs, and 20 (95.2%) of 21 SCLCs.

Table 4: Role of Thyroid Transcription Factor-1 (TTF-1) and p63


Immunostainings in Lung Tumors. [5, 111-114].

Series and nature of material used Diagnosis No of Antigen and % of cases


for immunostaining cases with positive reaction
TTF-1 P63
Di Loreto et al (1997) 112 SCLC 41 92.7 ND
Adenocarcinoma 8 62.5 ND
Sq.CC 9 11.1 ND
Folpe et al (1999) 111 SCLC 21 95.2 ND
LCNEC 8 75.0 ND
Adenocarcinoma 9 100.0 ND
Carcinoids 51 35.3 ND
Strum et al (2002) 113 SCLC 55 85.5 ND
LCNEC 64 48.4 ND
NEH 15 0.0 ND
Tumorlets 23 0.0 ND
Carcinoids 50 0.0 ND
Wu et al (2003) 114 SCLC 23 87.0 0.0
PD Sq.CC 13 0.0 100.0
Wu et al (2005) 5 SCLC 16 93.8 6.2
Adenoca.(Lung) 4 100.0 0.0
Adenoca.(EP) 4 0.0 0.0
PDSqCC 4 0.0 100.0
SCLC = small cell lung cancer, Sq.CC = squamous cell carcinoma, LCNEC = large cell neuroendocrine
carcinoma, NEH = neuroendocrine cell hyperplasia, Adenoca = adenocarcinoma, EP =
extrapulmonary.
66 Dilip K. Das

In SCLC and its metastatic deposits, the neoplastic cells are positive for cytokeratin and
neuroendocrine markers like neurone specific enolase, synaptophysin, and chromogranin
(Figure 2 C and D, Figure 3 C, D, and E). Chhieng et al [85] in a study of pleural effusion
specimens, positive or suspicious for involvement by SCLC, revealed positive reaction for
both chromogranin and TTF-1 in 5 out of 6 (83.3%), synaptophysin in 3 of 6 (50.0%) and
Ck20 in none. In 9 cases of cytologically proved SCLC in pleural fluid, Tamiolakis et al
[115] observed that the tumor cells were positive for neuron-specific enolase (NSE) in all the
9 cases (100%), whereas in 6/9 (66.7%) cases the tumor cells expressed synaptophysin, TTF-
1, and chromoranin A antigens.

SCLC vs. EPSCC

The distinction between SCLC and SCC from other sites may be difficult by routine
cytology but immuno-histochemical/-cytochemical stainings show promising results in this
endeavor (Table 5) [100, 116-118]. In a study of 82 SCC from various sites (28 lung, 18 skin,
12 GIT, 8 sinonasal, 5 bladder, 3 prostate, 3 uterine cervix, 2 thyroid, 2 salivary gland, and 1
pancreas), Ordonez [100] observed TTF-1 positivity in 27 of 28 (96.4%) SCLCs, and 4 of the
54 (7.4%) nonpulmonary SCC, that included 2 (16.7%) from GIT, 1 (20%) from bladder, and
1 (33.3%) from cervix. On the other hand 16 of 18 (88.9%) Merkel cell carcinomas (MCC)
and 1 (3.6%) SCLC cases were positive for Ck20. The author concluded that although TTF-1
is not a specific marker for SCLC, it may assist in distinguishing SCLC from some
nonpulmonary small cell carcinomas, particularly MCC, especially when it is used in
conjunction with Ck20. Byrd-Gloster et al [116] observed immunostaining for TTF-1 in 97%
of 36 SCLCs and none of the 21 Merkel cell tumors (MCC). Immunoreactivity for Ck20 was
demonstrated in 76% of Merkel cell tumors and 3% of SCLCs. These data indicate that TTF-
1 is a sensitive (97%) and specific (100%) marker for SCLC and can be used to differentiate
SCLCs from MCC. Leech et al [117] observed that the use of both anti-Ck20 and anti-TTF1
can reliably distinguish between MCC and metastatic small cell carcinoma (SCC), thus
avoiding the need for a detailed clinical investigation of patients with MCC in whom
metastatic SCC must be excluded. Among MCC cases, 10 (90.9%) of 11 were positive for
Ck20 and none was positive for TTF-1 and of the 10 SCC cases, none was positive for Ck20
and all were positive for TTF-1 in their material. Cheuk et al [118] also found that
immunostaining for TTF-1, especially when combined with immunostaining for Ck20, can
aid in the distinction between MCC and SCC (both pulmonary and extrapulmonary).
However, in individual cases, these markers can not be used to distinguish between
pulmonary and extrapulmonary SCC due to extensive overlap in immunophenotypes. In their
study [118] involving 52 pulmonary SCC, 50 extrapulmonaty SCC, and 23 MCC cases, TTF-
1 was expressed in 82.7% of pulmonary SCC, 42% of extrapulmonary SCC (range, 33.3%--
53.3% for various sites), and 0% of MCC. Ck20 was positive in 0% of pulmonary SCC, 4%
of extrapulmonary SCC, and 100% of MCC.
Small Cell Carcinomas: Contribution of Cytologic Tools... 67

Table 5. Role of Thyroid Transcription Factor-1 (TTF-1) and Ck-20 Immunostainings


in Small Cell Lung Cancer (SCLC) Vs. Extrapulmonary
Small Cell Carcinoma (EPSCC). [100, 116-118].

Series Diagnosis No of cases Antigen and % of cases


with positive reaction
TTF-1 Ck20
Ordonez (2000) 100 SCLC 28 96.4 3.6
EPSCC* 54 7.4 -
MCC 18 0.0 88.9
Byrd-Gloster et al (2000) SCLC 36 97.2 3.0
116
MCC 21 0.0 76.2
Leech et al (2001) 117 Meta SCC 10 100.0 0.0
MCC 11 0.0 90.9
Cheuk et al (2001) 118 SCLC 52 82.7 0.0
EPSCC** 50 42.0 4.0
MCC 23 0.0 100.0
SCLC = small cell lung cancer, EPSCC = Extrapulmonary small cell carcinoma,
MCC = Merkel cell carcinoma, Meta SCC = Metastatic small cell carcinoma, *
Including MCC, ** Excluding MCC.

Merkel Cell Carcinoma (MCC) of Skin

The diagnosis of MCC of the skin by FNA can be made by applying cytologic features in
addition to ancillary studies and clinical information [68]. The malignant cells in MCC are
characteristically positive for cytokeratin (CK) 20 (punctuate, globular/perinuclear pattern of
staining), neuroendocrinethe markers like synaptophysin and chromogranin (focal positivity),
and negative for thyroid transcription factor (TTF-1, pulmonary or thyroid origin excluded)
and CD45 (lymphoma excluded) [36]. The MCC cells are also positive for CAM5.2 and
CD56 (neuroendocrine marker). ICC results in MCC are universally positive for Ck, which
show a paranuclear dot-like pattern; neurone-specific enolase, epithelial membrane antigen,
and S-100 protein are positive in varying degrees, but leucocyte common antigen is
universally negative [68]. Skoog et al [70] observed a peculiar dot-like cytokeratin positivity
and diffuse NSE positivity in four MCC diagnosed by FNA. A weak positivity for S-100 was
observed. Das et al [90] described a metastatic MCC, in which immunocytochemical
stainings on FNA smears revealed focal button-like Ck positivity, diffuse positive reaction
for chromogranin, and negative reaction for LCA (Figure 4 C and D).

SCC of Breast

The small cell carcinoma of the breast reported by Kitakata et al [80] was positive for
neuroendocrine differentiation markers such as synaptophysin, CD56, and neurone specific
enolase (NSE), but negative for TTF-1, leukocyte common antigen (LCA), estrogen receptor
68 Dilip K. Das

(ER), and progesterone receptor (PR). Interestingly, the tumor cells lacked immunoreactivity
for epithelial markers, including cytokeratin AE1/3, CAM5.2, and epithelial membrane
antigen (EMA) in this case. In a small cell anaplastic carcinoma of breast diagnosed by FNA
cytology, Sebenik et al [2] observed numerous lymphoglandular bodies. However, the tumor
cells were strongly positive for NSE focally and negative for keratin and LCA.
Flowcytometric studies did not reveal any lymphocytic marker.

SCC of Esophagus

Ultrastructural studies performed in 92 SCC of the esophagus revealed neurosecretory


granules in 54 (58.7%) [119].

SCC of Stomach

In a SCC of stomach, the tumor cells were positive for Grimelius, gamma-NSE,
chomogranin A, and serotonin [120]. Tumor cells of the case described by Matsui et al [49]
were argyrophilic and were and also positive for chromogranin A.

SCC of Ovary

Immunohistochemistry can differentiate the two different types of small cell carcinomas
of the ovary. Whereas the hypercalcemic type of small cell ovarian carcinoma cells show
positive reaction for parathyroid hormone-related protein [54], the pulmonary type neoplasm
may be positive for cytokeratin, EMA, NSE, and chromogranin [53].

SCC of Endometrium

None of the cases described by Huntsman et al [56] contained argyrophil or argentaffin


cells, although 9 of the 11 tumors were immunoreactive for NSE.

SCC of Cervix

All the 13 small cell undifferentiated carcinomas reported by Gersell et al [57] expressed
one or more epithelial markers and at least one neuroendocrine marker. Electron microscopy
demonstrated dense-core granules in 6 of 7 tumors. In the study by Conner et al [71] all the
23 cases were immunoreactive for Ck, 10 positive for chromogranin, 13 positive for
synaptophysin, and 10 expressed p53 protein.
Small Cell Carcinomas: Contribution of Cytologic Tools... 69

SCC of Urinary Bladder

11 of the 12 cases reported by Mills et al [60] were positive for one or more of the
neuroendocrine markers (NSE, chromogranin, Leu-7, vasoactive intestinal peptide, and
serotonin). Electron microscopy performed in 7 tumors demonstrated dense-core granules in
all cases. Van Hoeven and Artymyshyn [121] in a study of 13 SCC of urinary bladder,
confirmed neuroendocrine nature of the neoplasm in 11 cases (NSE positive in 11 of 12
cases, synaptophysin in 2/11, chromogranin in 2/13, leu7 in 2/7) and by ultrastructural
analysis in two. SCC carcinoma cases of the urinary bladder described in cytologic specimens
have also been confirmed by ultrastructural and/or immunocytochemical studies [75, 77].

SCC of Prostate

Immunohistochemical study performed by Okada et al [63] revealed intense CEA


positivity, focal NSE positivity and weak positive reaction for PSA in SCC of prostate
associated with adenocarcinoma and intense NSE positivity and weak chromogranin A
positivity in a pure SCC. In the two combined SCC/adenocarcinoma cases reported by
Sakuma et al [65], NE markers (CgA and SNP) were weakly expressed in the part of SCC.

SCC of Stomach

Yamaguchi et al [79] reported a SCC of stomach, in which the tumor cells from
peritoneal washing cytology were positive for NSE, and synaptophysin on
immunocytochemical stainings.

Role of Cytology in Management


Transthoracic FNAC can be used with confidence to select treatment modalities and
avoid unnecessary surgeries in patients with lung malignancies. When the diagnosis of small
cell carcinoma is reached in a patient with a lung mass, a surgical treatment approach is no
longer considered and chemotherapy becomes the treatment of choice [18]. As a result of
EUS-FNA, thoracotomy/thoracoscopy was avoided in 49% and mediastinoscopy was avoided
in 68% in a study by Larsen et al [122]. According to Yasufuku et al [24], cytologically
diagnosed all the 5 cases of small cell lung cancer in their study were referred for
chemotherapy. In the report by Larsen et al [122], the direct result of the cytological
diagnosis obtained by EUS-FNA was that a final diagnosis of small cell lung cancer was
made in eight patients resulting in referral for chemotherapy, and in another three patients
with benign disease specific treatment could be initiated (sarcoidosis, mediastinal abscess,
and leiomyoma of esophagus).
Extrapulmonary small cell carcinoma (EPSCC) differs from small-cell lung carcinoma
(SCLC) in respect of etiology (smoking history significantly less in EPSCC), clinical course,
70 Dilip K. Das

survival (significantly better in EPSCC), frequency of brain metastasis (higher in SCLC), and
early death (higher in SCLC). These possible differences may influence the choice of
therapeutic approach [123]. FNA enables an early and confident diagnosis of an aggressive
tumor like MCC and an early planning of surgery [82]. Among EPSCC, majority of patients
with limited disease are treated with surgery, followed by adjuvant chemotherapy or
chemoradiotherapy [33]. This is evident from a number of studies, wherein surgical approach
with or without radiation therapy and chemotherapy has been resorted to in small cell
carcinoma involving extrapulmonary sites such as parotid gland [40], nasal cavity [1], minor
salivary glands of the tongue [41]. However, Kim et al [32] observed that regardless of
primary site or disease stage, EPSCC of sites other than cervix was usually a fatal disease
with a discouraging outcome for various treatment modalities. Overall, the response to
various treatment modalities and the median survival time observed were discouraging for
EPSCC [33].

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78 Dilip K. Das

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In: Small Cell Carcinomas: Causes, Diagnosis and Treatment ISBN: 978-1-60741-787-3
Editors: J. G. Maldonado and M. K. Cervantes 2009 Nova Science Publishers, Inc.

Chapter 3

Radiotherapy for Small Cell


Lung Cancer

Don Yee*
Radiation Oncologist, Cross Cancer Institute and Associate Professor, Department of
Oncology, University of Alberta, 11560 University Avenue, Edmonton, Alberta,
CANADA, T6G 1Z2

Abstract
Small cell lung carcinoma (SCLC) comprises approximately 20% of all primary lung
cancers. Biologically, SCLC is characterized by rapid proliferation, widespread
dissemination, and despite good response rates to initial treatments, poor overall survival
rates. The most widely-used staging system for this cancer features a dichotomy between
what is termed "limited-stage" or "extensive-stage" disease. Traditionally, chemotherapy
has been the mainstay of therapy for SCLC, but in the past 10-15 years, radiotherapy
interventions have provided the largest improvements in survival outcomes for SCLC
patients. Patients with limited stage disease derive local control and overall survival
benefits from the addition of concurrent thoracic radiation treatments given with their
chemotherapy. Prophylactic cranial radiation treatments given to limited stage disease
patients whose disease responds completely to initial induction treatments provides
improved freedom from CNS disease relapse and further overall survival benefits. The
benefits of concurrent thoracic radiotherapy and prophylactic cranial radiotherapy for
limited stage disease patients have been confirmed in several published meta-analyses.
Patients with extensive stage disease have what is considered incurable disease. Usually,
these patients are treated with chemotherapy alone, with radiotherapy reserved to palliate
bothersome local symptoms attributable to disease as they arise. A recently-published
randomized trial has demonstrated the CNS control and overall survival benefits of
giving prophylactic cranial radiation treatments to extensive stage patients who respond
to their initial chemotherapy treatments. From a radiotherapy perspective, the weight of
the current evidence available from published clinical trials indicates a beneficial role of:

*
Corresponding author: Phone: +1 780 432 8783, Fax: +1 780 432 8380, Email: donyee@cancerboard.ab.ca
80 Don Yee

1) thoracic radiotherapy given concurrently with chemotherapy followed by prophylactic


cranial irradiation in complete responders for patients with limited stage disease and 2)
prophylactic cranial irradiation for extensive stage disease patients who respond to
chemotherapy. This chapter will outline results from seminal clinical trials that have
established the important role of radiotherapy in the management of SCLC patients.
Ongoing areas of controversy with regards to SCLC radiotherapy including ideal
radiotherapy target volume and dose/ fractionation regimen for both thoracic
radiotherapy and prophylactic cranial irradiation and the potential role of consolidation
chest radiotherapy for patients with extensive stage disease will also be discussed.

Introduction
Small cell lung carcinoma (SCLC) makes up approximately 10-20% of all primary lung
cancer histologies. This particular histology is almost always associated with smoking and is
the most aggressive histologic subtype of lung cancer, clinically characterized by widespread
metastases, rapid dissemination, and poor survival outcomes. At the time of diagnosis,
approximately 20-30% of patients will have what is considered limited stage disease (LS-
SCLC) with the remainder having extensive stage disease (ES-SCLC) [1]. Extensive stage
disease is considered incurable and is associated with a dismal average survival of
approximately 6-12 months. Patients with LS-SCLC are usually offered a potentially curative
combination of chemoradiotherapy which is associated with median survival rates of
approximately 16-24 months [2]. Historically, treatment for SCLC consisted mainly of
chemotherapy alone, but results from recently published clinical trials and meta-analyses
have established the important role of radiotherapy (RT) in the management of SCLC
patients. In the current-day management of SCLC, both limited stage and extensive stage
patients usually have RT treatment options available to them. This chapter will outline the
role RT has in the treatment of SCLC patients and highlight the published literature which
has established the integral role of RT in the treatment of SCLC patients.

Staging and Clinical Work-up


Though a TNM staging system exists for primary lung cancers, the most widely used
staging system for SCLC is the Veterans Administration Lung Study Group (VALG) system
which utilizes the dichotomy of limited stage versus extensive stage disease [3]. The VALG
system defines LS-SCLC as disease confined to one hemithorax which is encompassable
within a reasonable RT portal. This staging system for SCLC was proposed in an era of
radiation oncology where radiation treatments were planned using a simpler two-dimensional
fluoroscopy-based approach of RT planning. This staging system considers patients with
pleural effusions with or without confirmation of cytologic involvement, contralateral hilar,
mediastinal or supraclavicular nodal involvement to have extensive stage disease. Most
clinicians who treat SCLC would still consider those with pleural effusions to have extensive
stage disease, and modern clinical trials involving LS-SCLC patients exclude those with
pleural effusions, with or without confirmation of cytologic involvement.
Radiotherapy for Small Cell Lung Cancer 81

Evolution of RT planning and delivery software may alter the traditional definitions of
limited stage versus extensive stage SCLC. The past 10-15 years has been witness to the
development and implementation of image-based three-dimensional approaches to RT
planning which employ a variety of imaging modalities including CT, MRI and nuclear
medicine. Modern RT treatment planning software enables calculation of anticipated
radiation doses to three-dimensional structures including target volumes and normal critical
structures defined usually from a CT image set. Anticipated doses to defined structures are
usually represented graphically in the form of dose-volume histograms. This three-
dimensional volumetric approach to RT treatment planning has facilitated the study of dose-
volumetric correlates with observed clinical RT toxicity rates. Graham reported results from a
retrospective study of 99 patients with non-small cell lung cancer (NSCLC) treated with RT
alone which correlated various tumor-related and dose volume histogram parameters with
rates of clinically significant radiation pneumonitis observed in the patient cohort. On
univariate analysis, the four factors which predicted for development of grade >= 2 radiation
pneumonitis were: percent of total lung volume which received at least 20 Gy (V20), the
effective volume (Veff) and total lung volume mean dose, and location of primary lung tumor
(upper versus lower lobe). On multivariate analysis, the only independent predictor of grade
>=2 radiation pneumonitis was V20 [4]. Since this seminal publication, the association
between dose volume histogram parameters and risk of radiation pneumonitis has been well-
established [5]. With improved ability to predict risk of radiation pneumonitis based on
anticipated RT doses to normal lung tissue, what is considered to be a tolerable lung portal
may be more clearly defined than simply being able to draw a rectangular RT portal around
intrathoracic disease visualized on a plain chest film. More prospective clinical trials where
RT for lung cancer patients is planned with modern RT planning software and observed RT
toxicity rates are correlated with dosimetric and other patient- and disease-related factors are
needed to more accurately define what is or is not a tolerable thoracic RT plan for lung
cancer patients.
Initial clinical work-up of SCLC involves a complete history and physical examination to
identify signs and symptoms of locoregional extension and/or distant metastatic disease.
Histologic confirmation of the cancer subtype is required to confirm the pathologic diagnosis
and is usually obtained through one of bronchoscopy, transthoracic CT-guided biopsy,
sputum cytology or open lung biopsy of a lung mass or biopsy of a suspicious accessible
lesion such as a liver mass or supraclavicular node. Pathologic specimens which are
equivocal for SCLC may be subjected to immunohistochemical stains to help confirm the
diagnosis. Laboratory investigations include assessment of complete blood counts, liver and
renal function and serum LDH levels. Pulmonary function tests are required for patients
being considered for radical thoracic RT. Radiologic investigations required include CT scan
of the head, thorax and abdomen and bone scan. MRI of the brain and positron emission
tomography (PET) scan are additional radiologic investigations which are not routinely done
for all patients, but may be performed if routine radiologic tests are equivocal in their ability
to differentiate between limited versus extensive stage disease.
82 Don Yee

Radiotherapy for Limited Stage Disease


(LS-SCLC)
Treatment of LS-SCLC and Benefits of Thoracic RT for LS-SCLC

Role of chemotherapy
Since the 1980's SCLC has been well-known to be an exquisitely chemosensitive tumor.
The current standard chemotherapy regimen for LS-SCLC is a combination of cisplatin-
etoposide. Though concurrent chemoradiotherapy is the current standard of care for patients
with LS-SCLC, no accepted consensus regarding what the most appropriate chemotherapy
regimen for this patient group exists. Three separate published meta-analyses or systematic
reviews have attempted to clarify this issue. Pujol's meta-analysis studied 19 randomized
trials including over 4000 patients enrolled in studies published from 1966-1999 to examine
the role of cisplatin in treatment of SCLC. Trials compared cisplatin-containing regimens to
regimens without cisplatin. The authors reported that patients treated with cisplatin-
containing regimens had a lower risk of death at 1 year which translated to a significantly
increased 1-year survival probability by 4.4% [6]. Mascaux's meta-analysis of 36 randomized
trials included over 7000 patients. Randomized trials included in this report compared
cisplatin and etoposide either together or alone versus other chemotherapy combinations
which did not include either drug. The authors reported significant survival advantages
favoring etoposide when compared to chemotherapy regimens which contained neither
cisplatin nor etoposide, regimens of cisplatin plus etoposide compared to regimens with
neither agent or regimens containing etoposide without cisplatin [7]. The most recent
published article attempting to identify the optimal chemotherapy regimen for LS-SCLC is a
practice guideline from the Lung Cancer Disease Site group of the Cancer Care Ontario's
program of Evidence-based Care in Canada [8]. This systematic review included 50
randomized controlled trials, including all trials studied in the Pujol and Mascaux
publications. The authors identified the most commonly used chemotherapy regimens in
SCLC clinical trials were either CAV (cyclophosphamide/ doxorubicin/ vincristin) or EP
(etoposide-cisplatin). The practice guideline endorsed cisplatin-etoposide as the preferred
chemotherapy regimen for LS-SCLC patients undergoing combined modality therapy, but
pointed out several qualifying findings including: only 10 randomized chemotherapy trials
have accrued exclusively limited stage patients and most relevant trials were conducted and
published prior to 1995 in an era where limited stage patients were treated with sequential as
opposed to concurrent chemoradiotherapy.
For patients who cannot receive cisplatin, carboplatin has been shown to be associated
with fewer toxicities and is generally considered to be a reasonable alternative systemic
chemotherapy agent. While carboplatin is considered an acceptable substitute for cisplatin for
patients who cannot tolerate cisplatin, no evidence exists to definitively show the equivalence
of carboplatin to cisplatin in terms of efficacy for LS-SCLC. The only randomized trial
comparing cisplatin-etoposide to carboplatin-etoposide in SCLC patients included 143 SCLC
patients where 41 limited stage patients were randomized to each arm. No significant
differences in response or survival were reported, but the trial was underpowered to prove
equivalence in the patients with limited stage disease [9]. No evidence exists to support the
Radiotherapy for Small Cell Lung Cancer 83

use of maintenance chemotherapy or high-dose chemotherapy with stem cell rescue in these
patients, and these approaches remain investigational for LS-SCLC.
Patients who recur after chemoradiotherapy are potentially eligible for second-line
chemotherapy. Known predictors of response to second-line chemotherapy after recurrence
include: extent of response to initial chemotherapy regimen, duration of initial response, time
off chemotherapy, and patient performance status [10].

Role of external beam thoracic RT


Prior to 1970, surgical resection was the standard treatment for all lung cancers,
including SCLC. As the specialty of radiation oncology evolved and the use of external beam
RT for cancers expanded, questions about the optimal local treatment for SCLC arose. A
MRC trial randomizing SCLC patients between external beam RT or surgical resection failed
to demonstrate any benefit for surgery [11]. As RT was associated with less morbidity than
surgery, interest in the use of external beam RT for definitive local therapy for SCLC
increased.
The subsequent discovery that SCLC is very chemosensitive established systemic
chemotherapy as the cornerstone treatment modality for this disease [6]. Although platinum-
based chemotherapy agents were associated with high initial response rates, local failure rates
between 75-90% were still observed [2]. These poor local control rates led to the
investigation of thoracic RT in addition to chemotherapy to try improve local control rates.
The local control and overall survival benefits conferred by the addition of concurrent
external beam thoracic RT to chemotherapy regimens for LS-SCLC have been quantified in 2
published meta-analyses. Warde's meta-analysis of 11 randomized trials of chemotherapy
alone versus chemotherapy combined with thoracic RT reported the addition of thoracic RT
to chemotherapy for LS-SCLC significantly improves 2-year overall survival by 5.4% and
local intrathoracic control by 25.3% [12]. Pignon's meta-analysis included 13 randomized
trials of over 2000 LS-SCLC patients. They reported the addition of thoracic RT to
chemotherapy for LS-SCLC resulted in a 14% reduction in mortality compared to
chemotherapy alone. Similar to results from Warde's meta-analysis, this corresponded to an
overall 3-year survival benefit of 5.4% [13]. The overall survival and local control benefits
conferred by addition of local thoracic RT to chemotherapy regimens for LS-SCLC patients
demonstrated in these 2 meta-analyses have established thoracic RT as part of the standard
management of LS-SCLC patients.

Relative Integration of Thoracic RT with


Chemotherapy for LS-SCLC
While Warde's and Pignon's meta-analyses established the role of thoracic RT in the
management of LS-SCLC, the question of what the optimal timing and integration of thoracic
RT with chemotherapy remained unanswered. Murray et al. reported results from a trial on
behalf of the National Cancer Institute of Canada (NCIC) which randomized 308 LS-SCLC
patients receiving three cycles of CAV (cyclophosphamide, doxorubicin, vincristine)
alternating with EP (cisplatinum and etoposide) to 40 Gy / 15 fractions of thoracic RT given
84 Don Yee

with either the first or final (third) chemotherapy cycle. Patients without progressive disease
after completion of all chemoradiotherapy treatments were given prophylactic cranial
irradiation (25 Gy/ 10 fractions). With a median follow-up time of almost five years,
significantly improved progression-free survival (median 15.4 months versus 11.8 months,
p=0.036) and overall survival (median 21.2 months versus 16.0 months, p=0.008) was
observed in patients given early RT [14]. Other trials from the same era attempting to answer
this question were hampered by low numbers and the use of what would now be considered
inferior chemotherapy regimens and/ or antiquated RT techniques [15]. In addition, different
clinical trials used different definitions of 'early' versus 'late' RT, making results of individual
trials difficult to interpret and the issue of early versus late RT largely unresolved at the time.
In the past 3 years, 5 published meta-analyses have tried to determine the optimal timing
of thoracic RT relative to chemotherapy for LS-SCLC [16-20]. All reported at least statistical
trends for improved survival associated with earlier RT. In addition to a meta-analysis, Spiro
replicated Murray's 1993 NCIC trial in his 2006 report. Unlike Murray however, Spiro was
unable to demonstrate a survival benefit in patients given RT with an earlier cycle of
chemotherapy. This may be due to fewer patients (69%) in his trial completing all prescribed
chemotherapy compared to Murray's trial, inclusion of patients with a performance status of 3
and/or less stringent staging criteria used in Sprio's trial [21]. The results from Spiro's trial
did however point out the importance of LS-SCLC patients receiving adequate systemic
therapy.
Another potential parameter of potential prognostic importance in LS-SCLC treatment is
the elapsed time from the first day of any treatment to the completion of thoracic RT. De
Ruysscher's recently published systemic review demonstrated this time factor was the most
important prognosticator of patient survival outcomes in LS-SCLC, with the best survival
outcomes occurring when thoracic RT is completed within 30 days from the start of
chemotherapy. De Ruysscher's review indicated each week thoracic RT extends beyond 30
days from the start of chemotherapy is associated with an absolute decrease in 5-year overall
survival of about 2%[22].

Thoracic RT Dose-Fractionation
Though the role of thoracic RT for LS-SCLC has been established, the ideal RT dose/
fractionation parameters for LS-SCLC thoracic RT have not yet been defined. LS-SCLC
patients treated in most Canadian centres receive between 45-50 Gy using conventional daily
fractionation schemes (180-200 cGy/ fraction), but these patients still have significant rates of
local failure ranging from 30-60% [2]. These poor local control outcomes may be at least in
part related to the natural biology of SCLC. Non-small cell lung cancers are considered to be
rapidly growing tumors with doubling times ranging from 72-146 days [23]. Such rapid
doubling times are believed to enable these tumors to repopulate rapidly during RT [24],
which likely worsens local control rates. Since SCLC is generally thought to be a more
rapidly growing histology than non-small cell [23], the impact tumor repopulation occurring
during RT which worsens local control may be even greater with SCLC.
Radiotherapy for Small Cell Lung Cancer 85

From a radiation oncology perspective, a radiobiologic concern regarding treatment of


SCLC is the phenomenon of accelerated tumor repopulation during RT, given that SCLC is
characteristically a rapidly-proliferating tumor [23]. Repopulation can decrease tumor control
rates in tumors treated with RT especially if delays in treatment occur [25]. The phenomenon
of accelerated tumor repopulation may apply to SCLC as published evidence suggests that
treatment interruptions worsen outcomes in LS-SCLC [26] and prolonging the time from the
start of any treatment to the end of thoracic RT past 30 days is associated with poorer
outcomes for LS-SCLC patients [20,22,27]. These observations need confirmation in future
prospective trials involving LS-SCLC patients.
Two RT-related paradigms are now being recognized as crucial to optimizing RT for LS-
SCLC by improving tumor sterilization and preventing tumor repopulation: 1) the need to
intensify RT dose and 2) the need to minimize the overall time to complete thoracic RT. To
achieve these goals, investigators have studied the effectiveness of different RT dose/
fractionation regimens. An RT dose-response relationship has been demonstrated for SCLC
[28,29], and external beam thoracic RT doses of up to 70 Gy using conventional once-daily
fractionation have been shown to be tolerable with promising results [30]. The CALGB
conducted a phase 1 RT dose escalation trial intended to define the maximal tolerated
thoracic RT dose for LS-SCLC (given with concurrent chemotherapy), using both once-daily
and twice-daily RT dose-fractionation regimens. Toxicities on this trial were scored
according the NCI Common toxicity criteria endpoints of grade 3 acute esophagitis and
pneumonitis. This trial found that the maximal tolerated dose of twice-daily RT was 45 Gy.
Patients were treated with up to 70 Gy using daily RT fractionation, which was well-tolerated
and the authors reported that the maximal tolerated dose of daily RT was not reached. Long-
term survival data from this trial were presented at the 2002 meeting of the American Society
of Clinical Oncology, demonstrating a median survival of 29.8 months and 24 months in the
twice-daily and daily RT arms with 5-year survival of 36% and 20% in the daily and twice-
daily arms, respectively[31]. This data suggests that twice-daily RT may not be the ideal
thoracic RT dose/fractionation strategy if one is able to give biologically equivalent RT
regimens using once-daily fractionation schedules.
A randomized study has compared twice-daily thoracic RT with standard once-daily
fractionation for SCLC. Turrisi randomized 417 LS-SCLC patients to receive either 45 Gy /
25 daily fractions or 45 Gy / 30 fractions (2 fractions given per day) starting with cycle one
of cisplatin/ etoposide chemotherapy [32]. With a median follow-up of almost 8 years, Turrisi
reported significantly improved median survival (19 months versus 23 months), overall
survival (16% versus 26% at five years) and local control (48% versus 64%) favouring
patients who received the twice-daily RT regimen, but at the cost of significantly increased
rates of grade 3 or higher acute radiation esophagitis (27% versus 11%). Since this seminal
trial was published, Patterns of Care studies in Japan and the United States [33,34] suggest
that twice-daily RT has not become a part of routine clinical practice for most radiation
oncologists who treat SCLC, as concerns exist regarding toxicity of this regimen and its true
efficacy in the face of contradictory findings from other randomized trials of twice daily RT
for LS-SCLC [35,36]. Schild and Bonner reported results from randomized clinical
comparisons of 50.4 Gy given in 28 daily fractions with 48 Gy given in 32 fractions given
with a twice-daily fractionation schedule. Both clinical trials incorporated planned 2.5 week
86 Don Yee

breaks in the twice-daily RT arms and thoracic RT which commenced with cycle 4 of
chemotherapy. Neither trial demonstrated any significant differences in local progression,
progression-free or overall survival, but patients in the twice-daily fractionation arms in both
trials experienced increased acute grade 3 or worse esophagitis.
Hypofractionated external beam RT is another fractionation strategy used for lung cancer
patients whereby each daily fraction of RT is larger than conventional. This strategy offers
several potential radiobiological and practical advantages [37-39]:

1. Larger daily fraction sizes may increase tumor control probability by increasing the
tumor cell kill and counteracting tumor cell repopulation during RT
2. Hypofractionated RT regimens allow for delivery of escalated, potentially more
efficacious doses of RT without increasing overall treatment time.
3. Hypofractionated regimens would decrease the number of trips a patient is required
to make to have RT. Furthermore, in a busy radiotherapy department, fewer RT time
slots would be required to deliver a hypofractionated regimen.

The potential disadvantage of hypofractionated RT is increased RT toxicity. Yee et al.


recently reported results of a phase 1 study of thoracic RT dose escalation for LS-SCLC
where the boost phase of RT was hypofractionated [40]. The maximum tolerated dose of
external beam thoracic RT given with concurrent chemotherapy in this trial was 50 Gy/ 25
fractions with the dose limiting toxicity being radiation esophagitis. In addition, a significant
trend for worsening of patient quality of life during a course of chemoradiotherapy was
observed in patients treated in this trial, especially in those patients given dose escalated
hypofractionated thoracic RT to 58 Gy. One factor limiting the thoracic RT dose given on
this trial was the relatively large volumes of tissue irradiated. All patients on this trial initially
received RT to sites of intrathoracic gross disease along with uninvolved mediastinal nodal
stations at risk for harboring micrometastases. To include these mediastinal nodal stations,
much of the thoracic esophagus was treated, likely contributing to the excessive rates of acute
radiation esophagitis observed in this trial.

Thoracic RT Target Volume


Traditionally, thoracic RT target volumes for LS-SCLC include clinically visible disease
along with uninvolved mediastinal node stations. The practice of elective nodal irradiation
(ENI) for LS-SCLC is controversial in modern radiation oncology practice. The traditional
inclusion of uninvolved mediastinal nodal regions in RT portals for LS-SCLC is based on
results from older studies which suggest that regional nodal failures occur more often in
patients whose uninvolved mediastinal nodes are not irradiated or are given a suboptimal
dose RT [41-43]. These results should be interpreted cautiously as the chemotherapy and RT
techniques used in these clinical trials would today be considered substandard and antiquated.
More recent data indicates the majority of intrathoracic recurrences in SCLC occur within the
RT target volume, suggesting an inadequate RT dose [44,45]. Promising local control results
from recent RT dose escalation trials for LS-SCLC support this hypothesis. The omission of
Radiotherapy for Small Cell Lung Cancer 87

ENI from RT regimens for LS-SCLC would decrease the amount of esophagus irradiated,
potentially decreasing rates of acute RT esophagitis, helping to maintain patient quality of
life and increasing the likelihood that patients complete all treatments as scheduled.
For NSCLC, most radiation oncologists are adopting a strategy of irradiating only visible
intrathoracic disease and leaving the chemotherapy component of a patients combined
modality treatment regimen to eradicate any micrometastatic disease [46]. This strategy of
limited volume RT allows for delivery of higher RT doses to target volumes while still
respecting normal structure RT dose tolerances. Several clinical trials of hypofractionated
dose-escalated thoracic RT which excluded ENI for NSCLC have recently been published.
Patients in these trials received dose-escalated thoracic RT up to 87 Gy planned and delivered
with modern 3D-conformal techniques with no excessive rates of RT-related acute toxicities
observed [47-49]. Toxicity results from these trials cannot be directly applicable to SCLC
patients, as concurrent chemotherapy was not used for the NSCLC patients and though not
specified, RT target locations in the NSCLC trials likely differed from the typical LS-SCLC
patient who has a centrally-located tumor in very close proximity to at least a portion of the
thoracic esophagus.
Exclusion of ENI is currently an investigational RT strategy for LS-SCLC patients.
Results from De Ruysscher's phase 2 trial of twice-daily RT concurrent with chemotherapy
for 27 LS-SCLC patients in which ENI was omitted showed a higher than expected rate of
isolated regional nodal failures with similar acute grade 3 RT esophagitis rates compared to
previous trials that included ENI [50]. In this trial, 3 patients developed an isolated nodal
recurrence, all in the ipsilateral supraclavicular region. Baas et al have reported results from a
phase 2 trial which included 38 LS-SCLC patients treated with chemotherapy concurrent
with involved-field thoracic RT (45 Gy/ 25 fractions). Two of 37 evaluable patients
developed isolated out-of-field regional nodal recurrences and another two patients
developed simultaneous in-field and out-of-field nodal failures [51]. The results from these
two relatively small clinical trials are not conclusive due to the limited numbers of patients
treated, and the omission of ENI for LS-SCLC patients requires further study to define the
true efficacy and safety of this RT strategy for LS-SCLC.
Efforts to improve accuracy of RT target volume definition in lung cancer patients are
ongoing. PET scanning exploits the differential uptake of a radio-labeled glucose analogue
into neoplastic tissue to provide three dimensional images of tumor tissues. This nuclear
imaging modality has been shown to have high sensitivity and specificity for NSCLC, and
this relatively high diagnostic accuracy has lead to the completion of several clinical trials
investigating the feasibility of using PET imaging to supplement information from CT image
sets to plan RT treatments for NSCLC patients [52,53]. Results from early PET clinical trials
of small numbers of SCLC patients indicate small cell tumors are also PET-avid and PET
scanning may contribute to the initial staging of SCLC patients [52,54]. Further clinical trials
are needed to study the utility of using information from PET scans to plan RT treatments for
SCLC patients.
As efforts continue to identify the ideal thoracic RT dose-fractionation regimen for LS-
SCLC, the disadvantages of using dose-intense thoracic RT schemes have been characterized.
One potential disadvantage of using dose-intense RT is increased RT toxicity rates. Dose-
intensified thoracic RT trials for LS-SCLC uniformly report increased RT toxicity rates,
88 Don Yee

namely acute radiation esophagitis. While toxicities of dose-intense thoracic RT for LS-
SCLC are well-documented, quality of life changes associated with these toxicities are not
well studied. Danielson's quality of life analysis of LS-SCLC patients treated with
hypofractionated dose-escalated thoracic RT indicated that patient-reported swallowing and
physician-reported dysphagia scores significantly worsened during RT. In addition, patients'
fatigue and pain significantly deteriorated during RT[55]. Patients in this trial treated with
higher doses of RT (58 Gy versus 50 Gy) were observed to experience worsened quality of
life parameters as indicated by patient-reported validated questionnaires . Ongoing trials of
dose-intense thoracic RT for LS-SCLC will require inclusion of patient-reported quality of
life as study endpoints to validate these preliminary findings.

Ongoing Clinical Trials


With the accumulation of published evidence indicating SCLC has a RT dose response,
efforts to identify more efficacious dose-intensified thoracic RT regimens for SCLC are
ongoing. Strategies including twice-daily RT and dose-escalated RT using standard daily
fractionation and hypofractionation have been studied, as summarized above. Two
randomized clinical trials comparing different RT dose-fractionation schemes for LS-SCLC
have recently been activated. The CONVERT clinical trial is lead by the National Cancer
Research Institute in Manchester, England is being conducted in Europe and in various
Canadian cancer centres via the National Cancer Institute of Canada (NCIC trial BR.28). This
trial is a randomized comparison of 45 Gy given in twice-daily fractionation (30 fractions)
versus 66 Gy given in 33 daily fractions. Radiotherapy will commence with cycle 2 of
cisplatin/ etoposide chemotherapy, and patients who have stable disease or better after
induction chemotherapy will be offered prophylactic cranial irradiation. Feasibility and
toxicity data from a group of 27 patients treated in this study (11 daily RT, 16 twice-daily
RT) have recently been presented at the 2008 meeting of the American Society of Clinical
Oncology[56]. This initial patient group experienced a 48% rate of acute grade 2 radiation
esophagitis with only one patient experiencing acute grade 3 radiation esophagitis. The target
accrual for the CONVERT trial is 532 patients with a primary endpoint of survival.
The new North American intergroup trial (CALGB 30610/ RTOG 0538) features a
standard arm of 45 Gy given with twice-daily fractionation and two experimental arms (70
Gy in 35 daily fractions and 61.2 Gy given with a concomitant boost schedule). An interim
analysis is planned for this study and the experimental arm with more acute RT toxicities will
be dropped to leave one experimental arm to compare with the standard arm of 45 Gy given
in twice-daily fractions in a randomized phase III fashion. Patients on this trial will receive
concurrent cisplatin/ etoposide chemotherapy and complete/ near-complete responders to
induction treatments will be offered prophylactic cranial irradiation.
While these two new randomized trials will answer specific questions related to RT dose-
fractionation schemes for LS-SCLC, the ideal RT dose-fractionation scheme for this patient
group will not be identified in these trials. Other strategies such as hypofractionated thoracic
RT are also currently being investigated. Ongoing RT dose escalation trials featuring RT
targeting only sites of gross intrathoracic disease thus far suggest that doses higher than 50
Radiotherapy for Small Cell Lung Cancer 89

Gy are tolerable, likely in part related to the fact that less thoracic esophagus is irradiated
with this targeting strategy[57,58].

Prophylactic Cranial Irradiation

Long-term SCLC survivors are at high risk of eventually developing cerebral metastases
[59,60]. As an analogy to patients with acute lymphoblastic leukemia, prophylactic cranial
irradiation (PCI) has been investigated for SCLC patients as a treatment modality intended to
decrease the rate of development of cerebral metastases. Early clinical trials of PCI reported
decreased intracranial recurrence rates, but PCI was initially not incorporated into routine
clinical practice as no survival benefits were reported in these early PCI clinical trials and
concerns about cerebral toxicity from the RT existed. Two recently published meta-analyses
have demonstrated that PCI given to LS-SCLC patients who attain complete responses to
chemoradiotherapy significantly decreased intracranial failure rates and increased overall
survival [61,62]. The Auperin meta-analysis analyzed data on 987 SCLC patients in complete
remission after their induction treatments. Auperin et al. reported PCI conferred a 16%
relative reduction in mortality, lower 3-year rates of developing brain metastases (33.3% vs.
58.6%), corresponding to a 5.4% absolute increase in overall survival at 3 years (20.7% vs.
15.3%). The Meert meta-analysis included data on 1547 patients from 5 clinical trials giving
complete responders PCI, 5 trials where PCI was given at the start of chemotherapy and 2
trials where PCI was given regardless of response status. Trials included both patients with
limited (5 studies) and extensive stage disease (7 studies). This analysis showed PCI
significantly decreases the incidence of cerebral recurrence in all trials considered and
improves survival only among complete responders.
To date, there is no prospective evidence to show that currently accepted RT dose/
fractionation regimens are associated with increased rates of unacceptable RT-related
neurocognitive toxicities. Thus, current standard practice for LS-SCLC patients who attain a
complete or near-complete response to chemoradiotherapy treatments is to proceed with PCI.

RT Dose/Fractionation for PCI


The RT dose-fractionation regimens used in the clinical trials included in Auperin's meta-
analysis were not uniform, but another finding from this meta-analysis was as statistical trend
for decreased rates of development of brain metastases in patients given PCI doses of at least
36 Gy in 2 Gy daily fractions. This finding prompted the initiation of an international multi-
centre randomized clinical trial comparing different RT dose-fractionation schemes for PCI
(PCI99-01, IFCT 99-01, EORTC 2203-08004, RTOG 0212). This trial was a phase 2/3 trial
for LS-SCLC patients who achieved a complete response to their induction treatments. The
trial featured 3 PCI treatment arms: a standard arm of 25 Gy in 10 daily fractions, and two
experimental arms of 36 Gy in 18 daily fractions and 36 Gy given in 24 twice-daily fractions.
The primary endpoint of this trial was the incidence of brain metastases development with
secondary endpoints of survival, quality of life and late RT-related neurotoxicity. Between
90 Don Yee

September 1999 and December 2005, 720 patients were accrued to this trial and the results of
the primary endpoint analysis were presented at the 2008 meeting of the American Society of
Clinical Oncology (ASCO) [63]. With a median follow-up of 38 months, a non-significant
trend for lower incidence of brain metastases in patients given higher-dose PCI was observed.
However, higher chest relapse and mortality rates were observed in the higher-dose PCI
patients, a result which is yet to be explained. The authors of this trial have concluded from
these results that 25 Gy in 10 daily fractions remains the standard PCI dose-fractionation
scheme for LS-SCLC.

Radiotherapy for Extensive Stage Disease


Patients with ES-SCLC are considered to have incurable disease. Extensive stage
patients face a dismal prognosis with a median survival between 6-12 months with virtually
no long-term survivors [64]. Traditionally, chemotherapy has been the mainstay of treatment
for ES-SCLC patients, but a recently-published randomized clinical trial has established the
role of PCI for extensive stage patients who respond to chemotherapy. In addition, ongoing
clinical trials are investigating the role of RT to extra-cranial sites in ES-SCLC patients.

Prophylactic Cranial Irradiation

SCLC patients are at high risk of developing brain metastases during their disease
course, with up to 80% of patients eventually developing brain metastases at 2 years. This
high rate of development of brain metastases led to several small clinical trials being
conducted to evaluate the efficacy of PCI. The Meert PCI meta-analysis included patient data
from five PCI clinical trials where PCI was given to only complete responders, five trials
were PCI was given at the start of chemotherapy treatments and two clinical trials where PCI
was administered to patients regardless of their response to induction treatments [61]. The
clinical trials analyzed in this meta-analysis included patients with both limited and extensive
stage disease. The results from Meert's meta-analysis indicated PCI significantly decreases
the incidence of brain metastases and improved survival in complete responders. Due to the
heterogeneity of patients included in this meta-analysis, the exact magnitude of benefit of
PCI for ES-SCLC patients remained unclear.
Since the publication of Meert's meta-analysis, more clinical trial data which quantifies
the benefits of PCI in ES-SCLC have been reported. The European Organization for
Research and Treatment of Cancer (EORTC) has completed a randomized clinical trial
(EORTC 22993) evaluating the efficacy of PCI in ES-SCLC patients who obtain any
objective response to their induction chemotherapy treatments [65]. Between February 2001
and March 2006, 286 ES-SCLC patients who attained an objective response to their
chemotherapy treatments were accrued and randomized to receive either PCI or no PCI.
Routine brain imaging as part of the staging work-up was not required as part of the protocol,
unless patients had symptoms suggestive of cerebral metastases. The primary endpoint of this
trial was the development of symptomatic brain metastases. Patients randomized to receive
Radiotherapy for Small Cell Lung Cancer 91

PCI received one of a variety of RT dose-fractionation schemes: 20 Gy/ 5 fractions (89


patients), 30 Gy/ 10 fractions (23 patients), 30 Gy/ 12 fractions (9 patients), 25 Gy/ 10
fractions (7 patients). The authors of this trial reported patients randomized to receive PCI
had significantly lower risk of development of symptomatic brain metastases, lower rate of
development of brain metastases at 1 year (14.6% versus 40.4%), longer median disease-free
survival (14.7 weeks versus 12.0 weeks) and superior median overall survival (6.7 months
versus 5.4 months). Overall survival at 1 year was significantly improved for patients who
received PCI (27.1% versus 13.3%). No significant deterioration in patient-specific quality of
life was detected in PCI patients using validated EORTC quality of life questionnaires. Since
up to 15-20% of SCLC patients have brain metastases at the time of diagnosis, a significant
proportion of patients on this clinical trial likely had subclinical brain metastases which were
undetected because routine brain imaging was not required for enrollment onto the study.
This suggests that the magnitude of benefit of PCI may have even been greater than what was
observed in the EORTC randomized trial had patients with radiologic evidence of brain
metastases detected with brain imaging been excluded from the trial. The impressive results
from this trial which favour PCI have helped to establish PCI as a standard treatment option
for ES-SCLC patients who achieve an objective response to their induction chemotherapy
treatments.

Thoracic RT for ES-SCLC

Current initial treatment of ES-SCLC consists of chemotherapy alone. Though modern


chemotherapy regimens used for ES-SCLC are associated with objective response rates of up
to 80% [66-69], relapses are inevitable. Local intrathoracic relapse rates of at least 50% are
observed in ES-SCLC patients after completion of initial chemotherapy [66,70] and are
usually associated with distressing symptoms such as dyspnea, cough, dysphagia, pain and
hemoptysis. These clinical manifestations of local thoracic recurrences negatively impact
patients quality of life and in some situations contribute to their death.
For ES-SCLC patients, extracranial RT has traditionally been used only to palliate
bothersome local symptoms caused by localized sites of disease in ES-SCLC as the
symptom(s) arise. Results from Slotman's recently-published PCI clinical trial for ES-SCLC
indicate that a pro-active approach with local RT in ES-SCLC patients may improve both
local control and overall survival in ES-SCLC patients [65]. Slotmans randomized clinical
trial demonstrated that RT given to common site of disease recurrence (brain) improves local
control and overall survival. The efficacy of PCI in this patient population is due to the
ability of local RT to decrease recurrence rates in a region where recurrences associated with
this disease commonly occur. As locally-persistent and/or recurrent intrathoracic disease
post-chemotherapy occurs very commonly in ES-SCLC patients, the addition of local
thoracic RT may be a valuable treatment which may provide improved local disease control,
patient quality of life and possibly survival for ES-SCLC patients.
Five randomized trials have investigated the utility of thoracic RT in ES-SCLC patients.
Brinckers clinical trial randomized 96 SCLC patients to receive either chemotherapy alone
or chemotherapy plus upper and lower half-body irradiation. No benefits were observed in
92 Don Yee

the irradiated patients [71]. Nous trial randomized 54 ES-SCLC patients to chemotherapy
alone or chemotherapy with 40 Gy of thoracic RT. No benefits were reported in patients give
chemotherapy plus RT [72]. Rosenthal randomized 48 ES-SCLC patients to receive either
chemotherapy alone or chemotherapy with 40 Gy of thoracic RT. Patients in this trial who
responded to chemotherapy and then proceeded to receive thoracic RT were observed to have
lower local recurrence rates [73]. Another trial compared 27 SCLC patients given
chemotherapy alone with 26 patients given chemotherapy with thoracic RT. No differences in
patient outcomes were reported in this trial [74]. These four trials, though randomized in
nature, are hampered by low patient numbers and the use of what is now considered
substandard chemotherapy regimens and/or antiquated RT and thus, their results should be
considered cautiously.
The most recent randomized trial of thoracic RT for ES-SCLC included 118 ES-SCLC
patients randomized to either 6 cycles of chemotherapy or 6 cycles of chemotherapy with 54
Gy of hyperfractionated thoracic RT. Chemotherapy used in this trial is considered the
current standard of care (cisplatin/ etoposide). Randomization on this trial required that
patients achieve an objective complete response to 3 cycles of chemotherapy. The authors of
this trial reported a trend for improved local control and significantly improved overall
survival in patients randomized to receive thoracic RT. High-grade acute RT toxicity, namely
esophagitis, was more prevalent in irradiated patients [75]. Since this publication, no further
published trials of chest RT for ES-SCLC have appeared, and questions such as which ES-
SCLC patient subgroups would benefit most from chest RT and ideal thoracic RT
dose/fractionation for ES-SCLC remain unresolved.

Ongoing Clinical Trials


RTOG 0834 will investigate the role of consolidation chest RT directed to areas of
residual disease in ES-SCLC patients after completion of chemotherapy. Similarly, the Dutch
Lung Group will initiate a clinical trial of consolidation chest RT for ES-SCLC patients who
respond to chemotherapy. Currently, a Canadian clinical trial of consolidation chest RT is
accruing ES-SCLC patients who achieve an objective radiologic response to chemotherapy to
undergo consolidation chest RT directed at residual intrathoracic disease. Patients in this trial
will receive 40 Gy in 15 daily fractions directed to encompass residual chest disease post-
chemotherapy along with prophylactic cranial irradiation [76].

Conclusion
The largest improvements in patient outcomes in SCLC in the past 10-20 years are
attributable to radiotherapy. Results from several seminal randomized clinical trials and meta-
analyses published in the past 15 years have helped to establish the integral role of RT in the
management of patients with both limited and extensive stage small cell lung cancer. Patients
with LS-SCLC derive local control and overall survival benefits from addition of thoracic RT
and PCI to their chemotherapy regimens. PCI given to ES-SCLC patients who respond to
Radiotherapy for Small Cell Lung Cancer 93

chemotherapy improves CNS control and overall survival. Despite these improvements
conferred by RT, further clinical trials are needed to define the optimal thoracic and PCI RT
dose-fractionation regimens. In addition, the potential role of RT to treat sites of extra-cranial
disease in ES-SCLC patients is being investigated.

Table 1. Summary of published meta-analyses confirming the benefits of addition of


thoracic RT to chemotherapy for LS-SCLC and PCI for LS-SCLC.

Chest RT
Author # studies # patients Local Control Overall Survival
Warde [12] 11 1911 25.3% improvement in 5.4% improvement in
J Clin Oncol local control favouring 2-yr OS favouring RT
1992 RT
Pignon [13] 13 2140 not reported 5.4% improvement in
NEJM 1992 3-yr OS favouring RT
Prophylactic Cranial RT
Author #studies # patients CNS Control Overall Survival
Auperin [62] 7 987 25.3% decrease in 3-yr 5.4% improvement in
NEJM 1999 rate of CNS failure 3-yr OS favouring PCI
Meert [61] 12 1547 hazard ratio 0.48 hazard ratio 0.82
BMC Cancer favouring PCI favouring PCI
2001

Table 2. Summary of published meta-analyses examining initiation of RT


with either an early or late cycle of chemo for LS-SCLC.

Author # Trials # Patients Outcome


Huncharek [16] 8 1574 Early RT improves survival
Oncologist 2004
Fried [17] 7 1524 Early RT improves 2-year survival
J Clin Oncol 2004
DeRuysscher [18] 7 1514 In trials using platinum-etoposide chemo,
Ann Oncol 2006 early RT improves 5-year OS, especially
if RT completed in <30 days
Spiro [19] 8 1849 Trend favouring early RT; early RT
J Clin Oncol 2006 significantly improves OS if chemo
administered successfully
Pijls-Johannesma [20] 7 1706 Significantly improved 2- and 5-year
Cancer Treat Rev 2007 survival if RT started within 30 days of
chemo start, and especially if RT
completed in <30 days
94 Don Yee

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In: Small Cell Carcinomas: Causes, Diagnosis and Treatment ISBN: 978-1-60741-787-3
Editors: J. G. Maldonado and M. K. Cervantes 2009 Nova Science Publishers, Inc.

Chapter 4

Small Cell Carcinoma: Distinction from


Large Cell Neuroendocrine Carcinoma

Kenzo Hiroshima*
Department of Diagnostic Pathology, Graduate School of Medicine, Chiba University,
1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan

Abstract
Neuroendocrine carcinomas of the lung include the three grades of low-grade typical
carcinoid, intermediate-grade atypical carcinoid, high-grade large cell neuroendocrine
carcinoma (LCNEC), and small cell lung carcinoma (SCLC). SCLC is a malignant
epithelial tumor consisting of small cells with scant cytoplasm, ill-defined cell borders,
finely granular nuclear chromatin and absent or inconspicuous nucleoli. The cells are
round, oval or spindle-shaped and nuclear molding is prominent. The mitotic count is
high. SCLC requires a light microscopic diagnosis and does not require a demonstration
of neuroendocrine differentiation by electron microscopy or immunohistochemistry.
Because the distinction between SCLC and LCNEC is difficult in some cases, some
propose that these carcinomas should be classified as one high-grade neuroendocrine
carcinoma (HGNC).
We reviewed the histological findings of HGNC and found that there was a definite
LCNEC and a tumor with characteristics of both LCNEC and SCLC. The latter tumor
cells had polygonal shape, a small amount of cytoplasm, and relatively large nuclei. The
nuclear chromatin was finely granular. The nucleoli were usually observed but were
small or inconspicuous. The tumors had organoid, trabecular, and palisading patterns,
and a rosette-arrangement was frequently observed. We temporarily defined this subtype
as intermediate cell neuroendocrine carcinoma (ICNEC), and examined biological
features of each group of HGNC using morphometry, immunohistochemical staining,
loss of heterozygosity (LOH), and methylation status of the p16 gene. Tumor cells of
ICNEC were positive for CD56, but negative for chromogranin A and synaptophysin.
The frequency of expression of NeuroD and p63 was higher in LCNEC than in SCLC,

*
Corresponding author: Email: kenzo@faculty.chiba-u.jp; Tel: 81-43-226-2178; Fax: 81-43-226-2180
102 Kenzo Hiroshima

and that of mASH1, p16, and TTF-1 was higher in SCLC than in LCNEC. The nuclear
size of ICNEC cells was between those of LCNEC and SCLC. The expression of CD56,
mASH1, and p16 was high, and that of NeuroD and p63 was low in ICNEC. The LOH
analysis suggested that ICNEC was close to SCLC. Our data suggest that ICNEC is
closer to SCLC morphologically, phenotypically, and genetically. Although further
studies are needed to analyze the biological behavior of LCNEC, ICNEC, and SCLC
including sensitivity to chemotherapy, the tentative recognition and inclusion of ICNEC
into SCLC may reduce the interobserver discrepancy in discriminating HGNC.

Introduction
Tumors of the lung with neuroendocrine morphology by light microscopy comprise a
spectrum of tumor types with different biology and clinical features. The morphologic types
include low-grade typical carcinoid, intermediate-grade atypical carcinoid, high-grade large
cell neuroendocrine carcinoma (LCNEC), and small cell lung carcinoma (SCLC).
The first World Health Organization (WHO) classification of SCLC in 1967 included the
four subtypes of lymphocyte-like, fusiform, polygonal, and others [1]. The second WHO
classification in 1981 changed lymphocyte-like type to oat cell carcinoma; fusiform
cell type and polygonal cell type were combined and changed to intermediate cell type;
and others was changed to combined oat cell carcinoma[2]. The third WHO
classification in 1999 [3] and the WHO classification in 2004 [4] dropped the terms oat cell
carcinoma and intermediate cell type, and all tumors with pure histology were called small
cell carcinomas.

Clinical Presentation
SCLC is a histologic subtype of lung cancer with a distinct biology and clinical course.
SCLC is found almost exclusively in smokers. This cancer has a rapid doubling time and a
more aggressive clinical course than non-small cell lung carcinoma (NSCLC), and it often
disseminates at presentation. SCLCs respond remarkably to chemotherapy initially, but they
frequently relapse and metastasize. Govindan et al. reported on the incidence of SCLC using
the surveillance, epidemiology and end results database [5]. The incidence of SCLC as a
percentage of the number of patients diagnosed with all types of lung cancer decreased from
17.26% in 1986 to 12.95% in 2002. Possible explanations for the decreased incidence include
the decrease in the percentage of smokers and the change to low-tar filter cigarettes.
However, one of the reasons for the decreased incidence of SCLC may be how pathologists
distinguish between SCLC and NSCLC [6].
The incidence of LCNEC is very low. It was reported to range from 2.4% to 3.1% in
resected lung cancers [7, 8]. Patients with LCNEC are predominantly male [7, 9], older, and
heavy smokers [7, 8]. All-stage five-year overall survival for LCNEC ranges from 13% to
57% [10]. The prognoses for LCNEC are comparable to those for SCLC [9, 11]. Iyoda et al.
analyzed 335 cases of pathologic stage Ia NSCLC treated by complete resection, and found
Small Cell Carcinoma: Distinction from Large Cell Neuroendocrine Carcinoma 103

that patients with LCNEC have a worse prognosis than patients with non-neuroendocrine
NSCLC [12].
SCLCs respond initially to chemotherapy, but they frequently relapse and metastasize;
whereas surgery is advocated for the treatment of patients with LCNEC if it is resectable
[13]. Dresler et al. reported that there was no prolongation of survival in patients with
LCNEC who received adjuvant therapy compared with those who did not, even in Stage I
[14]. However, in a retrospective study, Iyoda et al. reported that adjuvant chemotherapy
prolonged survival in early stage LCNEC but not in stage II or III disease [15]. They recently
reported a prospective study that found that adjuvant chemotherapy, consisting of cisplatin
and VP-16, after surgery appears promising for the improvement of the prognosis for patients
with completely resected LCNECs [16]. The study by Rossi et al. clearly confirms the role of
a SCLC-based chemotherapy in an adjuvant setting even for stage I disease [10].

Pathological Features
Architectural patterns of SCLC include nesting, trabecular, peripheral palisading, and
rosette formation. Sheet-like growth without these neuroendocrine morphologic patterns is
common. Tumor cells of SCLC are round, oval, or spindle-shaped; they usually are less than
the size of three small resting lymphocytes, however, there is no absolute size for the tumor
cells of SCLC [3]. They have scant cytoplasm, finely granular chromatin, and absent or
inconspicuous nucleoli (Figure 1a). The cells have a high mitotic rate that averages over 60
mitoses per 2 mm2. Necrosis is common and often extensive. Basophilic staining of vascular
walls due to encrustation by DNA from necrotic tumor cells is frequent in areas of necrosis.
The cell size of surgical specimens of SCLC often appears larger than typically seen in SCLC
from bronchoscopic biopsy specimens. Once tumor cells with conspicuous nucleoli or
pleomorphic giant tumor cells are identified, these are interpreted as large cell carcinoma
elements; and when these constitute 10% or more of the tumor volume, the tumor is
diagnosed as combined small cell and large cell carcinoma (SC/LC) [17].
Tumor cells in the tumor shown in Figure 1b are polygonal with a small amount of
cytoplasm. The nuclei are relatively large, and the nuclear to cytoplasmic (N/C) ratio is high.
The nuclear chromatin is finely granular. The nucleoli are usually observed but are small or
inconspicuous. The mitotic rate is high. The tumors have organoid, trabecular, and palisading
patterns, and a rosette-arrangement is common. We tentatively diagnose this tumor as
intermediate cell neuroencocrine carcinoma (ICNEC). All ICNEC had large infarct-like areas
of necrosis. Nicholson et al. diagnosed the tumor as SCLC that had similar histological
findings in their paper [17].
LCNEC has histological features such as organoid nesting, trabecular, palisading, and
rosette-like patterns. The tumor cells of LCNEC are generally large and polygonal with
moderate to abundant cytoplasm; nuclear chromatin is coarsely granular or vesicular and
nucleoli are prominent (Figure 1c). Mitotic counts are typically 11 or more (average 75) per 2
mm2 of viable tumor. A large infarct-like zone of necrosis is commonly present.
Confirmation of neuroendocrine differentiation is required using immunohistochemistry or
electron microscopy.
104 Kenzo Hiroshima

(a)

(b)

(c)

Figure 1. (a) Small cell lung carcinoma (SCLC). Cell size is small. Tumor cells have scant cytoplasm so
that the cellularity is high. The nuclei are round or oval. The chromatin of the nucleus is finely granular,
and nucleoli are inconspicuous. (b) Intermediate cell neuroendocrine carcinoma. Cell size is larger than
in SCLC, but smaller than in large cell neuroendocrine carcinoma (LCNEC). Tumor cells are polygonal
and have a moderate amount of cytoplasm. Rosette-like structures are present. The chromatin of the
nucleus is finely granular, and nucleoli are seen. Necrosis is present. (c) LCNEC. This tumor has
organoid nesting and palisading patterns. Tumor cells have abundant eosinophilic cytoplasm, coarsely
granular chromatin, and prominent nucleoli. Necrosis is present.
Small Cell Carcinoma: Distinction from Large Cell Neuroendocrine Carcinoma 105

Pelosi et al. studied bronchial biopsies from seven patients with typical carcinoid and
atypical carcinoid that had been over-diagnosed as SCLC based on evaluation of bronchial
biopsy specimens [18]. Carcinoid tumors were composed of tumor cells that had granular and
sometimes coarse nuclear chromatin patterns, high levels of chromogranin A/synaptophysin
immunoreactivity, and a low (<20%) Ki-67 labeling index. In contrast, SCLCs had finely
dispersed nuclear chromatin, lower levels of chromogranin A/synaptophysin immunoreactivity,
and a high (>50%) Ki-67 labeling index. The authors concluded that there was an over-
diagnosis of the carcinoid tumor as SCLC in small crushed bronchial biopsy specimens.
Distinguishing LCNEC from SCLC can be difficult in some cases. In a study by Travis et
al., there was unanimous diagnostic agreement on 70% of SCLCs and 40% of LCNECs in
surgically resected neuroendocrine tumors of the lung that were reviewed independently by five
pulmonary pathologists [19]. Most of the disagreements concerned the distinction between
SCLC and LCNEC. Marchevsky et al. reported that SCLC and LCNEC had a continuum of cell
size [20]. They and others suggested that the tumors should be combined into a single group in
daily practice as a high-grade neuroendocrine carcinoma (HGNC) [20-22].
We reviewed the histologic features of HGNC reported in our recent paper [23] and
found that there is heterogeneity in the pathological findings in SCLCs and that there is a
group of tumors temporarily categorized as ICNEC, which has histological characteristics of
both LCNEC and SCLC. The aim of this study was to elucidate the morphological,
phenotypical, and genetic characteristics of ICNEC and compare them with those of LCNEC
and SCLC, seeking its most appropriate categorization in the spectrum of HGNC.

Materials and Methods


Tumor Specimens

We reviewed the histologic features of SCLC and LCNEC reported in our recent paper [23].
Tumors were classified according to the WHO classification system for lung carcinoma[3, 4].
ICNEC was temporarily defined as a HGNC having cells with polygonal shape, large nuclei,
features of LCNEC, and a feature of SCLC consisting of a relatively small amount of cytoplasm
and a high N/C ratio. We analyzed 18 cases of LCNEC and 9 cases of ICNEC in stage I
[T1N0M0 or T2N0M0]: a tumor that does not invade the adjacent organs [T1 or T2], has no
regional lymph node metastasis [N0], and no distant metastasis [M0]. One case of LCNEC was
combined with ICNEC, one was combined with squamous cell carcinoma, and three cases of
ICNEC were combined with SCLC. We also analyzed 12 cases of classic large cell carcinoma
(CLCC) in stage I, 9 cases of carcinoid tumors (7 typical carcinoids and 2 atypical carcinoids) in
stage I, and 13 cases of SCLC with limited disease (7 cases in stage I and 6 cases in stages II to
IV). Three SCLC were combined SCLC with squamous cell carcinoma. All tumors, except one
LCNEC, were resected surgically at Chiba University Hospital between 1987 and 2005. The
one LCNEC was resected at Narita Red Cross Hospital in 2004. The tumors in all cases were
resected completely. We performed immunohistochemical staining for all LCNECs, ICNECs,
SCLCs, CLCCs, and carcinoid tumors studied. Neuroendocrine differentiation was detected for
LCNEC by positive immunohistochemical staining for chromogranin A, synaptophysin, or
106 Kenzo Hiroshima

CD56. We confirmed lack of neuroendocrine differentiation in all CLCCs. Informed consent


was obtained from all patients.
One of 12 patients with CLCC, and seven of 13 with SCLC underwent preoperative
chemotherapy. One patient with CLCC, four with LCNEC, four with ICNEC, and eight with
SCLC underwent postoperative adjuvant chemotherapy, and one patient with LCNEC and
two with SCLC underwent postoperative radiotherapy.

Morphometric Study

The nuclear diameter (ND) was measured in each case of LCNEC, ICNEC, and SCLC
with a CAS 200 cellular imaging system using a 40x objective lens (Becton Dickinson and
Company, San Jose, CA). The accuracy of the image measurement was confirmed with the
standard objective micrometer (Nikon, Tokyo, Japan). The NDs of 100 tumor cells and the
NDs of 20 mature lymphocytes were measured for each specimen. The mean and standard
deviation were then calculated.

Immunohistochemistry

All tumors were examined by immunohistochemical staining. Sections (4 m) were cut


from formalin-fixed paraffin-embedded tissues and placed on silanized slides
(DakoCytomation, Glostrup, Denmark). They were stained with polyclonal anti-
chromogranin A antibody (pre-diluted; Nichirei, Tokyo, Japan), polyclonal anti-
synaptophysin antibody (pre-diluted; DakoCytomation), polyclonal anti-Neuro D antibody
(1:200; N-19, Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal anti-phosphatase and
tensin homologue (PTEN) antibody (1:200; PN37, Zymed Laboratories, South San Francisco,
CA), monoclonal anti-CD56 antibody (pre-diluted; 123C3, Zymed Laboratories), monoclonal
anti-human cytokeratin high molecular weight (CK34E12) (prediluted; 34E12,
DakoCytomation), monoclonal anti-thyroid transcription factor-1 (TTF-1) antibody (1:200;
8G7G3/1, DakoCytomation), monoclonal anti-p63 protein antibody (1:400; 4A4,
DakoCytomation), and monoclonal anti-p16 protein antibody (1:50; G175-405, Pharmingen,
San Diego, CA). We used monoclonal anti-mASH1 antibody (1:50; 24B72D11.1, Becton
Dickinson Biosciences, Franklin Lakes, NJ) to detect hASH1 because this antibody cross-
reacts with hASH1 of human SCLC in the paraffin-embedded tissues [24, 25].
We used a Histofine Simple Stain Kit (Nichirei, Tokyo, Japan) for immunostaining. To
improve the staining pattern, the tissues were pretreated with microwaves for 15 minutes in
citrate buffer (10 mM pH 6.0) before staining with anti-CD56 antibody, anti-CK34E12
antibody, anti-Neuro D antibody, and anti-p16 antibody; or were heated in an autoclave at
121 C for 15 minutes before staining with anti-synaptophysin antibody, anti-PTEN antibody,
anti-TTF-1 antibody, and anti-mASH1 antibody. The tissues were heated in an autoclave at
121 C for 15 minutes in DAKO Target Retrieval Solution (S1700) (DakoCytomation) before
staining with anti-p63 protein antibody. Reactivity was considered negative if less than 10%
of tumor cells stained.
Small Cell Carcinoma: Distinction from Large Cell Neuroendocrine Carcinoma 107

Microdissection and DNA Extraction

DNA was extracted from paraffin-embedded materials as described previously [26].

Polymorphic DNA Markers and PCR-LOH

We examined previously the same cohort for the loss of heterozygosity (LOH) at 13
microsatellite markers: D3S1234(3p14.2), D3S1481(3p14.2), D3S1295(3p21.1), D3S1581
(3p21.3), D5S407(5q11), D5S410(5q31.3), D5S422(5q33), D9S171(9p21), IFNA(9p21),
D10S249(10p15.3), D10S1686(10q22.3), D13S153(13q14), ALE3/P53ivs1b(TP53). LOH
was defined as a reduction of an alleles peak height by at least 50% in the tumor, compared
with the normal sample [26].

Calculation of Fractional Regional Loss Index

The fractional regional loss (FRL) index was calculated as follows:

FRL = (total number of chromosomal regions with LOH)/(total number of informative


regions)

Methylation Analysis

We examined previously the same cohort for the methylation state of the p16 gene by
methylation-specific PCR [26, 27].

Survival Analysis

Because all cases with CLCC, LCNEC, ICNEC and carcinoid tumors and 7 of 13 cases
with SCLC were in stage I, survival of these cases was determined.
The interval between the date of surgery and the date of the first local and distant
recurrence was defined as disease-free survival, and the time after surgery until death was
defined as the overall survival. Kaplan-Meier curves and survival estimates were calculated
[28], and the Brestow-Gehan-Wilcoxon test was used to test for differences between groups.

Statistical Analysis

All statistical calculations were done with the StatView software package (SAS Institute
Inc., Cary, NC). Differences of the ratio of the ND of the tumor cells to ND of the
lymphocytes among lung cancer subtypes were analyzed by a one-way factorial ANOVA and
multiple comparison test (Fisher PLSD test). A chi-square test was used to evaluate the
108 Kenzo Hiroshima

immunohistochemical and methylation analysis for the lung cancer subtypes and the
differences of the frequency of LOH at specific regions among lung cancer subtypes.
Differences in FRL indices among lung cancer subtypes were tested by the Kruskal-Wallis
test. Probability values of p < 0.05 were regarded as statistically significant.

Results
Microscopic Study

In a case with combined LCNEC and ICNEC, the predominant histological type was
LCNEC, and in a case with combined LCNEC and squamous cell carcinoma, the
predominant histological type was LCNEC. In all cases with combined ICNEC and SCLC,
the predominant histological type was ICNEC. In two cases with combined SCLC and
squamous cell carcinoma, the predominant histological type was squamous cell carcinoma,
and in one case, the predominant histological type was SCLC.

Morphometric Study

The frequency distribution of the ratio of ND of the tumor cells to ND of lymphocytes


(T/L) is shown in a histogram (Figures 2a-c). The peak of the T/L ratio was between 2 and 3
for SCLC and ICNEC and between 3 and 4 for LCNEC. The average T/L ratio was 2.62
0.90 for SCLC, 2.91 0.76 for ICNEC, and 3.22 0.86 for LCNEC. The differences
between T/L ratios of LCNEC and ICNEC, ICNEC and SCLC, and LCNEC and SCLC were
significant (all p < 0.0001) according to the Fisher PLSD test.

Immunohistochemistry

Immunohistochemical study showed that 61% of LCNEC and 75% of SCLC stained with
antibody for chromogranin A, but none of ICNEC stained with it (p=0.0016). 78% of
LCNEC and 85% of SCLC stained with antibody for synaptophysin, but none of ICNEC
stained with it (p<0.0001). 50% of LCNEC and 92% of SCLC stained with antibody for
CD56, however, all of ICNEC stained with it (p=0.0050) (Table 1). ICNEC has
immunological phenotype of CD56 positive, chromogranin A negative, and synaptophysin
negative. Fourteen of 16 pure LCNECs, five of 9 ICNECs, and all of pure SCLCs were
completely CK34E12 negative. However, tumor cells of two of pure LCNECs and four of
ICNECs were stained focally with anti-CK34E12. In LCNECs and SCLCs combined with
squamous cell carcinoma, the last component showed marked immunoreactivity with
CK34E12.
Small Cell Carcinoma: Distinction from Large Cell Neuroendocrine Carcinoma 109

(a) (b)

(c)

Figure 2. The frequency distribution of tumor nuclear diameter/lymphocyte size (T/L) ratios. The
nuclear diameters (NDs) of 100 tumor cells and the NDs of 20 mature lymphocytes were measured for
each specimen. The T/L ratio of each tumor cell was calculated as the ND of each tumor cell divided by
the mean ND of lymphocytes. A histogram was made of all T/L ratios measured in each subtype.
(a) Small cell lung carcinoma. The peak of the T/L ratio is between 2 and 3.
(b) Intermediate cell neuroendocrine carcinoma. The peak of the T/L ratio is between 2 and 3.
(c) Large cell neuroendocrine carcinoma. The peak of the T/L ratio is between 3 and 4.
110 Kenzo Hiroshima

Table 1. Summary of immunohistochemical staining for neuroendocrine markers

LCNEC ICNEC SCLC


Chromogranin 11/18(61%) 0/9(0%) 9/12(75%) 0.0016
Synaptophysin 14/18(78%) 0/9(0%) 11/13(85%) <0.0001
CD56 9/18(50%) 9/9(100%) 11/12(92%) 0.0050
LCNEC, large cell neuroendocrine carcinoma;
ICNEC, intermediate cell neuroendocrine carcinoma;
SCLC, small cell lung carcinoma.

Table 2. Summary of immunohistochemical staining results

CLCC LCNEC ICNEC SCLC Carcinoid p value


mASH1 0/12(0%) 10/18(56%) 7/9(78%) 12/13(92%) 0/9(0%) <0.0001a 0.0715b
NeuroD 4/12(33%) 10/18(56%) 1/9(11%) 1/13(8%) 5/9(56%) 0.0203 0.0061
TTF-1 4/12(33%) 5/18(28%) 1/9(11%) 10/13(77%) 0/9(0%) 0.0012 0.0030
p63 6/12(50%) 3/18(17%) 0/9(0%) 0/13(0%) 0/9(0%) 0.0017 0.1378
p16 7/12(58%) 11/18(61%) 8/9(89%) 12/13(92%) 2/9(22%) 0.0066 0.0790
PTEN 9/12(75%) 5/18(28%) 1/9(11%) 1/13(8%) 9/9(100%) <0.0001 0.2956
CLCC, classic large cell carcinoma; LCNEC, large cell neuroendocrine carcinoma;
ICNEC, intermediate cell neuroendocrine carcinoma; SCLC, small cell lung carcinoma;
a, difference of the frequency of expression among five subtypes of lung carcinoma;
b, difference of the frequency of expression among high-grade neuroendocrine carcinoma of the lung.

Cytoplasm of bronchial gland cells was stained positively for mASH1 in normal lung
tissue, and no cells of the bronchial epithelium were stained; both cytoplasm and nucleus of
the tumor cells were stained. A few cells in normal bronchial epithelium had cytoplasmic
granules that stained positively for NeuroD; both cytoplasm and nucleus of tumor cells were
stained with that antibody. Type II alveolar epithelial cells in normal lung tissue were stained
with the antibody for TTF-1; only nuclei of tumor cells were stained. Nuclei of basal cells of
bronchial epithelium were stained with anti-p63 antibody, and only nuclei of tumor cells
were stained. Tumor cells were stained focally with anti-p63 antibody in two ICNEC, two
SCLC, and one carcinoid tumor. Because we defined negative reactivity as fewer than 10%
of tumor cells stained, these cases were negative for p63. This pattern contrasted with tumor
cells that were stained uniformly in CLCC and LCNEC. Only cells with nuclear activity for
anti-p16 antibody were recorded. Prostatic hyperplasia tissue was used for the positive
control for PTEN; both cytoplasm and nucleus of the tumor cell stained with the antibody.
Table 2 lists the total number and percentage of positive cases in each histological
category for each antibody. For cases with combined neuroendocrine carcinoma with
NSCLC, immunohistochemical staining of the neuroendocrine carcinoma is described, and
for a case with LCNEC combined with ICNEC and cases with ICNEC combined with SCLC,
immunohistochemical results of the predominant histological types are described in Table 2.
The differences of the frequency of the expressions of mASH1, NeuroD, TTF-1, p63, p16,
and PTEN were statistically significant between CLCC, LCNEC, ICNEC, SCLC, and
carcinoid tumors. The differences of staining among HGNCs were analyzed. The frequencies
of the expressions of mASH1 and p16 were higher in SCLC and in ICNEC than in LCNEC,
Small Cell Carcinoma: Distinction from Large Cell Neuroendocrine Carcinoma 111

and those of the expressions of NeuroD and p63 were higher in LCNEC than in SCLC and in
ICNEC. The frequency of the expression of TTF-1 was higher in SCLC than in LCNEC and
ICNEC. The differences of the frequency of the expressions of NeuroD and TTF-1 were
statistically significant among HGNCs. The results of immunohistochemical staining for
combined neuroendocrine carcinomas according to each component are shown in Table 3.
mASH1 was expressed in the component of ICNEC in a case of combined LCNEC and
ICNEC and in the component of SCLC in all cases of combined SCLC and squamous cell
carcinoma. TTF-1 was expressed in the component of SCLC in one case of combined ICNEC
and SCLC and in the component of SCLC in all cases of combined SCLC and squamous cell
carcinoma.

Polymorphic DNA Markers and PCR-LOH

Four specific 3p regions (D3S1234, D3S1481, D3S1295, D3S1581), three specific 5q


regions (D5S407, D5S410, D5S422), two specific 9p regions (D9S171, IFNA), one specific
10p region (D10S249), one specific 10q region (D10S1686), one specific 13q region
(D13S153), and one specific 17p region (ALE3/P53ivs1b) were analyzed. The frequencies of
LOH in specific regions are shown in Figure 3. The frequency of LOH at 10q is not shown
because the number of informative cases at 10q was small. The frequency of LOH at 3p was
higher in HGNC than in CLCC and the difference was statistically significant (p < 0.0005).
The frequency of LOH at 13q was also higher in HGNC than in CLCC, but the difference
was not statistically significant. The frequency of LOH at 5q and 17p was higher in SCLC
and ICNEC than in LCNEC and CLCC, and the frequency of LOH at 9p was higher in
LCNEC and CLCC than in SCLC and ICNEC, but the differences were not significant. The
frequency of LOH at 10p was higher in ICNEC than in SCLC, LCNEC, and CLCC, but the
differences were not significant.

Figure 3. Relations between percentage of loss of heterozygosity (LOH) by chromosomal regions and
histologic categories. SCLC, small cell lung carcinoma; ICNEC, intermediate cell neuroendocrine
carcinoma; LCNEC, large cell neuroendocrine carcinoma; CLCC, classic large cell carcinoma.
112 Kenzo Hiroshima

Table 3. Results of immunohistochemical results in combined LCNEC,


combined ICNEC, and combined SCLC

Case mASH1 NeuroD TTF-1 p63 p16 PTEN


Combined LCNEC
1 LCNEC* - - + - +
ICNEC + - + + + -
2 LCNEC* - + - - +
SQ - + - + - +
Combined ICNEC
1 ICNEC* + - - - + -
SCLC + - + - + -
2 ICNEC* + - - - + -
SCLC + - - - + -
3 ICNEC* - - - - + -
SCLC - - - - + -
Combined SCLC
1 SCLC* + - + - + -
SQ - + - + + +
2 SCLC + - + - + -
SQ* - - - + -
3 SCLC* + + + - - -
SQ - - - + -
LCNEC: component of large cell neuroendocrine carcnoma,
ICNEC: component of intermediate cell neuroendocrine carcinoma,
SCLC: component of small cell lung carcinoma, SQ: component of sqamous cell carcinoma,
*: predominant histological type, +: positive, -: negative, : decreased.

Mean FRL indices of the tumors were 0.38, 0.65, 0.83, and 0.72 for patients with CLCC,
LCNEC, ICNEC, and SCLC, respectively. The differences of FRL indices among the four
types of the tumor were significant (p = 0.0003); however, those among HGNCs were not (p
= 0.0924). Mean 3p FRL indices of the tumor were 0.20, 0.76, 0.89, and 0.96 for patients
with CLCC, LCNEC, ICNEC, and SCLC, respectively. The differences of FRL indices
among the four types of tumor were significant (p = 0.0007); however, those among HGNCs
were not (p = 0.3678).

Methylation Analysis

Hypermethylation of the p16 gene was observed in 4 cases of 12 CLCC (33.3%), 3 of 10


LCNEC (30.0%), 3 of 6 ICNEC (50.0%), and none of 8 SCLC (0%). CLCC, LCNEC, and
ICNEC tended to be methylated more frequently than SCLC, but the differences were not
significant (p = 0.1900).
Small Cell Carcinoma: Distinction from Large Cell Neuroendocrine Carcinoma 113

(a)

(b)

Figure 4. (a) The overall survival curves of p63-positive and p63-negative LCNEC cases. (b) The
overall survival curves of cases with TTF-1-positive and TTF-1-negative small cell lung carcinoma and
intermediate cell neuroendocrine carcinoma.

Survival Analysis

Five-year overall survivals of patients with CLCC, LCNEC, ICNEC, SCLC, and
carcinoid tumors were 63.6%, 73.3%, 57.1%, and 75.0%, 88.9%, respectively. The
differences of overall survival among the five subtypes were not significant. Five-year
disease-free survivals of patients with CLCC, LCNEC, ICNEC, SCLC, and carcinoid tumors
were 63.6%, 58.3%, 57.1%, 75.0%, 88.9%. The differences of disease-free survival among
the five subtypes were not significant.
We examined the correlations between survival of patients with HGNC and the
expressions of the proteins examined in this study. Comparing patients with/without a
specific protein expression, patients with NeuroD expression had better five-year disease-free
(79.5%/45.7%, p = 0.0336) and overall (90.9%/49.6%, p = 0.0355) survivals. Patients with
mASH1 expression had poorer five-year disease-free (46.4%/79.5%, p=0.2016) and overall
(60.2%/79.5%, p=0.4395) survivals, but the difference was not significant. Patients with p63
expression had poorer five-year disease-free (33.3%/60.7%, p = 0.0583) and overall
(33.3%/70.8%, p = 0.0428) survivals. Patients with TTF-1 expression had poorer five-year
114 Kenzo Hiroshima

disease-free (23.4%/68.6%, p=0.0419) and overall (18.8%/80.8%, p=0.0117) survivals. Other


biomarkers did not have prognostic significance for either disease-free or overall survivals in
HGNC.
We examined subset analysis of the correlations between survival of patients with HGNC
and the expressions of the proteins. ICNEC and SCLC were combined, because the number
of each subtype analyzed in this study was too small for statistical evaluation. Patients with
NeuroD expression had better five-year disease-free (74.1%/33.3%, p = 0.0413) and overall
(88.9%/50.0%, p = 0.0887) survivals in LCNEC. Patients with mASH1 expression had
poorer five-year overall survival (47.7%/100%) in ICNEC and SCLC, but the difference was
not significant. Patients with p63 expression had poorer five-year disease-free (33.3%/64.3%,
p = 0.0394) and overall (33.3%/83.3%, p = 0.0157) survivals in LCNEC (Figure 4a). Patients
with TTF-1 expression had poorer five-year disease-free (0%/77.1%, p=0.0269) and overall
(0%/77.1%, p = 0.0208) survivals in ICNEC and SCLC (Figure 4b).

Discussion
Achaete-scute homolog 1 (termed mASH1 in rodents, hASH1 in humans) is a basic
helix-loop-helix (bHLH) transcription factor that is important in early development of neural
and neuroendocrine progenitor cells in many tissues [29]. The NeuroD gene is a bHLH gene
and regulates neurogenesis, and it is reported that it maps to chromosome 2q32 [30]. It is
reported that hASH1 is expressed selectively in normal fetal pulmonary neuroendocrine cells
[31] as well as in pulmonary LCNEC (56.7%) and SCLC (71.8%) [32]. The frequency of
expression of mASH1 in our study was 56% in LCNEC, 78% in ICNEC, and 92% in SCLC.
The frequencies of expression of mASH1 in LCNEC and SCLC are comparable to those
reported in a previous paper [32]. The expression of NeuroD in our study was 56% in
LCNEC, 11% in ICNEC, and 8% in SCLC. The frequency of the expression of NeuroD was
higher in LCNEC, but it was also observed in carcinoid tumor and CLCC. NeuroD may be
related to neuroendocrine differentiation in NSCLC including LCNEC.
TTF-1 is a tissue-specific transcription factor that mediates cell determination and
differentiation in thyroid, lung, and brain. It is expressed in thyroid follicular cells, human
fetal lung, and alveolar Type II epithelial cells after birth. TTF-1 was detectable in pulmonary
adenocarcinomas [33, 34], SCLCs, LCNECs, carcinoid tumors, and CLCCs but not in
pulmonary squamous cell carcinomas [34, 35]. The prognostic value of TTF-1 was evaluated
in NSCLCs, and contradictory results were reported. However, TTF-1 positivity is reported
to be associated with better survival in NSCLC with meta-analysis [36]. We found that TTF-
1 was detectable in 33% of CLCC cases, 28% in LCNEC, 11% in ICNEC, and 77% in SCLC.
TTF-1 had a poor prognostic implication in SCLC and in ICNEC in our study.
p63 is a p53 homologue mapped to chromosome 3q27-28. p63 plays a role in stem cell
commitment in squamous epithelium. Some reported that p63 was expressed in pulmonary
squamous cell carcinoma but not in SCLCs [37, 38], whereas others reported that LCNEC
and SCLC had p63 expression [39, 40]. The reported differences of frequency of p63
expression in SCLC may be due to different identification methods for positive cells. Zhang
et al. reported that they defined the tumor as p63-negative if less than 10% of tumor cells
Small Cell Carcinoma: Distinction from Large Cell Neuroendocrine Carcinoma 115

were stained [38]. Pelosi et al. reported that they defined the tumor as p63-positive if nuclear
staining of the tumor cells was observed [39]. It is reported that p63 staining cases have a
better outcome in patients with squamous cell carcinoma [41, 42] but a poor outcome in
patients with neuroendocrine tumors [40]. In our study, half of CLCC and 17% of LCNEC
cases stained with anti-p63 antibody, but none of ICNEC and SCLC cases was p63-positive.
Patients with p63 expression had poorer five-year overall survival (33.3%) than those without
its expression (83.3%) in LCNEC (p = 0.0157).
A putative tumor suppressor gene, PTEN, was identified at 10q23 and is thought to play
an important role in the control of cell proliferation, apoptosis, differentiation [43, 44], and
break cell cycle progression by inhibiting the PI3K pathway [45, 46]. PTEN was mutated or
deleted homozygously in glioma, endometrial, and breast carcinoma tumors [47-49]. It was
reported that PTEN mutations were identified in SCLCs, but not in NSCLCs [50, 51]. We
found that the frequency of the loss of expression of PTEN was higher in HGNC than in
CLCC or carcinoid tumor, and the differences were significant; however, the differences
were not significant among subtypes of HGNC.
Recently, a genome-wide high-resolution search for LOH was made on SCLC and
NSCLC cell lines [52, 53]. These studies found that some loss of LOH was common in both
SCLC and NSCLC subtypes, whereas loss was subtype-specific in others. It has been
suggested that the genetic alterations undergone by SCLC and NSCLC are different. There
are contradictory reports about the differences of genetic changes between SCLC and
LCNEC. For example, LOH at 3p, 5q, 11q, 13q, 17p and mutations in the p53 and ras genes
were present both in SCLCs and LCNECs [54]. c-myc amplification was observed with a
similar frequency in SCLC (20%) and LCNEC (23%) [55]. SCLC and LCNEC were
indistinguishable by gene expression profiles, and HGNC of the lung can be classified into
two groups independent of SCLC and LCNEC [21]. However, although both SCLC and
LCNEC shared several chromosomal aberrations including losses of 3p, 4q, 5q, and 13q and
gains of 5p, a gain of 3q and a loss of 10q, 16q, and 17p were observed frequently in SCLC
but not in LCNEC, and a gain of 6p occurred more frequently in LCNEC [56]. Our study
found that the frequency of LOH at 3p and 13q was high in all subtypes of HGNC, it was
higher at 5q and 17p in SCLC and ICNEC than in LCNEC, and it was higher at 9p in
LCNEC than in SCLC and ICNEC. We observed methylation of the p16 gene in LCNEC and
ICNEC but not in SCLC. We conclude that there are different genetic changes in SCLC and
LCNEC and also common abnormalities between these two subtypes.
Chromogranin A and synaptophysin are the most reliable immunohistochemical markers
to detect neuroendocrine differentiation in neuroendocrine lung tumors [3, 4, 57, 58]. CD56
or neural cell adhesion molecule (NCAM) is also a sensitive and specific marker in
confirmation of neuroendocrine differentiation in malignant neoplasms [58, 59]. It is reported
that CD56 is useful for the immunohistochemical differentiation of SCLC and LCNEC from
nonneuroendocrine carcionomas [59-61]. ICNEC has a unique immunohistochemical
phenotype, such that all ICNECs were CD56 positive, chromogranin A negative, and
synaptophysin negative (n=9). CD56 was expressed in 11 SCLCs (92%), however, it was
expressed in nine LCNECs (50%) in our study.
It is reported that difficulty of diagnosis in HGNC occurs as a result of variety of reasons,
including the continuum of cell size and morphology between SCLC and LCNEC, and poor
116 Kenzo Hiroshima

sampling and tissue artifacts [17]. It is reported that there are borderline cases between
LCNEC and SCLC in high-grade neuroendocrine carcinoma [9]. We propose that one of the
main reasons why there are differences between observers in histological diagnosis of HGNC
may be the presence of ICNEC [23]. Some pathologists may diagnose ICNEC as SCLC
because the N/C ratio is high and the cytoplasm is not abundant. For example, Figure 10 in
the paper by Nicholson et al. represents ICNEC according to our criteria, and they diagnosed
this case as SCLC [17]. However, some pathologists may diagnose the same specimen as
LCNEC because the shape of the tumor cell is polygonal, the ND is larger than that of SCLC,
and nucleoli are usually observed. Our results suggest that ICNEC has the characteristics of
both LCNEC and SCLC. The average T/L ratio in ICNEC (2.91) is significantly smaller than
that in LCNEC (3.22). Although methylation of the p16 gene and the expression of TTF-1
suggested that ICNEC was close to LCNEC, the small nuclear size, the expression of CD56,
mASH1, NeuroD, p63, and p16, and the LOH analysis suggested that ICNEC was close to
SCLC. Thus, the tumor temporarily categorized as ICNEC was closer to SCLC
morphometrically, phenotypically, and genetically rather than to LCNEC.
In the present study, the five-year overall survivals of patients with CLCC, LCNEC,
ICNEC, SCLC, and carcinoid tumors were 63.6%, 73.3%, 57.1%, 75.0%, and 88.9%,
respectively. The differences of overall survival among the five subtypes were not
significant. Five-year survival for SCLC in this study was good, because we evaluated the
survival of SCLC in stage I. These cases were operated on in early stage and underwent
preoperative and/or postoperative chemotherapy. We had previously analyzed SCLC resected
surgically at our institute and reported that the five-year overall survival for patients with
SCLC in all stages was 16.4% [11].

Conclusion
The discordance of the histological diagnosis of HGNC among pathologists is partly due
to the existence of ICNEC. We suggest that LCNEC as defined in WHO classification [3, 4]
is an entity distinct from SCLC, and ICNEC is morphologically, phenotypically, and
genetically close to SCLC. Furthermore, the survival of patients with ICNEC is not
statistically different from that of patients with SCLC. Thus, we propose the tentative
categorization of ICNEC into SCLC. However, additional studies in larger series are needed
to consolidate the present conclusion by analyzing the biological behavior of LCNEC,
ICNEC, and SCLC, including sensitivity to chemotherapeutic agents.

Acknowledgments
The author thanks Ms. Tamiyo Taniguchi, Ms. Ayaka Sato, and Ms. Kazuko Abe for
their technical assistance.
Small Cell Carcinoma: Distinction from Large Cell Neuroendocrine Carcinoma 117

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In: Small Cell Carcinomas: Causes, Diagnosis and Treatment ISBN: 978-1-60741-787-3
Editors: J. G. Maldonado and M. K. Cervantes 2009 Nova Science Publishers, Inc.

Chapter 5

CT/MR-Based Movement Analysis


of Lung Tumors: Impact of Tumor
Motion in the 3D-Based Radiotherapy
of Lung Cancer

Arpad Kovacs
Dept. of Diagnostics and Oncoradiology, University of Kaposvar, Hungary

1. Introduction
Lung cancer is the leading cause of death in tumor-related morbidity, both in the male
and female populations [1]. The complex therapy of this malignant disease is based on
surgery, chemo-, and radiotherapy. Radiotherapy is often used in the treatment of lung cancer
either postoperatively or in a definitive setting depending on the tumor stage and general
condition of the patient. Local tumor control is a crucial question in the treatment process,
since the possibility of lung cancer recurrence is very high, even in early stages [2].
In 3D-based conformal radiotherapy of lung cancer, accurate delineation of PTV
(planning target volume) is critical. Many factors have to be taken in account during the
definition of PTV (e.g., microscopic spread of the tumor cells, daily setup errors, tumor
motion). In common practice, standard safety margins are added to clinical target volumes
(CTV), which are derived from a spiral CT scan. These safety margins are estimated
arbitrarily, potentially resulting in either excessive exposure of normal tissues (especially in
the case of combined chemo-radio therapy) or insufficient target volume coverage [3,4].
Overestimation of the PTV can result in a higher side effect profile, especially in combined
treatment settings. With the use of inappropriate PTV volume delineation, the delivery of an
ideal tumor-destroying dose becomes doubtful [5,6].
According to the International Commission on Radiation Units and Measurements
(ICRU) Report, Recommendation Nos. 50 and 62 [7,12], planning target volume includes the
gross tumor volume (GTVvisible tumor mass), the clinical tumor volume (CTV, GTV plus
124 Arpad Kovacs

microscopic tumor spread), the internal margin (IM, uncertainties resulting from intra- and
interfractional tumor and organ motions) and the set-up margin (SM, uncertainties resulting
from daily setup and positioning errors). Uncertainty resulting from tumor movement must be
considered in 3D therapy planning, especially in case of IMRT or stereotactic therapy.
Moreover, normal tissue toxicity must be considered in the definiton process, too. This factor
has high importance in the treatment of lung cancer patients (lung toxicity, heart toxicity,
spinal cord).
The charaterisation of the internal margins needed for the radiotherapy of upper and mid
lobe lung cancers was developed following a complex study at our institution. The main goals
were to detect tumor movements, to analyze uncertainties in treatment planning arising from
tumor motions and to study the effectiveness of the fixation system used in our department for
lung cancer radiotherapy. This chapter aims to demonstrate the results of this study.

2. Purposes
2.1. Dynamic MR-based Analysis of Tumor Movement

Fast MR acquisition techniques allow direct dynamic visualization of respiratory motion,


including assessment of parenchyma, chest wall and diaphragm, with high spatial and
temporal resolution [8,9,10]. In the first step, a dynamic MR-based study was designed. The
main goal was to accomplish a high precision characterization of tumor movements of upper
and mid lobe localized tumors (in as high a patient number as possible), and to calculate
numerical data for safety margins to be considered in 3D planning of lung cancer patients.

2.2. The Role of Tumor Movements in the Treatment Planning

The next aim of the study was to detect the possible uncertainties arising from tumor
movements in the daily treatment planning and under extreme breathing conditions. We used
CT fusion for the characterization of tumor motion.

2.3. The Influence of Thermoplastic Patient Fixation on Tumor Motions

Finally, the effects of our patient-positioning system on the chest wall and tumor motions
under extreme breathing conditions were analyzed using multislice CT.
CT/MR-Based Movement Analysis of Lung Tumors... 125

3. Materials and Methods


3.1. Dynamic MR-based Analysis of Tumor Movement

Twenty-four patients with newly-diagnosed (no previous surgery, chemo- or


radiotherapy) stage IIIV lung cancer were enrolled into the first part of the study (Table 1).

Table 1. Patient characteristics for Dynamic MR exams [40]

Patient Gender Age Localization TNM Tumor Size Hystology


1 Male 59 Right S3 T3N0M0 60x30x15mm Planocell.
2 Female 48 Right S3 T4N2M1 38x32x20mm Adenocc.
3 Female 48 Rigth S2 T4N2M1 25x10x9 mm Adenocc.
4 Female 61 Right S2 T4N0M0 43x30x23mm Adenocc.
5 Female 54 Rigth S3 T2N2M0 17x12x10mm Adenocc.
6 Female 53 Right S6 T2N2M0 35x25x21mm Adenocc.
7 Male 50 Right S3 T2N2M0 20x13x7 mm Adenocc.
8 Male 56 Left S6 T3N2M1 32x25x30mm Adenocc.
9 Male 58 Right S10 T2N2M1 35x32x27mm Adenocc.
10 Male 58 Left S2 T2N2M1 30x32x24mm Adenocc.
11 Female 67 Left S10 T2N1M0 43x25x30mm Planocell.
12 Male 54 Right S1 T4N2M1 66x55x42mm Adenocc.
13 Female 58 Left S1/2 T3N3M1 76x55x61mm Adenocc.
14 Female 57 Left S3 T3N2M1 36x26x26mm Planocell.
15 Male 62 Right S1 T2N0M0 43x39x25mm Adenocc.
16 Male 57 Left S3 T2N2M0 25x20x20mm Adenocc.
17 Male 69 Left S6 T3N2M0 30x30x24mm Adenocc.
18 Male 54 Left S1/2 T1N2M1 38x43x21mm Microcell.
19 Male 54 Right S1 T1N2M1 22x15x17mm Microcell.
20 Male 63 Left S2 T3N0M0 38x43x32mm Planocell.
21 Female 60 Left S6 T4N0M0 85x58x42mm Adenocc.
22 Male 49 Left S3 T4N2M1 57x37x41mm Planocell.
23 Male 49 Left S1/2 T4N2M0 25x18x25mm Planocell.
24 Male 49 Left S3 T4N2M0 88x49x34mm Planocell.

The patient selection criteria were the following:

good general condition (ECOG 0-1, Karnofsky score 10070%)


visibility of tumor on CT-MR
no contraindication of MR examination
good compliance

Tumor localization in the patients revealed nine lesions in the right S1-S3 segments, two
lesions in the right S4 segments, nine lesions in the left S1-S3 segments and four lesions in
the left S4-S10 segments (Figure 1).
Following a detailed explaination about the nature of the procedure, all patients provided
written informed consent under an institutionally approved subject research protocol. The
126 Arpad Kovacs

study was performed in accordance with the ethical standards of the responsible committee
on human experimentation and with the Helsinki Declaration of 1975 and 1983.

Figure 1. Tumor localization in Dynamic MR examinations. Color spots demonstrate tumor center
localizations [40].

Dynamic MR examinations
All MR examinations were performed using a clinical 1.5T whole-body scanner
(Magnetom Avanto, Siemens Medical Solutions, Erlangen, Germany) equipped with eight
receiver channels and a high-performance gradient system (30 mT/m). Patient preparation
involved precise patient positioning using a phased array torso coil followed by five minutes
relaxing time with the patient laying in the normal treatment position (supine position on the
flat cradle, with hands on a hand holder). After the localization series, in the coronal, sagittal,
and transversal planes were examined in each patient. Series were acquired through the
center of the tumor. The total acquisition time was 30 seconds for each plane, acquiring three
frames/s (100 slices/30 sec, TE/TR: 1.81/3.61 ms; flip angle: 71; receiver bandwidth: 501
Hz/pixel; field of view (FOV): 400; matrix 256x256; slice thickness: 10 mm; voxel size:
2.71x1.6x10 mm3).

Tumor movement analysis


Following the MR examination, the acquired data were transfered digitally to the
planning computer. All data were analysed with eRAD PACS software (eRAD PACS
Medical Solutions, USA). Tumor displacement was measured in the axial, sagittal and
coronal plane. To calculate the difference in position primary, the center of the tumor was
marked in all slices. By our definition the tumor center was the point of intersection of the
tumors greatest diameters on the actual slice. The tumor center definition was carried out
manually by two independent physicist (radiation oncologist and radiologist) (Figure 2).
The center coordinates of all the tumors were registered on all slices in all three planes. A
total of 24 x 100 x 9 = 2.16 x 104 coordinates were collected, studied, and visualized (Figures 3
and 4).
CT/MR-Based Movement Analysis of Lung Tumors... 127

Figure 2. Tumor center definition on dynamic MR series. The x mark shows the midposition of the
tumor center [40].

Figure 3. Orientation of the tumor center in all three planes [40].


128 Arpad Kovacs

deviation

430,000
425,000
coordinate values
420,000
415,000
410,000
405,000
400,000
395,000
390,000
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79 82 85 88 91 94 97 100

basic occasions

Figure 4. Visualization of the tumor center coordinates. The x axis highlights the basic occasions
(100/30 sec) while the y axis shows the registered basic occasion related coordinate values [40].

The tumor deviation was calculated from the coordinates; 12 coordinate unit difference
equal to 1 cm deviation, (defined by eRAD PACS measurement tool), the difference was
measured from the median coordinate values in all three planes. The independent deviation of
each tumors direction was calculated as:

x2 + y 2 + z 2 (x: cranio-caudal, y: antero-posterior, z: medio-lateral deviation in cm).

Finally in all three main directions - and direction independently- the probability of the
displacement was visualized (percentage of the basic occasions related to the distance in
cmFigure 5).

3.2. The Effect of the Tumor Movements for the Treatment Planning

Patient selection

Ten patients with stage II-IV lung cancer were enrolled into this part of the study (Table 2).
Pateint selection criteria were the following:

- good general condition (ECOG 0-1, Karnofsky score 100-70%)


- visibility of tumor on CT-MR
- no contraindication of MR examination
- good compliance
CT/MR-Based Movement Analysis of Lung Tumors... 129

summed deviations

120,00

100,00

80,00

60,00

percent

40,00

20,00

0,00
0,00 0,50 1,00 1,50 2,00 2,50

-20,00

direction-independent deviations in cm

Figure 5. Direction independent deviations of the tumors. In this figure the distance related percentages
were visualized. The graph demonstrates that the possibility of absolute deviations greater than 1 cm is
very low in tumors located in the upper-and mid lobe [40].

Table 2. Patient characteristics [39].

Patient Age Gender Localization Hystological Type TNM Stage Chemotherapy


1 51 male left central cc. Microcellulare T3N2Mx IIIa yes
2 53 male right central Adenocarcinoma TxNxM1 IV no
3 74 female right S6 Adenocarcinoma T2N0M1 IV yes
4 59 male left upper lobe cc. Planocellulare T3N2M0 IIIa no
5 61 male left upper lobe cc. Planocellulare T2N2M0 IIIa no
6 43 male right lower central cc. Planocellulare T2N2M0 IIIa yes
7 53 male left central cc. Microcellulare T4N1M1 IVb yes
8 72 male right upper lobe cc. Planocellulare T2N0M0 II no
9 71 male left upper lobe cc. Planocellulare T3N1M0 III no
10 69 female right central cc. Microcellulare T2N0M1 IVb yes
130 Arpad Kovacs

After data collection possible differences were investigated in the following factors:

- difference in tumor movements between left-side lesions


- difference in tumor movements between S1-2 and S3-10 lesions
- difference in tumor movements between patients aged under and over 55 years
- difference in tumor movements between the maximum diameter under and over 40
mm.

Eight males and two females with a mean age of 60.6 years (4374 years) participated in
the study. Five patients were diagnosed with centrally located and five with peripherally
located tumors. Three patients had microcellular, and seven non-small cell lung carcinoma.
As part of their complex treatment, five patients received chemotherapy as well. All patients
were informed about the examination and asked for written consent accepted by the local
ethics committee.
Following the normal planning CT scan two more scans were made with the same CT
parameters in maximum exspiration and maximum inspiration. For planning the normal
breathing scans were used with the fusion of the maximum inspiration and maximum
exspiration scans. After the fusion in all breathing phases, the gross tumor volumes were
contoured (GTV1,GTV2,GTV3). Around the GTV1 (normal breathing phase GTV) three
planning target volumes (PTV) were generated with a margin of 0.5 cm, 1.5 cm and 2.5 cm
(PTV1, PTV2, PTV3). Individual plans were generated to all PTV.

Patient positioning, planning CT


For patient positioning, a commercial ORFIT thermoplastic mask system was used
combined with a hand holder unit (Figure 6).
The mask was prepared individually for each patient. Masking process was followed by
the planning CT.
CT examinations were performed on a Siemens Somatom Sensation Cardiac 16 scanner,
(Erlangen, Germany), using the routine planning CT technique. After the topogram the
measurements were performed with the following parameters (standard for all patinets):

- CT thoracic basic program


- 5 mm slice thickness
- 2 mm interspacing
- uniform localization, uniform measurement range

After the mask preparation and 10 minutes of relaxation time, the breathing protocol was
explained for the patients (in the treatment room, treatment position). The protocol was the
following:

- Five minutes of normal breathing


- After maximum inspiration (sound sign) for 2025 secounds breathold in maximum
inspiration
- Maximum exspiration (sound sign) for 1015 seconds in maximum exspiration
CT/MR-Based Movement Analysis of Lung Tumors... 131

Figure 6. Individual thermoplastic patient fixation [22].

Table 3. Patient characteristics [22].

Patient Age Gender Localization Hystological Type TNM Stage Chemotherapy


1 51 male left central cc. Microcellulare T3N2Mx IIIa yes
2 53 male right central Adenocarcinoma TxNxM1 IV no
3 74 female right S6 Adenocarcinoma T2N0M1 IV yes
4 59 male left upper lobe cc. Planocellulare T3N2M0 IIIa no
5 61 male left upper lobe cc. Planocellulare T2N2M0 IIIa no
6 43 male right lower cc. Planocellulare T2N2M0 IIIa yes
lobe central
7 53 male left central cc. Microcellulare T4N1M1 IVb yes
8 72 male right upper cc. Planocellulare T2N0M0 II no
lobe
9 71 male left upper lobe cc. Planocellulare T3N1M0 III no
10 69 female right central cc. Microcellulare T2N0M1 IVb yes

Only patients who were able to perform the breathing process without any difficulties
were selected into the study. Three measurements were perfomed

with mask at normal breathing,


at maximum tidal volume inspiration,
at maximum tidal volume exspiration,

The CT series data were sent to the planning software (ONCENTRA MASTERPLAN,
USA) using the PACS system.

Planning, image fusion, DVH analysis


Prior to the contouring process, the acquired three image series (normal breathing,
maximum inspiration, maximum exspiration) were fused for all patients. During the fusion
process the series presenting the two extreme positions (maximum in- and exspiration) were
fused onto the normal CT series (normal breathing basic information). The automatic fusion
tool (based on mutual information) was used in the fusion process. After the fusion in all
three series (with identical window levels, WW:500, WL:70) the gross tumor volumes
(GTV1-normal breathing, GTV2-inspiration, GTV3-exspiration) were contoured. Planning
132 Arpad Kovacs

target volumes (PTV1, PTV2, PTV3) were generated around the normal GTV (GTV1) with a
0.51.52.5 cm margin. The consensus of three independent radiation oncologists was used
for the planning target volume.
To comply with tumor movement-related uncertainties (resulting in PTV coverage),
individual treatment plans were generated for all patients in 23 fields. These plans were
designed with the optimal field energy and configuration to reach the best PTV coverage with
minimal normal tissue exposure. The dose was prescribed to reach the best PTV coverage.
Dose volume histogramms were used for comparison and analysis of the plans. The
coverage index was used for the different GTV coverage analysis. The coverage index is
defined as: CI=PTVref /VPTV (PTV ref: volume of PT covered by the prescribed dose, Vptv:
volume of the PTV) (11). Higher coverage index means better coverage.

3.3. The Influence of Thermoplastic Patient Fixation on Tumor Motions

Patient selection
Ten patients with stage II-IV lung cancer were enrolled in the final part of the study
(Table 3).
Patient selection criteria were the following:

- good general condition (ECOG 0-1, Karnofsky score 10070%)


- visibility of tumor on CT-MR
- no contraindication of MR examination
- good compliance

Eight males and two females with a mean age of 60.6 years (4374 years) participated in
the study. Five patients were diagnosed with centrally located tumors and five with
peripherally located tumors. Three patients had microcellular and seven non-small cell lung
carcinoma. As part of their complex treatment, five patients received chemotherapy as well.
All patients were informed about the examination and asked for written consent accepted by
the local ethics committee.

Patient positioning, CT scan


For patient positioning we used the thermoplastic mask fixation system combined with
arm support which is used in daily routine work at our institution (Figure 6). The arm support
included three hand holders and a shoulder rest. The individual thermoplastic masks were
stabilized by four cuts on the maskfixing board. Before the examination the patients were
placed on the central part of the board (using laser-guided positioning aimed at keeping the
position of the body straight), hanging with both hands on a hand holder. The patient position
was also controlled in the x-y-z axis using the laser lights. Four radiopaque markers were
temporally positioned on the chest wall, as follows:

the first marker in the longitudinal center body axis, at the level of the second
intercostal space
CT/MR-Based Movement Analysis of Lung Tumors... 133

the second marker also in the longitudinal center body axis, positioned two
fingerbreadths cranially from the xiphoid process
the third and fourth markers at the level of the second marker, on the left and right
side, in the outer axillary line (Figure 7)

Figure 7. Marker placement on the chestwall [22].

Figure 8. Measurement process of tumor and chest wall movements [22].

Following careful positioning of the markers, a tailored ORFIT thermoplastic thoracic


mask was prepared for each patient. The masks were heated up to 78C and then tailored onto
the patients body precisely fixed to avoid any displacement within the mask. It was essential
that the mask perfectly follow the body contour without any free space between the patients
body and the mask. Laser signs corresponding to all three axis were marked for the positioning.
The training of the breathing exercises was conducted in the therapy simulator (Siemens
Simeview Simulator). Following a 5 min period of acclimatisation, all patients had to breath in
normal amplitude, withholding their breath for 30 seconds following maximum inspiration, and
then holding the breath expired at maximum exspiration. Only patients who were able to breathe
quietly and to withhold their breath for 30 seconds at maximum inspiration and 15 seconds at
maximum exspiration were assigned to participate in the study.

CT imaging
After patient preparation, the patients were examined with the multislice CT scanner
(SIEMENS Somatom Sensation 16 Cardiac). The patient immobilization system was placed
on the flat treatment table and the patients were localized in the original position, set in the
simulator. The accuracy of positioning was controlled with the lasers in all three planes.
134 Arpad Kovacs

Following the topogram, the measurements were made with the same settings (continuous
slice thickness of 8 mm with 2 mm interspaces, same starting points and endpoints, range of
same length), without marker and patient displacement. In addition to the routine procedure,
four further measurements were performed for each patient:

normal breathing with the mask


at maximum tidal volume inspiration
at maximum tidal volume exspiration
all measurements repeated in the same position without the mask.

With the use of fast CT scanning, the duration of one measurement was < 20 seconds and
the total examination time did not exceed three minutes. Using the acquired CT data, 2 mm
slices were depicted and reconstructed axially, coronally and sagittally; three dimensional
reconstructions were also made. Measurements were undertaken using eRAD Practice
Builder and PACS Software eRAD/ImageMedical. Using standardized windowing levels
(WL = 440 HU, WW = 45 HU), the serieses were paired and the following parameters were
analyzed (Figure 8).
(1) Analysis of chest wall motions:

upper marker displacement (as compared to the table board),


lower medial marker displacement (as compared to the table board),
lower lateral marker displacement (distance between the markers).

(2) Analysis of diaphragmatic movements:

Deviations of both diaphragms (distance between the intersection of clavicle and


second rib and the cranial pole of diaphragm in millimeters).

(3) Analysis of tumor motions (the measurements were performed starting from the point
of intersection of the greatest tumor diameters reconstructed in three planes):

distance between the tumor center and the longitudinal center body axis,
distance between the tumor center and the lateral chest wall,
distance between the tumor center and the anterior chest wall,
distance between the tumor center and the posterior chest wall,
craniocaudal displacement of the tumor center.

Statistical analysis
For data evaluation, the paired t-test was used. When comparing the data series, the mean
values were confronted in all cases and, during evaluation, a significance level of p 0.05
was considered to be a significant difference.
CT/MR-Based Movement Analysis of Lung Tumors... 135

Table 3. Differences in AP Direction [40].

Differences in AP Direction (cm) mean SD median P value


Left side (n=11) 0.09 0.04 0.07
Right side (n=13) 0.12 0.05 0.1 p0.2
Age<55 years (n=12) 0.1 0.04 0.09
Age>55 years (n=12) 0.11 0.05 0.1 p0.5
Men (n=14) 0.10 0.04 0.07
Women (n=10) 0.12 0.05 0.11 p0.5
Tumors in S1-S2 (n=10) 0.09 0.04 0.07
Tumors in S3-S6 (n=14) 0.12 0.05 0.11 p0.2
Tumor maximum diameter <40mm (n=13) 0.11 0.04 0.11
Tumor maximum diameter >40mm (n=11) 0.1 0.05 0.08 p0.5

Table 4. Differences in ML Direction [40].

Differences in ML Direction (cm) mean SD median P value


Left side (n=11) 0.10 0.04 0.08
Right side (n=13) 0.12 0.05 0.12 p0.5
Age<55 years (n=12) 0.12 0.04 0.11
Age>55 years (n=12) 0.11 0.05 0.08 p0.5
Men (n=14) 0.11 0.05 0.09
Women (n=10) 0.11 0.04 0.11 p0.5
Tumors in S1-S2 (n=10) 0.09 0.02 0.09
Tumors in S3-S6 (n=14) 0.13 0.05 0.12 p0.2
Tumor maximum diameter <40mm(n=13) 0.13 0.06 0.12
Tumor maximum diameter >40mm (n=11) 0.1 0.02 0.08 p0.2

Table 5. Differences in CC Direction [40].

Differences in CC Direction (cm) mean SD median P value


Left side (n=11) 0.22 0.18 0.16
Right side (n=13) 0.31 0.22 0.28 p0.5

Age<55 years (n=12) 0.26 0.21 0.18


Age>55 years (n=12) 0.27 0.19 0.17 p0.5

Men (n=14) 0.27 0.24 0.17


Women (n=10) 0.26 0.141 0.18 p0.5

Tumors in S1-S2 (n=10) 0.15 0.05 0.14


Tumors in S3-S6 (n=14) 0.35 0.22 0.30 p0.05

Tumor maximum diameter <40mm (n=13) 0.31 0.24 0.18


Tumor maximum diameter >40mm (n=11) 0.22 0.13 0.15 p0.5
136 Arpad Kovacs

Table 6. Direction Independent Differences [40].

Direction independent differences (cm) mean SD median P value


Left side (n=11) 0.14 0.1 0.11
Right side (n=13) 0.2 0.11 0.21 p0.2

Age<55 years (n=12) 0.17 0.11 0.14


Age>55 years (n=12) 0.16 0.1 0.14 p0.5

Men (n=14) 0.18 0.12 0.14


Women (n=10) 0.16 0.08 0.14 p0.5

Tumors in S1-S2 (n=10) 0.13 0.03 0.12


Tumors in S3-S6 (n=14) 0.2 0.13 0.18 p0.1

Tumor maximum diameter <40mm (n=13) 0.19 0.13 0.14


Tumor maximum diameter >40mm (n=11) 0.14 0.06 0.11 p0.2

4. Results
4.1. Dynamic MR-based Analysis of Tumor Movement

In all patients, dynamic MRI series showed regular synchronous tumor motion with good
mobility, displacements were periodic and reproducible. The acquisition of three images per
second allowed for continuous recording during the breathing cycle. Mean antero-posterior
deviation was 0.109 cm (range: 0.063 cm0.204 cm, SD: 0.04). Greatest AP deviation was
0.204 cm (patient 7). The mean medio-lateral deviation was 0.114 cm (range: 0.06 cm0.204
cm, SD: 0.05). Greatest medio-lateral deviation was 0.204 cm (patient 7) The greatest
deviation was measured in the cranio-caudal direction (mean: 0.27 cm, range: 0.079 cm
0.815 cm). Mean direction independent deviation was 0.18 cm (range: 0.09 cm0.48 cm)
(Figure 4). In Figure 5, distance related percentages (number of occasions related to the
distance -in cm- /100) were visualized. In the comparison of the tumor motion (antero-
posterior, cranio-caudal, medio-lateral and absolute deviation) between the age groups, the
tumor size, the laterality and the sex, no significant differences were found (Tables 3, 4, 5
and 6). Significant difference (in the level of p0.05) was observed only in thecase of the
lesion localization between the S12 and S36 segments, in the cranio-caudal direction
(Table 5).

4.2. The Effect of the Tumor Movements for the Treatment Planning

All GTV volumes were registered. In all cases, volume deviations were administered in
different breathing phases (min: 1.5%, max: 35.6%). The comparison Coverage Index (CI)
was used for GTV coverage. In case of extreme breathing conditions, the use of a 0.5 cm
margin was sufficient to maintain good coverage for central tumors. In the case of peripheral
CT/MR-Based Movement Analysis of Lung Tumors... 137

tumors, a 1.5 cm margin had to be used in order to maintain acceptable coverage (CI: 0.85
1.00). Coverage index values registered in the normal breathing position were 1.0.

CI GTV2

1,20

1,00

0,80

0,60
CI

0,40
0,20

0,00
1 2 3 4 5 6 7 8 9 10
beteg

Figure 9. GTV coverage indexes in maximum exspiration [39].

Figure 9 shows the GTV coverages in maximum exspiration. In three cases no sufficient
coverage was achieved despite the 2.5 cm margin planning.
Figure 10 shows the GTV coverages in maximum inspiration. In the case of patient 1,
only the 2.5 cm margin PTV allowed sufficient coverage.

CI GTV3

1,20

1,00

0,80

0,60
CI

0,40
0,20

0,00
1 2 3 4 5 6 7 8 9 10
beteg

Figure 10. GTV coverage indexes in maximum inspiration [39].


138 Arpad Kovacs

4.3. The Influence of the Thermoplastic Patient Fixation on Tumor Motions

Movements of chest wall (Figure 11), diaphragm (Figure 12) and tumor (Figure 13), with
and without mask fixation, were registered. When comparing the acquired data, the average
of maximum deviation was taken into account. For maximum deviation, the difference of
maximum inspiration and maximum exspiration was evaluated.

low er-m edial m arker displacem ent (as upper m arker displacem ent (as
com pared to the table board) com pared to the table board)
deviations in

deviations in
30 30
20 20
mm

mm
10 10
0 0
1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11

patients patients

low er-lateral m arkers displacem ent


(distance betw een the m arkers)
deviations in

20
mm

10

0
1 2 3 4 5 6 7 8 9 10 11

patients

Figure 11. Marker motions. Left column represents motion without mask fixation, right column shows
motions with the fixation system. Column 11 demostrates the mean values [22].

left diaphragm right diaphragm

80 100
deviations in mm

deviations in mm

60 80

60
40
40
20
20
0 0
1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11

patients patients

Figure 12. Diaphragm motions. Left column represents motion without mask fixation, right column
shows motions with the fixation system. Column 11 demostrates the mean values.
CT/MR-Based Movement Analysis of Lung Tumors... 139

tum or m otions m edial tumor motions lateral

deviations in mm

deviations in mm
20 20
15 15
10 10
5 5
0
1 2 3 4 5 6 7 8 9 10 11 0 1 2 3 4 5 6 7 8 9 10 11

patients patients

tumor motions craniocaudal tumor motions AP

deviations in mm
deviations in

30
40
mm

20
20 10

0 1 2 3 4 5 6 7 8 9 10 11
0 1 2 3 4 5 6 7 8 9 10 11

patients patients

tumor motions PA

30
deviations in mm

20

10

0
1 2 3 4 5 6 7 8 9 10 11

patients

Figure 13. Tumor motions. Left column represents motion without mask fixation, right column
represents motions with the fixation system. Column 11 demostrates the mean values [22].

- Upper marker displacement


Mean maximum displacement of upper marker, with and without mask, was 10.57.7
mm (p 0.05). In respect to the upper markers, the difference was found to be significant
with the use of the immobilizing system.

- Lower medial marker displacement


Mean maximum displacement of lower medial marker, with and without mask, was 10
7.4 mm (p 0.05. In respect to the lower medial markers, a significant difference was
observed.

- Lower lateral displacement


Mean maximum transverse displacement of markers in the lower outer axillary line, with
and without mask, was 8.14.2 mm (p 0.05). A significant difference was observed in this
case as well.

- Diaphragmatic movements
Mean maximum displacement of the right diaphragm, with and without mask, was 41.7
40.5 mm (p 0.5), and of the left diaphragm 40.536.8 mm (p 0.1). In respect to the
diaphragmatic movement, no significant difference was observed.
140 Arpad Kovacs

- Tumor motions
Median displacement of tumor motions, with and without mask, was 15.312.4 mm in
the craniocaudal direction (p 0.2), 11.58.8 mm in posteroanterior direction (p 0.15), 4.6
4.1 mm in medial direction (p 0.5), and 7.25 mm in the lateral direction (p 0.1). In these
cases significant differences were not observed. In case of the anteroposterior tumor motions
(mean: 8.96.3 mm), a significant difference was registered at a significance level of p
0.05.

5. Discussion
5.1. Dynamic MR-based Analysis of Tumor Movement

According to the International Commission on Radiation Units and Measurements


(ICRU) Report, Recommendation No. 62, when using 3D based radiotherapy the planning
target volume (PTV) has to include the uncertainties arising from internal organ motion,
patient movements and positioning errors [7]. In modern 3D radiotherapy of lung cancer the
question of organ motion is a very important factor. As it has been described in many
previous studies, movement of the tumor during the breathing cycle is a potential cause of RT
failure in lung cancer [13,14,15,16]. During RT, tumor mobility may be responsible for
insufficient coverage of the clinical target volume and delivery of an insufficient total dose to
destroy the tumor cells. In particular, high-precision techniques are very sensitive to failures
in patient setup and internal motion. Here, the concept of the optimal target volume adoption
bears the high risk of field border and out-of-field relapses if tumor mobility is not
sufficiently integrated into the treatment planning [17,18]. Up to now, several authors
discussed the monitoring of tumor motions. In the past, the respiratory cycle was examined
by fluoroscopic measures, however, these techniques include some disadvantages. There is
the possibility of an over- or underestimation of the actual movement, if the tumor is not
located in a defined distance from the focus caused by magnifying and lessening effects, and
sometimes (central or small tumors) the exact definition of the tumor may be difficult [19].
Other authors applied beam imaging systems for monitoring tumor motions. A major
advantage of this procedure is the real-time imaging, however, portal imaging did not become
very popular, furthermore, visualization qualities need to be improved as well [18].
CT-based definition of the tumor motion is the simplest and most popular method.
During this commonly used procedure, the PTV definition is performed on the basis of CT-
images taken under normal breathing conditions. Generally, the anatomical position
visualised on the image taken under normal breathing conditions is considered to be a mid-
position between in- and exspiration. However, the examination results clearly showed that
the position of diverse thoracic structures during normal breathing can by no means be
regarded as an interposition between in- and exspiration [20,21,22]. Incorrect anatomical
definition may lead to geometric inaccuracy. One study investigated the use of CT to measure
tumor mobility in 17 patients at two institutions [23]. The results demonstrated that a precise
and reproducible documentation of intrathoracic organ motions is possible using CT. In their
study, three CT series were performed: free-breathing, inspiration, and exspiration. The main
CT/MR-Based Movement Analysis of Lung Tumors... 141

disadvantage of this technique was that only snapshots can be taken of the tumor position and
the patient is exposed to additional radiation.
To reduce the uncertainties coming from tumor motions, Kutcher et al. presented four
options [24]:

monitoring of tumor motions and consideration of the results thereof at outlining of a


definitive therapeutic plan
patient training: breath control technique
deliveryof a triggered, breathing synchronized irradiation
active blocking of patients respiration

According to the literature up to now, several authors described their investigations


related to respiratory gated techniques, active blocking of patient respiration and movements.
Presently, several forms of positioning systems (mask fixation, vacuum systems, arm holders,
etc.) are known [25]. In the breathing synchronized, manually triggered LINAC treatments
also in use, the therapy is controlled by respiratory positions transmitted by video equipment,
laser or plethysmograph [26,27]. Some authors reported on treatments in deep breath-hold
with active control allowing a CTV reduction of 0.25 cm, resulting in a decrease of the
dosage-induced damage of normal lung tissues [28,29]. Several papers were published related
to the employment of active breath control (ABC) technique [30,31]. These equipments and
techniques are not widely used in the daily practice.
The main goal of the first part of our study was to make a precise dynamic MR based
analysis of lung cancer patients with upper and mid lobe tumors. In the literature available,
only a few articles can be found using Dynamic MR for tumor and organ motion monitoring
in lung cancer patients, and even in the traditional fluoroscopy or CT based studies mainly
720 patients were investigated. Seppenwoolde et al. described motion with gold implants
taken into or near to the tumor, using fluoroscopy [16]. Thirteen tumors were in the upper
and mid-lobe region and due to this analysis, similar results were found (21mm in AP and
medio-lateral direction, 0.711.1 mm in cranio-caudal direction) compared to our data shown
in the figure. In the CT-based study of van Sornsen de Koste JR et al., 14 tumors in the upper
localization were studied and a safety margin of 5 mm was proposed in general [34]. Other
groups used a safety margin of 1 cm (seven patients) (35). Shinichiro M et al. used a 256
multislice CT for 4D measurement of tumor motion, and in their 14 patient study (upper-mid
and lower lobe localization) they recommend the same value of 3.5mm in the left, right,
superior, anterior and posterior directions and 21 mm for internal margin in the inferior
direction [36]. According to dynamic MR based data of C. Palthow et al., when taking in
account the most extensive tumor displacement in quiet respiration, a general safety margin
of 3.4 mm for tumors of the upper region (six patients), 4.5 mm for the middle region (four
patients) must be considered [37]. Our study of 24 tumors is one of the highest number in the
context of dynamic MR based tumor motion analysis. This amount of data is comparable to
the motion analysis of Plathow et al. (24/35 tumors in the upper and mid lobes [38]. Our
numerical data give exact safety margins in all three main directions and in general too
(direction independent deviations). According to our results a mean safety margin of 0.25 cm
in the AP direction, 0.3 cm in the medio-lateral direction and 0.85 cm in the cranio-caudal
142 Arpad Kovacs

direction should be enough for upper and mid lobe lung cancers. Using our method a high
precision tumor motion definition can be delivered [40]. The tumor center coordinate
definition, slice by slice in all three directions, gives the opportunity of 3D reconstruction and
visualization of the tumor center movement (Figure 15). The main disadvantage of our
method is the time consumption; 300 coordinate points must be defined for each tumor to
make the analysis. In Figure 4, we have visualized all the 100 points registered during the 30
sec period in relation to the x-y-z coordinates. Around the median point spheres were
generated with 0.5 cm and 1cm diameter. Using this option, tumor center positions moving
out from these barriers can be visualized (Figure 14).

5.2. The Rule of the Tumor Movements in the Treatment Planning

Nowadays, in most radiotherapy departments, irradiation of lung cancer patients is


delivered by CT-based 3D planning. The main purpose of this study was to analyze the tumor
movement effects in the daily used routine planning process. In this study, we simulated
extreme breathing conditions to analyze the highest possible mistakes resulting from
breathing-related tumor motions. We generated a 0.5 cm, a 1.5 cm and a 2.5 cm safety margin
for the breathing-related planning uncertainty analysis. Our results show that in the case of
central tumors a 0.5 cm margin, and in the case of peripheral tumors a 1.52 cm margin is
needed to prevent breath-related tumor movements [39]. This question is more difficult in
cases of combined PTVs (central node volume + periferial tumor).

5.3. The Influence of Thermoplastic Patient Fixation on Tumor Motions

Our purposes with this study were to detect, describe and analyze tumor motions and to
assess the effectiveness of our immobilization system regarding tumor motion restriction.
During this study, we had to eliminate two critical problems. The first was the question of
patient immobilization associated with the reduction of tumor motions, the second the
selection of reference points. The selection of the comparison points in the motion analysis
was a critical question. During the respiratory cycle almost all parts of the chest are in
motion, making it difficult to determine fixed points of reference. DeNeve et al. defined
diaphragms, chest wall, carina and intervertebral discus as bases of comparison [32]. In the
study of Plathow et al., cranio-caudal displacement was measured from the T6/T7 disk space
to the proximal external tumor edge. Antero-posterior displacement was measured in relation
to a line tangential to the anterior edge of the vertebrae. Medio-lateral displacement was
measured from the midline through the spinal process [33]. In our method there is no need to
use such comparison points (which are also in motion) because the coordinates can be
defined easily using the software defining tool.
CT/MR-Based Movement Analysis of Lung Tumors... 143

Figure 14. 3-D visualization of the 100 tumor center position during the 30 sec period. Spheres
measuring 0.5 and 1 cm were generated around the median point. Using this option, tumor center
positions moving out from these barriers can be visualized [40].

6. Conclusion
6.1. Dynamic MRI-based Movement Analysis

Dynamic MR is a sensitive and well-tolerated method for tumor motion monitoring in the
case of high precision 3D therapy planning of lung cancer. Our results demonstrate that
tumors located in the upper and mid lobes have moderate breath synchronous movements.
The greatest deviation should be considered in the cranio-caudal direction. In tumors
144 Arpad Kovacs

localized in the upper and mid lobe, the investigated factors (age, sex, tumor size, laterality,
even location) were shown to be minor factors influencing tumor motions. In future studies,
our analyzing process can be converted into other modalities (CT-based motion tracking) and
may be integrated into complex CT/MRI-based gating techniques.

6.2. The Rule of Tumor Movements in the Treatment Planning

In our study, extreme breathing conditions were analyzed. According to our results, CT
scans used in the daily routine do not represent accurately the tumor midposition and the true
tumor volume. Due to breathing, synchronous extreme 0.5 cm margin tumor movements
must be used for planning in central localization. In the case of peripheral tumors a wider
margin should be used.

6.3. The Influence of Thermoplastic Patient Fixation on Tumor Motions

The main advantages of the immobilization system used by our team include its simple
construction, uncomplicated operation and, according to our experience, its relatively high
effectiveness when positioning lung cancer patients. Our measurements show that, besides
the advantage of optimal patient positioning, the movements of the skeletal chest wall can be
considerably reduced using this system. During the use of this mask fixation system, we
noted a decrease in tumor motions as well; however, it was not a significant level. This
system is not able to reduce breath-related tumor motions. Our results refer to the fact that the
planning CT examination applied in daily routine work cannot give exact details about the
actual tumor position, because it only takes a snapshot of the tumor location in a single
moment; therefore, wide margins are to be taken into account when defining the PTV.

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Chapter 6

Small Cell Lung Cancer: Clinical


Presentation and Diagnostic Modalities

Anant Mohan*1, Manisha Bhutani 2, Subhash Budania1,


Samir Naik3 and Randeep Guleria1
1
Department of Medicine, All India Institute of Medical Sciences, New Delhi, India
2
Department of Internal Medicine, Michigan State University,
East Lansing, MI 48824, USA
3
Department of Respiratory Medicine, Royal Preston Hospital, Preston, Lancashire, UK

Introduction
Lung cancer is second most common non-cutaneous cancer and leading cause of cancer
deaths in the United States. According to the SEER statistics, the total number of new cases
and deaths in 2008 were estimated to be 215,020 (accounting for approximately 15% of all
cancer diagnoses) and 166,280 (accounting for around 29% of all cancer deaths),
respectively. Mortality among men decreased by 1.3% per year from 19901994 and by 2.0%
per year from 19942004 [1]. Death rates are approaching a plateau after continuously
increasing for several decades in women. These trends in lung cancer mortality reflect the
decrease in smoking rates over the past 30 years. Lung cancer is classified into two broad
varieties for treatment purposes: Non Small Cell Lung Cancer (NSCLC) and Small Cell Lung
Cancer (SCLC). The proportion of SCLC among all lung cancers has fallen from 2025% in
the past to approximately 13% currently. However, it assumes significance due to its
relatively aggressive course and differences in clinical presentation from NSCLC.

*
Corresponding Author: Department of Medicine, All India Institute of Medical Sciences, New Delhi-110029,
India. Ph: 91-11-26593006. Fax: 91-11-26588641. E-mail: anantmohan@yahoo.com, anant_mohan@
rediff.com
150 Anant Mohan, Manisha Bhutani, Subhash Budania et al.

Clinical Presentation
SCLC has a highly variable clinical presentation. Most patients are asymptomatic in the
early stages since the lungs have significant respiratory reserve capacity and sparse nerve
supply. SCLC presents mainly with cough, chest pain and shortness of breath, but it must be
suspected in a person who presents with any of the following symptoms:

A cough that doesnt go away


Shortness of breath
Chest pain that doesnt go away
Wheezing
Coughing up blood
Hoarseness
Swelling of the face and neck
Loss of appetite
Weight loss for no known reason
Unusual tiredness

Clinical manifestations of SCLC can be divided into four categories: (1) those due to
local tumor growth, 2) those due to regional spread, (3) those due to distant metastases and
(4) paraneoplastic syndromes and generalized effects.

Clinical Features Due to Local Growth


Cough, dyspnea and chest pain are most common clinical manifestations of SCLC. In
patients with lung cancer, there are several possible causes of cough and dyspnea, such as:

Direct tumor effects such as intrinsic or extrinsic airway obstruction, pleural


involvement, parenchymal involvement in advanced stage, and superior vena cava
syndrome (SVCS).
Indirect tumor effects due to pneumonia, pleural effusion or a pulmonary embolus.
Treatment-related causes such as pulmonary fibrosis secondary to radiation therapy
or chemotherapy, or chemotherapy-induced cardiomyopathy.
Causes unrelated to the cancer. These include chronic obstructive airway disease and
congestive heart failure.

Chest pain due to a lung tumor usually occurs at an advanced stage and is caused not by
the physical presence of a tumor in lung parenchyma but as a result of local extension into the
adjacent chest wall or nerve fibers. Localized wheezing could be produced by intrinsic or
extrinsic airway obstruction by the tumor or enlarged lymph nodes.
Small Cell Lung Cancer: Clinical Presentation and Diagnostic Modalities 151

Figure 1.

Clinical Features Due to Regional


Spread of Tumor
Dyspnea, dysphagia, and hoarseness occur as a result of compression of trachea,
esophagus and recurrent laryngeal nerve respectively by a centrally placed tumor or enlarged
mediastinal lymph nodes, or due to lymphatic obstruction. The longer intra thoracic course of
the left recurrent laryngeal nerve makes it more susceptible to compression from left-sided
tumors compared to right-side lesions. Dysphagia usually occurs with both solids and liquids,
and may be complicated by recurrent aspiration due associated recurrent laryngeal nerve
palsy. Involvement of the phrenic nerve is associated with hiccups early in the disease; this
later leads to paralysis and elevation of the hemidiaphragm with resulting dyspnea.

Pancoast Syndrome
This is also known as superior pulmonary sulcus carcinoma. The clinical features consist
of Horners syndrome (ptosis, miosis, anhidrosis, and enophthalmos; Figure 1., destruction of
adjacent bone, atrophy of hand muscles and pain in the shoulder and ulnar distribution of the
arm), although all these four features dont always coexist always [1]. These symptoms are
secondary to involvement of sympathetic chain, stellate ganglion and the bronchial plexus
(C8, T1) by the tumor that runs in the superior outlet of thorax near the apex of lung. It is an
uncommon presentation in SCLC, perhaps because of the early propensity for widespread
dissemination and the typical central location.

Superior Vena Cava Syndrome (SVS) in SCLC


Lung cancer, especially SCLC, is the most common cause of SVS along with non-
Hodgkin lymphoma. This is because a majority of these tumors arise in the central airways.
As a result, the superior vena cava may get obstructed either due to invasion of the vein,
152 Anant Mohan, Manisha Bhutani, Subhash Budania et al.

extrinsic compression by the tumor, or due to intraluminal thrombosis. Lung cancer accounts
for 65 to 90% of SVS, with tumors involving the right main bronchus or right upper lobe
being the most notorious [2]. The most common symptoms of SVS are shortness of breath,
cough, and swelling of the face, neck, upper body, and arms. Other clinical features include
(Figure 2):

Hoarse voice
Chest pain
Difficulty in swallowing and/or talking
Hemoptysis
Swollen veins over the chest or neck
Bluish color to the skin
Drooping eyelid

The presence of SVS features indicates that the patient is no longer a candidate for
surgical removal of tumor mass.

Esophageal Compression
Compression of the esophagus can lead to dysphagia and odynophagia.

Tracheal Compression
Compression of the main stem bronchi and trachea can cause severe shortness of breath
and stridor.

Figure 2. Superior vena cava syndrome


Small Cell Lung Cancer: Clinical Presentation and Diagnostic Modalities 153

Pleural Effusion
Lung cancer accounts for 40% of symptomatic pleural effusions although SCLC
contributes less due to its relative central location. However, not all pleural effusions in lung
cancer are malignant. These patients are also prone to develop congestive heart failure,
pneumonia, pulmonary embolism, and malnutrition, each of which can cause pleural effusion.
Clues pointing malignancy are the presence of a massive effusion, hemorrhagic, and rapidly
refilling after being drained by thoracocentesis or through a chest tube.

Pericardial Effusion
Pericardial involvement arises from direct extension of the tumor or as a result of
retrograde spread through mediastinal and epicardial lymphatics. Lung cancer is the most
common neoplasm that produces pericardial metastases; accounting for 37% of the reported
cases. Fifty percent of these have concomitant pleural effusions. Dyspnea, orthopnea, fatigue,
or asthenia may be the only initial symptoms, while some patients may present with upper
abdominal distention due to downward hepatic distention, hiccups due to pressure on the
diaphragm, or pleuritic pain due to stretching of the pericardium (especially when lying flat).
Signs of effusion include Kussmauls sign (increased distention of jugular veins with
inspiration), Freidreichs sign (rapid diastolic descent of the venous pulse), and pulsus
paradoxus (decrease of more than 10 mm Hg in the diastolic pressure on inspiration). The
important signs of pericardial tamponade include tachycardia, pulsus paradoxus, elevated
jugular venous pressure, and hypotension; however, some patients may develop tamponade
without this clinical pattern. Approximately one-third of patients with pericardial metastases
eventually die from this complication.

Clinical Features Secondary to Metastatic Disease


Small cell lung cancer has a high propensity for dissemination to various organs (Table 1).

Table 1. Extent of disease at presentation.

Metastatic sites: Frequency


Liver 25%30%
Bone 25%40%
Bone marrow 20%
Adrenal 5%30%
Brain 10%
Extrathoracic lymph nodes 5%
Subcutaneous masses 5%
154 Anant Mohan, Manisha Bhutani, Subhash Budania et al.

The common symptoms arising from metastasis are the following:

Headache, papilloedema, seizures, syncope, mental status change and weakness


indicate brain metastasis or spinal cord compression, while pain indicates bone
metastasis. The bone marrow can also be involved and may cause pancytopenia.
Liver metastasis, which is more common with SCLC compared to other lung cancer
subtypes, presents with right upper quadrant pain, hepatomegaly and other non
specific symptoms.
Adrenal metastasis is relatively uncommon in SCLC compared to other histological
subtypes and usually remains asymptomatic. Extensive metastasis, however, may
lead to adrenal insufficiency and right flank pain.
Lung cancer is most common cancer causing skin metastasis although SCLC
contributes little to this. The skin of the chest, abdomen, and back are the usual sites,
although the umbilicus, lips, tongue, and foot may also be involved. The nodular
variety is the commonest dermatological presentation seen. The presence of
cutaneous metastasis portends a grave prognosis2.

Paraneoplastic Syndromes
Paraneoplastic syndromes are caused by factors produced by cancer cells that often act at
a site distant from both the primary site and its metastases. Lung cancer, particularly SCLC,
is the most common malignancy causing paraneoplastic syndromes. The common
paraneoplastic syndromes associated with SCLC are:

Cachexia and Anorexia: Presence of a tumor can be clinically suspected when patients
report anorexia accompanied by marked weight loss. These symptoms may occur as a result
of production of proteolysis-inducing factor (causes skeletal muscle breakdown), lipid-
mobilizing factors, and a number of pro-inflammatory cytokines, including tumor necrosis
factor-alpha (TNF- ), interleukin-1 (IL-1), and interleukin-6 (IL-6) as a consequence of
interactions between host cells and tumor cells3.

Syndrome of Inappropriate Antidiuretic Hormone Secretion (SIADH): SIADH is


present in 7% to 11% of patients with SCLC. Paraneoplastic SIADH results from the ectopic
production of antidiuretic hormone (ADH) or from other tumor-related mechanisms leading to
increased pituitary ADH secretion, which produces hyponatremia, hypervolemia, increased
renal sodium loss and inappropriately high urinary osmolality. The manifestations of
hyponatremia (mental status changes, lethargy, or seizures) are often absent despite very low
sodium levels, as the rate of decline is typically prolonged. The overt SIADH is characterized by
neurological and psychiatric symptoms attributable to cerebral edema and cellular swelling.

Cushings Syndrome: Cushings syndrome occurs in 3% to 7% of patients with SCLC


is due to ectopic production of immunoreactive adrenocorticotropic hormone (ACTH). The
clinical manifestations are less prominent than in Cushings disease; biochemical
Small Cell Lung Cancer: Clinical Presentation and Diagnostic Modalities 155

abnormalities predominate, whereas the physical changes are less prominent (30 to 70%
versus 3 to 7%). Distinctive laboratory features of ectopic ACTH syndrome include severe
hypokalemic alkalosis (70 to 90% of patients have serum potassium level < 3.0 mEq/L),
greatly elevated urinary 17-hydroxycorticoids excretion, and high serum ACTH levels (> 200
pg/mL in 65% of patients).

Lambert-Eaton Myasthenic Syndrome (LEMS): This condition manifests as weakness


of proximal muscles of the lower and upper extremities with relative sparing of respiratory
and bulbar muscles. In contrast to patients with other myasthenic syndromes, motor strength
in patients with LEMS may initially improve after exercise, subsequently weakening if
activity is sustained, a phenomenon known as post-exercise facilitation. On
electromyography, low compound muscle action potential after nerve stimulation with
decrement at low frequency stimulation (3 Hz) of more than 10% and increment after high
frequency stimulation (more than 20 Hz or preferably maximal voluntary contraction) of
more than 100%4. Pathogenesis involves immunologic cross-reactivity between tumor-
associated antigens and P/Q and Ntype voltage-gated calcium ion channels4.

Cancer Associated Retinopathy (CAR): Cancer Associated Retinopathy (CAR) is a


rare paraneoplastic syndrome, most commonly associated with SCLC. This occurs due to
production of antibodies against recoverin (23-kD CAR antigen), a Ca2+-binding protein
which controls phosphorylation of the visual receptor rhodopsin by inhibiting rhodopsin
kinase (GRK-1)5. CAR is characterized by rapid vision loss, visual field contraction, ring
scotomata, photosensitivity, night blindness, and color vision loss.

Subacute Sensory Neuropathy: Subacute sensory neuropathy is the most characteristic


peripheral neuropathy associated with SCLC. The main complaints are asymmetric
distribution of pain and paresthesias involving the arms more than the legs4. Later, numbness,
limb ataxia, and pseudoathetotic movements of the hands replace pain4. The deep tendon
reflexes are abolished and all modalities of sensation are involved, particularly joint
position4. This may precede the diagnosis of SCLC by several months. Type 1 antineuronal
nuclear antibody (ANNA-1), also known as anti-Hu, is a valuable serologic marker for
SCLC in patients with peripheral neuropathy. These antibodies are occasionally detected in
patients who have SCLC without neurologic symptoms but are not found in normal persons.

Subacute Cerebellar Degeneration: Subacute cerebellar degeneration is characterized by


the rapid development of severe pancerebellar dysfunction (gait difficulty and limb ataxia) due
to an extensive loss of Purkinje neurons with relative preservation of other cerebellar neurons.

Limbic Encephalopathy: Limbic encephalopathy is characterized by profound loss of


short-term memory, seizures, and behavior changes, including dementia, which often
antedate the diagnosis of cancer. It is associated with inflammatory infiltrates in the
hippocampal and medial temporal lobe regions of the brain and may be reversible with
successful treatment of the cancer. 50% of patients have Hu-Ab (type 1 antineuronal nuclear
antibody) and a few have CV2-Ab or amphiphysin-Ab.
156 Anant Mohan, Manisha Bhutani, Subhash Budania et al.

Figure 3. Digital clubbing.

Subacute Necrotic Myelopathy: Necrotizing myelopathy is an unusual neurologic


paraneoplastic syndrome characterized by a relatively acute, rapidly ascending paraplegia
that culminates in rapid deterioration and death.

Opsoclonus-Myoclonus (OPM): Opsoclonus is defined by the presence of spontaneous,


arrhythmic and large amplitude conjugate saccades occurring in all directions of gaze,
without saccadic interval. Opsoclonus is usually associated with myoclonus of the limbs and
trunk, and sometimes, with encephalopathy. There is no immunological marker to diagnose
OPM.

Clubbing (Figure 3): Clubbing is far less common with SCLC than with NSCLC (4%
versus 35%).6 Hypotheses have revolved around hypoxemia, hypercapnea, and humoral
mediators such as platelet derived growth factor, tumor necrosis factor, and hepatocyte
growth factor.

Acanthosis Nignicans: Acanthosis nigricans is a skin condition characterized by dark,


thick, velvety skin in body folds and creases. Most often, it affects armpits, groin and neck. It
is most commonly associated with insulin resistance and stomach cancer but is also reported
with lung cancer.

Dermatomyositis: Dermatomyositis (DM) is an idiopathic inflammatory myopathy with


characteristic cutaneous manifestations including heliotrope rash of the periorbital skin,
erythematous scaly plaques on dorsal hands with periungual telangiectasia and photosensitive
poikilodermatous eruption. The myopathy is generally symmetrical and slowly progressive
during a period of weeks to months affecting mainly the proximal muscles and characterized
by specific inflammatory lesions in muscle biopsy. It is caused by vasculitis determined by
humoral factors with subsequent inflammatory cell accumulation, mainly T CD4+ and B
cells, which infiltrate myocytes leading to its vacuolization and degeneration (mainly in the
skeletal muscles, rarely in the smooth muscles). Lung cancer is the most common type of
cancer associated with DM and among lung cancer subtypes; SCLC is most frequently
associated, accounting for approximately 50%.
Small Cell Lung Cancer: Clinical Presentation and Diagnostic Modalities 157

Table 2. Summary of the common paraneoplastic manifestations in SCLC [8].

Other than the above, paraneoplastic syndromes that are less frequently associated with
SCLC include hypercalcemia, deep vein thrombosis, marantic endocarditis, disseminated
intravascular coagulation, and the full syndrome of hypertrophic osteoarthropathy. In a
patient, the presence of one or more of these clinical findings may assist in early suspicion of
the type of lung cancer.

Comparison of Clinical Presentation


of SCLC versus NSCLC (Table 4)
Although there is considerable overlap between the clinical presentation of SCLC and
NSCLC, some features that favour SCLC are the following:

central location of the tumor


Hilar and mediastinal lymphadenopathy
Rapid tumor growth; and
Certain paraneoplastic conditions (SIADH, ectopic adrenocorticotrophic hormone,
Eaton-Lambert syndrome, cortico-cerebellar degeneration, and limbic
encephalopathy).
158 Anant Mohan, Manisha Bhutani, Subhash Budania et al.

Figure 4: The comparative prevalence of common clinical features of SCLC and NSCLC.

Screening of Lung Cancer


Screening means testing for a disease when there are no symptoms or history of that
disease. Screening for lung cancer is looking for lung cancer before a person develops
symptoms to detect it at early stage when the tumor is still small and localized to increase
resectability.

The U.S. Preventive Services Task Force (USPSTF) found fair evidence that screening
with LDCT (low dose computed tomography), CXR (chest radiograph), or sputum cytology
can detect lung cancer at an earlier stage than lung cancer would be detected in an unscreened
population; however, the USPSTF found poor evidence that any screening strategy for lung
cancer decreases mortality [9].
According to National Cancer Institute (NCI) On the basis of fair evidence, screening
using CXR and sputum cytology, does not reduce mortality from lung cancer, screening
would lead to false-positive results and unnecessary invasive diagnostic procedures and
treatments. Low dose Computed tomography is six times more likely than chest x-ray to
diagnose stage I lung cancer, and four times more likely to detect non calcified lesions and
cancerous tumors, it is not cost effective and leads to a high false positivity rate and
unnecessary diagnostic procedures and treatments. Until further conclusive evidence is
available, the above investigations are not currently recommended for screening.
The American College of Chest Physicians (ACCP) recommends that individuals
undergo screening only when it is administered as a component of a well-designed clinical
trial with appropriate human subjects protections [10].
Small Cell Lung Cancer: Clinical Presentation and Diagnostic Modalities 159

Screening Tools

(A) Autofluorescence Bronchoscopy (AFB) [11]


AFB is a bronchoscopic procedure in which a blue light rather than a white light is
employed for illumination, and premalignant and malignant tissue is distinguished by a
change in colour from normal tissue without the need for fluorescence-enhancing drugs. It is
currently being developed as a method for detecting early lung cancers, carcinoma in situ,
and dysplastic lesions of the tracheobronchial tree that are not visible by standard white light
bronchoscopy. Other than primary screening in high risk individuals, it is also indicated in
suspected lung cancer by abnormal sputum cytology findings, inspection for synchronous
tumors, surveillance following cancer resection. The high cost and relative low availability,
however, limits its routing use.

(B) Other Newer Techniques


Several newer screening tools are currently under investigation, such as
immunocytochemical analysis of sputum with monoclonal antibodies, identification of
genetic mutations, abnormal DNA methylation, abnormal patterns of immunostaining, and
other molecular changes. Several other potential targets in sputum, bronchial fluid, and
expired air may have a role in early lung cancer detection and are also being evaluated.

Genetic abnormalities

The development of both SCLC and NSCLC occurs through stimulation of proliferation and
mutagenesis occurring over several years, with exposure to tobacco and other carcinogens
playing an important role. Multiple genetic defects have been detected; some are
characteristic and probably associated with oncogenesis, while others are probably random or
unrelated. A detailed discussion of the molecular genetic changes associated with small cell
lung cancer is beyond the scope of this chapter. The most common characteristics of SCLC
are summarized below [12, 13]:

3 mutations are detected in 75 to 90 percent of SCLC.


Loss of heterozygosity of chromosomes 9p and 10q (the site of the PTEN gene) is
present in the majority of tumors.
Loss of the retinoblastoma gene function at 13q14 is nearly ubiquitous in SCLC.
Telomerase is a ribonucleoprotein enzyme that compensates for telomere shortening
during cell division by synthesizing telomeric DNA, thereby maintaining telomere
length. In normal somatic cells, telomerase activity is usually undetectable.
Activation of telomerase is detected in approximately 90 percent of SCLC.
Deletion of 3p, including 3p21-22, leading to inactivation of three putative tumor-
suppressor genes.
Upregulation of wild type c-kit and its phosphorylated form is present in up to 80 to
90 percent of SCLC.
In contrast to NSCLC, mutations in the K-ras oncogene and p16 abnormalities are rare.
160 Anant Mohan, Manisha Bhutani, Subhash Budania et al.

Staging of SCLC [14]


SCLC is typically staged according to the Veterans Administration Lung Cancer Study
Group (VALCSG) staging system.

1. Limited Disease (LD): Involvement restricted to the ipsilateral hemithorax (one lung,
the mediastinum and regional lymph nodes) that can be encompassed within a safe
radiation treatment plan. This corresponds, in part, to TNM stages I through IIIB.
2. Extensive Disease (ED): Spread of tumor outside the ipsilateral hemithorax.

Approximately 70% of patients have extensive disease at the time of initial presentation.

Diagnosis of Small Cell Carcinoma of Lung


Complete Blood Count and Peripheral Blood Smear

Bone marrow metastasis occurs in 10 to 13% patients with SCLC. A complete blood count
(CBC), therefore, is important in guiding the need for bone marrow examination. The presence
of variable degrees of cytopenias or immature white blood cells or red blood cells in peripheral
blood smear (leukoerythroblastic blood picture) raises a suspicion of bone marrow spread.

Serum Chemistry

Serum electrolytes should be obtained to look for paraneoplastic syndromes. Elevated


serum calcium and alkaline phosphatase raise the suspicion of bone metastasis, and bone scan
should be ordered even in the absence of symptoms. Elevated serum lactate dehydrogenase
(LDH) indicates increased tumor mass and cell turnover. Elevated serum LDH and
hyponatremia are considered adverse prognostic indicators for SCLC. Abnormal liver
function raises the possibility of hepatic metastasis.

Sputum Cytology

Sputum cytology is the least invasive means of obtaining a diagnosis in a patient


suspected of having lung cancer. The diagnostic accuracy of sputum cytology is dependent
on rigorous specimen sampling (early morning samples give highest yield, number of
samples (at least three), preservation techniques, as well as on the location and size of the
tumor (i.e., central versus peripheral). In experienced hands, the average sensitivity and
specificity of sputum cytology may approach 66% and 99%, respectively [15]. The sensitivity
is higher for central lesions (71%) compared to peripheral lesions (49%) [15].
Radiology
Small Cell Lung Cancer: Clinical Presentation and Diagnostic Modalities 161

A Chest radiogram is usually the first imaging investigation advised, followed by a CT


scan. Radiographic features suggestive of malignancy include the absence of a benign pattern of
calcification in the detected lesion, an enlarging nodule or mass, a nodule with a spiculated or
lobulated border, a larger lesion (greater than 3 cm = malignant unless proven otherwise), and a
thick-walled cavitary lesion. However, a plain chest radiograph may miss lesions smaller than 1
cm. Early abnormalities may include a hilar prominence or a small peripheral lung nodule; late
findings may show a large mass, consolidation/collapse of a lobe, pleural effusion, or
diaphragmatic elevation in phrenic nerve palsy. Small cell lung cancer rarely cavitates.
Computed tomography (CT) has now become pivotal in the diagnosis of lung tumors. Its
superior contrast resolution enable precise localization of suspicious masses and superior
lesion characterization. CT is able to delineate the morphological features of peripheral
nodules such as a corona radiata (in which soft tissue spicules radiate into the surrounding
parenchyma) and coarse spiculation, which are associated with a higher risk of malignant
infiltration. In addition, it can well visualize the mediastinal structures including lymph
nodes, as can demonstrate chest wall or rib involvement by the primary tumor. Hence, a
contrast enhanced CT scan is now considered the investigation of choice for staging of lung
cancer. A CT scan must include images of the neck and upper abdomen to detect cervical
lymph nodes as well as metastasis in the adrenals and liver.

MRI

Considering the predilection of SCLC to metastasize to the brain, MRI is advised in


patients with focal neurological signs although it is not recommended routinely. In addition,
it provides useful information regarding spinal cord involvement and in suspected pancoast
tumor and pericardial invasion.

Flexible Bronchoscopy (FB)

Flexible bronchoscopy is an invasive procedure that is utilized to visualize the nasal


passages, pharynx, larynx, vocal cords, and tracheal bronchial tree. FB (employing bronchial
washings, brushings, and biopsies) is diagnostic procedure of choice for suspected central
lesions. Factors that influence the diagnostic yield of FB include the size of the lesion, its
location, and the presence of a bronchus sign on CT. Smaller, more-peripheral lesions,
without a visible bronchus within or leading directly to them, are unlikely to be diagnosed by
FB. The overall sensitivity of flexible bronchoscopy biopsy for central endobronchial lesions
is 88% [16]. For visible central lesions, the sensitivity of direct forceps biopsy, washings and
brushings is74%, 48%, and 59%, respectively [16]. The sensitivity of FB in diagnosing
peripheral lesions depends primarily on the size of the lesion. In patients with lesion size < 2
cm, the sensitivity is only 33%, whereas it is 62% in patients with lesion size > 2 cm [16].
Bronchial aspirates may significantly increase the diagnostic yield in suspected lung cancer.
The overall sensitivity of bronchial washings in central and peripheral lesions approaches
59% and 52%, respectively [16].
162 Anant Mohan, Manisha Bhutani, Subhash Budania et al.

Diagnostic flexible bronchoscopy is generally a safe procedure; complications such as


bleeding, respiratory depression, cardiorespiratory arrest, arrhythmia, and pneumothorax may
occur in <1% of cases.

Trans-Thoracic Needle Aspiration (TTNA) and Transthoracic Needle Biopsy


(TTNB)

TTNA using fluoroscopy or CT guidance is the investigation of choice to sample


peripherally placed pulmonary lesions. The overall sensitivity and specificity of TTNA for
diagnosing peripheral lung cancers is 90% and 97%, respectively. The sensitivity for smaller
(>2 cm) and larger tumors (<2 cm) is statistically similar (95% and 91%, respectively).16
Sensitivity is higher for CT scan guided than for fluoroscopy-guided procedures (92% versus
88%). Adding automated cutting (core needle biopsy) to TTNA may increase the yield.
Relative contraindications to this procedure are the presence of severe pulmonary
hypertension, coagulopathy or a bleeding diathesis, severe COPD, or vascular malformations.
The most frequent complication of TTNA is pneumothorax in 25 to 30% of patients, with 5
to 10% of these patients requiring a chest tube. There can be up to a 10% incidence of
hemoptysis and hemorrhage, which is increased by the use of cutting needles. Air embolus
and tumor seeding are rare, 0.1% and 0.05%, respectively.

Positron Emission Tomography (PET) Scan [17]

PET is a physiologic metabolic imaging approach in which lesions are identified by the
increased uptake in neoplastic tissue of the glucose analog 5-fluorodeoxyglucose (FDG [2-
deoxy-2-fluoro-18F--d-glucopyranose]). PET scanning has proven to be an excellent modality
for evaluating solitary pulmonary nodules. The average sensitivity and specificity of FDG-PET
scanning for detecting a malignancy are 97% and 78%, respectively. However, PET is limited
by its inability to reliably pick lesions that are < 1 cm in size. False-positive PET scan results
may be obtained because of metabolically active but benign conditions involving inflammation
or infection (e.g., tuberculous granulomas, coccidioidomycosis, aspergillosis).
Even though the cumulative evidence suggests that PET added to conventional staging
improves the sensitivity in detecting extracranial disease, the frequency of changes in stage
attributable to PET are still unknown and is plagued by wide confidence intervals (CIs) in the
estimates of diagnostic and staging accuracy. Therefore, outside of a clinical trial, the routine
use of PET in SCLC cannot be recommended at present.

Bone Scan

Bone metastases from small cell lung cancer are predominantly osteoblastic, and a bone
scan is superior to plain radiographs in detecting such lesions. Bone scans should be, therefore,
Small Cell Lung Cancer: Clinical Presentation and Diagnostic Modalities 163

obtained in all patients with small cell lung cancer at diagnosis or during follow-up if new bone
symptoms develop or if serum calcium or alkaline phosphatase levels are elevated.

Endobronchial Ultrasound-Needle Aspiration (EBUS-TBNA)/Biopsy [18]

Endobronchial ultrasound (EBUS) utilizes the passage of ultrasound devices attached to


a fibreoptic bronchoscope inside the airways and the lung for exploration of the structures of
airway walls, the surrounding mediastinum, and the lungs. Histological diagnosis of small
peripheral lesions can be achieved more efficiently by EBUS guidance than with blind needle
aspiration, without the need for radiological equipment or risk of radiation exposure.
EBUS-TBNA has 75% sensitivity and 83% accuracy for peripheral lesion diameter < 3
cm, which is significantly higher than conventional TBNA (30.7 and 53 percent,
respectively) for similar types of lesions. There are no significant differences in
complications between patients who undergo bronchoscopy with or without EBUS guidance.

Medistinoscopy [19]

Mediastinoscopy is a surgical procedure that allows physicians to view areas of the


mediastinum. Standard cervical mediastinoscopy is widely employed for surgical exploration
of superior retrovascular mediastinum. This allows biopsy of paratracheal and subcarinal
lymph nodes as well as direct biopsy of retrovascular mediastinal tumors. At present, it is
regarded as the gold standard for diagnosing lesions in the anterior mediastinum, since EBUS
cannot image this region due to the air-filled trachea. Mediastinoscopy plays a fundamental
role for both diagnosis and staging purposes of SCLC. Video assisted mediastinoscopy
(VAM) has better sensitivity than standard cervical mediastinoscopy, and is proven to be safe
and effective in nodal assessment of the mediastinum

Video-Assisted Thoracoscopy

This is a relatively newer modality in which a video-attached scope is inserted into the
thorax and used to sample small peripheral tumors (less than 2 cm in diameter) and pleural
tumors, drain suspected malignant pleural effusions, and perform pleurodesis when required.

Trans-Esophageal Endoscopic Ultrasound Scanning (EUS)

This is a new minimally-invasive method that provides high resolution imaging of the
mediastinum using high frequency ultrasound probes attached to the tip of a flexible
endoscope and offers the facility of fine needle aspiration (EUS-FNA) or tru-cut biopsy under
real-time ultrasound guidance. EUS-FNA allows access to the posterior mediastinum and
tissue acquisition under real-time ultrasound guidance through the esophageal wall.
164 Anant Mohan, Manisha Bhutani, Subhash Budania et al.

Compared to CT, PET, mediastinoscopy as well as EBUS TBNA, EUS-FNA provides higher
diagnostic accuracy for lesions in the posterior mediastinum.

Immunohistochemistry (IHC)

Immunohistochemistry refers to the process of localizing proteins in cells of a tissue


section exploiting the principle of antibodies binding specifically to antigens in biological
tissues. This technique plays an important role in the diagnosis of SCLC. Nearly all small cell
cancers are positive for the epithelial markers keratin, epithelial membrane antigen, and BER-
EP4. In addition, neuroendocrine markers such as chromogranin A (positive in 84%),
synaptophysin (64%), CD57 (Leu-7), CD45, neurone specific enolase, thyroid transcription
factor 1 (TTF-1) and CD56 are also useful markers. CD56, TTF- 1, and CD45 are useful in
the diagnosis of small cell lung carcinoma in biopsies with extensive crush artifact.

Algorithm for the diagnostic workup of a patient with a central (Box 1) and non-central
(Box 2) lung lesion.

BoxBox
1: Patient with
1. Patient suspected
with central
suspected lesion
central (clinically
lesion (clinicallyand
andradiologically)
radiologically)
Sputum cytology

Positive Negative

FB

Positive Negative

PET scan

Lung cancer Positive Negative

No Lung Cancer
Small Cell Lung Cancer: Clinical Presentation and Diagnostic Modalities 165

Box 2: Patient with suspected non-central lesion (clinically and radiologically)

Conclusion
A variety of invasive/non-invasive techniques are now available to assist the clinician in
achieving a definitive diagnosis of small cell lung cancer. The most appropriate test is usually
determined by the type of lung cancer (SCLC or NSCLC), the size and location of the tumor,
and the local diagnostic facilities available.

References
[1] Cancer Facts and Figures 2008.
[2] Hepper, NGG; Herskovic, T; Witter, DM; et al. Thoracic inlet tumors. Ann Intern Med.,
1966, 64, 979.
[3] Terashima, T; Kanazawa, M. Lung Cancer with Skin Metastasis. Chest, 1994, 106,
1448-1450.
[4] Laviano, A; Meguid, MM; Inui, A; Muscaritoli, M et al. Therapy Insight: cancer
anorexiacachexia syndrome when all you can eat is yourself. Nature Clinical Practice
Oncology, 2005, 2, 158-165.
[5] Honnorat, J; Antoine, JC. Paraneoplastic neurological syndromes. Orphanet Journal of
Rare Diseases, 2007, 2, 22.
166 Anant Mohan, Manisha Bhutani, Subhash Budania et al.

[6] Thirkill, CE; Tait, RC; Tyler, NK; et al. The Cancer-Associated Retinopathy Antigen is
a Recoverin-Like Protein. Investigative Ophthalmology & Visual Science, 1992, 33, 10.
[7] Sridhar, KS; Lobo, CF; Altman, RD. Digital Clubbing and Lung Cancer. Chest, 1998,
114, 1535-1537.
[8] Palka, K; Johnson, DH. Small Cell Lung Cancer. In: Eds Fishman AP, Elias JA,
Fishman JA, et al. Fishmans Pulmonary Diseases and disorders, 4th ed. New York,
McGraw Hill, 2008, 189-1915.
[9] U. S. Preventive Services Task Force. Lung Cancer Screening: Recommendation
Statement. Ann Intern Med., 2004, 140, 738-739.
[10] Bach, PB; Silvestri, GA; Hanger, M; et al. Screening for Lung Cancer: ACCP
Evidence-Based Clinical Practice Guidelines (2nd Edition). Chest, 2007, 132, 69S-77S.
[11] George, PJM. Fluorescence bronchoscopy for the early detection of lung cancer. Thorax,
1999, 54, 180-183.
[12] Wistuba, II; Gazdar, AF; Minna, JD. Molecular genetics of small cell lung carcinoma.
Semin Oncol., 2001, 28:3.
[13] Meyerson, M; Franklin, WA; Kelley, MJ. Molecular classification and molecular
genetics of human lung cancers. Semin Oncol., 2004, 31, 4.
[14] Micke, P; Faldum, A; Metz, T; Beeh, KM; Bittinger, F; Hengstler, JG; Buhl, R. Staging
small cell lung cancer: Veterans Administration Lung Study Group versus International
Association for the Study of Lung Cancerwhat limits limited disease? Lung Cancer,
2002 Sep, 37(3), 271-6.
[15] Rivera, MP; Detterbeck, F; Mehta, AC. Diagnosis of Lung Cancer: The Guidelines.
Chest, 2003, 123, 129-136.
[16] Schreiber, G; McCrory, DC. Performance characteristics of different modalities for
diagnosis of suspected lung cancer: summary of published evidence. Chest, 2003,
123(1 Suppl), 115S-28S.
[17] Rivera, MP; Detterbeck, F; Mehta, AC. Diagnosis of Lung Cancer; the Guidelines.
Chest, 2003, 123, 129S-136S.
[18] Paone, G; Nicastri, E; Lucantoni, G; et al. Endobronchial Ultrasound-Driven Biopsy in
the Diagnosis of Peripheral Lung Lesions. Chest, 2005, 128, 3551-3557.
[19] Ernst, A; Silvestri, G; Johnstone, D. Interventional Pulmonary Procedures: Guidelines
from the American College of Chest Physicians. Chest, 2003, 123, 1693-1717.
In: Small Cell Carcinomas: Causes, Diagnosis and Treatment ISBN: 978-1-60741-787-3
Editors: J. G. Maldonado and M. K. Cervantes 2009 Nova Science Publishers, Inc.

Chapter 7

Biotechnology and Cancer: Uses


of Biotechnology for Prevention,
Diagnosis and Treatment of Cancer

Matias E Valsecchi*
University of Buenos Aires, Argentina

Abstract
Introduction

This review will focus in the current uses of biotechnological tools, including
functional genomics, microarrays, proteomics, pharmacogenomics, gene therapy,
nanotechnology and bioinformatics, for the design and development of revolutionary
diagnostic and therapeutics modalities that will represent the new standards of care in the
near future.

Methods

Using Pubmed, Ovid and EBSCO a MEDLINE database search was perfomed from
January 2002 to June 2008. Keywords used include: cancer, biotechnology, functional
genomics, nanotechnology or nanoparticles, proteomic, gene expression signature and
pharmacogenomics. Multiple combinations were used to enhance the search.

*
Corresponding author: E-mail: meval78@yahoo.com
168 Matias E Valsecchi

Results

There are at least three areas where the advances in biotechnology will most likely
produce innovative changes. The first area is related to the improvement in diagnostic
and prognostic assessment. Gene-expression signature, using microarrays analysis, can
predict relapse-free and overall survival, while functional genomics can be used to
interrogate a vast number of informative cancer-relevant phenotypes. Seldi-Tof and
Maldi-Ms techniques allow the study of proteomic patterns in serum in order to identify
biomarkers that can be used for screening and diagnosis purposes, including early
detection of recurrences. The proper interpretation of these data, generate by high-
throughput modalities, in a reliable and reproducible manner requires the help of a new
discipline known as bioinformatics. However, the most spectacular progress can be
attributed to the domain of the nanotechnology, especially through the production of
multifunctional nanoparticles. Both organic and inorganic nanoparticles not only can
enhance significantly most of the previously mentioned in-vitro techniques, but also
represent the cornerstone for the progress of the other two identifiable areas, tumor
imaging and new therapeutic options. Improvements in radiological techniques, using
supermagnetic metal core nanoparticles or quantum dots, will increase the sensitivity of
the MRI allowing a non-invasive tumor labeling in-vivo; tumor cells or tumor blood
vessels could be tracked through the whole body. Lastly, all efforts are directed towards
the conquest of the precious personalized medicine. In this regard, three approaches are
fundamental: 1) exact prediction of toxicity, which is possible through the
pharmacogenomics studies 2) precise drug delivery (homing), obtainable through
nanoparticles coupled with multiple cancer-specific targeting ligands 3) highly specific
and accurate selection of molecular targets, made possible through the use of monoclonal
antibodies, small inhibitors particles and siRNA.

Conclusions

The exponential progress of the biotechnological techniques and the increasing


investment in new fields such as nanotechnology, make us foresee the rapid advent of a
new kind of therapeutic agents that will make real the dream of personalized medicine.

Introduction
The annual global prevalence of cancer is about 25 millions patients with half of them
living in developed countries. Each year, approximately 11 million people worldwide are
diagnosed with cancer, of whom 1.5 millions live in North America (14%).[1] In the U.S.
cancer is currently responsible for about one in four deaths (25%), with absolute numbers that
exceed the half million deaths annually.[2] In a recent publication, Yabroff, et al.[3] have
estimated, based on data from SEER and Medicare files, that the 5-year net cost of care for
elderly cancer patients will be approximately $21.1 billions. Given the exponential growth of
the elderly population, especially in developed countries, this problem is far from being
resolved. These numbers reflex the enormous burden that the epidemic of cancer represents
for modern society, both in terms of human lives lost as in the expenditure of economical
resources.
Biotechnology and Cancer (Review) 169

It is well-known that cancer is an extremely complex disease that results from the
accumulation of multiples genetic and epigenetic changes in a multi-step process called
tumorigenesis. The initial concept that cancer is a single disease is an old-fashioned idea that
has been erased from the minds of the oncologists. Every day is more evident that even
within a single sub-category of tumors, such as adenocarcinoma of the lung, small differences
can produce mixed results, ranging from complete response to treatment to very aggressive
and resistant tumors. In short, heterogeneity is the rule in cancer.
In order to tackle this complicated problem, the modern science has witnessed a series of
changes in the dominant paradigms. Beginning with the seventies, the first systematic
approach was based on the testing of compounds, most of which came from natural sources,
which served merely to destroy the cells of rapid growth. Thousands of chemicals were tested
and the result was the first generation of effective antineoplastic drugs, most of which still
represent the backbone of the current standard-of-care treatments. Despite of its high
efficiency in killing cancer cells in vitro, the lack of specificity, the high toxicity and the
rapidly developed resistance by the tumor cells put a ceiling on the clinical utility of these
drugs. In the last decade of the last century, the combination of advanced techniques of
molecular biology and a better understanding of the molecular pathophysiology of cancer,
allowed the identification and isolation of the key components of the cell growth machinery
which led to the development of much more selective molecules, now generically called
targeted-agents. During the last couple of years, we have seen the emergence in clinical
practice of this new category of antineoplastic being the two best examples the small
molecules inhibitors [4] (e.g.: imatimib, erlotinib, sunitimib, sorafinib) and monoclonal
antibodies [5] (e.g.: transtuzumab, cetuximab, bevacizumab). A similar phenomenon
occurred with the images modalities, evolving from the simple X-ray to the CAT scan and
MRI up to the CT/PET scans.
All this progress has been translated into better and more diverse treatment options, but
there is still a huge gap between the investment that is made in basic and clinical research and
cancer mortality. The exponential progress of the biotechnological techniques and the
increased investment in fields such us nanotechnology [6] predicts the rapid arrival of a new,
third generation, of agents who promise to realize the dream of truly personalized medicine
[7]
This review will focus in the current uses of biotechnological tools, including functional
genomics, high-throughput screening methods such us microarrays, proteomics,
pharmacogenomics, gene therapy, nanotechnology and bioinformatics for the design and
development of revolutionary diagnostic and therapeutics modalities that will likely represent
the new standard-of-care in a near future (Figure 1).

Study Design
Literature Acquisition

Using Pubmed, Ovid and EBSCO a MEDLINE database search was perfomed from
January 2002 to June 2008. Keywords used include: cancer, biotechnology, functional
170 Matias E Valsecchi

genomics, nanotechnology or nanoparticles, proteomic, gene expression signature and


pharmacogenomics. Multiple combinations were used to enhance the search. Only English-
written literature and scientific articles that specifically address the following 3 topics were
considered for this review: 1) cancer prevention 2) new diagnostic or radiological techniques
3) new therapeutic strategies or improvement of current agents.

Figure 1. Current applications of the biotechnology in cancer. This figure illustrates the uses of every
technique in cancer screening, diagnosis and treatment.

New Horizons: Cutting the Edge


Moving Toward Better Diagnostic Tests and Assessment of Prognostic

It is very clear that the key to the success in the management of cancer is to detect and
treat as early as possible, especially in those tumors with factors of bad prognosis. To this
end, biotechnology has opened the door for multiple approaches.
Micro-chip arrays and functional genomics
DNA microarray was the first innovation of this group of new techniques that showed
clinical applicability. The basic concept behind the arrays is that it is now possible to test the
expression of thousands of RNAm in a particular cell. The ability to differentiate cells
according to the pattern of gene expression is particularly useful for two purposes: 1) detect
Biotechnology and Cancer (Review) 171

gene-expression signatures that predict prognosis 2) identify new genes that could become
therapeutic targets.
The gene-expression signature has potentially enormous benefit in the clinical practice as
a simple test can predict whether a patient has higher chances of recurrence based on the type
of genes over-expressed by the tumor. In that sense, this tool enhances our ability to classify
our patients.[8] Gene-expression signatures have been described for acute myeloid leukemia
[9] , diffuse large-B-cell lymphoma [10] , Burkitts lymphoma [11] lung cancer [12] and
breast cancer.[13] In the case of breast cancer at least two commercial tests approved by
FDA are available: Oncotype DX (Genomic Health, Inc) and MammaPrint (Agendia, Inc).
They are especially useful, in patients with early stages tumors and node-negative, to help
make decisions regarding the use of adjuvant chemotherapy.
In addition to the characterization of global changes in expression levels, it is also
important to establish whether such changes are truly relevant for the tumorigenic process
and feasible to become pharmacological targets. Here is where functional genomic plays a
mayor role.[14] Currently, high-throughput and high-resolution assays can be used to
interrogate a vast number of informative cancer-relevant phenotypes. There are two basic
approaches. Gain-of-function where a cDNA library is introduced into a cell in order to
produce the action of the specific gene (e.g.: genes involved in tamoxifen-resistance [15] or
the over-expression of HER-2/neu) and loss-of-function where siRNAs (short RNA duplexs)
or shRNAs (RNA hairpin) are introduced into the cells to knock-down precise genes (p53 or
PITX1, just to name some examples).

Proteomics
During recent years, many authors have published reports on proteomics patterns in
biological fluids, especially in serum, using innovative techniques such us SELDI-TOF
(surface-enhanced laser desorption and ionization) or MALDI-MS (matrix-assisted laser
desorption ionization mass spectroscopy). The idea is to discover useful serum biomarkers,
such us specific proteins secreted by cancer cells when the tumor has only a few millimeters
or even in the pre-cancerous phase, which can be used to diagnose very early stages through a
simple blood test. Moreover, detection of tumor specific peptides could allow oncologists
greater control of the therapeutic monitoring and detect early recurrence, even many months
before the conventional radiological techniques can visualize the metastasis.[16]
Serum proteomic has been studied in several tumors including prostate, ovarian, breast
and lung cancer. In prostate cancer, Adam et al [17] , Petricoin at al [18] and Qu et al [19]
have reported the isolation of different peptides with 85-100% of sensitivity and 80-100% of
specificity. Despite the fact that more clinical trials are needed to validate these results and
some unresolved questions [20,21] it is expected that future generations will forget of the
old-fashioned PSA for prostate cancer screening. In ovarian cancer, Petricoin et al [22] have
identified a cluster of peptides that can reliable separate patients with cancer from non-cancer
patients even in stage I. Similar results were reported for breast cancer [23] whereas in lung
cancer the proteomic profiles have been used for noninvasive diagnosis of lung nodules
[24,25] , detection of high-risk groups [26] and prediction of response to drugs. [27]
172 Matias E Valsecchi

The help of the bioinformatics and the nanotechnology


The incredible expansion of the biological data that is generated through the
implementation of techniques such us micro-chip arrays and proteomic analysis or even other
high-throughput molecular tools such us serial analysis of gene expression (SAGE) or
expressed sequence tags (EST), in addition to the softwares required by the new radiological
modalities, creates the problem of the analysis and interpretation of these data in a reliable
and reproducible way. In order to meet these needs a new science, known as bioinformatics,
has emerged. Bioinformatics combines several disciplines such us computer science,
mathematics, physics and biology.[28] Moreover, the presence of free online databases with
hundreds of millions of sequence of DNA, RNAm and proteins (available through websites
such us National Center for Genome Resources, Genbank, Unigene, Ensembl, BioInform,
SWISS-PROT, and others) makes extremely important to create software capable of handling
this information. Kim B et al [29] , for example, used a complex bioinformatics analysis with
novel algorithms to identify candidate genes to predict prognosis in lung cancer using the
public gene expression database.
Nanotechnology is a rapidly developing area within the medicine that is changing the
war against cancer and other diseases. In cancer biology, this has led to significant progress
in early detection, tumor imaging and tumor drug delivery. The main concept is that it is now
possible to produce nanoparticles, ranging from 1 to 1000 nm, which can be used to deliver
drugs or other components into selected tissues, such us a tumor metastasis. There are two
main types of nanoparticles and both share the same principle. A central core, which has the
drug itself or metals, is surrounded by a protective organic surface that avoids the degradation
in the physiologically aggressive environment of the blood. The outer layer can be
conjugated with a highly specific antibody, an oligonucleotide or an affinity peptide
providing multiple cancer-specific targeting ligands that significantly enhance the delivery or
homing of the particle. According to its main component nanoparticles can be classified as
organic (liposomes, dendrimers, carbon nanotubes, emulsions and others) or inorganic
(quantum dots, Raman probes and supermagnetic nanoparticles with metal core such us iron,
cobalt or nickel). [30,31]

Improvements in Radiologic Techniques

At present, the most innovative area regarding radiological techniques is the ability to
track cells and provide a non-invasive tumour imaging in-vivo. The supermagnetic
nanoparticles are very promising because they can be easily detected by magnetic resonance
(MRI). In this regard, several supermagnetic MRI contrast agents, based on ferric-oxide,
perflourocarbon or quantum dots cores, have been developed and used in clinical diagnosis.
Human cancer prostate xenografts have been tracked in mice [32] and even in human beings.
[33] Several groups have also showed the possibility of labeling tumour blood vessels and
follow their migration to the neo-vasculature of the metastases. [34,35] Furthermore, labeling
tumour cells with quantum dots allowed Voura et al [36] to track the extravasation of
metastatic tumour cell.
Biotechnology and Cancer (Review) 173

Lastly, it is necessary to warn that extensive pharmacokinetics and safety studies are
needed to be carried out to establish the safety profile of these contrasts in humans since most
of them are composed of heavy metals.

New Treatments Option

As a consequence of the biotechnological advances, in particular the mastery of


techniques such us the production of monoclonal antibodies or the discovering of antisense
small siRNA, that we are currently witnessing the emergence of a whole new class of
antineoplastic drugs, most of them already marketed or in clinical phases of investigation. All
the efforts are directed towards the conquest of the precious personalized medicine. In that
sense, the paradigm has shifted to the production of molecular-targeted drugs with low
toxicity. Different types of approaches could be recognized.

Monoclonal antibodies
This is the most familiar category as several drugs are already being used in clinical
practice. They allow a therapy aimed at a single target or epithope, mainly located at the cell
surface, resulting in the inhibition of the activation of a growth factor or triggering the
destruction of the neoplastic cells through an immune mediated process. [37] There are
several FDA-approved monoclonal antibodies for various types of cancers, including breast
(trastuzumab, bevacizumab) colon (bevacizumab, cetuximab) lung (bevacizumab,
cetuximab), non-Hodgkins lymphomas (rituximab, 90Y-Ibritumomab, 131I-tositumomab)
acute myeloid leukemia (gentuzumab) and CLL (alemtuzumab). [38] There are also a
significant number of on-going phase I, II and III trials that could quickly flood the market
with new drugs.

Nanoparticles therapeutics and gene therapy


As mentioned above, the domain of the nanotechnology with the production of
nanoparticles is the most promising area for the foreseeable future. Targeted nanoparticles
have the potential to provide therapies not achievable with any other form of drugs. The
advantages of these particles, in comparison with the classic drugs are many. By altering the
size and the surface characteristics it is possible to improve and modify the pharmacokinetics
properties that allow them to remain in circulation for prolonged periods of time and
accumulate passively in the tumors via an enhanced permeation and retention effect (EPR).
The latter is possible because of the unique morphology of the tumour vasculature that allows
nanoparticles extravasate easily. This happens even with the non-conjugated particles such as
the simple liposomes or dendrimers but the delivery to organ-specific targets, such us distal
metastases, may be significantly enhanced by coupling them with cancer-specific targeting
ligands. Moreover, nanoparticles are large enough to contain multiple targeting ligands that
can provide multivalent biddings to cell surface receptors. Lastly they can also carry and
accumulate several types of drugs or large number of siRNA. [39]
Some therapies that are now classified as nanoparticles have been around for a long time.
Liposomal chemotherapy has been FDA-approved since mid-1990s. The best example is the
174 Matias E Valsecchi

liposomal anthracyclines: liposomal daunorubicin (DaunoXome; Gilead, Inc), liposomal


doxorubicin (Doxil; Ortho Biotech) and pegylated liposomal doxorubicin (PL-DOX; Alza
Pharmaceuticals). Most recently the FDA approved Abraxane (Abraxis, Inc) an albumin-
based particle with a core containing paclitaxel for stage IV breast cancer. The ability to
generate diverse sort of particles is currently being studied by several compounds such us
nanocrystals (e.g.: Emend; Merck & Co, Inc), polymeric micelles (e.g.: Genexol-Pm;
Samyang) now under phase II [40] , dendrimers of polyamidoamine (pre-clinical phase) and
even inorganic particles with metal core that are able to generate heat after being expose to
near infra-red light (phase I trials completed). [41]

Pharmacogenomics
The polymorphisms of the human genome, including the minimal differences such as the
single-nucleotide polymorphism (SNF), can affect dramatically the response of individual
patients to a single drug. The reason of this phenomenon is these minimal differences can
affect the pharmacokinetics or pharmacodinamics properties of the drugs. If a molecule is not
metabolized properly, its metabolites can accumulate resulting in significant and potentially
lethal toxic effects for the patient.
This is the case, for example, of the Irinotecan and the 5-Fluoro-Uracil, two very
commonly used drugs in clinical oncology. There are individual polymorphisms in
detoxifying enzymes that are associated with the development of severe, grade IV, toxicity.
An haplotype of the enzyme UDP-glucuronosyltransferase 1A1, called (TA) indel or (TA)7,
can predict severe hematological toxicity in patients exposed to this drug. [42] Currently,
there is a commercial test approved by FDA (Invader UGT1A1) that can be used for
screening of the patients before the drug is administered. A similar scenario has been
described for the 5-FU and capecitabine. The enzyme DPD (dihidropyrimidin
Dehydrogenase), which metabolizes the 5-FU and is normally expressed in normal tissues
and tumors, exhibits a genetic polymorphism. If there is a point mutation, the substitution of
guanine by adenine (GT instead of AT) in the sequence of the binding site between intron-
exon, the transcript of the entire exon is prevented. This mutation, called IVS14 +1 G-A was
the most frequently detected in a number of patients with malignant solid tumors and severe
toxicity associated with the use of 5-fluoro-uracil. [43]
Similar phenomena have been described with multiple drugs, including 6-
mercaptopurine, azathioprine, gencitabine, and others.

Conclusion
The exponential progress of the biotechnological techniques and the continued
investment in fields such us nanotechnology has opened the doors into a new era of the
Medicine and oncology is probably the medical sub-specialty that has benefited most.
These new techniques are being used now for a myriad of medical applications such as
more accurate diagnosis, screening, prevention and especially the development of new
therapeutic options with better, more precise and less toxic drugs.
Biotechnology and Cancer (Review) 175

Everything leads us to predict that far from being completed, this is only the beginning of
what appears to be a new paradigm in medicine. Given the tremendous results that we can
already see, it is expected that future investments in this area will grow exponentially in the
future.

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In: Small Cell Carcinomas: Causes, Diagnosis and Treatment ISBN: 978-1-60741-787-3
Editors: J. G. Maldonado and M. K. Cervantes 2009 Nova Science Publishers, Inc.

Chapter 8

Small Cell Carcinoma of the Urinary


Bladder: Morphology, Histogenesis,
Diagnosis, Immunohistochemical
Markers and Therapeutic Strategies;
Case Report and Review
of the Literature

Amelia Petrescu*1, Gabriela Berdan1, Daniel Damian1, Viorel Jinga1,


Valentin Ambert1, Narcisa Manea1, Liviu Niculescu2
and Florin Andrei3
1
Pathology and Urology Departments, Prof. Dr. Th. Burghele Hospital,
Bucharest, Romania
2
Pathology Department, Carol Davila University of Medicine and Pharmacy,
Bucharest, Romania
3
Pathology Department, Victor Babes National Institute for Research and Development
in Pathology and Biomedical Sciences, Bucharest, Romania

Abstract
Small cell carcinoma of the urinary bladder is a rare malignancy, comprising less
than 1% of urinary bladder carcinomas. It is highly aggressive, with a dismal prognosis,
usually presenting with advanced-stage disease. There is a male prevalence and the most
common symptom is hematuria. Morphologically, this tumor resembles its pulmonary
counterpart. There are many theories about the histogenesis of this tumor: urothelial,
neuroendocrine and from stem cells.

*
Corresponding author: Burghele Hospital, Bucharest, 20 Panduri Road, Sect. 5, 050653 Bucharest, Romania,
Phone: +4021-4106910, Fax: +4021-4111055, E-mail: etamy58@yahoo.com
180 Amelia Petrescu, Gabriela Berdan, Daniel Damian et al.

The aim of this chapter is to present our experience in the case of a 44-year-old man
with a history of smoking (10 cigarettes/day), hospitalized for one month with
intermittent hematuria in January 2007 at the Department of Urology, Prof. Dr. Th.
Burghele Hospital, Bucharest, Romania. Ultrasonography and cistoscopy revealed a
sessile mass, sized 37/30 mm. The tumor was removed by transurethral resection and the
fragments were processed by standard histopathological methods: fixed in 10%
formaldehyde, paraffin embedded and stained with HE and VG. We also performed
immunohistochemical tests including Cromo, EMA, NSE, CD56, NK1, P53 and HCG.
Follow up studies of the patient were conducted for 20 months. Echographic, CT,
cistoscopic examination and a new TUR revealed no tumor relapse and no metastases.
Histopathological examination showed a tumor proliferation composed mainly of
sheets of small cells, uniform and rounded, mitotically active and a component of a
typical low-grade urothelial carcinoma. Immunohistochemical markers emphasized that a
diffuse positive staining of the small cell component for Cromo, EMA, NSE, CD56, NK1
and P53 was strongly positive (80%) and the urothelial carcinoma component was
focally positive for HCG.
The microscopical diagnosis was small cell carcinoma of urinary bladder coexisting
with a low-grade papillary urothelial carcinoma, invading the lamina propria (pT1).
The aggressive behaviour of this entity is due to the presence of the small cell
component and possibly to the association with HCG positive immunoreactivity. Our
patient was treated by local instillation with farmarubicine and six series of intravenous
cisplatin. We mention that until now there is no agreement about a standard therapy
management. Some authors recommend only cistectomy, while others, a combined
therapy: cistectomy, chemotherapy and radiotherapy. Based on this case and data from
the literature, we consider that immunohistochemical profile is helpful in diagnosis, and
this type of cancer must be known not only by pathologist, but also by the urologist and
oncologist because of its aggressive behavior and its different therapeutic strategies.

Keywords: Small cell carcinoma, urothelial carcinoma, morphology, immunohistochemistry,


therapy.

Introduction
Prof. Dr. Th. Burghele Hospital in Bucharest, Romania is a small uropathological
medical center with 221 beds. Our annual cases show a prevalence of prostate cancer, the
second place being represented by bladder cancer with urothelial carcinoma accounting for
the overwhelming majority of the cases.
Primary non-urothelial bladder tumors are rare in Europe and North America,
representing less than 5% of all bladder lesions combined [1]. Primary small cell
neuroendocrine carcinoma (SCC) of the urinary bladder is a rare entity, first described in
1981 by S.F. Cramer [2], and accounts for less than 1% of all bladder carcinomas [3]. Since
then, fewer than 200 patients with bladder SCC have been reported in the English language
medical literature. To date only 10 reports have described series larger than 10 patients [5].
Although other authors [3-5] have analysed larger series of patients, we have reported only
one case until now.
Small Cell Carcinoma of the Urinary Bladder 181

The mean age of patients presenting with bladder SCC is 66 years (range 36 to 85) with a
predilection for males (4:1) [4].
Neuroendocrine carcinoma also comprises carcinoid tumors and large cell
neuroendocrine carcinomas.
A large number of risk factors could be implicated in the etiology: bladder calculi, long-
term cystitis and, as shown in one study, a positive smoking history was reported in 65% of
cases [3].
Presenting symptoms of patients with bladder SCCs are non-specific, including
hematuria, abdominal pain, recurrent urinary tract infection, ureteral obstruction with flank
pain, dysuria, obstructive voiding symptoms and weight loss [3, 6]. In some cases, SCC was
found incidentally during surveillance cystoscopy for urothelial carcinoma [7].
Concerning the size and location of the tumor, the authors describe a mean size of 5 cm
(range 1.513 cm), most tumors arising in the lateral and posterior walls and rarely in
diverticulum and urachal remnants [5, 7]. Macroscopically, these tumors may appear nodular,
polypoid, sessile, ulcerated and/or infiltrative.
From a morphological point of view, some authors consider that the majority of bladder
SCCs coexist with other types of carcinoma including urothelial, adenocarcinoma and
squamous cell carcinoma [3, 6, 7], while others identified a prevalence of pure SCCs
(61.4%), mixed SCC-urothelial carcinoma (29.5 %) and mixed SCC-adenocarcinoma (2.3%)
[5].
Regarding histogenesis, there are three main theories: urothelial origin by metaplastic
changes, malignant transformation of bladder neuroendocrine cells and stem cell theory [8].
Because of the later stages at the time of diagnosis and aggressive behavior, SCCs have a
dismal prognosis.
Although some cases of SCCs were associated with paraneoplastic syndromes, a positive
diagnosis is based on the histological pattern: sheets of uniformly small, round, mitotic active
cells with overlapping nuclei, lacking prominent nucleoli, nuclear molding and tumor
necrosis [6].
Management of this entity is not standardized and therapeutic methods vary, including
neoadjuvant and chemotherapy followed by cystectomy, integrated chemotherapy and local
radiotherapy [6].
We report a new case of SCC of the urinary bladder associated with low-grade urothelial
carcinoma and discuss relevant current literature.
Our patient was a 44-year-old man with a history of smoking, without other
comorbidities, hospitalized for one month with intermittent gross hematuria.
Ultrasonography and cystoscopic examination revealed a sessile tumoral mass.
Transurethral resection of the tumor mass was effected and tissue fragments were sent to the
pathologic department to establish the histologic type, the degree of differentiation and
invasion according to the 2004 TNM system and WHO stage grouping. Others clinical
investigations (CT, pulmonary X-ray, RMN) established that our case was free of metastasis.
After a four-month follow up, cystoscopy was done at the same location for the biopsy, and
the pathologic examination showed cystitis features.
182 Amelia Petrescu, Gabriela Berdan, Daniel Damian et al.

Other authors in a retrospective study of six CT (computed tomography) cases with SCC
revealed these tumors as large, broad-based polypoid intramural masses extending to the
perivesical area, with staging C and D at the time of diagnosis [37].

Material and Methods


Macroscopically there was a sessile tumoral mass, sized 37/30mm, located on the antero-
lateral right wall of the urinary bladder.
Histopathological and immunohistochemical procedures were as follows: Many tissue
samples were obtained during transurethral electro resection. The samples were fixed in 10%
formaldehyde, paraffine embedded, sectioned and standard HE and VG stained, and then
examined by light microscopy (Nikon Eclipse E 600) using the 2004 TNM system and WHO
stage grouping for bladder tumors. Representative photomicrographs were taking using
Nikon Plan 20x and 40x.
Immunohistochemistry was performed on 3 m thick sections from 10% formalin fixed
paraffin-embedded specimens, according to the Avidin-Biotin Complex method of the tissue
[9], modified by Bussolatti and Gugliotta [10], Miller K. [11] and Ardeleanu Carmen [12].
Briefly, the procedure entailed deparaffinization in xylen and alcohol series rehydration,
washing in phosphate saline buffer (PBS), incubation with normal serum for 20 minutes
incubation with primary antibody overnight, standard labeling with a streptavidin-antibody
biotin (LSAB) kit (DAKO), washing in carbonate buffer and development in 3,3DAB
hydrochloride/H2O2.
Selected tumoral fragments were tested by the following antibodies: CROMO 1:50
(Novokastra, UK); EMA 1:75 (Dako, Denmark); NSE 1:100 (Dako, Denmark); CD56 1:50
(Neomarkers, USA); NK1 1:50 (Dako, Denmark); p53 1:50 (Dako, Denmark); beta HCG
1:500 (Novokastra, UK ).
All specimens were counterstained with Mayers hematoxylin, examined and
photographed on a Nikon Eclipse 600 microscope.

Results
The histopathological features showed a tumor proliferation composed of two distinct
components: extensive small cells areas (Figure 1) and foci of typical low-grade papillary
urothelial carcinoma.
Small Cell Carcinoma of the Urinary Bladder 183

Figure 1. Small cell carcinoma component (Haematoxylin-Eosine, ob. x 10).

Figure 2. Transitional low grade papillary carcinoma component (Haematoxylin-Eosine, ob. x 10).

Figure 3. NSE diffuse positive staining of the small cell component (ob. x 10).

The small cells were uniformly round, with increased nucleo-cytoplasmic ratio,
eosinophyl cytoplasm, hyperchromatic nuclei, finely granular chromatin and inconspicuous
nucleoli. The cytoplasm was scanty with molding of nuclei.
Foci of typical low-grade (G2) papillary urothelial carcinoma (Figure 2) were composed
of multiple layer of cells that had acidophilic cytoplasm and elongated nuclei with finely
granular chromatin.
184 Amelia Petrescu, Gabriela Berdan, Daniel Damian et al.

The tumor appeared to have invaded the lamina propria of the urinary bladder. The stage
was pT1N0M0 according to the 2004 TNM system and stage according to WHO stage
grouping.
Immunohistochemical profile emphasized the following:

NSE diffuse positive staining of the small cell component (Figure 3)


EMA diffuse positive reaction in small cell areas (Figure 4)
CROMO low positive reaction in rare tumor cells (Figure 5)
CD 56 positive reaction in tumor cells
NK1 positive reaction in tumor cells (Figure 6)

Figure 4. EMA diffuse positive reaction in small cell areas (ob. x 40).

Figure 5. CROMO low positive reaction in rare tumor cells (ob. x 40).
Small Cell Carcinoma of the Urinary Bladder 185

Figure 6. NK1 positive reaction in tumor cells (ob. x 40)

The urothelial carcinoma component stained focally for beta-HCG in these areas for
neuroendocrine tumoral markers were negative (Figure 7).
The rate of cell proliferation was increased: P53-80% positive reaction in tumor cells
(Figure 8).

Figure 7. Urothelial carcinoma component stained focally for beta-HCG (ob. x 40).

Figure 8. P53 80% positive reaction in tumor cells (ob. x 40).


186 Amelia Petrescu, Gabriela Berdan, Daniel Damian et al.

Comments
Among the many sites for primary small cell carcinoma is the genitourinary tract.
According to literature data, the majority of cases have been observed in the bladder and
prostate [8]. It is now belived that the small cell carcinoma of the bladder originates from the
toti potent stem cells present in the submucosa of the bladder wall [8].
Atkin et al. [13] found in their chromosome studies a hypertriploid mainline and a
hypertriploid DNA index in the dysplatic surface epithelium. The study of Terracino [14]
showed a high number of genomic alterations:deletions at 10q, 4q, 5q, 13q ; gains of DNA
sequences at 8q, 5p, 6p, 20q; high level amplifications at 1p 22-32, 3q26,8q24,12q14-21. The
analyse of one tumor having SCC and transitional cell carcinoma (TCC) suggested that SCC
can develop from TCC through the aquisition of additional genetic alterations.
C. Leonard et al. [15] found that monosomy 9 is an early event, homozygous detection of
P16 is frequent, chromosome 17 is implicated in high grades/stages of SCC, and the profile
of P53 mutations is different from that of TCC: allelic losses of 3p, 8p, 9p, 9q, 17 are
frequent. Tumor suppressor genes play a prominent role in the modification and progression
of urinary bladder carcinogenesis.
Ekkerhard et al. [16] found an important role in cell differentiation of caveolin-1 gene
hypermethylation and of abnormal protein expression. These two modifications could also
explain the phenotypical conversion of TCC into nonurothelial carcinomas.
The expression of thyroid transcription factor 1(TTF-1) is common in lung SCC. Jones et
al. [17] did not find any correlation between TTF-1 expression in bldder SCC and survival
and clinicopathological characteristics.
Among the most useful markers in diagnosing SCC of the bladder are Chromogranine A,
Synaptophysine, gama-Enolase and CD 44 variant 6.
Iczkowski [18] showed that Chromogranine A and CD 44 variant 6 may discriminate
between SCC and TCC.
Epidermal growth factor receptor (EGFR) gene, one of the oncogenes on chromosome 7,
has been implicated by several authors in the pathogenesis and progression of many
malignancies. Immunohistochemical staining for EGFR in the study of Xiaoyan et al. [19]
and in situ hybridisation (FISH) analyse showed that EGFR gene amplification does not seem
to induce protein overexpression. The EGFR expression in 27% cases of this study suggested
the possibility that anti- EGFR therapy may be beneficial for a subset of patients.
Soriano et al. [4] in their study found that, in addition to expression of neuroendocrine
markers, immunostaining for P53 and c-erbB2 was positive in 80%, respectively, in 50% of
cases. They confirmed the aggresive behaviour of SCC in the bladder and they concluded that
c-erbB2 positivity opens up new possibilities for the use of immunotherapy in such cases.
Gaisa NT et al. [26] identified identical point mutations of TP 53 in invasive small cell
carcinoma and TCC in situ and they provided for the first time molecular evidence for the
development of invasive SCC out of TCC in situ.
There are no specific clinical features that differentiate these patients from transitional
carcinoma of the bladder. The most common symptomatology is hematuria [30].
Small Cell Carcinoma of the Urinary Bladder 187

Cancer registration statistics of economically advanced countries indicate that bladder


carcinoma incidence ranks fourth in men and eight in women, but a reliable tumor marker for
predicting the disease course is still lacking [30].
The tumor histology is fairly characteristic on low power light microscopic examination
with a diffuse, patternless arrangement of round, blue hypercromatic cells interspersed with
areas of focal necrosis. The cells have granular chromatin, inconspicuous nucleoli and
frequent mitoses.
The tumors are widely invasive and involve muscle [30].
Although many immunohistochemical antibodies have been reported in small cell
carcinoma of the bladder, from a diagnostic perspective, chromogranin, NSE, synaptophysin
and cytokeratin (often dot-like positivity) are usually sufficient [30].
We report the case of a primary small cell carcinoma of the urinary bladder coexisting
with low-grade papillary urothelial carcinoma component.
The differential diagnosis is vast and included a high-grade urothelial carcinoma (some
of them have smaller cells and a diffuse growth pattern but IHC tests help us to obtain the
right diagnosis), lymphoma, metastatic lesions, especially from the lung, carcinoid,
limfoepitelial like carcinoma, plasmocitoid carcinoma [29, 30].
Lymphomas of the bladder are rare, the cells are small but usually preserve the architecture
of the bladder and are widely invasive [29]. The nuclear features are distinct from SCC.
It is crucial importance to recognize it because the bladder lymphomas treatment is
different, survival is prolonged and have a good prognosis with a good response to
chemotherapy. Immunohistochemical stains allow us to distinguish these possibilities.
The most frequent sites of metastases are:lymph nodes ( pelvic and extrapelvic), liver,
bone, lung, brain. J. Capdevilla et al. [28] reported one case of TCC and SCC presenting
adrenal metastases with the neuroendocrine component only.
The features of metastatic small cell carcinoma of lung origin are indistinguishable from
primary small cell carcinoma of the bladder on the basis of histology alone [30]. However,
the presence of a low-grade urothelial carcinoma in our case was suggestive evidence of a
primary bladder lesion and the X-ray of the lung didnt describe any lesion.
A more problematic situation is ruling out a direct extension of a small cell carcinoma
from the prostate gland or uterus, but prostate specific antigen or prostate specific acid
phosphatase staining in the better differentiated areas are helpful [6].
The true incidence of this entity in the urinary bladder may be underreported if the
diagnosis is not considered and the appropriate diagnostic methods are not carried out.
Although bladder neuroendocrine carcinoma is an aggressive tumor, the prognosis is better
than for those patients with neuroendocrine carcinoma of other sites [29].
Because the optimal management of this tumor is not very well defined, after discussions
between the oncologist, pathologist and surgeon the patient was proposed for radical
cystectomy. For initial case series of bladder SCC the best available therapy was a
combination of surgical resection and adjuvant chemotherapy. Later reports suggested that
integrated chemotherapy and radiotherapy for bladder SCC limited to the pelvis offered
improved long term survival rates [5, 20]. Neoadjuvant chemotherapy followed by
cystectomy has shown improved survival [5, 21].
188 Amelia Petrescu, Gabriela Berdan, Daniel Damian et al.

Small cell carcinoma of the urinary bladder therapy is different from that of small cell
carcinoma of the lung [31]. L. Cheng et al. (2004) showed no significant survival rate
between patients who did and did not undergo cystectomy [32]. Holmang et al. (1995)
revealed that primary small cell carcinoma of the bladder is less aggressive than pulmonary
small cell carcinoma and that some patients can be cured by transurethral resection, or partial
or radical cystectomy combined with radiotherapy [33]. Some studies showed that cisplatin-
based chemotherapy appears to have a better survival and prognosis [34]. The Mayo Clinic
experience (Nicholas W.W. Choong et al.) revealed that treatment is stage-dependent. They
showed that patients with bladder SCC should undergo radical cystectomy except when
metastatic disease is present (M1), in which case systemic chemotherapy is indicated.
Adjuvant treatment is not indicated for patients with Stage II disease after radical cystectomy
but should be considered for patients with Stage III and IV disease. Chemotherapy should be
a platinum-based regimen [5]. First-line chemotherapeutic agents were cisplatin, etoposide
and cyclophosphamide. Adjuvant radiotherapy after radical cystectomy has no role in stage
III bladder SCC because the majority of failures are extrapelvic. Adjuvant chemotherapy may
have a role in decreasing the chances of metastases [5]. Complete resolution of metastatic
disease has been documented with the use of chemotherapy alone [22] or chemotherapy with
external-beam radiation [23]. The reported median radiation dose was 6000 cGy [24].
Despite improved outcomes in treating small cell carcinoma, the prognosis remains
dismal. According to the WHO, poor survival predictors are age greater than 65, high TNM
stage and metastatic disease. This cancer is often detected in an advanced stage. Some
authors in a retrospective study of six CT (computed tomography) cases with SCC revealed
these tumors as large, broad-based polypoid intramural masses extending to the perivesical
area, with staging C and D at the time of diagnosis [37].
Survival rates are stage-dependent but in general are low. Others suggest that there is no
correlation between survival rate and stage. Median survival for all patients is 20 to 23
months and five-year disease-specific survival rates vary from 16% to 40% [7]. Urothelial
carcinomas may produce hormones, the most common of which is beta-human chorionic
gonadotropin (beta-HCG). We found focally positive reaction for beta HCG in the urothelial
carcinoma component. Hormone immunoreactivity was frequently observed in highly
proliferating areas [35]. We found similar results in the study of G. Moutzouris et al., who
revealed a significant correlation between beta-HCG immunoreactivity and tumor category;
patients with invasive tumors and those with metastases have the strongest positive reaction
[36]. Normal urothelium, urothelial papillomas and carcinoma in situ showed no positive
reactions [35]. Recent structural and clinical studies suggest that this hormone might act as a
local tumor growth factor.

Conclusion
Small cell carcinoma of the urinary bladder is a distinct histological and biologic disease
entity with an aggressive clinical course, poor prognosis and average life expectancy of only
a few months. It should be viewed as a systemic disease because most patients present with
metastatic disease.
Small Cell Carcinoma of the Urinary Bladder 189

Light microscopic diagnosis of SCCUB can be challenging. The diagnosis is supported


by positive immunostaining for chromogranin, NSE and NK1 (CD57) and has prognostic and
therapeutic implications.
Therapeutic modalities vary from institution to institution and include transurethral
resection as well as combinations of cystectomy, radiation therapy and systemic
chemotherapy. Owing to the rarity of this malignancy, no prospective study has been
performed that establishes the efficacy and duration of chemotherapy or the relative efficacy
of platinum-etoposide versus other chemotherapeutic regimens.
Because of aggressive behavior and distinct treatment, the pathologist should watch out
for the diagnosis of primary pure small cell carcinoma of the urinary bladder or the presence
of a small cell carcinoma component in cases associated with other types of cancer.

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In: Small Cell Carcinomas: Causes, Diagnosis and Treatment ISBN: 978-1-60741-787-3
Editors: J. G. Maldonado and M. K. Cervantes 2009 Nova Science Publishers, Inc.

Chapter 9

Is the Curative Thoracic Radiotherapy


Benefit and Cost- Effective in Extensive
Stage Small Cell Lung Cancer?

Gulden Diniz
Associate Professor in Pathology, Dr.Behcet Uz Childrens Hospital, Izmir, Turkey
Student of Basic Oncology PhD program, Dokuz Eylul University, Oncology Institute
Voluntary Researcher in Izmir Oncology Center

Abstract
Lung cancer is the most common cause of cancer- related death and small cell lung
cancer (SCLC) comprises about 15- 20 % of all lung cancer cases. Majority of patients
present with metastatic disease at diagnosis. Therapy regimens of SCLC include systemic
chemotherapy, irradiation of primary tumor and/ or metastases, adjuvant and
prophylactic cranial radiotherapy, as well as medication of pain or other symptoms.
Treatment is primarily dependent on the stage of SCLC and presence of metastases.
Chemotherapy still is the cornerstone in treatment. The standard chemotherapy is the
combination of etoposide and cisplatin. Despite initial sensitivity to therapy, >80% of the
patients die from recurrent disease within 2 years.
In limited stage disease, curative radiation therapy is delivered with the intention of
eradication all tumor cells and thereby achieving cure. Minimal tumor doses in the range
of 40- 45 Gy or more by conventional fractionation are necessary to effectively control
tumors in thorax. There is a common perception that radiotherapy for extensive stage
SCLC is essentially palliative. Unfortunately all treatment regimens continue to show
only modest improvement in outcome and their effects remain small for metastatic
disease.
The therapy management of SCLC has been focus of extensive investigation over
the past two decades and several new drugs are currently been developed to improve the
survival in SCLC. It was reported to provide an additional survival benefit in advanced

Correspondence adress: Gulden Diniz, MD, Kibris Sehitleri Cad. 51/11 Alsancak 35220, Izmir, Turkey, Phone;
+90.232.3625547, Fax. +90.232.3625522, E-mail: agdiniz@gmail.com, www.guldendiniz.com
194 G. Diniz

disease and to be less toxic. In the most of these studies, the cost- effectiveness of these
drugs was not calculated. Moreover, in these reports, therapy benefits for some
population such as elderly patients and patients with poor performance status
underrepresented. If it is emphasized that the half of all lung cancers occurs in persons
aged over 60 years, it is easily estimated how difficult to decide therapeutic approach and
to make the right balance between expected benefits of treatment and potential toxicity.
The elderly patients are more likely to be given only supportive care or no therapy.
Therefore intensive, high dose polychemotherapy could not be planned for therapy of
most patients.
Contrary radiation therapy may be safely delivered to elderly patients with poor
performance status. In a recent study, the efficacy of curative and palliative radiotherapy
in the treatment of E- SCLC was evaluated and compared of therapy effect on survival in
with or without metastatic disease. According to the statistical findings; the gains in
duration of median survival with the curative thoracic irradiation are 151.97 days in all
128 patients. The gains in duration of median survival with the curative thoracic
irradiation are 125.75 days in metastatic patients and 190.6 days in others. This result
raises the question of whether treating with radical thoracic radiotherapy with
concomitant chemotherapy, consisting of first-line drugs might be more beneficial and
cost-effective as well as less toxic treatment of extensive stage SCLC.

Keywords: SCLC, curative radiotherapy, palliative radiotherapy, survival benefit.

Introduction
Lung cancer is the most common cause of cancer- related death [1- 3]. Small cell lung
cancer (SCLC) currently comprises about 15- 20 % of all lung cancer cases and majority of
patients present with metastatic disease at diagnosis [1- 4]. Prior to the 1970s, SCLC was
treated in the same manner as non-small cell lung cancer (NSCLC) [ 4, 5]. Despite the
similarities between SCLC and NSCLC, such as association with smoking, sensitivity for
same drugs and usually presence of extensive disease at diagnosis; current therapy
managements are different [1- 5]. Because these tumors have not same biologic behaviors:
SCLC has propensity for early hematojenous spread, has more chemo sensitivity and has
lower median survival without therapy than NSCLC [5, 6]. SCLC leads to death rapidly in 2
to 4 months without treatment [2]. SCLC is staged as limited and extensive disease. Limited
disease is also adopted as a systemic disease. For improving local control and overall
survival; it is treated with surgery, radiotherapy and combined chemotherapy and it is
initially responsive to treatment [2, 5- 8]. Despite initial sensitivity to therapy, >80% of the
patients die from recurrent disease within 2 years [1- 8].
The etiology of SCLC is strongly tied to cigarette smoking and there are many reports
concerning molecular abnormalities involved in the pathogenesis of SCLC (9). Dominant
oncogenes of the Myc family are frequently over expressed in both SCLC and NSCLC, while
the K-Ras oncogene is never mutated in SCLC, but it is in 30% of NSCLCs (9, 10). Similarly
EGFR mutations are never observed in SCLC but it is in 20% of NSCLC (10). The most
frequent genetic abnormalities involve tumor suppressor genes. For example p53 is mutated
in more than 90% of SCLCs and more than 50% of NSCLCs; Rb is inactivated in over 90%
Is the Curative Thoracic Radiotherapy Benefit and Cost- Effective... 195

of SCLCs but only 15% of NSCLCs, p16/ CDKN2A mutations are almost never presence in
SCLCs but are presence in more than 50% of NSCLCs (9, 10). One of the tumor suppressor
gene abnormalities, 3p deletions are also frequently observed in both SCLC and NSCLC
(10).
Therapy regimens of SCLC include systemic chemotherapy, irradiation of primary tumor
and/ or metastases, adjuvant and prophylactic cranial radiotherapy, as well as medication of
pain or other symptoms [1,4]. Treatment is dependent on the stage of SCLC, presence or
absence of metastases, underlying health of the patients, degree of symptoms and the bias of
the treating physicians [5-8]. Chemotherapy still is the cornerstone in treatment of patients
with SCLC. Systemic therapy is essential in the management of SCLC because of its
propensity for early hematogenous spread [1, 3, 11-13]. Cyclophosphamide was the first
cytotoxic drug that demonstrated a survival benefit [1]. Prospective studies comparing the
effect of chemotherapeutic drugs demonstrated that combination with etoposide and a
platinum agent, increased rate of response and decreased toxicity [1-4]. As a result, the
current standard chemotherapy for SCLC is the combination of etoposide and cisplatin for
four to six cycles [1, 12, 18, 19].
Limited stage disease is treated with curative intend with chemotherapy and radiation
therapy. Cure is achieved in approximately 20% of these patients [2]. Curative radiation
therapy is delivered with the intention of eradication all tumor cells and thereby achieving
cure [1, 4]. Minimal tumor doses in the range of 40- 45 Gy or more by conventional
fractionation are necessary to effectively control tumors in thorax [14-18]. There is a
common perception that radiotherapy for E- SCLC is essentially palliative and is regarded as
the treatment for local control. Therefore combined chemotherapy regimen of a platinum-
based drug and etoposide is considered the standard of care for systemic control in these
patients. Unfortunately all treatment regimens continue to show only modest improvement in
outcome and their effects remain small for patients with metastatic SCLC [1-4]. In limited
disease, median survival is about 12-20 months, with no more than 6%-12% of patients
surviving beyond 5 years. In extensive disease, median survival is 7- 12 months, with < 5%
of patients living beyond 2 years and a 5-year survival rate of just 1% [11, 19-23].
Cancer therapy aims to destroy the tumor cells while normal cells are spared. Based on
increasing understanding of the cancer cell biology, targeted therapies have been developed.
This therapy process usually destine to destroy a key protein implicated in tumor cell
proliferation, survival, invasion or drug resistance, thereby producing less toxicity than
conventional therapies [1-4, 19, 20]. The most important handicap of this therapy with these
agents is high price originated from increase of product cost. [1- 4, 19]. Several therapeutic
approaches and new targeted agents have been introduced into clinical trials in SCLC, and a
few phase III studies, including matrix metalloproteinase inhibitors, thalidomide, and
vaccines, have already produced definitive results. Currently, negative results are more
commonly reported than positive ones. Nevertheless, clinical research in this field is still in
progress considering that several new targeted agents, such as antiangiogenic agents and
mammalian target of rapamycin inhibitors, offer a promise of improved outcomes (19).
The therapy management of SCLC has been focus of extensive investigation over the
past two decades and several new drugs such as inhibitor of topoisomerase-1 enzyme are
currently been developed to improve the survival in SCLC (1-4, 14-23). It was reported to
196 G. Diniz

provide an additional survival benefit in advanced disease and to be less toxic strategy for
treatment of SCLC compared with earlier cisplatin- based regimens [1- 4, 20]. In the most of
these studies , the cost- effectiveness of these drugs were not calculated [1-4, 11-23].
Moreover, they having been shown to impact on response and survival compared to classical
etoposide- cisplatin combination in selected patient population. But in these reports, therapy
benefits for some population such as elderly patients and patients with poor performance
status underrepresented. If it is emphasized that the half of all lung cancers occurs in persons
aged over 60 years, it is easily estimated how difficult to decide therapeutic approach and to
make the right balance between expected benefits of treatment and potential toxicity [1-3,
17]. Moreover increasing age seems to be the most important determinant of receiving
chemotherapy or not. The elderly patients are more likely to be given only supportive care or
no therapy. Therefore intensive, high dose polychemotherapy could not be planned for
therapy of most patients. A number of studies demonstrated that radiation therapy may be
safely delivered to elderly patients L-SCLC patients with poor performance status. As well as
the radiation therapy option may overcome many of the performance- and age-related
drawbacks which prevent more aggressive and toxic therapies [11-23].
In a recent study, the efficacy of curative and palliative radiotherapy in the treatment of
E- SCLC was evaluated and compared of therapy effect on survival in with or without
metastatic disease. From January 1998 through December 2004, 128 patients with E- SCLC
were treated with radiotherapy and concomitants combined chemotherapy. Radical
radiotherapy consisting of approximately 60 Gy given in up to 30 fractions was performed in
53 (41.4 %) of these patients. Other 75 patients (58.6 %) were treated with palliative dose
radiotherapy. In all patients, chemotherapy was planned with cisplatin and etoposide for 3- 6
cycles. The median follow up of patients was 287.41 days and median survival was 354.87
days. One and 2-year survival rates were 35.8% and 16.9% respectively in the radical
radiotherapy group, while these rates were 26.6% and 8% in the others. According to the
statistical findings; the gains in duration of median survival with the curative thoracic
irradiation are 151.97 days in all 128 patients. The gains in duration of median survival with
the curative thoracic irradiation are 125.75 days in metastatic patients and 190.6 days in
others. This result raises the question of whether treating with radical thoracic radiotherapy
with concomitant chemotherapy, consisting of first-line drugs might be more beneficial and
cost-effective as well as less toxic treatment of extensive stage SCLC (23). In conclusion, it
was apparently seen that the pace of therapy progress for lung cancer is too slow and it is not
seem promising for near future. Therefore until the most beneficial and cost effective as well
as the least toxic therapeutic agents will be developed, curative tumor irradiation may be
added for the therapy of all patients with E- SCLC (23).

References
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198 G. Diniz

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In: Small Cell Carcinomas: Causes, Diagnosis and Treatment ISBN: 978-1-60741-787-3
Editors: J. G. Maldonado and M. K. Cervantes 2009 Nova Science Publishers, Inc.

Chapter 10

Optimal Treatment of Small


Cell Lung Cancer

Bernard Lebeau
Lung diseases department, St-Antoine hospital,
Pierre et Marie Curie University, Paris, France

Thirty years of clinical research activity in the field of small cell lung cancer (SCLC)
treatment get me to conclude that standard treatment for this disease has not yet been
precisely defined. Some recent editorial assertions are untrue, due to rapid evolution of
knowledge or to simplification risks. We will try here to highlight some ideas whose
application presently can optimise treatments of patients affected by this cancer.

Initial Chemotherapy Choice


Chemotherapy is basic treatment of SCLC, always used, even for the rare surgically
treated patients, according to a neoadjuvant or an adjuvant mode. For all other inoperable
patients, it is an emergency to begin it as soon as possible, the day of histologic diagnosis. It
cannot be the same for all patients like yet often recommended for cisplatin-etoposide as a
standard [1]. This statement has never been demonstrated! Patients with good clinical
prognosis factors must receive a four-drug protocol treatment. A phase III randomised trial
had already proved survival benefit of epidoxorubicin plus cyclophosphamide addition to
cisplatin-etoposide compared to cisplatin-etoposide alone in selected extensive SCLC [2].
This result is not contradictory with our results obtaining no survival difference between
cyclophosphamide-doxorubicin-etoposide (CDE) and the same plus cisplatin (PCDE) for 457
patients with poor prognosis data [3], because the choice of drugs has to be adapted to the
patient status. For extensive SCLC, no progress has recently been identified, as concluded in
a 2008 editorial analysing interest of irinotecan and carboplatin association [4]; we presently
prefer to keep our old CDE, effective and less toxic (in absence of cardiac contra-indication).
200 Bernard Lebeau

At the opposite, for limited forms patients with good prognosis factors research of optimal
dosage for first course is very important. We demonstrated in a 1280 patients multivariate
analysis of pretherapeutic and therapeutic prognosis factors that complete response after the
second treatment session was firstly predictive of longer survival (p<0.0001), more powerful
than performance status or initial extension [5]. Value of early response is so sustained by a
forgotten fifteen-years trial, never reproduced but well proving that single increase of 25%
cisplatin and cyclophophamide dosages at the first course of a PCDE regimen obtained a 2-
year accrual survival from 26% to 43%, comparing to a PCDE without dosage increase at the
first course [6]. Surely, it is more easy to associate cisplatin-etoposide to a concurrent
thoracic radiotherapy limiting irradiation toxicity, but this standard treatment is not optimal
for limited forms patients with good performance status. These ones can receive a dose-dense
chemotherapy supported by G-CSF, without exceeding haematological toxicity limits.

Heparin Addition
Several hypothesis assert for heparin use to improve clinical outcome of SCLC. We
firstly proved efficacy of calciparin in a multicenter randomised trial with 277 patients,
obtaining better complete response rate (37% versus 23%, p=0.004) and better survival [7].
Again, this work confirm importance of beginning treatment quality because heparin was
only administrated during the first five weeks of treatment, covering the two first courses of
chemotherapy. Interest of anticoagulant treatment for SCLC patients was previously touched
for warfarin [8] It has been confirmed with deltaparin during all chemotherapy courses in a
more recent randomised trial [9]. The modalities of anticoagulation have to be better specify
by in-coming trials but in terms of survival gain heparin use is as important as thoracic
radiotherapy for limited forms! Other non-cytotoxic targeted therapies have been recently
tested for SCLC, but no one has presently come to light.

Chemotherapy Adaptations
Response evaluation has to be done quickly. Often the first standard X-ray is sufficient to
demonstrate objective response or to change treatment if no response. For rapid complete
responders, chemotherapy may be limited to six courses [10].For partial responders,
chemotherapy has to be going on as long as progression disease appears because stop
treatment is dangerous in a so progressive cancer. A second-line chemotherapy, even a third-
line chemotherapy [11], can be used for non-responders or relapsing patients. A long time
elapsed after complete response and discontinuation of treatment lead to use again the initial
protocol [12]. For other patients, monotherapy with topotecan gives interesting results but
with a high medullar toxicity [13], so found in our personal experience. In this situation, we
have preliminary tested with some success an oral and low-cost association of lomustine-
etoposide-cyclophosphamide [14] which is now in evaluation comparing it with a
conventional intravenous chemotherapy in a phase III study [15]. Regular treatment of
Optimal Treatment of Small Cell Lung Cancer 201

relapse participate to optimisation treatment of poor prognosis population which can now
achieve a 7% 2-year survival rate for non-selected extensive forms [16].

Thoracic Radiotherapy
Its efficacy has been demonstrated. This fact firstly needed a meta-analysis [17].
Thoracic radiation improves local control and survival in limited diseases. Timing of this
treatment stays unclear. In the meta-analysis, early administration is not clearly better than
late administration but benefit appears in sub-groups analysis restricted to trials using
platinum-based chemotherapies [18]. In an old phase III study, we used a very delayed
thoracic radiotherapy for 53 patients in complete response randomised after eight courses of
chemotherapy; this late treatment seems no adapted looking median survivals of 316 days in
the radiotherapy group and of 496 days in the control group [19]. Optimal dosage is still to be
established; 55 Gy seem the minimum and twice-daily concurrent radiotherapy is probably an
effective mode of treatment [20], but difficult to organize in many countries and cause of a
high grades 3 and 4 oesophagitis rate. Better tolerance of an alternating schedule of
irradiation versus a concurrent one appears in a multicenter clinical trial of 164 limited forms
of SCLC patients [21]; this tolerance is so dependent on associated chemotherapy. Once
again, chemotherapy is essential and primal, but time between the first day of chemotherapy
and the last day of chest radiation is the most important predictor of survival in limited
disease SCLC [22]. For extensive forms, interest of thoracic radiotherapy stays to confirm
even if one study furnished significantly better survival rates with the combined modality for
the most favourable subset of selected patients [23].

Cephalic Radiation
It has two indications. One is curative when cerebral metastasis are present at the initial
statement, what is observed at CT-scan in 13% of 724 patients [24] and more often when
magnetic resonance imaging is used; same treatment is done when there is intra-cephalic
disease progression. Irradiation is always pan-cephalic with a 40 Gy in four weeks dosage.
The second indication is prophylactic (PCI) and is classically restricted to limited form
patients in complete remission due to results of its meta-analysis studies showing a 5.4%
increase of survival rate at three years; PCI decreased the cumulative incidence of brain
metastasis from 58.6% in the control group to 33.3% in the treated group with a pooled
relative risk of 0.46 (95% confidence interval 0.38-0.57; p<0.001) [25]. This survival benefit
is identical to those obtained by thoracic radiation and by heparin use. So these three
treatments have to be applied. PCI is sometimes feared due to potential long-term
neurotoxicity; optimal treatment has to control this risk to avoid these severe alter-effects by
a decision analysis [26], whose application is not easy. Effectively, PCI dosage is always
debated: if meta-analysis concluded that larger doses of radiation led to greater decrease in
the risk of brain metastasis [25], a more recent randomised trial of 720 patients showed that
patients in the high-dose group, 36 Gy, had no significant gain in brain metastasis incidence,
202 Bernard Lebeau

24% versus 30%, and a worse overall survival at two years, 37% versus 42%, compared with
patients in the standard-dose group, 25 Gy in 10 fractions every 12 days [27]. This standard
may be optimal and its timing is surely to be early combined with thoracic radiation in
objective responders limited form patients, even if not yet in complete response. By
extension, PCI indication seems to be extended in extensive form patients with complete
response.

What May Be Expected from an Optimal


Treatment?
Data analysis from all 239 patients treated for SCLC in our center from 1990 to 2002
were retrospectively reviewed [16]. Many authors have demonstrated that to have good
results you have to select good patients, playing with inclusion criteria. So selecting in this
personal population the 27 patients with limited forms of SCLC optimally treated by
heparin + PCDE chemotherapy + thoracic radiation +PCI, we obtained a 2-year actual
survival of 44.2% (IC95%=24.7-62.1). This data is not a scientific proof of our believes but
the above references argued this optimal association. This very optimistic result has not to hid
the remaining bad prognosis of SCLC on the whole. We hope and need new medical
treatments, perhaps vaccine in this disease suddenly explosive after a lot of years of tobacco
consumption. This would be due to a mutation factor of viral origin because we often
observed epidemic-like distribution looking at randomisation site variations during our years
of group coordination. This dream for future does not wipe out the present situation: majority
of SCLC patients will rapidly die from their cancer. Like for all other lung cancers, best
treatment for SCLC is preventive: DONT SMOKE or STOP TOBACCO (and hashish). This
tobacco withdrawal is so important that it stays an important prognosis factor even when the
disease is on-going [28]. It has to be included in the optimal treatment of this disease.
Physicians have not to look for easiness; they must search solutions for difficulties to win the
impossible.

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204 Bernard Lebeau

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In: Small Cell Carcinomas: Causes, Diagnosis and Treatment ISBN: 978-1-60741-787-3
Editors: J. G. Maldonado and M. K. Cervantes 2009 Nova Science Publishers, Inc.

Chapter 11

Primary and Pure Neuroendocrine


Carcinoma of the Bladder:
Anatomoclinical Report Cases, Review
of the Literature and Discussion
of the Therapeutic Strategy

S. Ketata*1, H. FakhFakh2, H. Ketata2, A. Bahloul2 and M. N. Mhiri2


1
Department of Urology, CH Sidi Bouzid, Sidi Bouzid, Tunisia
2
Department of Urology, CHU Habib Bourguiba, Sfax, Tunisia

Abstract
Objective

Neuroendocrine carcinoma arising in the bladder has been described in many case
series. However, primary and pure small cell carcinomas (PSCC) of the bladder are very
rare, and patients commonly present with metastatic disease. No prospective studies
evaluating the most efficient treatment have been published. We reviewed our experience
with treating these tumors to evaluate their histopathological characteristics and clinical
outcome.

Patients and Methods

This chapter presents our experience in 5 patients with PSCC of the bladder during a
7-year period. The patients tumor characteristics, therapy, follow-up and survival status
were documented.

*
Corresponding Author: Service dUrologie, Centre Hospitalier de Sidi Bouzid, 9100 Sidi Bouzid, Tunisia, Email:
sabeurketata@yahoo.fr.
206 S. Ketata, H. FakhFakh, H. Ketata et al.

Results

All patients were male with a mean age of 67 years. The main clinical presentation
was macroscopic hematuria. All tumors were invasive at the time of diagnosis. Systemic
chemotherapy was given in 4 patients, and one patient was treated by radical
cystectomy. The overall median survival was 17 months.

Conclusion

PSCC of the bladder should be considered a systemic disease, because most patients
present with metastases. Prospective studies are needed to determine the optimal
treatment.

Keywords: Bladder, chemotherapy, chromogranin, small cell carcinoma, synaptophysin.

Introduction
Extrapulmonary small-cell carcinoma is a highly malignant tumor. It occurs at different
sites, such as the gastrointestinal tract, thymus, larynx, salivary gland, skin, breast, prostate
and cervix [1]. In contrast to its high frequent occurrence in the lung, bladder PSCC accounts
for only 0.48 to 1% of all bladder malignancies. The immunohistochemical characterization
of this tumour is now well described [2]. PSCC of the bladder has an aggressive behaviour
pattern, similar to PSCC arising elsewhere in the body, with up to 90% of patients developing
metastatic disease [1].
The best treatment strategy for this rare tumour remains unknown. However, surgery,
chemotherapy and radiotherapy have all been used, either alone or in combination.
Based on our experience in 5 patients with PSCC of the bladder and a review of the
literature, we outline some common clinical characteristics and the management of this
disease.

Patients and Methods


From 2001 to 2007, 5 patients with a histopathologic diagnosis of PSCC of the bladder
made by transurethral resection of the bladder tumor (TURBT) were included in a
prospective chapter. The incidence in our hospital was 1% (5 cases among 496 primary
bladder tumors treated). In all patients, the lesion had the characteristic histological features
of small cell carcinoma (SCC) such as a high nucleus to cytoplasm ratio with no appreciable
cytoplasm, and coarse chromatin with absence of prominent nucleoli and nuclear molding.
Immunohistochemistry (neuron-specific enolase, chromogranin and synaptophysin) was
performed.
The patient records for all cases were retrieved, including patient age at diagnosis,
gender, smoking history, presenting symptoms, histology and staging details, results of initial
Small Cell Carcinoma of the Bladder 207

investigations, initial and subsequent management, disease status at last follow-up, date of
death and whether death was disease-specific.
PSCC from other organs was excluded using computed tomography of chest, abdomen
and pelvis and colonoscopy.

Results
Case 1

A 58-year-old man presented with irritative bladder symptoms and recurrent macroscopic
hematuria for 4 months. He had been operated for bladder stone at the age of 32. Bimanual
rectal examination under anaesthesia revealed an immobile bladder bladder. Cystoscopy
revealed a large tumor involving the trigone. Transurethral resection of the bladder tumor
(TURBT) was performed. Histopathological examination showed a PSCC of the bladder.
Computed tomography (CT) of chest, abdomen and pelvis revealed a bladder tumor with
pelvic lymphadenopathy and multiple bilateral coin-shaped pulmonary metastases. Bone scan
showed no skeletal metastases. Primary PSCC in other organs was excluded. The patient
underwent six cycles of etoposide and cisplatin chemotherapy, and he was alive with disease
at 18 months after diagnosis.

Figure 1. Magnification of transurethral resection specimen with diffuse immunohistochemical staining


for (left) synaptophysin and (right) corresponding hematoxylin-eosin stain from patient 2. Original
magnification x 400.

Case 2

A 72-year-old man presented with gross hematuria. Cystoscopy revealed a tumor


involving the left bladder wall. Complete TURBT was performed. Bimanual rectal
examination under anaesthesia revealed no palpable mass and a freely mobile bladder.
Histopathologic examination revealed myoinvasive PSCC. Immunohistochemistry showed
positive staining for synaptophysin and chromogranin A (Figure 1). CT of showed pelvic
208 S. Ketata, H. FakhFakh, H. Ketata et al.

lymphadenopathy and metastatic lesions in the adrenal glands, but CT of the chest and bone
scan showed no evidence of pulmonary or skeletal metastases. The patient received six cycles
of etoposide and cisplatin chemotherapy. Repeated bladder biopsies and imaging of the chest,
abdomen and pelvis showed no evidence of disease 24 months after diagnosis.

Case 3

A 75-year-old man presented with recurrent macroscopic hematuria. Cystoscopy revealed


a large mass on the right bladder wall, which was not palpable or immobile by rectal
examination under anaesthesia. Complete TURBT was performed. Histopathologic
examination showed myoinvasive PSCC with positive immunohistochemical staining for
synaptophysin and chromogranin, and with involvement of the prostatic urethra (Fig. 2).
Chest, abdominal and pelvic CT revealed no evidence of metastases. PSCC from other organs
was excluded. The patient underwent radical cystoprostatectomy, urethrectomy and Bricker-
type urinary diversion. Nine months post operatively, diagnosis of bone marrow metastasis
with fluid-fluid levels from SCC of the urinary bladder was made with magnetic resonance
imaging. The patient underwent six cycles of etoposide and cisplatin chemotherapy, and was
alive with disease at 28 months after the initial diagnosis.

Figure 2. Magnification of transurethral resection specimen with immunohistochemical staining for


(left) chromogranin and (right) corresponding hematoxylin-eosin stain from patient 3. Original
magnification x 400.

Case 4

A 60-year-old man presented with macroscopic hematuria. Physical examination and


blood biochemistry analyses were normal. Ultrasound revealed a bladder tumor with irregular
thickening of the left wall. Cystoscopy confirmed this and TURBT was performed.
Histopathology demonstrated a PSCC of the bladder. CT of the chest, abdomen and pelvis
showed no evidence of metastasis, but bone scan showed multiple skeletal metastases. PSCC
from other organs was excluded. The patient underwent six cycles of etoposide and cisplatin
chemotherapy with partial response, but died of progressive disease 10 months after the
initial diagnosis.
Small Cell Carcinoma of the Bladder 209

Case 5

A 69-year-old man presented with painless gross hematuria. The patients medical
history was significant for cigarette smoking and urinary tract infection. Ultrasound and CT
revealed an irregular soft-tissue mass in the dome with irregular thickening of the bladder
wall, and pelvic lymphadenopathy (Figure 3). Cystoscopy confirmed the above findings and
TURBT revealed an invasive PSCC of the bladder. The bone scan showed multiple skeletal
metastases. PSCC from other organs was excluded. The patient underwent 2 cycles of
etoposide and cisplatin chemotherapy, but developed an infection that precluded further
chemotherapy. The patient died 6 months after diagnosis.

Figure 3. Contrast-enhanced computed tomography scan revealing the bladder dome tumor, with
irregular wall contours stain from patient 5.

Table. Summary of patient details.

Patient Sex Age Treatment Site of relapse Outcome


(years)
1 male 58 Chemotherapy Lung metastases Alive with
at presentation disease at 18 months
2 male 72 Chemotherapy Adrenal gland No clinical evidence
metastases at of disease at 24
presentation months
3 male 75 Cystoprostatectomy Bone metastases Alive with disease at
Chemotherapy at 9 months 28 months
4 male 60 Chemotherapy Bone metastases Cancer death at 10
at presentation months
5 male 69 Chemotherapy Bone metastases Cancer death at 6
at presentation months
210 S. Ketata, H. FakhFakh, H. Ketata et al.

Discussion
Primary small-cell carcinoma occurs at several sites along the urinary tract, but the
bladder is the most frequently reported [1]. PSCC of the bladder is a rare tumor, typically of
older men who present with gross hematuria. Patients suffering from this cancer are usually at
an advanced stage by the time of diagnosis. Bladder PSCC metastasizes to local or distant
lymph nodes, liver, bone, lung, brain, adrenal gland, spleen and abdominal cavity, and
paraneoplastic syndromes are uncommon [3]. The sites of metastatic disease reported in our
chapter included lymph nodes, bone, lung, adrenal gland.
The differential diagnosis of PSCC of the bladder includes small cell metastasis from a
site outside of the bladder, a poorly differentiated transitional cell carcinoma (TCC), and
primary or secondary lymphoma. It is important to rule out other primary tumors with CT
scans of the chest and abdomen. On gross pathology, the tumors tend to present as large,
invasive, sessile or polypoidal, ulcerated, and frequently necrotic bladder lesions. The tumor
shows a characteristic microscopic appearance composed of sheets and nests of small round
tumor cells containing hyperchromatic nuclei. On electron microscopy intracytoplasmic
neurosecretory granules can be found in SCC cells. Immunohistochemically, they react to
neuroendocrine markers (chromogranin, synaptophysin, or both). Neuron-specific enolase is
almost always positive, although this marker is not regarded as specific [1]. The
histopathologic findings in our patients confirmed the diagnosis of SCC.
The histogenesis of bladder PSCC remains unclear. There are two possible explanations
for its origin. The tumor may arise from a neuroendocrine Kultschitzky-like stem cell that
exists in the urothelium of the bladder. The second possibility is that these tumors arise from
poorly defined submucosal or muscularis cells of neural crest origin [2].
The prognosis in patients with neuroendocrine tumors of the bladder remains poor
despite an aggressive surgical approach and improvements in systemic multi-agent
chemotherapy. Even though PSCC of the bladder is a rare tumor and optimal management
has not been defined, it is clear that patients are at high risk for systemic metastases and
should be considered for adjuvant protocols [4].
Systemic chemotherapy likely has a significant role in the management of this high-risk
disease. Chemotherapeutic regimen used in our chapter (six cycles of etoposide and cisplatin)
appeared to provide better control of the small cell component than regimens typically
reserved for TCC [2]. In a review in 1995 Abbas et al observed that the best disease-free
survival was obtained with cystectomy followed by chemotherapy (median survival 21.1
months) [3]. Given the small number of patients treated with chemotherapy, it is not possible
to account for other covariates, such as stage, which could affect response to chemotherapy.
It appears that radiation therapy is less effective in treating this cancer although it may
provide palliation in a patient with advanced disease.
Because of the rarity of neuroendocrine tumors of the bladder, definitive conclusions
regarding optimal management cannot be made. To our knowledge, 25 cases of PSCC of the
urinary bladder have been described in the literature [4-7]. These aggressive tumors tend to
present at an advanced stage and tend to recur and progress despite surgical resection and
systemic chemotherapy [4]. The only long-term survivors appear to be patients in whom the
tumor was organ confined. There is clearly a need for methods of earlier detection of these
Small Cell Carcinoma of the Bladder 211

tumors, when they are more likely to be localized. Prospective studies are necessary to define
the impact of combined modality therapy for primary small cell bladder carcinoma.

Conclusion
In conclusion, pure PSCC of the bladder is occasionally curable by radical cystectomy.
In patients with metastasis, systemic chemotherapy may induce remission sometimes lasting a
few years.

References
[1] Mangar, SA; Logue, JP; Shanks, JH; Cooper, RA; Cowan, RA; Wylie, JP. Small-cell
carcinoma of the urinary bladder: 10-year experience. Clin Oncol, 2004, 16(8), 523-
527.
[2] Karpman, E; Goldberg, Z; Saffarian, A; Gandour-Edwards, R; Ellison, L M; Devere
White, RW. Analysis of treatment for small cell cancer of the bladder and report of
three cases. Urology, 2004, 64, 494-498.
[3] Abbas, F; Civantos, F; Benedetto, P; Soloway, MS. Small cell carcinoma of the bladder
and prostate. Urology, 1995, 46, 617-630.
[4] Quek, ML; Nichols, PW; Yamzon, J; Daneshmand, S; Miranda, G; Cai, J; Groshen, S;
Stein, JP; Skinner, DG. Radical cystectomy for primary neuroendocrine tumors of the
bladder: the University of Southern California experience. J Urol, 2005, 174(1), 93-96.
[5] Tunc, B; Ozguroglu, M; Demirkesen, O; Alan, C; Durak, H; Dincbas, FO; Kural, AR.
Small cell carcinoma of the bladder: a case report and review of the literature. Int Urol
Nephrol, 2006, 38(1), 15-19.
[6] Abrahams, NA; Moran, C; Reyes, AO; Siefker-Radtke, A; Ayala, AG. Small cell
carcinoma of the bladder: a contemporary clinicopathological study of 51 cases.
Histopathology, 2005, 46(1), 57-63.
[7] Henno, S; Guiraud, P; Rioux-Leclercq, N; Caulet-Maugendre, S; Kerbrat, P; Lobel, B;
Rame, MP; Turlin, B. An uncommon tumor of the urinary bladder: the small cell
carcinoma. Bull Cancer, 2000, 87(4), 307-310.
Index

adrenal gland, 58, 208, 210


A
adrenal insufficiency, 154
adrenocorticotropic hormone, 154
ABC, 141, 146, 189
adult, 17, 120
abdomen, x, 51, 77, 81, 154, 161, 207, 208, 210
aetiology, 27
abnormalities, 19, 20, 43, 115, 155, 159, 161, 189,
agar, 36
194
age, xv, 53, 77, 130, 132, 137, 144, 181, 188, 196,
Acanthosis nigricans, 156
197, 206, 207
accounting, xii, 149, 153, 156, 180
agent, 15, 23, 24, 26, 33, 49, 82, 94, 195, 210
accuracy, 61, 87, 106, 134, 160, 162, 163, 164, 176
agents, xiii, 23, 30, 46, 83, 116, 168, 169, 170, 172,
acetylcholine, 13, 38
188, 195, 196
acid, 2, 7, 26, 34, 36, 44, 187
aggregates, 11, 57, 58
acidophilic, 183
aggressive behavior, xiv, 180, 181, 189
ACS, 35
aid, 62, 64, 66
ACTH, 154
air, 159, 163
actin, 13
airways, 151, 163
action potential, 155
albumin, 39, 174
activation, 3, 4, 8, 12, 13, 14, 15, 16, 17, 20, 28, 36,
alcohol, 64, 182
37, 38, 41, 42, 43, 173
alemtuzumab, 173
activation state, 13, 38
algorithm, 176
activators, 10
alkaline phosphatase, 160, 163
acute, 29, 33, 85, 86, 87, 88, 89, 92, 96, 156, 171,
alkalosis, 155
173, 175
allele, 107
acute lymphoblastic leukemia, 89
alpha, 28, 32, 46
acute myelogenous leukemia, 33
alternative, 12, 14, 23, 65, 82
acute myeloid leukemia, 29, 33, 171, 173, 175
alternative hypothesis, 65
adenine, 174
alters, 37
adenocarcinoma, 3, 16, 32, 53, 55, 61, 63, 65, 69, 74,
amplitude, 133, 156
169, 181
Amsterdam, 45, 46, 146
adenocarcinomas, 64, 114
anaesthesia, 207, 208
adenovirus, 14, 40
analog, 162
adenoviruses, 43
angiogenesis, 4, 15, 28, 34, 47, 73
ADH, 154
angiogenic, 39
adhesion, 2, 3, 11, 12, 13, 15, 16, 36, 37, 42, 49, 115,
anhidrosis, 151
121
anorexia, 154, 165
administration, 23, 33, 201
ANOVA, 107
214 Index

antagonist, 24, 49 basic fibroblast growth factor, 41


antiangiogenic, 195 Bax, 19, 46
anti-apoptotic, 4, 21, 22, 25, 47 B-cell lymphoma, 2, 4, 171, 175
antibody, 14, 25, 40, 106, 108, 110, 115, 121, 155, Bcl-2, 2, 4, 10, 11, 19, 20, 21, 22, 25, 28, 29, 30, 36,
172, 175, 182, 189 40, 45, 46, 47, 48, 49
anti-cancer, 1, 43, 46, 49 Bcl-xL, 10, 21, 22, 25
anti-chromogranin A, 106 beam radiation, 188
anticoagulation, 200, 203 behavior, xi, xiv, 10, 14, 15, 21, 102, 116, 155, 180,
antidiuretic hormone, 154 189
antigen, 25, 67, 74, 155, 164, 187 benefits, x, xiv, 26, 79, 83, 89, 90, 91, 92, 93, 194,
antineoplastic, 169, 173 196
antisense, 5, 7, 8, 9, 10, 14, 19, 20, 22, 25, 29, 30, benign, 69, 161, 162, 176
31, 32, 33, 34, 38, 40, 44, 46, 48, 49, 173 bevacizumab, 169, 173
antisense oligonucleotides, 31, 49 bias, 35, 195
antitumor, 24, 25, 33 bile duct, x, 51, 55, 71
apoptosis, ix, 1, 6, 12, 13, 15, 16, 17, 18, 19, 20, 21, binding, 8, 12, 13, 16, 38, 40, 49, 155, 164, 174
22, 25, 28, 37, 40, 41, 42, 43, 44, 45, 46, 47, 48, biochemistry, 42, 43, 208
115, 120 bioinformatics, xii, xiii, 167, 168, 169, 172, 176
apoptotic, 4, 20, 21, 22, 25, 44, 46, 47, 54, 59, 63 biological behavior, xi, 102, 116
apoptotic pathway, 20 biomarkers, xiii, 114, 168, 171, 176
appetite, 150 biopsies, 10, 59, 74, 105, 121, 161, 164, 208
application, xv, 8, 34, 199, 201 biopsy, x, 51, 52, 53, 60, 61, 65, 72, 76, 77, 78, 81,
Argentina, 167 103, 105, 118, 156, 161, 162, 163, 181, 191
arrest, 4, 12, 18, 37, 38, 162 biotechnological, xii, xiii, 167, 168, 169, 173, 174
arrhythmia, 162 biotechnology, xiii, 35, 43, 167, 168, 169, 170
aspergillosis, 162 biotin, 182, 189
aspiration, x, 51, 52, 53, 61, 62, 63, 70, 71, 72, 74, birth, 21, 114
75, 76, 77, 151, 163 bladder, x, xiii, xiv, xv, 51, 52, 55, 56, 57, 59, 63,
assessment, xiii, 71, 81, 94, 124, 163, 168, 197 66, 69, 73, 74, 75, 78, 179, 180, 181, 182, 184,
asthenia, 153 186, 187, 188, 189, 190, 191, 205, 206, 207, 208,
asymptomatic, 150, 154 209, 210, 211
ataxia, 155 bleeding, 162
atrophy, 151 blindness, 155
attachment, 11 blocks, 28
autocrine, 12, 13, 14, 38 blood, xiii, 4, 24, 52, 81, 150, 160, 168, 171, 172,
Autofluorescence, 159 208
autonomy, 4 blood vessels, xiii, 4, 168, 172
autopsy, 53, 61 bolus, 31
availability, 159 bone marrow, 154, 160, 208
avoidance, 9 bone scan, 81, 160, 162, 208, 209
awareness, 62 borderline, 116
bradykinin, 16
brain, 58, 70, 81, 89, 90, 91, 97, 98, 114, 154, 155,
B
161, 187, 201, 210
breakdown, 154
B cells, 21, 45, 156
breast cancer, 29, 120, 171, 174, 175, 176
back, 154
breast carcinoma, 47, 115, 119
bandwidth, 126
breast mass, 76
barriers, 142, 143
breathing, 124, 130, 131, 132, 133, 134, 136, 137,
base pair, 5
140, 141, 142, 144, 145, 146, 147
basement membrane, 11
Index 215

bronchial epithelium, 110 CEA, 69


bronchial tree, 161 cell adhesion, 3, 11, 12, 36, 49, 115, 121
bronchoscopy, 81, 159, 161, 162, 163, 166 cell culture, 10
bronchus, 3, 94, 152, 161 cell cycle, 3, 15, 18, 19, 25, 37, 47, 49, 115
Buenos Aires, 167 cell death, 4, 16, 28
buffer, 106, 182 cell differentiation, 186
bulbar, 155 cell division, 4, 159
buttons, 59, 60 cell fate, 18
cell growth, 4, 14, 16, 17, 18, 20, 21, 22, 41, 42, 120,
169
C
cell lines, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
24, 39, 40, 42, 44, 45, 46, 48, 115
Ca2+, 13, 16, 155
cell signaling, 17
cachexia, 165
cell surface, 25, 173
Caenorhabditis elegans, 34
cellular signaling pathway, 25
calcification, 161
cervicitis, 61
calcium, 155, 160, 163
cervix, x, 51, 52, 55, 56, 57, 59, 61, 66, 70, 74, 75,
CAM, 25, 36, 121
76, 206
Canada, 31, 33, 82, 83, 88, 94
channels, 126
cancer cells, 3, 4, 17, 18, 22, 28, 36, 37, 38, 39, 40,
chemicals, 169
41, 42, 43, 44, 45, 46, 47, 48, 49, 120, 154, 169,
chemokine, 2, 12, 13, 15, 37
171, 175
chemoresistance, 15, 21, 37
cancer screening, 170, 171
chemoresistant, 43
cancer stem cells, ix, 1, 26, 42, 50
chemosensitization, 21
cancer treatment, 8, 175
chemotherapeutic agent, 116, 188
candidates, 28
chemotherapeutic drugs, 195
caps, 58
chemotherapies, 201
CAR, 155
Chemotherapy, xiv, 83, 92, 94, 129, 131, 188, 193,
carbon, 172
195, 199, 200, 203, 209
carbon nanotubes, 172
chest, ix, xi, 80, 81, 90, 92, 95, 97, 124, 133, 134,
carcinogenesis, 27, 28, 186
138, 143, 144, 146, 147, 150, 152, 153, 154, 158,
carcinogens, 159
161, 162, 201, 204, 207, 208, 210
carcinoid tumor, 62, 65, 76, 105, 107, 110, 113, 114,
chest radiograph, 158, 161
115, 116, 118, 181
childhood, 64
carcinomas, xi, xiii, xv, 19, 21, 45, 52, 57, 58, 61,
children, 30
62, 64, 66, 68, 73, 75, 76, 78, 101, 102, 111, 114,
China, 197
117, 118, 119, 120, 121, 179, 180, 181, 186, 188,
chitosan, 23
190, 205
CHO cells, 16
cardiomyopathy, 150
cholesterol, 23
cardiovascular disease, 34
chromatin, x, xi, 51, 54, 57, 58, 59, 60, 61, 62, 63,
carrier, 5, 23, 24
64, 101, 103, 104, 105, 183, 187, 206
CAS, 106
chromosome, 45, 114, 119, 186
caspases, 46
chronic cough, ix
CAT, 169
chronic hypoxia, 96
CAT scan, 169
chronic lymphocytic leukemia, 22, 30
categorization, 105, 116
cigarette smoking, 53, 70, 71, 194, 204, 209
category a, 173
cigarettes, xiii, 53, 102, 180
C-C, 2, 13
circulation, 173
CCR, 2
CDK6, 47
cDNA, 49, 50, 171
216 Index

cisplatin, xiv, 15, 21, 32, 41, 46, 73, 82, 85, 88, 92, computed tomography, 158, 182, 188, 207, 209
94, 96, 99, 103, 177, 180, 188, 193, 195, 196, Computed tomography (CT), 161, 207
197, 199, 202, 207, 208, 209, 210 computer science, 172
Cisplatin, 99, 198 concentration, 5, 23, 41
classes, ix, 1, 10 confidence, 22, 69, 162, 201
classical, 11, 20, 25, 146, 196 configuration, 132
classification, 54, 62, 77, 102, 105, 116, 117, 118, confusion, 63
166 congestive heart failure, 150, 153
clavicle, 134 consensus, 82, 132
cleavage, 8 consent, 106, 125, 130, 132
clinical diagnosis, 172 consolidation, xi, 80, 92, 161
clinical oncology, 174 construction, 144
clinical presentation, xii, xv, 149, 150, 157, 206 consumption, 142, 202
clinical trial, xi, 17, 22, 23, 31, 34, 79, 80, 81, 82, continuity, 26
84, 85, 86, 87, 88, 89, 90, 91, 92, 95, 97, 158, contrast agent, 172
162, 171, 195, 201, 203, 204 control, x, xii, xiv, 3, 18, 19, 25, 40, 79, 83, 84, 85,
clinically significant, 81 86, 91, 92, 93, 94, 95, 110, 115, 123, 141, 145,
clinician, 165 146, 171, 190, 193, 194, 195, 201, 210
cloning, 36 control group, 201
clubbing, 156 controlled trials, 82, 95
clustering, 59, 120 convergence, 16
clusters, x, 51, 54, 58, 61 conversion, 186
c-met, 39 COPD, 162
c-myc, 36, 41, 44, 45, 115 corona, 161
c-Myc, 4, 20, 22, 44, 45, 47 correlation, 14, 20, 21, 41, 43, 46, 62, 74, 76, 186,
CNS, x, 28, 79, 93 188
coagulopathy, 162 correlations, 75, 76, 113, 114
cobalt, 172 cost-effective, xv, 194, 196
coccidioidomycosis, 162 cough, 91, 150, 152
coding, 4, 25, 46 coupling, 25, 173
cohort, 81, 107 covering, 200
coil, 126 COX-2, 39
collagen, 13 CpG islands, 119
colon, 3, 56, 173, 203 CPT-11, 46
colonoscopy, 207 CRT, 146
colony-stimulating factor, 99 CSF, 200
colorectal cancer, 27, 29, 31, 32 CT, vii, x, xii, xiv, 51, 56, 72, 74, 77, 81, 87, 96, 97,
common bile duct, 56 123, 124, 125, 128, 130, 131, 132, 134, 141, 142,
common symptoms, 152, 154 144, 145, 146, 147, 161, 162, 164, 169, 180, 181,
communication, 18 182, 188, 191, 201, 207, 208, 209, 210
community, 5 cultivation, 28
compensation, 95 culture, 15, 19
complementarity, 8, 9 CXC, 47
complete blood count, 81, 160 CXC chemokines, 47
complete remission, 89, 98, 190, 201, 204 cycles, 83, 92, 195, 196, 203, 207, 208, 209, 210
compliance, 125, 128, 132 cyclic AMP, 16
complications, 53, 162, 163 cycling, 41
components, 12, 15, 20, 23, 24, 45, 62, 65, 169, 172, cyclodextrin, 23
182 cyclopamine, 18
compounds, 169, 174
Index 217

cyclophosphamide, 30, 82, 83, 99, 188, 199, 200, dexamethasone, 29


202, 203 diaphragm, 124, 134, 138, 140, 145, 153
cystectomy, 181, 187, 188, 189, 190, 210, 211 diastolic pressure, 153
cystitis, 181 dichotomy, x, 79, 80
cystoscopy, 181 differential diagnosis, x, 51, 64, 70, 77, 187, 210
cytochrome, 22 differentiation, xi, 3, 4, 12, 14, 36, 44, 57, 64, 67,
cytogenetics, 190 101, 103, 105, 114, 115, 119, 120, 181, 186
cytokines, 154 diploid, 28
cytology, x, 51, 52, 53, 57, 59, 61, 62, 66, 68, 69, 70, direct measure, 14
71, 72, 75, 76, 77, 81, 158, 159, 160 discipline, xiii, 168
cytopathologists, 61 discordance, 116
cytopenias, 160 disease progression, 201
cytoplasm, x, xi, 51, 54, 57, 58, 59, 63, 101, 103, disease-free survival, 91, 107, 113, 210
104, 105, 110, 116, 183, 206 diseases, 5, 23, 199
cytoskeleton, 13 displacement, 126, 128, 133, 134, 135, 139, 140,
cytotoxic, 12, 21, 195, 200 142, 143, 147
cytotoxicity, 14, 21, 22, 24, 46, 49 disseminated intravascular coagulation, 157
distribution, 59, 71, 108, 109, 151, 155, 177, 202
divergence, 121
D
division, 159
DNA, 3, 5, 8, 12, 17, 18, 33, 34, 37, 44, 45, 47, 103,
data collection, 130
107, 111, 159, 170, 172, 186
database, xiii, 102, 117, 167, 169, 172
DNA damage, 12, 37, 45
death, xi, xiv, 2, 4, 20, 25, 43, 45, 47, 52, 70, 82, 91,
DNA repair, 18, 47
107, 123, 156, 193, 194, 207, 209
docetaxel, 16, 29, 30, 32, 42
decay, 20
doors, 174
decisions, 43, 171
dosage, 141, 200, 201
deficiency, 177
dose-response relationship, 85
definition, xii, 87, 123, 126, 127, 140, 141, 142, 145,
dosing, 31
175
downregulating, 44
degradation, 5, 9, 18, 26, 172
down-regulation, 47
dehydrogenase, 39, 160, 177
dream, xiii, 168, 169, 202
delivery, ix, xii, 1, 9, 22, 23, 25, 26, 42, 48, 81, 86,
drug delivery, xiii, 34, 35, 49, 168, 172
87, 123, 140, 172, 173, 197
drug design, 5
dementia, 155
drug discovery, 28
dendrimers, 172, 173, 174
drug resistance, 10, 12, 13, 37, 52, 195
dendritic cell, 35
drug-induced, 49
Denmark, 106, 182
drug-resistant, 43
density, 25, 146
drugs, ix, xiv, xv, 1, 12, 17, 23, 48, 159, 169, 171,
deposits, 58, 66
172, 173, 174, 193, 194, 195, 196, 199
depression, 162
duration, xv, 8, 53, 83, 134, 189, 194, 196
deregulation, 23, 44
dysphagia, 88, 91, 151, 152
dermis, 56
dysplasia, 76
desorption, 171, 176
dyspnea, 91, 150, 151
destruction, 151, 173
dysuria, 181
detection, x, xiii, 51, 61, 159, 166, 168, 171, 172,
176, 177, 186, 210
detoxifying, 174 E
developed countries, 3, 168
developing brain, 89, 90 ECM, 2, 11, 12, 15, 37
deviation, 128, 136, 138, 144 ectodermal dysplasia, 76
218 Index

edema, 154 estrogen, 67


effusion, 58, 153 ethical standards, 126
elderly, xiv, xv, 56, 168, 175, 194, 196, 198 ethics, 130, 132
elderly population, 168 etiology, x, 51, 69, 70, 181, 194
electrolytes, 160 Europe, 88, 180
electromyography, 155 evolution, ix, xv, 1, 199
electron, xi, 101, 103, 210 examinations, 126, 130
elongation, 8 excision, 11, 36, 45
embolus, 150, 162 exclusion, 62
embryonic development, 18 excretion, 155
emission, 81, 98, 177 exercise, 155
employment, 141 exposure, ix, xii, 123, 132, 159, 163
emulsions, 172 expressed sequence tag, 172
encapsulated, 6, 31 extracellular matrix, 2, 11, 18
encephalopathy, 155, 156, 157 extracranial, 91, 162
encoding, 15 extravasation, 172, 177
Endobronchial, 163, 166 eyelid, 58, 75, 152
endocarditis, 157
endocrine, 36, 119
F
endocytosis, 24, 25, 26
endometrial carcinoma, 120
factorial, 107
endometrium, x, 51, 52, 55, 74
failure, 83, 84, 89, 93, 97, 140
endonuclease, 5, 8
FAK, 2, 12, 14, 16, 25
endoscope, 163
false positive, 61
endothelial cell, 62
family, 4, 12, 13, 15, 16, 17, 20, 21, 22, 28, 44, 48,
end-to-end, 4
50, 194
energy, 9, 132
fatigue, 88, 153
England, 39, 88
F-box, 19
enolase, x, 39, 52, 59, 60, 66, 67, 121, 164, 190, 206,
FDA, 171, 173, 174
210
FDG, 97, 162
enrollment, 91
females, 70, 130, 132
enterprise, 175
fetal, 114, 120
environment, 12, 21, 172
FGF-2, 15, 41
enzymes, 5, 34, 174
fibers, 150
epidemic, 168, 202
fibroblast growth factor, 2, 14, 15, 41
epidemiology, 102, 117
fibronectin, 13, 16
epidermal growth factor receptor, 176
fibrosis, 150
epidermis, 56
filament, 59, 60
epigenetic, 169
film, 81
epithelial cells, 11, 17, 47, 110, 114
fine needle aspiration, x, 51, 52, 53, 63, 70, 71, 72,
epithelium, 64, 110, 114, 119, 186
74, 76, 77, 163
epitope, 65
fine needle aspiration biopsy, (FNA) 51, 53, 77
EPR, 173
fingerprinting, 176
Erk, 16, 17
first generation, 169
erythematous, 156
FISH, 186
esophagitis, 85, 86, 87, 88, 92
fixation, xii, 124, 131, 132, 138, 139, 141, 144, 146
esophagus, x, 51, 52, 55, 56, 57, 68, 69, 73, 75, 78,
flank, 154, 181
86, 87, 89, 151, 152
floating, 11
EST, 172
flood, 173
esters, 38
flow, 121
Index 219

fludarabine, 30 genetic disease, 3


fluid, 66, 159, 208 genetic mutations, 159
fluorescence, 159, 177 genetics, 27, 28, 117, 166, 175, 197
fluoroscopy, 80, 141, 162 Geneva, 117
FNA, x, 51, 53, 54, 56, 58, 59, 60, 61, 67, 68, 69, 70, genitourinary tract, 186
71, 72, 77, 163 genome, 3, 4, 115, 174
focal adhesion kinase (FAK), 2, 12, 42 genomic, 18, 27, 43, 121, 171, 175, 186
folate, 23 genomic instability, 43
follicular, 61, 114 genomic regions, 27
forceps, 161 genomics, xii, xiii, 167, 168, 169, 170, 175
Ford, 50 genotoxic, 20
formaldehyde, xiv, 180, 182 Germany, 126, 130
Fox, 39, 94, 99 GFP, 9
fractionation, xi, xiv, 80, 84, 85, 86, 87, 88, 89, 91, Gibbs, 120
92, 93, 95, 193, 195 gland, 56, 57, 58, 66, 110, 206, 209, 210
fragile site, 27 glioma, 115, 120
France, 146, 199 GLP-1, 38
freedom, x, 79 glucose, 87, 162
frequency distribution, 108, 109 glycoprotein, 11, 36
fusiform, 102 goals, xii, 85, 124
fusion, 3, 4, 5, 124, 130, 132 gold, 141, 163
gold standard, 163
gonadotropin, 188
G
Gore, 48
GPCR, 2, 13, 14, 15, 16
G protein, 17, 28, 38
G-protein, 2, 13
gait, 155
grades, xi, 101, 186, 201
gallbladder, 52, 55, 73
granules, x, 52, 68, 69, 110, 189, 210
ganglion, 151
granulocyte, 99
gastric, 57
granulomas, 162
gastrin, 14, 38
graph, 129
gastrointestinal, 56, 118, 206
green tea, 43
G-CSF, 200
grouping, 181, 182, 184
gender, 206
groups, 5, 60, 107, 115, 137, 142, 171, 172, 201
gene, ix, xi, xii, xiii, 1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13,
growth, x, 2, 3, 4, 12, 13, 14, 15, 16, 17, 18, 19, 20,
15, 17, 18, 19, 20, 21, 23, 24, 25, 26, 27, 34, 35,
21, 22, 24, 28, 34, 36, 37, 38, 39, 40, 41, 42, 45,
41, 43, 44, 48, 49, 101, 107, 112, 114, 115, 116,
47, 48, 49, 50, 51, 52, 95, 103, 120, 150, 156,
118, 119, 120, 159, 167, 169, 170, 171, 172, 173,
157, 168, 169, 173, 176, 186, 187, 188
175, 177, 186, 195
growth factor, 2, 3, 4, 12, 14, 15, 16, 17, 24, 34, 38,
gene amplification, 3, 10, 19, 20, 21, 44, 186
39, 40, 41, 42, 45, 47, 49, 156, 173, 176, 186, 188
gene expression, xiii, 5, 8, 10, 18, 25, 34, 49, 115,
growth inhibition, 12, 14, 15, 40
118, 167, 170, 172
growth rate, 95
gene silencing, ix, 1, 5, 6, 10, 11, 12, 13, 17, 19, 21,
guanine, 174
23, 24, 25, 26, 34, 49
guidance, 72, 162, 163, 198
gene therapy, xii, 27, 35, 43, 167, 169, 173
guidelines, 196
generation, ix, 1, 9, 39, 169, 175
genes, 3, 4, 5, 9, 10, 11, 17, 19, 20, 22, 25, 27, 44,
49, 115, 159, 171, 172, 175, 186, 190, 194 H
genetic abnormalities, 194
genetic alteration, 3, 75, 115, 186 half-life, 22
genetic defect, 159 handling, 172
220 Index

hands, 126, 133, 155, 156, 160 host, 154


hanging, 133 H-ras, 32
haplotype, 174 HSP27, 6
health, 195 human, 2, 3, 9, 10, 11, 13, 18, 22, 23, 24, 25, 27, 28,
heart, xii, 117, 124, 150, 153 30, 33, 36, 37, 38, 40, 41, 42, 44, 45, 46, 47, 48,
heartbeat, 145 49, 95, 106, 114, 118, 119, 120, 126, 145, 158,
heat, 65, 174 166, 168, 172, 174, 175, 176, 188, 191
heavy metal, 173 human chorionic gonadotropin, 188
heavy metals, 173 human genome, 3, 174
Hedgehog signaling, 18, 43 human subjects, 158
height, 107 Hungary, 123
helix, 34, 114 hybridization, 9, 49
hematological, 21, 174 hybrids, 5
hematology, 30 hydrogen bonds, 8
hematoxylin-eosin, 207, 208 hypercalcemia, 157
hematuria, xiii, xv, 179, 180, 181, 186, 206, 207, hypermethylation, 186
208, 209, 210 hyperplasia, 28, 65, 110, 176
hemoptysis, 91, 162 hyponatremia, 154, 160
hemorrhage, 162 hypotension, 153
Heparin, 200 hypothesis, 28, 65, 86, 200
hepatocyte, 2, 14, 39, 156 hypoxemia, 156
hepatocyte growth factor, 2, 14, 39, 156 hypoxia, 47, 96
hepatoma, 45 Hypoxia, 7
hepatomegaly, 154
Herceptin, 5
I
heterodimer, 12
heterogeneity, 90, 105, 169
IAP, 6
heterozygosity, xi, 101, 107, 111, 159
IARC, 117
high grade glioma, 120
ICC, 67
high resolution, 163
id, 19
high risk, 89, 90, 140, 159, 210
identification, 5, 64, 114, 159, 169, 176
high-level, 20
idiopathic, 156
high-throughput screening, 5, 169
IFN, 35
hippocampal, 155
IGF-1, 2, 14, 15, 40
Hiroshima, vii, 101, 117, 118
IL-1, 154
histochemical, 66, 73, 74
IL-6, 154
histogenesis, xiii, 179, 181, 210
IL-8, 39
histogram, 81, 94, 108, 109
illumination, 159
histologic type, 52, 53, 70, 71, 181
images, 87, 136, 141, 161, 169
histological, xi, 3, 71, 76, 101, 103, 105, 108, 110,
imaging, xiii, 52, 58, 81, 87, 90, 106, 134, 141, 145,
112, 116, 154, 181, 188, 206
146, 161, 162, 163, 168, 172, 177, 201, 208
histology, 52, 80, 84, 102, 117, 187, 190, 206
imaging modalities, 81
histopathology, 117
imaging systems, 141
homogeneity, 145
imbalances, 190
homolog, 2, 4, 11, 12, 114, 119
immobilization, 134, 142, 144, 146
Honda, 42
immune response, 22, 35
Hong Kong, 71
immunocompromised, 10
hormone, 29, 30, 31, 68, 154, 157, 188
immunocytochemistry, 52, 76
hospital, 71, 199, 206
hospitalized, xiii, 180, 181
Index 221

immunohistochemical, xi, xiv, 45, 46, 75, 77, 81, inspiration, 130, 131, 132, 133, 134, 137, 138, 141,
101, 105, 106, 108, 110, 112, 115, 118, 119, 120, 146, 153
121, 180, 182, 187, 190, 206, 207, 208 instability, 10
immunohistochemical markers, 115, 120, 180 institutions, 141
immunohistochemistry, xi, 101, 103, 121, 180 insulin, 2, 14, 40, 156
immunological, 108, 156 insulin-like growth factor, 2, 14, 40
immunophenotypes, 66 integration, 9, 83, 97
immunoreactivity, xiv, 68, 105, 108, 119, 180, 188 integrin, 12, 16, 36, 37
immunostimulatory, 9, 48 integrity, 18
immunotherapy, 175, 186 interactions, 36, 154
implants, 141, 145 interference, 2, 8, 9, 27, 34, 35, 44, 48
implementation, 81, 172 interferon, 8
in situ, 61, 63, 159, 186, 188, 190 interleukin, 39, 154
in vivo, ix, 1, 7, 14, 16, 20, 21, 22, 23, 25, 36, 37, interleukin-1, 154
38, 40, 42, 43, 46, 47, 145, 177 interleukin-6, 154
inactivation, 4, 20, 36, 44, 159 interleukin-8, 39
incidence, 3, 11, 20, 21, 53, 71, 72, 89, 90, 102, 162, internalization, 25, 26
175, 187, 201, 206 internalizing, 24
inclusion, xi, 84, 86, 88, 102, 202 internet, 99
incubation, 182 interstitial, 145
incurable, x, 79, 80, 90 interval, 107, 156
independence, 15, 49 intervention, ix, 1, 5, 10, 11, 12, 21, 22
India, 149 intestine, 52
Indian, 71, 77 intracranial, 89
indication, 199, 201 intravascular, 157
indicators, 160 intravenous, xiv, 29, 31, 32, 33, 180, 200, 203
indices, 108, 112 intrinsic, 17, 150
induction, x, 15, 17, 22, 25, 35, 40, 43, 49, 79, 88, intron, 174
89, 90, 203 invasive, xiii, xv, 3, 14, 54, 63, 158, 160, 161, 163,
induction chemotherapy, 88, 90 165, 168, 172, 186, 187, 188, 189, 206, 209, 210
infection, 162, 181, 209 inversion, 145, 146
inflammatory, 47, 61, 154, 155, 156 inverted repeats, 9
influenza, 44 investment, xiii, 168, 169, 174
informed consent, 125 ion channels, 155
inguinal, 59, 60 ionization, 171, 176
inherited, 17 ipsilateral, 63, 87, 160
inhibition, 4, 5, 8, 10, 12, 14, 15, 17, 20, 21, 22, 33, Irinotecan, 174
39, 40, 42, 173 iron, 172
inhibitors, xiii, 5, 10, 14, 16, 18, 20, 28, 37, 45, 168, irradiation, xi, xiv, xv, 80, 84, 86, 88, 89, 91, 92, 94,
169, 176, 195 95, 96, 97, 98, 99, 141, 142, 193, 194, 195, 196,
inhibitory, 4, 16 197, 200, 201, 204
inhibitory effect, 16 isoenzymes, 15, 121
initial state, 201 isoforms, 14, 15, 17, 42
initiation, 8, 44, 53, 89, 93 isolation, 169, 171
innovation, 170
innovative procedures, 146
J
inorganic, xiii, 168, 172, 174
insertion, 10, 49
Japan, 39, 53, 71, 85, 96, 101, 106, 197
inspection, 159
Japanese, 39, 40, 71
Jun, 47
222 Index

Jung, 70, 72 limitations, 10, 77


lipid, 12, 17, 23, 24, 34, 42, 44, 120, 154
liposomes, 6, 23, 24, 25, 42, 49, 172, 173
K
liquids, 151
liver, 56, 58, 59, 73, 78, 81, 160, 161, 187, 210
kappa, 28
loading, 16
keratin, 68, 164
localization, 15, 17, 25, 47, 125, 126, 130, 137, 142,
Ki-67, 14, 19, 45, 47, 105
144, 161
kidney, x, 51, 52, 55, 56, 73, 74
location, 81, 144, 146, 147, 151, 153, 157, 160, 161,
killing, 169
165, 181
kinase, 2, 3, 5, 6, 12, 14, 15, 16, 19, 28, 31, 32, 36,
London, 39, 95, 189
37, 38, 40, 41, 42, 46, 47, 155, 176
loss of heterozygosity, xi, 101, 107, 111
Kinase, 6
losses, 115, 186
kinase activity, 5, 14, 15, 42
low power, 187
kinases, 2, 13, 14, 15, 16, 41, 42, 117
lung metastases, 73
King, 38, 76
lungs, ix, 150, 163
knockout, 10
lying, 153
Korean, 45, 70, 74
lymph, 54, 56, 58, 59, 64, 71, 75, 77, 105, 150, 151,
Kuwait, 51
153, 157, 160, 161, 163, 177, 187, 207, 208, 209,
210
L lymph node, 54, 56, 58, 59, 64, 71, 75, 77, 105, 150,
151, 153, 160, 161, 163, 187, 210
L1, 36 lymphadenectomy, 74
labeling, xiii, 105, 168, 172, 182 lymphadenopathy, 58, 71, 157, 207, 208, 209
lactate dehydrogenase, 39, 160 lymphatic, 151
lamina, xiv, 180, 184 lymphocyte, 57, 102, 109
laminin, 15, 37 lymphocytes, 58, 103, 106, 107, 108, 109
language, 180 lymphoid, 64
larynx, 52, 55, 56, 73, 161, 206 lymphoma, x, 2, 4, 32, 51, 61, 62, 64, 67, 76, 77,
laser, 133, 141, 171, 176 151, 171, 175, 187, 210
laterality, 137, 144
LCA, 67
LDH, 81, 160 M
learning, 10
M1, 6, 188
leiomyoma, 69
machinery, 18, 169
lens, 106
magnesium, 8
lentiviral, 19
magnetic, 172, 177, 201, 208
lesions, x, 13, 28, 51, 52, 58, 71, 72, 78, 125, 130,
magnetic resonance, 172, 177, 201, 208
151, 156, 158, 159, 160, 161, 162, 163, 164, 180,
magnetic resonance imaging, 177, 201, 208
187, 208, 210
magnetization, 145, 146
lethargy, 154
maintenance, 17, 18, 83
leucocyte, 67
males, 130, 132, 181
leukaemia, 34
malignancy, x, xiii, 51, 52, 56, 153, 154, 161, 162,
leukemia, 2, 4, 21, 22, 29, 30, 33, 89, 171, 173, 175,
179, 189
197
malignant, x, xi, 4, 15, 51, 57, 58, 61, 64, 67, 71, 72,
leukocyte, 67
77, 101, 115, 123, 153, 159, 161, 163, 174, 181,
LGB, 64
206
life expectancy, 188
malnutrition, 153
ligands, xiii, 12, 13, 14, 23, 39, 49, 168, 172, 173
mammalian cells, 8, 9, 34, 35
likelihood, 87
Index 223

management, xi, xiv, 78, 80, 83, 92, 94, 118, 170, metastatic disease, xiv, xv, 81, 188, 193, 194, 196,
180, 187, 190, 191, 193, 195, 197, 206, 207, 210 205, 206, 210
MAPK, 13, 17, 47 methylation, xi, 101, 107, 108, 115, 116, 119, 159,
market, 173 190
marrow, 153, 160 Methylation, 107, 112, 119
mask, 130, 131, 132, 133, 134, 138, 139, 140, 141, mice, 10, 11, 17, 22, 23, 38, 48, 118, 172
144 micelles, 174
mast cells, 189 microarray, 44, 46, 49, 119, 120, 170
mastery, 173 microenvironment, 10
mathematics, 172 micrometer, 106
matrix, 2, 11, 16, 18, 37, 126, 171, 195 microRNAs, 27
matrix metalloproteinase, 16, 195 microscope, 182
matrix protein, 37 microscopy, x, xi, 52, 68, 69, 101, 102, 103, 177,
maxillary sinus, 56 182, 210
MCC, 56, 59, 60, 62, 66, 67, 70 microtubules, 47
Mcl-1, 10, 21, 22, 36 microwave, 78
measurement, 14, 106, 128, 130, 134, 142, 145, 147 microwaves, 106
measures, 140 middle-aged, 71
median, xv, 54, 61, 70, 80, 84, 85, 90, 91, 128, 135, migration, 16, 18, 172
136, 142, 143, 188, 194, 195, 196, 201, 206, 210 mimicking, 10, 62, 76
mediastinum, 59, 160, 163 minority, 17, 26
mediators, 12, 156 miosis, 151
Medicare, 168 miRNAs, 4, 5, 25, 27, 35
medication, xiv, 193, 195 misinterpretation, 62
medicine, xiii, 27, 28, 34, 35, 39, 81, 168, 169, 172, misleading, 22
173, 175, 176 mitochondrial, 22, 28, 46, 48
MEDLINE, xiii, 167, 169 mitochondrial membrane, 28
MEK, 13, 15, 16, 17, 37, 40 mitogen, 38
melanoma, 21, 22, 25, 46, 47, 48, 49 mitogen-activated protein kinase, 38
memory, 155 mitogenic, 14
men, ix, xii, 53, 55, 70, 71, 149, 176, 187, 210 mitosis, 3, 18, 47
meningioma, 63 mitotic, xi, 43, 47, 57, 58, 59, 101, 103, 181
mental status changes, 154 mitotic checkpoint, 43
Merck, 174 MMP, 11, 42
mesothelial cells, 60 mobility, 136, 140, 141, 145, 147
MET, 39 modalities, xiii, 69, 70, 81, 144, 155, 166, 167, 168,
Met receptor, 14 169, 172, 189, 200
meta-analysis, 82, 83, 84, 89, 90, 94, 95, 98, 114, modality, 82, 83, 87, 89, 94, 95, 99, 162, 163, 197,
119, 197, 201, 203 201, 203, 204, 211
metabolic, 4, 162 modeling, 5, 146
metabolism, 41 models, 10, 14, 16, 22, 40, 50
metabolites, 174 modern society, 168
metalloproteinases, 11, 37 modulation, ix, 1, 21, 44
metals, 172, 173 MOE, 2, 6, 7
metastases, xiv, xv, 21, 73, 80, 89, 90, 98, 150, 153, molecular biology, 34, 35, 169
154, 162, 172, 173, 177, 180, 187, 188, 190, 193, molecular changes, 159
195, 204, 206, 207, 208, 209, 210 molecular medicine, 27
metastatic, xiv, xv, 3, 11, 12, 13, 14, 29, 31, 32, 33, molecular weight, 106
58, 59, 62, 64, 66, 67, 76, 78, 81, 118, 172, 177, molecules, ix, 1, 5, 8, 10, 23, 25, 36, 45, 121, 169
187, 188, 193, 194, 195, 196, 205, 206, 208, 210
224 Index

monoclonal, xiii, 5, 23, 40, 42, 106, 121, 159, 168, NEC, 62
169, 173, 177 neck, 56, 72, 150, 152, 156, 161, 203
monosomy, 186 necrosis, 28, 59, 103, 154, 156, 181, 187
monotherapy, 200 needle aspiration, x, 51, 52, 53, 61, 62, 63, 70, 71,
morbidity, xi, 83, 123 72, 74, 76, 77, 163
morning, 160 needles, 162
morphogenesis, 18 neoplasia, 3, 28, 118
morphological, 105, 118, 161, 181 neoplasms, x, 51, 52, 61, 62, 63, 64, 76, 77, 95, 115,
morphology, 14, 102, 115, 117, 173, 180 117, 118, 119, 120, 189
mortality, xii, 21, 57, 83, 89, 90, 149, 158, 169, 175 neoplastic, x, 45, 52, 58, 66, 87, 162, 173
motion, xii, 123, 124, 136, 138, 139, 140, 141, 142, neoplastic cells, x, 52, 58, 66, 173
144, 145, 146, 147 neoplastic tissue, 87, 162
mouse, 36, 50, 119 nephroblastoma, 64
movement, xii, 5, 124, 126, 132, 140, 142 nerve, 150, 151, 155, 161
MRI, xiii, 81, 136, 144, 145, 146, 147, 161, 168, nerve fibers, 150
169, 172 nervous system, 98, 204
mRNA, 5, 7, 8, 9, 10, 17, 20, 23, 25, 30 nesting, 103, 104
multidrug resistance, 11, 18, 36 Netherlands, 45, 46
multinucleated cells, 58 network, 4, 10
multiple myeloma, 29 neural crest, 210
multiples, 169 neuroblastoma, 64, 72, 77
multiplicity, 10 Neuroblastoma, 64, 77
multivariate, 53, 81, 200 neuroendocrine, xi, xiii, 3, 19, 27, 36, 43, 45, 46, 57,
murine model, 10, 14, 16, 22, 24, 49 62, 65, 66, 67, 68, 69, 72, 73, 75, 76, 77, 101,
muscle, 154, 155, 156, 187 102, 103, 104, 105, 106, 109, 110, 111, 112, 113,
mutagenesis, 159 114, 115, 116, 117, 118, 119, 120, 121, 164, 179,
mutant, 10, 45 180, 181, 185, 186, 187, 190, 210, 211
mutation, 3, 4, 12, 174, 202 neuroendocrine cells, 114, 181
mutations, 3, 4, 10, 12, 14, 15, 27, 39, 40, 41, 115, neurogenesis, 114
120, 159, 177, 186, 194 neurologic symptom, 155
myasthenic syndrome, 155 neuron-specific enolase, 39, 66, 206
MYC, 3, 20, 44, 45 neuropathy, 155
myeloid, 29, 33, 34, 171, 173, 175 neuropeptides, 13, 16
myeloma, 29 neurosecretory, 68, 210
myoclonus, 156 neurotensin, 38
myocytes, 156 neurotoxicity, 89, 201
myopathy, 156 neurotransmitters, 13
neutropenia, 177
New England, 27, 28
N
New York, 27, 35, 166
NF-B, 4
nanocrystals, 174, 177
NHL, 64
nanoparticles, xiii, 34, 167, 168, 170, 172, 173, 176,
nickel, 172
177
Nielsen, 27
nanoscience, 26, 34
nodal involvement, 80
nanotechnology, xii, xiii, 26, 34, 167, 168, 169, 170,
nodes, 58, 86, 161, 210
172, 173, 174, 176
nodules, 161, 162, 171
nanotubes, 172
non-Hodgkin lymphoma, x, 51, 62, 151
nasal cavity, 52, 56, 70, 72
non-human, 23, 49
National Academy of Sciences, 27, 29
non-human primates, 23, 49
natural, 10, 14, 20, 23, 84, 169
Index 225

non-invasive, xiii, 165, 168, 172 organ, xii, 5, 26, 38, 63, 124, 140, 141, 146, 173,
non-small cell lung cancer (NSCLC), 81, 94, 96, 97, 210
194 organic, xiii, 168, 172
normal, xii, 3, 5, 38, 81, 87, 107, 110, 114, 123, 124, organism, 3
126, 130, 131, 132, 133, 134, 137, 141, 145, 146, orthopnea, 153
155, 159, 174, 182, 195, 208 osmolality, 154
North America, 88, 168, 180 osteoarthropathy, 157
NSCLC, xii, 15, 22, 25, 31, 41, 81, 87, 94, 96, 97, ovarian cancer, 16, 42, 171, 176
102, 110, 114, 115, 145, 149, 156, 157, 158, 159, ovary, x, 51, 55, 56, 59, 63, 68, 73, 76
165, 194 oxide, 172
NSE, x, xiv, 52, 66, 67, 68, 69, 180, 182, 183, 184, oxygen, 14, 39
187, 189
N-terminal, 47
P
nuclear, x, xi, 9, 28, 47, 51, 54, 57, 58, 59, 60, 61,
64, 81, 87, 101, 103, 105, 106, 109, 110, 115,
p53, 4, 27, 28, 33, 36, 45, 46, 68, 114, 115, 171, 182,
116, 118, 155, 181, 187, 206
189, 190, 194
Nuclear factor, 28
paclitaxel, 30, 97, 99, 174, 177, 190, 198
nuclease, 8, 23
PACS, 126, 128, 131, 134
nuclei, xi, 57, 58, 59, 101, 103, 104, 105, 110, 181,
pain, xiv, 88, 91, 150, 151, 152, 153, 154, 155, 181,
183, 210
193, 195
nucleoli, x, xi, 51, 54, 57, 58, 59, 61, 101, 103, 104,
palliate, x, 79, 91, 95
116, 181, 183, 187, 206
palliative, xiv, xv, 193, 194, 195, 196, 198
nucleotides, 2, 5, 7, 8
palpation, x, 51
nucleus, 16, 104, 110, 206
pancreas, 52, 56, 58, 59, 60, 66
pancreatic, 32, 60
O paracrine, 12, 14
paraffin-embedded, 106, 107, 182
oat, 57, 94, 97, 102 paralysis, 151
observations, 21, 65, 85, 119 parameter, 84
obstruction, 150, 151, 181 paraneoplastic syndrome, 150, 154, 155, 156, 157,
odds ratio, 53 160, 181, 210
ODN, 6, 7, 10, 14, 16, 19, 20, 21, 22, 25 parathyroid hormone, 68
odynophagia, 152 parenchyma, 124, 150, 161
old-fashioned, 169, 171 parenchymal, 145, 146, 150
olfactory, 63, 72 paresthesias, 155
oligodeoxynucleotides, 2, 5, 8, 34, 38, 46, 48 Paris, 145, 199
oligonucleotides, 7, 31, 34, 44, 49 parotid, x, 51, 52, 55, 56, 59, 70, 72
omission, 86, 87 parotid gland, 52, 56, 70, 72
oncogenes, ix, 1, 3, 5, 20, 25, 27, 28, 39, 186, 194 particles, xiii, 168, 173, 174
Oncology, 30, 39, 40, 46, 79, 85, 88, 90, 94, 96, 97, pathogenesis, 21, 28, 186, 194
145, 147, 165, 176, 177, 193, 197, 198 pathologist, xiv, 180, 187, 189
oncolytic, 18 pathologists, 64, 102, 105, 116
oncoproteins, 36 pathology, 34, 36, 43, 45, 46, 210
online, 172 pathophysiology, 169
oophorectomy, 59 pathways, 14, 15, 17, 18, 25, 26, 49
open lung biopsy, 52, 53, 81 patient management, 78
OPM, 156 PCR, 107, 111, 119
optimization, 146 PDK, 2
oral, 70, 200, 203 pediatric, 77
ores, 172 pelvic, x, 51, 187, 207, 208, 209
226 Index

pelvis, x, 51, 55, 187, 207, 208 pleura, x, 51, 55, 73, 117
Pennsylvania, 33 pleural, 52, 53, 58, 66, 75, 77, 80, 117, 150, 153,
peptide, 14, 24, 38, 49, 69, 172 161, 163
perception, xiv, 193, 195 pleural effusion, 58, 66, 75, 77, 80, 150, 153, 161,
pericardial, 153, 161 163
periodic, 136 pleurodesis, 163
Peripheral, 70, 160, 166 plexus, 151
peripheral blood, 160 pneumonia, 150, 153
peripheral neuropathy, 155 pneumonitis, 81, 85, 94
peritoneal, 57, 69, 75 pneumothorax, 72, 162
permeation, 173 point mutation, 3, 174, 186
PET, 81, 87, 97, 162, 164, 169 Poisson, 53
PET-CT, 97 polymers, 23
P-glycoprotein, 11, 36 polymorphisms, 174
pH, 8, 106 polypeptide, 41
pharmaceutical, 23, 47, 48 polysialic acid, 36
pharmacodynamics, 29 poor, x, xiv, xv, 11, 12, 15, 36, 41, 46, 57, 79, 80,
pharmacogenomics, xii, xiii, 167, 168, 169, 170 83, 84, 114, 115, 158, 188, 194, 196, 199, 201,
pharmacokinetic, 23, 29, 30, 31, 33 210
pharmacological, ix, 1, 171 poor performance, xiv, xv, 194, 196
pharmacology, 33, 34 population, xiv, 26, 42, 91, 158, 168, 194, 196, 201,
pharynx, 52, 161 202
phenotype, 3, 4, 5, 9, 10, 19, 26, 35, 36, 108, 115, Portugal, 1, 26
119, 121 positive correlation, 21
phorbol, 38 positron, 81
phosphate, 182 positron emission tomography, 81
phosphorylates, 15, 47 postoperative, 106, 116
phosphorylation, 15, 16, 42, 44, 46, 47, 155 potassium, 155
photomicrographs, 182 power, 187
photon, 145 pRB, 43, 47
photosensitivity, 155 preclinical, 40
Physicians, 158, 166, 202 pre-clinical, 174
physics, 172 prediction, xiii, 168, 171
physiological, 8, 18 preneoplastic lesions, 28
physiology, 4 pressure, 153
PI3K, 2, 12, 15, 16, 17, 28, 41, 42, 47, 115 prevention, 170, 174
pituitary, 154 preventive, 202
PKC, 15 primary tumor, xiv, 59, 161, 193, 195, 210
planning, xii, 70, 80, 81, 96, 97, 123, 124, 126, 130, primates, 23, 49
131, 132, 137, 140, 142, 144, 145, 146 pro-apoptotic protein, 21
plaques, 156 probability, 8, 20, 82, 86, 128
plasma, 17, 34, 176 production, ix, xiii, 14, 154, 155, 168, 173, 191
plasma membrane, 17 progenitor cells, 114
plasmids, 9 progenitor-like, 17
plasminogen, 3, 18 progenitors, 43
platelet, 40, 156 progesterone, 68
platelet derived growth factor, 156 prognosis, xiii, 3, 11, 15, 21, 41, 46, 57, 78, 90, 103,
platinum, 83, 93, 188, 189, 195, 197, 201 120, 170, 171, 172, 179, 181, 187, 188, 199, 201,
play, 13, 15, 115, 186 202, 204, 210
PLC, 2, 13 prognostic value, 10, 13, 14, 20, 114
Index 227

program, 26, 82, 130, 146, 193 radiation therapy, x, xiv, xv, 31, 52, 70, 95, 97, 99,
pro-inflammatory, 154 145, 150, 189, 190, 193, 194, 195, 196, 203, 204,
proliferation, x, xiv, 3, 4, 13, 14, 15, 16, 18, 19, 36, 210
37, 38, 40, 44, 46, 47, 49, 79, 95, 115, 159, 180, radical cystectomy, xv, 187, 188, 206, 211
182, 185, 195 radio, xii, 87, 123
promoter, 18, 43, 190 radiological, xiii, 163, 168, 170, 171, 172
property, 17 radiopaque, 133
prophylactic, x, xiv, 79, 84, 88, 89, 92, 98, 193, 195, radiotherapy, x, xi, xii, xiv, xv, 11, 79, 80, 86, 92,
201, 204 94, 95, 96, 97, 98, 99, 106, 123, 124, 125, 140,
prostate, x, 3, 16, 22, 29, 30, 31, 33, 47, 51, 52, 55, 142, 145, 146, 147, 180, 181, 187, 188, 191, 193,
59, 66, 69, 74, 171, 172, 176, 177, 180, 186, 187, 194, 195, 196, 197, 198, 200, 201, 203, 204, 206
203, 206, 211 Raman, 172
protein, 2, 3, 4, 5, 6, 9, 10, 11, 13, 15, 16, 17, 18, 19, random, 159
20, 21, 22, 25, 28, 31, 32, 36, 37, 38, 41, 45, 46, range, ix, xiv, 2, 9, 26, 53, 57, 62, 66, 102, 130, 134,
47, 64, 67, 68, 106, 113, 120, 155, 176, 186, 190, 136, 181, 193, 195
195 rapamycin, 37, 195
protein family, 17 ras, 32, 115, 159
protein kinase C, 31, 32, 38, 41, 46 RAS, 3, 5, 6, 17, 25, 28
protein synthesis, 16 rash, 156
proteolysis, 154 reactive oxygen species, 14, 39
proteome, 176 reactivity, 110, 155
proteomics, xii, 167, 169, 171, 176 reality, 10, 26
protocol, 90, 125, 130, 197, 199, 200 receptors, 5, 12, 13, 14, 15, 24, 25, 28, 38, 39, 173
protocols, 210 recognition, xi, 18, 102
proto-oncogene, 3, 39 recombination, 18
PSA, 36, 69, 121, 171 reconstruction, 142, 145
ptosis, 151 recoverin, 155
public, 172, 176 rectal examination, 207, 208
pulmonary embolism, 153 rectum, 56
pulmonary function test, 147 recurrence, x, xii, 12, 17, 51, 52, 83, 87, 89, 91, 92,
pulmonary hypertension, 162 97, 107, 123, 171
pulse, 153 red blood cells, 160
Purkinje, 155 Red Cross, 105
red light, 174
reflexes, 155
Q
refractory, 29, 30, 32, 34
regional, 86, 87, 95, 105, 107, 145, 146, 150, 160
quality control, 97
regression, 48, 53
quality of life, 86, 87, 88, 89, 91
regular, 136
quantum, xiii, 168, 172, 177
regulation, ix, 1, 9, 15, 16, 17, 18, 35, 40, 43, 44, 47
quantum dots, xiii, 168, 172, 177
regulators, 18, 28
questionnaires, 88, 91
rehydration, 182
relapse, x, xiii, xiv, 3, 79, 90, 91, 97, 102, 103, 168,
R 180, 201, 209
relapses, 91, 140
radiation, x, xiv, xv, 31, 52, 70, 79, 80, 81, 83, 85, relationship, 46, 85, 96
86, 87, 88, 94, 95, 96, 97, 99, 126, 132, 141, 145, relaxation, 130
150, 160, 163, 188, 189, 190, 193, 194, 195, 196, relaxation time, 130
197, 201, 202, 203, 204, 210 relevance, ix, 1, 5, 14, 25, 26, 40
remission, 89, 98, 190, 201, 204, 211
228 Index

renal, x, 33, 51, 55, 81, 154 sarcoidosis, 69


renal function, 81 Sartorius, 46
repair, 3, 11, 18, 22, 36, 45, 47 SAS, 107
replication, 18, 29 scatter, 39
repression, 20 scientific community, 5
Research and Development, 179 scores, 88
resection, xiii, 10, 83, 102, 159, 180, 181, 182, 187, scotomata, 155
188, 189, 206, 207, 208, 210 SD, 31, 35, 48, 135, 136, 177
reserve capacity, 150 SDF-1, 2, 13
residual disease, 92 search, xiii, 115, 167, 169, 202
resistance, 8, 10, 11, 12, 13, 15, 16, 18, 20, 21, 22, second generation, 32
36, 37, 41, 43, 46, 49, 52, 156, 169, 171, 175, secrete, 14
191, 195 secretion, 16, 42, 154
resolution, 115, 124, 161, 163, 171, 188 seed, 10, 28
resources, 168 seeding, 162
respiration, 141, 142, 145, 146 seizures, 154, 155
respiratory, 64, 124, 140, 141, 143, 150, 155, 162 selecting, 202
retention, 49, 173 Self, 4
retinoblastoma, 4, 64, 159 self-renewal, 17
retinoic acid, 44 semiconductor, 177
Retroviral, 17 senescence, 4
retrovirus, 3 sensation, 155
reverse transcriptase, 2, 18, 43 sensitivity, xi, xiii, xiv, 19, 28, 41, 45, 61, 63, 64, 87,
Reynolds, 35, 77 102, 116, 160, 161, 162, 163, 168, 171, 193, 194
rhodopsin, 155 sensitization, 25
ribosomal, 5 series, xiv, xv, 21, 71, 73, 116, 121, 126, 127, 131,
ribozymes, 8, 34 132, 135, 136, 141, 169, 180, 182, 187, 205
RISC, 2, 8 serine, 15
risk, ix, 3, 14, 21, 23, 40, 53, 71, 81, 82, 86, 89, 90, serotonin, 68, 69
91, 140, 159, 161, 163, 171, 177, 181, 201, 210 serum, xiii, 12, 14, 15, 23, 25, 41, 81, 155, 160, 163,
rituximab, 173 168, 171, 176, 182
RNA, 2, 5, 7, 8, 9, 18, 25, 27, 34, 35, 42, 44, 45, 47, services, vi
48, 171 sex, 137, 144
RNAi, 2, 8, 9, 10, 15, 16, 17, 20, 34, 35, 48, 49 shape, xi, 4, 101, 105, 116, 145
rodents, 114 shock, 6
Romania, xiii, 179, 180 shortness of breath, ix, 150, 152
Rouleau, 72 short-term, 155
round cells, 57 short-term memory, 155
routing, 159 shoulder, 132, 151
SIADH, 154, 157
side effects, 8, 23, 177
S
Siemens, 126, 130, 133
sign, 130, 153, 161
S phase, 18
signal transduction, 37, 41
saccades, 156
signaling, 2, 10, 12, 13, 14, 15, 16, 17, 18, 20, 25,
safety, xii, 32, 87, 123, 124, 142, 173
28, 37, 40, 43, 45, 47
saline, 182
signaling pathway, 15, 18, 47
salivary glands, x, 51, 52, 55, 70, 75
signalling, 38, 39, 41, 42, 43
salt, x, 51, 57, 58, 59, 61, 63
signals, 4, 12, 14, 15, 17, 41
sample, 107, 162, 163
significance level, 135, 140
sampling, 54, 75, 116, 160
Index 229

signs, 81, 133, 153, 161 statistics, xii, 27, 149, 175, 187
single-nucleotide polymorphism, 174 stem cells, ix, xiii, 1, 17, 18, 26, 42, 50, 179, 186
singular, 10 sterilization, 85
sinuses, 52, 72 stimulus, 16
siRNA, xiii, 2, 6, 7, 8, 9, 10, 14, 15, 16, 18, 19, 21, stomach, 52, 55, 56, 57, 63, 68, 69, 73, 75, 78, 156
22, 35, 48, 168, 173 strain, 145, 146
SIS, 31 strains, 28
sites, x, 3, 15, 24, 27, 51, 52, 55, 56, 58, 59, 62, 66, strategies, xiv, 4, 23, 25, 34, 35, 88, 170, 180
70, 76, 86, 88, 90, 91, 93, 95, 97, 119, 153, 154, strategy use, 86
186, 187, 206, 210 strength, 155
skeletal muscle, 154, 156 streptavidin, 182
skin, 52, 55, 56, 59, 66, 67, 72, 74, 152, 154, 156, stress, 22
206 stretching, 153
smokers, 3, 27, 53, 102 stridor, 152
smoking, ix, xii, xiii, 53, 69, 70, 71, 80, 149, 180, stroma, 12, 13
181, 194, 204, 206, 209 stromal, 2, 13
smooth muscle, 156 students, 26
SNP, 69 sub-cellular, 15
sodium, 6, 7, 22, 29, 30, 48, 154 subgroups, 92
software, 81, 107, 126, 131, 143, 172 submucosa, 186
soil, 28 substitution, 174
solid tumors, 29, 30, 31, 32, 33, 34, 174 substrates, 15, 16
solubility, 8 suffering, 210
somatic cell, 159 suicide, 18, 43
somatic cells, 159 sulphur, 7
Spain, 26 Sun, 27, 34, 62, 75, 76, 96
spatial, 124 superior vena cava, 150, 151
species, 14, 39, 177 supply, 150
specificity, ix, 1, 7, 9, 10, 22, 23, 61, 63, 87, 160, suppression, 4, 27, 46, 49
162, 169, 171 suppressor, 3, 4, 5, 15, 19, 25, 27, 115, 120, 186, 194
spectroscopy, 171 surgeries, 69
spectrum, 40, 65, 77, 102, 105, 117, 121 surgery, xi, 70, 83, 94, 103, 107, 123, 125, 194, 206
S-phase, 3, 19 surgical, x, 36, 52, 69, 70, 83, 103, 118, 152, 163,
spheres, 142 187, 210
spin, 145, 146 surgical pathology, 36
spinal cord, xii, 124, 154, 161 surgical resection, 83, 187, 210
spindle, xi, 62, 63, 101, 103 surveillance, 102, 117, 159, 181
spleen, 210 survival rate, x, 3, 79, 80, 187, 188, 195, 196, 201
sputum, ix, 52, 53, 81, 158, 159, 160 survival signals, 12
squamous cell, x, 3, 15, 51, 53, 55, 61, 63, 65, 77, surviving, 195
105, 108, 111, 114, 119, 181 survivors, 89, 90, 210
squamous cell carcinoma, x, 3, 15, 51, 53, 55, 61, susceptibility, 27
63, 65, 77, 105, 108, 111, 114, 119, 181 susceptibility genes, 27
stability, 7, 8, 9, 25 SV40, 43
stages, xii, 19, 105, 116, 123, 150, 160, 171, 181, swallowing, 88, 152
186 swelling, 60, 152, 154
standard deviation, 106 sympathetic, 151
standards, xiii, 126, 167 symptom, xiii, 91, 179
starvation, 12 symptoms, x, xiv, 79, 81, 90, 91, 150, 151, 152, 153,
statins, 17 154, 155, 158, 160, 163, 181, 193, 195, 206, 207
230 Index

synaptophysin, x, xi, 52, 66, 67, 68, 69, 101, 105, thyroid, 64, 66, 67, 76, 78, 106, 114, 119, 164, 186
106, 108, 115, 164, 187, 206, 207, 208, 210 time frame, 10
synchronous, 136, 144, 159 timing, 83, 84, 94, 95, 202, 203
synchronous tumors, 159 tissue, xii, 23, 37, 46, 50, 59, 77, 81, 86, 87, 110,
syndrome, 33, 60, 151, 152, 154, 157, 165 114, 116, 119, 120, 124, 132, 159, 161, 162, 163,
synergistic, 11, 15, 40 164, 181, 182, 209
synthesis, 9, 15, 16 TNF, 154
tobacco, 159, 202
Tokyo, 39, 106
T
tolerance, 197, 201
Topotecan, 203
tachycardia, 153
toxic, xiv, xv, 9, 35, 174, 194, 196, 199
Taiwan, 71
toxic effect, 174
tamoxifen, 171, 175
toxicities, 82, 87, 88, 89
tar, 102
toxicity, xii, xiii, xv, 7, 81, 85, 86, 87, 88, 89, 92, 95,
targets, xiii, 5, 6, 11, 12, 13, 14, 17, 20, 25, 26, 38,
98, 124, 168, 169, 173, 174, 177, 194, 195, 196,
159, 168, 171, 173
200
taxane, 29
TP53, 4, 7, 10, 19, 45, 107
TCC, 186, 187, 210
trachea, 151, 152, 163
TE, 30, 34, 97, 126
tracking, 144, 177
tea, 43
training, 133, 141
technical assistance, 116
trans, 44
telangiectasia, 156
transactions, 38
telomerase, 2, 4, 18, 42, 43, 120, 159
transcript, 5, 8, 35, 174
telomere shortening, 159
transcriptase, 2, 18, 43
telomeres, 4, 18, 28
transcription, 8, 19, 20, 43, 64, 67, 76, 77, 78, 106,
temperature, 177
114, 119, 164, 186, 190
temporal, 124, 155
transcription factor, 19, 20, 43, 67, 76, 77, 78, 106,
tendon, 155
114, 119, 164, 186, 190
therapeutic agents, xiii, 168, 196
transcriptional, 5, 44, 47, 175
therapeutic approaches, 3, 195
transcripts, 14, 34, 40
therapeutic targets, 14, 25, 171
transfection, 24, 26
therapeutics, xiii, 28, 34, 35, 48, 167, 169, 173, 175
transferrin, 23
therapy, x, xi, xii, xiv, xv, 10, 16, 21, 27, 30, 33, 34,
transformation, 17, 26, 27, 29, 181
38, 42, 43, 44, 45, 47, 48, 49, 78, 79, 82, 83, 84,
transgenic mice, 118
95, 103, 123, 124, 133, 141, 144, 145, 173, 180,
transition, 19, 28, 62
186, 187, 188, 193, 194, 195, 196, 197, 203, 205,
transitional cell carcinoma, 63, 186, 190, 191, 210
211
translation, 5
thermoplastic, 130, 131, 132, 133
translational, 40, 44
Thomson, 75
translocation, 21, 45
thoracic, x, xv, 79, 81, 83, 84, 85, 86, 87, 88, 91, 92,
transmembrane, 14
93, 94, 95, 96, 97, 98, 99, 130, 133, 141, 146,
transurethral resection, xiii, 180, 188, 189, 206, 207,
151, 194, 196, 197, 198, 200, 201, 202, 203, 204
208
thoracotomy, 69
trastuzumab, 5, 173
thorax, xiv, 77, 81, 151, 163, 193, 195
trial, x, 14, 22, 29, 30, 31, 32, 33, 34, 79, 82, 83, 84,
three-dimensional, 81, 97, 177
85, 86, 87, 88, 89, 90, 91, 92, 94, 95, 96, 97, 98,
threonine, 15
99, 158, 162, 177, 197, 198, 200, 201, 203, 204
thrombosis, 152, 157
triggers, 22, 35
thymidine, 20
tumor cells, x, xi, xii, xiii, xiv, 5, 10, 17, 18, 22, 24,
thymoma, 2, 12
25, 51, 54, 56, 57, 59, 60, 61, 62, 64, 66, 68, 69,
thymus, x, 51, 52, 55, 56, 73, 117, 206
Index 231

101, 103, 105, 106, 107, 108, 109, 110, 114, 123, values, 61, 108, 128, 135, 137, 138, 139
140, 154, 168, 169, 184, 185, 193, 195, 210 variation, 54, 71
tumor growth, 42, 50, 150, 157, 188 vascular endothelial growth factor (VEGF, 3, 14,
tumor metastasis, 172 34, 39, 45, 47
tumor necrosis factor, 28, 154, 156 vascular wall, 103
tumor proliferation, xiv, 180, 182 vasculature, 172, 173
tumorigenesis, 28, 41, 169 vasculitis, 156
tumorigenic, 3, 4, 21, 42, 171 vasoactive intestinal peptide, 69
tumour, 39, 50, 95, 117, 120, 172, 173, 206 VCAM, 13
Tunisia, 205 vector, 40
Turkey, 193 VEGF, 3, 5, 7, 14, 20, 39
turnover, 5, 160 vein, 151, 157
two-dimensional, 80 venous pressure, 153
tyrosine, 2, 5, 14, 16, 38, 42, 117, 176 vertebrae, 143
vincristine, 83, 99, 203
viral vectors, 9
U
virus, 18, 27, 29, 44
visible, xii, 54, 86, 87, 123, 159, 161
U.S. Preventive Services Task Force (USPSTF), 158
visual field, 155
ubiquitin, 19
visualization, 124, 141, 142, 143
UDP-glucuronosyltransferase, 174, 177
voice, 152
UGT, 174
voiding, 181
ultrasonography, 53, 76
vulva, 52, 55, 74
ultrasound, 71, 78, 163
uncertainty, 142
underreported, 187, 198 W
uniform, xiv, 58, 89, 130, 180
United States, xii, 27, 29, 85, 117, 149 warfarin, 200, 203
univariate, 63, 81 weakness, 154, 155
upper airways, 73 websites, 172
urethra, 59, 208 weight loss, ix, 154, 181
urinary, x, xiii, xiv, 51, 52, 55, 56, 57, 59, 63, 69, 74, Weinberg, 27
75, 78, 154, 155, 179, 180, 181, 182, 184, 186, wheezing, ix, 150
187, 188, 189, 190, 191, 208, 209, 210, 211 white blood cells, 160
urinary bladder, x, xiii, xiv, 51, 52, 55, 56, 57, 59, WHO, 62, 102, 105, 116, 181, 182, 184, 188
63, 69, 74, 75, 78, 179, 180, 181, 182, 184, 186, wild type, 159
187, 188, 189, 190, 191, 208, 210, 211 withdrawal, 202
urinary tract, 181, 209, 210 women, ix, xii, 53, 71, 72, 149, 187
urine, 10, 16 World Health Organization (WHO), 62, 102
urokinase, 3, 18 worm, 8
urologist, xiv, 180
urothelium, 188, 210
X
uterus, 187
xenografts, 17, 39, 48, 172
V xiphoid process, 133
X-linked, 6
vaccine, 202
vacuum, 141
Y
validation, 10, 20, 22, 26
validity, 176
yield, 53, 160, 161, 162