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ORAL CANCER RESEARCH ADVANCES

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ORAL CANCER RESEARCH ADVANCES

ALEXIOS P. NIKOLAKAKOS
EDITOR

Nova Biomedical Books


New York

Copyright 2007 by Nova Science Publishers, Inc.

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Oral cancer research advances / Alexios P. Nikolakakos, editor.
p. ; cm
Includes bibliographical references and index.
ISBN 13: 978-1-60741-924-2 (E-Book)
1. Mouth--Cancer. 2. Head--Cancer. 3. Neck--Cancer. I Nikoladados, Alexios P.
[DNLM: 1. Mouth Neoplasms. 2. Head and Neck Neoplasms. 3. Mouth Neoplasms-genetics. WU 280 0632 2007]
RC280. M60725 2007-11-07
616.99491--dc22
2007026603

Published by Nova Science Publishers, Inc.

New York

CONTENTS
Preface

ix

Chapter 1

Effective Administration Methods of 5-Aminolevulinic Acid as a


Photosensitizer in Photodynamic Therapy for Tongue Tumor
1
Toshiyuki Ogasawara, Norio Miyoshi, Kazuo Sano, Hidetaka Kinoshita,
Tetsushi Yamada, Toru Ogawa, Kazuki Miyauchi and Yoshimasa Kitagawa

Chapter 2

Relationships Between Biological and Clinicopathologic Features in


Esophageal Carcinoma
Takuma Nomiya, Kenji Nemoto and Shogo Yamada

Chapter 3

Prognostic Indicators in Oral Squamous Cell Carcinoma


Mrcio Diniz-Freitas, Eva Otero-Rey, Andrs Blanco-Carrin,
Toms Garca-Caballero, Jos Manuel-Gndara Rey and Abel GarcaGarca

Chapter 4

Tumor-Targeting Non-Viral Gene Therapy for the Treatment of


Oral Cancer
Yoshiyuki Hattori and Yoshie Maitani

11
51

95

Chapter 5

New Diagnostic Imaging Modalities for Oral Cancers


Yasuhiro Morimoto, Tatsurou Tanaka, Izumi Yoshioka,
Yoshihiro Yamashita, Souichi Hirashima, Masaaki Kodama,
Wataru Ariyoshi, Taiki Tomoyose, Norihiko Furuta, Manabu Habu,
Sachiko Okabe, Shinji Kito, Masafumi Oda, Hirohito Kuroiwa,
Nao Wakasugi, Tetsu Takahashi and Kazuhiro Tominaga

Chapter 6

The Role of the Percutaneous Endoscopic Gastrostomy in the


Management of Head and Neck Malignancy
CME Avery

155

The Biomechanical Basis for Internal Fixation of the Radial


Osteocutaneous Donor Site
CME Avery

183

Chapter 7

125

Chapter 8

Contents

vii

The Current Role of Prophylactic Internal Fixation of the Radial


Osteocutaneous Donor Site
CME Avery

195

Chapter 9

Cytologic Diagnosis of Oral Malignancies: Scope and Limitations


Dilip K. Das

Chapter 10

Benign and Malignant Tumors Occurring in the Pterygopalatine


Fossa and Adjacent Structures of the Pterygopalatine Fossa: Recent
Advances of Diagnosis and Surgical Management
Xin-Chun Jian

Chapter 11

Molecular Aspects of Oral Cancer: the Role of Phase I and II


Biotransformation Enzymes in Carcinogenesis
Karin Soares Gonalves Cunha and Dennis de Carvalho Ferreira

211

229

247

Chapter 12

TP53 Mutation, c-myc Amplification and Squamous Cell Carcinoma


Recurrence
263
J. Seoane, P. Varela-Centelles, M.A. Romero , A. De la Cruz, F. Barros,
L. Loidi and J.L. Lpez Cedrn

Chapter 13

Recent Advances and Future Prospects Upon the Arterial Framework


of the Face and Related Applications for Facial Flaps
Egidio Riggio

Index

275
285

PREFACE
Oral cancer is any cancerous tissue growth located in the mouth. It may arise as a
primary lesion originating in any of the oral tissues, by metastasis from a distant site of
origin, or by extension from a neighboring anatomic structure, such as the nasal cavity or the
maxillary sinus. Oral cancers may originate in any of the tissues of the mouth, and may be of
varied histologic types: teratoma, adenocarcinoma derived from a major or minor salivary
gland, lymphoma from tonsillar or other lymphoid tissue, or melanoma from the pigment
producing cells of the oral mucosa. Far and away the most common oral cancer is squamous
cell carcinoma, originating in the tissues that line the mouth and lips. Oral or mouth cancer
most commonly involves the tissue of the lips or the tongue. It may also occur on the floor of
the mouth, cheek lining, gingiva (gums), or palate (roof of the mouth). Most oral cancers look
very similar under the microscope and are called squamous cell carcinoma. These are
malignant and tend to spread rapidly. This new book presents important research from around
the world.
Chapter 1 - Objective: Photodynamic therapy (PDT) is a promising cancer treatment in
which a photosensitizing drug accumulates in tumors and is subsequently activated by visible
light of an appropriate wavelength matched to the absorption. The advantages of this method,
as compared to other conventional cancer treatment modalities, are its low systemic toxicity
and its ability to destroy tumors selectively. 5-aminolevulinic acid (ALA)-induced
protoporphyrin-IX (PpIX) has been used as a photosensitizer in PDT for oral cancer, which
advantage is low side effect compared to other photosensitizer. This study investigates the
optimal method of administrating ALA by analyzing PpIX fluorescence in tongue tumor
tissue.
Methods: PpIX intensities in the mouse (C3H) transplanted tongue cancer (NR-S1) were
compared with those in normal tongue after intraperitoneal (i.p.), oral (p.o.), or topical
administration of ALA. Tongues were sampled at various times after ALA administration.
PpIX intensities were obtained from frozen sections of each sample by using a
spectrophotometer.
Results: PpIX intensity in the tumor group peaked at 3 h after the i.p. and 5 h after the
p.o. administration of ALA, and these levels were about twice as high as those in the normal
group. Maximum PpIX accumulation in the tongue tumor tissue was seen at 5 h after the oral

Alexios P. Nikolakakos

administration of ALA. In contrast, the topical administration of 20% ALA cream was
associated with the lowest PpIX accumulation in the tumor throughout the experiments.
Conclusion: Based on these results, most effective administration route of ALA was oral
administration and 5 h after administration was regarded to be the optimal time for light
irradiation in ALA-PDT.
Chapter 2 - The clinical characteristics and radiosensitivity of esophageal cancer differ
individually, even in individuals with the same histopathological type. Several investigators
have reported that prognosis of patients with esophageal carcinoma differs according to its
macroscopic appearance, and it has been shown that macroscopically infiltrative type (like
scirrhous type in gastric cancer) is radioresistant and that its prognosis is extremely poor
compared to that of macroscopically localized type. The major factors that are thought to
have a potent impact on radiosensitivity of a tumor are cell proliferation activity, tumor
oxygenation, genetic repair, and intrinsic radiosensitivity.
In our study, Ki67, CD34, vascular endothelial growth factor (VEGF), thymidine
phosphorylase (TP) and metallothionein (MT) expressions and microvascular density were
evaluated using surgically resected esophageal squamous cell carcinomas without
preoperative treatment. Microvascular density (MVD) was evaluated in different ways:
average-MVD was estimated as an index of tumor oxygenation, and highest-MVD was
estimated as an index of the most active neovascularization in the tumor.
In the analysis of proliferation activity (Ki67 labeling index), proliferation activity of the
radiosensitive group of esophageal carcinomas was higher than that of the radioresistant
group of esophageal carcinomas. In the analysis of microvascular density, average-MVD of
macroscopically infiltrative type was significantly lower than that of localized type, whereas
highest-MVD of macroscopically infiltrative type was significantly higher than that of
localized type. The VEGF expression level of infiltrative type was significantly higher than
that of localized type. A significant positive correlation was found between highest
microvascular density and VEGF expression, and a borderline significant negative correlation
was found between average microvascular density and expression of VEGF. TP expression
showed a positive correlation with highest-MVD, but the correlation was not as strong as that
of VEGF expression. In the analysis of MT, which is recognized as a protein that has a
radioprotective effect, expression of MT was not increased in esophageal carcinoma of the
radioresistant group. Metallothionein expression was increased in the radiosensitive group.
Furthermore, expression of MT was not increased in preoperatively treated esophageal
carcinomas. These results suggested that MT does not have a great impact on clinical
radiosensitivity in esophageal carcinoma and also suggested that MT expression is not
induced by therapeutic irradiation or anticancer agents.
The results suggest that radioresistant type is poorly oxygenated by low average-MVD,
includes a large amount of hypoxic fraction that is refractory to treatment, shows induction of
angiogenic factors and activated neovascularization, and has a high rate of hematogenous
metastasis. Tumor oxygenation and presence of a hypoxic fraction seem to have great
importance for curability of esophageal carcinoma compared to various other factors the
authors have investigated.
Chapter 3 - Every year, more than 300,000 new cases of oral cancer are diagnosed
worldwide. Oral squamous cell carcinomas (OSCCs) make up about 90 - 95% of these cases.

Preface

xi

Despite intensive research into treatment modalities for oral cancer, the 5-year survival rate
has shown little improvement in recent decades. One of the reasons for this is that the TNM
classification system (the conventional basis for treatment decisions, in conjunction with
histological tumor grade) has proved not to be a consistently good predictor of prognosis.
There is thus a pressing need for research into new prognostic indicators, with the aim of
enabling the evaluation of the biological aggressiveness of each patient's particular tumor/s.
In recent decades, considerable research effort has been dedicated to the identification of
new markers of OSCC, with the aim of better predicting tumor behavior and clinical course.
Certainly, an improved knowledge of the different biological mechanisms participating in
carcinogenesis, as well as of cell proliferation, apoptosis, tumor growth and tumor invasive
capacity, may assist individual diagnosis, and help in the development of new treatment
strategies.
The aim of the present chapter is to briefly review the use of tumor markers for
prediction of the biological behavior of OSCCs. The review is divided into three parts,
considering first clinical markers, then histological markers, and finally
immunohistochemical markers.
Chapter 4 - Despite advances in surgery, radiotherapy, and chemotherapy, the survival of
patients with oral squamous cell carcinoma has not significantly improved over the past
several decades. Gene therapy has the potential for the treatment of oral cancer. Cancer gene
therapy is currently being met with the development of non-viral vectors, because non-viral
vectors have a much lower potential for an adverse inflammatory or immune reaction,
compared with viral vectors. For gene delivery, oral cancer is a particular appropriate target
since it can be applied by direct injection. Also since folate and transferrin receptors are
frequently overexpressed on oral tumors such as nasopharyngeal tumor and head and neck of
squamous cell carcinoma, folic acid and transferrin have been utilized as a ligand for tumortargeting gene delivery. Non-viral vectors conjugated to these ligands have been used as
carriers of therapeutic DNA to targeted oral tumor. The strategies are used for inactivation of
oncogene expression, introduction of tumor suppressor genes, and introduction of a gene that
enable to a prodrug to be activated into an active cytotoxic drug. In this review, the authors
outline tumor-targeting liposome and lipid-based nanoparticle vectors, and discuss the
effectiveness as these non-viral vectors for DNA transfection and for gene therapy to treat
human oral tumors.
Chapter 5 - This article reviews the use of imaging modalities; both commonly used and
recently introduced, to evaluate oral cancers and their lymph node metastases. Magnetic
resonance images (MRI) and X-ray computed tomography (CT) images are used to determine
the size, invasive area, and possible pathology of primary cancers. In addition, the two
modalities are useful for staging and detecting clinically occult lymph node metastases at
different levels of the neck. In particular, a follow-up MR examination method, dynamic MR
sialography, for patients with xerostomia after radiation therapy is introduced, and the use of
fusion images of the tumors and vessels using three-dimensional fast asymmetric spin-echo
(3D-FASE) and MR angiography is discussed. Furthermore, ultrasound imaging (US), in
addition to its use for staging and detecting clinically occult lymph node metastases, plays an
important role in confirming intra-operative surgical clearance of tongue carcinomas. In
addition, the role of US-guided, fine-needle aspiration biology is also reviewed. Finally, the

xii

Alexios P. Nikolakakos

role and limitations of fusion images obtained from positron emission tomography (PET) and
CT (PET-CT), which are currently used worldwide, are discussed.
Chapter 6 - This chapter reviews the role of the percutaneous endoscopic gastrostomy
(PEG) for providing nutritional support in the management of oral cancer. An assessment of
the current use of the PEG technique is based on an analysis of the prospective operating
series of the author.
Insertion of a PEG was attempted on 200 occasions, mainly for malignancy of the oral
cavity but also the oropharynx, and some benign pathology and trauma. Seventy-six percent
(152/200) of gastrostomies were inserted at the time of definitive surgical treatment and
19.5% (39/200) were inserted at an examination under anaesthesia, often prior to
radiotherapy.
Five percent (10/200) of procedures had significant endoscopic findings including one
synchronous malignancy. The rate of successful insertion was 97% (194/200). The incidence
of minor and major complications was 12.5% (25/200) and 3% (6/200) respectively. There
was no procedure related mortality. The overall 30-day mortality rate was 7% (10/200)
including deaths from terminal disease. Those at increased risk of death were 65 years and
over (P=0.005). The median PEG duration was 287 (SE 37) days. Duration was significantly
longer for stage T3-4 tumours (P=0.01), N1 or greater neck disease (P=0.02), following
surgery with radiotherapy when compared to surgery alone (P<0.001), particularly for
hemiglossectomy (P=0.02) and maxillectomy procedures (P=0.003), following a segmental
composite bone resection rather than a soft tissue resection, with or without a rim resection,
(P=0.03) and finally radiotherapy alone when compared to surgery alone (P=0.004). There
was no obvious relationship to age or the type of free flap. Four (2.1%) patients have a
gastrostomy that is likely to be permanent. Only two patients did not use the gastrostomy.
All patients with T3 and T4 oropharyngeal tumours undergoing radiotherapy or with oral
tumours that required reconstruction with a free or pedicled flap were offered a PEG on the
basis that nutritional support would be required for more than 2 to 4 weeks. This included T2
tumours without neck disease (stage II disease) if the site of the tumour is likely to have a
significant effect on function and hence a flap reconstruction is indicated.
The policy of early gastrostomy placement appears to be appropriate. However, the exact
pattern of use of the gastrostomy during the various phases of treatment remains to be
defined. The insertion of a PEG may be safely performed with a high degree of success and a
low incidence of complications by an experienced maxillofacial surgeon.
Chapter 7 - Fracture of the radial free flap osteocutaneous donor site is common and
causes considerable morbidity. Most fractures are probably caused by relatively low-energy
torsional forces. This complication has lead to reduced use of the flap in clinical practice.
However, the incidence of fracture may be reduced by placing a bone plate, at the site of the
section defect, at the time of harvesting the flap. Both anterior and posterior surgical
approaches have been described. The strengthening effect of different types of plate and
position were studied using the sheep tibia as a model for the radial osteocutaneous donor
site.
Fifty matched pairs of adult sheep tibias were tested in torsion and 4-point bending. The
weakening effect of an osteotomy was first assessed by comparing an osteotomised bone with
an intact bone. Then pairs of bones with an osteotomy were compared with and without

Preface

xiii

reinforcement with different types of 3.5mm plate. The plate was placed in either the anterior
(over the defect) or posterior (on the intact cortex) position.
An osteotomised bone was significantly weaker than an intact bone. A plate in either the
anterior or posterior position significantly strengthened an osteotomised bone. The dynamic
compression plate was the strongest reinforcement in both torsion and bending. In torsion the
mean strength of the intact bone was 45% greater than after osteotomy (P=0.02). The
reinforced bone was on average 61% stronger than the unreinforced bone (P<0.001). Plating
restored the strength of the osteotomised bone to that of an intact bone (100%) with a plate in
either the anterior (97%) or posterior position (101%).
The tibia was able to withstand much greater loads in bending. In bending the mean
strength of the intact bone was 188% greater than after osteotomy (P=0.02). The reinforced
bone was on average 184% stronger then the unreinforced bone (P<0.001). The posterior
plate (80%) had a significantly greater effect than an anterior plate (46%) in restoring the
strength of the osteotomised bone to that of an intact bone (100%) (P=0.01).
The use of prophylactic internal fixation is recommended for the routine management of
the radial osteocutaneous donor site. A plate in the anterior or posterior position will
significantly strengthen the donor site and the surgeon may choose either site. The additional
strengthening effect of the posterior plate in bending is probably not relevant in clinical
practice as the radius is likely to fracture first as a result of lower torsional forces.
Chapter 8 - The radial osteocutaneous donor site is dramatically weakened at the site of
the osteotomy despite bevelling of the osteotomy cut and limiting the amount of bone
removed. Fracture results in considerable morbidity particularly if healing is not ideal. The
incidence of fracture remains relatively high, ranging from 0% to 66%, with a mean of 25%.
The largest studies have recently reported fracture rates of 15% and 18% and the incidence of
secondary surgery, for fractures, was also high, 67% and 46% respectively.
Clinical and biomechanical evidence now supports the routine use of prophylactic
internal fixation of the radial donor site with a dynamic compression plate to reduce the
incidence of fracture and the need for secondary surgery. The plate is effective in either an
anterior or posterior position and the choice of site is a matter of surgeon preference. This
chapter describes the clinical experience of the author with the anterior approach to internal
fixation. In a retrospective review of a series of 28 donor sites the incidence of fracture was
3.6% (1 out of 28). The single fracture was undisplaced and secondary surgery was not
indicated. In the literature the incidence of fracture with a plate in the anterior or posterior
position is relatively low. To date 268 donor sites have been managed with prophylactic
internal fixation and only 7 have fractured, of which only one underwent secondary surgery.
The incidence of reported complications related to the technique is also low and very few
plates have been replaced or removed.
The technique of prophylactic internal fixation allows the surgeon to safely harvest a
modest volume of bone, which in conjunction with the excellent soft-tissue paddle means that
the radial osteocutaneous flap retains a wide range of potential applications. However, in the
current practice of the author, and many others, the radial flap remains a compromise choice
or back-up flap. Recent indications have included relatively small defects of the maxilla,
older patients that are unlikely to undergo dental implantation, patient preference and if there

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Alexios P. Nikolakakos

is significant peripheral vascular disease or poor general health that will be exacerbated by
use of an alternative donor site.
Chapter 9 - Cancer of mouth and pharynx is one of the ten most common cancers in the
world. Detection of a precancerous or cancerous lesion at an early stage is an important factor
to improve 5-year survival rate of oral cancer. A comprehensive physical examination aided
by imaging techniques like computed tomography (CT), and magnetic resonance imaging
(MRI) are the standard evaluation tools in patients with oral, and pharyngeal neoplasms.
Although surgical biopsy and histopathology is considered gold standard for diagnosing the
oral lesions, it is impractical to routinely subject large number of patients to biopsy. Whereas
oral exfoliative cytology is a useful, economical and practical tool in the diagnosis of oral
dysplasia and carcinoma involving cheek, lip and tongue, similar role is played by fine needle
aspiration (FNA) cytology for minor salivary gland tumors and other solid neoplasms of the
palate, cheek and pharyngeal areas. By brush cytology a spectrum of oral lesions including
dysplasia, carcinoma in situ, occult and clinically evident squamous cell carcinoma can be
diagnosed. FNA cytology, which collects samples from areas difficult to reach by surgical
biopsy, can differentiate benign from malignant tumors and classify them into subtypes.
Whereas pleomorphic adenoma is a common benign tumor, adenoid cystic carcinoma,
mucous cell carcinoma, acinic cell carcinoma, malignancy in pleomorphic adenoma, and
polymorphous low-grade carcinoma are the malignant neoplasms detected in the minor
salivary glands. The other oral neoplasms detected by FNA are non-Hodgkin lymphomas,
and some rare primary malignancies like sarcomas and chordoma. Metastatic lesions in oral
cavity too have been diagnosed by FNA cytology. The efficacy of brush cytology in detection
of oral squamous cell carcinoma is very high in majority of reports, which is as follows:
sensitivity (84.4 9.97%), specificity (78.6 29.36%), positive predictive value (71.4
31.39%), and negative predictive value (83.0 16.40%). The sensitivity, specificity, and
diagnostic accuracy of FNA cytology for oral malignancies are also high. However, false
negative reports are possible with the oral brush cytology technique and some palatal salivary
gland tumors are difficult to diagnose by FNA cytology. In difficult situations, ancillary
techniques such as cytomorphometry, DNA-cytometry, immunocytochemistry, and molecular
tools act as valuable adjunct to cytodiagnostic techniques.
Chapter 10 - Surgery in the pterygopalatine fossa region presents anatomic and surgical
problems related to the difficulty of access. When a tumor in the pterygopalatine fossa
involves the maxilla and extends into the maxillary sinus and a tumor of the deep lobe of the
parotid gland extends into the pterygopalatine foss, extensive resection is often necessary.
Because of this, there has been a tendency either not to operate on these cases at all or else to
carry out simply a partial or piecemeal removal. The current underlying principle of skull
base approaches is to minimize brain retraction while maximizing skull base visualization.
This concept facilitates three-dimensional tumor resection, tumor margin verification, and
functional reconstruction with appropriate esthetic concerns. Current many approaches have
been used for the tumor of the middle skull base or the pterygopalatine fossa.
With advancements in imaging, diagnostic technology, diagnostic pathology, surgical
technology and instrumentation, reconstructive techniques, the surgery of the lateral cranial
base or the middle cranial base is now receiving significant attention and interest. It is
purpose of this paper to provide readers with an overall review of benign and malignant

Preface

xv

tumors occurring in the pterygopalatine fossa and adjacent structures of the pterygopalatine
foss: Recent advances of diagnosis and surgical management.
Chapter 11 - Oral cancer is the most common malignant neoplasm of the head and neck
and over half of the people who develop this cancer die within five years after the diagnosis.
Carcinogenesis is a highly complex process involving both environmental, mainly tobacco
and alcohol use, and inherited risk factors. In recent years, inter-individual genetic
differences and individual susceptibility to human cancer triggered by environmental
exposures has been studied. This environment-gene interaction in carcinogenesis is well
reflected by phase I and II enzymes that are involved in the metabolism of carcinogens.
Cytochrome P450 family of enzymes (CYP), involved in phase I, converts many carcinogens
into DNA-binding metabolites in target cells and can modulate intermediate effect markers
such as DNA-adducts. Phase II enzymes, including glutathione S-transferase (GST), Nacetyltransferase (NAT) and others, play important roles in protecting cells from DNA
damage by carcinogens and reactive oxygen species. Genetic alterations of these two classes
of enzymes have been considered as risk modifiers of some major tobacco-related cancers,
including oral cancer. The aim of this chapter is to review the molecular aspects of oral
cancer, emphasizing the role of phase I and II enzymes in oral carcinogenesis. Propositions
for further researches are highlighted.
Chapter 12 - Purpose: to investigate TP53 mutation and c-myc amplification as markers
for tumour aggressiveness in terms of tumour recurrence in OSCCs.
Methods and materials: Thirty one incident cases of oral squamous cell carcinomas were
studied for tumour relapse. The variables considered were demographic, clinical, pathological
and genetic.
Results: the mean age of 62.09 years (range 36 to 88). Seventeen patients (54.8%) were
smokers. The tongue was the main affected area (54.8%). No distant metastases could be
identified. Most patients were at early stages of the disease with moderately differentiated
tumours and of grade I in Anneroths malignancy scale. The oncogene study showed
abnormalities in both TP53 (6/31; 19.2%) and c-myc (4/31; 12.9%), that distributed as
follows: TP53+/c-myc+ (n=1; 3.2 %); TP53+/c-myc- (n=5; 16.1%); TP53-/c-myc+ (n=3; 9.7
%); TP53-/c-myc- (n=21; 67.7%). TP53 mutations were significantly more frequent in
advanced stages. Statistically significant differences in node status were identified in terms of
oncogene alterations. Multivariate Cox regression analysis recognized prognostic value for
recurrence for alterations of TP53 and c-myc (p<0.05).
Conclusions: TP53 mutation is related to advanced stage of oral cancer and suggests the
usefulness of analysis of TP53 mutations and c-myc amplifications in order to identify those
OSCCs more prone to relapse.
Chapter 13 - Coverage of facial defects is frequently challenging. Despite the numerous
flaps described, the search for additional flaps with good color match and minimal donor-site
morbidity continues and attempts to find valid options to free flaps perhaps overused in the
last two decades, in particular for soft tissue replacement of the moderate-to-large perioral
resections. The reconstructive research runs through the study of functional topographic or
regional anatomy with all the scientific and clinical implications. The article reviews the last
reports that are basically focused upon the arterial supply derived from neglected branches of
the superficial temporal artery (transverse facial artery, zygomatic-orbital artery, and middle

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Alexios P. Nikolakakos

temporal artery) or the terminal branches of the frontal terminal branch, from the variants of
the terminal facial artery and a definite collateral named cutaneous zygomatic branch, or from
the submental artery. The up-to-date research embraces the study of the cutaneous perforators
of the face. Relevant anterograde or reverse flaps, axial or perforator flaps, and monolayered
or multilayered composite flaps are discussed as current, original or still imaginative chances.
Moreover, for the realization of totally new flaps in the field of compound facial
reconstruction, clinical research efforts should tend to merge with the future perspective of
bone and soft-tissue engineering research.

In: Oral Cancer Research Advances


Editor: Alexios P. Nikolakakos, pp. 1-10

ISBN 978-1-60021-864-4
2007 Nova Science Publishers, Inc.

Chapter 1

EFFECTIVE ADMINISTRATION METHODS


OF 5-AMINOLEVULINIC ACID AS A
PHOTOSENSITIZER IN PHOTODYNAMIC
THERAPY FOR TONGUE TUMOR
Toshiyuki Ogasawara1,, Norio Miyoshi2, Kazuo Sano1,
Hidetaka Kinoshita1, Tetsushi Yamada1, Toru Ogawa1,
Kazuki Miyauchi1 and Yoshimasa Kitagawa3
1

Division of Dentistry and Oral Surgery, Department of Sensory and Locomotor


Medicine, 2Division of Tumor Pathology, Department of Pathological Sciences, School
of Medicine, Faculty of Medical Sciences, University of Fukui, Fukui 910-1193, Japan;
3
Oral Diagnosis and Oral Medicine, Department of Oral Pathobiological Science,
Graduate School of Dental Medicine, Hokkaido University, Sapporo 060-8586, Japan.

ABSTRACT
Objective: Photodynamic therapy (PDT) is a promising cancer treatment in which a
photosensitizing drug accumulates in tumors and is subsequently activated by visible
light of an appropriate wavelength matched to the absorption. The advantages of this
method, as compared to other conventional cancer treatment modalities, are its low
systemic toxicity and its ability to destroy tumors selectively. 5-aminolevulinic acid
(ALA)-induced protoporphyrin-IX (PpIX) has been used as a photosensitizer in PDT for
oral cancer, which advantage is low side effect compared to other photosensitizer. This
study investigates the optimal method of administrating ALA by analyzing PpIX
fluorescence in tongue tumor tissue.

Correspondence concerning this article should be addressed to: Toshiyuki Ogasawara, Division of Dentistry and
Oral Surgery, Department of Sensory and Locomotor Medicine, School of Medicine, Faculty of Medical
Sciences, University of Fukui, 23-3 Matsuokahimoaizuki, Eiheiji, Fukui 910-1193, Japan. Tel: 81-776-613111(ex2409); Fax: 81-776-61-8128; E-mail: ogasa@u-fukui.ac.jp.

Toshiyuki Ogasawara, Norio Miyoshi, Kazuo Sano et al.


Methods: PpIX intensities in the mouse (C3H) transplanted tongue cancer (NR-S1)
were compared with those in normal tongue after intraperitoneal (i.p.), oral (p.o.), or
topical administration of ALA. Tongues were sampled at various times after ALA
administration. PpIX intensities were obtained from frozen sections of each sample by
using a spectrophotometer.
Results: PpIX intensity in the tumor group peaked at 3 h after the i.p. and 5 h after
the p.o. administration of ALA, and these levels were about twice as high as those in the
normal group. Maximum PpIX accumulation in the tongue tumor tissue was seen at 5 h
after the oral administration of ALA. In contrast, the topical administration of 20% ALA
cream was associated with the lowest PpIX accumulation in the tumor throughout the
experiments.
Conclusion: Based on these results, most effective administration route of ALA was
oral administration and 5 h after administration was regarded to be the optimal time for
light irradiation in ALA-PDT.

Keywords: 5-aminolevulinic acid, protoporphyrin-IX, photodynamic therapy, tongue cancer,


spectroscopy, pharmacokinetic

INTRODUCTION
Surgery with radiotherapy and / or chemotherapy has been used as the conventional
treatment for tongue cancer. However, this treatment causes cosmetic and functional
disturbances, especially in the head and neck region.
Photodynamic therapy (PDT) is a promising cancer treatment in which a photosensitizing
drug accumulates in tumors and is subsequently activated by visible light of an appropriate
wavelength matched to the absorption [1]. The advantages of this method, as compared to
other conventional cancer treatment modalities, are its low systemic toxicity and its ability to
destroy tumors selectively [2]. Photofrin is the most widely used photosensitizer in clinical
PDT trials and is the only agent that has been approved for cancer treatment in many
countries. However, photofrin remains in the skin and causes photosensitivity lasting several
weeks, and the tumor selectivity of this agent is poor [3]. 5-aminolevulinic acid (ALA) is a
precursor of protoporphyrin IX (PpIX) in the biosynthetic pathway for heme, and PpIX is an
efficient photosensitizer. Today ALAPDT is successfully used for the treatment of a variety
of neoplastic and nonneoplastic diseases [4]. ALA- derived PpIX can be cleared from the
body within 24-48 h after systemic ALA administration [5], and because of this rapid
clearance, ALA-based PDT would reduce the risk of prolonged skin phototoxicity [6].
The kinetics of ALA-induced PpIX production in different tissues has been studied,
typically by means of fluorescence spectroscopic techniques [7]. However, the relationship
between the PpIX fluorescent accumulation in oral tumor tissue and the ALA administration
methods has not been elucidated. This study investigated the optimal method for
administrating ALA in PDT by analyzing PpIX fluorescence in tongue tumor tissue.

Administration Methods of ALA in Tongue Tumors

MATERIALS AND METHODS


Animals and Tumors
Male C3H/HeNCrj mice, 6-8 weeks old, 22-26 g (Charles River, Osaka, Japan) were
used in all experiments.
An NR-S1 mouse squamous cell carcinoma [8] (National Institute of Radiological
Sciences, Chiba, Japan) was transplanted to the mouse tongue, and when the tumor reached a
size of at least 3mm x 3mm, the photosensitizer was administered. The photosensitizer was
also administered to normal mice as a control.

Chemicals and ALA Administration Route


ALA was obtained as a hydrochloride in 98.0% pure powder from Cosmo Oil (Tokyo,
Japan).
In the intraperitoneal ALA administration group, ALA was freshly dissolved in 0.2 ml of
saline and injected at a dose of 250 mg/kg or 500 mg/kg.
In the oral ALA administration group, animals were given 250 mg/kg or 500 mg/kg of
ALA freshly dissolved in 0.5 ml of saline by means of a gastric tube.
In the case of topical ALA administration, an oil-in-water emulsion containing 20% ALA
was freshly prepared prior to use. After topical administration of ALA cream to the tongue,
animals were maintained under deep anesthesia by pentobarbital sodium to in order to
prevent the ALA cream from being washed out or swallowed. Furthermore, two kinds of
ALA ester derivative (ALA methyl ester and ALA pentyl ester; Cosmo Oil) were also
administrated and compared with topical application. These ALA ester derivatives, are more
lipophilic than ALA, and thus may penetrate more easily through the keratinized layer and
deeper into tumors than ALA itself [9].
Mice were killed at 1, 3, 4, 5, 6 and 8 h after ALA administration (n=4 animals/time
point), and mouse tongue samples were excised. Serial frozen sections (10m-thick) of each
sample were prepared for exact histological localization and quantitative measurement of the
concentration of PpIX. PpIX localization was confirmed by fluorescence microscopy
(PROVIS-AV80type; Olympus, Tokyo, Japan) by comparing with a hematoxylin-eosin (HE)-stained section. The wavelength width of the excitation filter was in the blue violet region
(400-440 nm), and the observation wavelength was more than 475 nm.

Quantitative Measurement of PpIX Fluorescence


ProtoporphyrinIX intensities in the mouse transplanted tongue cancer were compared
with those in the normal tongue after intraperitoneal, oral, or topical administration of ALA.
Levels of PpIX fluorescence were measured with a spectrophotometer [10].

Toshiyuki Ogasawara, Norio Miyoshi, Kazuo Sano et al.

Fluorescence emission spectra excited by 410 nm light were obtained from a total of 5
serial frozen sections (10m-thick) from each sample by using a spectrophotometer (850
type; Hitachi, Tokyo, Japan) equipped with a holder for the particle sample.
The subtracted spectrum was obtained by subtracting the background spectrum from the
sample raw spectrum.
A typical fluorescence spectrum from a tumor showed prominent emission bands at
=635 nm and=705 nm, which corresponded to the standard PpIX spectrum (Figure 1).
The PpIX concentration (M) was calculated from the fluorescence intensity at the 635
nm peak of the sample emission spectrum and a calibration curve of the known
concentrations of standard PpIX solution. Standard PpIX aqueous solution was prepared with
phosphate-buffered saline solution, cationic surfactant, acetyl-trimethyl-ammonium-bromide
and PpIX.

Figure 1. Fluorescence emission spectra excited by 410 nm light are obtained from a total of 5 serial
frozen sections (10m-thick) from each sample by using a spectrophotometer. The no.3 subtracted
spectrum was obtained by subtracting the no.2 background spectrum from the no.1 sample raw
spectrum. In addition, we confirmed that the no.3 subtracted spectrum pattern corresponded to the no.4
standard PpIX spectrum.

Statistics
Groups of normally distributed data were compared using Student`s t-test, while the nonparametric Mann-Whitney test was otherwise employed. Values of <0.05 were considered to
indicate statistical significance.

Administration Methods of ALA in Tongue Tumors

RESULTS
The fluorescence microscopic image showed that the red fluorescence emission of PpIX
was distributed strongly and homogeneously in the tongue tumor tissue at 5 h after oral
administration of ALA. However, PpIX accumulation was not seen in the necrotic area of the
tumor tissue. In addition, there was very weak PpIX accumulation in the normal lingual
muscle after administration of ALA.

Figure 2. The PpIX concentration (M) was calculated from the fluorescence intensity at the 635 nm
peak of the subtracted spectrum and a calibration curve of the known concentrations of standard PpIX.

Figure 3. Fluorescence image and corresponding HE-stained image of tongue tumor tissue at 5 h after
oral administration of ALA.

The tumor group showed constantly higher PpIX intensities than the normal group
throughout the experiments following the i.p. and p.o. administration of ALA. PpIX intensity
in the tumor group peaked at 3 h after the i.p. and 5 h after the p.o. administration of ALA,
and these peak values were about twice as high as those in the normal group. However, the

Toshiyuki Ogasawara, Norio Miyoshi, Kazuo Sano et al.

PpIX intensitiy in the tumor group was not enhanced by an increase in the administrated dose
of ALA from 250 mg/kg to 500 mg/kg (Figures 4 and 5). Maximum PpIX accumulation in
the tongue tumor tissue was seen at 5 h after the oral administration of ALA (Figure 6). In
contrast, the topical administration of 20% ALA cream was associated with the lowest PpIX
accumulation in the tumor throughout the experiments (Figure 7). Furthermore, the topical
administration of 20% ALA ester derivatives cream (ALA methyl ester and ALA pentyl
ester) also resulted in low PpIX accumulation in the tumor, which was not different from the
case of topical administration of 20% ALA cream.

Figure 4. PpIX intensity in tongue tissue after intraperitoneal administration of ALA.

Figure 5. PpIX intensity in tongue tissue after oral administration of ALA.

Administration Methods of ALA in Tongue Tumors

Figure 6. PpIX intensity in tongue tumor tissue after various types of ALA administration.

Figure 7. PpIX intensity in tongue tissue after topical administration of ALA.

DISCUSSION
If the photosensitizer that is administered before light illumination accumulates more
highly in tumor tissue, the efficacy of PDT for cancer might be improved. Although photofrin
should be used only via intravenous administration, ALA can be used via various

Toshiyuki Ogasawara, Norio Miyoshi, Kazuo Sano et al.

administration routes. There have been numerous studies on ALA-induced PpIX fluorescence
after a variety of administration routes in various organs. Systemic (intravenous or oral)
ALA-based PDT has also been reported for treatment of oral neoplastic lesions [5,11].
However, the optimal administration method of ALA in PDT for oral cancer is still not
established. The present study was carried out to determine the most effective route and
optimal timing of ALA administration with respect to subsequent therapeutic illumination.
In most studies, the techniques used were based on a noninvasive method using a
spectrofluorometer or the chemical technique of high pressure liquid chromatography
(HPLC) to measure in vivo PpIX fluorescence after administration of ALA.
Spectrofluorometry is simple and noninvasive, but can detect only surface emission of the
tissue. HPLC can measure the whole tissue, but the resulting values are an average for the
whole tissue (more than 1 g) and have no relation to the histopathological findings.
Furthermore, the techniques required for this method are complicated [10]. We confirmed the
direct detection the PpIX concentrations from the frozen section always in combination with
histological staining using cryosamples.
Previous studies have shown that the peak of PpIX fluorescence intensity varied between
1 and 6 h after ALA administration in different tissues [5,12,13]. Our present results showed
that PpIX intensity in the tongue tumor tissue peaked at 3 h after the intraperitoneal (i.p.)
administration of ALA. Another study has also shown that PpIX fluorescence in rat tongue
cancer reached a maximum intensity at 3 h after ALA i.p. administration [13]. However, in
this study, the maximum PpIX accumulation in the tongue tumor tissue was confirmed at 5 h
after oral administration of ALA. Although the reason for this finding is unclear, Mustajoki et
al. [14] have shown that a high serum ALA level can be achieved in a human volunteer by
continuous enteral infusion of ALA solution. Loh et al. [15] reported that the temporal
fluorescence kinetics after oral administration were comparable with that after intravenous
injection in the stomach, colon and bladder mucosa of normal rats. Oral administration is
considered to be simpler, and it does not require full buffering. ALA can be undertaken by
patients themselves, prior to therapy and without supervision [15]. The results of the present
study suggested that oral administration was the most effective administration method in
ALA-PDT for oral cancer.
Furthermore, there was no obvious difference of PpIX intensity between 250 mg/kg and
500 mg/kg after both i.p. or p.o. administration of ALA. Accumulation of PpIX in tongue
tumor tissues reaches a plateau after administering at least 250 mg/Kg doses of ALA. Ma et
al. [13] reported that early malignant lesions in rat tongue showed complete response to the
i.p. administration of ALA-based PDT at both 250 mg/Kg and 1000 mg/Kg. These results
suggested that it is not necessary to administer a greater amount of ALA in order to achieve
sufficiently high PpIX levels suitable for PDT in oral cancer.
Topical ALA-based PDT has been widely used in treating neoplastic lesions of the skin
and bladder [4], because local administration of ALA might increase the PpIX concentration
in the tumor without unwanted general side effects. It is known that topical application of an
oil-in-water emulsion of ALA on the skin lesion can permit penetration of ALA into the
lesion and allow synthesis of PpIX [16,17]. Recently, in cases of oral cancer, topical
administration of ALA as a rinsing solution has also been tried for ALA-photodynamic
diagnosis. However, because there has been no report on the use of topical administration

Administration Methods of ALA in Tongue Tumors

ALA-PDT for oral cancer, we here tried topical administration of 20% ALA cream for tongue
tumor. Our results showed that PpIX intensity in the tongue tumor after topical administration
of ALA cream was not enhanced compared with that in the normal tongue. Several ALA
esters have been synthesized, and are more lipophilic than ALA. This higher lipophilicity
might result in better penetration into the skin, higher PpIX levels and a more uniform and
deeper PpIX distribution [18]. Therefore, we attempted to apply the two kinds of ALA esters
for topical administration. However, ALA esters also did not enhance the PpIX intensity in
the tongue tumor tissue. Although the reason for this result is unclear, the neutral pH of saliva
might cause the immediate degeneration of ALA in the oral mucosa [15,18]. Based on these
results, 5 h after oral administration of ALA was regarded to be the optimal time for light
irradiation in ALA-PDT.

ACKNOWLEDGEMENT
This work was supported by a Grant-in-Aid for Scientific Research (C) (15592104) from
Japan Society for the Promotion of Science.

REFERENCES
[1] Date M, Sakata I, Fukuchi K et al. (2003). Photodynamic therapy for human oral
squamous cell carcinoma and xenografts using a new photosensitizer, PAD-S31. Lasers
Surg Med 33: 57-63.
[2] Gaullier JM, Berg K, Peng Q et al. (1997). Use of 5-aminolevulinic acid esters to
improve photodynamic therapy on cells in culture. Cancer Res 57: 1481-1486.
[3] Peng Q, Berg K, Moan J et al. (1997). 5-aminolevulinic acid-based photodynamic
therapy: principles and experimental research. Photochem Photobiol 65: 235-251.
[4] Peng Q, Warloe T, Berg K et al. (1997). 5-Aminolevulinic acid based photodynamic
therapy. Cancer 79: 2282-2308.
[5] Grant WE, Hopper C, MacRobert AJ et al. (1993). Photodynamic therapy of oral
cancer:photosensitization with systemic aminolevulinic acid. Lancet 342: 147-148.
[6] Webber J, Kessel D, Fromm D (1997). Side effects and photosensitization of human
tissue after aminolevulinic acid. J Surg Res 68: 31-37.
[7] Stolic S, Tomas SA, Roman-Gallegos E et al. (2002). Kinetic study of -Ala induced
porphyrins in mice using photoacoustic and fluorescence spectroscopies. J Photochem
Photobiol B: Biol 68: 117-122.
[8] Usui S, Urano M, Koike S et al (1976). Effect of PSK, a protein polysaccharide, on
pulmonary metastasis of C3H mouse squamous cell carcinoma. J Natl Cancer Inst 56:
185-187.
[9] Juzenas P, Shafaei S, Moan J et al. (2002). Proitoporphyrin IX fluorescence kinetics in
UV-induced tumours and normal skin of hairless mice after topical application of 5aminolevulinic acid methyl ester. J Photochem Photobiol B: Biol 67: 11-17.

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[10] Miyoshi N, Ogasawara T, Nakano K et al. (2004). In light of recent developments,


application of fluorescence spectral analysis in tumor diagnosis. Appl Spectrosc Rev 39:
437-455.
[11] Fan KN, Hopper C, Speight PM et al. (1996). Photodynamic therapy using 5aminolevulinic acid for premalignant and malignant lesions of oral cavity. Cancer 78:
1374-1383.
[12] Henderson BW, Vaughan L, Bellnier DA et al. (1995). Photosensitization of murine
tumor, vasculature and skin by 5-aminolevulinic acid-induced porphyrin. Photochem
Photobiol 62: 780-789.
[13] Ma G, Ikeda H, Inokuchi T et al. (1999). Effect of photodynamic therapy using 5aminolevulinic acid on 4-nitroquinoline-1-oxide-induced premalignant and malignant
lesions of mouse tongue. Oral Oncol 35: 120-124.
[14] Mustajoki P, Timonen K, Gorchein A et al. (1992). Sustained high plasma 5aminolevulinic acid concentration in a volunteer: no porphyric symptoms. Euro J Clin
Invest 22: 407-411.
[15] Loh CS, MacRobert AJ, Bedwell J et al. (1993). Oral versus intravenous administration
of 5-aminolaevulinic acid for photodynamic therapy. Br J Cancer 68: 41-51.
[16] Kennedy JC, Pottier RH (1992). Endogeneous protoporphyrin IX, a clininically useful
photosensitizer for photodynamic therapy. J Photochem Photobiol B 14: 275-292.
[17] Szeimies RM, Sassy T, Landthaler M (1994). Penetration potency of topical applied 5aminolevulinic acid for photodynamic therapy of basal cell carcinoma. Photochem
Photobiol 59: 73-76.
[18] van den Akker JTHM, Lani V, Star WM et al (2000). Topical application of 5aminolevulinic acid hexyl ester and 5-aminolevulinic acid to normal nude mouse skin:
differences in protoporphyrin IX fluorescence kinetics and the role of the stratum
corneum. Photochem Photobiol 72: 681-689.

In: Oral Cancer Research Advances


Editor: Alexios P. Nikolakakos, pp. 11-50

ISBN 978-1-60021-864-4
2007 Nova Science Publishers, Inc.

Chapter 2

RELATIONSHIPS BETWEEN BIOLOGICAL AND


CLINICOPATHOLOGIC FEATURES IN
ESOPHAGEAL CARCINOMA
Takuma Nomiya, Kenji Nemoto and Shogo Yamada
Department of Radiation Oncology, Yamagata University School of Medicine, Japan.

ABSTRACT
The clinical characteristics and radiosensitivity of esophageal cancer differ
individually, even in individuals with the same histopathological type. Several
investigators have reported that prognosis of patients with esophageal carcinoma differs
according to its macroscopic appearance, and it has been shown that macroscopically
infiltrative type (like scirrhous type in gastric cancer) is radioresistant and that its
prognosis is extremely poor compared to that of macroscopically localized type. The
major factors that are thought to have a potent impact on radiosensitivity of a tumor are
cell proliferation activity, tumor oxygenation, genetic repair, and intrinsic
radiosensitivity.
In our study, Ki67, CD34, vascular endothelial growth factor (VEGF), thymidine
phosphorylase (TP) and metallothionein (MT) expressions and microvascular density
were evaluated using surgically resected esophageal squamous cell carcinomas without
preoperative treatment. Microvascular density (MVD) was evaluated in different ways:
average-MVD was estimated as an index of tumor oxygenation, and highest-MVD was
estimated as an index of the most active neovascularization in the tumor.
In the analysis of proliferation activity (Ki67 labeling index), proliferation activity of
the radiosensitive group of esophageal carcinomas was higher than that of the
radioresistant group of esophageal carcinomas. In the analysis of microvascular density,
average-MVD of macroscopically infiltrative type was significantly lower than that of
localized type, whereas highest-MVD of macroscopically infiltrative type was
significantly higher than that of localized type. The VEGF expression level of infiltrative
type was significantly higher than that of localized type. A significant positive
correlation was found between highest microvascular density and VEGF expression, and

12

Takuma Nomiya, Kenji Nemoto and Shogo Yamada


a borderline significant negative correlation was found between average microvascular
density and expression of VEGF. TP expression showed a positive correlation with
highest-MVD, but the correlation was not as strong as that of VEGF expression. In the
analysis of MT, which is recognized as a protein that has a radioprotective effect,
expression of MT was not increased in esophageal carcinoma of the radioresistant group.
Metallothionein expression was increased in the radiosensitive group. Furthermore,
expression of MT was not increased in preoperatively treated esophageal carcinomas.
These results suggested that MT does not have a great impact on clinical radiosensitivity
in esophageal carcinoma and also suggested that MT expression is not induced by
therapeutic irradiation or anticancer agents.
The results suggest that radioresistant type is poorly oxygenated by low averageMVD, includes a large amount of hypoxic fraction that is refractory to treatment, shows
induction of angiogenic factors and activated neovascularization, and has a high rate of
hematogenous metastasis. Tumor oxygenation and presence of a hypoxic fraction seem
to have great importance for curability of esophageal carcinoma compared to various
other factors we have investigated.

1. BACKGROUND AND EPIDEMIOLOGY


Esophageal cancer is a malignancy that has an extremely poor prognosis. Esophageal
carcinoma arises from squamous cells of the esophagus. Squamous cell carcinoma accounts
for most esophageal malignancies, and adenocarcinoma is the second-most frequently
occurring esophageal malignancy. The proportions of histological type differ according to
country and race. A recent survey has shown that the ratios of squamous cell carcinoma and
adenocarcinoma in esophageal malignancies in North America are 50-60% and 40-50%,
respectively, whereas the ratios of squamous cell carcinoma and adenocarcinoma in Japan are
more than 90% and less than 5%, respectively [1,2].
Smoking, alcohol, hot meals, having Barrett esophagus, and genetic inheritance are
thought to be the causes of esophageal carcinoma. The difference in the proportions of
squamous cell carcinoma and adenocarcinoma of the esophagus might be due to genetic
differences between races and to environmental factors, though the reasons have not been
clarified.
Recent clinical studies on esophageal squamous cell carcinoma have been shown that the
5-year survival rate of patients with esophageal squamous cell carcinoma regardless of stage
is about 20-30%. Esophageal carcinoma has thus been a refractory disease despite recent
advances in multimodal treatments. The reasons for the extremely poor prognosis are that
esophageal carcinoma easily extends to the submucosal layer, results in wide lymphogenous
metastases from the neck to abdomen, easily invades adjacent critical organs, easily
debilitates the host due to esophageal stenosis and eating disorder, and is difficult to resect
completely. It has been shown that prognostic factors of gastro-intestinal malignancies
include depth of invasion, extent of lymphogenous metastases, presence of distant metastasis,
tumor length, site, age, and extent of lymphatic/ blood vessel invasion [3-5].
In case of gastro-intestinal malignancies, it is known that the clinical characteristics and
malignant potential of the tumor differ according to its macroscopic appearance. For
example, gastric cancer is roughly classified into localized type and invasive type and is

Relationships Between Biological and Clinicopathologic Features

13

classified according to presence of ulceration [6]. In gastric cancer, it has been reported that
not only the abovementioned depth of invasion, extent of lymphogenous metastases, and
lymphatic/ blood vessel invasion but also macroscopic appearance greatly affect patients'
prognosis [3,4,7-11]. The prognosis of patients with macroscopically infiltrative type of
gastric cancer is generally regarded as poor, and the prognosis of patients with Borrmann's
type IV (scirrhous type, diffusely infiltrating type) is extremely poor [7,12,13]. These
findings suggest that the morphologic difference shows difference in biological features such
as frequency of metastasis, invasiveness, and refractoriness to treatment.
There are guidelines for treatment of esophageal carcinoma in Japan in which
macroscopic appearance is defined according to Borrmann's classification for gastric cancer
[14-16]. Many studies have shown that there is a difference in prognoses of patients with
esophageal carcinoma according to macroscopic type in Japan [5,13,17-21]. Similar to gastric
cancer, the prognosis of patients with macroscopically infiltrative type of esophageal
carcinoma is unfavorable, and the prognosis of patients with diffusely infiltrative type
(similar to Borrmann's type IV) is extremely poor [13].

Figure 1. A) Survival curves of patients with esophageal carcinoma treated by radiotherapy alone
according to macroscopic types (Stage II-III). The prognosis of patients with macroscopically
infiltrative type of esophageal carcinoma is significantly poorer than that of patients with localized type.
B) Response to radiotherapy alone. Response rate (CR+PR) of infiltrative type is also significantly
worse than that of localized type.

2. TREATMENT OUTCOME AND CLINICAL FEATURES


Figure 1A shows survival curves of patients with stage II-III esophageal squamous cell
carcinoma treated with radiotherapy alone in our institution from 1981 to 1991 (n=156; 144
males, 12 females; median age, 68.5 years; age range, 46-91 years) [22]. The prognosis of
patients with macroscopically infiltrative type of esophageal carcinoma was significantly
poorer than that of patients with macroscopically localized type (p<0.0001). Figure 1B shows
treatment responses of 134 of the 156 patients for whom data were available. The response to
radiotherapy of infiltrative type was significantly worse than that of localized type (p=0.001).
As shown in Figure 1A-B, our data also suggest that clinical characteristics and prognosis of

14

Takuma Nomiya, Kenji Nemoto and Shogo Yamada

patients differ according to macroscopic appearance in esophageal carcinoma. It can be said


that macroscopically infiltrative type of esophageal carcinoma not only has an extremely poor
prognosis but is also more radioresistant than localized type. However, it is not clear whether
the unfavorable prognosis of infiltrative type is due to metastasis tendency, poor local
controllability, or both of these. Based on these differences in clinical features of esophageal
carcinoma, we investigated tumor biological differences from the viewpoint of biological
characteristics such as cell proliferation activity, microvascular density, activity of
angiogenesis, and intrinsic radiosensitivity.

3. SIGNIFICANCE OF PROLIFERATION ACTIVITY IN


MALIGNANCIES
In our studies, cell proliferation activity, microvascular density, expressions of
angiogenesis factors, and expressions of factors that affect intrinsic radiosensitivity were
evaluated using surgically resected esophageal carcinoma specimens, not biopsy specimens.
Ki67 labeling index was used for evaluation of cell proliferation activity of the tumor by
immunohistochemistry. Ki67 antigen is one of the proteins that are expressed in the nuclei of
proliferating cells [23]. The Ki67 antibody combines with a nuclear antigen that is present in
proliferating cells but absent in resting cells. According to results of past experiments, Ki67
nuclear antigen is present in S, G2 and M phases of the cell cycle but is absent in G0 phase
[24,25]. Ki67 positivity in G1 phase differs depending on the cell line. Ki67 antibody can
sensitively detect cells in the proliferating phase. The ratio of Ki67-positive cells in
malignant tissue indicates growth rate of the tumor, and Ki67 labeling index is widely used as
an index of proliferation activity of the tumor in clinical pathology.
Expression of Ki67 antigen in various tissue has been reported, and it has been shown
that Ki67 positivity of a malignant tumor is generally higher than that of normal tissue [2632].
Many studies have shown a high Ki67 labeling index in various malignancies: brain
tumor [31], head and neck cancer [33,34], breast cancer [28,35-37], gastric cancer [30,38-42],
bladder cancer [27,43], rectal cancer [32] and prostatic cancer [44]. Ki67 (MIB-1) labeling
index is regarded as one of the indexes that show degree of malignancy of tumor in practical
work. In general, it is thought that a tumor that shows a higher level of proliferation activity
has a higher degree of malignancy.
Past studies have shown relationships between high Ki67 labeling index and poor
prognosis in breast cancer [28,35-37,45-47], bladder cancer [27,43], brain tumor [31], and
prostatic cancer [44]. However, other studies have shown that there are no relationships
between high Ki67 labeling index and poor prognosis in head and neck cancer [34], rectal
cancer [32], and gastric cancer [40-42]. A high level of cell proliferation activity of a
malignant tumor does not therefore appear to be simply correlated with poor prognosis of
patients. It appears that there is no correlation between high Ki67 labeling index and poor
prognosis for malignancies that have arisen from oral-digestive system (gastric cancer, rectal
cancer, head and neck tumor, etc.), and there might be something like organ dependence in
the relationships between high proliferation activity level and prognosis. In the relationships

Relationships Between Biological and Clinicopathologic Features

15

between other factors, histological grade [27,47], depth of tumor invasion [43], tumor size
[38], blood vessel invasion [41], expression of p53 [29], and lymphogenous metastases
[33,35,36] have been shown to be factors that correlate with Ki67 labeling index, but a
definite theory has not yet been established.
On the other hand, from the viewpoint of conventional radiation biology, it is known that
the higher the level cell proliferation activity becomes, the more radiosensitivity increases.
Based on this theory, a tumor with a high level of proliferation activity seems to be
radiosensitive but to have a higher degree of malignancy. It has not been determined whether
a high level of proliferation activity of a tumor is an advantage or disadvantage for patients
with esophageal carcinoma.
In our study, Ki67 labeling index was evaluated using sections of macroscopically
localized type and infiltrative type of esophageal carcinoma without preoperative treatment.
Ki67 labeling index was estimated independently by two skilled pathologists who had
received no clinical information other than the name of the disease, and several microscopic
fields were selected at random and the Ki67-positive cell rate (Ki67 labeling index) was
calculated by counting 1000 malignant cells. The average value of the two Ki67 labeling
indexes was taken as the Ki67 labeling index of the specimen (There was good agreement
between the Ki67 labeling indexes calculated by the two pathologists: mean S.D. values of
the two Ki67 labeling indexes were 53.3 20.7% and 55.1 21.3%, respectively (p=N.S.) and
the mean difference between two Ki67 indexes of the same specimen was 8.4 5.9%.).
The mean (S.D.) Ki67 labeling index of all specimens was 54.2% (20.4), and there
was a large variation (range, 8.5-88.5%). In comparison according to macroscopic type, Ki67
labeling index of the infiltrative type was significantly lower than that of the localized type
(46.9 20.4% vs. 61.5 18.1%, p=0.022, Figure 2A). The values of Ki67 labeling index
showed a large variation from 8.5% to 88.5% in this study, and the values showed an almost
normal distribution.

Figure 2. Comparison between localized type and infiltrative type in Ki67 labeling index (A), averageMVD (B), highest-MVD (C) and VEGF expression (D). Black circles and black bars: localized type,
white circles and white bars: infiltrative type, Ki67: Ki67 labeling index, average-MVD: averagemicrovascular density, highest-MVD: highest-microvascular density, VEGF: vascular endothelial
growth factor. Average-MVD: sum of vessel counts in four randomly selected fields in the tumor tissue

16

Takuma Nomiya, Kenji Nemoto and Shogo Yamada

at low magnification. Highest-MVD: microvessel count at high magnification in the area of highest
neovascularization in the tumor.

According to past studies on Ki67 positivity, the ranges of Ki67 positivity were 11-18%
in brain tumor [31], 14-64% in esophageal cancer [48], 4-87% in gastric cancer [41], 7-70%
in colorectal cancer [32], 10-50% in cervical cancer [49], 0-17% in bladder cancer [27,43],
and 0-17% in prostatic cancer [44]. A certain variation of cell proliferation activity is seen in
almost all malignancies, but a very large variation is seen in malignancies of the digestive
system. The distribution of Ki67 labeling indexes in this study nearly corresponded with
these in past studies, but the reason for these variations is unknown. It seems to be important
that there was a significant difference between cell proliferation activities in macroscopically
localized type and infiltrative type of esophageal carcinoma. A high Ki67 labeling index was
expected in infiltrative type, which has an unfavorable prognosis, but contrary to that
expectation, the Ki67 labeling index of the infiltrative type was significantly lower than that
of the localized type.
Several studies have also suggested that there is no relationship between high Ki67
labeling index and poor prognosis in esophageal carcinoma [50-52]. It has been reported that
the prognosis of patients with a high Ki67 labeling index was more favorable than that of
patients with a low Ki67 labeling index who received radiotherapy for esophageal carcinoma
[51]. However, there was a large overlap of Ki67 labeling index between localized type and
infiltrative type of esophageal carcinoma in this study, and it cannot be concluded that
patients with a high Ki67 labeling index have good prognosis. The mechanism and cause of
the difference in cell proliferation activity is discussed in the following section with the
results of other factors.

4. ANGIOGENESIS AND PIVOTAL ROLE OF VEGF


Tumor cells keep growing while they are within a microscopic size, but the tumor
requires oxygen and nutrition when it grows larger than a certain size. Growth of a tumor in
diameter temporarily stops when it has reached a certain size, and then angiogenesis precedes
the next tumor growth in diameter [53]. Several conditions are required for the process of
angiogenesis, including demand for oxygen by the tumor, release of angiogenic factors from
tumor cells, attenuation of angiogenesis-inhibiting factors by the host, and migration of
endothelial cells. These mechanisms consist of interaction between tumor cells, host tissue,
and endothelial cells, and then formed blood vessels accelerate further growth of the tumor
[54-56].
Various angiogenic factors, including bFGF (basic fibroblast growth factor), PD-ECGF
(platelet-derived endothelial cell growth factor) and PlGF (placenta growth factor) have been
identified, but VEGF (vascular endothelial growth factor) is considered to be one of the
strongest angiogenic factors [57]. VEGF is a 34-42-kDa heparin-binding, dimeric, disulfidebonded glycoprotein. VEGF is known as VPF (vascular permeability factor), and it has been
shown that VEGF is a potent mediator of angiogenesis and vascular permeability [58-60].
Expression of VPF/VEGF is seen in various animals, various normal organs, and various

Relationships Between Biological and Clinicopathologic Features

17

neoplasms. VEGF stimulates secretion of fibrinogen to the extra-vascular matrix and


contributes to the formation of an interstitial fibrin structure of the tumor. Activities of the
fibrinolytic system and plasminogen affect interstitial fibrin formation, and the activity differs
depending on the tumors. Development of tumor stroma activates inflammatory reaction and
migration of macrophages [61,62]. There is something in common between tumor stroma
generation, tumor neovascularization, and the process of wound healing [63].
bFGF is thought to be one of the potent angiogenic factors, but VEGF, unlike bFGF,
specifically interacts with endothelial cells. It is known that VEGF, unlike the angiogenic
factor PD-ECGF, stimulates not only migration of endothelial cells but also proliferation of
endothelial cells [64,65]. Several studies in which the expressions of VEGF and bFGF were
compared showed that VEGF is more inducible and highly expressed by hypoxia than is
bFGF in experiments using xenografts of melanoma and pancreatic tumor cell lines [66-68].
Other experiments showed that a monoclonal antibody specific for VEGF, the inactivated
recombinant soluble human VEGF receptor, and a dominant-negative mutant of the VEGF
receptor inhibited angiogenesis of a tumor [69-71]. These findings suggest that VEGF plays
an important role in angiogenesis, although there are many angiogenic factors, including
bFGF, Ang1/2 and PD-ECGF [72].

5. MICROVASCULAR DENSITY, OXYGEN TENSION, AND


HYPOXIC FRACTION
Tissue oxygen tension affects many intracellular environments such as glucose
metabolism, cell cycle, and angiogenesis. Oxygen tension is one of the important parameters
in tumor tissue, and various studies have been carried out to determine oxygen tension in a
tumor. Evaluation of microvascular density by immunohistochemistry [73-76], evaluation of
hypoxia by a hypoxia marker such as misonidazole and pimonidazole [66,77-80], evaluation
of blood perfusion by a perfusion marker Hoechst33342 [81], and measurement by
Eppendorf oxygen electrodes [75,82,83] have been performed for evaluation of tumor oxygen
tension. Oxygen tension in a tumor seems to be dependent on microvascular density and
blood vessel perfusion, and it has been shown that there is a significant correlation between
microvascular density and oxygen tension in human tumors [74,84].
It had been thought that cells within a certain distance from vessels were oxygenated and
that cells more distant from vessels were in a hypoxic state. However, not all hypoxic cells
exist in uniform distance from blood vessels because of heterogeneity in the actual tumor
[85,86]. Results of some studies have shown that there is a correlation between microvascular
density and tumor oxygen tension [75,76,82], while results of other studies have shown that
there is no correlation between microvascular density and tumor oxygen tension [87-89]. A
correlation between microvascular density and oxygen tension was seen in cervical cancer,
no correlation was seen in head and neck cancer, and both results that there were significant
correlation between microvascular density and oxygen tension and that there were no
significant correlation between them were observed in experiments using melanoma
xenografts. These different findings suggest that there is heterogeneity depending on the
organ or cell line. However, biopsy specimens were used for evaluating microvascular

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Takuma Nomiya, Kenji Nemoto and Shogo Yamada

density in the above studies, and it is problematic whether a biopsy specimen displays
characteristics of the whole tumor. The uncertainty of these different results of the studies
may caused by methodological problems.
On the other hand, an experiment in which the proportion of hypoxic cells and distance
from blood vessels were determined using breast cancer xenografts showed that the
proportion of the radioresistant hypoxic fraction increased in an area more than 140 m from
blood vessels [90]. To sum up, although there is individual heterogeneity due to oxygen
diffusion, blood perfusion, acidosis and glucose metabolism, it can be concluded that the
proportion of hypoxic cells increases with increase in intervascular distance in tumor tissue.

6. MICROVASCULAR DENSITY AND VEGF EXPRESSION


We discussed the concept of vessel density that affects tumor oxygenation and amount of
the hypoxic fraction of a tumor in the previous section. There seems to be another
significance in microvascular density. A local "vascular hot spot" induced by overexpression
of an angiogenic factor in tumor tissue seems to have another importance from the viewpoint
of tumor biology. Weidner et al. suggested this concept of "vascular hot spot" early on [73].
It is thought that a "vascular hot spot" indicates pathological angiogenic activity of the tumor.
The way to make vessel count to evaluate angiogenic activity is different from the way to
make vessel count to evaluate tumor oxygen tension. After the area of highest
neovascularization in a tumor specimen has been identified by observation under a
microscope at low magnification, vessel count is then made at high magnification in the
vascular hot spot. According to their study, a significant positive correlation between VEGF
expression and microvascular density (in the vascular hot spot) has been shown in human
breast cancer [91]. It is thought that microvessels in the vascular hot spot are induced by
VEGF overexpression. Correlations between VEGF expression and neovascularization have
also been found in breast cancer, hepatocellular carcinoma (HCC), ovarian cancer, germ cell
tumor, and melanoma xenografts [53,66,68,92-95].
In an experiment, the growth of VEGF-transfected xenografts was significantly faster
and the microvascular density of VEGF-transfected xenografts was significantly higher than
those of control xenografts [76]. Another study has suggested that genetic overexpression of
VEGF is more important than hypoxia-induced upregulation [66]. To sum up, microvascular
density of a local "vascular hot spot" in tumor tissue significantly correlates with VEGF
expression and indicates activity of tumor angiogenesis. In analysis of tumor tissue obtained
from human non-small cell lung cancer (NSCLC), expression level of a hypoxia marker
(hypoxia-inducible factor: HIF family) was very high in specimens with high microvascular
density or low microvascular density but was low in specimens with medium microvascular
density [96]. These findings suggest that there are two patterns of vessel structure: one affects
oxygenation and formation of a hypoxic fraction of the tumor, and the other is hypoxiainduced and angiogenic factor-activated microvessels.

Relationships Between Biological and Clinicopathologic Features

19

7. RESULTS OF OUR INVESTIGATIONS


We evaluated microvascular density of the tumor in two ways and expression of VEGF
by immunohistochemistry using surgically resected human esophageal squamous cell
carcinoma specimens. 1) Average microvascular density (a-MVD): a-MVD was estimated as
an index of tumor oxygen tension or amount of oxygenated cells. Using sections stained with
anti-CD34 antibody, four areas including tumor tissue were randomly selected, and
microvessel counts were made at low magnification, and then the sum of microvessels in the
four fields was taken as a-MVD of the section. 2) Highest microvascular density (h-MVD): hMVD was estimated as an index of the most active neovascularization in the tumor. Using
sections stained with anti-CD34 antibody, the area of highest neovascularization in the tumor
was identified and microvessel count of that field were made at high magnification.
Microvessels were counted on the basis of the methods described by Weidner [73]. 3) VEGF
expression: Several microscopic fields were selected at random, and VEGF-positive cell rate
was calculated by counting 1000 malignant cells.
In the analysis of average microvascular density, a-MVD of macroscopically infiltrative
type, which is thought to be radioresistant and have a poor prognosis, was significantly
higher than that of localized type (mean S.D.: 370 102 vs. 475 91, p=0.0014, Figure 2B),
[22]. In contrast, h-MVD of infiltrative type was significantly higher than that of localized
type (150 75 vs. 82 33, p=0.0006, Figure 2C). In the analysis of VEGF expression, VEGF
expression of infiltrative type was significantly higher than that of localized type (67.4 15
vs. 44.4 13, respectively, p<0.0001, Figure 2D). There was significant and strong positive
correlation between VEGF expression and h-MVD (r=0.73, p<0.0001, Figure 3A). A
negative correlation was found between a-MVD and VEGF expression, but it was borderline
significant (r=0.30, p=0.06, Figure 3C).

Figure 3. A) Positive correlation between VEGF expression and highest-MVD (microvascular density).
B) Negative correlation between VEGF expression and average-MVD. C) Positive correlation between

20

Takuma Nomiya, Kenji Nemoto and Shogo Yamada

TP expression and highest-MVD. D) Survival curves according to VEGF expression. The patients were
divided into two groups of equal size according to VEGF expression. E) Survival curves according to
TP/VEGF expressions. TP(-)/VEGF(-): n=11, TP(-)/VEGF(+) or TP(+)/VEGF(-): n=18,
TP(+)/VEGF(+): n=11.

Different results have been obtained regarding the relationship between microvascular
density and tumor oxygen tension, the correlation between microvascular density and
hypoxic fraction, the relationship between microvascular density and radiosensitivity, and the
relationship between microvascular density and VEGF expression [66,74-76,80-84,87-89,9295,97]. One possible reason for the difference in results is the difference in methods used to
evaluate microvascular density. Another possible reason for heterogeneity of data is the
difference in type of study: an experimental study using an animal model, an in vitro study, or
a clinical study using human tumor. There seems to be a tendency that uniform data can
easily be obtained in an experimental study using cell lines with identical clones or using a
model of uniform animal xenografts, whereas it is difficult to obtain uniform data in a clinical
study using specimens from human tumors that consist of heterogeneous clones. Accurate
definition in evaluating microvascular density should be required for avoiding confusion.
The results of our study showed paradoxical data that the inverse vessel counts were
obtained between average microvascular density and highest microvascular density in
comparison between macroscopically localized type and infiltrative type of esophageal
carcinoma. Based on known information, these data can be interpreted as follows. 1) The
number of hypoxic cells increases with increase in intervessel distance, and low averageMVD in tumor tissue therefore leads to an increase in the amount of hypoxic fraction. Low
average-MVD in infiltrative type of esophageal carcinoma suggests low oxygen supply to the
tissue and the presence of a hypoxic fraction. The finding of a lower Ki67 labeling index
(low cell proliferation activity) in the infiltrative type supports the presence of larger amount
of hypoxic fraction.

Figure 4. Presumptive mechanism of the formation of a hypoxic fraction in tumor tissue and
angiogenesis from the results of our study and known information. 1) There is underdeveloped tumor
vascularization (as suggested by low average-MVD). 2) The hypoxic fraction increases (as suggested

Relationships Between Biological and Clinicopathologic Features

21

by low Ki67 labeling index). 3) VEGF is induced by hypoxia. 4) Development of immature


microvessels is induced by VEGF overexpression (as suggested by high highest-MVD). 5) These
microvessels with high permeability increase the incidence of hematogenous metastasis (which seems
to be one of the reasons for unfavorable prognosis).

2) Hypoxia-inducible genes and hypoxia-inducible proteins are expressed in the hypoxic


fraction. The potent angiogenic factor VEGF is one of the hypoxia-inducible proteins
[67,72,98,99]. The results showing low average-MVD and high VEGF expression level in
infiltrative type of esophageal carcinoma suggest the presence of hypoxia and hypoxiainducible VEGF expression (Figure 4A). It was not statistically strong, but negative
correlation between a-MVD and VEGF expression is thought to be one of the supporting
findings of relationship between increase in hypoxic fraction and increase in expression of
angiogenic factor.
3) Angiogenesis is activated by VEGF overexpression, and it was found that irregular
microvessels locally and densely developed in tumor tissue (Figure 4B). Low average-MVD,
high VEGF expression level, and high highest-MVD were seen in the infiltrative type of
esophageal carcinoma. The strong positive correlation between VEGF expression and
highest-MVD also seems to support the above mechanism. Figure 5(A-F) shows microscopic
findings of typical cases of macroscopically localized type (A-C) and infiltrative type (D-F).
The microscopic photos in A-C and those in D-F are of the same specimens: Figures A and D
show average-MVD (CD34 stain at low magnification), Figures B and E show VEGF
expression and Figures C and F show highest-MVD (CD34 stain at high magnification).

Figure 5. Microscopic findings of typical cases of macroscopically localized type (A-C) and infiltrative
type (D-F) of esophageal carcinoma. The microscopic photos in A-C and those in D-F are of the same
specimens. A/D: CD34 stain at low magnification (for estimating average-MVD), B/E: VEGF stain,
C/F: CD34 stain at high magnification (for estimating highest-MVD). The case of macroscopically
infiltrative type of esophageal carcinoma (D-F) shows low average-MVD (D), overexpression of VEGF
(E) that seems to be induced by hypoxia, and activated neovascularization (F) that seems to be VEGFinduced vascularization.

22

Takuma Nomiya, Kenji Nemoto and Shogo Yamada

4) Furthermore, these immature vessels with high permeability stimulate the occurrence
of hematogenous or lymphogenous metastasis [91,92]. The presence of a hypoxic fraction not
only leads to radioresistance but also increases the frequency of hematogenous metastasis.
The mechanism such like this (abovementioned 1-4) seems to be one of the causes of
radioresistance and unfavorable prognosis in the infiltrative type of esophageal carcinoma.
There were large variations in the results of Ki67 labeling indexes and microvessel counts in
our studies. This is because the data were obtained from human specimens consisting of
heterogeneous clones and conditions. Although it is difficult to obtain uniform data in a study
using human tumor specimens consisting heterogeneous subjects, it is noteworthy that
significant differences were found despite the large variations.

8. HYPOXIC FRACTION AND CELL CYCLE


It has generally been thought that the amount of quiescent cells increases in the hypoxic
fraction [100,101]. The results of a study in which hypoxic fractions were compared using
melanoma xenografts showed there were relationships between low microvascular density,
increase in hypoxic fraction, and increase in the proportion of quiescent cells by flow
cytometry [97]. However, it has been reported that not all cells in the hypoxic fraction are in
quiescent phase and that some cells proliferate slowly even in the hypoxic area. Rate of
proliferating cells in the hypoxic fraction seems to differ depending on cell line, but it is
thought that malignant cells stop or delay their cell cycle at G0/G1 phase or at G2/M phase
[101,102].
The results of our study showed that the cell proliferation activity level of infiltrative
type of esophageal carcinoma, which seems to be radioresistant and have an unfavorable
prognosis, was significantly lower than that of localized type. This was unexpected because it
is generally thought that a tumor with a high growth rate is highly malignant. However, to
sum up following factors such as increase in hypoxic fraction, cell cycle stop/delay,
overexpressions of hypoxia-inducible proteins (VEGF) and being radioresistance, it is
consistent that infiltrative type of esophageal carcinoma that is thought to be radioresistant
and to be refractory to treatment could be poorly oxygenated and show low level of
proliferation activity.
A study has shown that mutation of p53 causes alteration in the cell cycle under a
condition of hypoxia and that this leads to resistance to hypoxia [103]. It has also been
reported that normal endothelial cells in S phase increase under a condition of hypoxia and
that the cell cycle of normal endothelial cells is delayed but does not stop under a condition
of hypoxia [104]. This seems to be rational mechanism, but it is interesting that endothelial
cells, which play a pivotal role in angiogenesis, do not stop their cell cycle and continue to
proliferate even under a condition of hypoxia.

Relationships Between Biological and Clinicopathologic Features

23

9. HYPOXIA-INDUCIBLE FACTORS (HIFS) AND METABOLIC


CHANGES
Metabolism under a condition of hypoxia that differs from metabolism under the
condition of normoxia. Under hypoxic conditions, an increase in blood perfusion is seen in
normal tissue by its ability of autoregulation [105]. However, it is not known whether tumor
tissue has the ability to autoregulate blood perfusion. It is generally thought that angiogenesis
and oxygen supply cannot meet oxygen consumption by preceding tumor growth, and then
the hypoxic fraction in the tumor usually increases with tumor growth.
An in vivo experimental study showed that there is a gradient of oxygen tension in tumor
tissue and that increases in lactate content and hypoxic fraction correlate with decrease in
metabolic activity [106]. Synthesis of angiogenic factors such VEGF, NADPH oxidase
metabolism, cytochrome P450 metabolism and synthesis of heme proteins are changed in
cells to adapt to hypoxia [107-109]. HIFs act as promoters or mediators of activation of
transcription of these hypoxia-inducible proteins. Hypoxia-inducible factor-1 alpha (HIF1alpha) and hypoxia-inducible factor-2 alpha (HIF-2alpha) are promoters involved in
angiogenesis. HIF-1 consists of HIF-1alpha of 120 kDa and hypoxia-inducible factor-1 beta
(HIF-1beta) of 94 kDa, and they form heterodimerized basic-helix-loop-helix proteins
containing a Per-AHR-ARNT-Sim (PAS) domain.
HIF-1 proteins are rapidly expressed in the cytoplasm under a condition of hypoxia, and
HIF-1alpha and HIF-1beta are then heterodimerized and are transported into the nucleus,
where they initiate transcription of hypoxia-inducible proteins. HIF-1alpha is rapidly
inactivated by hydroxylation or degraded by ubiquitin-mediated proteolysis under the
condition of normoxia (Figure 6, left). Under a condition of hypoxia, HIF-1alpha is stabilized
by avoiding inactivation by prolyl hydroxylase domain-containing enzyme (PHD) or factor
inhibiting HIFs (FIH) and starts transcription of specific genes (Figure 6, right). It is also
known that HIFs are induced in conditions of anemia, high altitude, and wound healing [110114]. HIF-1 itself is known as a hypoxia marker, and its corresponding expression with other
hypoxia markers such as pimonidazole or CAIX has been reported [115]. HIF-1alpha is
unique to HIF-1 whereas HIF-1beta (aryl hydrocarbon receptor nuclear translocator: ARNT)
can dimerize with the aryl hydrocarbon receptor [116]. Rapid expression of HIF-1alpha
subunit in the nucleus is thus thought to be important for its activation [96,117,118]. HIF-1
has independently been identified and reported as endothelial PAS-1 (EPAS-1), HIF-like
factor (HLF), a member of the PAS superfamily 2 (MOP2), and HIF-related factor (HRF), but
they have high homology [119-121].
HIF-1 plays an important role not only in activation of angiogenesis but also in
regulation of erythropoietin, glycolytic enzyme, and nitric oxide synthesis [122-128], and
there are two groups of hypoxia-inducible proteins, HIF-1-dependent inducible proteins and
HIF-1-independent inducible proteins [129]. The important point is that HIF-1 is a positive
mediator of VEGF, which is the most potent angiogenic factor. Overexpression of VEGF and
HIF-1 in the hypoxic fraction and correlation of VEGF expression and HIF-1 expression have
been shown in both in vitro and in vivo studies [67,72,96-99]. HIF-1 might be a target of
anti-angiogenic treatment.

24

Takuma Nomiya, Kenji Nemoto and Shogo Yamada

Figure 6. Mechanism of HIF-1alpha regulation in normoxia and hypoxia. HIF-1alpha: hypoxiainducible factor-1 alpha, HIF-1beta: hypoxia-inducible factor-1 beta, OH: hydroxyl residue, pVHL: von
Hippel Lindau protein, Ub: ubiquitin, 26S-P: 26S proteasome, PHD: prolyl hydroxylase, FIH: factorinhibiting HIFs, p300/CBP: coactivators of HIF-1alpha.

10. REPORTS ON REDUNDANCY


Several studies have shown significant delay in growth of HIF-1alpha knockout tumors
[130-133]. From result showing that there was no delay in tumor growth in the absence of
HIF-1 but presence of VEGF, tumor growth delay is thought to be caused by VEGF
downregulation due to absence of HIF-1alpha [134]. Interestingly, expression levels of HIF1-dependent proteins such as VEGF, phosphoglycerate kinase 1 (PGK1) and Glut-1 were
significantly decreased by HIF-1 knockout, but the expressions of these proteins did not
completely disappear [130,132,133].
Recently, the existence of redundancy, which compensates for loss of a major pathway
by activating other pathways, has been shown in several human metabolic pathways
[135,136]. It also seems to be a redundancy that there are HIF-1alpha-dependent proteins
(such as p53, p21 and Bcl-2) and HIF-1alpha-independent proteins (such as p27 and
GADD153) among hypoxia-inducible proteins [133]. As compensatory pathways that induce
VEGF expression other than hypoxia-HIF-1alpha pathways, these factors are also known as
upregulators of VEGF expression such as interferon-gamma (IFN-gamma), Escherichia coli
lipopolysaccharide (E. coli LPS), and inducible nitric oxide synthase (iNOS) [137].
HIF-2alpha is one of positive mediators of angiogenic factors that seems to compensate
for major angiogenic factor mediators. HIF-2alpha, which has a function similar to that of
HIF-1alpha, has recently attracted attention, and a correlation between microvascular density
and expression of HIF-2alpha has been reported. However, there are paradoxical reports of a
correlation between HIF-2alpha expression and high microvascular density and of a
correlation between HIF-2alpha expression and low microvascular density [136,138]. These
paradoxical data can be explained from the viewpoint of the relationship between low
microvascular density and increase in hypoxic area and from the viewpoint of the relationship
between HIF/VEGF expressions and HIF/VEGF-induced neovascularization as well as the
results of our studies.

Relationships Between Biological and Clinicopathologic Features

25

11. HYPOXIC FRACTION AND RESPONSE TO TREATMENT


Although there has been controversy for many years regarding the degree of a hypoxic
fraction in a tumor, it is now thought that most tumors include a hypoxic fraction to some
degree. Although the degree of the hypoxic fraction differs depending on the cell line, the
existence of an unexpectedly large hypoxic fraction in human tumor xenografts in mice has
been shown [85,139-141]. Direct measurement of oxygen tension by a computerized
electrode has indicated the existence of a hypoxic fraction in human cervical cancer and
human breast cancer [115,142,143].
It has been suggested that hypoxic status of cells is one of the causes to be refractory to
radiotherapy or chemotherapy, and several studies have shown a relationship between
hypoxic status of cells and resistance to anticancer agents [140,144-146]. There are some
theories that the refractoriness is due to weakness of the genome or due to metabolic
alteration under a condition of hypoxia, but the truth is unknown. It is not clear whether the
hypoxic fraction is acute or chronic and whether the cell cycle stops or progresses slowly
under a condition of hypoxia. It has been reported that the hypoxic fraction increases with
tumor growth in the early stage but that the size of the tumor and proportion of the hypoxic
fraction are not correlated when the tumor exceeds a certain volume and that growth rate is
also not correlated with amount of the hypoxic fraction [139].
On the other hand, it has been suggested that refractoriness to treatment depends on the
amount of severe hypoxic fraction rather than that of mild to moderate hypoxic fraction in the
tumor [88,83]. In an experimental study using human breast cancer xenografts, although
normal tissue was well oxygenated by hyperbaric oxygen, the hypoxic fraction in the tumor
was increased under a condition of hyperbaric oxygen [147]. A severe hypoxic fraction is
resistant to reoxygenation independent of the presence of glucose [148]. It has also been
shown that blood transfusion improved the hypoxic fraction and increased average oxygen
tension in a tumor but that the proportion of severe hypoxic fraction did not change [75].
These results suggesst that the hypoxic fraction in a tumor might be more resistant to
oxygenation than our expectation.
Tumor tissue under a condition of severe hypoxia with low level of angiogenic activity
becomes necrotic, and it is therefore questionable whether a severe hypoxic fraction around
the necrotic area has the ability to retain clonogenicity [149]. Further investigation is needed
to clarify whether a severe hypoxic fraction or a moderate hypoxic fraction is more
influential. Negative correlations between microvascular density/angiogenic activity and proapoptotic factors (caspase-3, Fas ligand) have been shown in human head and neck cancer
and human NSCLC [150,151]. A relationship between low microvascular density and
presence of necrosis has been shown in pancreatic cancer, and hypoxia-induced necrosis was
observed in human melanoma xenografts after administration of a VEGF-neutralizing
antibody [67,92]. Based on results of those studies, tumor tissue with a high level of
angiogenic activity can be more resistant to apoptosis or necrosis under a condition of severe
hypoxia. One of the possible mechanisms of radioresistance or chemoresistance is that
hypoxia decreases metabolic activity and that hypoxia-induced angiogenesis prevents cells
from apoptosis or necrosis.

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Takuma Nomiya, Kenji Nemoto and Shogo Yamada

12. MICROVASCULAR DENSITY AND RADIOSENSITIVITY


According to conventional radiation biology, radiosensitivity is affected by cell
repopulation (proliferation), (re)oxygenation, DNA repair and cell redistribution [152].
Oxygen tension in tumor tissue is one of the main factors affecting radiosensitivity [153]. The
presence of a hypoxic fraction had been known since 1950's, and it is known that hypoxic
cells are 3-times more radioresistant than are aerobic cells [85,103,140]. Tumor oxygenation
is affected by vascular density, blood vessel perfusion, cell proliferation, and apoptotic
activity [144], and there are wide variations in degree of tumor oxygenation, the degree of
tumor oxygenation differing individually even in the same histological type [87,154,155].
Some rare tumors show uniform oxygenation within a certain distance from blood
vessels [149], but most solid tumors show a heterogeneous distribution of aerobic/hypoxic
fractions, and there is no relationship between oxygenation and clinical characteristics such
as tumor volume and clinical stage [82,85,86,142,143]. As stated above, most tumors include
a hypoxic fraction to some degree, and the amount of the hypoxic fraction increases in
proportion to distance from blood vessels, though there is heterogeneity [79,81,86,147]. The
results of an experiment on radiosensitivity using melanoma xenografts suggested that
resistance of the hypoxic fraction to irradiation is due to a correlation between amounts of the
hypoxic fraction before and after irradiation [87]. Some studies have shown that the amount
of hypoxic fraction before treatment is more important than the amounts during treatment and
after treatment in human cervical cancer [83]. On the other hand, Wouters et al. suggested
that response to conventionally fractionated irradiation is highly dependent upon the cells at
oxygen levels intermediate between fully oxygenated and hypoxic [156]. In several clinical
studies, correlations between microvascular density, radiosensitivity, and local controllability
have been shown in human head and neck tumors [80,157,158]. The results of our studies
have also shown relationships between microvascular density, hypoxic fraction and
radiosensitivity. Selective sensitization of the hypoxic fraction therefore seems to be an
option for tumor control.

13. VEGF EXPRESSION AND RADIOSENSITIVITY


Although the relationship between the presence of a hypoxic fraction and radiosensitivity
has been mentioned above, the direct effect of VEGF expression on radiosensitivity of
esophageal squamous cell carcinoma has not been shown. We investigated the effects of
VEGF expression and VEGF receptor expression on radiosensitivity using transfected cell
lines. VEGF gene and VEGF-receptor 1 (Flt-1) gene were transfected to a cell line of human
esophageal squamous cell carcinoma (KYSE50, Figure 7). Three cell lines were established
by transfection: 1) VEGF(-)/Flt-1(-) as a control, 2) VEGF(+)/Flt-1(-) and 3) VEGF(+)/Flt1(+). Figure 8 shows the survival curves of the control, VEGF(+)/Flt-1(-) and VEGF(+)/Flt1(+) cells after irradiation (unpublished). No significant difference was found between the
radiosensitivities in vitro of the 3 cell lines. Consistent with the generally accepted view, the
results of this study suggested that the mechanism by which VEGF overexpression promotes

Relationships Between Biological and Clinicopathologic Features

27

in vivo tumor growth is alteration in the microenvironment by neovascularization and not a


direct effect of VEGF on the cell cycle or radiosensitivity in esophageal carcinoma.
It has been reported that VEGF-transfected xenografts showed significantly more rapid
growth than did control xenografts in vivo and that the rapid growth was inhibited by
inhibition of the angiogenic factor [159,160]. It has been reported that there was no
significant difference between radiosensitivity of a VEGF-transfected cell line and that of a
control cell line in fibrosarcoma [159], whereas there was significant difference between
radiosensitivity of normal endothelial cells with an angiogenic factor and that of normal
endothelial cells without an angiogenic factor [161]. Results of our study and other studies
suggest that VEGF does not have a direct effect on tumor cells and that both increase in
radioresistance and VEGF upregulation seem to occur as a consequence of the presence of a
hypoxic fraction.

Figure 7. A: control cell line (transfected without VEGF insertion). B: VEGF-transfected cell line
labeled with Fluorescein streptavidin. C: control cell line (transfected without Flt-1 insertion). D: Flt-1transfected cell line labeled with Texas Red streptavidin.

14. BIOLOGICAL FUNCTIONS OF VEGF RECEPTORS


VEGF and its receptors, VEGF-receptor 1 (Flt-1) and VEGF-receptor 2 (FLK/KDR),
have recently been shown to have an important role in normal and pathological angiogenesis
[162,163]. Flt-1 and FLK/KDR, members of the tyrosine kinase family, contain seven
immunoglobulin-like loops and are expressed in various human tissues and cell lines (Figure
9) [164,165]. VEGF-receptors, Flt-1 and FLK/KDR, are specifically expressed in vascular
endothelial cells in normal and tumor tissues, and Flt-1 is expressed in monocytes and
macrophages [166-168]. Expression of Flt-1 has been found in endothelial cells around tumor
tissue but not in endothelial cells in normal tissue. In the area of tumor tissue with high

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Takuma Nomiya, Kenji Nemoto and Shogo Yamada

vascular density, overexpressions of both Flt-1 and FLK/KDR have been observed
[95,169,170].

Figure 8. Survival curves of VEGF(-)/Flt-1(-) (control) cell line, VEGF(+)/Flt-1(-) cell line and
VEGF(+)/Flt-1(+) cell line. The cells were irradiated with single doses of 2, 4, 6, 8, 10 and 12 Gy, and
cell survival was estimated by clonogenic colony assay. Cell lines were cultured at 37C in air with
20% O2.

Figure 9. Mechanism of VEGF-receptor activation. VEGF-receptors are dimerized when ligand-bound,


and the intracellular domain is then activated and intracellular signaling pathways are activated. VEGF:
vascular endothelial growth factor, PlGF: placenta growth factor, PI3K: phosphoinositide 3-kinase,
AKT: one of protein kinases, Src: one of protein kinases.

Relationships Between Biological and Clinicopathologic Features

29

It has been shown that autophosphorylation of Flt-1 is very weak despite dimerization
with high affinity to its ligand VEGF. On the other hand, FLK/KDR shows stronger
autophosphorylation by binding to VEGF despite having much less affinity to VEGF. It has
been reported that mouse embryos homozygous for a targeted mutation in the Flt-1 locus died
of abnormal vascular formation that was excessive formation rather than undevelopment
[171-174]. Both Flt-1 and FLK/KDR seem to play essential roles in embryonal
vasculogenesis. However, based on the results of these studies, it is thought that FLK/KDR is
a positive regulator of angiogenesis in the VEGF/VEGF-receptor system and that Flt-1 is a
negative regulator in this receptor system.

15. THYMIDINE PHOSPHORYLASE AND ANGIOGENESIS


PD-ECGF, an angiogenic factor, has been shown to have catalytic activity for thymidine
phosphorylation [175]. It has been revealed that PD-ECGF is identical to thymidine
phosphorylase (TP) by an experiment that the protein obtained from a PD-ECGF-transfected
cell line was identified by an anti-TP antibody [176,177]. TP does not enhance tumor growth
in vitro but enhances tumor growth in vivo [178]. This different effect of TP between in vitro
and in vivo suggests that the enhancement of tumor growth by TP is due to activation of
angiogenesis.
TP increases thymidine uptake of endothelial cells and has a strong effect on cellular
thymidine metabolism. It is known that TP metabolites, dR-1-P (2-deoxyribose-1-phosphate)
and 2dR (2-deoxyribose), stimulate migration of endothelial cells [179]. TP has angiogenic
activity by stimulating migration of endothelial cells, but TP does not stimulate cell
proliferation because it is not a growth factor [178,180,181]. Although expression of TP has
hardly been seen in normal tissue, expression of TP has been shown in various malignancies
and normal tissue around malignancies such as hepatocellular carcinoma, (HCC) [182,183],
NSCLC [184,185], gastro-intestinal cancer [186,187], renal cell carcinoma, (RCC) [188],
breast cancer [178], bladder cancer [189], and ovarian cancer [190].
We investigated TP expression, which seems to have a different angiogenic pathway
from VEGF signaling pathways, in esophageal carcinoma. As shown in Figure 3C, a positive
correlation between TP expression and highest microvascular density was found [191]. This
correlation was borderline significant, and the correlation was not as strong as that between
VEGF expression and highest-MVD mentioned above. The difference in statistical
significance between TP and VEGF in our study suggests the angiogenic potential of TP and
VEGF. No significant correlation was found between TP expression and average
microvascular density or between TP expression and Ki67 labeling index. No definite
evidence of a relationship between hypoxia and TP expression was found in our study either.
Several investigators have reported a positive correlation between local microvascular
density and TP expression in various malignancies [183,188,192,193], and the results for TP
expression in our study are consistent with the results of these studies. It is clear that TP has
angiogenic activity in tumor tissue by inducing migration of endothelial cells. There is
controversy regarding the correlation between expressions of TP and VEGF, but no

30

Takuma Nomiya, Kenji Nemoto and Shogo Yamada

significant correlation was found between expressions of TP and VEGF in our studies
[194,195].

16. MICROVASCULAR DENSITY, VEGF/TP EXPRESSION


AND PROGNOSIS
We analyzed prognosis of the patients in this study according to VEGF/TP expression
using Cox's proportional hazards model and Kaplan-Meier methods [191]. The patients were
divided into two groups of equal size according to VEGF expression and also into two groups
of equal size according to TP expression. The prognosis of patients in the high VEGF
expression group was significantly poorer than that of patients in the low VEGF expression
group (3-year survival rate: 30% vs. 70%, p=0.01, Figure 3D), and the prognosis of patients
in the high TP expression group tended to be poorer than that of patients in the low TP
expression group, though the difference was not statistically significant (3-year survival rate:
40% vs. 62%, p=0.1, figure omitted).
The patients were also divided into three groups according to the combination of VEGF
expression and TP expression: TP(-)/VEGF(-) group (patients with low level of TP
expression and low level of VEGF expression), TP(-)/VEGF(+) or TP(+)/VEGF(-) group
(patients with low level of TP expression and high level of VEGF expression or patients with
high level of TP expression and low level of VEGF expression), TP(+)/VEGF(+) group
(patients with high level of TP expression and high level of VEGF expression). Three-year
survival rates of the TP(-)/VEGF(-) group, TP(-)/VEGF(+) or TP(+)/VEGF(-) group, and
TP(+)/VEGF(+) group were 73%, 55%, and 18%, respectively (p=0.005, Figure 3E). The
prognosis of patients in the TP(+)/VEGF(+) group was much worse than that of patients in
the other groups, and the prognosis of patients in the TP(-)/VEGF(-) group was significantly
better than that of patients in the other groups. No significant differences were found in
results of prognostic analyses according to gender, age, site, tumor length, and Ki67 labeling
index. Microvascular density and macroscopic type were excluded from prognostic analysis
because of their strong correlation with VEGF expression.
These results suggest that overexpression of VEGF is one of negative prognostic factors
and that prognosis of patients with co-expression of VEGF and TP is extremely poor. It has
been shown that expression of VEGF is one of steps of malignant transformation in various
malignancies [58,170,196-200]. Several clinical studies have also shown that expression of
VEGF is one of negative prognostic factors in brain tumor [64], gastric cancer [198], RCC
[201], breast cancer [94], and esophageal cancer [200]. From the viewpoint of TP expression,
some studies have shown no relationship between prognosis and expression of TP [182,183],
but several studies have shown that expression of TP is one of negative prognostic factors in
RCC [188], cervical cancer [202], colon cancer [192], and breast cancer [195]. From these
results and our results, it seems that TP expression is not a strong prognostic factor. Several
investigators have suggested that co-expression of VEGF and TP is a clinically unfavorable
prognostic factor in some malignancies [195,203]. The results of these studies are consistent
with results of our study.

Relationships Between Biological and Clinicopathologic Features

31

However, there is advantage of TP expression in metabolic activity, that is, TP catalyzes


conversion of 5'-DFUR to 5-FU in tumor tissue [176,204]. It has been shown that TPtransfected cells became 160-fold more sensitive to 5'-DFUR (5'-deoxy-5-fluorouridine,
prodrug of 5-FU) than did control cells [181,205]. It is therefore thought that an anticancer
drug is more effective in tumors that have high expression levels of TP than in tumors with
low TP expression levels. Several clinical and experimental studies have shown the outcomes
of anticancer treatment using 5-FU or its precursor [176,204-206]. Evaluation of TP and
VEGF expressions seems to be useful for predicting patients' prognosis and for selecting
effective anticancer agents.
On the other hand, microvascular density of a tumor also correlates with prognosis of
patients. There have been many studies in which microvascular density was evaluated as the
most active neovascularization in the tumor (highest-MVD), and these studies have suggested
that high vascularity is associated with significantly unfavorable prognosis for patients with
NSCLC [207], breast cancer [73,207], bladder cancer [169], RCC [201], and various
malignancies [208,209]. It has been reported that increase in the hypoxic fraction,
overexpression of angiogenic factors and prominent vascularization increase the probability
of hematogenous metastasis. Stackpole et al. investigated the relationship between amount of
hypoxic fraction and probability of metastasis using melanoma xenografts [97], and Weidner
et al. showed a relationship between higher microvascular density and increase in frequency
of metastasis using human breast cancer [91]. Rofstad et al. also showed relationships
between increase in hypoxic fraction, overexpression of angiogenic factors, and increased
frequency of lung metastasis using melanoma xenografts [92]. Well-developed microvessels
with high permeability, so-called "vascular hot spot", are an open gate to systemic
circulation. The mechanisms of metastasis are complex and have not been completely
revealed, but overexpression of angiogenic factors, migration of endothelial cells,
neovascularization, enhancement of vascular permeability, and migration of cancer cells are
thought to be essential for the occurrence of metastasis. To sum up, the presence of a hypoxic
fraction and activated angiogenesis seem to affect not only tumor controllability but also the
incidence of metastasis and prognosis of patients.

17. INTRINSIC RADIOSENSITIVITY AND METALLOTHIONEIN


EXPRESSION
Metallothioneins (MTs) are a family of low-molecular-weight (6-7 kDa) intracellular
proteins, and they are cysteine-rich proteins with high affinity for both essential and nonessential metals. There are four isoforms of MT (MT-I, -II, -III and -IV) in human organs,
and MT-I and -II are the most predominant isoforms. It has been about 50 years since MT
was first identified as a Cd-, Zn-binding protein in the horse kidney. It is known that MT-I
mRNA and MT-II mRNA are induced not only by heavy metals but also by many nonmetallic compounds including ethanol, alkylating agents, and physical or chemical oxidative
stress conditions [210,211]. In addition to its principal roles in detoxification of potentially
toxic heavy metals and in regulation of the homeostasis of essential trace metals, MT can also
act as an antioxidant and a free radical scavenger [210-212]. Cellular damage caused by

32

Takuma Nomiya, Kenji Nemoto and Shogo Yamada

radiation is mainly oxidative damage due to the formation of several types of oxygen free
radicals caused by ionization of water molecules. In an experimental study, radioresistance
acquired by MT induction by pretreatment of Cd salts was seen in a human epithelial line and
a mouse fibroblast line [213]. Similarly, another experiment demonstrated that MT-induced
tumor cells acquired radioresistance [214]. However, several investigators have suggested
that MT induction does not have an effect on radioresistance of tumor cells [215-218], and it
has remained controversial whether MT expression affects the clinical radiosensitivity of
tumor or not. MT induction by therapeutic irradiation is interesting because radiotherapy is
widely used for cancer treatment.
We evaluated the expression of MT by immunohistochemistry using specimens of
resected esophageal squamous cell carcinoma and estimated the significance of expression of
MT in clinical radiosensitivity [219]. The resected esophageal carcinomas included 20
macroscopically localized type without preoperative treatment (PT), 20 macroscopically
infiltrative type without PT and 5 macroscopically infiltrative type with PT (40 Gy of
preoperative irradiation and preoperative chemotherapy with CDDP and 5-FU). Analyses of
patients with esophageal carcinoma treated by only radiotherapy showed that
macroscopically infiltrative type had a significantly poor prognosis and was significantly
resistant to radiotherapy (Figure 1). If MT expression has a great impact on clinical
radiosensitivity in esophageal carcinoma, a higher level of MT expression level should be
seen in infiltrative type than in localized type, and if MT induction by therapeutic irradiation
affects clinical radiosensitivity, a higher level of MT expression level should be seen in
patients who have received preoperative treatment. Figure 10 shows the MT expressions in
each group. MT expression level of macroscopically localized type without PT was
significantly higher than that of infiltrative type without PT (p=0.026) and was significantly
higher than that of infiltrative type with PT (p=0.024). No significant difference was found
between the group without PT and the group with PT in infiltrative type of esophageal
carcinoma.
The results of this study showed, contrary to expectation, that MT expression level in the
infiltrative type of esophageal carcinoma was lower than that in the localized type of
esophageal carcinoma, suggesting that the cause of radioresistance in infiltrative type is
unlikely to be the radioprotective effect of MT expression. Similarly, the results of analysis
according to presence of preoperative treatment showed that it is unlikely that a tumor
acquires resistance to treatment due to MT induction by therapeutic irradiation or anticancer
agents. The results of our study are consistent with results of studies showing no correlation
between radiosensitivity and MT expression. It seems unlikely that MT will be a target
molecule of cancer therapy in the future.

Relationships Between Biological and Clinicopathologic Features

33

Figure 10. Expressions of MT. Localized type without preoperative treatment (PT): n=20, infiltrative
type without PT: n=20, infiltrative type with PT: n=5. Patients in the infiltrative type with PT group
received preoperative irradiation of 40 Gy and preoperative chemotherapy (CDDP and 5FU). High MT
expression level was not seen in radioresistant infiltrative type. There were no findings that MT
expression was induced by preoperative treatments.

18. CONCLUSIONS AND FUTURE VIEWS


We have investigated relationships between clinical features and biological features from
the viewpoints of cell proliferation activity, tumor oxygenation, angiogenic activity and
intrinsic radiosensitivity using resected human esophageal squamous cell carcinoma
specimens. The results of our studies suggested that the factors that have the greatest effects
on controllability and prognosis are tumor oxygenation and amount of hypoxic fraction. The
significance of a hypoxic fraction in tumors has been suggested previously, before the results
of our studies have suggested that the presence of a hypoxic fraction is one of the most
important factors. Many trials have already been performed, but to overcome hypoxic
fraction in tumor is a problem of the moment to be solved.
Cell proliferation activity was shown to be correlated with microvascular density, and not
all of the specimens with unfavorable prognosis showed a high level of cell proliferation
activity in our study. Although stress-inducible proteins that are biosynthesized and act as
radioprotectants are not only MTs, expression of stress-inducible protein was not consistent
with clinical radiosensitivity. Investigation of DNA repair, which has an important role in
radiosensitivity, was not performed in our previous studies. Activity of DNA repair is
essential for estimating radiosensitivity, and we will investigate this in a future study. Though

34

Takuma Nomiya, Kenji Nemoto and Shogo Yamada

angiogenic activity does not have a direct effect on radiosensitivity, it plays an important role
in tumor growth and occurrence of metastasis.
Inhibition of angiogenic factors is attracting much attention, and clinical trials have been
started. However, there has been no report of angiogenic factor-inhibiting agents dramatically
improving patients' prognosis. As stated above with regard to redundancy, metabolic
pathways in humans do not seem be so simple that inhibition of one factor can inhibit one
metabolic system. However, elucidation of cytokines and intracellular signaling pathways has
been rapidly progressing, and all of the proteins and metabolic systems in humans should be
elucidated in the near future.
Generally, basic experiments using a single cell line are suitable for determining the
influence of a difference in one factor because heterogeneity of the genome is excluded. On
the other hand, investigations using obtained human tumors include much heterogeneity in
the results because tumors have heterogeneous genomic backgrounds, and large variations in
results have seen in our studies. These methods seem to be suitable to extract several potent
factors from a population including many parameters and heterogeneity. Many genes have
been identified and expressions of thousands of genes can now be analyzed simultaneously
new techniques such as the microarray technique. Advances in research technology have
made it possible to perform an experiment in several months that would have taken several
years before. Further developments in cancer biology are expected.

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In: Oral Cancer Research Advances


Editor: Alexios P. Nikolakakos, pp. 51-93

ISBN 978-1-60021-864-4
2007 Nova Science Publishers, Inc.

Chapter 3

PROGNOSTIC INDICATORS IN ORAL


SQUAMOUS CELL CARCINOMA
Mrcio Diniz-Freitas1, Eva Otero-Rey1, Andrs Blanco-Carrin2,
Toms Garca-Caballero3, Jos Manuel-Gndara Rey2 and
Abel Garca-Garca4
1

Oral Surgery and Oral Medicine Departments, School of Dentistry, University of


Santiago de Compostela, Spain;
2
Department of Oral Medicine, School of Dentistry,
University of Santiago de Compostela, Spain;
3
Department of Morphological Sciences, School of Medicine and Dentistry, Clinical
University Hospital, University of Santiago de Compostela, Spain;
4
Department of Maxillofacial Surgery, Clinical University Hospital, University of
Santiago de Compostela, Spain.

ABSTRACT
Every year, more than 300,000 new cases of oral cancer are diagnosed worldwide.
Oral squamous cell carcinomas (OSCCs) make up about 90 - 95% of these cases. Despite
intensive research into treatment modalities for oral cancer, the 5-year survival rate has
shown little improvement in recent decades. One of the reasons for this is that the TNM
classification system (the conventional basis for treatment decisions, in conjunction with
histological tumor grade) has proved not to be a consistently good predictor of prognosis.
There is thus a pressing need for research into new prognostic indicators, with the aim of
enabling the evaluation of the biological aggressiveness of each patient's particular
tumor/s.
In recent decades, considerable research effort has been dedicated to the
identification of new markers of OSCC, with the aim of better predicting tumor behavior
and clinical course. Certainly, an improved knowledge of the different biological
mechanisms participating in carcinogenesis, as well as of cell proliferation, apoptosis,

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52

tumor growth and tumor invasive capacity, may assist individual diagnosis, and help in
the development of new treatment strategies.
The aim of the present chapter is to briefly review the use of tumor markers for
prediction of the biological behavior of OSCCs. The review is divided into three parts,
considering first clinical markers, then histological markers, and finally
immunohistochemical markers.

I. INTRODUCTION
In recent decades there have been significant advances in the treatment of oral cancer, in
radiotherapy and chemotherapy, and in surgical techniques for resection and reconstruction
[1]. Despite this, however, survival rates have not significantly improved over this period [2].
The number of deaths per year attributable to oral cancer and its complications totals
about 7800 in the USA (5100 men and 2700 women) and 900 in the UK [3]. In Brazil, deaths
attributable to oral cancer in 2002 totaled 2715 in men and 700 in women, giving mortality
rates of 3.15 and 0.78 deaths per 100,000 people per annum in men and women respectively
[4]. In Spain, Nieto and Ramos [5] reported an increase in mortality due to oral cancer
between 1975 and 1994. Note though that in this study we included cancers of the
oropharynx, the lips and the salivary glands within their definition of oral cancer.
Oral squamous cell carcinomas (OSCCs) make up about 90 - 95% of oral cancers [6].
Over recent decades, considerable research effort has been dedicated to the identification of
new markers of OSCC, with the aim of better predicting tumor behavior and clinical course.
Certainly, an improved knowledge of the different biological mechanisms participating in
carcinogenesis, as well as of cell proliferation, apoptosis, tumor growth and tumor invasive
capacity, may assist individual diagnosis, and help in the development of new treatment
strategies.
The aim of the present chapter is to briefly review the indicators currently available for
prediction of the biological behavior of OSCCs. The review is divided into three parts,
considering first clinical indicators, then histological indicators, and finally
immunohistochemical markers. Note that this chapter does not consider genetic markers of
potential utility in diagnosis and prognosis (including DNA microarray techniques): these are
considered in Chapter "Current Trends in the Molecular Diagnosis of Oral Squamous Cell
Carcinoma".

II. CLINICAL INDICATORS


II.1. Age
The prognosis for patients with OSCC is directly determined by tumor size, and in
particular by dissemination to the cervical lymph nodes. However, in geriatric patients the
prognosis may also be influenced by age-related factors, mainly because these patients often
have compromised pulmonary, cardiovascular, renal and/or endocrine function. As a result,

Prognostic Indicators in Oral Squamous Cell Carcinoma

53

treatment plans for older patients may be restricted by factors of this type: notably, the more
aggressive standard therapies may not be well tolerated, and may indeed reduce survival of
these patients [7].
However, the influence of age on prognosis remains rather poorly understood. Some
studies have not found any variation in survival with patient age [8,9], whereas a study by
Varela-Centelles et al. [10] found that age, together with tumor size, was the principal
determinant of survival in a sample of 94 patients with OSCC.
OSCC rarely presents in young patients, although its incidence in younger age groups
appears to be increasing [11]. In the literature, "young patients" have been diversely and
arbitrarily defined, for example as patients younger than 30, 40 or 45 years [12,13,14]. For
some authors young patients show more aggressive tumors with higher rates of relapse and
cervical metastasis, and higher mortality [15]. However, a study by Rennie et al. [16] did not
corroborate these findings. These authors found that the low incidence of OSCC in younger
subjects was insufficient to allow reliable inferences about mortality rates, and suggested that
the perception of the disease as more aggressive in younger patients may be influenced by
emotional factors (i.e. patients and health-care professionals alike are particularly impacted
by the appearance of a potentially fatal disease in a relatively young person).
Recently, Sasaki et al. [17] have studied the clinico-pathological characteristics of OSCC
in patients aged less than 40 years, but did not find any characteristics specific to this age
group; neither did they find any differences in survival between patients aged < 40 years and
40 years.

II.2. Sex
Due to the marked predominance of men in most series studied, few studies have
attempted to evaluate the possible importance of sex as a prognostic factor. In fact, there
appear to be no differences in prognosis between men and women [18,19,20], although some
authors have reported lower survival in women, attributing this to longer delays in seeking
medical care and to lower acceptance of treatment [21]. In contrast, Boffetta et al. [22]
obtained higher survival rates in women than in men.

II.3. Location
The limits of the oral cavity - unlike other areas of the body - are not always easy to
define. Not surprisingly, then, the definition of the term "oral cancer" as regards location has
proved very difficult for both clinicians and researchers. Often a variety of rather
homogeneous tumor types has been grouped under this term. In most studies, tumors of the
lips and salivary glands have not been included, due to their different histological structure,
and to the fact that the lips are exposed to solar radiation. In some studies tumors of the
oropharynx have been included.
Several authors [23,24,25] have stressed the importance of defining "oral cancer" more
precisely, in view of the rather lax use of this term in the research literature and textbooks. In

54

Mrcio Diniz-Freitas, Eva Otero-Rey, Andrs Blanco-Carrin et al.

line with the latest edition of the WHO International Classification of Diseases (ICD), Moore
et al. [24] have suggested that the term "oral cancer" should include tumors of the mobile part
of the tongue, the floor of the mouth, the jugal mucosa, the mucosa of the upper and lower
alveolar ridge, and the palate; tumors of the lips, oropharynx and salivary glands should be
considered separately.
As regards prognosis, it appears to be widely accepted that 5-year survival rates are
lower for patients with tumors in more posterior intraoral locations [26,27]. It is well known
that carcinomas of the lower lip have a more favorable prognosis than carcinomas in intraoral
and oropharyngeal locations [28,29], and that intraoral carcinomas have better prognosis than
oropharyngeal carcinomas [30]. These associations are explicable in terms of proximity to the
lymph nodes, and to a lesser extent in terms of clinical stage, histological grade and
characteristics of the invasive front (including mode of invasion, degree of perineural
invasion, and degree of vascular invasion), as well as the ease with which clean surgical
margins can be achieved, and the appearance of second primary tumors [31,32,33]. Thus,
these between-location differences in tumor course and prognosis are probably partially
(though not entirely) explained by differences in vascular and lymphatic networks [34].
As regards intraoral location, however, the results in the literature do not show close
agreement. Zelefsky et al. [35] found worse prognosis in patients with lesions in the anterior
2/3 of the tongue (oral tongue) by comparison with patients with lesions on the floor of the
mouth. Recently, Bell et al. [36] found no significant differences in prognosis when they
compared the anterior 2/3 of the tongue with other intraoral locations.

II.4. Tumor Stage (TNM Classification)


The TNM system is accepted worldwide as a classification system for diverse types of
neoplasm. This system evaluates the anatomic extension of malignant solid tumors, and is a
key indicator of best treatment, as well as an important prognostic factor.
Among the most widely used classifications in oral cancer is the staging classification
proposed by the American Joint Committee on Cancer (AJCC) [37]. In this classification,
stage is determined by the size of the primary lesion and by regional dissemination (to
cervical lymph nodes) or distant dissemination. Cure rates for oral cancer depend on stage:
the 5-year survival rate is 80% in initial stages, 40% in patients with regional involvement,
and less than 20% in patients with distant metastasis [38]. Early detection significantly
increases the possibility of a successful cure. At the moment of diagnosis, 36% of patients
show localized disease, 43% disease with regional involvement, and 9% distant metastasis; in
the remaining 12% of patients disease stage is not readily identifiable [39]. These findings
suggest that early diagnosis of this disease is poor, bearing in mind that squamous cell
carcinomas are formed in the superficial epithelium of the oral cavity, leading to visible
changes at an early stage.
Early detection of asymptomatic stages ensures not only improved survival, but also
better quality of life for the patient, since aggressive treatments will be less frequently
required [40,41].

Prognostic Indicators in Oral Squamous Cell Carcinoma

55

In a series of 47 patients with OSCC [42,43], we found that stages T and N


independently correlated with patient survival in univariate analyses; in multivariate analyses,
however, stages T and N were not statistically significant predictors of prognosis, and only
the final clinical stage correlated with survival. These results indicate that combined
consideration of tumor size and cervical lymph node involvement may allow more accurate
assessment of prognosis in patients with oral cancer.
Cervical metastasis is relatively frequent in oral cancer, and it has been suggested that
this is in fact the principal indicator of prognosis [44,45]. It seems that metastatic
dissemination to cervical lymph nodes is directly correlated with primary tumor size. It has
been demonstrated that T1 tumors of the tongue and floor of mouth undergo cervical
metastasis in 14% and 11% of cases respectively, versus 77% and 54% respectively for T4
tumors in these locations [46]. In our series of patients with OSCC, the presence of palpable
cervical adenopathies, and the incidence of regional relapse, were also correlated with the
size of the primary tumor. However, cervical metastases may also develop from relatively
small tumors (T1 or T2, n=12), while conversely large tumors may course without cervical
metastasis (T3 or T4, n = 7).
Since the initial development of the AJCC TNM classification it has been revised various
times. In relation to oral cancer, several modifications of the 5th edition have recently been
proposed. Additionally, the German-Austrian-Swiss group for the study of tumors of the
maxillofacial region (DSAK) has demonstrated in an extensive retrospective study of 1532
cases of primary oral cancer that modification of the TNM classification to include tumor
thickness significantly improves prediction of 5-year survival rates [47]. Other studies have
likewise demonstrated that tumor thickness is of prognostic value in patients with oral cancer
[48,49], but nevertheless this parameter has not yet been included in the TNM system.
Although it includes important prognostic factors, the TNM system does not accurately
predict the biological properties of tumors. For example, the system does not explain the
considerable proportion of small tumors that show poorer course than expected [50].

III. HISTOLOGICAL INDICATORS


III.1. Tumor Grade (Degree of Differentiation)
Histologically, OSCCs are characterized by the presence of invasive islets and/or cords
of malignant epithelial cells similar to those of the spinous layer. Neoplastic cells generally
present an abundant eosinophilic cytoplasm with large nuclei and increased nucleus-tocytoplasm ratio. Diverse degrees of cellular and nuclear pleomorphism are often observed
[51].
Although developed a long time ago, the classification of Broders [52] is still widely
used to evaluate histological grade, in terms of degree of similarity between the tumor and the
normal architecture of the epithelium. Carcinomas that produce significant quantities of
keratin and show some degree of maturation from the basal to surface layers are classed as
well differentiated. Carcinomas that do not produce keratin, but in which some degree of
stratification is apparent despite deviation from the normal architecture, are classed as

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56

moderately differentiated. When keratin is not produced the tumor bears little resemblance to
the normal architecture and shows frequent cellular anomalies; these carcinomas are classed
as poorly differentiated [53,54].
Histological grade is closely related to biological behavior. Well-differentiated tumors
typically show slower growth and produce metastases at later stages. In contrast, poorly
differentiated tumors typically grow more rapidly and metastasize in early stages. However,
the process of histological grading of OSCCs remains a subjective process that depends
strongly on the individual judgment of the observer. In most cases, clinical [TNM] staging
would appear to be more effective than histological grading as a prognostic indicator.
Histological grading of OSCCs is almost always done with conventional light
microscopy, although immunohistochemical techniques using cytokeratin markers may be
useful for distinguishing poorly differentiated or non-differentiated tumors from other
malignant lesions.
A special form of OSCC is verrucous carcinoma, characterized histologically by its broad
base of implantation and its papillary formation. Deep crypts containing keratin plugs are
typically observed between elongated surface projections. The epithelium is dysplastic, but
only rarely shows pronounced dysplastic traits. The basal membrane remains intact, and there
is often a chronic infiltrate of inflammatory cells in the underlying connective tissue. The
interface between the tumor and the adjacent normal epithelium is generally well defined,
with minimal or zero invasion of epithelial cells along the length of the broadly curving
invasive front of the bulb-shaped epithelial crest [55,56].
Histological grading has been used for decades to predict the prognosis of patients with
OSCC. The prognostic value of the different systems used has varied considerably [57].
Degree of differentiation as assessed by Broders' system or similar has been considered a
useful predictor of dissemination to the cervical lymph nodes [58]. Recently, Kademani et al.
reported a higher rate of cervical metastasis and reduced survival in patients with poorly
differentiated tumors. These authors also found that it is more difficult to achieve clean
surgical margins in resections of poorly differentiated tumors.

Figure 1. Examples of OSCCs showing different degrees of differentiation (a, well differentiated; b,
moderately differentiated; c, poorly differentiated).

Prognostic Indicators in Oral Squamous Cell Carcinoma

57

III.2. The Invasive Front


Pathologists have been aware for several decades that tumor cells in the most invasive
areas of malignant tumors differ substantially from those in other central and surface areas. In
view of this, in 1992 Bryne et al. [59] proposed a new approach to histological classification
of OSCCs, considering only those cell layers showing deep invasiveness, and their interface
with the host tissue (the "deep invasive front"). This approach, a simplification and
modification of the multifactorial approach proposed by Anneroth et al. [60], offers highly
significant additional information in patients with the same TNM stage. The approach is
based on quantitative evaluation of histological parameters including degree of
keratinization, nuclear polymorphism, pattern of invasion, and lymphocytic infiltration. Its
prognostic value has been confirmed in diverse studies of carcinomas of the head and neck
[61,62].

III.3. Pattern of Invasion


Pattern of invasion, a factor common to the various classifications of the invasive front,
has proved to be one of the most important parameters for predicting the course and
prognosis of OSCCs. Although the scales used for classification of pattern of invasion show
some differences, they mostly consider that increasing cellular dissociation is an indicator of
increased tumor invasiveness, more aggressive clinical behavior, increased number of local
and regional relapses, and poorer prognosis.
Spiro et al. [63] have suggested a simplification of Bryne et al.'s classification of the deep
invasive front (Figure 2). These authors studied pattern of invasion in squamous cell
carcinomas of the tongue. They found that the pattern was associated with both patient age
and TNM stage, but not with histological grade. The survival of patients with tumors
showing a more invasive pattern was significantly lower than in patients with less invasive
tumors.
Bundgaard et al. [64] studied 8 histological parameters in 78 stage-I OSCCs, finding that
pattern of invasion was the only factor with prognostic value in this patient group. We
concluded that evaluation of pattern of invasion was a useful alternative to other more
complex methods in which several histological parameters must be considered.
Tumors with a more invasive histological pattern are typically associated with the
appearance of local relapses [65], possibly because of the greater difficulty of achieving clean
surgical margins without neoplastic infiltration in tumor resections.
Together, these findings suggest that pattern of invasion is a criterion worth taking into
account when selecting postoperative adjuvant treatments with radio- and/or chemotherapy.

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58

Figure 2. a) Tumor with well-defined margins pressing against adjacent tissue; b) tumor with margins
infiltrating the adjacent tissue in solid cords; c) tumor with margins containing groups or cords of
infiltrating cells (> 15 tumor cells per group or cord); and d) tumors showing marked cellular
dissociation giving rise to small groups (<15 tumor cells per group) and even isolated single tumor
cells.

III.4. Tumor Thickness (Depth of Tumor Invasion)


Many authors have observed that tumor thickness (tumor depth) is better correlated with
metastasis to cervical lymph nodes and survival than surface diameter. Some studies have
tried to relate tumor thickness to local relapse and cervical metastasis [66,67,68]. Recent
studies have demonstrated that tumor thickness is a significant prognostic factor in squamous
cell carcinomas of the tongue, probably because access to the lymphatic system is a key
determinant of regional dissemination [69].
Although most studies have confirmed that tumor thickness is an important prognostic
indicator, there does not appear to be a consensus about cut-off values for discriminating
tumors with poor and better prognosis. Spiro et al. found poorer prognosis in patients with
tumor thickness greater than 2 mm. Brown et al. found poorer prognosis in patients with
tumor thickness greater than 3 mm, and Gonzlez-Moles et al. [70] likewise found a lower
survival rate among patients with tumor thickness greater than 3 mm. In addition, these latter
authors did not find differences in survival rate between patients with tumor thickness 4 - 7
mm and patients with tumor thickness > 7 mm. Urist et al. [71] proposed a tumor thickness

Prognostic Indicators in Oral Squamous Cell Carcinoma

59

cut-off value of 6 mm for distinguishing poor from better prognosis. We ourselves have
found that patients with tumor thickness 8 mm show significantly poorer prognosis than
patients with tumor thickness < 8 mm.
These discrepancies between previous studies may be partly due to the fact that the
methodology for measuring tumor thickness has varied considerably between studies.
Although most authors have used an optical micrometer, others have not specified how
measurements were obtained [72,73]. Some authors have defined tumor thickness as the
distance between the deepest point of invasion and the tumor surface, while others have
defined it as the distance between the deepest point of invasion and an imaginary line
representing the reconstructed continuation of the basal membrane of the adjacent normal
mucosa. In addition, in some studies the authors have explicitly excluded keratinized layers
or inflammatory infiltrate from the measurements of tumor thickness, while other authors do
not specify whether these layers were included or not.
Assuming that normal tissue shows a greater resistance to deep invasion than to lateral
tumor extension, it may be expected that the greater the capacity for deep invasion, the more
aggressive will be the tumor. Thus for Gonzlez-Moles et al. the tumor mass that reveals the
tumor's capacity for deep growth, and thus its aggressiveness, is that observed below the
imaginary line reconstructing the basal membrane of the adjacent normal mucosa (Figure 3),
since this mass reflects the potential for destruction by neoplastic cells. According to these
authors, exophytic growth of the tumor should not be taken into account, because it does not
represent the tumor's real capacity for deep invasion against strong resistance. On the same
grounds, these authors suggest that the space left below the basal membrane of ulcerated
tumors should be taken into account, since it reflects the tumor's destructive capacity.

Figure 3. Measurement of tumor thickness as the distance between the deepest point of invasion and an
imaginary line reconstructing the basal membrane of the adjacent normal mucosa.

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III.5. Surgical Margins


For some authors, one of the most important individual prognostic factors is degree of
extirpation of the tumor, with failure to completely remove the primary tumor tissue being the
principal cause of death in OSCC patients [74]. If neoplastic cells are present at the surgical
margin, the local relapse rate will increase substantially and the survival rate will decline
[75,76].
There is likewise disagreement in the literature about the effects of post-resection
adjuvant treatment with radio- and/or chemotherapy in patients with positive surgical
margins. Kovcs [77] found that postoperative treatment with radio- and/or chemotherapy did
not improve survival rate in patients with surgical margins showing tumor infiltration under
microscopy. However, Zelefsky et al. [78] reported good results from postoperative
radiotherapy of tumors with positive surgical margins. In another study, radiotherapy was
reported to reduce the risk of relapse, but not to the same degree as in patients with tumorfree margins.
A potential problem with many studies that have investigated the relationship between
surgical margin status and prognosis has been the use of univariate analyses. Tumor relapse
occurs in response to multiple interacting factors [79]. Some studies using multivariate
analyses have not been able to demonstrate an independent relationship between surgical
margin status and local relapse or survival [80].
In addition, the histological identification of positive surgical margins appears to be
problematic, and a system of molecular staging based on detection of genetic alterations in
neoplastic cells has been proposed. The detection of mutations in the p53 gene by PCR
allowed identification of neoplastic cells in 13/25 patients with histologically negative
surgical margins, and 5 of these patients suffered local relapse, versus no relapse in any of the
12 patients with negative margins by this technique [81]. In another similar study, mutations
of the p53 gene were detected in 72% of tumors with margins assessed as negative by
histological methods [82]. The presence of genetic alterations in the histologically normal
epithelium adjacent to the tumor may explain the lack of relationship between surgical
margin status and survival.

III.6. Perineural and Vascular Invasion


Perineural invasion is a recognized route of dissemination in tumors of diverse
histological types in the head and neck region. In the literature, reports of the prevalence of
perineural invasion in carcinomas of the head and neck have ranged from 6 to 30% [83]. This
wide variation may be one of the causes of the contradictory reports in the literature as
regards the prognostic value of perineural invasion.
Rahima et al. found perineural invasion to be associated with regional relapses, distant
metastases and reduced 5-year survival. Perineural invasion correlates with other histological
parameters, such as tumor grade and depth of invasion, suggesting that tumors showing
perineural invasion are more aggressive.

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Figure 4. a) Perineural invasion; b) vascular invasion.

As with perineural invasion, the prognostic value of vascular invasion in OSCC is


likewise controversial: some authors have found a relationship with prognosis [84,85,86,87],
while others have not [88,89]. These discrepancies between studies may be partially
attributable to differences in sensitivity in detection of perineural and vascular invasion
(Figure 4). Recently, Kurtz et al. [90] have demonstrated that immunohistochemical
techniques can increase the rate of detection of perineural invasion (mAb S100) and vascular
invasion (mAb CD31) in OSCC, suggesting that these techniques may have diagnostic and
prognostic value. In the same study, the authors found that vascular invasion (though not
perineural invasion) was significantly associated with prognosis.
The use of immunohistochemical markers in OSCC is discussed in the next section.

IV. IMMUNOHISTOCHEMICAL MARKERS


Immunohistochemistry is without doubt the technique that has had greatest impact on
disease diagnostics in recent decades [91]. The current importance of immunohistochemistry
is demonstrated by its application in more than 20% of tumor diagnoses [92]. However,
immunohistochemical demonstration of specific cell proteins is not only used as a diagnostic
technique, but also to predict the behavior of certain types of tumor (i.e. for prognostic) and
their response to specific new treatment modalities (i.e. for treatment selection) [93,94]. The
methodologies used may involve simple detection of stained tumor cells, or may use scales
for the quantitation of staining intensity. Immunostaining can also be used to identify and
highlight specific structures for prognosis, such as peritumoral blood vessels and microscopic
metastases.
The pathogenesis of oral cancer, like that of other types of malignant solid tumor,
depends on the acquisition of six fundamental capabilities by the neoplastic cells: 1) growth
independent of growth-promoting signals, 2) insensitivity to growth-inhibiting signals, 3)
avoidance of programmed cell death (apoptosis), 4) unlimited proliferative potential, 5)
induction of angiogenesis, and 6) capacity to invade tissues and metastasize (Figure 5) [95].
These behaviors are not independent, and are the result of a complex multi-stage process of
genetic alterations. In OSCC, immunohistochemical techniques have been used to identify
behaviors of this type and to predict biological behavior. Some of the more important
immunohistochemical markers used are discussed in what follows.

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Figure 5. Capabilities that are acquired by cells during malignant solid tumor development.

IV.1. Oncogenes
IV.1.a. Epidermal Growth Factor Receptor (EGFR)
EGFR is a glycoprotein forming part of the c-erbB receptor family, comprising
transmembrane receptors with intrinsic tyrosine kinase activity. The family has four
members: EGFR (c-erbB-1/HER-1), c-erbB-2 (HER-2/Neu), c-erbB-3 (HER-3) and c-erbB-4
(HER-4) [96]. EGFR is coded by the EGFR gene (erbB1) located on chromosome 7
(7p13-q22). It binds various ligands, including epidermal growth factor (EGF), transforming
growth factor (TGF-), amphiregulin, betacellulin, crypto and epiregulin. Ligand binding
to EGFR's extracellular domain induces homo- or heterodimerization, resulting in
autophosphorylation of tyrosine residues in the receptor's intracellular domain, and
consequent activation of intracellular signaling cascades. This autophosphorylation induces
activation of signal transduction cascades [97], leading eventually to cell proliferation,
inhibition of apoptosis, stimulation of invasion, and metastasis (Figure 6) [98].
Over-expression of EGFR in human cancer was first described in a vulvar epidermoid
carcinoma cell line [99]. Subsequently, over-expression of EGFR has been observed in a
wide variety of human tumors, including tumors of the breast, ovary, prostate, bladder, lung,
brain and pancreas [100]. Over-expression of EGFR is likewise frequent in oral cancer, and
30% of cases showing over-expression of the protein show EGFR gene amplification
[101,102].

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Figure 6. Carcinogenic involvement of the epidermal growth factor receptor. (Adapted with permission
from Harari PM. Epidermal growth factor receptor inhibition strategies in oncology. Endocrine-Related
Research 2004;11:689-708. Society for Endocrinology .

In premalignant lesions, EGFR expression seems to increase proportionally to the degree


of epithelial dysplasia. Grandis et al. [103] found increased expression of EGFR in dysplastic
mucosa adjacent to squamous cell carcinomas of the head and neck region, by comparison
with normal oral mucosa in control patients. In addition, they found that expression of EGFR
increased with increasing dysplasia severity, and that EGFR expression was markedly higher
in squamous cell carcinoma than in precancerous dysplastic mucosa. Similar findings were
reported by Shin et al. [104],who additionally observed that with increasing dysplasia
severity, EGFR over-expression extended into more superficial layers of the epithelium.
EGFR gene amplification and increased EGFR expression in oral cancer are associated
with tumor differentiation and aggressiveness [105]. It has been reported that OSCCs
showing EGFR over-expression respond better to chemotherapy than non-EGFRoverexpressing tumors. This is possibly because EGFR-overexpressing tumors show higher
proliferative activity, and thus greater sensitivity to cytotoxic drugs [106]. Recent research
suggests that anti-EGFR antibodies may be useful in the treatment of premalignant and
malignant lesions showing EGFR over-expression [107].
Cells showing EGFR over-expression show a growth advantage over normal cells,
especially when the EGFR over-expression is accompanied by increased expression of its
ligand TGF- [108]. The observation of increased EGFR expression in peritumoral tissues
adjacent to tumors may explain the early relapse and second primary tumors rather frequently
seen after surgery, and anti-EGFR therapies may be especially indicated in these patients.
Studies of the prevalence of EGFR immunoreactivity in OSCCs have obtained
contradictory results. Sakai et al. [109] found EGFR expression in 14.8% of cases of OSCC,
while Kusukawa et al. [110] detected EGFR expression in only 30.8% of cases.

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The prognostic significance of EGFR expression level in OSCCs is controversial. Some


authors have found that higher EGFR expression levels are significantly correlated with less
favorable prognosis, while others have reported the opposite relationship [111]. Still other
studies have found no correlation between EGFR expression and patient survival [112].
Partridge et al. were among the first researchers to investigate EGFR expression in
OSCC. They studied 20 cases and did not find any correlation between EGFR expression and
tumor grade, disease-free interval or survival. Subsequently, Bergler et al. found correlation
between EGFR expression and tumor stage. Maiorano et al. [113] evaluated EGFR
expression in the cytoplasm and membrane of neoplastic cells, finding expression in 36% of
cases. In addition, patients with tumors showing expression of EGFR in the cell membrane
and/or cytoplasm showed a more favorable prognosis. When EGFR expression was observed
only in the membrane, the correlation with improved prognosis was more significant.
Ulanovski et al. [114] found expression de EGFR in 34% of 23 SCCs of the tongue. These
authors found higher EGFR expression in tumors with lower histological grade. They did not
find any correlation between EGFR expression and tumor thickness or N stage, both
important predictors of tumor progression. They likewise found no association between
EGFR expression and the presence of cervical metastases, recurrence or survival. Bankfalvi
et al. [115] observed a correlation between EGFR expression and pattern of invasion in 75
cases of OSCC. In addition, they observed correlation between EGFR expression and T
stage. Over-expression of EGFR was associated with unfavorable prognosis. Strkel et al.
[116] studied the prognostic value of EGFR expression in 100 patients with OSCC. All cases
showed EGFR expression: in addition, EGFR expression levels showed a positive correlation
with a malignancy grading of the invasive tumor areas, and a strong negative correlation with
5-year survival. Yamada et al. [117] observed EGFR expression in 51% of OSCCs, but no
correlation with tumor grade. Laimer et al. [118], in a study of 109 cases of oral and
oropharyngeal SCC using tissue microarrays (TMAs), found that over-expression of EGFR
was significantly associated with reduced survival.
Xia et al. [119] evaluated the prognostic value of EGFR (HER-1), HER-2, HER-3 and
HER-4 in 47 cases of OSCC, and found significant relationships between all four markers
and survival. The combined presence of EGFR/HER-2/HER-3 was a better predictor of
survival than any of the individual markers.
Thus some studies have indicated that over-expression of EGFR has prognostic value in
OSCC, being associated with reduced survival. As noted, however, not all studies have
obtained this result: for example, Smid et al. [120] did not detect any relationship between
EGFR expression and survival in patients treated by surgery and subsequent radiotherapy.
Discrepancies between studies may be related to any of diverse factors, including the
technique used for determining EGFR expression level (sensitivity and specificity, tissue
fixation time, pretreatment procedures, antibodies), the small number of patients in some
studies, variation in the localization, stage and treatment of the lesions, short follow-up times,
and use of different statistical methods.
The apparently critical role of EGFR expression in carcinogenesis has led to extensive
research aimed at identifying selective inhibitors of EGFR-mediated signaling. Important
strategies currently at the clinical development stage include immunotherapy using
monoclonal antibodies and chemotherapy using tyrosine kinase inhibitors. The optimal use of

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therapies of this type requires quantitative determination of EGFR expression by each tumor,
since they can only be usefully applied to tumors showing EGFR over-expression.
In view of the contradictory data on the frequency of EGFR over-expression, there is a
clear need for standardized immunohistochemical procedures for diagnostic determination of
EGFR in OSCC. FDA approval of a commercial kit for this purpose would be a useful first
step. In a study of 47 patients by our group, using the EGFR PharmDX kit (Dako), 98% of
subjects showed EGFR expression, and over-expression (complete or incomplete moderateintensity staining of the cell membrane in >10% of neoplastic cells) was observed in 74% of
subjects. Using the same kit, Schartinger et al. [121] detected EGFR expression in 70.5% of
oral and oropharyngeal SCC samples. Due to the high frequency of over-expression of EGFR
obtained in these two studies, we agree with the view that therapies based on inhibition of
EGFR-mediated signaling may be of value in OSCC patients.
IV.1.b. Cyclin D1
The cyclins, together with the cyclin-dependent kinases (CDKs), are upregulators of cell
cycle progression. The product of the CCND1/cyclin D1 gene phosphorylates the product of
the retinoblastoma gene Rb, inducing transition from the G1 phase to the S phase of the cell
cycle. Cyclin D1 activity is inhibited by diverse tumor suppressor genes, including p16, p21
and p27 [122].
Increased expression of cyclin D1 in OSCCs is correlated with more advanced tumor
grade [123] Cyclin D1 over-expression has been detected in 32% [124] and 68% [125] of
OSCCs. Few studies have investigated the association between cyclin D1 expression and
OSCC prognosis. Some such studies have found a correlation between cyclin D1 overexpression and unfavorable prognosis (regional metastasis and poorer survival) [126], but
other studies have found no such relationship [127].

IV.2. Tumor Suppressor Genes


IV.2.a. The p53 Gene
Mutation of the tumor suppressor gene p53 is one of the most frequent and most studied
genome changes in human cancer [128]. The p53 gene is located on the short arm of
chromosome 17, and codes a 53-kDa phosphoprotein whose function is to regulate gene
transcription, DNA synthesis and repair, coordination of the cell cycle and programmed cell
death. [129] These functions reflect the capacity of p53 to modulate the expression of diverse
genes [130].
Under normal conditions, p53 detects DNA damage and interrupts cell cycle progression
at the G1-S transition. After DNA damage, p53 levels increase, stimulating expression of the
protein p21, coded by the gene WAF1/CIP1. This protein is an inhibitor of the CDKs that
block phosphorylation of pRB, which in turn blocks release of transcription factor E2F,
preventing DNA replication [131]. However, expression of WAF1/CIP1 can also be induced
via p53-independent routes, for example by growth factors including platelet-derived growth
factor (PDGF), fibroblast growth factor (FGF) and transforming growth factor beta (TGF-).

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If for any reason there is a failure in this control mechanism, p53 induces apoptosis, thus
preventing proliferation of cells with damaged DNA.
Mutations in the p53 gene allow neoplastic cells to pass from G1 to S phase, propagating
their genetic alterations, which may lead to the inactivation of other tumor suppressor genes
or the activation of oncogenes [132]. In OSCC, mutations of the p53 gene appear to occur
before the transition from superficial to invasive carcinoma [133]. Mutations of the p53 gene
are typically point mutations or deletions. Point mutations may give rise to a structurally
altered protein that sequesters and inactivates the wildtype protein; deletions simply give rise
to partial or total loss of p53 expression and function. Additionally, some p53 mutations may
give rise to p53 over-expression, frequently seen in epithelial dysplasia and OSCCs.
Dysplastic lesions showing p53 over-expression have increased risk of malignant
transformation [134], and p53 mutations in cancers of the head and neck region are
associated with high tumor aggressiveness and poor prognosis [135,136], so that p53 is
certainly a useful prognostic marker in OSCC. However, studies of p53 expression in OSCC
have obtained contradictory results: some studies have found no relationship with survival
[137,138,139], while others have found that p53 overexpression is associated with lower
survival [140,141]. These different results are attributable to variation among studies in
factors like sample size and heterogeneity, type of tissue analyzed, tissue pretreatments,
antibodies used, and the threshold used to define overexpression.
IV.2.b. The p27 Gene
The p27/KIPL gene, located on chromosome 12 (12p12-12p13.1), likewise has an
important role in detaining the cell cycle in G1 phase, regulating proliferation via binding and
inhibition of the G1 cyclin/Cdk protein kinase. Mutations of p27 are rare, but have been
described in diverse types of malignant human tumor [142]. In addition, several studies have
reported loss or reduction of p27 in diverse types of neoplasia. Down-regulation of p27 has
been associated with increased tumor aggressiveness and reduced survival [143].
Although p27 mRNA levels do not change during the cell cycle, p27 protein levels do
vary, peaking during the G1 and G0 phases. The lower p27 levels at other phases are
principally due to increased degradation rates.
In normal oral epithelium, the cells of the spinous and granular layers show intense
expression of p27 in the nucleus [144]. Several studies have reported that p27 expression is
reduced in lesions with severe epithelial dysplasia, and that expression is further reduced in
lesions that progress to OSCC [145,146]. Reduced p27 levels have also been reported in early
stages of OSCC invasion [147]. These studies have indicated that the reduction in p27
expression occurs at very early stages of oral carcinogenesis.
Various studies have suggested that reduced p27 expression may be a useful prognostic
marker in patients with OSCC. Kudo et al. [148] reported reduced p27 levels in 87% of cases,
and these reduced levels were correlated with more aggressive tumor behavior, including
increased metastatic potential and reduced patient survival.

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IV.3. Cell Proliferation Markers


Cell proliferation can be defined as an increase in cell number resulting from cell
division, i.e. cell cycle termination [149]. The rate of proliferation of a cancer cell population
depends on various factors such as the proportion of cells that are proliferating (the growth
fraction), the duration of the cell cycle (cell cycle time), and the rate of cell loss due to cell
death and differentiation (cell loss factor). Study of the growth fraction is particularly
important: the higher the growth fraction, the faster the growth of the tumor.
Hyperproliferation is an early (though not specific) marker of tissue growth disorders. It
is generally accepted that an increase in proliferation is associated with more advanced
lesions, and that the distribution of proliferative cells in the tissue may give more information
on the regulatory mechanism that has failed during the process of multistep carcinogenesis.
Various methods have been described for quantifying cell proliferation rate. Among the
immunohistochemical markers most commonly used to this end is Ki-67/MIB-1 [150].

IV.3.a. Ki-67 (MIB-1)


The antigen Ki-67 is a non-histone nuclear protein expressed in all phases of the cell
cycle (G1, S, G2 and M), but absent from resting cells (G0) [151]. The gene for this antigen
is located on the long arm of chromosome 10 (10q25) [152]. Ki-67 expression begins in
phase G1, increases gradually during phases S and G2 and peaks during mitosis (M) [153].
During interphase, Ki-67 can be detected only during the nucleus, while during mitosis much
of the protein is transferred to the chromosomal surface. It is rapidly degraded when the cell
enters the non-proliferative state [154], and there does not appear to be Ki-67 expression
during the DNA repair process [155]. The precise function of this protein remains unknown
[156]
Ki-67 can be detected with polyclonal anti-Ki-67 antibody or with the anti-Ki-67
monoclonal MIB-1: the former works only in freeze-cut sections, while the latter works in
paraffin sections, and is thus more widely used [157]. Ki-67 immunostaining (often called
Ki-67/MIB-1 staining) generally has less background and more contrast than staining for
proliferating cell nuclear antigen (PCNA), so that staining with Ki-67 immunostaining is
easier to interpret and quantify. Thus immunodetection of Ki-67 is a useful way of assessing
the growth fraction in normal and malignant tissues [158] and assessments of the density of
Ki-67-positive cells (referred to as "Ki-67 proliferative index" or similar) are widely used.
Premalignant lesions in diverse anatomical locations are characterized by increased cell
proliferation [159,160], generally related to the degree of epithelial dysplasia. Thus cell
proliferation markers may be useful for evaluation of the type and stage of oral premalignant
lesions. The changes in proliferative capacity of oral premalignant lesions may reveal early
preneoplastic changes and indicate their potential for malignant transformation. It has also
been demonstrated that Ki-67 immunostaining intensity is associated with the histological
grade of leukoplakic lesions of the oral cavity, increasing with increasing severity of
dysplasia [161,162]. These observations suggest that disturbances of proliferation may be an
early consequence of exposure to carcinogenic agents. However, Ki-67 immunostaining in
premalignant lesions has not yet been shown to have prognostic value [163].

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Ki-67 immunostaining was recently studied in malignant and premalignant lesions of the
oral cavity [164]. In hyperkeratotic lesions and dysplastic lesions Ki-67/MIB-1 is seen both
in the basal and suprabasal layers of the epithelium, while in normal oral mucosa it is
generally restricted mostly to the basal layer. In OSCC lesions most malignant cells show
intense Ki-67 immunostaining (Figure 5). Cell proliferation as measured by Ki-67 expression
at the tumor invasion front has been shown to be closely correlated with tumor grade [165].
Intense Ki-67 immunostaining in OSCCs is associated with poor prognosis [166,167]. In
fact, not only quantitative but also qualitative evaluations may be indicative of OSCC
behavior. Suprabasal expression of Ki-67 in dysplastic oral lesions and malignant oral lesions
is correlated with poor prognosis, with recurrences and with cervical metastasis [168].
It has been reported that Ki-67 immunostaining is an indicator of treatment failure. In a
large series of SCCs of the oral cavity and oropharynx treated by surgery and radiotherapy,
intense Ki-67 immunostaining was indicative of early recurrence [169]. Similar results were
described in OSCCs of the tongue. Bortoluzzi et al. [170] found that Ki-67 immunostaining
in OSCCs declined with advancing tumor grade. However, Sittel el al. did not detect any
relationship between Ki-67 immunostaining level and tumor grade, though staining level was
higher in tumors showing subsequent recurrence than in tumors not showing recurrence.
Furthermore, tumors with above-average Ki-67 immunostaining showed a significantly
shorter time to recurrence. All patients were treated with surgery and radiotherapy, so the
authors concluded that proliferation capacity as measured by Ki-67 immunostaining is a
useful indicator of early recurrence risk in patients treated in this way. De Vicente et al.
found that higher proliferation capacity as indicated by Ki-67 immunostaining was associated
with more advanced tumor grade. However, the authors did not find any relationship between
proliferation capacity and survival. Xie et al. did not find any relationship between Ki-67
immunostaining and tumor size, N stage, clinical stage or tumor grade. However, Ki-67
immunostaining was more intense in tumors subsequently showing a lower disease-free
interval. Silva et al. [171] found that Ki-67 immunostaining was lower in head and neck SCC
patients with shorter survival.
Tumuluri et al. analyzed proliferation capacity as indicated by Ki-67 immunostaining at
the tumor invasion front in OSCCs. Their index of cell proliferation was obtained by
counting number of positive cells per mm2 of epithelium using an automatic image analysis
system specifically designed for the study. They found higher proliferation at the invasion
front of tumors larger than 5 mm and in advanced-stage tumors (stages III and IV). In
addition, N2-stage tumors showed higher proliferation than N0- and N1-stage tumors.
Interestingly, proliferation was significantly higher in N0 tumors than in N1 tumors. This
fact, which initially appears contradictory, was attributed by the authors to the possibility that
in some patients with N0 tumors, positive lymph nodes may not be detected by palpation.
Finally, the authors found more intense Ki-67 immunostaining in tumors showing distant
metastasis. Note though that other studies have not found any relationship between
proliferation (as indicated by Ki-67 immunostaining) at the tumor invasion front and cervical
lymph node involvement or survival [172].
Bettendorf and Herrmann [173] studied Ki-67 immunostaining in 329 OSCCs. Staining
intensity was positively correlated with histological tumor grade, pattern of invasion, tumor
size and invasion depth, cervical lymph node status, and 5-year survival rate. Ki-67

Prognostic Indicators in Oral Squamous Cell Carcinoma

69

immunostaining intensity in individual patients did not show any association with prognosis.
Stoll et al. [174] studied Ki-67 immunostaining in 107 SCCs of the oral cavity and
oropharynx, and did not find any relationship between Ki-67 immunostaining and diseasefree interval or survival.. A study of 47 OSCCs by our group did not detect any relationships
between Ki-67 immunostaining and histological parameters, recurrence, disease-free interval
or survival.
Thus Ki-67 immunostaining, generally using MIB-1, has been widely used to evaluate
cell proliferation (growth fraction); however, the precise method used has varied widely.
Some authors have recommended quantification of number of positive cells per square
millimeter of section, or in the case of dysplastic lesions per millimeter length of the basal
layer [175,176]. Another problem is precisely which part of the tumor to study, since Ki-67
expression can show a heterogeneous distribution within a given tumor. Specifically, Ki-67
immunostaining has been found to be higher at the tumor invasion front than in more central
and superficial areas of the tumor. Tumor cells at the invasion front also show more
aggressive behavior, so that this area can be expected to give better information on tumor
progression and patient prognosis.
In view of our review of the literature, we can conclude that although cell proliferation
indices based on Ki-67/MIB-1 immunostaining are correlated with the degree of cell
differentiation, they have not proved to be good indicators of prognosis in most studies. As
noted, the growth of a tumor cell population depends on at least three factors: the percentage
of cells in the cell cycle, the duration of the cycle, and the cell loss factor. Thus some authors
have suggested that it is not possible to evaluate tumor growth using a single marker [177],
which would explain the rather contradictory results reported in the literature.

Figure 7. Oral squamous cells carcinoma showing a) low proliferation index (low Ki-67/MIB-1
immunostaining) and b) high proliferation index (high Ki-67/MIB-1 immunostaining).

IV.4. Apoptosis Markers


IV.4.a. Bcl-2
The proto-oncogene Bcl-2, located on chromosome 18, was the first anti-apoptotic gene
discovered [178], and is involved in the regulation of apoptosis by p53, with expression
levels inversely related to those of this protein [179].

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Bcl-2 and other proteins of this family are the central elements in the apoptotic program
and the principal effectors of programmed cell death. All proteins of this family have two
domains, Bh1 and Bh2, that regulate the formation of dimers between the antagonists (Bcl-2
and Bcl-XI) and agonists of apoptosis (Bax) [180]. The activity of this family of proteins is
regulated by competition between pro-apoptotic and anti-apoptotic dimers. Thus if the level
of Bcl-2 (anti-apoptotic) is greater than that of Bax (apoptosis-inducing), then Bcl-2/Bcl-2
homodimers will prevail and cells will be protected from programmed cell death. Conversely,
if Bax is in excess, this will favor the formation of Bcl-2/Bax heterodimers and induce
apoptosis. Thus the relationship (relative expression level) between apoptosis-inducing and
apoptosis-protective proteins will determine the cell's eventual fate [181].
Loro et al. [182] found that Bcl-2 expression in OSCCs was lower than in normal oral
mucosa, and that Bax expression was associated with tumor grade. Another study found that
neither Bcl-2 expression nor apoptosis index had significant prognostic value in a series of 57
patients with OSCC. However, increased Bax expression was associated with unfavorable
prognosis [183].
In conclusion, reduced Bcl-2 expression in premalignant and malignant human
keratinocytes suggests that preferential expression of apoptosis-protective members of the
Bcl-2 family is a key early phenomenon in the development of OSCC [184], making cells
sensitive to the appearance of mutations and to tumor progression. Under normal conditions,
the process of apoptosis efficiently eliminates genetically damaged cells and prevents their
proliferation and progression toward malignancy. If this process fails, cells with damaged
DNA may survive, leading to hyper-responsiveness to proliferation signals. This continued
persistence of apoptosis-resistant cells increases the likelihood that additional mutations will
arise, leading eventually to a malignant phenotype.
IV.4.b. Survivin
In addition to pro- and anti-apoptotic Bcl-2 proteins, another apoptosis-inhibitory protein
has been identified, surviving [185,186]. This protein is undetectable in most normal adult
tissues, but is expressed in human cancer cells, showing correlation with increased tumor
aggressiveness and reduced patient survival. There is evidence that inhibition of apoptosis by
survivin may be a useful predictor of poor prognosis in human cancer, and that survivin may
be a useful diagnostic and therapeutic target in malignant tumors [187].
Tanaka et al. [188] studied the expression of survivin in OSCCs, finding absence or weak
expression of this protein in normal oral mucosa. Of the OSCCs studied, 58% showed
survivin immunoreactivity in the cytoplasm. However, the authors did not detect significant
differences between survivin expression and the clinicopathological characteristics of the
lesions. In this same study, the authors found that 37% of leukoplakias showed survivin
expression. Survivin levels in leukoplakias and in malignant tissues were significantly higher
than in normal oral mucosa. However, no significant difference in survivin expression was
detected between leukoplakias and OSCCs.
Lo Muzio et al. [189] investigated the predictive potential of survivin immunostaining
for identifying premalignant lesions (with epithelial dysplasia) at higher risk for malignant
transformation. Survivin immunostaining was detected sporadically and with low intensity in
normal oral mucosa, in the basal and suprabasal layers. However, 94% of premalignant

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lesions that progressed to carcinoma showed survivin immunostaining, versus only 33% of
lesions that did not subsequently undergo malignant transformation. No correlation was
detected between survivin immunostaining and degree of epithelial dysplasia. All OSCCs
were survivin-positive.
Subsequently, this group studied the expression of survivin in a series of 78 OSCC
patients, and found overexpression (defined as expression in > 75% of cells) to be a predictor
of poor prognosis [190].

IV.5. Angiogenesis Markers


Tumor growth is associated with raised cellular activity, so increased blood supply is
essential for continued tumor development. Angiogenesis (i.e. formation of new blood
vessels) is a process comprising multiple steps regulated by both stimulatory and inhibitory
factors. The critical steps for neovascularization include degradation and remodeling of the
extracellular matrix and proliferation of endothelial cells. Angiogenesis has been associated
with metastasis and reduced survival in various types of tumor, including OSCCs [191,192].
Although direct evaluation of angiogenesis in histological sections is a difficult
procedure, it has been suggested that microvessel density, quantified on the basis of
immunostaining of microvessels, may be a useful index [193]. Unfortunately, different
studies have used different antibodies to identify microvessels, so that among-study
comparisons are difficult.
IV.5.a. Vascular Endothelial Growth Factor (VEGF)
Of the different angiogenic factors, vascular endothelial growth factor (VEGF) is
particularly important. VEGF induces proliferation, differentiation and migration of
endothelial vascular cells, increases the permeability of capillary vessels [194], and increases
endothelial cell survival by preventing apoptosis [195]. Some studies have demonstrated that
VEGF is an independent prognostic factor in patients with cancer of the breast [196], colon
[197], and esophagus [198]. However, few studies have investigated the correlation between
VEGF expression and prognosis in OSCC.
Uehara et al. [199] did not find any correlation between VEGF expression and cervical
metastasis in OSCC patients, but tumors with poor prognosis showed higher expression of
VEGF. Moriyama et al. [200] found a relationship between VEGF expression and incidence
of cervical metastasis in a series of 44 patients with OSCC. VEGF appears to be involved in
the process of angiogenesis in oral cancer, but its possible utility as a prognostic indicator has
not yet been assessed [201]. Increased vascularization in malignant tissues may provide a
basis for anti-angiogenesis therapies directed against tumor cells.

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IV.6. Markers of Invasiveness and Metastasis


OSCC is characterized by its high degree of local invasiveness, and its marked tendency
to metastasize to cervical lymph nodes [202] leading to high rates of local and regional
recurrence.
The mechanism of invasion and metastasis is complex, and consists of multiple
sequential steps [203]. To achieve invasion and metastasis, the neoplastic cells must detach
from the primary tumor and invade the extracellular matrix. During this first step, loss of
intercellular adhesion and cell-extracellular matrix adhesion are essential for tumor
progression. Recent studies suggest that aberrant expression of intercellular adhesion
molecules like E-cadherin, and of cell-extracellular matrix adhesion molecules like laminin 5,
is related to the biological behavior of the tumor, leading to acquisition of an invasive
phenotype, so that these proteins may be of value for predicting prognosis.
Adhesion molecules regulate the growth and differentiation of epithelial cells and play an
important role in the maintenance of the structural integrity and organization of the stratified
squamous epithelium. Reduced integrity of intercellular adhesion molecules has been
implicated in loss of cell differentiation, accompanied by greater mobility and invasiveness,
by neoplastic epithelial cells in diverse human carcinomas [204].

IV.6.a. Laminin 5 2
The laminins are a large family of basal-membrane glycoproteins with multiple
biological functions, including adhesion and roles in cell dispersion, migration, proliferation,
and differentiation [205].
A laminin molecule is made up of an chain, a chain and a chain that combine to
form a heterodimer. There are various isoforms of each chain, and their diverse combinations
give rise to a great variety of laminin isoforms [206]. The different biological activities of the
different laminins in part simply reflect differences in their tissue expression patterns.
Laminin 5, made up of an 3, a 3 and a 2 chain, is initially synthesized as a 460-kDa
precursor, which after secretion into the extracellular matrix undergoes specific proteolytic
processing to produce a smaller form. Chain 2 of laminin 5 is of particular interest, since its
expression is limited to epithelial tissues, where it has roles in the epithelial attachment
system and in cell motility. This chain is essential for adhesion of basal keratinocytes to the
underlying basal membrane, and acts as an adhesion ligand for integrins 31, 61, 64,
and 21 [207] (Figure 8).
Results to date suggest that laminin 5 2 expression is increased in a various human
carcinomas, and that expression of this monomer is characteristic of cancer cells with an
invasive phenotype. Several studies have indicated that expression of laminin 5 2 may serve
as a marker of invasiveness in diverse types of SCC. It has been suggested that laminin 5 2
secreted by tumor cells stimulates cell motility and thus invasiveness [208]. Experimental
studies have demonstrated that laminin 5 2 promotes cell dispersion when added to
epithelial cell cultures [209,210].

Prognostic Indicators in Oral Squamous Cell Carcinoma

73

Figure 8. a) Expression of laminin 5 2 is limited to epithelial tissues, where it has roles in epithelial
attachment and in cell motility. B) Increased laminin 5 2 expression is seen at the tumor invasion front,
and has been associated with increased invasive potential.

The higher expression of laminin 5 2 within the tumor invasion front, the zone in which
tumor cells show most aggressive phenotype [211] supports the possibility that this molecule
might be a useful marker of tumor progression and malignancy in diverse types of cancer of
epithelial origin [212]. At the invasion front, the tumor tissue frequently shows more
advanced undifferentiation and greater cell dissociation [213]. The expression of laminin 5 2
in the invasion front has been studied in diverse types of cancer and has been associated with
tumor recurrence and poor prognosis in SCCs of the esophagus [214], and colon [215], and in
lung adenocarcinomas [216].
Nordemar et al. [217] studied expression of laminin 5 2 in premalignant lesions that
subsequently underwent transformation to OSCC or not. The authors found laminin 5 2
expression in 60% of lesions that underwent transformation by contrast with 23% of lesions
that did not. They concluded that premalignant lesions showing laminin 5 2 expression have
a higher risk of undergoing malignant transformation.
To date, there have been few studies of the relationship between laminin 5 2 expression
and prognosis in patients with OSCC, and most of these studies have considered only lingual
SCCs.
Ono et al. [218] were the first researchers to investigate the expression of laminin 5 2 in
oral cancer. In a study of 67 SCCs of the tongue, these authors did not find any relationship
between laminin 5 2 expression and tumor stage, cervical metastasis or depth of invasion.
They did observe a relationship between laminin 5 2 expression and both tumor grade and
mode of invasion: with increasing laminin 5 2 expression the tumor showed more advanced
undifferentiation and a more invasive pattern. In addition, increasing laminin 5 2 expression
was associated with poorer prognosis.
In a subsequent study [219] of 108 SCCs of the tongue, the same authors confirmed their
initial conclusions. Again, increased laminin 5 2 expression was associated with increased
tumor invasion depth and poorer prognosis. The authors also investigated possible
relationships between EGFR and laminin 5 2 expression, finding that tumors with high
EGFR expression also showed high laminin 5 2 expression.

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Stoltfuz et al. [220] studied laminin 5 2 expression in 36 T1 tumors of the tongue. They
did not find any significant relationship with tumor histological grade; however, increased
laminin 5 2 expression was correlated with increased risk of recurrence. Lim et al. [221] did
not find any association between laminin 5 2 expression and cervical metastasis or prognosis
in stage-I and stage-II SCCs of the tongue. In a study by our group of 47 patients, we did not
find any significant relationship between laminin 5 2 expression and survival. However, we
observed increased laminin 5 2 expression in dysplastic epithelium adjacent to OSCCs
(Figure 9) [222], suggesting that laminin 5 2 may be useful as a marker of invasion or
malignant transformation by dysplastic lesions of the oral cavity; however, these conclusions
need to be confirmed by further studies.

Figure 9. Expression of laminin 5 2 in dysplastic epithelia adjacent to an OSCC.

IV.6.b. E-Cadherin
The cadherins are a family of cell-surface glycoproteins that act as intercellular adhesion
molecules via calcium-dependent interactions. The classical cadherins, originally named in
view of their tissue specificities (E-cadherin, epithelial; N-cadherin, neural; P-cadherin,
placental), have been used as markers in the identification of normal and neoplastic tissues.
These cadherins comprise a relatively large extracellular segment and short transmembrane
and cytoplasmic domains [223].
E-cadherin is a 124-kDa transmembrane glycoprotein, coded by the CDH1/cadherin-E
gene located on chromosome 16 at 16q22.1. This is a key molecule for intercellular adhesion.
The extracellular domains of E-cadherin molecules of adjacent cells link together to create
intercellular adhesion. This adhesive function of cadherins is dependent on their association
with cytoplasmic proteins called catenins, which anchor cadherins to the cytoskeleton [224].
The catenin family includes the , and catenins. The cytoplasmic domain of E-cadherin
binds directly to and , and the resulting E-cadherin/-catenin/-catenin is anchored to the
actin cytoskeleton via catenin [225]. E-cadherin also participates in signal transduction

Prognostic Indicators in Oral Squamous Cell Carcinoma

75

pathways controlling diverse cellular phenomena, including polarity, differentiation, growth


and cell migration [226].
In the normal or hyperplastic oral epithelium, E-cadherin is expressed in the inferior zone
of the spinous and basal layers [227], except at the basal surface of basal cells [228].
Under-regulation of E-cadherin-mediated cell-cell adhesion has been associated with
progression of diverse malignant human tumors [229,230], including cancers of the head and
neck region [231,232] and oral cancers [233]. Loss of E-cadherin expression has also been
associated with increased invasiveness, advanced T and N stage and poor prognosis [234].
(Figure 10) It seems that abnormal expression of E-cadherin can be caused by multiple
mechanisms, including loss of heterozygosity at the CDH1 locus, and somatic mutations.
Transcriptional silencing by hypermethylation of CpG islands in the promoter region has also
been described in diverse tumors and cell lines [235].

Figure 10. a) The extracellular domains of E-cadherin molecules of adjacent molecules link together,
thus creating cell-cell adhesion. This adhesive function of E-cadherin depends mainly on the association
with cytoplasmic proteins, called catenins, that bind the E-cadherin to the cytoskeleton. b) Loss of
cadherin expression has been associated with OSCC progression to more invasive stages.

Bnkfalvi et al. [236] studied the role of various molecules involved in cell adhesion in
the epithelium (CD44, E-cadherin, -catenin) during oral carcinogenesis oral. They
concluded that in early stages there may be a transient increase in E-cadherin expression,
finally reversed with loss of expression when the cells acquire invasive phenotype (i.e. in the
late stages of carcinogenesis).
Hung et al. [237] found that E-cadherin immunoreactivity was lower in premalignant
lesions than in normal oral mucosa, and lower in OSCC than in premalignant lesions. The
reduction with respect to normal oral mucosa was statistically significant in advanced OSCC
stages. Note though that 60% of metastatic lesions showed higher E-cadherin
immunoreactivity than the primary OSCC.
Bagutti et al. [238] found a correlation between E-cadherin immunostaining and tumor
grade: less differentiated tumors showed reduced expression of E-cadherin. Shinohara et al.
[239] did not find any correlation between E-cadherin immunostaining and tumor grade, but
observed that reduced immunostaining was associated with more invasive histological
patterns. In addition, they found reduced immunostaining in tumors with cervical metastases.

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In view of E-cadherin's important role in maintenance of intercellular connections, it has


been suggested that alterations in E-cadherin expression may be an early event in the process
of metastasis [240]. Tanaka et al. [241] studied E-cadherin expression in lymph node
metastatic processes in 159 patients with OSCC. They found a significant relationship
between reduced expression of E-cadherin in primary tumors and associated cervical
metastases at the moment of diagnosis. In addition, they found reduced survival in patients
showing reduced E-cadherin expression.
Chow et al. evaluated E-cadherin expression in 85 cases of SCC of the tongue. They
found reduced expression in 85% of cases. The reduction was not correlated with sex, age,
histological differentiation or clinical stage. However, reduced E-cadherin expression was
correlated with the presence of clinical and subclinical metastases to the cervical lymph
nodes. In addition, local and regional recurrences were significantly more frequent in tumors
with weak or absent E-cadherin immunostaining.
Chang et al. found reduced E-cadherin expression in 83% of cases of carcinoma of the
tongue. Prognosis was significantly poorer in cases with strong E-cadherin immunoreactivity.
Lim et al. investigated the utility of various histological and immunohistochemical
markers (p53, Ki-67, EGFR, cyclin D1, CD31, Cox-2, MUC1, laminin 5 2, E-cadherin, catenin) for predicting the appearance of late cervical metastases in stage-I and -II tumors. In
multivariate analyses, E-cadherin was the only immunohistochemical marker associated with
the appearance of cervical metastases.
In a study by our group of 47 patients with OSCC, E-cadherin expression declined
significantly with increased invasiveness (Figure 11). In addition, we found a close
association between reduced expression of cadherin-E and local recurrences, and a
significantly shorter disease-free interval. Both univariate and multivariate analyses indicated
that absent or weak E-cadherin immunostaining indicative of poor prognosis.
The mechanism of intercellular adhesion mediated by E-cadherin remains incompletely
understood. Different possible causes have been suggested for the reduced E-cadherin
expression in tumor tissues, including suppression of the E-cadherin promoter gene,
destabilization of the protein leading to disruption of binding to catenins, or mutations and
deletions in the E-cadherin gene. Loss of heterozygosity on chromosome 16, on which the Ecadherin gene is located, is frequent in hepatocellular carcinoma [242] and carcinoma of the
stomach [243] and breast [244]. In SCC of the head and neck region loss of heterozygosity on
chromosome 16 is infrequent and translocations appear only occasionally. In OSCC the Ecadherin gene does not show mutations [245].
As noted above, one of the most widely known pathways for inactivation of E-cadherin
function is transcriptional silencing by hypermethylation of CpG islands in the E-cadherin
gene promoter region [246]. Nakayama et al. [247] found that 94.4% of cases of OSCC with
reduced E-cadherin expression showed such hypermethylation. These findings were
supported by in vitro studies of methylation in cultures of OSCC cell lines without Ecadherin expression: when a demethylating agent was added to cultures, the cells recovered
E-cadherin expression and showed a more cohesive growth pattern.

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Figure 11. Expression of E-cadherin in oral squamous cell carcinomas. a) A low-invasiveness OSCC:
most cancer cell nests show intensive E-cadherin immunoreactivity. b) A high-invasiveness OSCC: loss
of E-cadherin immunoreactivity is observed in small groups of infiltrating cells.

One of the possible mechanisms through which DNA methylation to promoter regions
may regulate gene expression is by preventing binding of transcription factors to the
methylated promoter. Current evidence suggests that binding of transcription factors essential
for E-cadherin expression is inhibited by methylation of the promoter region.
Thus reduced expression of E-cadherin appears to be a useful marker of dissociation of
neoplastic cells from the primary tumor. In line with this, reduced E-cadherin expression in
OSCC is associated with increased risk of local and regional metastasis, and poorer
prognosis.

CONCLUSIONS
Although clinical staging systems cannot precisely evaluate the biological properties of
tumors, and thus cannot precisely predict their evolution, tumor size and most importantly the
existence of metastases at the moment of diagnosis remain the most important prognostic
factors.
Of histological indicators, both tumor thickness and characteristics of the tumor invasion
front give better prognostic information than histological grade alone.
Of immunohistochemical markers, E-cadherin has proved to be of great utility for
predicting course and prognosis in patients with OSCC. This is one of the markers that has
shown most consistent results in the literature.
Bearing in mind that carcinogenesis is a multifactorial process that progresses by steps, it
seems likely that a multitude of biological markers will be associated with prognosis, and that
single markers alone will not be effective for predicting prognosis. Thus techniques that
allow simultaneous quantitation of multiple markers, such as tissue microarrays [248,249]
and DNA microarrays [250,251] seem particularly promising. This question will be discussed
in more detail in the Chapter Current trends in the molecular diagnosis of oral squamous cell
carcinoma.

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In: Oral Cancer Research Advances


Editor: Alexios P. Nikolakakos, pp. 95-124

ISBN 978-1-60021-864-4
2007 Nova Science Publishers, Inc.

Chapter 4

TUMOR-TARGETING NON-VIRAL GENE


THERAPY FOR THE TREATMENT OF
ORAL CANCER
Yoshiyuki Hattori and Yoshie Maitani
Institute of Medicinal Chemistry, Hoshi University, Ebara 2-4-41, Shinagawa-ku, Tokyo,
142-8501 Japan.

ABSTRACT
Despite advances in surgery, radiotherapy, and chemotherapy, the survival of
patients with oral squamous cell carcinoma has not significantly improved over the past
several decades. Gene therapy has the potential for the treatment of oral cancer. Cancer
gene therapy is currently being met with the development of non-viral vectors, because
non-viral vectors have a much lower potential for an adverse inflammatory or immune
reaction, compared with viral vectors. For gene delivery, oral cancer is a particular
appropriate target since it can be applied by direct injection. Also since folate and
transferrin receptors are frequently overexpressed on oral tumors such as nasopharyngeal
tumor and head and neck of squamous cell carcinoma, folic acid and transferrin have
been utilized as a ligand for tumor-targeting gene delivery. Non-viral vectors conjugated
to these ligands have been used as carriers of therapeutic DNA to targeted oral tumor.
The strategies are used for inactivation of oncogene expression, introduction of tumor
suppressor genes, and introduction of a gene that enable to a prodrug to be activated into
an active cytotoxic drug. In this review, we outline tumor-targeting liposome and lipidbased nanoparticle vectors, and discuss the effectiveness as these non-viral vectors for
DNA transfection and for gene therapy to treat human oral tumors.

96

Yoshiyuki Hattori and Yoshie Maitani

ABBREVIATIONS
AS-ODN, Antisense oligodeoxynucleotide;
CHEMS, Cholesteryl hemisuccinate;
CMV,
Cytomegalovirus;
Chol,
Cholesterol;
DC-Chol, 3([N-(N',N'dimethylaminoethane)-carbamoyl] cholesterol;
DMRIE,
Dimyristyloxypropyldimethyl hydroxyethyl ammonium;
DOPE,
Dioleoyl phosphatidylethanolamine;
DPPE,
Dipalmitoyl phosphatidylethanolamine;
DSPE,
Distearoyl phosphatidylethanolamine;
DOTAP,
Dioleoyltrimethylammonium;
DOTMA, Dioleoyltrimethylammonium;
FR,
Folate receptor;
GCV,
Ganciclovir;
HSV-tk,
Herpes simplex virus thymidine kinase;
IL,
Interleukin;
OH-Chol, Cholesteryl-3-carboxyamidoethylene-N- hydroxyethylamine;
PEG,
Polyethyleneglycol;
PEI,
Polyethylenimine;
PLL,
Poly-L-lysine;
PEG-DSPE, Polyethyleneglycol-distearoylphoshatidylethanolamine;
RFC,
Reduced folate carrier;
RNAi,
RNA intereferance;
SCC,
Squamous cell carcinoma;
siRNA,
Small interfering RNA;
Tf ,
Transferrin;
TfR,
Transferrin receptor

1. INTRODUCTION
Oral cancers constitute about 3-5 percent of all cancer in the United State and are more
common in persons over age 50 [1]. Most oral malignant tumors are classified as squamous
cell carcinoma (SCC), while the incidence of malignant melanoma and osteosarcoma is
comparatively low. Early-stage (I and II) oral SCC can be treated with surgery or radiation.
However, in the majority of oral cancers present at an advanced stage (III and IV), a
combination of surgery and radiation therapy provides the best survival rate. In chemotherapy
for oral cancer, no evidence of increased survival when chemotherapy alone was used.
Therefore, chemotherapy is usually combined with radiation therapy. Currently used
chemotherapeutic agents include cisplatin, carboplatin, 5-fluorouracil (5-FU) and the taxanes
(paclitaxel and docetaxel). However, patients with recurrent oral cancer that is refractory to
chemotherapy and/or radiation therapy have a median life expectancy of several months [2],

Tumor-Targeting Non-Viral Gene Therapy for the Treatment of Oral Cancer

97

and the response rate to chemotherapeutic regimens is approximately 15%. Two-thirds of


patients dying of this disease have no evidence of distant metastases.
Gene therapy is currently being investigated as a new therapeutic approach for oral
tumor, which is a particular appropriate target since it can be applied by direct injection. Also
since folate and transferrin receptors are frequently overexpressed on oral tumors such as
nasopharyngeal tumor and head and neck of squamous cell carcinoma, folic acid and
transferrin have been utilized as a ligand for tumor-targeting gene delivery. Cancer gene
therapy has been intensively developed using non-viral vectors, among which cationic
liposomes and nanoparticles are the most investigated, because non-viral gene therapy has a
much lower potential for an adverse inflammatory or immune reaction, compared with viral
vectors. However, the clinical application of non-viral gene therapy for treatment of oral
cancer will require optimization of gene delivery in tumor specificity and transfection
efficiency. We focus on tumor-targeting lipid-based nanoparticle vectors, and discuss the
effectiveness as a non-viral vector for DNA transfection and for gene therapy to treat human
oral tumors.

2. GENE EXPRESSION MECHANISMS


Plasmids for gene expression systems contain a cDNA coding for either a full gene or
minigene and several other genetic elements, including introns, polyadenylation sequences
and transcript stabilizers to control transcription, translation, protein stability and secretion
from the host cells [3]. The minimal transcription unit required for expression of a therapeutic
protein consists of a 5 promoter upstream of the gene encoding the therapeutic protein (for
example, HSV-tk or cytosine deaminase) and a polyadenylation signal downstream of the
gene. The presence of a polyadenylation signal in a transgene construct helps in the correct
processing of the mRNA generated by transcription. Some transgene constructs have introns
that increase pre-mRNA processing and nuclear transport [4]. Promoter sequences play a
vital role in initiating gene transcription. Promoter sequences offer recognition sites for the
RNA polymerase to initiate the transcription process. Enhancers are regions in the plasmid
DNA that enhance the production of the gene of interest by as much as several hundred times
[5]. Enhancers can be tissue specific and can be present in the plasmid either upstream or
downstream from the promoter region. Transcriptional efficiency can be substantially
improved by the choice of suitable enhancers. Commonly used enhancer and promoter
sequences are derived form viral origins such as cytomegalovirus (CMV), simian virus 40
(SV40), Moloney murine leukemia virus (MoMLV), and Rous sarcoma virus (RSV) [5].
Higher efficiency can be obtained by engineering the plasmid with strong tissue- or
tumor-specific promoters. However, there are few reports about oral tumor-specific
promoters. Promoter of human papilloma virus (HPV) [6], mouse mammary tumor virus
(MMTV) [7,8], multi-drug-resistance gene (mdr-1) [9] and ED-L2 promoter of Epstein Barr
(EB) virus [10] are known to be active in oral cancer cells or in other squamous epithelial
cells. Promoter from virus such an EB virus might have a useful for nasopharyngeal tumorspecific promoter, because EB virus is present exclusively in the nasopharyngeal tumor cells,
but not in the surrounding normal tissues [11]. Recently, S100A7 gene has been shown to be

Yoshiyuki Hattori and Yoshie Maitani

98

markedly over-expressed in SCC, and up-regulatory elements for transcription activity of the
S100A7 in oral SCC was identified [12]. This promoter might have potential for oral SCCspecific therapeutic strategies.
The actual level of strength that will needed for most gene therapy applications is still
not determined. In oral tumor cells, the order of promoter activities of CMV, SV40, MMTV,
HPV and mdr-1 was CMV > SV40 > HPV > mdr-1 > MMTV [9]. The RSV promoter was
reported to be active in the CHU-2 line of oral cancer cells at low level than the SV40
promoter [13]. In suicide gene therapy with herpes simplex virus thymidine kinase (HSV-tk)
gene, HSV promoters were not strong enough to induce the suicide phenotype in the HSV-tk
gene-introduced cells after addition of ganciclovir (GCV) [9]. Therefore, CMV and SV40
promoters appear to be the first choice for gene therapy.
Table I. Viral vectors and therapeutic genes for treatment of oral tumor
Therapeutic gene

Adenoviruses

Retroviruses
AAV
Lentivirus

Cell lines

Tissue

Ref.

Nasopharyngeal carcinoma (NPC)


NPC
SCC of head and neck
SCC of head and neck
Oral SCC
NPC
NPC
NPC
Clinical trial for NPC
SCC of head and neck
NPC
Tongue carcinoma
Oral SCC, SCC of tongue
Oral tumor
Oral tumor
Tongue carcinoma
NPC
Oral SCC
Oral SCC
Oral cancer

[15]
[16]
[17]
[18]
[19]
[20]
[21,22]
[24]
[23]
[25-28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]

p21

Oral SCC

[14]

HSV-tk

Oral SCC

[39]

Oral cancer

[40]

p16
p16
p27
p53 or p27
p53
p53
p53
P53 + cisplatin
p53 + radiation
HSV-tk
HSV-tk
HSV-tk
IL-2
IL-2
HSV-tk + IL-2
Endostatin
Endostatin
Rb
IB
Anti-bcl2 ribozyme

HIV-1 VPR

C666-1
CNE-1, CNE-2Z
SNU-1041, -1066, -1076
SNU-1041, -1066, -1076
HSC-2,-3, -4, SAS
CNE-1
CNE-1, CNE-2Z
CNE-1, C666-1
KB
Tca8113
HSC-2, -3, OSC70
SCC VII
SCC VII
Tca8113
NE-2
012
SCC15, SCC5
686LN, 1483, Tu183

AT-84

3. VIRAL VECTOR SYSTEMS


Viruses used in oral cancer gene therapies include retroviruses [14], adenoviruses [1538], adeno-associated viruses (AAV) [39] and lentivirus [40]. The majority of viral-mediated
gene therapy for oral cancer has used adenoviruses (Table I). Adenoviruses are DNA viruses
that infect a cell and transfer DNA into the nucleus. This DNA does not integrate into the

Tumor-Targeting Non-Viral Gene Therapy for the Treatment of Oral Cancer

99

host genome. Multiple administrations of the vector are usually required since expression of
therapeutic gene is transient. The advantage of adenoviral vectors is that most cells are
susceptible to infection, regardless of their position in the cell cycle. In addition,
adenoviruses can be produced at a relatively higher titer, thus increasing the efficiency of
their administration. However, approximately 90% of humans have already formed
antibodies against the virus. Pre-existing antibodies can limit the effectiveness of this
strategy, particularly upon a second exposure to the vector. Several genetic alterations have
been described in oral cancer, including mutations of p53 [18-24], p16 [15,16], and p27 [17].
The most extensively studied mutations in oral cancer are those of p53, and p53 gene transfer
was tested in SCC patients by injecting the primary or regional tumor with an adenoviral
vector expressing wild-type p53 (Table I).

4. NON-VIRAL VECTOR SYSTEMS


Viral vectors are efficient in transfection, but pose risks to the host due to the
immunogenicity of viral proteins, the potential for oncogenesis due to chromosomal
integration, and the generation of infectious viruses due to recombination. Non-viral vectors
are an attractive alternative method for gene transfection. Particulate systems are needed for
the delivery of DNA, which is unstable and highly hydrophilic, and has high molecular
weight, because of the high nuclease levels present in serum and its inability to cross intact
endothelial barriers. Advantages of using cationic particles include stabilization of the DNA
by protecting it, for example, from serum nucleases and enhancing cellular uptake via
endocytosis as compared to neutral or anionic particles. This is a result of the favorable
electrostatic interaction of cationic particles with the negatively charged moieties on
biological membranes.
There are two types of methods: physical method such as electrotransfection, and
chemical methods, such as vector-mediated transfection. Among them, particulate vectors,
e.g., polymeric particles and lipid-based particles, possess specific advantages and
disadvantages. Lipid-based nanoparticles can be divided in three groups, liposomes,
emulsions and particles. Liposomes contain an inner water phase, emulsions contain an inner
oil or water phase, and nanoparticles are here defined as having no inner phase. The
advantages of lipid-based nanoparticles are, for instance, the ease of modifying the surface of
the particles for tissue-specific targeting, their lack of immunogenicity, their relative safety,
and relative ease of large-scale production. The disadvantages include poor efficiency of
transfection.

4.1. Physical Methods


Naked plasmid DNA provides a promising mechanism for gene delivery, as it is less
immunogenic than most non-vial vectors currently used. Although naked plasmid DNA has
no target-specificity and is more susceptible to nuclease degradation in serum than
encapsulated DNA, high levels of gene expression in oral solid tumor can be obtained by

100

Yoshiyuki Hattori and Yoshie Maitani

intratumoral injection of naked plasmid DNA [41]. Delivery of naked DNA has been
prompted through the application of electric pulses to the target areas. Electroporation
involves injection of the naked DNA followed by the application of an electric pulse over the
target tissues [42]. The electric pulses increase the permeability of the cell membranes and
allow for increased uptake of the naked DNA into tumor cells. Transfection by
electroporation into oral tumor B88 xenografts resulted in consistently efficient transduction
of a higher number of the cells than that by naked DNA alone [43]. Hydrodynamic injection
allows DNA delivery to larger target regions, and is not limited to superficial tissue [44]. This
method involves high-pressure injection of a large volume of solution containing the DNA of
the interest. Gene expression was found to increase, particularly in hepatocytes, as a result of
defects in the cells resulting from the high-pressure injection. When plasmid DNA coding for
gene of secretable protein as a therapeutic protein was injected by hydrodynamic injection, it
can lead to appearance of the mRNA in the liver and the protein in the serum.

4.2. Particle Vectors


Non-viral particle systems for DNA can be classified into two major types based on the
nature of the synthetic material: 1) liposomal delivery systems (DNA entrapped in and/or
complexed to liposomes) [45-47], 2) nanoparticle delivery systems (DNA/nanoparticle
complexes). Cationic polymers, liposomes and nanoparticles are commonly used in gene
delivery because they can easily complex with the anionic DNA molecules [48].
Polymer/DNA complexes (polyplexes), liposome/DNA complexes (lipoplexes) or
nanoparticle/DNA complexes (nanoplexes) are used to deliver DNA into cells. The general
mechanism of action of these complexes is based on the generation of a cationic complex
owing to electrostatic interaction of cationic polymers or lipid with anionic DNA [49]. The
cationic complex can then interact with the negatively charged cell surface to improve DNA
uptake.
Liposomes
Liposomes are spherical, polymolecular aggregates with a bilayer shell configuration.
Depending on the method of preparation, lipid vesicles can be uni- or multilamellar,
containing one or many bilayer shells, respectively. Liposomes typically vary in size between
20 nm and a few hundred micrometers. Their core is aqueous in nature, its chemical
composition corresponding to that of the aqueous solution in which the vesicles are prepared.
The advantages of liposome vector are to have ability to entrap DNA in liposome or to
complex DNA with cationic liposomes.
To increase the overall efficiency of DNA delivery into the cells, cationic liposomes
were designed and used as vectors. Cationic liposomes form complexes with DNA, i.e.,
lipoplexes, through charge interactions. Cationic liposomes are generally composed of a
cationic
lipid,
such
as
dioleoyltrimethylammonium
chloride
(DOTMA),
dioleoyltrimethylammonium propane (DOTAP), dioleoyldimethylammonio propane
(DODAP), dimyristyloxypropyldimethyl hydroxyethyl ammonium (DMRIE) or
dimethylaminoethanolamine carbamoyl cholesterol (DC-Chol), and a helper lipid, such as

Tumor-Targeting Non-Viral Gene Therapy for the Treatment of Oral Cancer

101

dioleoyl phosphatidylethanolamine (DOPE) or cholesterol (Chol), which provides


fusogenicity and stability to the lipoplex (Figure 1). In gene therapy for treatment of oral
tumor, cationic liposomes formulated with DC-Chol/DOPE [50-53], DOTAP/Chol [54],
DOTMA/Chol [55-58] and DMRIE/DOPE [59,60] have been used in clinical trials (Table II).
In in vitro DNA transfection for oral tumor cells, commercially available cationic liposome,
Metafectene (Biontex Loboratories Gmb, Planegg, Germany) [61], Genejammer (Stratagene,
CA, USA) [61], Oligofectamine (Invitrogen Corp., Carlsbad, CA, USA) [62] and
lipofectamine2000 (Invitrogen) have also been used (Table II).
Anionic liposomes have also been used as vesicles to entrap DNA for gene transfer for in
vitro and in vivo transfection. However, these liposome formulations present some limitations
associated with low efficiency of DNA encapsulation, DNA degradation induced by
sonication and the requirement to remove the free DNA from liposome-entrapped DNA. To
overcome these limitations, stabilized antisense-lipid particles (SALP) were developed for
improved efficiency of encapsulation of DNA [63]. These systems utilize cationic lipids such
as DODAP or N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), DC-Chol and an
ethanol-containing buffer system for encapsulating large quantities of DNA in lipid vesicles.
DOTMA
O

+
N
O

X
O

DOTAP

Chol

X=

HO-

DC-Chol
O

DODAP

X = (CH3)2NCH2CH2NHCO

O
O

OH-Chol
+

+ N

X = HOCH2CH2NHCH2CH2NHC

O
O

DMRIE

HO

N
O
O

DOPE
+
H3N

O
O

P
O-

O
O

O
O

Figure 1. Structures of commonly used cationic lipids and helper lipids in gene therapy. DOTAP;
dioleoyltrimethylammonium propane, DODAP; dioleoyldimethylammonio propane, DOTMA;
dioleoyltrimethylammonium chloride, DODAP, ioleoyldimethylammonio propane, DMRIE;

Yoshiyuki Hattori and Yoshie Maitani

102

dimyristyloxypropyldimethyl-hydroxy ethyl ammonium, Chol; cholesterol, DC-Chol;


dimethylaminoethanolamine carbamoyl cholesterol, OH-Chol; cholesteryl-3-carboxyamidoethyleneN-hydroxylamine, and DOPE; dioleoyl phosphatidylethanolamine.

Table II. Gene delivery and therapy by non-viral vectors. All vectors were used in in
vitro transfection
Transfection method/
Formulation

Naked DNA

Liposome

Nanoparticle

DNA

Promoter

Gene

Combination

Cell lines or tumor

in vivo

AS-ODN

EGFR

Hydrodynamic
injection

Plasmid

CAG

IL-21 and 15

1483 (SCCHN)
UM-22B
(Hypopharyngeal tumor)
SCC-VII (SCCHN)

Electroporation

Plasmid

CMV

p27

DC-Chol/DOPE

Plasmid

U6

TGF- AS-ODN

DC-Chol/DOPE

Plasmid

U6

EGFR AS-ODN Endostatin

DC-Chol/DOPE

Plasmid

CMV

DMRIE/DOPE

Plasmid

RS

Ref.

i.t.

[41]

i.v.

[55]

B88 (Oral tongue cancer) i.t.

[43]

1483

i.t.

[50]

Docetaxel

1483

i.t.

[51,52]

E1A

Clinical trial for SCCHN

i.t.

[53]

HLA-B7

Clinical trial for SCCHN

i.t.

[59,60]

i.t.

[57]

DOTMA/Chol

Plasmid

CMV

IL-2

Surgery

SCC VII

DOTMA/Chol

Plasmid

CMV

IL-2

Cisplatin

SCC VII

i.t.

[144]

DOTMA/Chol

Plasmid

CMV

IL-2 and IL-12

SCC VII

i.t.

[56]

DOTMA/Chol

Plasmid

CMV

IL-2 and IL-12

Radiation

SCC VII

i.t.

[58]

DOTAP/Chol

Plasmid

SV40

LacZ

[54]

Plasmid

CMV

HSV-tk

HMG (Oral malignant


melanoma)
HSC-3 and H357
(oral SCC)

Metafectene
and Genejammer

[61]

Oligofectamine

AS-ODN

telomerase

Tca 8113 (Tongue


carcinoma)

[62]

CD-TK

CNE-2 (NPC)

[152]

Calcium Phosphate Plasmid

Retinoic acid

CMV; cytomegalovirus, RS; respiratory syncytial virus, CAG; chicken -actin promoter with CMV enhancer, SV40; simian virus 40, Squamous cell carcinoma;
SCC, Squamous cell carcinoma of head and neck; SCCHN.

Nanoparticles
The definition of nanoparticle is a formula containing no inner phase, in contrast to
liposomes. Cholesterol derivatives are usually unable to form stable bilayers unless used in
combination with DOPE or some other neutral lipids. Therefore, these particles composed of
cholesterol derivatives and surfactants are nanoparticles without bilayers. Cationic
cholesterol derivatives have been used because of their high transfection activity and low
toxicity. A series of second-generation cholesterol-based cationic lipids have been developed
and studied. Among them, cholesteryl-3-carboxyamidoethylene-N-hydroxyethylamine (OHChol), having a hydroxyethyl group at the amino terminal (Figure 1) is a cationic lipid
showing the most efficient transfection efficiency [64,65]. Nanoparticles consisting of
cationic cholesterol derivatives, DC-Chol or OH-Chol as a cationic lipid and Tween 80 can
be prepared by a modified ethanol injection method, and are about 100-200 nm in size and
show no significant change in size for at least 1 year [66,67]. DNA/nanoparticle complexes

Tumor-Targeting Non-Viral Gene Therapy for the Treatment of Oral Cancer

103

(nanoplex) were formed after mixing the particle and DNA solutions. In other cases, to form
multicomponent vectors, DNA was compacted with polymers or detergent to form
DNA/polymer or DNA/detergent complexes.

5. NON-VIRAL VECTORS FOR ORAL TUMOR TARGETING


The challenge for tumor-specific targeting using particulate gene delivery systems is to
decrease this nonspecific gene transfer in the normal tissues while simultaneously
maintaining or increasing the level of gene transfer to the tumor tissues. Selective targeting of
ligand-linked non-viral vectors to cell surface receptors expressed on tumor cells is a
recognized strategy for improving the therapeutic effectiveness of gene therapeutics. For nonviral gene delivery into oral tumor, folic acid and transferrin have been used as a tumortargeting ligand.

5.1. Folic Acid


Coenzyme derivatives of folic acid (reduced folate) are necessary for the synthesis of
purine and pyrimidine precursors of nucleic acids, for the metabolism of several amino acids,
and for the initiation of protein synthesis in mitochondria. Acquisition of folic acid, therefore,
is critically important to the viability of proliferating cells. Humans and other mammals
cannot synthesize folic acid, and thus must obtain the vitamin from exogenous sources via
absorption in the intestine [68]. Reduced folate is a structurally related compound that has the
biochemical activity of folic acid. Unless otherwise indicated, the use of the term reduced
folate throughout this review refers to the principal plasma folate, 5-methyltetrahydrofolate,
whereas the use of folic acid or folate strictly refers to the vitamin in its oxidized form or
folic acid derivatives, respectively.
Two functionally different systems exist for cellular uptake of folates: (1) membranebound folate receptor, which is linked to the cell surface via a glycosylphosphatidylinositol
(GPI) anchor and internalizes folates by receptor-mediated endocytosis [69] and (2) reducedfolate carrier (RFC), which uses a bidirectional anion exchange mechanism to transport folate
into the cytoplasm [70]. The RFC is a low-affinity, high capacity system that mediates the
uptake of reduced folate into cells, predominantly at pharmacologic (micromolar)
extracellular folate concentrations [71]. The RFC transports monoglutamyl reduced folate
across tissue membranes.
Cellular folate transport can also be mediated by 38- to 44-kDa membrane associated
folate-binding proteins (FBPs) or FRs (these terms are used synonymously throughout),
which bind physiologic folate with high affinity in the nanomolar range [71]. Three isoforms
of FR have been identified and two, FR- and -, are attached to the cell by a GPI-anchor,
while FR- is secreted due to the lack of an efficient signal for GPI modification [72]. FRs
were found to be clustered in membrane regions called rafts [73] or caveolae [74,75] which
are rich in cholesterol and glycosphingolipid. The role of FRs in the cellular transport of

104

Yoshiyuki Hattori and Yoshie Maitani

folate is not well understood [76,77], although a potocytosis (rafts [73] or caveolin-coated
endocytosis [74,75]) model has been proposed.
FRs have been found to be overexpressed in a wide range of tumors. While elevated
expression of FR has frequently been observed in various types of human tumors, the
receptor is generally absent in normal tissues with the exception of the proximal tubules of
the kidney, the choroid plexus, intestinal brush-border membranes, type 1 and type 2
pneumocytes of the lung, and placental tissue [71,78-84]. FR- is frequently overexpressed in
tumors, including ovarian, colorectal, breast, lung, renal cell carcinomas and brain metastases
derived from epithelial cancers [72,85]. FR- is frequently overexpressed in tumors of nonepithelial cell lineages such as sarcomas and acute myeloid leukemia [86], and FR- is
overexpressed in malignant hemopoietic cells [87]. The causes of FR overexpression in
tumors are unclear, but high levels of FR are associated with increased biological
aggressiveness of carcinomas.
The major route of reduced folate entry into nonmalignant cells is RFC, which will not
transport folate conjugates of any type. Thus, folate-linked pharmaceuticals only enter cells
via the FR, which is overexpressed on cancer cells [88]. This inability of folate conjugates to
penetrate the RFC contributes to the low toxicity of folate-linked agents toward normal cells.
Therefore, FR presents an attractive target for tumor-selective delivery. FR-targeting
materials can continuously accumulate in cells due to receptor recycling. FR-targeting
imaging agents arrived on the market in 2004.

Figure 2. Schematic diagrams of FR- or TfR-targeting liposome and nanoparticle. A) FR-targeting


liposomes, B) FR-targeting nanoparticles, C) Tf-liposomes, D) TfR-targeting immunoliposomes.

Tumor-Targeting Non-Viral Gene Therapy for the Treatment of Oral Cancer

105

5.2. Folate-Linked Particle Vectors


Folic acid offers many potential advantages as a targeting ligand: (1) small size of the
targeting ligand, which often leads to favorable phamacokinetic properties of the folate
conjugates and reduced probability of immunogenicity; (2) convenient availability and low
cost; (3) relatively simple and defined conjugation chemistry; (4) high affinity for FR and
lack of FR expression in normal tissue; (5) the receptor and ligand complex can be induced to
internalize via endocytosis and (6) high frequency of FR overexpression among human
tumors. Therefore, folate-linked targeting systems show great potential for clinical and
therapeutic application
The liposomes used in recent studies have been coated with folate-polyethyleneglycol
(PEG)-lipid to facilitate tumor-targeting by an active mechanism (via FR) (Figure 2A).
PEGylated lipids can significantly reduce the nonspecific gene transfer activity in the lung,
and conjugation of the targeting ligand, folate, to the PEG chain can restore the gene transfer
activity toward FR-positive tumors in vivo [89]. The incorporation of a long PEG spacer
between folate and the lipid is important for efficient FR-targeted gene delivery. Modifying
the length of the PEG-spacer between folate and the lipid optimized the targeting activity of
the liposomes, and PEG spacers ranging from molecular weight 1,000 to 3,400 could function
as effective spacers [90]. This is believed to the need for folate to enter the binding pocket of
FR on the cell surface.
Table III. Tumor-targeted non-viral lipid-based vectors. All vectors were used in in vitro
transfection
Ligand

Formulae

Folic acid (Folate-PEG-Lipid


or Folate-Lipid)

Chol/DODAP/PEG-CerC16

Liposome
entrapping DNA

Lipoplex

Lipoplex

Transferrin
receptor antibody
Liposome

Lipoplex

EFGR

KB

in vivo

Ref.

[101]

EggPC/Chol

AS-ODN

EFGR

KB

DODAP/DSPE/Chol/PEG-DSPE
DC-Chol/eggPC/PEG-DSPE

AS-ODN

Bcl-2

KB

[98]

EggPC/Chol

AS-ODN

ICAM-1, H-Ras

KB

i.v.

[90]

EFGR

KB

[99]

DOTAP/Chol

Plasmid

SV40

Luciferase

KB

[93]

DOTAP/DOPE

Plasmid

CMV

p53

JSQ-3

i.v.

[91]

DOTAP/DOPE

Plasmid

CMV

p53

JSQ-3

i.v.

[92]

CHEMS/DOPE
DOPS/DOPE
LPDII type CHEMS/DOPC

Plasmid

RSV

Luciferase

KB

[95]

Plasmid

CMV

Luciferase

KB

[100]

Plasmid

CMV

Luciferase

KB

[103, 104]

Cationic
liposome

C14Corn
C14Corn

Plasmid

CMV

HSV-tk

KB

i.t.

[66,67,105]

OH-Chol/Tween80

Plasmid

CMV

Luciferase

KB

[106]

DOTAP/Chol

Plasmid

CMV

HSV-tk

HSC-3, SCC-7 i.t.

DOTAP/DOPE

Plasmid

CMV

p53

JSQ-3

[115]

DOTAP/DOPE

Plasmid

RSV

p53

JSQ-3

i.t.

[116]

DOTAP/DOPE
DDAB/DOPE

Plasmid

CMV

p53

JSQ-3

i.v.

[118]

DC-Chol/Tween80
Lipidnanoparticle

Transferrin
Liposome

Tumor

AS-ODN

Tetradecylornithinylcystein (C14Corn)
Nanoplex

Gene

[96]

Diolein/CHEMS

Nanoparticle

AS-ODN

Promoter

Chol/DODAP/DSPC/PEG-CerC16

Liposome
Lipopolyplex

DNA

Cationic
liposome

Cationic
liposome

i.v.: intravenous injection, i.p.: intraperitoneal injection; i,.t.:intratumoral injection.

[114]

106

Yoshiyuki Hattori and Yoshie Maitani

FR-targeting cationic liposomes were coated with folate-derivatives, such as folateDOPE [91], folate-PEG-DOPE [92], folate-PEG-phosphatidylethanolamine (PE) [93], folatePEG-DOPE [94,95], folate-PEG-distearoyl phosphatidylethanolamine (DSPE) [90,96-99],
folate-PEG-Chol [100] (Table III). A folate-cationic liposome system could mediate gene
therapy with p53 antisense DNA in head and neck tumor cells (JSQ-3 cells) [92]. For
liposome-encapsulated DNA, FR-targeting liposomes coated with a folate-derivative, folatePEG-DSPE, showed efficient gene transfer into KB cells [90,96,98,99,101] (Table III).
LPDII-type lipoplexes (lipopolyplexes) consist of a ternary complex of anionic
liposomes, DNA-condensing polycation, and plasmid DNA (Table III). To prepare a
formulation of LPDII-type vector, DNA was first attached to poly-L-lysine (PLL) and then
mixed with pH-sensitive anionic liposomes composed of DOPE/CHEMS/folate-PEG3350DOPE [95,100]. PH-sensitive liposomes are fusogenic at acidic pH and thus can be used to
facilitate the endosomal disruption and subsequent release of plasmids in the cytoplasm. An
LPDII vector that incorporated polyethylenimine (PEI) as a DNA-condensing agent and a
cationic/anionic lipid pair, composed of dimethyldioctadecylammonium bromide
(DDAB)/CHEMS/polyoxyethylene sorbitan monoolate (Tween80)/folate-PEG3350-DSPE
showed efficient gene delivery into KB cells [102].
An FR-targeting cationic nanoparticle incorporating folate-PEG3400-dipalmitoyl
phosphatidylethanolamine (DPPE) and a cationic dithiol-detergent (dimerized tetradecylornithinyl-cysteine, (C14Corn)2) showed efficient FR-dependent cellular uptake and
transfection [103]. C14Corn was capable of monomolecular DNA condensation, and
modification of the surface of monomolecular DNA with distamycin-PEG3400-folate
conjugate with 2 equivalents of bisbenzimidazole fragment increased cellular uptake in KB
cells [104].
FR-targeted cholesterol-based nanoparticles consisting of Tween 80 and DC-Chol (NPIF) or OH-Chol (NPII-F) with 1-2 mol% folate-PEG2000-DSPE were produced (Figure 2B)
[66,67,105,106]. The use of NPI-F increased transfection efficiency 44-fold in KB cells
compared with NPI without folate-PEG-lipid [67]. In contrast, NPII without PEG-lipid
exhibited the highest level of transfection activity into the cells. PEG-lipid in NPII reduced
the transfection activity 30-fold, but folate-PEG of NPII-F increased the activity 6.6-fold
compared to PEG-coated NPII [106].

5.3. Transferrin
Iron is required for the activity of ribonucleotide reductase, a key enzyme involved in
DNA synthesis. Transferrins comprise a family of large non-hem iron-binding glycoproteins.
The three major types of transferrins have been characterized. Serum transferring (Tf) occurs
in blood and other mammalian fluids including bile, aminitotic fluid, cerebrospinal fluid,
lymph, colostrom, and milk. Ovotransferrin (oTf) is found in avian and reptilian oviduct
secretions and in avian egg white [107,108], and lactoferrin (Lf) is found in milk, tear, saliva,
and other secretion [109,110]. Tf is mainly synthesized by hepatocytes, with a concentration
of 2.5 mg/ml and 30% occupied with iron in blood plasma [111]. The principal biological
function of transferrins is thought to be related to iron binding properties. Serum Tf has the

Tumor-Targeting Non-Viral Gene Therapy for the Treatment of Oral Cancer

107

role of carrying iron from the sites of intake into the systemic circulation to the cells and
tissues.
The transferrin receptor (TfR) is a key cell surface molecule that regulates uptake of ironbound transferrin by receptor-mediated endocytosis. For more than 20 years, there has been a
known correlation between the number of cell surface TfR and the rate of cell proliferation.
TfR expression is higher in oral cancer cells than in normal cells [112,113]. TfR expression
correlates with cellular proliferation and is found higher in rapidly dividing cells. The density
of TfR has also been correlated with the rate of DNA synthesis and metastatic potential of
tumor cells. Therefore, TfR are considered to be useful as a prognostic tumor maker and as a
potential target for gene delivery in therapy of oral cancer.

5.4. Transferrin-Liposome Vectors


Tf has demonstrated that its ability to direct cationic liposome to the receptor-bearing
cells. Cationic liposomes composed of positively charged lipid bilayers can be complexed to
negatively charged DNA and Tf by simple mixing (Figure 2C) [114-116]. Tf-lipoplex was
negatively charged ternary complexes of cationic liposome, plasmid DNA and Tf. The in
vitro transfection efficiency of cationic liposomes can be dramatically increased when
complexed with Tf, employing ligand-receptor mediated endocytosis mechanism. Tfliposome showed 60-70% and 20-30% of in vitro and in vivo transfection efficiency,
respectively, for JSQ-3 cells. The optimal Tf-lipoplex particle size on gene transfection
efficiency was found to be 50-90 nm [117]. The particle has a highly compacted structure,
and resembles a virus particle both in archtitecture and its uniformly small size. This viruslike compact nanostructure is likely to be the key to their high gene transfection efficiency
and efficacy both in vitro and in vivo. A model of self-assembly process of this nanoparticles
was proposed [117].
Modification of liposome with TfR antibody also enables active targeting to the receptor
bearing tumor cells. Immunoliposome for TfR has been developed as a gene delivery vehicle
(Figure 2D). Xu et al. reported a cationic immunolipoplex system directed by a single chain
antibody variable region fragment (scFv) against the TfR enhanced the transfection efficiency
for JSQ-3 cells both in vitro and in vivo [118].

6. DNA GENE THERAPY


Cancer gene therapy has become an increasingly important strategy for treating a variety
of human diseases [119]. These strategies of gene therapy for oral cancer in clinical trials
include inactivation of oncogene expression, gene replacement for tumor suppressor genes,
and cytokine transfer.

108

Yoshiyuki Hattori and Yoshie Maitani

6.1. Tumor Suppressor Gene


Several genetic alterations have been described in oral cancer, including p53, the
retinoblastoma gene (Rb1), p16, and p21. The most extensively studied mutations in oral
cancer are those of p53. Dysfunction of p53 is associated with the progression of
tumorigenesis [120]. The presence of mutant p53 has also been shown to be associated with
an unfavorable prognosis for many human cancers, including lung, colon and breast cancers.
Effective restoration of p53 function in tumor cells is expected to re-establish normal cell
growth control and restore appropriate responses to DNA damage.
When folate-linked cationic liposomes were used as a vector for human SCC of the head
and neck, the folate ligand increased the transfection efficiency and transient p53 gene
expression both in vitro and in vivo [91,92,121]. The systemic delivery of p53 into tumors
resulted in efficient expression of functional p53, sensitizing the tumors to chemotherapy and
radiotherapy [92]. Tf-lipoplex has also demonstrated high efficiently in tumor-target gene
delivery and long-term therapeutic accuracy in systemic p53 gene therapy for head and neck
cancer [115,116]. Tf significantly increased the transfection efficiency for JSQ-3 cells when
compared with the liposome alone even in the presence of high levels of serum. Moreover,
when combined with radiation, the Tf-lipoplex-p53-treated group exhibited significant tumor
regression in head and neck cancer animal model [116].
p27 kip1 is cyclin-dependent kinase inhibitor that regulates progression of cells from G1
into S phase in a cell cycle [122,123], and p27 kip1 protein has attracted as an important
prognostic factor in various malihnancies. Loss of p27 kip1 has been associated with disease
progression in several malignancies [124]. Transfection with p27 kip1 gene into malignant
human oral cancer cells leads to inhibition of proliferation, invasion and metastasis,
suggesting that p27 kip1 acts as a tumor suppresser gene [125]. Reduced expression of p27 kip1
in cancer cells occures due to an increase in the rate by ubiqutin-mediated degradation [126].
Therefore, mutant-type p27 kip1 gene, which was not influenced by ubiqutin-mediated
degradation, was constructed for gene therapy [127]. When the transfection of mutant-type
p27 kip1 gene was directly injected into human oral tongue cancer B88 xenografts by
electroporation, mutant-type p27 kip1 exhibited suppression of tumor growth [43].
Deregulation of connexin (Cx) expression is believed to play a part in carcinogenesis
[128]. Cx proteins have an essential role in gap junction intercellular communication (GJIC),
which is often impaired among tumor cells and between tumor cells and surrounding normal
cells. Connexin 43 (Cx43) is a tumor-suppressor [129], and its expression is reduced in
various tumors including oral tumors [130]. Forced expression of the Cx43 gene in several
Cx43-deficient tumor cell lines attenuated their malignant [131,132]. When Cx43 plasmid
DNA was transfected into KB cells by NPI-F, Cx43 exhibited suppression of tumor growth
[133]. The transfection into KB cells induced up-regulated mRNA expression of p16, which
is known as a tumor growth suppressor [16]. The expression of Cx43 in KB cells also
increased apoptosis via down-regulation of anti-apoptotic bcl-2 mRNA expression and upregulation of apoptosis-associated enzyme caspase-3/7 activity.
E1A, a gene derived from adenovirus type 5, has been shown to have potent antitumor
activity through a variety of mechanisms, including down-regulation of HER-2 expression
[134,135], induction of apoptosis [136], inhibition of metastasis [137,138], and reversion of

Tumor-Targeting Non-Viral Gene Therapy for the Treatment of Oral Cancer

109

tumor cells toward a differentiated epithelial phenotype [139]. The EIA gene has been
successfully transfected into SCC of head and neck cells using cationic liposome comprised
of DC-Chol/DOPE complexed with the E1A plasmid [53]. The E1A gene has demonstrated
antitumor activity in vitro and in xenografts models. Intratumoral injection of the lipoplex
with E1A plasmid has been shown to be safe and well-tolerated and produce tumor responses
comparable to other biologic agents in phase II trials [53].
6.2. Immunotherapy
The immunologic gene therapy approach to oral cancer involves either increasing the
immunogenic potential of tumor cells or augmenting the paitients immune response to a
tumor. Although oral cancer is not classically immunogenic, there is abundant evidence for
immune recognition. Tumor-specific T-cell mediated immunity has been recognized as a key
mechanism of the antigen specific immune response against tumors [140]. Cytotoxic T
lymphocyte (CTL) responses are essential for the recognition of tumor cells and can
eliminate established tumors in animal models and humans [141,142]. The induction of
strong antigen specific CTL response is, therefore, the major goal of many current cancer
gene therapies [143].
Interleukin (IL)-2 stimulates the proliferation and activation of several types of
leukocytes with anti-tumor activities, including natural killer (NK) cells, lymphokineactivated killer cells and antigen-specific T-helper cells and cytotoxic lymphocytes, as well as
macrophages and B cells [32,33]. The anti-tumor activity of IL-2 has been demonstrated in
clinical studies, but the mechanism which IL-2 treatment results in tumor regression in
human is not fully understood. For the clinical use, local production of low levels of IL-2
should produce limited or no systemic side effects, because systemic therapy with IL-2 is
associated with significant toxicity. Non-viral gene therapies with IL-2 have been studied as
an alternative to the potentially toxic adenovirus or other viral vectors. A plasmid/cationic
lipid system for IL-2 gene therapy has been described that DOTMA/Chol formulated with IL2 plasmid (pCMV-IL-2) reduced tumor size by intratumoral injection [57,58,144,145].
Treatment of tumors with formulated pCMV-IL2 produced IL-2 protein levels that were 5fold over background, and increased IFN- by 32-fold and IL-12 by 5.5-fold compared with
control plasmid formulation [144]. The phase II studies have initiated and focus on either
comparing the novel non-viral IL-2 gene immunotherapy formulation alone to methotrexate
or comparing IL-2 gene therapy in combination with cisplatin in recurrent or unresectable
patients with SCC of head and neck [144]. The use of combinated IL-2 and IL-12 gene
therapy for SCC also resulted in significant anti-tumor effect, most likely due to increased
activation of CTL and NK cells [58].
IL-21 also plays important roles in the regulation of T, B, and NK cells, and provides an
immunotherapy strategy for cancer gene therapy by stimulating both Th1 and Th2 immune
responses [146]. Significant antitumor effect was observed by repeated transfection with IL21 by hydrodynamic injection into the mice bearing subcutaneous SCC of head and neck, and
co-administration of IL-21 and IL-15 genes resulted in increased suppression of tumor
growth, significantly prolonging the survival periods [55].
Attempts have also been made to generate tumor specific (HLA-restricted) immune
responses. Recognition of foreign tumor antigens by the immune system requires presentation

110

Yoshiyuki Hattori and Yoshie Maitani

of antigen peptide fragments in context of MHC-class I or class II molecules. CD8+ CTLs are
activated by MHC-class I bearing cells, while CD4+ CTLs are stimulated primarily by tumor
peptides presented by cell surface MHC-class II molecules. MHC-class I levels in oral tumor
cells are often decreased or undetectable [147,148]. This may represent one mechanism by
which tumor cells escape immune rejection. Avellovectin-7 consisted of a DNA plasmid
(VCL-1005) containing the gene for allogeneic MHC-class I protein, human leukocyte
antigen B7 (HLA-B7), and 2-microglobulin complexed with cationic lipid mixture
(DMRIE/DOPE) that aids in the uptake of VCL-1005 into tumor cells [59,60]. Intratumoral
injection of Avellovectin-7 to SCC of head and neck resulted in potential tumor growth
suppression in 20 of the 60 patients 6 weeks after the injection and in persistent tumor
regression lasting 16 weeks or longer in 11 patients [59].

7. DNA THERAPY IN CHEMOTHERAPY


Suicide gene therapy is a strategy whereby a gene is introduced into cancer cells, making
them sensitive to a drug that is normally non-toxic. The suicide genes used often encode
enzymes that metabolize non-toxic prodrugs into toxic metabolites. Among them, two of the
best characterized systems are herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir
(GCV) [149] and cytosine deaminase (CD)/5-fluorocytosine (5-FC) [150]. These suicide
gene-prodrug systems are currently being evaluated in clinical trials. HSV-tk converts the
antiviral drug, such a GCV, to the monophosphorylated forms that are then metabolized to
the toxic form (GCV triphosphate) by cellular phosphokinases [149]. GCV triphosphate
interacts with cellular DNA polymerase, causing interference with DNA synthesis and
leading to the death of dividing cells. CD converts 5-FC into the toxic anabilite 5-FU, which
is subsequently processed either to 5-fluorouridine triphosphate (FUTP) or to 5-fluoro-2deoxyuridine-monophospate (FdUMP) [150]. Whereas FUTP is incorporated into RNA and
interferes with RNA processing, FdUMP irreversibly inhibits thymidylate syntheses and thus
interferes with DNA synthesis. A powerful characteristic of both suicide gene therapies is
that the transduction of a small fraction of tumor cells with the suicide gene can result in
widespread tumor-cell death (bystander effect). The cell-to-cell transfer of enzyme-activated
prodrug between enzyme expressed tumor cells and neighboring unmodified cells via gap
junctions is a major mechanism of the bystander effect [151,152]. This is attractive therapy
since the transduction of a small fraction of the tumor cells with the suicide gene can result in
widespread tumor-cell death.
Calcium phosphate nanoparticle (CPNP) was developed as non-viral vector, and the
CPNP/DNA complex could deliver DNA into nasopharyngeal CNE-2 tumor xenografts by
intratumoral injection [153]. When CNE-2 cells were treated with 5-FC plus the CPNP/
CDglyTK plasmid coding for CD and HSV-tk fusion protein, the potent antitumoral activities
was observed in vitro. In FR-targeting nanoparticle vector, tumor growth of xenografts was
significantly inhibited when a NPI-F nanoplex of the HSV-tk gene was injected
intratumorally and GCV was administered intraperitomeally [67].

Tumor-Targeting Non-Viral Gene Therapy for the Treatment of Oral Cancer

111

8. ANTISENSE DNA AND SIRNA THERAPY


Posttranscriptional gene silencing approaches using various nucleic acid based molecules
such as antisense oligodeoxynucleotides (AS-ODNs) have drawn much attention because of
their potential therapeutic usefulness for treating various genetic disorders and infectious
diseases, including cancer. AS-ODNs are short single-stranded segments of DNA that upon
cellular internalization can selectively inhibit the expression of a single protein [154]. The
AS-ODN forms a duplex with the mRNA or pre-mRNA and inhibits their translation or
processing, consequently inhibiting protein biosynthesis. Since the targets for antisense
applications are in the cytoplasm, AS-ODN does not need to enter the cell nucleus [155].
Several applications of AS-ODN have been described in treatment of oral tumor,
including EGFR, bcl-2, TNF- and HER-2 AS-ODNs (Table II and III). The epidermal
growth factor receptor (EGFR) is commonly overexpressed in a variety of solid tumors, and
clinical trials indicate that this antigen has important roles in cancer progression [156]. When
naked EGFR AS-ODN was directly injected into SCC of head and neck 1483 xenografts,
tumor volume was significantly reduced in the mice treated with a combination of EGFR ASODN and docetaxel, suggested that blocking EGFR in conjunction with cytotoxic
chemotherapy, cancer cells undergo apoptosis [41]. Direct injection of the EGFR AS-RNA
expressing plasmid DNA into SCC of head and neck xenografts with liposome resulted in
inhibition of tumor growth, suppression of EGFR protein expression, and an increased rate of
apoptosis [52]. A combination of anti-angiogenic endostatin and EGFR AS-RNA expressing
plasmid DNA also led to significantly enhanced inhibition of SCC of head and neck growth
in nude mice [51].
FR-targeted liposomes (SALP) entrapping EGFR AS-ODN efficiently mediated
intracellular delivery of the AS-ODN to KB cells, resulting in significant down-regulation of
EGFR expression and cell growth inhibition [96,99]. Bcl-2 is one of the most important
mammalian regulators of apoptosis and is overexpressed in the majority of human neoplasms,
including breast, prostate and lung carcinomas [157]. FR-targeted liposomes containing bcl-2
AS-ODN showed promising transfection activity in KB cells and induced down-regulation of
bcl-2 expression and an increase of the sensitivity to daunorubicin [98].
The ability of gene-specific double-stranded RNA to trigger the degradation of
homologous cellular RNAs is known as RNA intereferance (RNAi). RNAi is a powerful
gene-silencing process that holds great promise in the field of cancer therapy [158]. RNAi
can be induced in mammalian cells by the introduction of synthetic small interfering RNA
(siRNA) 2123 base pairs in length or of plasmids that express short hairpin RNAs (shRNAs)
that are subsequently processed to siRNAs by the cellular machinery [159,160]. Doublestranded RNA (dsRNA) suppresses the expression of a target gene by triggering specific
degradation of the complementary mRNA sequence. For therapeutic use in cancer, candidate
target genes for RNAi-mediated knockdown have been identified [161]. To promote their
gene inhibition effect, folate-linked cationic nanoparticles have been employed to form
complexes with negatively charged synthetic siRNA. HER-2 is a member of the EGFR
family that participates in tumor growth and proliferation [162]. We found that the NPIIF/HER-2 siRNA nanoplex efficiently mediated intracellular delivery of synthetic siRNA to
KB cells, resulting in a significant down-regulation of HER-2 expression and cell growth

Yoshiyuki Hattori and Yoshie Maitani

112

inhibition (70% cell viability compared with that of control siRNA) (unpublished data). The
development of RNAi delivery system has the potential in cancer therapy.

9. CONCLUSION
In further, gene diagnosis will be prevailed, corresponding to obtained gene
informations, gene therapy may open a new dimension of treatment of cancer, resulting in
better tumor-targeting gene therapy. In this review, we showed that non-viral vectors could
deliver DNA with high transfection efficiency and selectivity, inhibiting tumor growth
following intratumoral injection into oral tumor. Tumor-targeted liposomes and lipid-based
nanoparticles have potential as a clinically effective vector in cancer gene therapy. However,
there is generally little correlation between in vitro and in vivo gene transfer efficacies of
vector formulations, due to very different parameters. Further efforts aimed at optimizing
tumor-targeting vector formulations for local administration should lead to the clinical
evaluation of these vectors for cancer gene therapy delivery.

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In: Oral Cancer Research Advances


Editor: Alexios P. Nikolakakos, pp. 125-153

ISBN 978-1-60021-864-4
2007 Nova Science Publishers, Inc.

Chapter 5

NEW DIAGNOSTIC IMAGING MODALITIES FOR


ORAL CANCERS
Yasuhiro Morimoto1,, Tatsurou Tanaka1, Izumi Yoshioka2,
Yoshihiro Yamashita2, Souichi Hirashima2, Masaaki Kodama2,
Wataru Ariyoshi2, Taiki Tomoyose2, Norihiko Furuta2,
Manabu Habu2, Sachiko Okabe1, Shinji Kito1, Masafumi Oda1,
Hirohito Kuroiwa1, Nao Wakasugi1, Tetsu Takahashi2 and
Kazuhiro Tominaga2
1

Dep. of Oral Diagnostic Science, Kyushu Dental College, Kitakyushu, Japan;


Dep. of Oral and Maxillofacial Surgery, Kyushu Dental College, Kitakyushu, Japan.

ABSTRACT
This article reviews the use of imaging modalities; both commonly used and recently
introduced, to evaluate oral cancers and their lymph node metastases. Magnetic
resonance images (MRI) and X-ray computed tomography (CT) images are used to
determine the size, invasive area, and possible pathology of primary cancers. In addition,
the two modalities are useful for staging and detecting clinically occult lymph node
metastases at different levels of the neck. In particular, a follow-up MR examination
method, dynamic MR sialography, for patients with xerostomia after radiation therapy is
introduced, and the use of fusion images of the tumors and vessels using threedimensional fast asymmetric spin-echo (3D-FASE) and MR angiography is discussed.
Furthermore, ultrasound imaging (US), in addition to its use for staging and detecting
clinically occult lymph node metastases, plays an important role in confirming intraoperative surgical clearance of tongue carcinomas. In addition, the role of US-guided,

Correspondence concerning this article should be addressed to: Yasuhiro Morimoto DDS PhD, Division of
Diagnostic Radiology, Department of Oral Diagnostic Science, Kyushu Dental College, 2-6-1 Manazuru,
Kokurakita-ku, Kitakyushu 803-8580, JAPAN. TEL: 81-93-582-1131 (Ext. 2111); FAX: 81-93-581-2152; Email: rad-mori@kyu-dent.ac.jp.

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fine-needle aspiration biology is also reviewed. Finally, the role and limitations of fusion
images obtained from positron emission tomography (PET) and CT (PET-CT), which are
currently used worldwide, are discussed.

Keywords: PET-CT; X-ray CT scanning; MRI; Dynamic MR sialography; MR angiography;


ultrasonography; intra-oral ultrasonography

INTRODUCTION
Malignant neoplasms of the mouth constitute appropriately 1% of all cancers and about
3% of head and neck cancers [1-3]. Seven percent of oral cavity tumors are malignant, and
over 90% of malignant neoplasms are squamous cell carcinomas [1,3]. Since other
malignancies can occur in the mouth, such as minor salivary tumors, malignant lymphomas,
and a variety of other rare tumors, these are also discussed [1,2]. The second half of the last
century generated much knowledge about the human body, including mapping of the human
chromosome, but the occurrence, development, and diagnosis of oral cancer still remain
obscure. It is thought that both a genetic predisposition and environmental factors, including
alcohol abuse and tobacco chewing, lead to the development of oral cancers [1-3]. A
particular characteristic of oral cancers is that most lesions are amenable to direct clinical
examination and can be easily biopsied [1-3]. Therefore, the most important purpose of
imaging these lesions is to identify the extent of invasion into surrounding tissues, including
the mucosa, fatty tissues, vessels, nerves, muscles, salivary glands, mandible, and maxilla [2].
In addition, lymph node metastases in primary neoplasm-related regions can be detected
using various kinds of imaging modalities [1,2]. Of course, the sizes, shape, and inner
characteristics of lesions should also be delineated. Consequently, primary mouth
malignancies are staged according to the TNM system of the American Joint Committee on
Cancer Staging (AJCCS) [1,2].
In the present article, basic and recent clinical applications of various imaging
modalities, such X-ray CT scan, MRI, US, and PET-CT, as they relate to diagnosing mouth
cancer, are reviewed.

X-RAY CT SCANS AND MRI USED FOR DETECTING PRIMARY


ORAL CANCERS AND LYMPH NODE METASTASES
X-ray CT scan and MRI are the most common and the most suitable modalities for
evaluating oral cancers [1-5]. CT scans are performed using whole body type machines
(Figure 1); for example, in our dental hospital, a Toshiba X Vision RE machine (Toshiba
Co. Ltd., Tokyo, Japan) is used. For X-ray CT scans, it is commonly advocated that scanning
be performed in the axial plane without angulation in 5-mm-thick contiguous sections. X-ray
CT scan is the most appropriate for identifying and evaluating the lesions location and size,
as well as bone and surrounding soft tissue invasion [1-5]. CT scans are performed after the
patient has been given an intravenous dose of 50 mL iohexol (300 mgI/mL; Omnipaque

New Diagnostic Imaging Modalities for Oral Cancers

127

300, Daiichi Pharmaceutical Co. Ltd., Tokyo, Japan) at the start of scanning, followed by
an additional 50-mL intravenous infusion during scanning to allow better visualization of the
vascular structures [1,4,5]. Images are photographed based on standard algorithms and softtissue windows. In cases where an exact evaluation of erosive changes in the mandible and
maxilla is required, coronal plane views should be produced using multi-planar
reconstruction (MPR) techniques after acquisition of axial planes with a 1-2-mm section
thickness [1,5]. Furthermore, the CT scan can encompass the area from the cavernous sinuses
to the thoracic inlet in order to examine the primary cancer and possible lymph node
metastases in the neck.

Figure 1. The whole body type X-ray CT scanning machine showing gantry and patient bed (Toshiba X
Vision RE, Toshiba Co. Ltd., Tokyo, Japan).

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Yasuhiro Morimoto, Tatsurou Tanaka, Izumi Yoshioka et al.

Figure 2. 1.5 Tesla whole body type MR system (VISART, Toshiba Co. Ltd., Tokyo, Japan) showing
the bore and patient bed.

MR images are acquired using a 1.5-T full-body MR system; for example, in our dental
hospital, a VISART machine (Figure 2) (Toshiba Co. Ltd., Tokyo, Japan) is used. Head coils
are most sensitive for visualizing primary lesions around the oral cavity, but they are less
useful for visualizing lymph nodes located in the neck. Thus, a circular polarized neck coil
(Figure 3) is essential for visualizing the neck. MR images are commonly obtained using the
following 6 sequences: 1) STIR or fat-saturation coronal T2-weighted images; 2) STIR or fatsaturation axial T2-weighted images; 3) coronal T1-weighted images without contrast
medium; 4) axial T1-weighted images without contrast medium; 5) coronal T1-weighted
images with contrast medium; and 6) axial T1-weighted images with contrast medium
[1,2,4,5]. MR image slice thickness should be 5-7 mm [1,4,5]. Similar to X-ray CT scans,
MR images can encompass the area from the cavernous sinuses to the thoracic inlet in order
to examine the primary cancer and possible lymph node metastases in the neck.

Figure 3. One type of RF coils, circular polarized neck coils are used for examinations of the neck,
showing.

DIAGNOSIS USING X-RAY CT SCANS FOR PRIMARY ORAL


CANCERS AND THEIR LYMPH NODE METASTASES
The X-ray CT scan imaging findings of representative cancers of the mouth, such as
squamous cell carcinomas and minor salivary gland malignancy, commonly include soft
tissue density masses with mild contrast enhancement (Figure 4) [1,2,4,5]. Of course, tumor
margins are often difficult to discern when the tumor abuts or invades adjacent muscle or the
lymphoid tissue located in Waldeyers ring [5]. Furthermore, masses affected by dental metal
streak artifact are often undetectable on X-ray CT scanning (Figure 5). It has been reported

New Diagnostic Imaging Modalities for Oral Cancers

129

that particular radiological findings and parameters using dynamic CT could also be useful
[6-8]. Wakasa et al. reported that the peak height, which is the relative CT value measured
from the base CT value to the point where the curve reaches its peak, is useful for
distinguishing between inflammation and tumors [7]. Transit time, which is the time between
two transit points on the time-density curve, has been reported to be significantly longer in
benign tumors than in malignant tumors [7]. Michael et al. showed that dynamic CT scanning
was valuable for the differential diagnosis, management, and follow-up of hemangiomas [6].

Figure 4. A 52 -year-old female with a right gingival squamous cell carcinoma. The soft tissue density masse
(arrowheads) with the mild contrast enhancement by iohexol (300 mgI/mL; Omunipaque 300, Daiichi
Pharmaceutical Co. Ltd., Tokyo, Japan) is shown at gingival gums and alveolar bone of the right premolar
regions. The mass invades and destructs the cortical and spongy bone around the right premolar regions.

Figure 5. Steak artifact (arrowheads) caused by dental crowns is shown on an X-ray CT scanning image.

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Correctly diagnosing metastatic lymph nodes is important for determining the prognosis
of patients with oral squamous cell carcinoma (SCC) [2,9-14]. The use of CT scanning
continues to be the best diagnostic method for preoperative detection of metastatic neck
disease [2,15-23]. Criteria have been developed for the CT diagnosis of lymph node
metastases. With respect to size, the major axes of the jugulodigastric nodes and
submandibular nodes are 15 mm; for the other nodes, the major axes are 11 mm. The minor
axes of the jugulodigastric nodes and submandibular nodes are 10 mm; for the other nodes,
the minor axes are 8 mm. Furthermore, lymph node sizes on the affected size are two times
larger than those on the contralateral side. The shape of lymph nodes metastases is round;
lymph node swelling can result in the formation of a conglomerate. In addition, the presence
of central nodal necrosis (CNN) is strongly correlated with malignancy [2,9,13-23]. In our
dental hospital, when CNN is present, lymph node metastasis is diagnosed (Figure 6). If CNN
is absent, then lymph node metastases are diagnosed if two other criteria are present.

Figure 6. A 39-year-old female with a right gingival squamous cell carcinoma. The lymph nodes with
the central nodal necrosis (CNN) on an X-ray CT scanning image (arrowheads) are diagnosed as
metastases. A) Two submandibular and a jugulodigastric lymph nodes with CNN is diagnosed as
metastasis (arrowheads). B) A submental and two submandibular lymph nodes with CNNs are
diagnosed as metastasis (arrowheads).

It is important to recognize that there is a significant relationship between the extent of


SCC differentiation in lymph nodes and the incidence of CNN in metastatic lymph nodes
[24]. In well-differentiated cancers, metastatic lymph nodes tend to produce CNN, whereas
moderately- and poorly-differentiated cancers do not tend to result in CNN. If patients with
moderately differentiated or undifferentiated primary oral SCC have metastatic lymph nodes,
then CNN would not be able to form in lymph nodes with a maximum diameter of less than
25 mm. Therefore, CT scans should be examined for lymph node density changes in order to
determine whether a biopsy is needed to rule out metastatic lymph nodes in patients with
moderately differentiated or undifferentiated SCC in the primary sites [24]. Though it is

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advocated that PET-CT should be used to determine whether there are lymph node
metastases, this approach is not that useful, as explained below.

NEW X-RAY CT MACHINE FOR DIAGNOSING ORAL CANCERS


An X-ray CT scanning machine is now being developed, and recently multidetector CT
(MDCT) and cone-beam CT have been introduced for imaging mouth-related regions. Both
machines have advantages for diagnosing oral cancers. It has been reported that cone-beam
CT can be used to evaluate and measure three-dimensional bone defects and the presence of
periapical lesions more accurately than intraoral X-rays [25-27]. To the best of our
knowledge, though there have been no reports dealing with the identification, measurement,
and assessment of bone resorption caused by oral cancers, based on previous reports, conebeam CT appears to be a useful tool for accurately evaluating three-dimensional bone
resorption.
The development of the MDCT machine has allowed faster CT scanning and rapid
acquisition of numerous thin axial images. Therefore, more accurate reconstruction images of
bones can be obtained than with older X-ray CT scanning machines. It is possible that
MDCT, much like cone-beam CT, could also be useful for accurately evaluating threedimensional bone resorption. The MDCT should improve the performance of CT angiograms
and dynamic contrast and maneuver imaging [28,29]. MDCT angiography is used to
delineate the great vessels and to provide information about the exact location of neoplasms,
lymphadenopathy, and their vascular infiltration or spread.

DIAGNOSIS USING MR IMAGES FOR PRIMARY ORAL


CANCERS AND THEIR LYMPH NODE METASTASES
MR imaging of representative oral cancers, such as squamous cell carcinomas,
commonly shows mild to moderate hyperintensity signals on fat suppression T2-weighted
images, and isointense signals to muscles on T1-weighted images without contrast
enhancement (Figure 7). Therefore, fat suppression T2-weighted images might be the most
appropriate sequence for determining the presence of tumors. Based on the likely treatments
that will be used, particular fat-suppression techniques, such as the frequency-selective fat
saturation method (FS) and the short inversion time inversion recovery (STIR), should be
chosen. Compared to STIR, FS sequences have a better resolution and are generally thought
to be more useful [30-33]. However, when using FS-sequences, the uniformity of the
magnetic field is severely disturbed; this results in more cases of insufficient fat suppression
than with STIR. In particular, in our previous study, we demonstrated that insufficient fat
suppression is more likely to occur in the oral region when using FS than when using STIR,
and that, with FS, the degree of fat suppression homogeneity in the head and neck region
depends on the location (Figure 8). In addition, the degree of fat suppression instability with
FS may change between pre- and post-reconstruction images when metal plates and
myocutaneous flaps are used. Therefore, we should recognize that instability of the amount of

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fat suppression, which appears to depend on location and reconstruction with metal plate and
myocutaneous flap in patients with oral cancer on MR images using, is generally seen in
patients with primary squamous cell carcinomas [30].

Figure 7. A 39-year-old female with a right gingival squamous cell carcinoma. The masse (arrowheads)
with isointensity signal to muscles in gingival gums and alveolar bone of the right molar regions on T1weighted images without the contrast enhancement (A), and with moderate hyperintensity signal is
shown on fat suppression T2-weighted images (B).

Figure 8. Axial T2-weighted images with STIR (short inversion time inversion recovery) in
subcutaneous space at the submental level showing a sufficient fat suppression (arrowheads) (A), but
insufficient fat suppression (arrowheads) with fat saturation (FS) (B).

Recently, it has been shown that particular imaging findings and parameters of dynamic
contrast-enhanced MR images could be used as diagnostic tools for primary oral cancers [3436]. Takashima et al. suggested that dynamic MR imaging contributes to helping predict
whether head and neck lesions are malignant, though it can help limit the differential

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diagnosis and has the potential of predicting vascularity and recurrence [34]. Asaumi et al.
showed that dynamic MRI might be useful in differentiating malignant from benign tumors,
and in detecting the extent of the tumors in sublingual carcinomas [35]. However, there are
reports that question the utility of dynamic MR imaging [37-39]. Arakawa et al. showed that
dynamic MR imaging was not significantly better than T2-weighted imaging for identifying
areas of invasion [37]. Murakami et al. also reported that, though large tumors extending
around tissues are relatively well delineated on dynamic images, the images were no better
than T2-weighted images [38]. In the oral cavity, unlike other closed regions, most of the
lesions are amenable to direct clinical examination and biopsy. Therefore, it is unnecessary
for radiologists to determine the pathological diagnosis of masses in the mouth using imaging
modalities. In addition, since there is a relatively large amount of fatty tissue around the oral
cavity, it is more difficult for radiologists to diagnose areas of cancer invasion in the mouth
using T1-weighted images than using T2-weighted images. Therefore, dynamic MR imaging
may not necessarily be a useful technique for identifying the size of oral cancers or for
determining their pathological diagnoses.
On the other hand, there are reports stating that dynamic contrast-enhanced MR images
are useful for diagnosing lymph node metastases [40-43]. Noworolski et al. studied multiple
factors, including peak time and peak enhancement, in 21 patients with squamous cell
carcinoma of the head and neck and found that, on dynamic imaging, lymph node metastases
had heterogeneous contrast enhancement, while normal lymph nodes had homogeneous
enhancement [43]. Fischbein et al. also showed that metastatic lymph nodes had a longer time
to peak enhancement and a lower peak enhancement than reactive lymph nodes [42]. Thus,
overall, metastatic lymph nodes have a longer time to peak, a lower peak enhancement, a
lower maximum slope, and a slower washout slope than normal lymph nodes. Furthermore,
this technique can be used to distinguish between normal and malignant tissue and to
differentiate a malignant lymphoma from other lymph node enlargements. Asaumi et al.
demonstrated that metastatic lymph nodes in cases with squamous cell carcinoma had greater
and faster peak enhancement than malignant lymphoma [44]. One of the newest techniques
involves the use of both MR lymphangiography and carbon dye in patients with mucosal
head and neck cancers to detect sentinel lymph nodes; this technique has been found to be
useful [45].
Very recently, diffusion-weighted images have been used to diagnose lymph node
metastases [46-51]. Diffusion-weighted MRI with ADC mapping is a promising, new
technique that can differentiate metastatic from benign lymph nodes [48]. On the ADC map,
malignant lymph nodes usually show low signal intensity, and benign lymph nodes usually
show high signal intensity. The mean ADC values of metastatic and lymphomatous lymph
nodes were significantly lower than the mean ADC value of benign cervical lymph nodes
[46-51]. In our dental hospital, ADC values mapping images are represented by the color
indication (Figure 9). In particular cases that are difficult to diagnose the expanse of diseases,
ADC values mapping are represented as fusion images of T2-weighted imaging (Figure 9).
However, since the ADC values are dependent on the particular MR system, a threshold
value for differentiating malignant from benign lymph nodes must be determined in each
hospital for each machine. Nevertheless, there have been only a few reports of the use of

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these techniques in primary oral cancers, since it is unnecessary for radiologists to determine
the precise pathological diagnosis of mouth lesions.

Figure 9. Axial diffusion-weighted image and ADC map of a 60-year-old male with phlegmon. a) Axial
ADC map indicated by color representation shows an abscess formation (arrowhead) as a red and the
abscess-free area as the colors of except red. ADC map is prepared from FASE-DWI results obtained
with a b factor of 900 sec/mm2. The ADC of the abscess in Figure 9A is 0.95x10-3 mm2/s and that of the
abscess-free area was 2.75x10-3 mm2/s. b) Superimposed view of one part of the axial ADC map
indicated in Figure 9A and a T2-weighted image.

In the last decade, iron oxide-enhanced MRI has been used, which involves the use of
ultra-small superparamagnetic iron oxide (USPIO) particles that are administered through an
intravenous injection. The USPIO particles are concentrated in the reticuloendothelial system
by functioning histiocytes located in normal lymph nodes. The changes occur over a 6 to 24
hour period after the administration of the contrast agent. Therefore, normal lymph nodes
demonstrate a reduced signal intensity on T2*-weighted gradient-echo and T2-weighted MR
images. In contrast, metastatic lymph nodes do not take up the USPIO particles and, thus,
maintain their pre-contrast signal intensity. It has been reported that this technique improves
the accuracy of detecting lymph node metastases [52,53].
The next technique has no direct diagnostic use for oral cancers but was developed and
introduced as a new technique for use during follow-up of patients with xerostomia after
radiotherapy. This technique is called dynamic MR sialography, and it is a new noninvasive diagnostic technique that evaluates the physiological function of the salivary glands
[54]. The technique monitors the time-dependent changes in saliva after citric acid
stimulation using continuous magnetic resonance (MR) sialographic images [54,55]. Using
this technique, we found that dynamic MR sialographic images and parameters have a very
high potential of being used as a diagnostic tool for xerostomia and Sjgrens syndrome
[55,56]. We also used this technique in patients with xerostomia after radiotherapy to
examine salivary function and obtain salivary flow rate data in physiologic states
(unpublished data). The maximum change ratio and detectable duct area using dynamic MR
sialographic parameters decreased in parallel with the decreases of salivary flow rates in 3
patients after radiotherapy. These data could be used to determine when treatment for

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Figure 10. Dynamic MR sialographic images (A and B) and graphs (C and D) from the left parotid glands
ducts of a 67-year-old male with decrease of salivary flow rate before (A and C) or after (B and D)
radiotherapy for the tongue squamous cell carcinomas. A) The main duct and its side branches in parotid
gland became clearer in a time-dependent fashion immediately after citric acid stimulation until 90 seconds
(arrowheads). After 90 seconds, the main duct in parotid gland became obscure in a time-dependent manner.
The detectable areas in the main duct and the side branches in parotid gland before and after citric acid
stimulation were prominently changed. B) After the radiotherapy for tongue, the detectable areas in the main
duct and the side branches in parotid gland before and after citric acid stimulation were relatively a little
clearer (arrowheads) than before the radiotherapy. C) The graph demonstrating the relationship between the
time course after citric acid stimulation and the changing ratio of the detectable area in the parotid gland
ducts for the patient of Figure 10A. The changing ratio was higher. D) The graph demonstrating the
relationship between the time course after citric acid stimulation and the changing ratio of the detectable area
in the parotid gland ducts for the patient of Figure 10B. The maximum changing ratio and detectable ductal
area after radiotherapy decreased evidently.

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xerostomia should begin. The changes in the dynamic MR sialographic images and the data
of one case before, during, and after radiotherapy are shown in Figure 10. The dynamic graph
produced using MR sialography demonstrated that the maximum change ratio decreased
markedly 4 weeks after radiotherapy. These data, together with the clinical data and salivary
flow rates, could be used to determine when treatment for xerostomia should begin. However,
the possible limitation of that study was the relatively small sample size. Therefore, our next
trial will include a larger sample size to more firmly establish our results and the criteria for
starting irrigation treatment.

ORAL CANCER-RELATED, SPECIAL NEW MR TECHNIQUES


In this final section dealing with MR techniques, we introduce two new special
techniques that we developed and recently reported: 1) The identification of vessels in the
mouth and neck using MR angiography; 2) The identification of the trigeminal nerve in the
root entry zone using MR cisternography.

Figure 11. Superimposed view of a three dimensional (3D)-phase contrast (PC) MR angiography
(MRA) image and a 3D-fast asymmetric spin echo (FASE ) sequenced heavy T2-weighted image in 38year-old patient with a haemangioma in the left cheek. This image allows both the haemangioma
(arrowhead) and the external carotid arterial system to be seen on different rotations.

MR angiography (MRA) without contrast medium injection is a technique that is used to


delineate the relationship between vessels and tumors located in the oral region. This
technique is simple and noninvasive. The main external carotid artery and its branches,
including the lingual and facial arteries, can be imaged on MRA using 3-dimensional phase
contrast (Figure 11). In addition, we devised a new method that involves the superimposition
of MRA and T2-weighted images. This approach successfully demonstrates both the presence

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of the hemangioma and the course of the feeding arteries in 3D without the need to use
contrast medium (Figure 11) [57]. However, at present, tumors that do not have much fluid or
vessels cannot be depicted as one fusion image. In the future, we plan to resolve this and
would like to be able to visualize the relationship between various kinds of cancers and the
vessels around tumors without the need for contrast medium.
MR cisternography is a technique that outlines the cisterns of the brain, including the
cerebellopontine angle cistern, using MR sequences without contrast medium. In our dental
hospital, MR cisternography is used in patients with trigeminal neuralgia to detect
neurovascular compression in the root entry zone of the trigeminal nerve (Figure 12) [58].
Rarely, patients with neuralgia of unknown etiology have tumors of the cerebellopontine
angle region that invade the nerve [2]. In our previous reports, we showed that 4% (6 of 150
patients) of trigeminal neuralgia patients had brain tumors, which is relatively high [58].
Patients with a tumor of the cerebellopontine angle region have been relatively young in
previous reports, including our report [2,58]. Therefore, dentists should consider the
possibility of a brain tumor in relatively younger patients who have continuous trigeminal
neuralgia of unknown etiology; MRI or X-ray CT examinations should be done to rule out
space-occupying lesions as soon as possible in such patients. Figure 13 shows a brain tumor
that can be seen invading the root entry zone of the trigeminal nerve in a 67-year-old female
who had been previously diagnosed as having trigeminal neuralgia of unknown etiology.

Figure 12. Transverse original MR cisternographic image with axial (A) and reformatted coronal (B)
3D- fast asymmetric spin echo (FASE) images of a 52-year-old female with trigeminal neuralgia in a
left side caused by neurovascular compression (NVC) in the anterior inferior cerebellar artery. The
trigeminal nerve (arrow) surrounded of blood vessels (arrowhead) was visualized on MR
cisternography (A, B).

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Figure 13. Transverse original images of blood vessels and the trigeminal nerve in the root entry zone
(REZ) by MR cisternography with axial (A) 3D-fast asymmetric spin echo (FASE) images and T2weighted (B) images of a 67-year-old female with trigeminal neuralgia in a right side. The brain tumor
(arrowheads) invaded the REZ region of the trigeminal nerve.

DIAGNOSIS USING ULTRASONOGRAPHIC IMAGES OF


PRIMARY ORAL CANCERS AND THEIR LYMPH NODE
METASTASES
Ultrasonography can be used to assess lymph nodes in patients with oral cancers. The
usefulness of ultrasonography for the diagnosis of lymph node metastases has been
previously reported [59-63]. In one of the reports, ultrasonography performed by experienced
ultrasonographers had a diagnostic accuracy rate of about 90% in cervical lymph node
staging [62]. In other reports, sonography was significantly better than CT in depicting
cervical metastatic nodes; [64] overall, the diagnostic accuracy rate for lymph node
metastases was almost 75-85%, which is similar to our data. Ultrasonography is the method
of choice for evaluating tumor infiltration into the great vessels walls. In addition, Power
Doppler ultrasonographic images with B mode have a higher accuracy rate than B-mode
alone [65-68]. Doppler ultrasonography provides information about internal lymph node
blood flow, including hilar and peripheral parenchymal nodal flow, and improves the
diagnostic accuracy for detecting lymph node metastases from oral cancers. In addition,
Chikui et al., based on an analysis of pathology data, suggested that, after irradiation, the
enhanced Doppler signals contribute to better visualization of the vessels and better detection
of any vascular abnormalities [66,67]. Thus, particular attention should be paid to follow-up
imaging examinations of patients with oral cancers before and after radiotherapy to detect
lymph node metastases [66].
As already mentioned, ultrasonography is a non-invasive and easy to use imaging
modality for patients with various diseases of the soft tissues of the head and neck. Thus, in
addition to detection of lymph node metastases on initial examination, it is very useful for

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following oral cancer patients after surgical treatment and/or radiotherapy. In areas that have
been excised and exposed to radiation, normal tissues are replaced with cicatrix produced by
granulation tissues. Therefore, the diagnosis of metastases by direct palpation of the
remaining lymph nodes in the neck becomes more difficult after cancer treatment, and
ultrasonography becomes increasingly more useful to detect subclinical lymph node
metastases.
On the other hand, US-guided, fine-needle aspiration biopsy including cutting needle
biopsy of lymph nodes is easy to perform and has a high sensitivity and specificity, which
results in accurate diagnosis of subclinical lymph node recurrences (Figure 14) [69-76].
Studies have reported that the pathological diagnoses of lesions obtained using US-guided
needle biopsy agreed with the final pathological diagnoses after surgical dissection in about
90% of cases. However, in about 10-20% of cases, adequate pathological specimens could
not be obtained. There have been few reports of major complications, but hematomas have
been reported, including a report from our dental hospital [69,73]. Recently, Soudack et al.
suggested that in pre-biopsy color Doppler sonography should be routinely used to guide the
cutting needle to areas of the lesion showing sufficient vascularity [76]. Power or color
Doppler ultrasonography may be associated with fewer vascular-related complications and
may equal X-ray CT scan and MR images for identifying vascular-related findings. When
doing US-guided needle biopsy as part of the preoperative assessment of head and neck
lesions, including diagnosing lymph node metastases, the newly developed Monopty biopsy
instrument (MBI) (Monopty, Bard Urologic Division; Covington, GA, USA) has been used;
few of the pathological samples had rush artifacts and/or were obscured by blood, both of
which are problems that are commonly associated with manual biopsy techniques.

Figure 14. View of ultrasonography (US)-guided fine-needle aspiration biopsy including cutting needle
biopsy of lymph node (A). US image showing success centesis (arrowhead) of needle into the mass as
metastatic lymph nodes suspected (B).

There have been some reports on the increasing use of US-guided needle biopsy for
parotid glands [77-79], thyroid glands [80,81], and others. In the future, X-ray CT- [82-84]
and MRI-guided needle biopsy [85-88] will likely be used clinically to diagnose
lymphadenopathy.

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Figure 15. View of examination for tumor thickness of tongue using intraoral ultrasonography (A). US image
showing size including thickness of squamous cell carcinoma (arrowheads) in tongue using intraoral
ultrasonography (B).

Figure 16. A) A removal squamous cell carcinoma before produce of an embedded specimen. B) An
embedded specimen produced using our peculiar technique by gelatin. C) Ultrasonographic image precisely
demonstrates the figure, thickness, and size of squamous cell carcinoma embedded by glatin of Figure 17-B
using intraoral ultrasonography. D) One of the pathological sections in the same specimen of Figure 17-B, C. The excellent coincidence between the ultrasonograhic image and finding of the pathological section can
be demonstrated.

In general, ultrasonography should be used to diagnose lymph node metastases in the


head and neck regions, as mentioned above, but the technique is often used to accurately
estimate a tumor's size and to define adequate resection margins of tongue cancer cases with
tumor extension and deep infiltration [89-95]. In particular, tumor thickness in oral squamous
cell carcinomas is highly related to the occurrence of cervical metastasis; thus, accurate

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preoperative assessment is indispensable to improve therapeutic effects. Many reports


suggest that intraoral ultrasonography offers the most exact assessment of oral tumor
thickness (Figure 15) [91-94]. The accuracy of tongue tumor thickness can be measured to
within 1 mm with intraoral ultrasonography. Very recently, we confirmed this finding while
studying the use of intraoral ultrasound to easily allow the operator to assess surgical
clearance intraoperatively (Figure 16) [95].
Usually, as in our dental hospital, intraoral ultrasonography of the tongue is performed
with an 8- to 12-MHz linear array transducer. The probe is placed directly on the surface of
the tumor, as shown in Figure 15. Therefore, intraoral ultrasonography can evaluate tumor
thickness of cancers that occur in frontal areas of the tongue, but not in those that occur in
posterior areas.

DIAGNOSIS USING PET-CT IMAGES FOR PRIMARY ORAL


CANCERS AND THEIR LYMPH NODE METASTASES
Recently, a considerable amount of research has focused on the use of positron emission
tomography (PET) as an important oncologic imaging tool in the oral and maxillofacial
region that can help in primary tumor staging, evaluation of treatment response, recurrence
detection, and restaging [96-114]. In particular, PET is useful for evaluating recurrent or
residual cancers [97-99], especially when MRI and/or X-ray CT scans cannot distinguish
tumor changes caused by chemotherapy and/or radiotherapy. At present, fluorine-18-labeled
(18F) fluoro-2-deoxy-D-glucose (FDG) is most commonly used for patients with various
diseases. FDG, a glucose analogue, is transported into the cells by glucose transporters and
then is phosphorylated intracellularly, but not further metabolized [100- 102]. The
distribution of FDG throughout the body mainly reflects glucose metabolism of individual
tissues. In most cancer cells, the combination of an increased concentration of glucose
transporters, increased glucose phosphorylation, and low phosphatase activity results in
relatively high FDG concentrations [100-102]. Therefore, in general, malignant tumors show
high FDG uptake; therefore, FDG-PET is useful for differentiating between benign and
malignant disease. In order to distinguish between benign and malignant tumors on the basis
of the degree of FDG uptake (Figure 17), many reports have stated that the standardized
uptake value (SUV) should be 3.0-3.5 [103]. However, since inflammatory cells also have
increased glucose metabolism, it is difficult to differentiate between inflammation and
malignant tumors on PET imaging [102,104]. Moreover, even normal regions in and around
the oral cavity show substantial variations in uptake that can present difficulties for
identifying pathological changes. Thus, a permissible range of variation should be established
for particular tissues. In particular, intense FDG uptake is found in organs and tissues that
contain abundant lymphoid tissue, such as the pharynx, pharyngeal tonsil, palatine tonsils,
and lingual tonsillar tissue (Figure 18). On the other hand, FDG uptake is low in normal
lymph nodes, even though they contain abundant lymphoid tissue. Furthermore, Bogsrud and
Lowe noted that a characteristic V-shaped high uptake area in the floor of the mouth along
the medial borders of the mandible, which is a consistent finding on FDG-PET, most likely
represents the sublingual glands.

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The most important clinical use of FDG-PET imaging stems from its ability to include
the entire body and so detect metastases distant from the neck in patients with oral cancers
[105,106]. This advantage is of particular interest when considering nuclear medical
investigations. Figure 19 shows that distant metastases from the neck to the ilium could be
detected using FDG-PET imaging in a patient from our dental hospital with a 1-cm-size
tongue cancer. Another important use of FDG-PET imaging is to provide data on lymph node
metabolism. Thus, FDG-PET imaging should be done in cases with suspected lymph node
metastases based on X-ray CT scan, MR images, and ultrasonography. Therefore, in our
dental hospital, FDG-PET is done in cases with suspected lymph node metastases.

Figure 17. Positron emission tomography (PET) image using fluoro-2-deoxy-D-glucose (FDG) in
whole body of a 79-year-old male showing intense FDG uptakes in a left tongue squamous cell
carcinoma (arrowhead).

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Figure 18. PET images using FDG in oral and maxillo-facial regions of a patient with left gingival gum
squamous cell carcinoma showing intense FDG uptakes in organ and tissues containing the abundant
lymphoid tissue as likely the pharynx, pharyngeal tonsil, palatine tonsils (arrowheads in A), and lingual
tonsillar tissue (arrowheads in B) except carcinoma (arrow in A and B).

Figure 19. PET images using FDG in whole body showing that metastasis distant can be detected to the
lymph nodes in inferior internal jugular chain (arrowhead in A) and the ilium metastasis (arrowhead in
B) in a 59-year-old male with a right tongue cancer having size of the only 1 cm.

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Figure 20. PET-CT fusion images (C) using FDG in head and neck regions showing identifications of
the exact location (arrowhead) of the lymph node, which is suspected as the lymph node metastasis
(arrowhead in B) using X-ray CT scanning (A) and PET image (B) in a 79-year-old male with a left
tongue squamous cell carcinoma.

The normal anatomy of the oral and maxillofacial regions including the neck is very
complex, and it is difficult to identify the exact location of tissues with abnormal findings,
due to the low resolution of current PET machines. However, PET can be combined with CT
scanning of the head and neck to increase the accuracy (89-97%) compared to X-ray CT
scanning alone (69-75%) [107]. Therefore, by combining PET with computed tomography
(CT), the diagnostic accuracy in head and neck regions can be increased. In particular,
PET/CT allows the locations of metastatic lymph nodes to be more exactly and easily
determined. Figure 20 shows that PET/CT can be used to diagnose lymph node metastases
and identify their location when they have been identified as suspicious of lymph node
metastases on X-ray CT scanning and ultrasonography. However, some studies of the use of
FDG-PET or PET/CT for the evaluation of neoplasms and lymph nodes metastases in oral
cancers have not been favorable [108-113]. In about 5-10% of patients, PET and PET/CT
yielded false-positive results for cervical metastases, due to the presence of inflammatory
cells that also have increased glucose metabolism, as discussed above. Figure 21 shows
representative false-negative results in patients with oral cancers. In one case, the lymph node
was diagnosed as metastasis-negative based on X-ray CT scanning and MR images, but
metastasis-positive using ultrasonography. Therefore, FDG-PET/CT imaging was performed.
The lymph nodes showed high FDG uptake, and the SUV was 3.0-3.5 [103,114]. However,
after neck resection surgery, it was found that the lymph node did not have any metastases.
Thus, the advantages and limitations of PET and PET/CT imaging need to be clearly
understood. In particular, a high FDG uptake, such as an SUV over 3.5, does not necessarily
imply that the lesion is neoplastic.

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Figure 21. The representative case regarded as the result of false-positive results using a PET image
with FDG in an 89-years-old female with squamous cell carcinomas of a right tongue. It is very difficult
for the lymph node (arrowhead) in superior internal jugular chain to diagnose metastasis or not using Xray CT scanning (A) and MR images (B). Therefore, the lymph node (arrowhead) on an additional FDG
PET imaging (C) is shown as high FDG uptake, and SUV also is 3.8. We diagnosed the lymph node as
metastasis. However, the result was metastasis negative after neck resection surgery.

CONCLUSION
This article has reviewed the commonly used and newly introduced imaging modalities
that are used to evaluate oral cancers and their metastases to lymph nodes. Imaging is
advancing very rapidly in both medicine and dentistry. Therefore, the accuracy of detecting
oral cancers and of lymph node metastases using modern modalities is also continuing to
improve. In this review article, MDCT, new MR imaging techniques, and PET-CT have been
discussed as particularly noteworthy. Two particular applications of MR imaging, which are
routinely performed in our hospital, were discussed: dynamic MR sialography, which can be
used as a follow-up examination method for patients with xerostomia; and the production of
fusion images between tumors and vessels using 3D-FASE and MR angiography. Finally, we
reviewed the uses and limitations of PET-CT based on the literature and our dental hospitals
cases.

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In: Oral Cancer Research Advances


Editor: Alexios P. Nikolakakos, pp. 155-181

ISBN 978-1-60021-864-4
2007 Nova Science Publishers, Inc.

Chapter 6

THE ROLE OF THE PERCUTANEOUS


ENDOSCOPIC GASTROSTOMY IN THE
MANAGEMENT OF HEAD AND NECK
MALIGNANCY
CME Avery
University Hospitals of Leicester, Leicester, UK.

ABSTRACT
This chapter reviews the role of the percutaneous endoscopic gastrostomy (PEG) for
providing nutritional support in the management of oral cancer. An assessment of the
current use of the PEG technique is based on an analysis of the prospective operating
series of the author.
Insertion of a PEG was attempted on 200 occasions, mainly for malignancy of the
oral cavity but also the oropharynx, and some benign pathology and trauma. Seventy-six
percent (152/200) of gastrostomies were inserted at the time of definitive surgical
treatment and 19.5% (39/200) were inserted at an examination under anaesthesia, often
prior to radiotherapy.
Five percent (10/200) of procedures had significant endoscopic findings including
one synchronous malignancy. The rate of successful insertion was 97% (194/200). The
incidence of minor and major complications was 12.5% (25/200) and 3% (6/200)
respectively. There was no procedure related mortality. The overall 30-day mortality rate
was 7% (10/200) including deaths from terminal disease. Those at increased risk of death
were 65 years and over (P=0.005). The median PEG duration was 287 (SE 37) days.
Duration was significantly longer for stage T3-4 tumours (P=0.01), N1 or greater neck
disease (P=0.02), following surgery with radiotherapy when compared to surgery alone
(P<0.001), particularly for hemiglossectomy (P=0.02) and maxillectomy procedures
(P=0.003), following a segmental composite bone resection rather than a soft tissue
resection, with or without a rim resection, (P=0.03) and finally radiotherapy alone when
compared to surgery alone (P=0.004). There was no obvious relationship to age or the

CME Avery

156

type of free flap. Four (2.1%) patients have a gastrostomy that is likely to be permanent.
Only two patients did not use the gastrostomy.
All patients with T3 and T4 oropharyngeal tumours undergoing radiotherapy or with
oral tumours that required reconstruction with a free or pedicled flap were offered a PEG
on the basis that nutritional support would be required for more than 2 to 4 weeks. This
included T2 tumours without neck disease (stage II disease) if the site of the tumour is
likely to have a significant effect on function and hence a flap reconstruction is indicated.
The policy of early gastrostomy placement appears to be appropriate. However, the
exact pattern of use of the gastrostomy during the various phases of treatment remains to
be defined. The insertion of a PEG may be safely performed with a high degree of
success and a low incidence of complications by an experienced maxillofacial surgeon.

INTRODUCTION
The technique of percutaneous endoscopic gastrostomy (PEG) was first described by
Gauderer [1] and Ponsky [2]. It was used in the management of children and adults to create a
gastrocutaneous fistula without the need for a laparotomy. Originally intended as a method of
delivering long-term enteral nutrition it has since been employed in an increasingly wide of
conditions for the provision of temporary nutritional support [3]. The most common
indications are neurological disorders, head and neck malignancy, severe facial or head
trauma and gastrointestinal decompression [4].
Cancer Patient

GI Tract Patent

Undergoing antineoplastic tx
and
moderate/severe malnutrition

Offer PEG placement if


long-term tx anticipated >4 weeks
and intact GI tract

Figure 1. Based on Rabeneck 1997.

GI Tract Not Patent

Anticipated life span >6 - 8 weeks


(and unable to place stent)

If life span anticipated <6 - 8 weeks

Can offer PEG for decompression

Do not offer PEG Placement

Advanced cancer with


severe malnutrition and
life expectancy <2months
(cancer cachexia)

Advanced cancer unresponsive to


chemo/radiation therapy
/steady deterioration of performance
with severe malnutrition anticipated
life span >2 months

Do not offer PEG Placement

Do not offer PEG Placement

The Role of the Percutaneous Endoscopic Gastrostomy

157

A PEG is often the method of choice for the enteral feeding of patients with head and
neck malignancy. It was initially used mainly with end-stage disease [5,6] but is now more
commonly inserted whenever nutritional support is required for more than 2 to 4 weeks. The
indications include painful or ineffective mastication or swallowing, oropharyngeal and
oesophageal obstruction, or supplemental nutrition after surgery and during chemotherapy
and radiotherapy. The ethical issues surrounding insertion of a gastrostomy, particularly
when an illness may be in the terminal phase, has been reviewed [7,8] and an algorithm for
assisting in the decision-making process published [7]. A PEG should only be inserted for
patients likely to derive physiological benefit from nutritional supplementation and respond
to cancer treatment. It should not be offered when life expectancy is less than two months or
no improvement in the quality of life may be expected. If in doubt a trial period with a
nasogastric tube may be appropriate.
The PEG is commonly inserted by a gastroenterologist [9] or a gastrointestinal or general
surgeon [5,10-12]. It is occasionally inserted by a specialist nurse [13], otolaryngologist [1416] or maxillofacial surgeon [17]. A radiologically inserted gastrostomy (RIG) or
percutaneous radiologic gastrostomy (PRG) is less commonly inserted [18] using a variety of
guidance techniques including fluoroscopy [19-22], computerised tomography and
fluoroscopy [23] and ultrasound [24].
The gastrostomy may be performed under local anaesthetic, with or without sedation, or
under general anaesthesia. The various methods of gastrostomy insertion, their relative merits
and complications have been discussed in excellent review articles by Schapiro [25], Safadi
[4] and Mellinger [13]. Most of the literature is composed of retrospective cohort studies.
There are few prospective studies of the PEG technique and most deal with the issue of
nutritional support for dysphagia after a cerebrovascular accident [21,26-34]. The author is
aware of only two retrospective series by a single surgeon managing head and neck cancer
patients [5,11]. The influence of tumour site and stage within the oral cavity and the type of
surgical treatment has an unpredictable effect upon speech and swallowing function. This
chapter reviews the prospective experience of the author with the first 200 PEG procedures
performed for mainly oral malignancy.

The Pull Technique of PEG Insertion


The author used the conventional pull technique [35] and the Freka (Fresenius Kabi
Ltd, UK) feeding system with a size 9 FR tube. Antibiotic prophylaxis with 1.5 grams of
Cefuroxime was given at induction of general anaesthesia [36-38]. The endoscope was
inserted under direct vision to minimise the risk of contact with the tumour or perforation.
The latter is more likely to happen in the presence of anterior cervical osteophytes, a
pharyngeal pouch or abnormalities of the oesophagus such as a stricture, malignancy or
eosinophilic oesophagitis [39]. The upper gastrointestinal tract was examined as far as the
proximal duodenum with an Olympus (Keymed Ltd, UK) double-lumen or paediatric size
gastroscope. The abdomen was palpated to detect organomegaly prior to insufflation. The
oropharynx was thoroughly suctioned. Contact between the equipment and the tumour was
avoided by shielding the tumour with a hand. The gastrostomy site was selected with a

158

CME Avery

combination of transillumination and palpation. The most favourable angle for insertion of
the trocar created a well-defined indentation of the stomach wall. Indentation is more
important than transillumination, which is often unsatisfactory and less reliable [4]. If
indentation is equivocal then the safe tract technique described by Foutch [40] has been
advocated [4,35]. The routine use of all three techniques has recently been advised in
guidance issued by the British Society of Gastroenterology [39]. The author uses this
technique in the obese or when there has been previous abdominal surgery. A syringe of
water is advanced while aspirating. If air enters the syringe before reaching the stomach it has
punctured another hollow viscous. If in doubt a RIG should be obtained. A second look
gastroscopy was performed and the stomach decompressed. All patients were commenced on
an antacid drug and given 30 mls of sterile water per hour overnight, via the PEG. Feeding
was usually commenced within 12 to 24 hours of surgery.

METHODS
Data was prospectively collected and included; patient details, the stage and site of the
tumour, the timing of insertion and removal, duration of procedure, incidental findings, the
surgical procedure, administration of radiotherapy with or without chemotherapy and
complications. Radiotherapy, if indicated, was usually given after surgery.

Statistical Analysis
Kaplan-Meier survival methods were used to estimate the percentage of cases with a
PEG still in place after one year and the median duration. PEG duration was calculated as the
number of days from insertion to either (a) removal (b) patient death with the PEG in place or
(c) May 31st 2006. For patients having more than one PEG, but a continuous period of use,
the duration was computed from the date of initial insertion. The log-rank test was used to
compare PEG duration curves.

RESULTS
Insertion of a PEG was attempted on 200 consecutive occasions on a total of 187 patients
between May 2000 and May 2006. All patients undergoing major surgery and reconstruction
with a free or pedicled flap were offered a PEG on the basis that recovery of oral function
was not expected within two to four weeks. Three patients preferred to have a nasogastric
tube, of which one subsequently required a PEG during radiotherapy treatment. One
additional patient was referred for a RIG because of a previous oesophagectomy. The patient
data is tabulated in Table 1. The main indication was malignant disease of the oral cavity.
Twelve oncology patients had the PEG replaced either because of a second tumour, recurrent
disease or disintegration of the tube, of which 7 had continuous use that ranged from 475 to

The Role of the Percutaneous Endoscopic Gastrostomy

159

2029 days. Two patients did not use the PEG, of which the first recovered quickly after
surgery and the second received only palliative care.
Table 1. Patient data and indications for attempted PEG insertion (N=200)
Number of patients (n)
Male:female
Mean age in years (SD)1
Malignant tumour2
Replacement only
Facial fractures
Benign tumour
Severe dysplasia
Osteoradionecrosis
Secondary reconstruction
Neurological deficit
1
2

187
115:72
63 (14)
184
7
3
2
1
1
1
1

Age at time of first PEG for those with more than one PEG;
Including second tumour or recurrent disease.

Table 2. PEG insertion procedures (N=200)

Successful PEG insertion


Failed PEG insertion
At examination under anaesthesia
At definitive surgical procedure
After definitive surgical procedure
During radiotherapy treatment
Median duration of procedure in minutes (IQR, range, n)
1
2

% (n)
97 (194)
3 (6)
19.5 (39)
76 (152)
4 (8)1
0.5 (1)2
10 (8-12, 4-21, 197)

Definitive surgery elsewhere (1) and replacement tubes;


Initially refused PEG insertion.

Seventy-six percent (152/200) of insertions followed a tracheostomy at the time of


definitive surgical treatment and 19.5% (39/200) were at an initial examination under
anaesthesia (Table 2). The median procedure time was 10 minutes. The rate of successful
insertion was 97% (194/200). One patient had a successful insertion at the second attempt,
with a paediatric gastroscope, because of an oesophageal stricture. A RIG was obtained for 2
patients because of a post-cricoid web and morbid obesity. Eleven percent (22/200) of
patients had incidental endoscopic findings, of which 10 (5%) were significant. This included
one malignant oesophageal tumour and a Barretts oesophagus (Table 3).
The incidence of minor complications was 12.5% (25/200) (Table 4). A clinically
detectable infection occurred at 7.5% (15/200) of gastrostomy sites, of which 5 were infected
with Methicillin resistant Staphylococcus aureus (MRSA). The incidence of major
complications was 3% (6/200). There was no mortality directly related to the PEG procedure.

CME Avery

160

However one elderly patient with a large pneumoperitoneum and phrenic nerve injury
succumbed to a chest infection exacerbated by these complications. The overall 30-day
mortality rate was 7% (14/200) including deaths from terminal disease. Those at increased
risk of death were 65 years and over (13% v 2%, P=0.005).
Table 3. Incidental findings during endoscopy (N=200)
Incidental finding
Post cricoid web
Barratts oesophagus
Oesophageal adenocarcinoma
Oesophageal stricture
Benign oesophageal lesion biopsy
Gastro-oesophageal reflux
Hiatus hernia
Gastritis
Benign gastric ulcer biopsy
Retained suture at gastric resection site

(n)
1*
1*
1*
3*
2*
1
1
9
2*
1

* Significant clinical finding.

Table 4. Major and minor complications


Major Complications
Aspiration
Peritonitis
Dislodged tube passed per rectum
Tube migration into gastric wall
Perforation
Gastrocolic fistula
Haemorrhage
Major infection
Tumour implantation
Large pneumoperitoneum
Minor Complications
Peristomal wound infection
Peristomal bleed
Peristomal leakage
Tube obstruction or fragmentation
Tube migration in to small bowel

(n)
0
0
2
1
0
0
2
0
0
1
15
1
4
5
0

Based on Schapiro 1996.

The PEG durations were analysed with regard to age, stage of disease, type of resection
and reconstruction, and modality of treatment (Table 5 and Figure 2). The median duration

The Role of the Percutaneous Endoscopic Gastrostomy

161

was 287 (SE 37) days. Duration was not significantly different for those less than 65 years of
age (P=0.63). It was significantly longer for stage T3-4 tumours (P=0.01), N1 or greater neck
disease (P=0.02) and following surgery with radiotherapy when compared to surgery alone
(P<0.001) but not when compared to radiotherapy alone (P=0.50). Duration was also
significantly longer for radiotherapy alone when compared to surgery alone (P=0.004). The
radiotherapy alone group were primarily T3 or T4 oropharyngeal tumours and/or the patient
was not fit for surgery.
Table 5. Differences in median PEG duration between treatment and operation
subgroups
Variable

T 1-2
N0
Surgery and radiotherapy
Radiotherapy
2 separate surgical
procedures and radiotherapy
Primarily soft tissue resection

Median (SE)
duration of
PEG in days
226 (22)
232 (18)
360 (31)
478 (93)
713 (479)
226 (14)

Variable

T 3-4
N1 and greater
Surgery
Surgery
Surgery
Primarily
composite bone
resection

Median (SE)
duration of
PEG in days
358 (36)
360 (63)
166 (18)
166 (18)
166 (18)

Statistical
significance
log-rank test
P=0.01
P=0.02
P<0.001
P=0.004
P=0.02

358 (29)

P=0.03

Patients receiving additional chemotherapy are included within the radiotherapy group.

100
90
80
Radiotherapy

70

% of PEG

60
50
40

Surgery+Radiotherapy

30
Surgery

20
10
0
0

100

200

300

400

500

PEG duration in days


Figure 2. Duration of PEG by modality of treatment.

600

700

800

CME Avery

162

Table 6. Relationship between duration of PEG placement and other factors for
oncology patients 2000-2006 (N=183)
Nos
of
cases
93
90
75
100
80
94

% (SE) with
PEG after 12
months
41 (5)
39 (8)
34 (6)
47 (7)
49 (7)
34 (7)

Median (SE)
duration of
PEG in days
252 (51)
307 (43)
226 (22)
358 (36)
360 (63)
232 (18)

95
18
15
11

47 (6)
40 (14)
36 (14)
41(16)

360 (31)
239 (9)
337 (24)
212 (30)

6
12
12
6

50 (25)
47 (15)
58 (16)
67 (19)

257 (225)
308 (119)
713 (352)
503 (143)

Retromolar +/- rim resection


Multiple sites of resection

3
6

60 (22)

91
-

Other resections

80 (18)

Two separate surgical procedures


and radiotherapy
Surgery alone
Hemiglossectomy
Hemimandibulectomy

57 (19)

713 (479)

67
27
9

22 (7)
21 (9)
-

166 (18)
162 (32)
-

Anterior floor of mouth +/- rim


resection
Anterior mandibulectomy
Oropharynx
Partial and hemimaxillectomy

11

15(13)

161 (46)

2
3
8

176
115 (15)

Buccal resection
Retromolar +/- rim resection
Radiotherapy (no surgery)
By Principle Type of Resection
Primarily soft tissue resection
Anterior floor of mouth +/- rim
Hemiglossectomy
Oropharynx
Buccal
Retromolar +/- rim
Primarily composite bone resection
Hemimandibulectomy
Partial & Hemimaxillectomy
Anterior mandibulectomy

4
3
11

69 (19)

211
478 (93)

102
22
45
16
10
6
58
26
21
8

34 (5)
30 (11)
29 (8)
45 (13)
59 (18)
0
44 (9)
42 (14)
43 (14)
42 (22)

226 (14)
173 (30)
219 (14)
308 (93)
503 (147)
211 (131)
358 (29)
337 (31)
357 (106)
257 (183)

Age less than 65 years


Age of 65 years and over
T1-2
T3-4
N stage 1 or greater disease
N0 stage disease
By Treatment Modality & Operation
Surgery and radiotherapy
Hemiglossectomy
Hemimandibulectomy
Anterior floor of mouth +/- rim
resection
Anterior mandibulectomy
Oropharynx
Partial & hemimaxillectomy
Buccal resection

Individual PEG durations


for when <10 cases

46*, 70*, 74, 257, 262*, 749

281, 360, 475*, 503, 642*,


2029*
25*, 91, 229
232, 246, 358*, 383*, 1016*,
1876*
73*, 164, 210*, 262*, 287*,
507*
134, 176, 246, 475*, 713,
749, 2029*

5*, 7*, 9*, 17*, 28*, 75*,


113*, 286*, 385*

83*, 85
64, 176, 499
6*, 10*, 77, 94*, 98, 112,
155*, 224
55*, 105, 205*, 225*
57, 211, 274

25*, 57, 91, 211, 229, 274

46*, 70*, 74, 83*, 85, 257,


262*, 749

Includes oncology patients with osteoradionecrosis (1), secondary surgery (1) and severe dysplasia (1);
Excludes oncology patients with a nasogastric tube (3) and lost to follow-up (1).
* PEG in place at death or on 31st May 2006.

The Role of the Percutaneous Endoscopic Gastrostomy

163

Patients that underwent two separate surgical procedures and radiotherapy had longer
durations on average than those having a single surgical procedure (P=0.02) but the duration
was similar to those undergoing a single surgical procedure and radiotherapy (P=0.82). The
duration following a primarily soft tissue resection, with or without a rim resection, was
significantly shorter than after a composite bone resection (P=0.03) (Table 5 and Figure 2).
Within the surgery with radiotherapy group and the surgery alone group there was no
statistically significant difference (P=0.47) between operation subgroups with at least 6
patients (Table 6). For hemiglossectomy (P=0.02) and maxillectomy procedures (P=0.003)
the median duration was significantly longer with surgery and radiotherapy when compared
to surgery alone. The limited numbers prevent statistical testing within other subgroups. Four
patients that had undergone buccal resection (1), multiple sites of resection (2) and two
separate surgical procedures with radiotherapy had a PEG in place for over 1000 days and are
unlikely to have it removed. One hundred and sixty patients were reconstructed with a free
flap including radial, DCIA, fibula, radial composite, rectus abdominus and latissimus dorsi
flaps. There was no obvious relationship between the type of free flap and duration of the
PEG.

DISCUSSION
Nutritional Considerations
Good nutritional status is important to optimise the potential for recovery from illness but
is often not achieved and leads to a delay in recovery, an increased incidence of
complications and significantly increased costs [41]. Progressive weight loss and malnutrition
occurs commonly in the elderly and those with cancer and is a major source of morbidity and
mortality [8,42]. Patients who undergo major surgery, particularly those with malignant
disease, are at risk of malnutrition due to starvation, the stress of surgery and an increase in
metabolic rate. Nutritional support does not prevent weight loss but may significantly reduce
the extent of the loss [43]. The nutritional support of the cancer patient has been well
reviewed by Rivadeneira [44].
Patients with malignancy of the head and neck region often have underlying nutritional
problems because of a history of excessive smoking, poor diet, alcohol abuse [45]. The local
effects of the tumour may cause painful mastication and swallowing, and obstruction of the
oropharynx or oesophagus [46]. The nutritional status is also compromised by the effects of
treatment, particularly radiotherapy, which causes mucositis, xerostomia, altered taste,
dysphagia and loss of appetite [47-50].
An increase in survival of adequately nourished, rather than malnourished, patients with
head and neck cancer has been claimed [51]. It was speculated that this resulted from an
action on the immune system. However, an improved response to tumour treatment has not
generally been found, although good nutrition will reduce the incidence of complications,
morbidity and mortality [42,49,52-54].

164

CME Avery

The Enteral Route of Nutritional Supplementation


Enteral feeding is preferred to parenteral feeding because it preserves the architecture of
the gastrointestinal tract and prevents bacterial translocation. The enteral route has fewer
complications and is less expensive than intravenous supplementation, which is no longer the
method of choice for the treatment of head and neck carcinoma [44,52].
A review of the evidence for enteral tube feeding by the National Collaborating Centre
for Acute Care [55] concluded that enteral feeding should be considered for those who are
malnourished or at risk of malnutrition and have an inadequate or unsafe oral intake but a
functional accessible gastrointestinal tract (Table 7).
Table 7. Criteria for malnutrition based on National Collaborating Centre for Acute
Care (55)

At risk of malnutrition

Malnutrition

Criteria
have eaten little or nothing for more than 5 days and/or
are likely to eat little or nothing for the next 5 days or
longer and have a poor absorptive capacity.
and /or have high nutrient losses and/or have increased
nutritional needs from causes such as catabolism.
a BMI* less than 18.5 kg/m2
unintentional weight loss greater than 10% within the last
3 6 months or
a BMI* less than 20 kg/m2 and unintentional weight loss
of greater than 5% within the last 3 6 months.

* Body mass index.

In general enteral tube feeding increased the nutritional intake over and above that with
standard care and/or oral supplements. This usually led to an improvement in nutritional
status but inconsistent benefit in terms of length of hospital stay or mortality rates. The
evidence of benefit related to complications, quality of life, costs and cost-effectiveness was
limited. The cost-effectiveness of preoperative enteral nutrition was unclear [55].

The Nasogastric Route


Nasogastric (NG) tubes are useful if nutritional support is required for less than two to
four weeks. The problems with these tubes are numerous and include; a poor aesthetic
appearance and patient dissatisfaction, discomfort, suture line disruption, interference with
speech and swallowing function, alar necrosis, sinusitis, replacement with radiological
confirmation of position for displacement or tube obstruction, an association with
oesophagitis, gastro-oesophageal reflux and aspiration pneumonia [56,57].

The Role of the Percutaneous Endoscopic Gastrostomy

165

The Gastrostomy Route


The percutaneous gastrostomy has largely supplanted the open gastrostomy as the
preferred route of access to the functioning gastrointestinal tract [9]. However, the particular
surgical approach may vary with referral patterns and local expertise [58,59]. Open surgical
gastrostomy has had a comparable morbidity to PEG in small retrospective [60] and
prospective studies [61] but often requires a general anaesthetic, may cause more respiratory
problems and is more expensive. Wollman et al [9] considered that the procedure took
significantly longer to perform and had a significantly higher rate of major complications and
30-day mortality than a PEG or RIG. Complications with a laparoscopic gastrostomy
approach may also be comparable [62,63] or higher [59,64].

Comparison of Feeding with the Oral, Nasogastric or Percutanous


Gastrostomy Routes
The benefit of PEG feeding for long-term nutritional support has been established. The
comparison of initial feeding with an NG tube or PEG has been of particular interest in the
management of dysphagia after acute stroke. A poor outcome may occur despite early PEG
feeding [65]. The largest prospective multi-centre trial concluded that it was very unlikely
there was a significant benefit with an early PEG but it was preferred because it was more
discrete, comfortable and less easily dislodged than an NG tube. A PEG was recommended if
the NG tube was required for more than 2 to 4 weeks or could not be tolerated [31,33,34,55].
However, the initial use of a PEG has been advocated on the basis of prospective studies that
have demonstrated significantly increased delivery of feed, greater weight gain and lower
mortality [27-29]. However, the improvement in the quality of life, functional, nutritional or
subjective health status measures may be limited [29,30] and a PEG places a burden on the
patient as well as the carers.
There are relatively few studies of this issue in the management of head and neck
oncology. Significantly greater weight retention has been demonstrated in prospective studies
of early nutritional support with an NG tube [53] or PEG [66,67] rather than the oral route.
The PEG may also help to maintain quality of life scores [67] and be the choice of patients
[26] but this is not a consistent finding [66]. Magne [68] noted that an NG tube was as
effective as a PEG tube in maintaining body weight but the PEG had a lower incidence of
aspiration and the advantages of better cosmesis, mobility and quality of life considerations.
The PEG also has a role in the rehabilitation of post-surgical or inoperable patients with
dysphagia [69].
Gibson [56] retrospectively studied 89 patients with advanced stage III and IV tumours.
Hospital stay was up to 61% shorter with a PEG. This finding was statistically significant for
tumours of the larynx and pharynx but not the oral cavity. It was speculated that function was
better preserved with anterior tongue and floor of mouth tumours but the series only included
15 oral tumours and only 26 defects were reconstructed with either a pedicled or free flap.
The extent of the resection and type of reconstruction may be expected to affect function.

166

CME Avery

Mekhail [70] identified hypopharyngeal and stage T4 tumours, female gender and
treatment with chemoradiation therapy as factors that predicted the need for a feeding tube.
The study of 158 patients only included 4 oral cancers. Patients with a PEG had greater
dysphagia at 6 months than those with NG tube. This may have been a selection bias as the
duration of NG tube use was significantly shorter, median of 8 and 28 weeks respectively. It
was speculated that a PEG stimulated less swallowing effort leading to disuse of the muscles
of deglutition with an increased incidence of pharyngeal stenosis and dependency on the
PEG.

Contra-Indications to Percutaneous Gastrostomy


The absolute contraindications to PEG placement include complete obstruction of the
oropharynx or oesophagus, diffuse peritonitis, anorexia nervosa and a significantly limited
lifespan. The relative contraindications include significant ascites, peritoneal dialysis,
coagulopathy, gastric varices, portal hypertension, large hiatus hernia, hepatomegaly, morbid
obesity, subtotal gastrectomy and neoplastic or infiltrative diseases of the gastric or
oesophageal walls [4,60,71]. A PEG can often be placed despite marked trismus or partial
oropharyngeal obstruction by using a paediatric endoscope or using the manoeuvres of a
straight laryngoscope and a nasal or open pharyngeal approach [72]. Previous [73] or recent
[74] abdominal surgery does not preclude insertion if the site of puncture is carefully
selected. The incidence of failure may be higher after partial gastrectomy and the safe tract
technique should be used [40]. With a coagulopathy the platelet count should be greater than
100 X 109/L and the INR less than 1.4 [39].
It is important to try and identify patients that will not benefit from a PEG. The ethical
issues surrounding insertion of a gastrostomy particularly when the illness may be in a
terminal phase has been reviewed [7,8], and an algorithm developed for assisting in the
decision-making process (Figure 1) [7]. A PEG should only be inserted for patients likely to
benefit physiologically from nutritional supplementation and respond to cancer treatment. It
should not be offered when life expectancy is less than two months or no improvement in the
quality of life may be expected. The risk factors for early death in other studies include; age
over 75 years, urinary tract infection, diabetes mellitus, cardiac failure, severe functional
impairment and dementia [32,75]. There is little information on quality of life considerations
[8,75]. In the chronically ill geriatric population with primarily neurological disease there is
limited evidence that insertion of a PEG improves nutritional or functional parameters [30]
and it should not be inserted for palliative reasons without sufficient justification.

Types of Percutaneous Gastrostomy Technique


The various methods of gastrostomy insertion, their relative merits and complications
have been reviewed [4,13,25]. In head and neck practice the Push [58] or Pull [76-78]
techniques have their advocates based on relatively small comparative reports often with few

The Role of the Percutaneous Endoscopic Gastrostomy

167

oncology patients. The choice of technique will often depend on the training and personal
preference of the surgeon or operator.

Rate of Successful Percutaneous Gastrostomy Placement


The earliest large series that included oropharyngeal pathology reported PEG insertion
success rates of 95 to 100% [79-82] but rates may be lower with head and neck malignancy
[83]. The success rate in the current study of 97% is comparable with a meta-analysis of the
gastroenterological literature by Wollman [9] (95.7%) and current head and neck practice (90
to 98.5%) [10,11,14-17,84]. The majority of patients with common gastrointestinal
pathology, including partial gastrectomy, were successfully treated. Patients with severe
trismus or an oesophageal stricture were managed with a paediatric gastroscope. A RIG was
the first choice for a patient with a previous oesophagectomy and was necessary after 2 failed
insertions caused by a post-cricoid web and morbid obesity. A PEG may be safely placed
with a high degree of success by an appropriately trained otolaryngologist or maxillofacial
surgeon [14-17].

The Complication Rates of Percutaneous Gastrostomy


The classification and incidence of procedure related complications is variable and
largely based on retrospective reports of differing groups of patients, often including a mix of
neurological and oropharyngeal malignancy [25]. Schapiro & Edmundowiez [25] reported
two reviews with mean incidences of major complications of 2.7% to 2.8% and minor
complications of 6% to 7.1%. Wollman et al [9] reported a major complication rate of 9.4%
and minor complication rate of 5.9%, with a procedure related mortality of 0.53%. In the
recent Guidelines of the British Society of Gastroenterology [39] the incidence of major
complications are stated to be 3% with the exact incidence depending on the patient
population and generally being higher with malignant disease. The complication rates are
similar irrespective of the technique used, whether this is the common Pull (Ponsky) method,
the Push (Sachs-Vine) method or the direct introducer (Russell) method [39]. Particular
gastrostomy tubes may be associated with a higher incidence of complications [85]. Mortality
is closely related to the severity of underlying disease. Cardiopulmonary complications
account for about 50% of the potentially serious morbidity and procedure-related deaths
associated with endoscopy, often as a result of over sedation of the elderly or at-risk patient.
In head and neck surgical practice the incidence of major complications ranges from 0%
to 35% and minor complications from 8% to 17.5% [10,11,14-17,84]. The incidence of major
complications appears to be higher when the operator is a trainee surgeon or
gastroenterologist [12,39,84]. In the experience of the author and Lloyd et al [17] the major
complication rate of an experienced maxillofacial surgeon is relatively low, 3.0% and zero
respectively.
Aspiration and resultant pneumonia remain common major complications [25].
Aspiration may occur during the procedure or later as a result of oropharyngeal aspiration or

168

CME Avery

gastro-oesophageal reflux. The risk of aspiration is not entirely eliminated with a PEG [39,
86]. The supine position during PEG placement and an oropharyngeal tumour may increase
the risk of aspiration, especially when the gag reflex is obtunded. The incidence of
respiratory distress under intravenous sedation may be as high as 7% [83] to 10% [87].
Airway obstruction occurred in 1% [83] and 2 out of 29 patients with an unsecured airway
required an emergency tracheostomy [87]. It has been recommended that sedation or general
anaesthesia should be avoided unless the tumour has been removed or the airway secured,
sometimes with a tracheostomy, and the PEG performed in theatre [5,10,17,87].
Head and neck surgeons often prefer to take the opportunity to place the PEG either
during an initial examination under a general anaesthetic or at the time of definitive surgery
when the airway is protected by an endotracheal tube or tracheostomy [15,16]. This may
reduce the immediate risk of aspiration or cardio-respiratory complications [10] and avoids
an additional separate treatment episode.
Enteral feeding may commence as early as three or four hours after insertion in an
uncomplicated patient [55,88,89] but is not without risk of aspiration and death [90]. The risk
of late aspiration may be reduced by initially keeping the tracheostomy cuff inflated, feeding
in an upright or semi-upright position, retaining that position for 30 minutes and later
performing aspiration of the residual gastric volume. The author defers feeding after major
oncology surgery for twelve hours because gastric emptying is delayed, the airway reflexes
are obtunded and evaluation of the abdomen after some free flap procedures is required.
Feeding is delayed further if examination of the abdomen is equivocal. Most patients
commence feeding within 12 to 24 hours of surgery. Guidance on managing the symptomatic
abdomen after PEG placement is available [25,39,55]. Surgeons undertaking the gastrostomy
procedure must be familiar with all aspects of patient management from pre-surgical consent
through to the detection and management of post-surgical complications.
In the current series infection of the stoma site was the commonest minor complication.
Specific infections may occur at up to 30% of stoma sites [89]. Infection with MRSA may be
increasingly more common [91] but was of no serious consequence in this series. Two
patients developed a buried bumper syndrome. This complication occurs after 1.9% of
procedures [92] and typically presents as a late event with difficulty flushing and leakage.
Excessive tension on the flange has been implicated in this and other major complications
[4]. The buried bumper may be removed endoscopically or with a mini-laparotomy approach
[92]. The author now has a lower threshold for routine gastroscopy and replacement of a PEG
that has been in place for over one year.
The 30-day mortality rate after PEG insertion varies with many factors. Janes [75] noted
an increasing mortality rate, from 8% to 22%, with greater use of the technique but the
procedure-related mortality decreased from 2% to zero. In the meta-analysis by Wollman [9]
the mortality rate was 14.7% and in a later report it had fallen to 10% [18]. More recently
rates of 4.5% with a PEG and 3.1% with a RIG been reported [22]. In the current study the
overall 30-day mortality rate was 7%, which included those managed for terminal care. The
risk of death was significantly higher in those aged 65 years and over.

The Role of the Percutaneous Endoscopic Gastrostomy

169

The Timing of Gastrostomy Insertion in Head and Neck Surgery


The value of early placement of a gastrostomy tube prior to radiotherapy and
chemotherapy, for mostly advanced oropharyngeal and laryngeal malignancy, has been
established in several retrospective studies on the basis of a reduction in weight loss [93-97],
reduced duration and frequency of hospitalisation [93,94,96], decreased treatment
interruptions [94,97] and a variable effect on quality of life measures [66,67]. There may also
be value in insertion before the onset of severe hypoalbuminaemia [98].
The exact timing of gastrostomy insertion prior to surgery remains contentious. Head and
neck surgeons, usually otolaryngologists, often have the tube inserted as a separate episode
prior to definitive surgical treatment [46] [16,56] or at varying times including examination
under anaesthesia or both during and after surgery [14,15]. Fewer complications may occur
when the PEG is placed for oropharyngeal and laryngeal tumours during definitive surgery,
either before or after tumour resection, and after surgery rather than when placed
preoperatively or for inoperable tumours [57]. This highlights the importance of considering
the tumour site when analysing a report.
Recent publications from maxillofacial surgeons, managing primarily oral carcinoma,
have favoured early PEG insertion, sometimes at the time of examination under anaesthesia
but principally at the time of definitive surgery and after intubation when a safe airway has
been obtained [10,11,17]. These patients are likely to be at increased risk of respiratory
compromise because of a high incidence of grade 2 to 4 scores on the American Society of
Anaesthesiologists intubation scale [10,11]. Insertion after surgery or during radiotherapy is
less frequently performed.
In the current study 19.5% of patients had the PEG placed at the time of an examination
under anaesthesia or prior to definitive treatment. The histological diagnosis had already been
obtained. These were mainly oropharyngeal tumours managed with radiotherapy, with or
without chemotherapy, or patients with inoperable disease, poor medical condition or marked
weight loss including a few that underwent a trial period of feeding before the treatment
modality was decided. Seventy-six percent of patients had a PEG inserted immediately after
the tracheostomy and before the tumour resection. The tumour is identified and carefully
shielded from the endoscopic equipment to minimise the risk of tumour implantation. A PEG
was not inserted after tumour removal because resection does not usually significantly
improve access and would require re-draping of the surgical field. Placement immediately
after reconstruction is also undesirable because of the risk of damaging the flap. Only 4.5%
of gastrostomies were inserted after definitive surgery or during radiotherapy.

The Non-Endoscopic Gastrostomy Approach


The endoscopic insertion of a gastrostomy is currently the gold standard technique [21].
However, many gastrostomy tubes are placed using additional image guidance such as
fluoroscopy [19] [20,21] [22], computerised tomography and fluoroscopy [23] and ultrasound
[24]. The relative merit of each technique is beyond the scope of this article but the outcome
is variable and probably operator dependent [21]. In a meta-analysis of the gastroenterology

170

CME Avery

literature the rate of successful gastrostomy placement was significantly higher for a RIG
(99.2%) than a PEG (95.7%) and major complications were significantly less frequent, 5.9%
and 9.4% respectively [9]. A subsequent series supported these findings but importantly there
were no differences in 30-day mortality or complication rates [18]. More recently higher rates
of successful PEG insertion of 96% to 100%, together with a lower incidence of
complications, have been reported in the head and neck literature [10,11,22], including those
inserted by a maxillofacial team [17] and the findings of the current series. Although the rates
of success reported for the RIG technique may remain marginally higher with an experienced
operator [99] the procedure is less readily available and not infallible, as two of the patients
in the current series also failed a RIG procedure.
A RIG has been advised on the basis of several additional factors including the limited
diagnostic yield of routine gastroscopy [21]. The prevalence of incidental findings during
endoscopy ranges between from 10% to 71% [18]. Wollman & DAgostino [18] noted a 30%
rate of incidental endoscopic findings in their series, of which 10% had an intervention,
mostly for peptic disease. In their opinion the clinical importance of many incidental findings
is unproven. However, in the head and neck oncology group there is a relatively high
incidence of metachronous and second primary tumours [100]. In the current series the
significant incidental pathology detected included a synchronous oesophageal
adenocarcinoma and a Barretts oesophagus. Chandu [11,101] also reported a synchronous
gastric carcinoma and Barretts oesophagus in a series of patients with oral carcinoma.
Foutch [85] preferred a PEG because the endoscopic information did alter patient
management, although no malignancies were detected [102]. A further consideration is that a
RIG may avoid tumour implantation at the stoma site [19,21,103] as the result of direct
implantation [104]. However, this rare complication is now recognised and the head and neck
surgeon is ideally trained to both identify and shield the tumour during the procedure [14,15].
The availability of the RIG technique and the degree of local expertise may vary. By
performing a PEG the surgeon avoids the logistical issues and inconvenience of a separate
procedure. There is a short delay between diagnosis and surgical resection hence the issue of
pre-surgical feeding is usually not important. A thorough examination of the oropharynx for
recurrent disease is also performed when later removing the gastrostomy. The procedure may
be undertaken within a theatre environment, with an anaesthetist, as many patients will have
a compromised airway after flap reconstruction and radiotherapy. It is for these reasons that
the author prefers a PEG. In my practice a RIG is the procedure of choice only when
endoscopy is unlikely to succeed. Occasional additional indications may include the need to
avoid disturbing a recent surgical flap or suture line [19,20].

The Duration of the Gastrostomy


The exact pattern and duration of gastrostomy use during treatment is unknown. Some
gastrostomies are only placed at a late stage when oral intake is not being maintained and a
number are never removed. The typical time that a gastrostomy is in place is difficult to
ascertain because of differing methods of collating and presenting data. The mean or median
values in the head and neck literature range from 13.8 to 67.1 weeks [6,11,12,17,70,93,105].

The Role of the Percutaneous Endoscopic Gastrostomy

171

In the current series the median duration was 287 (SE 37) days or 41 weeks. The delay
between the provisional decision to remove a gastrostomy and the procedure was less than 6
weeks in most cases. Only 2.1% of patients had a PEG in place for over 1000 days and are
unlikely to have it removed. Unlike Mekhail [70] there was no evidence of prolonged
dependency on the PEG as a result of atrophy of the pharyngeal musculature. Dependency
was more likely to be related to massive or repeated surgery together with radiotherapy.
There is only one other detailed analysis of PEG duration in patients treated for oral
cancer. In a series of 49 patients Chandu [11] usually inserted the PEG at the time of
definitive surgery. The mean duration of gastrostomies that were removed electively was 114
days and in those that died with the PEG in-situ it was 470 days. A PEG was still in-situ for
14% of patients because of dysfunctional swallowing and a risk of aspiration. In comparison
Schweinfurth [106] inserted the gastrostomy at varying times in 142 patients with primarily
laryngeal or pharyngeal pathology. The incidence of long-term gastrostomy was 27%, with
only 24% of gastrostomies being removed.
The analysis of PEG duration and comparison with other publications is complicated by
many factors as gastrostomies are inserted at different stages of treatment, are replaced for
deterioration or complications, re-inserted or retained for recurrent disease, and recently
inserted gastrostomies must be accounted for while data is also skewed by very long-term
durations. However, the likely length of gastrostomy duration and the incidence of permanent
dependence on the tube is valuable information when counselling a patient.

Indications for Gastrostomy and Predictors of Prolonged Dependence


The need for a gastrostomy and likely duration is difficult to predict because of an
uncertain relationship to various factors including age, medical and nutritional status, speech
and swallowing function, tumour site and stage, the surgical resection and type of
reconstruction [107]. The indications for insertion have not been systematically studied and
variable criteria have evolved with experience. When considering a report it is important to
identify the tumour group and treatment modality being considered as publications from
otolaryngology institutes often have a minority of oral carcinoma sites and a preponderance
of non-surgical treatment.
Gastrostomy has been advised for Stage III and IV disease of the oropharynx treated
primarily with radiotherapy [95] or surgery [56] and also for combined modality treatment,
previous radiotherapy or significant pre-treatment weight loss [105,108]. A significant
reduction in hospitalisation was seen for pharyngeal and laryngeal sites but not oral tumours
[56]. The study by Gardine [108] reviewed a mixture of PEG, nasogastric and
oesophagostomy routes and was unusual as it contained a majority of oral tumours. These
patients had a slightly higher incidence of prolonged dependency on tube feeding but the
factors associated with a significantly increased risk of long-term nutritional support included
stage IV disease, pharyngeal tumours, combined modality treatment and previous
radiotherapy or significant pre-treatment weight loss. For primarily oropharyngeal and
laryngeal malignancy Schweinfurth [106] also identified several predictive factors including;
heavy alcohol use, base of tongue tumours treated with radiotherapy, reconstruction with a

172

CME Avery

myocutaneous rather free flap, post-operative radiotherapy, mandibulectomy but not


mandibulotomy alone, moderately or poorly differentiated tumours, large tumour size but not
TMN stage or surgical resection of the floor of mouth and oral tongue. This study had an
unusual preponderance of male patients. Ringstrom [109] noted that only 55% of
gastrostomies placed after surgery were temporary. An oropharyngeal tumour site and
advanced T stage were predictive of the need for gastrostomy.
In the surgical treatment of mainly oral carcinoma Chandu [11] listed the indications for
gastrostomy insertion as Stage III and IV disease treated with surgery and radiotherapy, and
Stage I and II disease in association with major neck dissections with or without distant flap.
For benign disease the indications were large composite resections with free flap
reconstruction. Although there was a 14% incidence of long-term PEG dependency due to
abnormal swallowing and aspiration there was no analysis of other potentially linked factors.
In the current study the duration of gastrostomy was significantly longer for stage T3-4
tumours (P=0.01), N1 or greater neck disease (P=0.02), following surgery with radiotherapy
when compared to surgery alone (P<0.001) and for radiotherapy alone when compared to
surgery alone (P=0.004) (Table 5 and Figure 2). The radiotherapy alone group were primarily
stage T3 or 4 oropharyngeal tumours. Unlike previous studies [56,106] there was a
significantly increased median duration for hemiglossectomy (P=0.02) and maxillectomy
procedures (P=0.003) with radiotherapy when compared to surgery alone. Patients that
underwent two separate surgical procedures and radiotherapy had longer durations on
average than those having a single surgical procedure (Table 5, P=0.02). The duration
following a primarily soft tissue resection, with or without a rim resection, was significantly
shorter than after a segmental composite bone resection (P=0.03) (Table 5 and Figure 3).
There was no obvious relationship with age or the type of flap reconstruction but the latter
will be amenable to further analysis with greater patient numbers. A limitation of this study is
the lack of a control group and changes in weight have not been considered. However, with
the role of PEG now well established it may be difficult to undertake a prospective
randomised comparison of PEG with NG tube feeding except perhaps in targeted groups.

Current Indications for PEG Insertion


If the fundamental criteria for PEG insertion have been met then all patients with T3 and
T4 oropharyngeal tumours undergoing radiotherapy and patients with oral tumours that
require reconstruction with a free or pedicled flap are offered a PEG on the basis that
recovery of oral function is not expected within two to four weeks. This includes T2 tumours
without neck disease (stage II disease) if the site of the tumour is likely to have a significant
effect on function and hence a flap reconstruction is indicated. Those undergoing a smaller
oral procedure or extra-oral resection, particularly in conjunction with a neck dissection, that
may compromise oral function are also offered a PEG. Other factors may also impact upon
this decision making process (Table 8). Walton [84] has advocated a more selective insertion
policy on the basis of an unusually high incidence of major complications, probably as the
result of not having an experienced endoscopist. A more restrictive insertion policy would
probably have led to more gastrostomies being performed at a later stage, when the patient

The Role of the Percutaneous Endoscopic Gastrostomy

173

may be in a more debilitated state. The current threshold for PEG insertion is lower than in
other studies because it is difficult, at present, to identify a subgroup of patients that would
not benefit from a PEG. Nevertheless, it is particularly important to try to identify patients
that are likely to die soon after the procedure.
100
90
80
70

% of PEG

60
50

Composite bone

40
30

Soft tissue

20
10
0
0

100

200

300

400

500

600

700

800

PEG duration in days

Figure 3. Duration of PEG by principle type of resection.

Table 8. Current indications for insertion of a PEG


Current indications
Fundamental criteria for insertion have been met and PEG not contra-indicated
Recovery of oral function within 2 to 4 weeks is not expected
Malnutrition or at risk of malnutrition during treatment
T3 and T4 oropharyngeal tumours undergoing surgery and/or radiotherapy
Intra-oral reconstruction with free or pedicled flap
Smaller oral procedure or extra-oral surgery, particularly in conjunction with a
neck dissection, likely to adversely affect oral function
Other factors may be relevant ie reduced oral function, previous radiotherapy

174

CME Avery

CONCLUSION
This study has confirmed that a percutaneous endoscopic gastrostomy may be inserted
with a high degree of success and minimal complications by an experienced maxillofacial
surgeon. All patients with T3 and T4 oropharyngeal tumours undergoing radiotherapy or with
oral tumours that require reconstruction with a free or pedicled flap are offered a PEG on the
basis that recovery of oral function is not expected within two to four weeks. This includes
T2 tumours without neck disease if the site of the tumour is likely to have a significant effect
on function and hence a flap reconstruction is indicated. Those undergoing a smaller oral
procedure or extra-oral resection, particularly in conjunction with a neck dissection, that may
compromise oral function are also offered a PEG.
In the opinion of the author most gastrostomies may be inserted at the time of definitive
surgery. Those requiring insertion at an initial examination under anaesthesia may usually be
identified as having advanced oral or oropharyngeal disease, are likely to receive
radiotherapy with or without chemotherapy as the primary modality of treatment, are in poor
general health, unsuitable for major surgery or there has been significant weight loss and
demonstrable weight gain is desirable prior to the decision about the final treatment modality.
The incidence of late gastrostomy insertion should be relatively low.
The author is now able to more accurately advise patients about the likely duration of the
PEG. Prolonged dependency is associated with T3 and T4 tumours, N1 or greater neck
disease, the combination of surgery and radiotherapy and particularly two surgical
procedures, and a segmental composite bone resection. However, the incidence of permanent
gastrostomy is low. The exact pattern of gastrostomy use during the different phases of
treatment should be studied.

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In: Oral Cancer Research Advances


Editor: Alexios P. Nikolakakos, pp. 183-194

ISBN 978-1-60021-864-4
2007 Nova Science Publishers, Inc.

Chapter 7

THE BIOMECHANICAL BASIS FOR INTERNAL


FIXATION OF THE RADIAL OSTEOCUTANEOUS
DONOR SITE
CME Avery
Department of Maxillofacial Surgery, University Hospitals of Leicester, Leicester, United
Kingdom.

ABSTRACT
Fracture of the radial free flap osteocutaneous donor site is common and causes
considerable morbidity. Most fractures are probably caused by relatively low-energy
torsional forces. This complication has lead to reduced use of the flap in clinical practice.
However, the incidence of fracture may be reduced by placing a bone plate, at the site of
the section defect, at the time of harvesting the flap. Both anterior and posterior surgical
approaches have been described. The strengthening effect of different types of plate and
position were studied using the sheep tibia as a model for the radial osteocutaneous
donor site.
Fifty matched pairs of adult sheep tibias were tested in torsion and 4-point bending.
The weakening effect of an osteotomy was first assessed by comparing an osteotomised
bone with an intact bone. Then pairs of bones with an osteotomy were compared with
and without reinforcement with different types of 3.5mm plate. The plate was placed in
either the anterior (over the defect) or posterior (on the intact cortex) position.
An osteotomised bone was significantly weaker than an intact bone. A plate in either
the anterior or posterior position significantly strengthened an osteotomised bone. The
dynamic compression plate was the strongest reinforcement in both torsion and bending.
In torsion the mean strength of the intact bone was 45% greater than after osteotomy
(P=0.02). The reinforced bone was on average 61% stronger than the unreinforced bone
(P<0.001). Plating restored the strength of the osteotomised bone to that of an intact bone
(100%) with a plate in either the anterior (97%) or posterior position (101%).
The tibia was able to withstand much greater loads in bending. In bending the mean
strength of the intact bone was 188% greater than after osteotomy (P=0.02). The

184

CME Avery
reinforced bone was on average 184% stronger then the unreinforced bone (P<0.001).
The posterior plate (80%) had a significantly greater effect than an anterior plate (46%)
in restoring the strength of the osteotomised bone to that of an intact bone (100%)
(P=0.01).
The use of prophylactic internal fixation is recommended for the routine
management of the radial osteocutaneous donor site. A plate in the anterior or posterior
position will significantly strengthen the donor site and the surgeon may choose either
site. The additional strengthening effect of the posterior plate in bending is probably not
relevant in clinical practice as the radius is likely to fracture first as a result of lower
torsional forces.

INTRODUCTION
The incidence of fracture of the radial osteocutaneous donor site ranges from zero to
66%, mean 25% (28 fractures at 114 donor sites) [1]. In two recent large series the incidences
of fracture were 15% [2] and 18% [3], with secondary surgery required for 67% and 46% of
these fractures, respectively. Fracture of the radius often causes significant morbidity and
functional deficit [4]. This frequent complication and the restricted volume of bone available
led to a reduction in the popularity of the flap.
During axial loading of the forearm the radius bears most of the forces [5]. Osteotomy of
the preserved radius has been shown to reduce the strength in bending to 24% of that of an
intact radius [6] (Table 1). The sheep tibia model of the radial donor site has been validated
by comparison with the preserved human radius [7]. The retained strength in torsion after
removal of 30% of the cross-sectional area (1/4 diameter) of the tibia was 26%, and after
removal of half of the circumference was 15%. Bevelling the osteotomy had a statistically
significant but small strengthening effect of only 5%. This is unlikely to be of significant
benefit in clinical practice [7]. The dramatic weakening after osteotomy results from the loss
of cortical integrity [8] and creation of an open-section defect [7], which reduces the
energy-absorbing capacity of the bone [9,10]. The effect is greatest on bones such as the
radius, which have relatively thin cortical walls and with long transcortical defects [11].
It is good practice to minimise the risk of fracture by using a careful surgical technique,
for example avoiding over-cutting, using a bevelled osteotomy and removing 50% or less of
the circumference of the radius. However, the weakening effect of an osteotomy is so
dramatic that the ideal solution would be to strengthen the remaining radial bone, particularly
against low-energy forces, which are thought to be responsible for most fractures [6].
Prophylactic internal fixation of the radial osteocutaneous donor site was first recommended
by the author in 1999 [12] on the basis of a small clinical series. A dynamic compression
plate (DCP) was placed in an anterior position (across the section defect) to act as a bridging
plate. Since then the strengthening effect of a plate in the anterior position has been reported
in larger clinical series with a low incidence of fracture [13,14]. Bowers et al [1] also
demonstrated the strengthening effect of a plate in the posterior position (on the intact
opposite cortex) in a biomechanical study. The use of the posterior position has also been
reported in clinical studies with a low rate of fracture [15,16].

The Biomechanical Basis for Internal Fixation

185

In this chapter the author investigated the effect of varying the type of plate and position
using the sheep tibia as a model for the radial osteocutaneous donor site.
Table 1. The weakening effect of an osteotomy

Model
Number of pairs of bone
Type of osteotomy
Amount of bone removed
Length of bone removed
Mean strength in torsion of bone
with osteotomy compared with
intact bone (100%)
Mean strength in bending of bone
with osteotomy compared with
intact bone (100%)

Swanson 1990
Preserved human radius
20
Right-angle and bevel
1/3 of diameter
9 cm

Meland 1992
Sheep tibia
45
Right-angle, keel and
bevel
30%-50% cross-section
1-4 diameter length

14.7 to 25.9

24

METHODS
Bone Pairs
Fifty matched pairs of intact freshly frozen adult sheep tibias of a similar size and age
were tested. The bones were divided into 5 groups, each of 10 matched pairs. Within each
group 5 pairs were tested in torsion and 5 by bending.
Group 1 compared pairs of bones that were intact with those after osteotomy. Groups 2 to
5 compared pairs of osteotomised bones with and without reinforcement (Table 2). The
different positions were simulated by putting the plate over the section defect (anterior) or on
the intact cortex opposite the section defect (posterior) (Figures 1 and 2). Three different
types of 3.5 mm plate were tested. A DCP (STRATEC Medical Ltd, UK) placed anteriorly or
posteriorly. A pelvic reconstruction plate (STRATEC Medical Ltd, UK) and a titanium plate
(DePuy International Ltd, UK) placed anteriorly. The forces at failure during torsion or
bending were recorded, and a photographic record was taken.

Preparation of Bone
The bones were stripped of soft tissue except for the periosteum, and stored in moist
sealed packages at 28oC. A defect 60 mm long of 40% of the circumference was marked out
at three points using a measuring tape (Figure 3). The defect was created in the flat
midsection of the shaft, with a right-angled osteotomy at each end, using a fine saw (EXAKT.
Germany). A right-angled osteotomy was easier to standardise, and drilling out the corner

CME Avery

186

eliminated over-cutting or points where stress may be concentrated [6]. This technique may
also be stronger than bevelling, as stress is defined as force per unit area and the removal of
load-bearing bone creates greater stress on the remaining structure [17,18]. This section
defect is a broadly similar proportion to that harvested in clinical practice [6]. As the crosssection of the tibia is not circular 40% of the circumference was removed as a reproducible
standard. This is within the recommended range of 40% to 50% of the circumference [19,20],
or one quarter [7] to one third [6] of the diameter.
An 8-hole plate was placed with 2 bicortical screws at each end in a non-compressive
position. This is the minimum number of screws required for stability (Figures 1 and 2). The
constructs were mounted in an aluminium mould to create the attached cement endings
required for the testing apparatus. A bulky diaphysis was cut down to size. Orthopaedic grade
polymethylmethacrylate cement (Palacos LV-40. Schering-Plough, Europe) covered the
entire diaphysis and contracted to grip the bone [21].
Table 2. Comparison of the matched bone pairs
Group
1
2
3
4
5

Pairs
5
5
5
5
5
5
5
5
5
5

Type of bone
Intact
Intact
Osteotomy
Osteotomy
Osteotomy
Osteotomy
Osteotomy
Osteotomy
Osteotomy
Osteotomy

Type of bone/ plate/position


Osteotomy
Osteotomy
Osteotomy / DCP/cortex
Osteotomy / DCP/cortex
Osteotomy / DCP/section
Osteotomy / DCP/section
Osteotomy / Reconstruction/section
Osteotomy / Reconstruction/section
Osteotomy / titanium/section
Osteotomy / titanium/section

Test
Torsion
Bending
Torsion
Bending
Torsion
Bending
Torsion
Bending
Torsion
Bending

In each matched pair one bone was compared with the other. For example in Group 2 one bone will
have an osteotomy but not be reinforced and the other will have an osteotomy and be reinforced with a
dynamic compression plate (DCP) on the cortex.

Figure 1. Plate over defect (anterior position) with 4 bicortical screws.

The Biomechanical Basis for Internal Fixation

187

Figure 2. Plate on cortex (posterior position) with 4 bicortical screws.

Figure 3. Standardised section defect.

Torsion Testing
A torsion apparatus (Crofts Engineering Ltd, UK) with a force transducer gave a direct
reading of 1 millivolt (mv), equivalent to 1 Newton (N), and the force in Newton metres
(Nm) was calculated. Rotation of the apparatus at 1 degree/second induced a uniform torque
over the length of the bone. The degree of rotation was recorded every 1 to 2 seconds.

Four-Point Bending Test


A Hounsfield H50KM apparatus (Hounsfield Test Equipment Ltd, UK) was fitted with a
DO30 Universal Type attachment and a 50 KN load cell. The bones with an osteotomy were
loaded with the section defect on the opposite side of the superior loading point. Nylon
blocks were placed over the steel supports of the apparatus to avoid crushing the bone. The
equipment was calibrated with wooden dowels. The rate of displacement was 10 mm/minute.
A constant bending movement was applied across the length of the bone. The force in
Newton metres (Nm) was automatically recorded at a frequency of 1Hz.

Statistical Analysis
Raw data and summary results were tabulated, using means, standard errors (SE), and
percentages. The number of pairs of bones in each group was small for the purpose of
statistical inference as the variation between bones may not have been captured sufficiently.
Approximate 95% confidence intervals (CI) were calculated for mean strength as a ratio

CME Avery

188

(x100) of bone after osteotomy. Parametric methods were used to test for increased strength
relative to a bone after osteotomy (Students 2 sample t test) and for differences between the
strengths of different plates (ANOVA). Approximate 95% confidence intervals (CI) were
calculated for mean strength as a ratio (x100) of bone after osteotomy and evidence that bone
or bone and plate were stronger was tested using the one-sample t test.

RESULTS
The statistical analysis is presented in Tables 3 and 4. The results in Table 3 are
expressed as a ratio 100 X (intact bone/bone with osteotomy) to obtain a percentage value for
the pair, with the baseline value for bone with an osteotomy being 100. The wide confidence
intervals with some results reflect the small number of pairs, and the variability in the
increased strength ratios of individual pairs. The one sample t test examines whether the
mean ratio is significantly different from 100, where 100 implies that the intact bone or the
bone and plate has the same strength as the bone after osteotomy. In Table 4 the two sample t
test compares the mean of the bone and plate with the mean for the intact bone.
Table 3. Increased strength of the bone with an osteotomy and reinforced with a plate

Torsion groups
1 Intact
2 DCP cortex
3 DCP section
4 Reconstruction section
5 Titanium section
Torsion groups 2 to 5
All torsion
Bending groups
1 Intact
2 DCP cortex
3 DCP section
4 Reconstruction section
5 Titanium section
Bending groups 2 to 5
All bending

No of
pairs

How much stronger than a bone with an


osteotomy
Mean
SE
95% CI for
ratio x100*
mean

Evidence of bone or
bone and plate
stronger than bone
with an osteotomy
(One-sample t test p
value)

5
5
5
5
5
20
25

145
173
179
166
127
161
158

13
24
28
14
6
10
9

110 to 181
107 to 240
101 to 257
126 to 206
110 to 143
140 to 183
140 to 176

0.02
0.04
0.05
0.01
0.01
<0.001
<0.001

5
5
5
5
5
20
25

288
578
231
182
142
284
283

50
38
36
13
17
42
34

150 to 420
473 to 684
131 to 331
147 to 217
94 to 190
196 to 370
213 to 355

0.02
<0.001
0.02
0.003
0.07
<0.001
<0.001

DCP = Dynamic compression plate.


* For example, an intact bone has 145% of the mean strength of a bone with an osteotomy or is
approximately 45% stronger.

The Biomechanical Basis for Internal Fixation

189

Table 4. Percentage of strength restored by reinforcement of a bone with an osteotomy

Torsion groups
1 Intact
2 DCP cortex
3 DCP section
4 Recon section
5 Titanium section
Bending groups
1 Intact
2 DCP cortex
3 DCP section
4 Recon section
5 Titanium section

Nos of
Pairs

Mean
moment
(Nm)

SE

Percentage
strength
restored

2-sample
t test
(p value)

5
5
5
5
5

23.0
23.2
22.4
16.4
17.4

2.0
2.0
2.4
1.1
0.7

101
97
71
76

0.95
0.85
0.02
0.03

5
5
5
5
5

173
139
80.4
64.3
45.9

25
11
13
7.2
7.3

80
46
37
27

0.25
0.01
0.003
0.001

DCP = Dynamic compression plate.

Torsion Testing
The mean strength of the intact bone was 45% greater than the bone after osteotomy
(P=0.02). The reinforced bone, with an osteotomy, was on average 61% stronger than the
unreinforced bone (P<0.001) for groups 2 to 5. The mean increase in strength with an anterior
or posterior DCP was 73% and 79%, respectively. The reconstruction (66%) and titanium
(27%) plate constructs were weaker (Table 3).
The mean strength of a reinforced bone, with an osteotomy, was compared with an intact
bone (100%). The anterior (97%) and posterior (101%) DCP constructs were of similar
strength. The reconstruction (71%) and titanium (76%) plate constructs were weaker (Table
4). Spiral or oblique fracture occurred at the angle of the osteotomy, the section defect,
between screw holes, or within the shaft of an intact bone.

Four-Point Bending Test


The mean strength of the intact bone was 188% greater than that of the bone after
osteotomy (P=0.02). The reinforced bone, with an osteotomy, was on average 184% stronger
than the unreinforced bone (P<0.001) for groups 2 to 5. The mean increase in strength with a
posterior DCP (478%) was greater than with an anterior DCP (131%). The reconstruction
(82%) and titanium (42%) plate constructs were weaker (Table 3).
The mean strength of a reinforced bone, with an osteotomy, was compared with an intact
bone (100%). The posterior DCP (80%) was significantly stronger than an anterior DCP
(46%) (2 sample t test P=0.01; Mann-Whitney U test P=0.03). The reconstruction (37%) and

190

CME Avery

titanium (27%) plates were weaker (Table 4). The difference in mean strengths between the 4
constructs was significant (ANOVA P<0.001, Kruskal-Wallis P=0.005).
Intact bones failed with a transverse fracture across the shaft. Bones with an osteotomy
failed with a transverse fracture at the angle of the osteotomy or within the section defect.
Bones that had been reinforced failed at the screw holes or at the angle of the osteotomy site.

DISCUSSION
The intact tibia is significantly stronger than the osteotomised tibia in both bending and
torsion. The tibia is able to withstand much greater bending forces. This is consistent with the
hypothesis that most radial fractures are caused by relatively low-energy forces [6,7], and
with torsional forces being responsible for most of these fractures. A bone plate in either the
anterior or posterior position significantly increases the strength of an osteotomised bone.
The DCP is the strongest form of reinforcement and a DCP in either the anterior or posterior
position restores the strength, in torsion, of an osteotomised bone to that of intact bone.
Reinforcement with a reconstruction or a titanium plate was weaker than an intact bone. A
posterior DCP restored 80% of the bending strength of intact bone and was significantly
(P=0.01) stronger than an anterior DCP (46%). This is consistent with the tension band
principle [22]. A tension band restores the load-bearing capacity of an eccentrically loaded
bone, whilst minimising the forces borne by the fixation device. To be effective the tensile
forces must be converted to compressive forces, as bone is strongest when loaded in
compression. In this case the plate would exert a force equal in magnitude but opposite in
direction to the bending moment created by the tensile force. Hence a plate on the intact
cortex should be the strongest form of reinforcement.
There are a number of factors that contribute to an assessment of the potential strength of
a particular form of reinforcement. The weakest reinforcement in both torsion and bending
was the titanium plate, despite a high yield strength and modulus of elasticity closest to that
of bone [23]. Bone is more flexible than either stainless steel (E 200 GPa) or titanium (E 110
GPa) [24]. There should be greater sharing of the load between the bone and titanium plate,
with the potential for less stress shielding in the longer-term [23,24]. However, it was
difficult to adapt the titanium plate passively to the surface of the bone. This probably
induced stress within the bone and at the screw holes. Once a load was applied the bone
deformed faster than the plate. The screws should transfer part of the load to the plate, which
protects the bone until the defect fractures. However, fracture occurred first at the screw
holes because of the concentration of stress [25]. This then unloads the plate and transfers the
entire load to the bone, which then fractures at the section defect. The reconstruction plate
was more readily adapted to the contour of the bony surface as it is more malleable and may
be manipulated around the long axis but the design is not as strong as the DCP [26].
Comparisons between the groups of paired bones and other studies are constrained by the
relatively small numbers and lack of direct linkage between the groups. In addition care
should be taken when comparing reports using different techniques of osteotomy and variable
section defects. In the current study the mean bending strength retained by the osteotomised
tibia was similar to that reported by Meland et al [7] and Bowers et al [1] but the retained

The Biomechanical Basis for Internal Fixation

191

strength in torsion was much greater (Tables 1 and 5). Drilling out the corner of the
osteotomy and removing 40%, rather than 50%, of the circumference may have contributed
to the greater retained strength but the difference was quite large. These factors were not
independently tested. Meland et al [7] claimed that an intact tibia was stronger in torsion than
the human radius, but this effect was lost when bones with an osteotomy were compared.
However, the ratio of the torque strength values for bones that were intact (22%) and after an
osteotomy (21%) was actually similar, so this statement was incorrect. Therefore the sheep
tibia is probably an adequate but not an ideal model for the radius. This is not surprising as
the sheep tibia is relatively short and stout in comparison to the human radius.
Table 5. Reinforcement of the radius and tibia after osteotomy
Bowers 2000
Avery 2005
Type of bone
Fresh human radius
Preserved sheep tibia
Number of pairs
20
50
Length of osteotomy (cm)
8
6
Amount of bone removed
50% cross-section
40% circumference
Type of plate and position
DCP cortex
DCP cortex & section
Percentage retained strength of bone with osteotomy: intact bone (100%)
Torsion
18
69
4-point bending
24
35
Percentage retained strength of bone with osteotomy + DCP cortex: intact bone
(100%)
Torsion
63
101
4-point bending
73
80
Percentage retained strength of bone with osteotomy + DCP section: intact bone
(100%)
Torsion
97
4-point bending
46
DCP = Dynamic compression plate.

Bowers et al [1] showed that the human radius with an osteotomy and a DCP in the
posterior position was 4 times stronger in torsion than an unreinforced bone; it restored a
mean of 63% of the strength of an intact bone (Table 5). In the current study reinforcement
increased the mean strength by a factor of 1.6. The restoration of strength with a DCP in
either position was greater than that reported by Bowers et al [1] (Table 3). This may partly
reflect the relative strength of the sheep tibia. Another factor is that, unlike Bowers et al [1],
monocortical screws were not placed in the section defect, to avoid concentration of stress
and removal of load-bearing bone [25]. Placing monocortical screws weakened the construct.
In addition the radial bones used were from elderly patients and may have been
correspondingly weak, particularly if from female patients [27,28]. In the same study the
mean bending strength with reinforcement was 2.7 times greater than that of an osteotomised
radius. This is comparable with a factor of 2.8 in the current study. The bending strengths
retained with a posterior DCP were also similar, 80% and 73%, respectively (Table 5).

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This biomechanical study and that of Bowers et al [1] has demonstrated that prophylactic
internal fixation restores sufficient strength to reduce the risk of fracture of the residual
radius. However, this effect has not been quantified with an in vivo study. The strengthening
effect is supported by the clinical experience of the author [29] and others who have reported
relatively low rates of fracture ranging from zero [13,14,16] to 9.6% [15] at a total of 246
plated donor sites. The latter figure of 9.6% was reduced to zero once the practice of inserting
monocortical screws within the defect was discontinued [15]. Only one fracture was treated
with secondary surgery. The most appropriate position for the plate remains debatable. The
anterior and posterior positions are equally effective in resisting torsional forces. A posterior
plate resists greater bending forces but requires additional dissection. This strengthening
advantage is probably not relevant in clinical practice as it is likely that a fracture will occur
first as the result of a lower torsional force. There is no study that compares the morbidity of
the two surgical approaches and position used is a matter of personal preference.

CONCLUSION
The sheep tibia is an acceptable model for studying the biomechanics of the radial donor
site. It is probably a relatively strong bone in comparison to the human radius. The tibia was
able to withstand much greater bending loads than torsional forces. An osteotomy
significantly weakens a bone and placing a plate on either the anterior or posterior surface
significantly strengthens an osteotomised bone. The dynamic compression plate was the
strongest reinforcement in both torsion and bending. A plate in either the anterior or posterior
position restored the strength of the osteotomised bone to that of an intact bone when
subjected to torsional forces. The posterior plate had a significantly greater strengthening
effect than an anterior plate under bending loads.
On the basis of these findings the use of prophylactic internal fixation is recommended
for the management of all radial osteocutaneous donor sites. The additional strengthening
effect of the posterior plate in bending is probably not relevant in clinical practice as the
radius is likely to fracture first as a result of lower torsional forces.

REFERENCES
[1] Bowers KW, Edmonds JL, Girod DA, Jayaraman G, Chua CP, Toby EB. Osteocutaneous
radial forearm free flaps. The necessity of internal fixation of the donor-site defect to
prevent pathological fracture. J Bone Joint Surg Am 2000;82(5):694-704.
[2] Thoma A, Khadaroo R, Grigenas O, Archibald S, Jackson S, Young JE, et al.
Oromandibular reconstruction with the radial-forearm osteocutaneous flap: experience
with 60 consecutive cases. Plast Reconstr Surg 1999;104(2):368-78; discussion 379-80.
[3] Clark S, Greenwood M, Banks RJ, Parker R. Fracture of the radial donor site after
composite free flap harvest: a ten-year review. Surgeon 2004;2(5):281-6.

The Biomechanical Basis for Internal Fixation

193

[4] Richardson D, Fisher SE, Vaughan ED, Brown JS. Radial forearm flap donor-site
complications and morbidity: a prospective study. Plast Reconstr Surg 1997;99(1):10915.
[5] Markolf KL, Lamey D, Yang S, Meals R, Hotchkiss R. Radioulnar load-sharing in the
forearm. A study in cadavera. J Bone Joint Surg Am 1998;80(6):879-88.
[6] Swanson E, Boyd JB, Mulholland RS. The radial forearm flap: a biomechanical study of
the osteotomized radius. Plast Reconstr Surg 1990;85(2):267-72.
[7] Meland NB, Maki S, Chao EY, Rademaker B. The radial forearm flap: a biomechanical
study of donor-site morbidity utilizing sheep tibia. Plast Reconstr Surg 1992;90(5):76373.
[8] Timmons MJ, Missotten FE, Poole MD, Davies DM. Complications of radial forearm
flap donor sites. Br J Plast Surg 1986;39(2):176-8.
[9] Frankel VHB, A.H. Load capacity of tubular bone. In: Kenedi RM, editor. Biomechanics
and related bio-engineering topics. New York: Permagon Press; 1965. p. 381-396.
[10] Clark CR, Morgan C, Sonstegard DA, Matthews LS. The effect of biopsy-hole shape and
size on bone strength. J Bone Joint Surg Am 1977;59(2):213-7.
[11] Hipp JA, McBroom RJ, Cheal EJ, Hayes WC. Structural consequences of endosteal
metastatic lesions in long bones. J Orthop Res 1989;7(6):828-37.
[12] Nunez VA, Pike J, Avery C, Rosson JW, Johnson P. Prophylactic plating of the donor
site of osteocutaneous radial forearm flaps. Br J Oral Maxillofac Surg 1999;37(3):210-2.
[13] Villaret DB, Futran NA. The indications and outcomes in the use of osteocutaneous
radial forearm free flap. Head Neck 2003;25(6):475-81.
[14] Kim JH, Rosenthal EL, Ellis T, Wax MK. Radial forearm osteocutaneous free flap in
maxillofacial and oromandibular reconstructions. Laryngoscope 2005;115(9):1697-701.
[15] Werle AH, Tsue TT, Toby EB, Girod DA. Osteocutaneous radial forearm free flap: its
use without significant donor site morbidity. Otolaryngol Head Neck Surg
2000;123(6):711-7.
[16] Militsakh ON, Werle A, Mohyuddin N, Toby EB, Kriet JD, Wallace DI, et al.
Comparison of radial forearm with fibula and scapula osteocutaneous free flaps for
oromandibular reconstruction. Arch Otolaryngol Head Neck Surg 2005;131(7):571-5.
[17] Wittkampf AR, Starmans FJ. Prevention of mandibular fractures by using constructional
design principles. I. Computer simulation of human mandibular strength after segmental
resections. Int J Oral Maxillofac Surg 1995;24(4):306-10.
[18] Wittkampf AR, Wittkampf FH, van den Braber W. Prevention of mandibular fractures by
using constructional design principles. II. A tension strength test on beagle mandibles
with two different types of segmental resections. Int J Oral Maxillofac Surg
1995;24(4):311-2.
[19] Soutar DS, Scheker LR, Tanner NS, McGregor IA. The radial forearm flap: a versatile
method for intra-oral reconstruction. Br J Plast Surg 1983;36(1):1-8.
[20] Urken MC, ML. Sullivan, MJ. Biller, HF. Radial forearm. In: Urken M, editor. Atlas of
regional and free flaps for head and neck reconstruction. First ed. New York: Raven;
1995. p. 149-168.
[21] Rimnac CM, Wright TM, McGill DL. The effect of centrifugation on the fracture
properties of acrylic bone cements. J Bone Joint Surg Am 1986;68(2):281-7.

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[22] Perren SM. Basic aspects of internal fixation. In: Muller MEA, M. Schneider, R.
Willenegger, H., editor. Manual of internal fixation. Berlin: Springer; 1991. p. 1-158.
[23] Wright TML, SL. Principles and methods. In: Buckwalter JE, T. Simon, S., editor.
Orthopaedic basic science: biology and biomechanics of the musculoskeletal system. 2nd
ed. Rosemont: American Academy of Orthopaedic Surgeons; 2000. p. 181-215.
[24] Uhthoff HK, Bardos DI, Liskova-Kiar M. The advantages of titanium alloy over stainless
steel plates for the internal fixation of fractures. An experimental study in dogs. J Bone
Joint Surg Br 1981;63-B(3):427-84.
[25] Brooks DB, Burstein AH, Frankel VH. The biomechanics of torsional fractures. The
stress concentration effect of a drill hole. J Bone Joint Surg Am 1970;52(3):507-14.
[26] Schatzker J. Screws and plates and their application. In: Muller MEA, M. Schneider, R.
Willenegger, H., editor. Manual of internal fixation. 3rd ed. Berlin: Springer; 1991. p.
179-290.
[27] Sowers MR, Clark MK, Hollis B, Wallace RB, Jannausch M. Radial bone mineral
density in pre- and perimenopausal women: a prospective study of rates and risk factors
for loss. J Bone Miner Res 1992;7(6):647-57.
[28] Itoh S, Tomioka H, Tanaka J, Shinomiya K. Relationship between bone mineral density
of the distal radius and ulna and fracture characteristics. J Hand Surg [Am]
2004;29(1):123-30.
[29] Avery CM, Danford M, Johnson PA. Prophylactic internal fixation of the radial
osteocutaneous
donor
site.
Br
J
Oral
Maxillofac
Surg
2006,
doi:10.1016/j.bjoms.2006.08.025.

In: Oral Cancer Research Advances


Editor: Alexios P. Nikolakakos, pp. 195-210

ISBN 978-1-60021-864-4
2007 Nova Science Publishers, Inc.

Chapter 8

THE CURRENT ROLE OF PROPHYLACTIC


INTERNAL FIXATION OF THE RADIAL
OSTEOCUTANEOUS DONOR SITE
CME Avery
Department of Maxillofacial Surgery, University Hospitals of Leicester, United
Kingdom.

ABSTRACT
The radial osteocutaneous donor site is dramatically weakened at the site of the
osteotomy despite bevelling of the osteotomy cut and limiting the amount of bone
removed. Fracture results in considerable morbidity particularly if healing is not ideal.
The incidence of fracture remains relatively high, ranging from 0% to 66%, with a mean
of 25%. The largest studies have recently reported fracture rates of 15% and 18% and the
incidence of secondary surgery, for fractures, was also high, 67% and 46% respectively.
Clinical and biomechanical evidence now supports the routine use of prophylactic
internal fixation of the radial donor site with a dynamic compression plate to reduce the
incidence of fracture and the need for secondary surgery. The plate is effective in either
an anterior or posterior position and the choice of site is a matter of surgeon preference.
This chapter describes the clinical experience of the author with the anterior approach to
internal fixation. In a retrospective review of a series of 28 donor sites the incidence of
fracture was 3.6% (1 out of 28). The single fracture was undisplaced and secondary
surgery was not indicated. In the literature the incidence of fracture with a plate in the
anterior or posterior position is relatively low. To date 268 donor sites have been
managed with prophylactic internal fixation and only 7 have fractured, of which only one
underwent secondary surgery. The incidence of reported complications related to the
technique is also low and very few plates have been replaced or removed.
The technique of prophylactic internal fixation allows the surgeon to safely harvest a
modest volume of bone, which in conjunction with the excellent soft-tissue paddle means
that the radial osteocutaneous flap retains a wide range of potential applications.
However, in the current practice of the author, and many others, the radial flap remains a

CME Avery

196

compromise choice or back-up flap. Recent indications have included relatively small
defects of the maxilla, older patients that are unlikely to undergo dental implantation,
patient preference and if there is significant peripheral vascular disease or poor general
health that will be exacerbated by use of an alternative donor site.

INTRODUCTION
The potential complications at the osteocutaneous donor site are the same as those
encountered after harvest of a soft-tissue flap but also include fracture of the remaining
radius. The incidence of fracture in early reports varied from 28.5% to 43% [1-6]. Following
fracture of the radius movement of the wrist is often restricted and grip or pinch strength is
reduced, particularly if healing is not ideal [4,5]. It has been claimed that a keel or bevelled
shaped osteotomy may reduce the frequency of fracture. Swanson [7] reported an incidence
of 8% (1 out of 13) and Weinzweig [8] 5.2% (1 out of 19) but the number of donor sites were
small. Fractures were attributed to excessive removal of bone, creation of points of stress
concentration by over-cutting and inadequate immobilisation.
Table 1. The weakening effect of an osteotomy

Model
Number of bone pairs
Type of osteotomy
Amount of bone removed
Length of bone removed
Mean strength in torsion of osteotomised
compared to intact bone (100%)
Mean strength in bending of
osteotomised compared to intact bone
(100%)

Swanson 1990
Preserved human
radii
20
Right-angle and bevel

Meland 1992
Sheep tibiae

1/3 of diameter
9 cms

45
Right-angle, keel and
bevel
30-50% cross-section
1-4 diameter lengths

14.7 to 25.9

24

The patterns of physiological loading of the radius and the dissipation of forces by the
musculature have not been studied in vivo. In biomechanical studies of the cadaveric forearm
the radial bone bears most of the forces in axial loading [9]. The weakening effect of an
osteotomy is substantial. The mean strength in bending of an osteotomised preserved human
radius is 24% of an intact radius [7] (Table 1). A bevelled osteotomy was not significantly
stronger than a right-angled osteotomy. The preserved human radius is a reasonable model as
the biomechanics are similar to those of a fresh bone [10]. The sheep tibia model has been
validated by comparison with the preserved radius [11]. The retained strength in torsion after
removal of 30% ( diameter) of the cross-sectional area of the tibia was 25.9% and after
removal of 50% was 14.7%. A bevelled osteotomy was statistically stronger but the actual

The Current Role of Prophylactic Internal Fixation

197

effect was minimal (5%) and of no clinical importance. Alternative donor sites were
recommended because the small changes in the stability of the remaining cortical bone
associated with variations in shape, width, depth or length of osteotomy defect are irrelevant
in comparison with the 70 to 80 percent decrease in strength and stiffness caused by even the
smallest ostectomy defect [11].
The main weakening effect of an osteotomy comes from the disruption of the integrity of
the cortex [2] and creation of an open-section defect [11,12]. This causes a significant loss
of strength in torsion by reducing the ability of the bone to absorb energy [10,13]. The
greatest effect is on bones, such as the radius, with thin cortical walls or long transcortical
defects [14]. Most fractures of the radius are spiral and are probably caused by relatively lowenergy torsion forces, as the radius is able to withstand much greater bending forces
[7,11,15].
Table 2. Morbidity at the osteocutaneous donor site

Type of study
Number of donor sites
Mean age in years (range)
Male:female
Type of osteotomy
Mean bone length in cms (range)
Percentage of radial circumference
External support cast (weeks)
Number of donor sites with PIF (type)
Incidence of fracture % (n)
Incidence of secondary surgery % (n)

Thoma 1999
Retrospective
60
60 (26-86)
38:22
Keel
9.4 (5-14)
Below-elbow (-)
0
15 (9)
67 (6)

Clark 2004
Retrospective
71
62* (-)
49:22
Bevel and right-angle
7.7 (5-11)
Above-elbow (8)
3 (2 plates, 1 nail)
18 (13)
46 (6)

* Mean age of fracture group is 60 years and no fracture group is 62 years.

In the first prospective study of morbidity at the radial donor site Richardson [16]
reported a fracture rate of 17% (6 out of 35). The type of osteotomy used was not specified.
Function of the hand was restricted at 36% of donor sites without fracture and 100% with
fracture. Fractures that fail to unite or become significantly displaced often require secondary
surgery, with external fixation and non-vascularised [17] or vascularised bone grafting [18].
In a recent review of the literature the incidence of fracture at the radial donor site remained
relatively high, ranging from 0% to 66%, with a mean of 25% [15]. The two largest
retrospective series have also recently reported high rates of fracture and secondary surgery.
In 1999 Thoma [19] reviewed 60 donor sites managed with a keel shaped osteotomy and a
below-elbow cast, the incidence of fracture was 15%. The recognised causes of fracture were
excessive weight bearing or activity. Fracture was not linked to age, gender or size of the
bone section. Internal fixation and bone grafting was necessary for 67% of the fractured
donor sites yet all were reported as having a successful outcome. Despite the additional
morbidity of a secondary surgical procedure the flap was considered a first line choice for
reconstruction of the mandible. In 2004 Clark [12] reported on 71 donor sites, of which 68

CME Avery

198

were managed without internal reinforcement and 3 had internal fixation. The incidence of
fracture was 18%, of which 46% required secondary surgery. Fractures occurred despite an
above-elbow back-slab cast and were attributed to excessive removal of bone. The type of
osteotomy was not a significant factor. Prophylactic internal fixation (PIF) of the radial donor
site was advised, particularly for female patients (Table 2).
The radial donor site may be strengthened to reduce the risk of fracture. The technique of
PIF, using a dynamic compression plate (DCP), was first described in 1999 by the author
[20]. A DCP was placed across the section defect using a conventional anterior surgical
approach. The significant strengthening effect of internal fixation at both the posterior
(opposite intact cortex) and anterior (over section defect) position has since been confirmed
in biomechanical studies by Bowers [15] and the author [21]. This chapter describes the
clinical experience of the author with the anterior approach to prophylactic internal fixation
of the radial osteocutaneous donor site.

Figure 1. The osteocutaneous radial flap.

THE SURGICAL TECHNIQUE OF INTERNAL FIXATION


The osteocutaneous flap is harvested using the conventional anterior approach [22]
(Figure 1). A subfascial dissection technique was used until superseded by the incomplete
suprafascial technique [23] (Figure 2). A cuff of the flexor pollicis longus muscle is removed
together with a third to one half of the circumference of the radius. An instrument is used to
measure the distance to the posterior border of the radius to avoid excessive bone removal,
particularly in the mid-section where the radius is curved (Figure 3). In addition, the crosssection of the radius is triangular and 40% of the circumference approximates to the
minimum width of radius on a radiograph in the anteroposterior plane [24]. The osteotomy
cuts are bevelled or keel shaped to improve the surgical access and reduce the risk of overcutting (Figure 4). The distal osteotomy cut is made at least 2 cms from the radial styloid to
allow sufficient space for two distal screws. The proximal and distal muscle attachments are

The Current Role of Prophylactic Internal Fixation

199

stripped and then reattached at the end of the procedure. A 3.5 mm plate is placed on the
anterior or antero-medial surface and retained with a minimum of 2 bicortical screws at each
end (Figure 5). The screw holes are tapped out to the correct length and the screws are placed
in a neutral (non-compressive) position with the plate acting as a bridging plate. Care is taken
to approximate the flexor pollicus longus and brachioradialis muscles to cover the plate
(Figure 6) and the radial defect is then repaired with a skin graft. The author prefers to use a
negative pressure wound dressing to encourage healing of the skin graft [25]. For long
section defects a reconstruction plate may be placed as it is more malleable and easier to
adapt to the radius but it is a weaker form of reinforcement [21]. When there is insufficient
distal space a T-shaped plate may be inserted (Figures 7 9). A complete above or belowelbow plaster cast is applied and the arm initially supported in a sling. A below-elbow
lightweight cast is placed when the skin graft at the radial donor site is inspected, usually by
the 10th day after surgery A prefabricated cast may be used [26]. External support is provided
for a total of 6 weeks while gentle physiotherapy of the wrist and hand is commenced. Strict
advice is given on avoiding load bearing.

Figure 2. Suprafascial dissection over the ulna aspect and subfascial dissection on to the radius.

Figure 3. Posterior border of radius identified.

200

CME Avery

Figure 4. A bevelled or keel-shaped osteotomy is preferred.

Figure 5. A 3.5 mm DCP on the antero-medial surface.

METHODS
The operations were performed between December 1995 and July 2001 at the Royal
Surrey County Hospital, Guildford, United Kingdom and between May 2000 and April 2007
at the University Hospitals of Leicester, Leicester, United Kingdom. The case-records were
reviewed retrospectively, although more recent data were collected prospectively. Data
included; patient details, age, gender, type of osteotomy and length of bone section, type of
fixation plate, site of fracture, size of radial donor site skin defect, type of skin graft and
wound complications. The length of the bone section was measured on a lateral radiograph of
the forearm. A radiograph was obtained each time the cast was removed to exclude a fracture.

The Current Role of Prophylactic Internal Fixation

201

Figure 6. Approximation of the flexor muscles to cover the plate.

Figure 7. A reconstruction plate over a long section defect.

RESULTS
Thirty-one consecutive patients were reconstructed with osteocutaneous flaps for oral
malignancy involving the mandible [21] and maxilla [10] (Table 3). Three patients did not
have PIF because of lack of a suitable plate and one of these donor sites fractured. These
patients are excluded from further analysis. Five patients died, without fracture, within 30
days of surgery and this reflects the often elderly and infirm nature of this subgroup of
patients. The 3.5 mm plates used included 24 steel DCP, 2 titanium DCP, 1 reconstruction
and 1 T-shape plate (STRATEC Medical Ltd. UK and DePuy International Ltd. UK).

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CME Avery

The incidence of fracture was 3.5% (1 out of 28). A single fracture occurred in a 51-year
old male 10 weeks after surgery. The fracture was undisplaced and managed conservatively.
Insufficient distal space had been left when creating the osteotomy site and because a Tshaped plate was unavailable a DCP was inserted with only 1 distal screw, which weakened
the reinforcement. There were no significant wound infections, none of the plates were
removed and all of the skin grafted donor sites healed without delay.

Figure 8. A minimum of two proximal and distal bicortical screws.

Figure 9. A T-shaped plate.

The Current Role of Prophylactic Internal Fixation

203

Table 3. Prophylactic internal fixation of the osteocutaneous donor site


Author
Type of study
Number of
donor sites
Donor sites
with PIF
Mean age in
years (range)
Male: female
Mean followup in months
(range)
Type of
osteotomy
Mean length
of bone in
cms (range)
Radial
circumferenc
e%
Site of plate
fixation
Type of
fixation plate
External cast
(weeks)
Incidence of
fracture % (n)
Secondary
surgery % (n)

Werle
2000
Retrospective
54

Villaret
2003
Retrospective
34

Militsakh
2005
Retrospective
108

Kim
2005
Retrospective
52

Avery
2007
Retrospective
31

52

34

108

52

28

62 (16-89)

- (54-76)

62 (16-93)

63 (16-87)

67 (25-89)

30:24*
16 (0-45)

28:6
28 (7-54)

68:40
29 (1-116)

34:18
- (4-33)

13:15
32.5 (0-86)

Bevel

Bevel

Bevel

Keel

Bevel or Keel

7.6 (5.5-12)

6.6 (3-12)

6.3 (3-11)

7 (4-9.5)

50

40

50

33-50

Posterior

Anterior

Posterior

Anterior

Anterior

Steel DCP,
LC-DCP,
reconstruction
Below-elbow (1)

Steel DCP

Steel DCP

Steel DCP

9.6 (5)

Below-elbow
(1)
0 (0)

Below-elbow
(1)
0 (0)

Below-elbow
(1)
1.9 (1)

Steel DCP,
titanium DCP,
reconstruction
Below-elbow
(6)
3.6 (1)

0 (0)

0 (0)

0 (0)

100 (1)

0 (0)

- Data not available;


LC-DCP = limited contact dynamic compression plate.

DISCUSSION
Prophylactic internal fixation has been shown to prevent pathological fracture of the long
bone [27-30]. The first description of PIF at the radial donor site was a case-report utilising
an intramedullary nail [31] but the nail was too short to resist rotational forces [32]. The
current author later reported the use of PIF with a steel DCP at 8 radial donor site defects at
the time of flap harvest [20]. The DCP was placed in an anterior or antero-medial position. A
moderate amount of bone was harvested without fracture. A subsequent report of 22 plated
donor sites supported these findings [33]. The incidence of fracture at that time was 4.5% (1
out of 22).

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CME Avery

The strengthening effect of a plate in the posterior position, on the intact cortical surface
opposite the donor site section defect, was reported in a biomechanical study by Bowers [15].
A comparable strengthening effect of a plate in either the anterior or posterior position has
been reported by the author in a study using the sheep tibia model [21]. An osteotomised tibia
was significantly weaker than an intact bone. A plate in either the anterior or posterior
position significantly strengthened an osteotomised bone. The dynamic compression plate
was the strongest reinforcement in both torsion and bending. In torsion a plate in either
position restored the strength of the osteotomised bone to that of an intact bone. The tibia was
able to withstand much greater loads in bending. The posterior plate had a significantly
greater strengthening effect, than an anterior plate, under bending loads but this is probably
not relevant in clinical practice as the radius is likely to fracture first at a lower torsional
force.
Four retrospective clinical series have been published following the original description
of the technique, with an incidence of fracture that ranges from zero to 9.6% (Table 3). The
posterior cortex was plated in two articles. Werle [34] reported an incidence of fracture of
9.6% (5 out of 52). The fractures all occurred in the initial part of the series when
monocortical screws were inserted within the section defect. This practice removed loadbearing bone, created areas of stress concentration and weakened the reinforcement [35,36].
However, none of the fractures were displaced and all were managed conservatively. In the
largest operative series Militsakh [37] omitted some technical details but clarified these in a
personal communication [38]. There were no significant fractures at 108 donor sites. This
comment is based on the observation that some forearms were not radiographed after surgery
but none of these patients were symptomatic. In the other two articles the plate was placed in
an anterior position. Villaret [39] managed 34 donor sites without fracture and Kim [40]
reported one fracture out of 52 donor sites (1.9%). The amount of the radius removed in these
reports was similar to the current series. The high proportion of males in the series by Villaret
[39] is noted but only Clark [12] has linked females to an increased risk of fracture. In the
current series the fracture rate was also low (3.6%) and the single fracture was caused by a
technical error.
The relatively inaccessible posterior cortex has been plated for two reasons. Firstly, it is a
strong reinforcement, particularly under bending loads, and secondly it was an attempt to
avoid problems with healing of the skin graft. However, the incidence of skin graft loss
reported by both Werle [34] and Militsakh [37] was high, 43% and 30% respectively. In the
current series healing at the donor sites occurred without complication or delay and no plates
were removed. The author prefers to use an incomplete suprafascial dissection technique and
a negative pressure wound dressing to improve graft healing [25]. Nevertheless, it is
important to carefully approximate the flexor musculature and ensure the plate is adequately
covered. There are no studies that directly compare the morbidity of the anterior and posterior
surgical approaches.
An additional advantage of PIF is that fractures typically remain undisplaced and
secondary surgery is often unnecessary. In contrast the rates of secondary surgery reported by
Thoma [19] and Clark [12] were relatively high, 67% and 46% of fractures respectively
(Table 2). Thoma [19] claimed healing was successful after surgical repair but offered no

The Current Role of Prophylactic Internal Fixation

205

objective evidence. The incidence and severity of functional loss is likely to be lower with
PIF but this is unproven [34].
Both Werle [34] and Villaret [39] used a below-elbow cast as an external support for
only one week. The author prefers to provide external support for 6 weeks as the remodelling
of bone takes several weeks [41-43]. However, the ideal duration of external support remains
unknown. A variety of techniques have been used at the unreinforced donor site including; a
full above-elbow plaster cast for 6 weeks with fracture rates of 23% (4 out of 17) [5] and
17% (6 out of 35) [16] or 8 weeks support with a fracture rate of 8% (1 out of 13) [7] and a
thermoplastic below-elbow cast with no fractures (0 out of 15) [26]. The type and duration of
external support may be an important factor in the prevention of fracture but the effectiveness
of these techniques cannot be assessed. A survey of orthopaedic surgeons in the United
Kingdom recommended 6 weeks in a complete above-elbow cast [44]. However, a belowelbow cast allows earlier mobilisation of the elbow and would appear to be sufficient if
internal fixation has been applied and load-bearing is avoided.
The technique of PIF has been criticised by Rockwell [45] who claimed that fractures
were becoming less frequent and the method was not cost-effective in the Canadian health
care system. However, the fracture rate in their report was still quite high, 15% [19].
Militsakh [37] noted the length of stay in intensive care facilities was shorter, and therefore
the expense was lower, with a radial osteocutaneous flap rather than other free flap donor
sites. There are many variable factors involved in a cost analysis but the insertion of a
permanent plate on the radius is a small element. In the longer term a plate has the potential
for causing a stress protection effect leading to localised osteopenia and late fracture. The
mechanism may be mechanical unloading [41,46-48] or reduced perfusion [43,49,50] of the
cortical bone. In the current study 3.5 mm plates were used as they are of sufficient strength
to resist fracture but less likely to cause osteopenia than heavier plates [51-54]. A limited
contact DCP may reduce the risk of osteopenia [49] but this is controversial [48]. Werle [34]
used limited contact plates and reported radiographic evidence of bone remodelling without
osteopenia. In the current study thirteen donor sites were radiographed six months or later
after operation and all had a degree of bone-infill and remodelling of the cortex. Attempts to
quantify the extent of the remodelling with ultrasound and magnetic resonance imaging were
not sufficiently reproducible. Finally, the removal of a plate from the forearm is associated
with a small but significant risk of nerve injury and late fracture, therefore asymptomatic
plates should not be routinely removed [45,53-56]. To date of the 274 plates inserted for PIF
in the literature, including this chapter, only two have required removal and one was replaced
for an early fracture.
The clinical role of the osteocutaneous radial flap has been compromised by two main
factors. To reduce the risk of fracture the volume of bone harvested is restricted to 40 to 50%
of the circumference [5,22,57], or one third of the diameter [7], or 30% of the cross-sectional
area [11]. This volume of bone is often insufficient for dental implants and other sites now
provide greater bone stock [58-60]. However, the incidence of fracture remains high because
even a small osteotomy leads to a dramatic weakening of the radial bone. In the opinion of
the author the most serious complication at the osteocutaneous donor site is relatively
frequent and early fracture. The benefits of PIF in reducing the incidence of fracture and the
need for secondary surgery outweigh the potential complications of plate infection, stress

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protection and late fracture. The use of PIF significantly strengthens the remaining radial
bone and allows the surgeon to harvest a modest volume of bone. A subgroup of patients that
would not benefit from PIF has not yet been identified. A direct comparison of different
management strategies is unlikely because a multicentre trial would probably be needed. The
osteocutaneous radial flap has become increasingly popular again following the introduction
of PIF. Villaret [39] believes that the clinical indications for the flap have been expanded.
Militsakh [37] has used it as the flap of choice for reconstruction of the mandible including
the treatment of osteoradionecrosis. However, for many other surgeons it remains less
important as alternative donor sites with greater bone stock are usually preferred [61]. In the
practice of the author the radial osteocutaneous flap currently constitutes approximately 5%
of the flaps used. The introduction of the technique of PIF has consolidated the role of the
osteocutaneous radial flap as a compromise or back-up flap. At the beginning of this
operative series the flap was often used for reconstruction of the mandible. More recent
indications have included relatively small defects of the maxilla, particularly for older
patients that are unlikely to undergo dental implantation, patient preference, and if there is
significant peripheral vascular disease or poor general health that will be exacerbated by the
use of an alternative donor site.

CONCLUSION
The radial osteocutaneous donor site is dramatically weakened by an osteotomy. Clinical
and biomechanical evidence supports the routine use of prophylactic internal fixation with a
dynamic compression plate. The incidence of fracture and secondary surgery is relatively low
in comparison to that at the unreinforced donor site. The plate is effective in either an anterior
or posterior position and the choice of site is a matter of surgeon preference. The incidence of
complications reported in the literature is low and very few plates have been replaced or
removed. The technique allows the surgeon to safely harvest a modest volume of bone, which
in conjunction with the excellent soft-tissue paddle means that the flap retains a wide range
of potential applications. In the current practice of the author, and many others, the radial flap
remains a compromise choice or back-up flap. Recent indications have included relatively
small defects of the maxilla, particularly in older patients that are unlikely to undergo dental
implantation. Additional considerations include patient preference, and if there is significant
peripheral vascular disease or poor general health that will be exacerbated by a procedure at
an alternative donor site.

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[22] Soutar DS, Scheker LR, Tanner NS, McGregor IA. The radial forearm flap: a versatile
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[58] Martin IC, Cawood JI, Vaughan ED, Barnard N. Endosseous implants in the irradiated
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In: Oral Cancer Research Advances


Editor: Alexios P. Nikolakakos, pp. 211-228

ISBN 978-1-60021-864-4
2007 Nova Science Publishers, Inc.

Chapter 9

CYTOLOGIC DIAGNOSIS OF ORAL


MALIGNANCIES: SCOPE AND LIMITATIONS
Dilip K. Das
Department of Pathology, Faculty of Medicine, Kuwait University.

ABSTRACT
Cancer of mouth and pharynx is one of the ten most common cancers in the world.
Detection of a precancerous or cancerous lesion at an early stage is an important factor to
improve 5-year survival rate of oral cancer. A comprehensive physical examination aided
by imaging techniques like computed tomography (CT), and magnetic resonance imaging
(MRI) are the standard evaluation tools in patients with oral, and pharyngeal neoplasms.
Although surgical biopsy and histopathology is considered gold standard for diagnosing
the oral lesions, it is impractical to routinely subject large number of patients to biopsy.
Whereas oral exfoliative cytology is a useful, economical and practical tool in the
diagnosis of oral dysplasia and carcinoma involving cheek, lip and tongue, similar role is
played by fine needle aspiration (FNA) cytology for minor salivary gland tumors and
other solid neoplasms of the palate, cheek and pharyngeal areas. By brush cytology a
spectrum of oral lesions including dysplasia, carcinoma in situ, occult and clinically
evident squamous cell carcinoma can be diagnosed. FNA cytology, which collects
samples from areas difficult to reach by surgical biopsy, can differentiate benign from
malignant tumors and classify them into subtypes. Whereas pleomorphic adenoma is a
common benign tumor, adenoid cystic carcinoma, mucous cell carcinoma, acinic cell
carcinoma, malignancy in pleomorphic adenoma, and polymorphous low-grade
carcinoma are the malignant neoplasms detected in the minor salivary glands. The other
oral neoplasms detected by FNA are non-Hodgkin lymphomas, and some rare primary
malignancies like sarcomas and chordoma. Metastatic lesions in oral cavity too have
been diagnosed by FNA cytology. The efficacy of brush cytology in detection of oral

Correspondence concerning this article should be addressed to: Dr. Dilip K. Das, MBBS, MD, PhD, DSc,
FRCPath. Department of Pathology, Faculty of Medicine, Kuwait University, P.O.Box: 24923, Safat 13110,
Kuwait. Tel: 00965-5319476; Fax: 00965-5338905; E-mail: dilip76@hotmail.com.

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Dilip K. Das
squamous cell carcinoma is very high in majority of reports, which is as follows:
sensitivity (84.4 9.97%), specificity (78.6 29.36%), positive predictive value (71.4
31.39%), and negative predictive value (83.0 16.40%). The sensitivity, specificity, and
diagnostic accuracy of FNA cytology for oral malignancies are also high. However, false
negative reports are possible with the oral brush cytology technique and some palatal
salivary gland tumors are difficult to diagnose by FNA cytology. In difficult situations,
ancillary techniques such as cytomorphometry, DNA-cytometry, immunocytochemistry,
and molecular tools act as valuable adjunct to cytodiagnostic techniques.

Keywords: Oral cavity, mouth, neoplasm, cancer, carcinoma, minor salivary gland,
malignancy, lymphoma, sarcoma, chordoma

INTRODUCTION
Cancer of the oral cavity is among the top ten cancers of the world and accounted for
274,000 new cases in 2002 [1]. The World Health Organization (WHO) predicts a continuing
worldwide increase in the number of patients with oral cancer, extending the trend well into
the next several decades [2]. The well known high-risk factors for oral cancer are
tobacco/alcohol use in western Europe, southern Europe, and southern Africa, the chewing of
betel quid in south-central Asia and Melanesia, and solar irradiation in Australia (mostly lip
cancer) [1]. In addition, a strong evidence for an etiological relationship between human
papillomavirus and a subset head and neck cancer has recently been noted [2]. Several oral
lesions such as leukoplakia, erythroplakia, actinic keratosis, and lichen planus are considered
to be premalignant lesions for oral squamous cell carcinoma, since an increased risk of
malignant transformation is associated with them [3,4]. These lesions are often subtle and
asymptomatic, requiring a high index of suspicion on the part of clinician, especially if the
risk factors such as tobacco use or alcohol abuse are present [5].
The detection of a precancerous or cancerous lesion when small is one of the most
important factors to improve 5-year survival rates of oral cancer [6]. Although surgical
biopsy is the most definitive method for diagnosing oral lesions, it is impractical to routinely
subject large number of patients to biopsy [6,7]. Diagnostic oral exfoliative cytology, on the
other hand, is a useful, economical and practical tool in the diagnosis of oral dysplasia and
carcinoma, but is not yet used so extensively as in cervico-vaginal cytology [8]. Brush
cytology of all visible oral lesions, if they are clinically considered suspicious for oral cancer,
are an easily practicable, cheap, non-invasive, painless, safe, and accurate screening method
for detection of oral precancerous lesions, carcinoma in situ or invasive squamous cell
carcinoma in all stages [9]. However, the great variation in technical quality in cytological
smears increases the chance for a diagnostic failure on microscopic examination. With
improvement in cytologic techniques that have resulted in the development of liquid-based
preparations, the use of this approach as an auxiliary diagnostic tool for oral mucosal lesions
has gained a renewed interest [10].
Besides oral squamous cell cancer, a variety of malignant neoplasms involving minor
salivary glands, sarcomas, lymphoma, and those invading from adjoining areas or
metastatizing from distant sites, affect the oral cavity. Inflammatory lesions presenting as

Cytologic Diagnosis of Oral Malignancies: Scope and Limitations

213

ulcers and swellings may create confusion with oral cancer and other common neoplasms of
this area [11]. Tumors arising from minor salivary glands of the palate may exhibit an overlap
of clinical and biologic features that may produce diagnostic and therapeutic dilemmas since
surgical treatment can be very different, depending on the dimensions and malignant or
benign nature of the tumors [12]. A large benign neoplasm of soft palate, e.g. pleomorphic
adenoma, can cause sleep apnea syndrome [13]. Under these circumstances, FNA cytology,
as a simple, economic, and painless technique, can help to diagnose such cases in an outpatient set up. This tool can play an important role in differentiating inflammatory from
neoplastic lesions and also benign from malignant neoplasms of the oral cavity [11], leading
to their proper management.

DIAGNOSTIC TECHNIQUES
A comprehensive physical examination aided by imaging techniques like computed
tomography (CT), positron emission tomography (PET), and magnetic resonance imaging
(MRI) are the standard evaluation tools in patients with head and neck carcinomas including
occult malignancies [14]. However, management of such lesions requires a prior tissue
diagnosis. The various biopsy techniques utilized in the diagnosis and treatment of mouth
diseases and tumors include incisional and excisional biopsy, needle (drill) biopsy, aspiration
biopsy, curettage, tissue imprints, exfoliative cytology and frozen section [7].
Exfoliative cytology is a simple, noninvasive procedure for studying epithelial cells of
mucosal surfaces. For conventional cytologic smears, a wooden spatula has been used as a
standard tool for collection of sample by scrapping motions from oral precancerous and
cancer. However, the cytobrush is found to be significantly more efficient than the wooden
spatula, in terms of both cell yield and cell dispersion [15]. Jones et al [16] have advocated
the use of cytobrush for obtaining diagnostic cytology smears from the oral mucosa, taking
into consideration the factors such as degree of patient discomfort, the convenience to the
clinician, and the quality and distribution of epithelial cells collected. Kujan et al [17]
recommended the use of a special custom-designed oral cytobrush for liquid based cytology
(LBC). According to these authors [17], LBC which shows good sample perversion,
specimen adequacy, and visualization of cell morphology, has potential as a screening tool
for oral cancer and precancer. Hayama et al [10] performed both conventional and liquidbased cytology, and concluded that both these tools were diagnostically reliable but the LBC
showed overall improvement in sample preservation, specimen adequacy, visualization of
cell morphology and reproducibility. When both these tools are used, the conventional smear
is first prepared by stroking the brush along the glass slide, which is immediately wet-fixed in
95% ethanol, to be stained by Papanicolaou stain. Then the brush containing the remaining
sample is inserted into the transport medium, an alcohol based preservative, to be processed
for LBC smears as per the following steps: vortexing, density reagent centrifugation,
decanting and resuspension of cell pellets followed by gravity sedimentation on poly-l-lysine
coated slides, and subsequent staining with Papanicolaou stain. The LBC sample can also be
used for immunocytochemical assays. It is found that the cell morphology as well as
immunocytochemical stainings are better visualized in liquid based preparations.

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Dilip K. Das

Fine needle aspiration (FNA) cytology can play an important role in the diagnosis of oral
lesions, especially for those that present as a mass or growth [11]. For this a 10 ml disposable
plastic syringe and a 23 gauge disposable needle fitted with a handle (Franzen syringe pistol)
is used for aspiration cells from a mass lsion in the oral cavity. By this simple and
inexpensive technique, not only lesions of the oral cavity but also those in the pharyngeal
area can be punctured with minimal inconvenience to the patients. Maghami et al [18]
demonstrated that MRI-guided FNA of the retropharynx is feasible, safe, and sensitive
enough to obviate the need for open biopsies in large percentage of cases. According to
Cerulli et al [19], FNA appears to be a very useful tool for preoperative diagnosis, which
helps in planning the surgical management. Whereas the air-dried smears prepared from the
aspirated material are stained by May-Grnwald-Giemsa stain (Diff-Quik), wet-fixed smears
are stained by Papanicolaou method, and can be subjected to immunocytohemical staining.
The aspirated material can be made into a cell block to study morphology, architectural
pattern, and immunohistochemical characteristics. The aspirated material can also be utilized
for molecular oncologic studies.

CYTOMORPHOLOGICAL FEATURES
Only a few articles describe the cytomorphological features of a wide spectrum of oral
neoplasms [11,20]. Das et al [11] diagnosed 45 cases of oral and pharyngeal lesions by FNA
cytology, which included 15 benign neoplasms, 16 malignancies, 11 inflammatory, and 3
inadequate cases. The benign tumors were pleomorphic adenoma (11 cases), schwannoma
(2), odontogenic tumor (1), and neoplasm, not otherwise specified (1). The malignancies
were malignant salivary gland tumors (7 cases), squamous cell carcinoma (6 cases), NHL (2
cases), and malignant odontogenic tumor (1case). Shah et al [20] performed transmucosal
FNA for oral and pharyngeal lesions from 79 sites in 76 patients. In their material [20], the
sites for 38 malignant lesions were buccal (11 cases), tongue (11), alveolar ridge (3), floor of
mouth (3), palate (3), tonsil (2), pharynx (2), lip (1), retroareolar ridge (1), and maxilla (1).
The cytodiagnosis of malignant lesions were squamous cell carcinoma (24 cases), melanoma
(4), mucoepidermoid carcinoma (3), lymphoma (3), adenoid cytic carcinoma (1),
adenocarcinoma (1), metastatic adenocarcinoma (1), and sarcoma (1).

Oral Precancerous Lesions and Squamous Cell Carcinoma:


Oral cancer is likely to develop from antecedent dysplastic oral mucosal lesions, if an
early diagnosis is not made and treatment given. Leukoplakia is the most common oral
premalignant lesion but erythroplakia is particularly relevant considering its almost certain
relationship with dysplasia and invasive squamous cell carcinoma [2]. The early,
asymptomatic oral and oropharyngeal cancers differ markedly from advanced cancers in their
clinical presentation, course, and outcome [21]. Maraki et al [22] classified the cytological
diagnoses in 98 cases of oral lesions into 75 cases of tumor cell negative (including reactive
and inflammatory), four of doubtful tumor cells (mild and moderate dysplasia), four cases of

Cytologic Diagnosis of Oral Malignancies: Scope and Limitations

215

suspicious for tumor cells (sparse abnormal or severe dysplastic cells, and those with vague
criteria for malignancy), and 15 tumor cell positive cases (unequivocal malignant cells). In
these cases the final diagnoses were 15 cases of squamous cell carcinoma, 21 leukoplakias,
three eythroplakia, and 59 other, inflammatory oral lesions.
Leukoplakia:
The smears from leukoplakia contain hyperkeratotic surface cells and many tightly
packed cells with nuclei, which may include those representing an abnormal maturation
pattern [23].
Oral Carcinoma in Situ (CIS) and Squamous Cell Carcinoma:
In a study of 77 oral CIS [24], the high-risk sites were floor of the mouth (23.2% of all
lesions), tongue (22%), and lips (in males only, 19.5%). In another study, among 229
aspirates of squamous cell carcinomas of the head and neck region, 187 (81.7%) were
cervical or submandibular sites and 42 (18.3%) were oral cavity sites [25]. Fifty three
squamous cell carcinoma of the oral cavity diagnosed by Remmerbah et al [9] had the
following gross anatomical distribution: floor of the mouth (6 cases), floor of the mouth and
tongue (12), tongue (11), lip (0), tonsil and palate (10), alveolar ridge (7), and cheek (7). In a
study by Miyamoto et al [26], the 50 oral squamous cell carcinomas were derived from the
tongue (31 cases), lower gingival region (9), upper gingival region (2), buccal mucosa (4),
and the floor of mouth (4). Enlarged nuclei, variation in nuclear size and shape
(pleomorphism), irregular nuclear border, increased nuclear cytplasmic ratio, hyperchromasia
with abnormal chromatin pattern and distribution, multiple prominent and irregular nucleoli,
and discrepancy in maturation are the characteristics of malignant cells in oral cancer [23]. In
a study of six squamous cell carcinoma cases of the oral cavity diagnosed by FNA cytology
[11], the tumor cells were present in compact clusters, discohesive groups and in singly
dispersed form, with varying degrees of keratinization (Figure 1). Degenerative changes,
necrosis, and giant cell reaction were observed in one or more cases. These cytologic
features, when present in a lymph node aspirate from the cervical region, may point towards
an occult primary in the catchment areas including oral cavity. Rajab et al [27] described a
glycogen-rich clear cell carcinoma in the tongue, which was diagnosed by biopsy and FNA
cytology of neck swelling. Patients with metastatic cancer detected by excisional biopsy or
fine needle aspiration cytology of cervical lymph nodes in cases with occult primary tumor in
the head and neck region can be benefited by positron emission tomography (PET), which
has a sensitivity of 66.0% and specificity of 92.9% [28].
Radiation Changes in Oral Cancer:
The treated oral cancer cases also require cytological evaluation for detection of radiation
response and recurrence of their lesions. The radiation-induced changes in oral cancer cells
include micronucleation, multinucleation, binucleation and nuclear budding, which become
evident in the initial few days of radiotherapy [29]. The statistically significant dose-response
relationship and the high intertumoral variation suggest that serial assay of these changes has
a potential use for radiosensitive prediction. Mehrotra et al [30] also found a dose-related
increase in multinucleation, micronucleation, nuclear budding, binucleation and cytoplasmic

Dilip K. Das

216

granulation, which was observed after various fractions of radiotherapy in both normal and
malignant oral cells. However, these changes were more marked in malignant cells.

Figure 1. Keratinizing squamous cell carcinoma of tongue: FNA smear from a small growth and ulcer
in the middle and left side of tongue of a 42-year-old woman. A: Smear shows loose cohesive clusters
of malignant cells with evidence of keratinization (Papanicolaou x 200). B. Higher magnification of
malignant cells shows distinct nucleoli and an epithelial (keratin) pearl (Papanicolaou x 400).

Malignant Salivary Gland Tumors:


The malignant salivary gland tumors of the oral cavity diagnosed by FNA cytology
include malignancy in pleomorphic adenoma, adenoid cystic carcinoma, acinic cell tumor,
mucoepidermoid tumor, mucous cell carcinoma, and squamous cell carcinoma. FNA
cytology of 151 patients with salivary gland tumors by Cajulis et al [31] showed the
following distribution: 125 (83%) from the parotid gland, 23 (15%) from the submandibular
gland and only 3 (2%) from the soft palate. Palate is the most common site for salivary gland
tumors in oral cavity, both benign and malignant. Six of the 11 pleomorphic adenomas
diagnosed by Das et al [11] were also located in palate. Of the seven malignant oral salivary
gland tumors diagnosed by them [11], five were located in the palate, one in the cheek, and
the remaining one in cheek and palate. The cytodiagnosis of these seven malignant salivary
gland tumors were adenoid cystic carcinoma (2 cases), malignancy in pleomorphic adenoma
(2), acinic cell carcinoma (1), mucous cell carcinoma (1), and squamous cell carcinoma (1).
Of the 51 cases of palatal salivary gland tumors aspirated by Sahai et al [32], the reviewed
FNA cytology diagnoses included 27 cases of pleomorphic adenoma, seven polymorphous
low grade carcinomas (PLGA), eight adenoid cystic carcinoma, three mucoepidermoid
carcinoma, three oncocytoma, and three undetermined type malignancies.

Cytologic Diagnosis of Oral Malignancies: Scope and Limitations

217

Adenoid Cystic Carcinoma:


The smear contains globules and cylinders of mucoid material surrounded by closely
packed, uniform cells with round nuclei and scanty cytoplasm (Figure 2). Although the
cytologic features of adenoid cystic carcinoma is very characteristic, terminal duct carcinoma
of the soft palate, at times, may be difficult to distinguish from this neoplasm in FNA smears
[33].

Figure 2. Adenoid cystic carcinoma. A: FNA smear from the palatal swelling in a 50-year-old man.
Cylinders of mucoid material are surrounded by small and monomorphic neoplastic cells (MayGrnwald-Giemsa x 200). B: FNA smear from a swelling in the posterior part of the soft palate in a 45year-old woman. Small monomorphic tumor cells surround the mucus globules (May-GrnwaldGiemsa x 436).

Mucous Cell Adenocarcinoma:


The smears show pleomorphic malignant cells with round nuclei and abundant cytoplasm
containing intracytoplasmic reddish, granular areas suggesting mucin or intracytoplasmic
mucin globules.
Acinic Cell Carcinoma:
Tumor cells are present in cohesive clusters as well as dissociated form in the smears.
They have eccentric nuclei and abundant, homogenously, dense cytoplasm with fine
granularity, resembling oncocytes to some extent. Many bare nuclei, the size of lymphocytes
are scattered in the background.
Malignancy in Pleomorphic Adenoma:
The smears contain loosely cohesive tumor cells showing mild to moderate
pleomorphism and mitotic activity. History of a pre-existing pleomorphic adenoma may be

218

Dilip K. Das

forth coming and/or background may show varying amount of fibrillar mesenchymal tissue
reminiscent of a mixed salivary gland tumor.
Basal Cell Adenocarcinoma:
It is an extremely rare low-grade malignant tumor of the salivary glands, particularly of
minor salivary glands, which has cytologic features of basal cell adenoma, together with
infiltrative growth [12], difficult to appreciate in cytology.
Epithelial-Myoepithelial Carcinomas:
FNA cytology of this minor salivary gland tumor of the hard palate show a biphasic
population consisting of cells of ductal epithelial and myoepithelial origin arranged in small
clusters and sheets [34]. The myoepithelial cells have small, uniform nuclei; ample, clear
cytoplasm and distinct cell border, while the ductal epithelial cells have larger, mildly
pleomorphic nuclei and scanty cytoplasm. These ductal cells tend to form tubules among
background sheets of clear myoepithelial cells. Hyaline material surrounding cell clusters and
adenoid cystic carcinoma-like areas with orangeophilic globules are also not uncommon.
Polymorphous Low-Grade Adenocarcinoma (PLGA):
It is a minor salivary gland arcinoma usually arising intraorally, primarily in the palate.
Of the 61 palatal tumors diagnosed by Sahai et al [35] in FNA sears, 10 were PLGA. Evans
and Batsakis [36] described 14 cases, which were intraoral in location, involving the palate in
11, the buccal mucosa in two, and the posterior mandibular area in one. According to Sahai et
al [35] there are no established FNA cytologic features of PLGA. The smears may show
branching papillary pattern with small round to oval cells having scant to moderate
cytoplasm, round nucleus, fine chromatin, and inconspicuous nucleoli. FNA cytologic
features of a PLGA, arising at the base of the tongue, included cuboidal epithelial cells and
short, spindle-shaped myoepithelial-like cells [37]. This tumor also contains large clusters of
cells with myxoid material at the center, and occasionally palisading tumor cells surround
them.

Lymphoma:
FNA cytology is a useful tool for diagnosis of nonHodgkin lymphoma (NHL) in general
[38] and oral NHL as well [11]. However, the result may not be always conclusive [39]. The
lymphomas of the oral cavity show a more or less mono-typic population of atypical
neoplastic lymphoid cells (Figure 3A). The other cytomorphologic features depend upon the
subtype of NHL. For example, The FNA cytologic features of Burkitts lymphoma (Figure
3B), which commonly involves the jaw bones and adjoining soft tissues, include abnormal
lymphoid cells with cytoplasmic vacuolations due to lipid, interspersed with nonneoplastic
histiocytes containing cell debris [40]. Arotiba et al [41] reported a case of multiple
malignancies involving gingiva, lip, frontal bone, and parotid gland, which was diagnosed as
NHL of gingiva and lymphoblastic lymphoma of the parotid by FNA, and he had a
concomitant carcinoma of the prostate.

Cytologic Diagnosis of Oral Malignancies: Scope and Limitations

219

Figure 3. Non-Hodgkin lymphoma. A: Centroblastic lymphoma: FNA smear from the right tonsillar
growth in a 68-year-old man. Smear shows distinct nucleoli in centroblasts (May-Grnwald-Giemsa x
500). B: Burkitt-type lymphoma: FNA smear from the upper jaw tumor in 4-yr-old male child. Small
non cleaved lymphoma cells contain cytoplasmic vacuolations (May-Grnwald-Giemsa X 1000).

A
Figure 4. Embryonal rhabdomyosarcoma. A. Oral and left maxillary swelling in a boy. B. FNA smear
from the swelling shows small round tumor and tadpole-shaped cells (May-Grnwald-Giemsa x 250).
C. Tumor cells are positive for desmin (x 400).

Dilip K. Das

220
Sarcomas:

Oral cavity is a known site for embryonal rhabdomyosarcoma, and FNA cytology can be
utilized for its diagnosis [42,43]. FNA smear from embryonal rhabdomyosarcoma shows
tadpole-shaped cells, besides small round cell population with round to oval nuclei and one to
two small nucleoli (Figure 4). The FNA cytologic features of leiomyosarcoma sarcoma of the
oral cavity have also been reported in the literature [44]. The smears from this rare neoplasm
are cellular, with the cells arranged in fascicles and dispersed form and the nuclei are
elongated with blunt ends, imparting a cigar-shaped appearance (Figure 5).

Figure 5. Leiomyosarcoma. A: FNA smear from a recurrent fungated oral swelling that extended to the
lower jaw in an 11-year-old male child. Smear shows bundles of spindle shaped cells with elongated
nuclei against a back ground of collagenous material (May-Grnwald-Giemsa x 250). B. The neoplastic
cells have cigar shaped nuclei with blunt ends (May-Grnwald-Giemsa x 500).

Chordoma:
Chordoma is an uncommon neoplasm of intra-osseous notochord remnants derivative,
which is characterized by slow progressive growth, recurrences after incomplete removal,
and late metastasis in about one third of cases [45]. It most commonly arises from the
sacrococcygeal region and somewhat less frequently from speno-occipital region.
Koibasioglu et al [46] described an oropharyngeal chordoma diagnosed by FNA cytology. In
FNA smears of sacro-coccygeal chordoma shows classic physaliferous cells with bubbly
appearance and a myxoid fibrillary background, which is intensely metachromatic [47].
Similar cytologic features are observed in oropharyngeal chordomas, involving the oral
cavity (Figure 6).

Cytologic Diagnosis of Oral Malignancies: Scope and Limitations

221

Figure 6. Chordoma: FNA smear from the palatal mass in a 35-year-old woman. A. Smear shows
typical physaliferous cells with abundant vacuolated cytoplasm imparting a bubbly appearance. Bright
fibrillar magenta colored material is observed in the background (May-Grnwald-Giemsa x 400). B.
Intense PAS positive reaction is seen in cytoplasm of tumor cells. Background matrix is positively
stained by Alcian-blue (PAS- Alcian blue x 400).

Metastatic Malignancies:
Oral cavity can be a site of metastatic cancers and FNA is an ideal tool for diagnosis of
these lesions without any complications. FNA cytologic diagnosis of metastatic lesions in the
oral cavity from sites such as breast [48] and liver [49] has been reported in the literature.

DIAGNOSTIC EFFICACY
The efficacy of exfoliative (brush) cytology for oral precancerous and cancerous lesions,
as revealed from five recent studies [8,9,50-52], was as follows (table I): The sensitivity
ranged from 71.4 to 94.6% with an average of 84.4%. The specificity showed a wide
variation, which ranged from 32.0 to 100.0% with an average of 78.6%. The averages of
positive and negative predictive values were 71.4 and 83.0%, respectively. In an earlier
review of cytological diagnoses in 1306 cases from 14 studies [53] the sensitivity for
diagnosing oral cancer ranged from 73.8% to 100%, with an average of 87.4%. According to
Navone et al [8], oral cytology can improve the accuracy of histology, and may be a useful
screening tool for the diagnosis of oral neoplasia/dysplasia.
There are very few studies highlighting the efficacy of FNA cytology in oral lesions. Das
et al [11] diagnosed 45 cases of oral lesions by FNA cytology and correlation with
histopathology was observed in 77.9% cases. Shah et al [20] demonstrated a high degree of
sensitivity (93%) and specificity of 86% for intraoral FNAB, when compared with biopsy by

Dilip K. Das

222

conventional means. In a study by Gross et al [12], FNA cytology had an accuracy of 91.6%
and an error rate of 8.4% for tumors arising from minor salivary glands of the palate.
Concurrence with histopathology was observed in 22 of 24 of their cases.
Table I. Efficacy of oral smear for detection of oral dysplasia, carcinoma in situ, and
squamous cell carcinoma
Oral Lesions

Sensiti
vity %
94.6

Specifi
city %
99.5

Accura
cy %
-

PPV
%
98.1

NPV
%
98.5

Dysplasia, CIS, ca
(Toluidine blue staining)
Squamous cell carcinoma

77.0

67.0

43.5

88.9

92.5

100.0

100.0

84.6

Dysplasia, squamous ca

71.4

32.0

44.1

60.0

Dysplasia and/or ca

86.5

94.3

89.6

Mean S.D

84.4
9.97

78.6
29.36

89.6

71.4
31.91

83.0
16.40

Cancer

Authors
Remmerbach et
al, 2001
Onofre et al,
2001
Remmerbach et
al, 2003
Poate et al,
2004
Navone et al,
2004

Ca= carcinoma; PPV= positive predictive value; NPV= negative predictive value; CIS= carcinoma in
situ.

DIAGNOSTIC DIFFICULTIES/ DILEMMAS


False negative reports are possible with the oral brush cytology technique. Poate et al
[52] observed poor results with brush biopsy results and concluded that not all potentially
malignant lesions are detected by this noninvasive investigative procedure. In a study
conducted in 1970s, the false negative rate of cytodiagnosis of oral cancer was found to be
12.5% as against an average of 14.5% in previous reports [54]. The recent studies as shown
in table I also indicate an average sensitivity of 84.5%. Potter et al, [55] who found very good
result, i.e., 4 (3.5%) negative cases by brush cytology among 115 histologically proved oral
squamous cell carcinomas, suggested that persistent lesions should undergo tissue biopsy for
definitive diagnosis.
Palatal salivary gland tumors are difficult to diagnose cytologically and this is more so in
case of newer entities such as polymorphous low grade adenocarcinoma (PLGA). Sahai et al
[32] found that 18 of the 51 palatal tumors (35.3%) could not be typed at initial cytologic
examination. It is known that certain diagnostic problems can occur in differentiating
pleomorphic adenoma from adenoid cystic carcinoma, monomorphic adenoma, and
mucoepidermoid carcinoma [56]. Ustundag et al [57] described an adenoid cystic carcinoma
of tongue arising in the minor salivary gland, which was initially diagnosed as a pleomorphic
adenoma by FNA cytology. According to Kim et al [58], carcinoma ex pleomorphic adenoma

Cytologic Diagnosis of Oral Malignancies: Scope and Limitations

223

of the palate may be missed by FNA cytology and even by histopathologic examination of
the open biopsy. Negahban et al [59] also faced diagnostic challenge in a case of clear cell
carcinoma arising in pleomorphic adenoma of minor salivary gland of the palate. A neoplasm
like primary ectopic meningioma, located submucosally in the floor of the mouth, may pose a
diagnostic challenge to clinicians and cytopathologists as its FNA cytologic feature may be
confused with low-grade salivary gland neoplasm and light microscopic findings require the
support of ancillary studies like immunohistochemitry and electron microscopy to arrive at
the final diagnosis of primary ectopic meningioma [60].

ANCILLARY STUDIES
Recently, cytomorphometric assessments improved by advanced computer-assisted
image analysis systems have gained importance in the diagnosis of malignant and
premalignant oral lesions. Cytomorphometric analysis via oral brush biopsy is a valuable
adjunct to biopsy for identification of premalignant and early cancerous oral lesions. It is a
rapid and minimally invasive procedure with high specificity and sensitivity rates, requiring
no topical or local anesthetic [6]. A study on expression of antigens in cytologic preparations
obtained from macroscopically normal oral mucosa of patients with tongue carcinoma and
controls showed that oral mucosa of cancer patients had a more than three-fold increased
expression of cytokeratin 19 among a panel of antigens, as compared to controls (36.0 versus
11.3%; P<0.01) [61]. Using a panel of keratin antibodies in smears and biopsies of 34 oral
cancer patients, Ogden et al [62] observed that the sensitivity of K19 was greatest but its
specificity was poor; and the keratin antibodies with best positive predictive values were K8
and LH 8. These authors [62] concluded that for exfoliative cytology to be of value as a
diagnostic test, it remains necessary to employ assays using more than one keratin antibodies.
Acording to Remmerbach et al [9], application of AgNOR technique to cytologic
preparations is found to be a useful adjunct to other methods in routine cytological diagnosis
of oral cancer, since it can help to solve cytologically suspicious and doubtful cases. The
same group of authors [9], in a study of oral squamous cell carcinoma from brush smears
found that the best cut off value of the mean number of AgNOR dots per nucleus
distinguishing benign from malignant cells was 4.8. Applying these methods they achieved a
positive and negative predictive value of 100% each. Mao [63] reported a mean AgNOR
count per nucleus in cancer group to be 4.7 0.72 vs. 2.4 0.37 in normal mucosa (p<
0.005) with no overlap between the two groups.
Cytology with DNA-cytometry is also a highly sensitive, specific, and objective adjuvant
as well as non-invasive tool for the early diagnosis of oral epithelial neoplasia, showing
excellent compliance among patients [22,50]. Remmerbach et al [50] detected DNA
aneuploidy (Feulgen stained smears examined under a TV image analysis system) in 96.4%
of carcinoma in situ and invasive carcinoma. Sensitivity of DNA-aneuploidy for detection of
cancer cells in oral smears was 96.4%, specificity 100%, positive predictive value 100%, and
negative predictive value 99%. Combination of cytological diagnosis and DNA-aneuploidy
raised the sensitivity to 98.2%, specificity to 100%, positive predictive value to 100%, and
negative predictive value to 99.5%. Maraki et al [22] have shown that the sensitivity of

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Dilip K. Das

cytological diagnosis combined with DNA image cytometry may be as high as 100% and
specificity 97.4%. It is also found that cytology along with DNA-cytometry is a highly
sensitive, specific, and non-invasive method for periodical follow up oral lichen planus (LP)
lesions in order to detect early or exclude malignancy [64].
Oral cytology sample collected with liquid-based technology has been utilized not only
for immunocychemical staining but in Hybrid Capture-2 for detection of HPV as well as the
PCR-based Roche AMPLICOR HPV test [17]. Oral CDx (OralScan Laboratories Inc.), a
computer-assisted method for analysis of the oral brush biopsy, is reported to be a highly
accurate method (sensitivity 100%, false negative rate 0%) for detection of oral precancerous
and cancerous lesions. The specificity of OralCDx positive result was 100%, while the
specificity for OralCDx atypical results was 92.9% [65]. Fluorescent in situ hybridization
(FISH), performed on FNA biopsies of 50 primary oral squamous cell carcinomas (OSCCs),
using a BAC clone specific for cyclin D1 gene (CCND1), revealed numerical aberrations in
21 (42.0%) [26]. In this study, the CCND1 aberration was associated significantly with
histopathologic grading (p= 0.032), the mode of invasion (p= 0.047), pathologic lymph node
status (p= 0.045), disease recurrence (p= 0.004), and survical (p= 0.004). In another study on
41 primary oral squamous cell carcinomas (OSCCs) based on fluorescent in situ
hybridization (FISH) on FNA biopsies and immunohistochemistry, the same group of authors
[66] found CCND1 amplification as a more reliable prognostic indicator than CCND1 over
expression in OSCCs. Aberrations in cyclin D1 gene (CCND1), which was detected by
fluorescence in situ hybridization (FISH) in 15 (33.3%) of 45 FNA biopsies of oral squamous
cell carcinomas (OSCCs) and was associated with mode of invasion of primary tumor
(p=0.01) and the presence of occult lymph node metastasis (p<0.001), appear to be valuable
in identifying patients at high risk of late lymph node metastasis in stage I and II OSCCs
[67].

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In: Oral Cancer Research Advances


Editor: Alexios P. Nikolakakos, pp. 229-246

ISBN 978-1-60021-864-4
2007 Nova Science Publishers, Inc.

Chapter 10

BENIGN AND MALIGNANT TUMORS OCCURRING


IN THE PTERYGOPALATINE FOSSA AND
ADJACENT STRUCTURES OF THE
PTERYGOPALATINE FOSSA: RECENT ADVANCES
OF DIAGNOSIS AND SURGICAL MANAGEMENT
Xin-Chun Jian
Department of Oral and Maxillofacial Surgery, Xiangya Hospital, Central South
University, Changsha, 410008, Hunan, Peoples Republic of China.

ABSTRACT
Surgery in the pterygopalatine fossa region presents anatomic and surgical problems
related to the difficulty of access. When a tumor in the pterygopalatine fossa involves the
maxilla and extends into the maxillary sinus and a tumor of the deep lobe of the parotid
gland extends into the pterygopalatine foss, extensive resection is often necessary.
Because of this, there has been a tendency either not to operate on these cases at all or
else to carry out simply a partial or piecemeal removal. The current underlying principle
of skull base approaches is to minimize brain retraction while maximizing skull base
visualization. This concept facilitates three-dimensional tumor resection, tumor margin
verification, and functional reconstruction with appropriate esthetic concerns. Current
many approaches have been used for the tumor of the middle skull base or the
pterygopalatine fossa.
With advancements in imaging, diagnostic technology, diagnostic pathology,
surgical technology and instrumentation, reconstructive techniques, the surgery of the
lateral cranial base or the middle cranial base is now receiving significant attention and
interest. It is purpose of this paper to provide readers with an overall review of benign
and malignant tumors occurring in the pterygopalatine fossa and adjacent structures of
the pterygopalatine foss: Recent advances of diagnosis and surgical management.

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In the region of the face, sinuses, or palate, perineural tumor spread usually follows the
maxillary division of the trigeminal nerve. Neoplasm can follow the branches of the
trigeminal back to the pterygopalatine fossa(PPF), a key landmark in detection of perineural
extention. Tumor arising in the face can follow the intraorbital nerve. Tumors of the palate
follow the palatine nerves through the greater and lesser palatine foramen and the
pterygopalatine canal, passing superiorly from the palate to the PPF. Tumors of the maxillary
sinus follow the superior alveolar nerves, perforating the lateral maxillary sinus wall before
passing along the posterior wall of the maxillary sinus and entering the PPF. Alternately, a
tumor of the maxillary sinus may erode directly into the infraorbital cannal or PPF, gaining
access to the neural element. From the PPF, tumor can follow the trigeminal nerve through
the foramen rotundum to gain access to the middle cranial fossa at the gasserian ganglion in
Meckel cave. Surgery in the pterygopalatine fossa region presents anatomic and surgical
problems related to the difficulty of access.
With advancement in imaging, diagnostic technology, diagnostic pathology, surgical
technology and instrumentation, reconstructive techniques, skull base surgery is now
receiving significant attention. It is the purpose of this paper to provide readers with an
overall review of diagnosis and surgical management of benign and malignant tumors arising
in the PPF and in adjacent structures of the PPF.

ANATOMIC STRUCTURES OF THE PTERYGOPALATINE FOSSA


To safely separate the tumors from the PPF, knowledge of the anatomic structures of the
PPF region is very important to surgeons.
The pterygopalatine fossa is a narrow funnel-shaped space below the cranial base that is
bounded anteriorly by the medial part of the maxillary tuberosity, posteriorly by the anterior
or sphenomaxillary surface of the pterygoid process of the sphenoid bone, and medially by
the lateral surface of the vertical plate of the palatine bone. Its roof is formed by the root of
the greater sphenoid wing. A lateral boundary is missing; here the pterygopalatine fossa
communicates with the infratemporal fossa through the pterygomaxillary fissure (Figure 1).
The pterygopalatine space is widest in its upper part and narrows downward and
continues into the pterygopalatine canal between the medial surface of the maxilla and the
lateral surface of the vertical plate of the palatine bone. The canal opens into the oral cavity
through the greater and lesser palatine foramina. The pterygopalatine fossa contains the
ramification of the maxillary nerve, the terminal branches of the maxillary artery, and the
pterygopalatine ganglion.
The maxillary nerve enters the pterygopalatine fossa through the foramen rotundum.
Below the opening of this canal and medial to it opens the pterygoid canal, leading the
pterygoid or Vidian nerve to the pterygopalatine, or Meckels ganglion. A pterygoid artery,
one of the terminal branches of the internal maxillary artery, can be traced posteriorly into the
Vidian canal. The palatine nerves and the descending palatine artery reach the oral cavity
through the pterygopalatine canal. Through the sphenopalatine foremen between the orbital
and sphenoid processes of the palatine bone and the inferior surface of the body of the
sphenoid bone the pterygopalatine nerves and artery pass into the oral cavity.

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231

Figure 1. Pterygopalatine fossa.

Table 1. Benign and malignant tumors occurring in PPF and adjacent structures of PPF
Benign tumors
Angiofibroma
Chondroma
Chordoma
Craniopharyngioma
Hemangioma
Lymphangioma
Meningioma
Neurilemoma
Neurofibroma
Osteoma
Pericytoma
Schwannoma
Inverted papilloma
Reactive granulomatous lesion

Malignant tumors
Acinic cell adenocarcinoma
Adenocarcinoma
Adenoid cystic carcinoma
Fibrous histocytoma
Olfactory neuroblastoma
Rhabdomyosarcoma
Sarcoma

BENIGN AND MALIGNANT TUMORS OCCURRING IN THE PPF


AND ADJACENT STRUCTURES OF THE PPF
A variety of benign tumors may involve the pterygopalatine fossa and pterygoid plates.
The epithelial or inverting papilloma usually presents as unilateral soft tissue mass
accompanied by simultaneous bone expansion and facial destruction [1,2]. Juvenile
nasopharyngeal angiofibromas demonstrate a nasopharyngeal soft tissue mass and expansion
of the pterygopalatine fossa [1]. Since these lesions tend to spread locally by extension along
natural foramina and fissures, enlargement and/or destruction of these openings into the fossa

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may be demonstrated by careful tomography [3]. Other benign tumors such as large pituitary
adenomas or neurinomas of the trigeminal ganglion may erode the sphenoid alae extending
into the base of the pterygoid plates and apex of the fossa.
Primary malignant tumors of the paranasal sinuses usually produce opacification and
bone destruction without evidence for focal expansion of the sinus walls [4]. Squamous cell
carcinoma, adenocarcinoma, melanoma, rhabdomyosarcoma and the uncommon
esthesioneuroblastoma may all involve the pterygopalatine fossa and adjacent structures by
direct extension.
A large group of benign and malignant tumors in the PPF and adjacent structures of the
PPF all could be encountered (Table 1) [5-19].

Figure 2. Sources and routes of direct spread of malignant tumors in the PPF. A) Tumor spreads to the
maxillary sinus by destroying the posterior wall; B) Tumor extends into the orbit through the inferior
orbital fissure; C) Tumor extends into the oropharynx through the palatovaginal canal; D) Tumor in the
PPF extends into the middle fossa through foramen rotundum; E) Sources of the tumor in the PPF.

ROUTES OF DIRECT SPREAD OF MALIGNANT TUMORS IN THE


PPF
According to anatomic characteristics of the pterygopalatine fossa and reports in the
literature report, we know that the routes of spread of the tumors in the PPF are direct. These
tumors are not metastatic to the middle cranial fossa, the maxillary sinus, the orbit, the oral
cavity and the paraphylaryngic area, the most common method of spread is by tumor erosion
through skull base bone, such as meningiomas and neurofibromas occurring in the

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233

pterygopalatine fossa, may extend into the middle cranial fossa through the foramen
rotundum, oval, or both. A tumor arising in the PPF would grow into the maxillary sinus by
destroying the posterior wall (Figure 2A). These tumors arising in the PPF also may extend
into the orbit through the medial part of the inferior orbital fissure (Figure 2B). The tumors
accurring in the PPF extend into the nasopharynx and the oropharynx through the
palatovaginal canal, into the infratemporal fossa through the pterygomaxillary fissure, into
the oral cavity by the greater and lesser palatine canals (Figure 2C). The many tumors
accurring in adjacent anatomic structures of the PPF also may extend into the PPF, such as
tumors in the meddle cranial fossa may extend into the PPF through the foramen rotundum,
ovale, or both (Figure 2E). Tumors of the orbit extend into the pterygopalatine fossa by way
of the inferior orbital fissure and/or orbital apex. The tumors in the maxillea and in the
maxillary sinuses extend into the pterygopalatine fossa through the posterior wall of the
maxillary sinuses. The tumors occurring in the upper palatine and/or the oral cavity may
extend into the PPF through the greater and lesser palatine foramina. Other common tumors,
such as tumors of the deep lobe of the parotid gland, including adenocarcinomas,
mucoepidermoid carcinomas, or adenoid cystic carcinomas, may extend into the middle skull
base through the pterygopalatine fossa. In our cases, there are extracranial meningioma from
the middle skull base or mucoepidermoid carcinomas and adenoid cystic carcinomas
occurring in the deep lobe of the parotid gland (Figure 2E).

RADIOLOGY AND RADIOLOGIC DIAGNOSIS OF THE


PTERYGOPALATINE FOSSA
The pterygopalatine fossa is a major distribution center for the parasympathetic
innervation and vascular supply of deep facial structures. Therefore this important structure
provides a natural pathway for dissemination or spread of disease processes to contiguous
structures. The normal radiographic anatomy of the pterygopalatine fossa and the adjacent
pterygoid plates are considered in detail. Variation in these structures as well as their
alterations in a variety of pathologic entities is described.
The pterygopalatine fossa itself is variable in size and shape. The transverse diameter as
well as overall length of the fossa varies considerably from side to side and from patient to
patient. The configurations range from an elongated, slitlike fissure to a somewhat more
triangular teardrop-shaped opening. In all normal instances, the surrounding cortical margins
of the fossa are well delineated.
Plain film radiographs and lateral hypocydoidal tomograms through the pterygopalatine
fossa and medial pterygoid plate show the broader superior portion of the fossa as it narrows
inferiorly to become the pterygopalatine canal. Tomograms through the medial pterygoid
plate show a somewhat bulbous inferior prominence, the pterygoid humulus. The lateral
pterygoid plate is rarely seen in its entirely on a single lateral tomographic section because of
its more oblique orientation. The lateral pterygoid lamina is scimitar-shaped, with a
posteriorly directed, slightly concave surface [1].
The base of the pterygoid plates and the pterygoid fossa, the V-shape space contained
between the two pterygoid plates, are both best appreciated on either anteroposterior or

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submentovertex tomograms. The foramen rotundum and pterygoid canal can also be
identified on these views. The palatinovaginal or pharyngeal canal is anatomically
insignificant and is rarely visualized.
Computed tomographic studies of the pterygopalatine fossa have indicated, in axial CT,
the pterygopalatine fossa had different shapes at different levels. In an axial CT section 5mm
more rostral, the upper most part of the PPF is seen adjacent to the infraorbital fissure, which
forms an angle of about 45 degrees with heads transverse diameter. The posterior-medial
margin of the PPF at its uppermost extent appears flat. In a CT image at this level the
foramen rotundum appears as a sagittally oriented channel about 1mm wide connecting the
middle fossa and the PPF. The pterygoid canal is not usually visualized in axial CT. The PPF
contains fat in which vessels are identified. Maxillary artery branches appear as multiple
small round enhancing structures in axial CT sections through the lower portion of the PPF.
The maxillary nerve and the pterygo-palatine ganglion usually are not visualized in routine
patient scanning due to partial volume averaging of the skull base.
In a coronal CT section 5mm more posterior to the PPF, the triangular openings of the
pterygoid canals appear at the base of the sphenoid bone. Superolateral to the pterygoid
canals are the round openings of the foramen rotundum. In coronal CT sections 5mm more
posterior, the pterygoid canals, with a round opening about 1mm in diameter, are seen. At
this level, small troughs at the upper aspect of the sphenoid bone and below the superior
orbital fissures mark the posterior ends of the foramen rotundum. In coronal CT sectioning,
the maxillary nerve and sometimes the pterygopalatine ganglion are seen as a round softtissue structure within fat at the upper part of the PPF. Maxillary artery branches appear on
CT as multiple small round or serpiginous enhancing structures [20].
In patients with tumor infiltrating the PPF, CT shows fat replaced with a denser tissue
that usually enhances significantly with intravenous contrast media. The nerves and blood
vessels are not distinguished from the tumor. Tumors can enlarge or erode the foramen
rotundum and pterygoid canal [20].
To visualize the PPF optimally, 5 and/or 1.5-mm thick axial and coronal sections are
recommended [20]. Neoplastic infiltration of PPF is recognized by erosion of osseous
margins, replacement of fat by higher-density tissue, and obscuring of vascular and neural
tissues in the PPF.
The combination of CT and magnetic resonance (MR) imaging has been the standard
evaluation for 2 decades. The technologic advances in CT and MR have improved the
resolution and efficiency of the data that can be acquired (Figure 3). Computed tomography
is critical in analyzing the cranial base bone anatomy whereas MR gives better soft tissue
definition in certain areas [21]. CT also can detect enlargement of foramina and erosion of
cranial base bone, MR is superior in detecting small lesion and is capable of identifying
perineural spread of malignant lesions (Figure 4). Such information can be important in
predicting outcomes of therapy [22]. Recent advances in CT technology such as multiclector
volumetric scanners and 3-D reconstruction techniques have improved the resolution of
smaller bone landmark [23]. Computed tomography angiography is useful technique that
allows an assessment of the critical vascular structures. Specifically, it can show the precise
relationship between the internal carotid arteries and other cranial base structures and can
determine the relationship of such arteries to the lesion [24].

Benign and Malignant Tumors Occurring in the Pterygopalatine Fossa

Figure 3. CT features of tumor in the PPF.

Figure 4. MR features of tumor in the PPF.

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Xin-Chun Jian

SIGNIFICANCE OF FINE-NEEDLE ASPIRATION CYTOLOGY IN


THE DIAGNOSIS OF DEEP LESIONS IN THE PPF
Most lesions of the head and neck are readily accessible, and biopsy for diagnostic
purposes is a relatively simple procedure. Generally, a portion of every lesion suspected of
malignancy should be removed for microscopic examination before definitive therapy is
attempted. The simplest method of biopsy is the excision of a small portion of tissue, using a
scalpel and fine forceps. This is satisfactory for lesions of the skin, lip and anterior portion of
the oral cavity. Open biopsy is, however, extremely difficult, and is almost impossible for
tumors occurring in the pterygopalatine fossa [17].
The initial report concerning fine-needle aspiration cytology (FNAC) is from Memorial
Hospital for Cancer and Allied Diseases by Martin and Ellis in 1930 [25] . The formal use of
FNAC in the head and neck began in 1974 and did not achieve wide-spread acceptance until
about a decade ago, when its effectiveness and accuracy were eventually realized [26].
Usually, FNAC can provide clinically useful information that exceeds that obtained by
palpation or imaging alone. The 90% to 95% sensitivity obtained with FNAC for the
diagnosis of palpable head and neck lesions is comparable to that obtained with traditional
open biopsy techniques [27]. But deep-seated lesions may be difficult to access using FNAC
and can be confused with complex anatomic structures in the PPF because of the lack of
accuracy in needletip localization and possible risk of injury to surrounding vital tissues [2832]. In such instances, the advent of image-guided aspiration has broadened its applicability.
Fine-needle aspiration combined with imaging guidance such as radiography,
ultrasonography, computed tomography, emission computed tomography, and magnetic
resonance imaging may enhance the accuracy of the diagnosis.
An 8-mm, 20-gauge MR-compatible needle (Cook Company) that is high in nickel
content, thereby reducing the ferromagnetic properties of the alloy and decreasing the
magnetic susceptibility differences between the alloy and surrounding tissue, was used under
MR guidance to enhance accuracy of the aspiration. He et al [32] achieved an access rate of
100% (12 of 12 patients), with an accuracy of 91.67% (11 of 12 patients), sensitivity of
85.71%, and specificity of 100%. This is the same as the reports of MR-guided aspiration in
other head and neck regions [33] and superior to ultrasound-guided and CT-guided cytology
in other head and neck regions [34,35]. MR can offer superior soft tissue contrast and
multiplanar imaging capability compared with CT and ultrasonography [36,37]. The MRguided needletip can be localized within masses with the diameter of 2 to 3mm [38]. The
above-mentioned studies strongly suggest a preferential role of MR over CT in guidance for
diagnosis of the deep-seated lesions in the head and neck.

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237

SURGICAL APPROACHES BENIGN AND MALIGNANT TUMORS


IN THE PPF
The rationale for resection of benign PPF tumors has been well established by improved
preoperative imaging assessment, new surgical techniques, better postoperative care, and
fruitful cooperation between multidisciplinary teams. Despite the technical reproducibility of
the classic techniques (ie, the craniofacial and subcranial approaches), modifications are
continually being designed to enhance access to this anatomic region [39].
Surgery in the pterygopalatine fossa area presents anatomic and surgical problems
related to the difficulty of access. When a lesion involves the maxilla, maxillectomy is often
necessary. An anterior approach involving a Weber-Ferguson incision, or one of its
modifications, is often used (Figure 5A). The problem with this particular approach, however,
is limited posterior exposure, because separation of the maxilla from the pterygoid plates is
normally done last and is performed blind, often using a chisel in an already blood-filled field
[40-43]. Access to lesions extending into pterygoid plates and associated structure is even
more limited with use of this approach.
Barbosa [44], in 1963, developed an extended anterior approach (Figure 5B). A WeberFerguson incision was extended from the lateral canthus of the eye posteriorly to the root of
the helix of the ear. This allowed a large inferiorly based flap to be raised, which includes the
parotid gland within the incision. The masseter and temporalis muscles were divided
horizontally, a portion of the ascending ramus of the mandible was resected, and the
temporomandibular joint was disarticulated. The then allowed direct lateral access to the
pterygomaxillary region, and the pterygoid muscles and plates could be removed. Barbosa
originally suggested this technique as a method for performing extending maxillectomy,
which could include the orbit and even the anterior cranial fossa in addition to structures of
the pterygomaxillary region.
Crockett, in 1963, modified this approach by adding a lateral extension from the
commisure of mouth (Figure 5C). Following reflection of this flap, access was gained to the
pterygomaxillary region by raising two osteoplastic flaps. The inferior flap consisted of the
arch and the orbital process of the zygomatic bone attached on the masseter muscle. The
superior flap contained the divided coronoid process still attached to the temporalis muscle.
Retraction of these two flaps provided a window to the pterygomaxillary region [45].
When lesions involve only the structures of the pterygomaxillary fossa and not the
maxilla, access from an anterior approach requires unnecessary maxillectomy. At this time, a
number of lateral approaches have been suggested for gaining direct access to
pterygomaxillary region.
Conley proposed, in 1956, an extended preauricular incision extending down into the
neck, with a second submandibular incision extending to the angle of the mouth (Figure 5D).
With these incisions, an extensive superior and a smaller inferior flap could be developed.
The inferior part of the temporalis muscle and its attachment to the coronoid process could be
removed, as could the zygomatic arch with the attached masseter muscle, as well as the
ascending ramus of the mandible. The parotid gland and facial nerve were left undisturbed,
but the facial incision and bony defect were not cosmetically pleasing, and many of the
deeper structures had to be sacrificed [46].

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Xin-Chun Jian

Figure 5. Outline of incisions. A) Weber-Ferguson; B) Barbosa; C) Crockett; D) Conley; E) Dingman


and Conley; F) Attenborough; G) Jian.

In 1970, Dingman and Conley [47] described an inferior approach via a submandibular
incision, which was extended anteriorly to include a midline lip-splitting incision and
posteriorly to run to the mastemporal incision (Figure 5E). After the extensive skin-flap was
raised, the parotid gland and facial nerve were retracted inferiorly. The zygomatic arch was
sectioned and reflected inferiorly on the masseter muscle, and the ascending ramus of the
mandible was divided horizontally, as in the prior approaches.
According to comouflaging the skin incision, care of the facial nerve, and sufficient
exposure to accomplish the task at hand are the keys to a successful result. In 1998, we [48]
modified the Barbosa approach by adding in a lateral incision in the mandibular
gingivobuccal fold from the canine tooth to the retromolar area. This allows a large,
inferiorly based flap to be raised, which includes the parotid gland. The masseter and
temporalis muscles are divided horizontally, and the ascending ramus of the mandible is

Benign and Malignant Tumors Occurring in the Pterygopalatine Fossa

239

osteotomided between the mandibular angle and the sigmoid notch and reflected to expose
the tumor in the pterygopalatine fossa and maxillary sinus. We believed that this technique is
especially useful for tumors in the pterygopalatine fossa extending into the maxillary sinus
(Figure 5G).
For small lesions, the endoscopic approach with or without external approaches offers an
effective mean for tumor resection [49]. Standard approaches to the PPF require
transmaxillary techniques that violate the anterior and posterior walls of the maxillary sinus,
with the risks of facial edema and pain, infraorbital nerve injury, oroantral fistula and
vascular injury. An endoscopic approach to the PPF can potentially reduce these risks, along
with providing better visualization than headlight-or microscope-directed approaches.
DelGaudio [50] reported an endoscopic approach to the PPF for definitive resection of a
schwannoma.
The procedure was begun with a large maxillary antrostomy, ethmoidectomy, and wide
sphenoidotomy to expose the medial and anterior aspects of the tumor. The mucosa of the
posterior maxillary sinus was elevated from superomedial to inferolateral. The thinned
posterior wall of the maxillary sinus was easily removed from the anterior and superior
surfaces of the PPF mass to expose the capsule. The sphenopalatine artery was dissected from
the surface and medial aspect of the mass, cauterized, and transected medially to completely
free the medial aspect of the tumor. The tumor was then bluntly dissected off of the pterygoid
plates posteriorly. Because of the tight confines of the PPF and the dense inferior attachments
of the tumor to the vasculature of the PPF, the tumor could not but removed en bloc. The
capsule was therefore opened to allow complete removal of the tumor. The inferior portion of
the tumors was removed last, after identification and clipping of the main trunk and branches
of the internal maxillary artery. After confirmation of complete tumor removal and irrigation
of the PPF, the surgical area with exposed pterygoid periosteum was covered with a
dissolvable hyaluronic acid pack [50].
The endoscopic approach to the PPF is a safe and effective surgical procedure. This
approach can be used for both diagnostic biopsy and definitive tumor removal where
appropriate. The approach described herein proceeds from medial to lateral, allowing for
identification of the sphenopalatine vasculature early in the procedure to reduce the risk of
vascular injury, which could obscure the endoscopic view. Lateral extension can provide
access to the inferior orbital fissure. The use of an image-guided system is a useful adjunct to
this surgical approach.

SURGICAL APPROACHES BENIGN AND MALIGNANT TUMORS


OCCURRING IN ADJACENT STRUCTURES OF THE PPF
For larger lesions occurring in the PPF, the transfacial and submandibular approaches
offer excellent exposure and tumor control. However, for tumors extending to adjacent
structures, above attention approaches alone can not provide adequate exposure, and
therefore other techniques should be added to allow safe tumor extirpation. In 1986, Jackson
et al use craniofacial osteotomies to facilitate skull base tumor resection. A zygomatic arch
osteotomy was performed together with an osteotomy of the ascending ramus of the mandible

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Xin-Chun Jian

and a total parotidectomy. A orbital osteotomy was performed involving the lateral wall, the
inferior orbital margin, and the floor of the orbit. This latter segment was not removed but
simply moved anteriorly in order to gain better exposure of the retromaxillary and ethmoid
areas. In order to excise the tumor totally, it was necessary to resect the posterior half of the
maxilla and also a portion of the ethmoid sinuses using a Weber-Ferguson approach. The
orbit and the zygomatic arch were wired back into position, but the ascending ramus of the
mandible was not, since the lower portion of the resection communicated directly with the
maxillectomy area, and the chances of inflection and sequestration of the ascending ramus
were high [51].
Janecka et al, in 1990, developed an approach to nasopharynx, clivus and cavernous
sinus by using facial soft tissue translocation and craniofacial osteotomies. Surgical field
obtained at the skull base can extend from the contralateral eustachian tube to ipsilateral
geniculate ganglion. It includes the nasopharynx, clivus, sphenoid and cavernous sinus, as
well as the entire infratemporal fossa and superior orbital fissure (Figure 6A). They believed
that the greatest advantage of their approach was in the direct access to a neoplasm in this
area, previously accessible to surgery in only limited fashion. The excellent visualization and
the potential for surgical control of important anatomic structures (carotid artery, optic nerve
or the facial nerve), as well as complete visualization of practically all surgical margins is the
hallmark of this approach [43] when the pterygopalatine fossa tumor extends into the middle
skull base and the intracranial area, however, all the approaches described have limitations.
We, in 2003, described a new approach to the pterygomaxillary fossa and the cranial base,
using facial translocation. This approach offers excellent visualization. The first incision of
this approach is divided the upper lip in the midline, passes under the nasal pyramid and
extends laterally, reaching the level of the temporomandibular joint, at which point it exists to
meet the vertical coronal/preauricular incision. An incision is then made along the maxillary
buccogingival fold in the involved side, running from the midline to the retromolar area.
Another incision is made along the mandibular buccogingival fold on the involved side,
running from canine to retromolar area. Our surgical approach to extensive tumors in the
pterygomaxillary fossa and the skull base is a combined technique that exposes the cranial
base from the temporal bone to the contralateral eustachian tube. Note that with this
approach, the corresponding intracranial anatomy can be easily accessed. If it is anticipated
that the dura of central skull base will be exposed, then vascularized tissue will be needed to
reconstruct this area. The temporalis muscle provides regional tissue for this purpose and can
be accessed via a frontotemporal incision made anterior to the tragus and extending into the
hairline above the temporal region (Figure 6B).

Benign and Malignant Tumors Occurring in the Pterygopalatine Fossa

241

Figure 6. Outline of incisions used in facial translocation approach. A) Janecka, 1990; Skin incision; B)
Jian, 2003; Skin incision.

This technique was designed to provide direct access to the central skull base and/or
infratemporal fossa and corresponding intracranial anatomy. It is indicated for benign and
malignant lesions that involve 2 or more of the following anatomic areas: pterygopalatine
fossa, sphenoid sinus, nasopharynx, infratemporal fossa, cavernous sinus and/or floor of the
middle fossa and clivus. Because exposure of the cranial base is wide and direct, this
approach facilitates en bloc resection of many skull base neoplasms [16]. To prevent facial
incisions, a unilateral or bilateral medial maxillectomy can be carried out from above via the
subcranial approach [52]. Combination of the craniofacial approach with the facial
translations approach can help resection of large juvenile angiofibromas infiltrating the
nasopharynx or pterygopalatine fossa while adding a LeFort I down-fracture allows excision
of large chordomas involving the clivus.

RECONSTRUCTION AFTER EXCISION OF BENIGN AND


MALIGNANT TUMORS OCCURRING IN ADJACENT STRUCTURES
OF THE PPF AND IN THE PPF
The ultimate success of contemporary lateral skull base procedure is as dependent on
reliable reconstructive methods as the technical competency of the proceeding resection. As
procedures have evolved, one reconstructive principle has continued to maintain validity
through the years. Vascularized tissue provides the strongest foundation for a stable
reconstruction [53]. In open procedures involving subcranial approaches or craniotomies,
vascularized tissue can be supplied by local flaps, the most commonly used being the reliable
pericranial flap, harvested with or without galea. Temporal fascia or temporalis muscle is one
of them. Depending on the size of the dural defect, either a free fascial graft or a pedicled
vascularized rotation flap of temporal fascia can be used for closure. Small defects of less
than 6 mm are covered with free fascia or plugged with fat. For larger defects, a rotation flap
of vascularized superficial or deep temporal fascia is used [54]. The temporalis muscle is also

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Xin-Chun Jian

freely mobile from its original position and can be placed over the pericranial flap, covering
the anterolateral cranial base, sphenoid sinus, nasopharynx, orbit, space of previous maxillary
sinus and the pterygopalatine, as well as infratemporal fossa regions (Figure 7). The
temporalis muscle, facing the nasal cavity and nasopharynx may be covered with free grafts
of normal mucosa or can be permitted to be remucosalized by healing of secondary
intention. For smaller defects, this flap may be adequate for reconstruction alone. Large
defects require more robust tissue flaps, the microvascular free tissue transfers of
fasciocutaneous tissue or muscle being used most commonly [53,54]. The volume of the
defect is critical to flap selection to avoid brain compression by excessive flap tissue. Hence,
de-epithelialized radial forearm free flaps are useful to seal smaller lateral skull fossa defects
when pericranium is not available or inadequate, and rectus abdominis muscle useful or
larger defect [54,55]. Both microvascular flaps are useful due to their reliability, long pedicle
and low donor site morbidity.

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243

Figure 7. Reconstruction of the central skull base with the temporalis muscle and fascia. A) A patient
with neurofibroma of the right maxillary sinus extending into the PPF, frontal view; B) Frontal view
postoperatively; C) Incisive design, lateral view; D) After exposure of the right maxilla and PPF, lateral
view; E) Preparation of the temporalis muscle and fascial flap, lateral view; F) After the temporalis
muscle and fascial flap transfers to the central skull base for repairing defect of the central skull base,
lateral view; G) After sutures, lateral view; H) Lateral view after stures were removed.

244

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[39] Mehta RP, Cueva RA, Brown JD, et al: Whats new in skull base medicine and surgery?
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[42] Janecka IP: Classification of facial translocation approach to the skull base. Otolaryngol
Head Neck Surg 1995; 112: 579-585.
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1151-1160.

In: Oral Cancer Research Advances


Editor: Alexios P. Nikolakakos, pp. 247-262

ISBN 978-1-60021-864-4
2007 Nova Science Publishers, Inc.

Chapter 11

MOLECULAR ASPECTS OF ORAL CANCER:


THE ROLE OF PHASE I AND II
BIOTRANSFORMATION ENZYMES IN
CARCINOGENESIS
Karin Soares Gonalves Cunha1,2,3 and Dennis de Carvalho Ferreira4
1

Department of Oral Pathology and Diagnosis, School of Dentistry, Federal University of


Rio de Janeiro, Rio de Janeiro, Brazil;
2
School of Dentistry, University Center Serra dos rgos (UNIFESO), Terespolis,
Brazil;
3
Fluminense Federal University, Niteri, Brazil;
4
Department of Microbiology, Sector of Sexually Transmitted Diseases, Federal
Fluminense University, Niteri, Brazil.

ABSTRACT
Oral cancer is the most common malignant neoplasm of the head and neck and over
half of the people who develop this cancer die within five years after the diagnosis.
Carcinogenesis is a highly complex process involving both environmental, mainly
tobacco and alcohol use, and inherited risk factors. In recent years, inter-individual
genetic differences and individual susceptibility to human cancer triggered by
environmental exposures has been studied. This environment-gene interaction in
carcinogenesis is well reflected by phase I and II enzymes that are involved in the
metabolism of carcinogens. Cytochrome P450 family of enzymes (CYP), involved in
phase I, converts many carcinogens into DNA-binding metabolites in target cells and can
modulate intermediate effect markers such as DNA-adducts. Phase II enzymes, including
glutathione S-transferase (GST), N-acetyltransferase (NAT) and others, play important
roles in protecting cells from DNA damage by carcinogens and reactive oxygen species.
Genetic alterations of these two classes of enzymes have been considered as risk
modifiers of some major tobacco-related cancers, including oral cancer. The aim of this

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Karin Soares Gonalves Cunha and Dennis de Carvalho Ferreira


chapter is to review the molecular aspects of oral cancer, emphasizing the role of phase I
and II enzymes in oral carcinogenesis. Propositions for further researches are
highlighted.

1. INTRODUCTION
Oral cancer is a serious health problem. It is the sixth most common malignancy, with
over 200,00 new cases reported annually worldwide [41]. Squamous cell carcinoma accounts
for approximately 90% of all malignancies diagnosed in oral cavity. It is the most common
malignant neoplasm of the head and neck and half of the people who develop oral cancer die
within five years after the diagnosis [5,45].
Carcinogenesis is a highly complex process involving both environmental and inherited
risk factors. In oral cancer, as well as in other head and neck cancers, predominantly tobacco
and alcohol consumption are the most significant external factors involved in tumor
formation [20].
The higher incidence of cancer in first-degree relatives of patients with squamous cell
carcinomas of the head and neck confirms the presence of genetic susceptibilities in the
development of this disease [14]. Moreover, within a population, it is remarkable that only
some people develop cancer, even with similar exposures.
In recent years, inter-individual genetic differences and individual susceptibility to
human cancer triggered by environmental exposures has been studied. This environmentgene interaction in carcinogenesis is well reflected by phase I and II enzymes that are
involved in the metabolism of carcinogens [39].
Cytochrome P450 family of enzymes (CYPs), involved in phase I, converts many
carcinogens into DNA-binding metabolites in target cells and can modulate intermediate
effect markers such as DNA-adducts. Phase II enzymes, including glutathione S-transferase
(GST), N-acetyltransferase (NAT) and others, play important roles in protecting cells from
DNA damage by carcinogens and reactive oxygen species [16,41,57].
Susceptibility to oral cancer in a particular individual may depend in part on the
metabolic balance between phase I and phase II enzymes [20]. Genetic alterations of these
two classes of enzymes have been considered as risk modifiers of some major tobacco-related
cancers, including oral cancer. The aim of this chapter is to review the molecular aspects of
oral cancer, emphasizing the role of phase I and II enzymes in oral carcinogenesis.

2. CHEMICAL CARCINOGENESIS
Carcinogenesis is a multistep process that involves three distinguishable but closely
connected stages: initiation (normal cell transformed or initiated cell), promotion (initiated
cell preneoplastic cell), and progression (preneoplastic cell neoplastic cell) [59].
Accumulation of genetic changes that occurs during carcinogenesis allows for the
disruption of normal cellular functions and enables a clonal expansion of abnormal cells to
form a malignant neoplasm [34].

Molecular Aspects of Oral Cancer

249

Initiation is the first step of carcinogenesis and it is a result of rather rapid and irreparable
damage to the cell. Chemical carcinogenesis initiates when DNA in a cell or population of
cells is damaged by exposure to exogenous or endogenous carcinogens [27].
Carcinogens can react with DNA to form adducts (carcinogen metabolites bound
covalently to DNA) and result in DNA alteration, which is an important step in chemical
carcinogenesis [1]. According to their ability to react to DNA to form DNA-adducts,
chemicals carcinogens can be divided into two groups. The first group comprises chemical
compounds that are direct-acting agents and can react to DNA without metabolic activation
to form DNA-adducts. The second group comprises chemical carcinogens that are indirect
agents and need to be activated by phase I enzymes to react to DNA [34]. As these chemicals
do not react directly with cellular constituents and require enzymatic conversion into their
ultimate carcinogenic forms, they are termed procarcinogens [40].
Among known human carcinogens, only a few chemicals belong to the class of 'direct
carcinogens', including ethylene oxide, bis(chloromethyl)ether and some aziridine or
nitrogen-mustard derivatives used in anticancer chemotherapy. On the other hand,
nucleophilic or chemically inert compounds that need to be activated to form DNA-adduct
represent the great majority of human carcinogens and comprise aromatic and heterocyclic
amines, aminoazo dyes, polycyclic aromatic hydrocarbons (PAHs), N-nitrosamines,
halogenated olefins and others [40].

2.1. Tobacco Products and Oral Cancer


Tobacco products contain a diverse array of chemical carcinogens and most of them
require metabolic activation to exert their carcinogenic effects. Tobacco can be consumed in
both smoking and smokeless form. Tobacco smoke comprises nearly 60 known carcinogens,
but smokeless tobacco contains fewer carcinogens than tobacco smoke because most of them
are formed during combustion [67]. The major carcinogens present in tobacco are PAHs,
nitrosamines and aromatic amines. In smokeless tobacco, levels of PAHs are typically low,
but there is a high concentration of nitrosamines [10,67].

2.2. Alcohol and Oral Cancer


Alcohol, particularly in association with tobacco, has been recognized as an important
risk factor for OSCC [48]. Approximately 75% of all oral cancers arise in association with
alcohol and tobacco consumption [48]. An independent role of alcohol in carcinogenesis has
also been identified [66].
The mechanisms by which alcohol can cause cancer are still poorly understood [3,66].
Ethanol and water are the main component of most alcoholic drinks, but pure ethanol does
not act as a carcinogen in animal studies [3,66].
It has been proposed that ethanol may increase the penetration of carcinogens across oral
mucosa [3]. This may be through intercellular passage of carcinogens entering the oral
mucosa or perhaps by increasing the permeability of the oral mucosa [3,42,43,66]. This may

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Karin Soares Gonalves Cunha and Dennis de Carvalho Ferreira

explain the synergistic action of alcohol drinking and tobacco use in oral carcinogenesis, but
it does not explain the increased risk of oral cancer in alcohol drinkers and never-smokers
[3].
Acetaldehyde, the primary metabolic of ethanol, is a candidate for the carcinogenic effect
of alcohol. The main pathway of oxidation of ethanol to acetaldehyde is via the enzyme
alcohol desidrogenase (ADH) [66]. It is known that acetaldehyde can form DNA-adducts in
human cells in vitro and also in rats, thus resulting in DNA alterations [3].
Production of reactive oxygen species and nitrogen species is another possible
mechanism of alcohol-related carcinogenesis [3]. Ethanol is the most important inducer of a
specific phase I enzyme (CYP2E1), which is important in the metabolic activation of PAHs
and nitrosamines [38,52]. Beyond being an inducer, ethanol is also a substrate of CYP2E1
[52].
Other components in alcoholic beverages, including impurities and contaminants, might
also increase the risk of oral cancer [3]. PAHs have been found in hard liquors (e.g. whiskey)
and N-nitrosamines have been identified in beers [28].

3. XENOBIOTIC METABOLISM
The biochemistry of mammalian cellular metabolism is a complex array of enzymes and
metabolic products whose purpose is to generate energy for life and protect fidelity for DNA
replication. In humans, these activities occur in a hostile environment where there is a
continued exposure to a wide variety of foreign compounds (xenobiotics) [21].
During evolution process, organisms developed mechanisms to protect them against
chemical insults [21]. When chemical carcinogens are internalized by cells, they are often
metabolized, by phase I enzymes. These resulting metabolic products form the substrates of
phase II enzymes, which participate in reactions that involve the conjugation of these
products with endogenous molecules, such as glutathionine, facilitating their elimination
[10].
Nevertheless, this natural process that serves to excrete xenobiotics also activates
chemical procarcinogens [34]. Phase I enzymes convert relatively inert chemicals into
electrophilic intermediates via oxidation reactions. Electrophilic chemical species are
naturally attracted to nucleophiles like DNA and protein, and through DNA-adduct
formation, DNA genetic damage results [34].

3.1. P450 Enzymes (CYPs)


Over 90% of phase I metabolisms is mediated by cytochomes P450 (CYPs) [37]. CYPs
are intracellular monomeric hemoproteins that belong to the monooxygenase gene
superfamily and activate molecular oxygen for the oxidative metabolism of a great variety
of lipophilic organic chemicals [17,21].

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The difference between CYPs and other hemoproteins is the presence of thiol-group
functioning as a ligand that alters the electron density of the resonant porphyrin ring of the
heme and provide an electronic center for the activation of molecular oxygen [21].
There are many reactions catalyzed by CYPs and the number of chemicals that can serve
of substrates metabolized by CYPs is enormous and is greater than 1000 and includes
endogenous substrates, such as steroids, fatty acids, and prostaglandins, as well as exogenous
chemicals, including drugs and lipohilic xenobiotics [21,32].
Based on the nucleotide sequences, all CYP-encoding genes occurring in the mammalian
genome were divided into 10 gene families which were further divided into subfamilies.
Currently, it is believed that there are 50 different genes encoding CYPs in human genome
[17].
CYPs were categorized by Families and Subfamilies based on the principle that a CYP
protein sequence form on gene family is defined as usually having less than 40% of
resemblance to that from any other family. Subfamilies grouped together proteins have less
than 60% sequence similarity [21].
CYPs are localized in smooth endoplasmic reticulum and mitochondrial membrane in
liver and in extra-hepatic localization [17,21,68]. Although the liver represents the major site
for CYP-dependent metabolism, the extrahepatic tissues also express significant activity
[19,65,68].
CYPs are distributed in almost every organ of the human body, although the type of CYP
in a tissue appears to be specific [19,21,68]. The cellular expression of many CYPs is
regulated by transcriptional factors which become activates during exposure to various
chemicals. The ability of a chemical to serve as an inducer is generally linked to a CYP
family [21].
There is a paucity of studies that aimed to evaluate the expression of CYPs in oral cavity.
Farin et al. (1995) evaluated the expression of CYPs in oral epithelial cells immortalized by
human papilloma virus type 16E6/E7 genes and many mRNAs transcripts for several CYPs
was observed [13].
Yokose et al. (1999) performed an immunohistochemical study to identify CYP2C and
3A in human non-neoplastic and neoplastic tissues, including normal tongue and OSCC
occurring in tongue. Neither CYP2C nor 3A were expressed in normal and neoplastic tongue
[68].
Vondracek et al. (2001) studied the CYPs expression (CYP1A1, CYP1A2, CYP2A6,
CYP2B6, CYP2C, CYP2D6, CYP2E1, CYP3A4/7 and CYP3A5) in primary cultures of
normal keratinocytes obtained from buccal mucosa. A RT-PCR based analysis demonstrated
consistent expression of mRNA for CYPs 1A1, 1A2, 2C, 2E1, 3A4/7 and 3A5 [65].

3.2. Phase II Enzymes


Phase II enzymes include glutathione S-transferases (GSTs), N-Acetyl transferases
(NATs) and others [39]. As already explained before in this chapter, these enzymes are
important in detoxification of active metabolites produced by phase I enzymes.

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GSTs are a supergene family of ubiquitous multifunctional enzymes that play an


important role in drug, carcinogens, and reactive species detoxification and act both as
peroxidases and as catalysts of glutathionine transfer to electrophiles [10,17]. GSTs are
known to play important role in detoxification of several carcinogens found in tobacco,
especially benzo[a]pyrene and other PAHs, epoxybutanes, ethylene oxide and halomethanes
[5,22]. These enzymes catalyze the conjugation of reduced glutathionine with reactive
electrophilic intermediates formed during phase I, resulting in increased water solubility and
allowing renal excretion of carcinogenic metabolites [10,17].
The GST family in humans can be divided into four classes: Alpha (), Mu (), Pi (),
and Theta () enzymes, and they consist of several isoenzymes with overlapping substrate
specificity [24, 39].
NATs are cytosolic enzymes present in liver and other tissues in majority of mammals.
Human NATs comprise two isoenzymes, the monomorphic NAT1 and the polymorphic
NAT2. Both isoforms catalyze the reaction in which xenobiotics containing amine or
hydrazime groups are transformed into aromatic amines and hydrazides. This reaction, named
N-acetylation, is the major biotransformation pathway of such compounds [17].

4. ASSOCIATION BETWEEN PHASE I AND II POLYMORPHIC


VARIANTS AND THE RISK OF ORAL CANCER
Since xenobiotic substances metabolism can affect the potency of most carcinogens by
activating or detoxificating them, carcinogen-metabolizing enzymes polymorphisms have an
important role in susceptibility to such xenobiotic related cancers [11].
Genetic alterations in CYPs have been related to individual susceptibility to various
types of malignancies, such as lung, breast, prostate, ovarian, and head and neck cancer
[4,17,30,36,41,53,55,57,64]. Cancer risk is determined by the degree of expression and/or
activity of CYP enzymes [12]. An elevated activity of these enzymes increases the risk of
cancer whereas a lower or absent activity reduces cancer risk.
Similarly, variation in the expression and activity of phase II enzymes due to heritable
genetic polymorphisms have also been associated to the susceptibility of various cancers
(lung, colorectal, bladder, head and neck cancers and others), resulted from the altered ability
to face biological insult caused by exposure to carcinogens [7,9,25,47,50].
It also has been postulated that certain genotype combinations can increase the risk of
cancer by acting synergically [10]. For e.g. enhanced activation of procarcinogens by phase I
enzymes, accompanied by reduced or loss of phase II enzymes function, both caused by
genetic polymorphisms, can lead to greater risk for cancer than attributed by single gene
variant alone [10].

4.1. CYPs Polymorphisms and the Risk of Oral Cancer


To date, three families of CYPs could be associated to the development of OSCC and
they are listed bellow.

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253

4.1.1. CYP1 Gene Family Studies


There are three genes (CYP1A1, CYP1A2 and CYP1B1) belonging to the CYP1 gene
family [17]. CYP1A1 and CYP1A2 catalyzed chemical reactions and substrates for which are
PAHs and cicyclic/heterocyclic aromatic amines, respectively, and thus resulting in the
activation of these procarcinogens and formation of mutagenic and genotoxic metabolites
[17].
The human enzyme CYP1A1 is the most active among the CYPs in metabolizing
procarcinogens, like PAHs and aromatic amines, into active species forming DNA-adducts
[44]. Several important single nucleotide polymorphisms have been identified in the CYP1A1
gene, located on chromosome 15q22.
The CYP1A1 m1 allele has a thymine/cytosine point mutation in the 3-noncoding region
at nucleotide T6235C, which has been associated with elevated enzyme activity [33,55].
Another polymorphic variant of CYP1A1 gene (m2), which has also been associated with
elevated enzyme activity, is the isoleucine/valine substitution in heme-binding region in exon
7 at nucleotide A4889G [33,55]. The variant CYP1A1 m3 has a mutation in intron 7 and
appears to be African-American specific. Another polymorphism (m4), located two bases
upstream of the m2 site, also causes amino acid substitutions of Thr for Asn in the hemebinding regions of the enzyme [55].
The majority of the studies that investigated the association between OSCC risk and
polymorphisms of loci involved in metabolic pathways of xenobiotics, has included the study
of CYP1A1 genetic variants [51,57]. Park et al., in 1997, first reported the association of
CYP1A1 polymorphism and an increased susceptibility to OSCC in USA [51]. Many other
studies, performed in different countries (Japan, Korea, Brazil, India, Twain), have also
obtained the same results [6,15,30,41,53,56,57,58].
Conversely, other studies performed in Germany, Japan, Brazil and Netherlands could
not find this association [20,31,35,41,49].
CYP1B1 also contributes to aromatic hydrocarbon hydroxylase activity, and interindividual variation in the expression of CYP1B1 has been observed. Human CYP1B1
catalyzes the oxidation of PAHs to yield electrophilic intermediated capable binding
covalently to DNA [44]. Several polymorphisms have been identified in the coding region of
CYP1B1 gene. Most polymorphisms resulted in the formation of a truncated or nonfunctional
protein [33]. Four sense polymorphisms were found in the CYP1B1 gene: at position 48 (Arg
to Gly), at position 119 (Ala to Ser), at position 432 (Val to Leu), and at position 453 (Asn to
Ser) [33].
The first study that associated CYP1B1 polymorphisms and head and neck cancers was
performed in 2001, in Germany, by Ko et al [33]. These authors observed that CYP1B1
Val432Leu polymorphism is an inheritable predisposing factor for smoking-induced head and
neck squamous cell carcinomas. Nevertheless, another study performed in the USA found
that CYP1B1 Val432Leu polymorphisms are not associated with an increased risk of
squamous cell carcinoma of the head and neck [38]. To our knowledge, there is not any other
study which investigated CYP1B1 polymorphisms in patients with head and neck cancer.

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4.1.2. CYP2 Gene Family Studies


CYP2 family is the largest studied so far and comprises six subfamilies: CYP2A,
CYP2B, CYPEC, CYP2E and CYP2F [17].
Among xenobiotic metabolizing enzymes, the CYP2A family is characteristic of its
catalytic properties to nitrosamines [61]. Several genetic alterations within the CYP2D6 gene
were found responsible for attenuation or lack of the enzyme activity [17].
A study performed in Indian Subcontinent (Sri Lanka) suggested that deficient CYP2A6
activity due to genetic polymorphisms reduces oral cancer risk in betel quid chewers [61].
The lesser risk of oral cancer in these patients may be explained by the fact that individuals
with this genotype are incapable to bioactivating betel quid specific procarcinogens to
carcinogens. Another study, performed in Croatia, could not find statistically significant
difference between the occurrence of CYP2D6 genetic alterations in head and neck patients
and the control population [62].
CYP2E1 catalyzes metabolic activation of several compounds found in cigarette smoke,
such as N-nitroso-dimethylamine, benzene, N-nitrosonornicotine, and ethanol [38]. More
than 25 polymorphisms of CYP2E1 have been identified, one of which is G1532C located
upstream of the CYP2E1 transcription start site that is believed to affect CYP2E1 expression
[23,38].
Due to its ability to bioactivate compounds which are potentially carcinogenic, CYP2E1
polymorphisms have been linked to the development of human cancers. Sugimura et al.
(2006) observed that CYP2E1 polymorphisms affect the risk of OSCC in Japanese
population. They concluded that these polymorphisms had significant interactions with
smoking but there was not any interaction with heavy drinking [57]. Liu et al. (2001) also
observed that CYP2E1 polymorphisms may contribute for an increased risk for oral cancer in
the USA [39]. Similar results were also observed in a Brazilian study which observed that the
CYP2E1 genetic alteration increased the risk for oral cancer [15]. However, another Brazilian
study could not find an association between CYP2E1 polymorphism and head and neck
cancer risk [41]. Li et al. (2005) also did not observe a relationship between CYP2E1 genetic
alterations and squamous cell carcinomas of the head and neck, in the USA [38].
4.1.3. CYP3 Gene Family Studies
The human CYP3 gene family is located on chromosome 7q and comprises four genes:
CYP3A3, CYP3A4, CYP3A5 and CYP3A7. Enzymes encoded by these genes are involved in
oxidative metabolism of aflatoxins, N-nitrosamines and others carcinogens [17].
In a study performed in the USA, with patients with squamous cell carcinoma of the head
and neck, it was observed that the expression of CYP3A4 was significant lower in the tumor
tissue than in adjacent normal tissue [2]. To our knowledge, it is the only study that
investigated the role of CYP3 gene family in OSCC development.

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4.2. Phase II Genes Polymorphisms and the Risk of Oral Cancer


4.2.1. Glutathinine-S Transferase Class Studies
GST class includes five isoenzymes (GSTM1 though GSTM5) with high genetic
similarity [18,19,20,24,49,53].
Most of the studies that evaluated GSTs polymorphisms and their association with oral
cancer risk has focused on the GSTM1 gene alterations. A non-functional null-allele
(GSTM1*O) and two functional alleles (GSTM1*A and GSTM1*B) of GSTM1 have been
characterized. It has been hypothesized that the lack of these enzymes decreases the ability to
detoxify carcinogens specifics from the tobacco. Therefore, these genetic alterations have
been associated to an increased risk for different types of cancer, including head and neck
cancer [54].
Sato et al. (2000), in Japan, investigated the association of risk for OSCC and genetic
alteration of GSTM1 and CYP1A1 genes in relation to cigarette-smoking dose. The results
showed that individuals with specific polymorphisms in both GSTM1 and CY1A1 genes have
a high risk of OSCC and combined genotyping of the susceptible GSTM1 and CYP1A1 genes
revealed higher risk than that ascribed to a single susceptible gene, in the lowest cigarettedose level [53].
In a study performed in Japan, it was also observed a significantly higher prevalence of
inactive GSTM1 in patients with oral cancer with alcohol-drinking habit when compared with
non-cancer group with alcohol-drinking habit. GSTM1 deficiency was 2.5 times more
prevalent in oral cancer patients and it was particularly high in patients with cancer of the
tongue, lower gingiva and floor of the mouth [47].
Hahn et al. (2002) investigated the association of GSTM1 polymorphisms and
susceptibility to oral cancer in German patients. The results demonstrated no statistically
difference in the prevalence of that GSTM1 homozygous deletion (null genotype) between
oral cancer patients and the controls [20]. Another study also performed in Germany obtained
similar results [18]. This study showed that GSTM1 null-genotype was almost equally
frequent in patients with oral carcinoma and control group. Nevertheless, the simultaneous
null genotype of GSTM1 and GSTT1 was significantly more common in oral cancer patients
than in controls [18].
Park et al. (2000) investigated the potential role of GSTM polymorphisms in risk for oral
cancer in an African-American population compared to Caucasians [50]. It was suggested
that GSTM1 null and GSTM3 intron 6 polymorphisms play an important role in risk for oral
cancer among African-Americans, showing that the class of GSTs are an important tobacco
carcinogen detoxifying enzymes in this population. On the other hand, no significant
associations were observed between GSTM genotype and oral cancer risk in Caucasians.
In a study performed in the Netherlands, no difference could be found in relation to the
occurrence of polymorphic variants in the GSTM1 between head and neck cancer patients
and controls [49].
The association of GSTM1 null polymorphism and the risk for oral leukoplakia in
individuals with tobacco-smoking habit in Brazilian population was investigated [12]. The
results showed a positive association between the presence of GSTM1 null genotype and the

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development of oral leukoplakia. GSTM1 polymorphisms were also demonstrated to be a risk


factor for developing oral leukoplakia in ethnic Indian betel quid/tobacco chewers [46].
4.2.2. Glutathinine-S Transferase Class Studies
GST (GSTP) codifies functionally different GSTP1 variant proteins. GSTP1 gene has
two different polymorphisms: a single nucleotide polymorphism (SNP) in the coding
sequences at codon 105 A G (Ile105Val), ad another at codon 114 C T (Ala114Val)
[22]. Both polymorphisms result in changes of catalytic activity and have been associated to
increasing the risk of cancer development. GSTP1 metabolizes carcinogens among them
benzo[a]pyrene diolepoxide, which is one of the most important carcinogenic metabolites in
tobacco smoke [22,54].
GSTP1 and also GSTM1 and GSTT1 allelic and genotype distributions were studied
among Brazilians from Rio de Janeiro with oral cancer. The results did not support the
hypothesis of increased risk of GSTP1 G/G, GSTM1 or GSTT1 null genotypes for developing
cancer in oral cavity. Differences in GSTM1 and GSTT1 genotype frequencies among ethnic
groups could be observed. The frequency of GSTM1 null genotype was lower in non-withes
(34-38%) than in whites (46-48%) in case patients and controls. The frequency of GSTT1 null
genotype was higher in non-whites (26%) than in whites (21%) in the control group [22].
4.2.3. Glutathinine-S Transferase Class Studies
In relation to GSTT1, a nonfunctional (GSTT1*2) and a functional (GSTT1*1) allele have
been identified [33]. GSTT1 is involved in a conjugation of several low-molecular-weight
toxins, such as ethylene oxides or methyl-halogenids [54]. The null genotype of GSTT1 has a
decreased capacity in detoxifying carcinogens present in tobacco smoke, leading the
formation of DNA-adducts and DNA damage [11].
Drummond et al. (2005) evaluated the association between GSTT1 polymorphism and
risk for OSCC in a Brazilian population from Minas Gerais state. The prevalence of GSTT1
deficiency (null) was significantly higher in OSSC patients with oral cancer of the floor of
the mouth [11].
The link between polymorphism at the GSTT1, GSTM1, GSTM3 genetic alterations and
susceptibility to oral cancer among Indian tobacco users were also investigated. The dataset
demonstrated that the GSTT1 and GSTM3 genotypes did not influence susceptibility to cancer
of the buccal mucosa, while the GSTM1 null genotype emerged as a significant risk factor
among Indian tobacco chewers as well as bidi and cigarette smokers [5].
In a study performed with Taiwan population, it was investigated the association of
GSTT1 and GSTM1 genotypes with risk for, age of onset, and neck lymph node metastasis in
areca-associated OSCC. The data indicated that the null genotypes for GTM1, GSTT1 or both
isoforms were not associated with the risk of OSCC. GSTT1 null patients presented at a
significant older age than did those with the GSTT1 non-null genotype. The GSTM1 null
genotype was significantly associated with the presence of lymph node metastasis. Patients
with a combined GSTM1/GSTT1 null genotype appeared to have a higher risk for lymph node
metastasis compared to those with the opposite genotypes [39].

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4.3. N-Acetyl Transferases (NATs) Studies


NATs catalyze the acetylation of the aryl- and heterocyclic amine, which are major
carcinogens present in tobacco [8]. NAT1 and NAT2 have been shown to be polymorphic
enzymes that segregate independently into a large number of of polymorphic genotypes that
correspond to rapid and slow acetylator genotype [31]. The distribution of genotypes of this
family was characterized in homozygous wild-type, homozygous mutant or heterozygous and
the translated of nucleotide changes into functional NAT2 alleles, genotypes and inferred
acetylator phenotypes (rapid, intermediate and slow) [8,63].
Few studies have investigated the association between NAT polymorphisms and the risk
for head and neck cancers [41,20,8,31,29]. Authors have suggested that people with
genotypes known to be associated with the slow acetylator phenotype would be less able to
detoxify tobacco smoke metabolites, thus having an increased susceptibility to OSCC [8,31].
Katoh et al (1998) investigated NAT1 and NAT2 genetic polymorphisms in OSCC
patients from Japan. The results suggested that NAT1*10 allele may be a genetic determinant
among Japanese people, but no risk was associated with NAT2 slow acetylator polymorphism
[31].
A case-control study performed by Jourenkova-Mironova et al. (1999), in France,
showed moderated increase in risk for oral cancer associated with the slow acetylator
genotypes [29]. Conversely, Hung et al. (1993), investigating Japanese population, showed
that NAT2 rapid acetylator genotype was not a significant risk factor for oral cancer [26].
Similarly, another case-control study performed in the USA did not observed an association
between slow acetylator phenotypes and OSCC risk [8].

5. CONCLUSION
The conflicting results from the studies which investigated the association of CYPs,
GSTs and NATs polymorphisms and the development of oral cancer may be due to several
confounding factors. One important factor is that there are significant ethnic differences in
frequency distribution of genetic polymorphisms of these genes [41,62]. Other factors include
the sample sizes, the choice of the control group and the lack of data on diet and time or
frequency of exposition to the environmental factors [12,41,54,60].
Although many studies have been performed to identify an association between OSCC
and phase I and II enzymes polymorphisms, little attention has been paid to the genes that are
actually expressed in oral cavity. Greater knowledge of the expression pattern and activity of
these enzymes in different oral sites should be useful to better understand the role these
polymorphisms in oral carcinogenesis.
Although many studies have suggested a correlation between oral cancer and drugmetabolizing enzymes genetic alterations, they cannot elucidate the casual relationships
between xenobiotic exposure, phase I and II polymorphisms and cancer. Only prospective
studies of cohort followed over time and assayed for the development of cancer can
distinguish the susceptible phenotype definitively [21].

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Longitudinal studies investigating the role of these polymorphisms on malignant


transformation of premalignant lesions, such as leukoplakia, would be interesting for
targeting patients with malignant lesion at high risk for future malignant lesions [12].
Better understanding the relationship between phase I and phase II polymorphisms and
OSCC would be important to identify biomarkers of susceptibility to oral cancer. This may
have several implications and include the possibility of developing chemoprevention
programs for highly susceptible patients, allowing early intervention and the implementation
of efficient prevention and treatment strategies [41].

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In: Oral Cancer Research Advances


Editor: Alexios P. Nikolakakos, pp. 263-274

ISBN 978-1-60021-864-4
2007 Nova Science Publishers, Inc.

Chapter 12

TP53 MUTATION, C-MYC AMPLIFICATION AND


SQUAMOUS CELL CARCINOMA RECURRENCE
J. Seoane1, P. Varela-Centelles1,2, M.A. Romero1 , A. De la Cruz3,
F. Barros4, L. Loidi4 and J.L. Lpez Cedrn5
1

Department of Stomatology, School of Medicine and Dentistry, University of Santiago


de Compostela, Santiago de Compostela, Spain;
2
CS Praza Ferrol, XAP Lugo, Galician Health Service, Spain;
3
Pathology Service, Galician Cancer Centre, A Corua Spain;
4
Unit of Molecular Medicine, INGO, Galician Health Service, Santiago de Compostela,
Spain;
5
Head of Oral and Maxillofacial Surgery Service, Hospital Juan Canalejo, Galician
Health Service, A Corua, Spain.

ABSTRACT
Purpose: to investigate TP53 mutation and c-myc amplification as markers for
tumour aggressiveness in terms of tumour recurrence in OSCCs.
Methods and materials: Thirty one incident cases of oral squamous cell carcinomas
were studied for tumour relapse. The variables considered were demographic, clinical,
pathological and genetic.
Results: the mean age of 62.09 years (range 36 to 88). Seventeen patients (54.8%)
were smokers. The tongue was the main affected area (54.8%). No distant metastases
could be identified. Most patients were at early stages of the disease with moderately
differentiated tumours and of grade I in Anneroths malignancy scale. The oncogene
study showed abnormalities in both TP53 (6/31; 19.2%) and c-myc (4/31; 12.9%), that
distributed as follows: TP53+/c-myc+ (n=1; 3.2 %); TP53+/c-myc- (n=5; 16.1%); TP53/c-myc+ (n=3; 9.7 %); TP53-/c-myc- (n=21; 67.7%). TP53 mutations were significantly
more frequent in advanced stages. Statistically significant differences in node status were
identified in terms of oncogene alterations. Multivariate Cox regression analysis
recognized prognostic value for recurrence for alterations of TP53 and c-myc (p<0.05).

J. Seoane, P. Varela-Centelles, M.A. Romero et al.

264

Conclusions: TP53 mutation is related to advanced stage of oral cancer and suggests
the usefulness of analysis of TP53 mutations and c-myc amplifications in order to
identify those OSCCs more prone to relapse.

INTRODUCTION
Oral squamous cell carcinoma (OSCCs) behaviour in a patient is extremely variable, and
its prediction is of great importance in order to determine the prognosis and select the
appropriate therapy [1,2]. In current oncological practice, the treatment of OSCCs is based on
the histological grade and TNM stage but these parameters have proved a limited predictive
capacity in terms of therapeutic outcome. This is why the scientific community acknowledges
the need for exploring additional prognostic markers that help to predict the biological
behaviour of these tumours.
The myc family of oncogenes has been linked with neoplasia, particularly the c-myc gene
that codes for a nuclear protein that is involved in cell growth, differentiation and
programmed cell death [3]. However, the most frequently documented genetic change that
appears in human cancer is that occurring on the short arm of chromosome 17 (17p) in the
region that contains the TP53 gene [4]. In squamous cell carcinoma of the head and neck
(SCCHN), 40-50% of the tumours studied have shown a mutation in this gene [5].
The TP53 mutations result either in no expression of the wild-type p53 or in overexpression of the mutant p53 protein [6]. The TP53 mutation causes both a loss of its tumour
suppressor function and a gain of its oncogenic function by means of p53 controlled genes
[7,8]. Most of investigations have generally analysed head and neck cancer not oral
cancer. Moreover, there are some controversial data on the prognostic value of the TP53
mutation in patient which OSCCs [9], which was associated with a negative prognosis by
some authors [10-12], but others found no correlation with poor outcome [13,14].
The involvement of p53 and its association with other genetic factors is poorly
understood in oral squamous cell carcinoma [15], especially the association of c-myc with
p53 which has been less explored, however important [15,16].
The aim of this study was to investigate TP53 mutation and c-myc amplification as
markers for tumour aggressiveness in terms of recurrence in oral squamous cell carcinomas.

METHODS AND MATERIALS


Study Design
Thirty one incident cases of oral squamous cell carcinomas diagnosed at the Galicia
Cancer Centre (A Corua, Spain) in the 1995-1999 period entered the study. This centre is
the reference hospital for radiotherapy for all five general hospitals in the North of Galicia
(Spain). The patients included represented stages I-IV, none had distant metastasis and were
all given intended curative treatment. Based on the stage of the disease and the primary site,

TP53 Mutation, c-myc Amplification and Oral Cancer Recurrence

265

all 31 patients underwent radiotherapy alone (n=12; 38.7%) or combined with surgery or/and
chemotherapy (n=19; 61.3%).
Local recurrence was considered when tumoral growth reappeared after complete
disappearance of tumours confirmed with histological biopsies after treatment.
A survival study was designed aimed at determining the influence of TP53 mutations and
c-myc amplifications on tumour relapse.
The variables considered were demographic (age and sex), clinical (location of the
lesion, clinical presentation, tumour size, lymph node status, distant metastases and tumour
stage), treatment (date of treatment), evolution (recurrence, date of recurrence, exitus, date of
exitus and cause of exitus), pathological (Anneroth score of malignancy and degree of
differentiation) and oncogene status (TP53 mutations and c-myc amplifications).

Analysis for TP53 Mutations


Ten-micrometer sections were obtained, and appropriate areas from tumour were selected
by light microscopy and microdissected for DNA extraction. Necrotic areas were avoided.
Digestion with proteinase K followed by boiling was used as the extracting DNA method.
Polymerase chain reaction amplification of p53 exons 4 to 8 was performed: for a reaction of
25 L, 2L of extracted DNA was added to a mix of 10 mmol/L HCl (pH 8.3), 50 mmol/L
KCl, 1.5 mmol/L MgCl2, 200 mol/L of each dNTP (deoxynucleotide triphosphate), 1L of
each primer (15mM concentration) and 1.25 U of Taq-polymerase (Promega). Primers are
described in table 1 and amplification conditions were: 40 cycles (95C 20 sec.; 60C (55C
for exon 5) 30 sec.; 72C 60 sec). A screening of mutations was performed through SSCP
analysis using an automatic electrophoresis system (Phast System; Amerham, Biotech,
Sweden) followed by a silver staining method for the detection of amplified products. The
single-stranded PCR products were separated on PhastGel homogeneous polyacrilamide gels
(20%) with native buffer strips (0.88 M L-alanine, 0.25 M Tris, pH 8.8) (Amersham
Biothech). The electrophoretic runs were performed at 15C for 200 to 400 Vh according to
the length of the fragments. A sample of normal tissue for each exon studied was used as a
negative control.
All fragments with anomalous migration as detected by SSCP were sequenced in an
ALFexpress sequencer (Amersham Biothech) following standard protocols.

Analysis for Amplification of c-myc Gene


The amplification of the oncogene c-myc was analysed using differential PCR, where the
target gene and a reference gene (tissue plasminogen activator, t-PA) ware co-amplified by
PCR in the same reaction vessel. The reference gene t-PA is linked to c-myc gene, avoiding
misinterpretation of chromosomal. Normal DNA was titrated with previously characterized
myc-amplified DNA (cell line HL60) to verify the relationship between myc and TPA
signals.

J. Seoane, P. Varela-Centelles, M.A. Romero et al.

266

2L of extracted DNA was added to a mix of 10 mmol/L HCl (pH 8.3), 50 mmol/L KCl,
1.5 mmol/L MgCl2, 200 mol/L of each dNTP (deoxynucleotide triphosphate), 1L of each
primer (for c-myc and t-PA genes, all 15mM concentration) and 1.25 U of Taq-polymerase
(Promega), in 50 l total volume. Primers (one primer for each pair was fluorescently
labelled with Cy5) are described in table 1 and amplification conditions were: 35 cycles
(95C 30 sec.; 68C 30 sec.; 72C 30 sec.).
Table 1. Sequence of primers used in the analysis
Gene

Exon

Sequence

TP53

5 ATCTACAGTCCCCCTTGCC 3
5 GCAACTGACCGTGCAAGTCA 3
5 TTCCTCTTCCTACAGTACTC 3
5 GCAAATTTCCTTCCACTCG 3
5 CCATGAGCGCTGCTCAGAT 3
5 AGTTGCAAACCAGACCTCAG 3
5 GTGTTATCTCCTCGGTTGGC 3
5 CAAGTGGCTCCTGACCTGGA 3
5 CTGCCTCTTGCTTCTCTTTT 3
5 GAGGCAAGGAAAGGTGATAA 3
5 GTTTCATCGTGTTGGCCAGGATGGT 3
5 CCAAAGAGCCACTCTAAGCCTGGT 3
5 AGGCCCGTGTGTAAACATAGGTG 3

5
6
7
8
c-myc
t-PA

1L of the PCR product was mixed with 4L formamide solution, denatured, and
electrophoresed in a denaturing polyacrylamide gel (5.7% acrylamide, 0.3% bisacrylamide, 7
M urea) using an ALFexpress sequencer. The running conditions were 1500 V and 25 W at a
constant temperature of 55C for 130 minutes with a running buffer of 0.6 X TBE. The data
were processed using the AlleleLinnks software (Amersham Biotech). Three values were
obtained for each PCR product: fragment size, peak height and peak area. The results
expressed as a ratio of c-myc to t-PA peak areas were compared to the controls to provide a
measure fo the number of c-myc haplotypes.

Statistical Analysis
A descriptive study was performed by means of Students t and chi square tests to
analyse associations between the data. The probability of treatment failure was calculated for
the endpoint of relapse-free survival and tumour relapse by the Kaplan-Meier analysis with
the log rank test for curves comparison. All time estimates were done using the date of
primary treatment as initial value.
A multivariate Cox proportional hazards analysis was also performed using the forward
stepwise conditional method for including parameters in the model.

TP53 Mutation, c-myc Amplification and Oral Cancer Recurrence

267

The significance level chosen for all tests was 5%. Analysis was performed on a SPSS
8.0 statistical package. The mean observation time was 34.54 months. No patients were lost
for follow-up.
Table 2. Relationships between clinico-pathological variables and oncogene status. Chi
square test

Node status

T category

Clinical stage

Degree of
differentiation
Degree of
malignancy

N0
N1
N2
p-value
T1-T2
T3-T4
p-value
I-II
III-IV
p-value
Good
Moderate/Poor
p-value
I
II-III
p-value

Cases
21
3
7
22
9
18
13
10
21
17
14

p53+
2 (9.5%)
2 (66.7%)
2 (28.6%)
0.05
3 (13.6%)
3 (33.3%)
0.21
1 (5.6%)
5 (38.5%)
0.0034
2 (20%)
4 (19%)
0.65
3 (17.6%)
3 (21.4%)
0.57

c-myc+
2 (9.5%)
1 (33.3%)
1 (14.3%)
0.512
3 (13.6%)
1 (11.1%)
0.67
2 (11.1%)
2 (15.4%)
0.56
2 (20%)
2 (9.5%)
0.38
2 (11.8%)
2 (14.3%)
0.62

p53-/c-myc4 (19%)
2 (66.7%)
3 (42.9%)
0.155
17 (77.3%)
5 (55.6%)
0.21
15 (83.3%)
7 (53.8%)
0.08
7 (70%)
15 (71.4%)
0.62
12 (70.6%)
10 (71.4%)
0.63

RESULTS
Descriptive Analysis
A total of 31 patients entered the study (25 male, 6 female) with a mean age of 62.09
(range 36 to 88). Seventeen patients (54.8%) were smokers. Most lesions (61.39%) were
ulcerated-type, followed by exophytic (25.8%) and mixed-type (12.9%). The tongue was the
main affected area (54.8%). No distant metastases could be identified.
Most patients were at early stages of the disease, mainly at stage II (38.7%, n=12),
followed by stage IV (25.8%, n=8) and stages I and III (16.1%, n=5), and stage 0 (3.2%,
n=1). The sample was composed chiefly of small tumours (T0 (3.2%, n=1); T1 (22.6%, n=7);
T2 (45.2%, n=14); T3 (22.6%, n=7) and T4 (6.5%, n=2)) with no lymph node affectation (N0
(67.7%, n=21); N1 (9.7%, n=3); N2a (3.2%, n=1); N2b (12.9%, n=4) and N2c (6.5%, n=2)).
The tumours studied were predominantly moderately differentiated (51.5%, n=16), with
32.3% (n=10) well differentiated and 16.1% (n=5) poorly differentiated. According to
Anneroths malignancy score, 54.8% (n=17) were grade I; 38.7% (n=12) were grade II and
6.5% (n=2) were grade III.

268

J. Seoane, P. Varela-Centelles, M.A. Romero et al.

The oncogene study showed abnormalities in both TP53 ( 6/31; 19.2%) and c-myc (4/31;
12.9%), that distributed as follows: TP53+/c-myc+ (n= 1; 3.2 %); TP53+/c-myc- (n= 5;
16.1%); TP53-/c-myc+ (n= 3; 9.7 %); TP53-/c-myc- (n=21; 67.7%). In total two TP53
mutations were found in exon 5; two in exon 6 and two in exon 7.
We found no significant relation between the mutational status of TP53 and age, gender,
primary tumour site and Anneroths malignancy grade. The TP53 mutations were
significantly more frequent in advanced stages than in the early ones. Statistically significant
differences in node status were identified when cases were distributed according to TNM
classification criteria, (table 2).

Survival Analysis
Kaplan Meier analysis showed that oncogene alterations were not translated into
significantly different recurrence rates (table 3) although the survival plots show quite
independent curves (figures 1-3). Multivariate Cox regression analysis recognized prognostic
value for recurrence when alterations of TP53 and c-myc were considered (Exp = 10.74;
p=0.0223). Oral cancers without TP53 and c-myc alterations have showed longer relapse-free
survival periods than those cancers with these modifications, although this difference did not
reach signification.
Table 3.Relationship among TP53 mutations, c-myc amplification and tumour relapse
(Kaplan Meier)
Cases
TP53+
c-myc+
TP53- & c-myc-

6
4
21

Median relapse-free
time (months)
14.46
8.33
53.48

3 year relapsefree
41.67%
50%
60.19%

Log-rank test
0.35
0.689
0.67

DISCUSSION
TNM classification of oral carcinomas is a classical method for estimating a prognosis
and establishing treatment strategies. However, it does not take into account the biological
properties of a single tumour that would explain the number of stage I and II tumours with a
rapid lethal course [17].
As age, race and gender influence the stage of disease at presentation, and the choice of
therapy, survival differentials based on these factors may be illusionary and may represent
secondary associations with clinical variables. Grading of squamous cell carcinoma is
unreliable as a predictor of behaviour as a solitary criterion. Major reasons quoted for its
unreliability include the knowledge that the differentiation may vary in different sites of the
tumour, the biopsy fragment may not be representative, and observer interpretation of the
findings may vary [18].

TP53 Mutation, c-myc Amplification and Oral Cancer Recurrence


1,1

1,0

,9

,8

Cum Recurrence

,7

p53
,6

abnormal

,5
normal
,4
0

20

40

60

80

months
Figure 1. Recurrence plot for TP53 mutation.

1,1
1,0

,9
,8

,7

Cum Recurrence

,6

c-myc
abnormal

,5

,4

normal

,3
0

20

40

months
Figure 2. Recurrence plot for c-myc amplification.

60

80

269

J. Seoane, P. Varela-Centelles, M.A. Romero et al.

270
1,2

1,0

,8

p53 & c-myc combined

Cum Recurrence

,6

Non concordant
p53 & c-myc
,4
Normal p53 & c-myc
,2
0

20

40

60

80

months
Figure 3. Recurrence plot for TP53 and c-myc.

Despite these shortcomings several compound prognostic models have been proposed
including different tumour features according to their weight on the sample analysed. Ever
since the scientific community acquired the ability to explore carcinogenesis at a molecular
level, genetic variables were considered in the survival analyses and supposed a qualitative
change in this area.
One of the most commonly studied genes in oncongenesis is the TP53 gene that is
involved in the first transformations of a cell to the cancer cell stage [19]. The reported
frequency of TP53 mutation in OSCCs varies widely, from 21% [20] to 64% [21]. This may
be a reflection of the techniques used to detect mutations, the regions of the TP53 subjected
to analysis and differences in mutation rates between anatomical sites [22,23]. Moreover, the
mutational spectrum has been shown to vary between countries and races [5]. Some studies
have reported an association between smoking and alcohol use and the frequency of TP53
mutations [5,24]. In this sense, there are reports describing frequencies of TP53 mutations of
around 14-17% in OSCCs of non-smoker patients [8,25]. The low frequency of TP53
mutations identified in this study might well be explained by the high proportion of nonsmoker patients in this series.
As it happens in this report, many studies analysing gene sequence have focused on
exons 5-8 of TP53, since the majority of mutations occur there [22]. However, it is possible
that additional mutations might have been detected by direct sequencing of the entire gene
(exons 2-12).
There have been conflicting conclusions of the stage in oral carcinogenesis at which
TP53 mutation occurs. It has been reported that the incidence of TP53 mutations increased

TP53 Mutation, c-myc Amplification and Oral Cancer Recurrence

271

from early lesions (dysplasias) to invasive OSCC [26]. However, other authors support a late
role for p53 in the sequential expression of the invasive phenotype [27,28], because p53positive lymph node metastases may develop from p53-negative primary tumours [29].
The finding that clinical stage III-IV cancers harboured more TP53 mutations than
clinical stage I-II cancers supports the theory that it is the accumulation of mutations and not
necessarily its order what determines cancer progression [30]. Moreover, TP53 mutations of
advanced diseases might be an early event which contributes to a more aggressive behaviour
of a given cancer compared with more innocent tumours without genetic lesions [31].
The correlation between TP53 mutation and poor prognosis has been identified [10-12]
and the contradictory results reported [13,14] seem to be due to the different anatomical
locations, tumour stages and treatments considered in the analyses. However, there is now an
emerging consensus on that TP53 mutation is predictive of clinical outcome for at least some
treatment regimens [22].
A high prevalence of TP53 mutations (68%) has been identified in recurrent squamous
cell carcinomas [32,33]. In addition, several studies have proved that mutations in TP53 are
strongly associated with recurrence in those patients receiving radiotherapy as their main
treatment option [10,11,32,34]. However, this association could not be confirmed when the
patients were treated surgically and received postoperative radiotherapy [35]. The survival
analysis for recurrence of our patients, treated mainly with radiotherapy, show an
independent behaviour of TP53 mutation positive and TP53 mutation negative tumours
(figure 1), which may point at the direction described by other research groups [36,37].
The c-myc gene codes for a nuclear protein p62 which is involved in cell growth,
differentiation and programmed cell death [38], although the proportion of cases in OSCC
where the gene is amplified is around 15% [39], similar to the one in our series. It has been
hypothesized that the over-expression of c-myc could be due to a lack of wild-type p53, and
could be an attempt by the cell to restore its overexpression [40]. The overexpression of both
genes has been suggested to play a vital role in the progression of cancer and apoptosis
inhibition in cancer treatment resistance [41].
Our results show a scarce presence of combined alterations of TP53 / c-myc in OSCC and
disclose a higher recurrence rate of OSCCs that harbour TP53 mutations or c-myc
amplifications than those OSCCs whose TP53 and c-myc genes are normal.
The ideal marker in tumour prognosis is one that when present indicates tumour
development and when absent excludes this possibility. However, it is unlikely that such a
marker exists. It is therefore more likely that TP53 could be one factor in a panel of factors
with importance for outcome, rather than the single prognostic factor [5].

CONCLUSION
This study shows that TP53 mutation is related to advanced stage of oral cancer and
suggests the usefulness of combined analysis of TP53 mutations and c-myc amplifications in
order to identify those OSCCs more prone to relapse.

272

J. Seoane, P. Varela-Centelles, M.A. Romero et al.

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FX. TP53 DNA contact mutations are selectively associated with allelic loss and have a
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[34] Alsner, J; Sorensen, SB; Overgaard, J. TP53 mutation related to poor prognosis after
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[35] Sittel, C; Ruiz, S; Volling, P; Kvasnicka, HM; Jungehulsing, M; Eckel, HE. Prognostic
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[36] Koch, WN; Brennan, JA; Zahnrak, M. p53 mutation and locorregional treatment failure
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FX. TP53 DNA contact mutations are selectively associated with allelic loss and have a
strong clinical impact in head and neck cancer. Oncogene, 1998 16, 1671-1679.
[38] Baral, RN; Patnaik, S; Das, BR. Co-overexpression of p-53 and c-myc proteins linked
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Oral Sci, 1998 106, 907-913.
[39] Grabenbauer, GG; Mhlfriedel, C, Rdel, F. Squamous cell carcinoma of the
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In: Oral Cancer Research Advances


Editor: Alexios P. Nikolakakos, pp. 275-283

ISBN 978-1-60021-864-4
2007 Nova Science Publishers, Inc.

Chapter 13

RECENT ADVANCES AND FUTURE PROSPECTS


UPON THE ARTERIAL FRAMEWORK OF THE
FACE AND RELATED APPLICATIONS FOR FACIAL
FLAPS
Egidio Riggio
Plastic Surgery Division, Fondazione IRCCS Istituto Nazionale dei Tumori,
Milano, Italy.

ABSTRACT
Coverage of facial defects is frequently challenging. Despite the numerous flaps
described, the search for additional flaps with good color match and minimal donor-site
morbidity continues and attempts to find valid options to free flaps perhaps overused in
the last two decades, in particular for soft tissue replacement of the moderate-to-large
perioral resections. The reconstructive research runs through the study of functional
topographic or regional anatomy with all the scientific and clinical implications. The
article reviews the last reports that are basically focused upon the arterial supply derived
from neglected branches of the superficial temporal artery (transverse facial artery,
zygomatic-orbital artery, and middle temporal artery) or the terminal branches of the
frontal terminal branch, from the variants of the terminal facial artery and a definite
collateral named cutaneous zygomatic branch, or from the submental artery. The up-todate research embraces the study of the cutaneous perforators of the face. Relevant
anterograde or reverse flaps, axial or perforator flaps, and monolayered or multilayered
composite flaps are discussed as current, original or still imaginative chances. Moreover,
for the realization of totally new flaps in the field of compound facial reconstruction,

Correspondence concerning this article should be addressed to: Egidio Riggio Plastic Surgery Division,
Fondazione IRCCS Istituto Nazionale dei Tumori, Milano via Venezian 1, 20137 Milano, Italy. Email:
egidio.riggio@istitutotumori.mi.it.

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Egidio Riggio
clinical research efforts should tend to merge with the future perspective of bone and
soft-tissue engineering research.

INTRODUCTION
The face is supplied by branches of the facial, superficial temporal, and infraorbital
arteries and terminal branches of the ophthalmic artery in the forehead. The superficial
temporal artery, its branches as transverse facial, zygomatico-orbital, middle temporal
arteries, and the terminal branches supply the preauricolar, jugal superior, malar, and lateral
forehead regions and, part of buccal, orbital, infraorbital and temporal regions. The facial
artery and its branches, including superior and inferior labial, submental supply lips, mental
and jugal inferior regions and part of the buccal, infraorbital, orbital, and nasal regions. The
infraorbital artery provides the homonymous region together with the zygomaticofacial artery
(from the maxillary artery) and the angular artery. The forehead supraorbital regions are
nourished by the supratroclear and supraorbital (frontal) arteries originating from the
ophthalmic.
Facial flaps are generally supported by axial vessels originating multiple perforators
directed towards the skin. Research for axial island flaps based on unknown/neglected
arteries or uncommon anatomical variants as well as for the application of a single-perforator
flap is developing. Based on facial and superficial temporal vascular frameworks, new flaps
with better color/texture match and minor donor-site damage can replace distant flaps for
reconstruction of facial defects from moderate to large.
The skin is supported by two vascular networks in the body but, here, the subdermal
layer permits the elevation of a viable random flap (SVNF, subdermal vascular network flap)
without including the deeper subcutaneous layer. The richly dense arteriolar network has a
honeycombe-like architecture of anastomoses that distribute the flow in certain directions on
different zones. Xiong et al. [1] propose to plan the flap major axis according to the flow
direction of the main local vessels without including them in the flap. That means that SVNF
should be drawn as transverse in malar and superior jugal areas (based on the transverse
facial artery direction) and oblique in buccal and lower jugal areas (from anterior-superior to
posterior-inferior, based on the facial artery axis). A lateral genicervical island flap is
described, drawn vertically, slightely obliquely along the mandibular arch, from the lower
parotid-masseteric area to the neck. The flap pedicle is preauricolar, 1-1.5 cm wide and 3-4
cm long. The flap surface ranges from 6x6 to 9x8 cm while the thickness is 2-4 mm. Surgical
anatomy is poorly described but the scientific implications are worthwhile for the preparation
of well-planned SVNF on definite perforators supplying large skin areas, with thin pedicle
and large rotation arc.

FACIAL ARTERY
The classic anatomy is insufficient. Knowledge of the variations in terminal course and
in number/type of collateral branches, also in the same individual, is essential for the correct

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277

choice of any loco-regional flap used for facial and buccal reconstruction. As a first approach
to reconstruction, it should always be important to preserve the main axial artery's continuity,
and to use one of its branches or one of its perforators as well. Such a concernment embraces
all arteries besides the facial.
Five variations in terminal course are described by Niranjan [2]: the classic ending as
angular artery at the medial canthus; the labialis superior at the upper lip; the alar at the alar
nasi; the nasal at the dorsum nasi; the long course running vertically towards the infraorbital
foramen and transversally towards the nose, eventually ending as angular. The distribution
pattern has been recently classified in five types and more subtypes by the study performed
on 284 hemifaces by Loukas et al. [3]. Knowing the subtypes of the superior labial artery can
properly allow the selection of the indication for the Abb flap. Furthermore, it is noticeable
that examination of subtype C-1 and, mostly, C-2, where the facial artery terminates solely as
superior labial artery, branching off vertically towards the columella bifurcating in bilateral
alar nasal vessels could suggest the creation of an axial flap including subcolumellar zone
and bilateral nasojugal zone which represent a wide surface in elderly patients. The incidence
of these variations occurs in less than 8% of population.
A small branch, cutaneous zygomatic branch, is presented by Gardetto et al. [4,5] for a
new axial island zygomatic flap. This artery has been constantly found in all 62 hemifaces
examined. It runs upwards over the buccinator muscle bifurcating at the major zygomatic
muscle's border. One branch ascends laterally and ends in the subcutaneous tissue. The
second continues through the major zygomatic muscle and penetrates the overlying
subcutaneous tissue. A critical point is the venous blood drainage, since the tributaries of the
facial vein do not run precisely alongside the artery in the cheek region. Unfortunately, the
collateral vein has to be cut to make the elevation possible. However, in clinical application,
major venous congestion has not been observed, likely due to drainage by small veins present
alongside the artery. Another critical point is the relationship with the zygomatic and buccal
branches of the facial nerve. Near the inferolateral border of the zygomatic muscle, buccal
branches could cross the facial artery and are to be preserved. The nasolabial island can be
extended to the infraorbital skin due to arteriolar anastomoses.
Hofer et al. [6] describe a new nasolabial island flap based on one facial artery perforator
meanly large at 1.2 mm. A perioral defect from 1.5x1.5 to 2.5x5 cm can be repaired by
rotation (120-180 degrees) of a skin island flap with donor-site direct closure. Two
suggestions are essential. The first is that the distribution of facial perforators is equally
frequent in all facial artery variations. They range from 3 to 9 units for the segment mandiblenasal alar rim and most of the perforators are 4-8 cm from the mandibular angle and,
therefore, located below the zygomatic and risorius muscles and lateral to the depressor
anguli oris muscle. The second is that the flap survival is reliable even if the pivot point
(perforators site) is peripheral but preserve a small cuff of subcutaneous tissue around the
perforator in order to facilitate the venous drainage, to prevent trouble, and to avoid some
vessel kinking. A technique disadvantage involves the nearness of the facial artery that
excludes the Doppler identification of the perforator and makes necessary its exposure
through the wound edges of contemporary tumor resection.
Kawai et al. [7] find new suggestions for perioral flaps starting from the architecture of
the lower lip arteries. The supply is mostly derived by three branches of the facial artery:

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inferior labial artery, horizontal labiomental artery, and vertical labiomental artery. The first
that is classically described as arising near the buccal angle has been found to have three
different origins. The most frequent origin is not that most known, called by the authors as
type B, but that branching off the facial artery near the mandibular border (type A). It can
uncommonly diverge from the superior labial artery near the commissure (type C). On the
other hand, Pinar et al. [8] found type B in 60% of 50 cadaver specimens and the inferior
labial artery as absent in 10%. The second (horizontal labiomental artery) arises near the
lower mandibular border proximally to the origin of the inferior labial artery and runs on the
mucosal side between depressor labii inferioris and orbicularis oris muscles. The third, i.e.
the vertical labiomental artery, diverges from the submental artery near the mentum running
deep to the depressor labii muscle and bifurcating into superficial and deep branches close to
the orbicularis oris muscle edge. Two records are relevant: a) the horizontal labiomental
artery caliber can be larger than the inferior labial artery, b) flow and caliber of the horizontal
and vertical labiomental arteries appear to be complementary to each others. Small vertical
branches of these arteries form a plexus in both submucosal and subcutaneous layers
surrounding the orbicularis muscle. It becomes possible to harvest mucosal/skin vertical flaps
without muscle interruption as well as planning new flaps like a submental flap with vertical
mental extension or any reverse-flow vertical labiomental flap.
The facial artery gives off the submental artery at 3-15 mm below the mandibular arch
excepting in one of 56 dissections reported by Martin et al. [9] where the origin is from the
carotid artery. It supports one of the current flaps that better matches the requisites for
midface reconstruction in one stage. It may be used for coverage of perioral, intraoral,
periorbital, and other defects and leave an acceptable donor-site scar. The ipsilateral anterior
belly of the digastric muscle must be included in the flap especially when extended to the
upper half of the neck, because the artery run mostly deep to the anterior belly and give skin
perforators. It also improves the venous drainage [10,11]. The only problem is given by the
inclusion of submental lymph nodes into the flap. Contemporary use in case of cancers
potentially metastasizing should be avoided or delayed at a second stage after minimal
follow-up. The reverse-flow flap, its free variant, and the perforator-based reverse flap, even
if has introduced a new perspective for facial flap, are to be used when the normal flow is
unavailable, taking care of possible facial nerve injury and venous congestion. The distal
pedicle crosses the overlying marginal nerve and, more cranially, the facial vein passes
beneath the midfacial nerve branches. It is challenging the possibility of harvesting a double
flap including a mucosal-buccinator muscle flap over the facial artery [12,13].

SUPERFICIAL TEMPORAL ARTERY


Studies concerning terminal course and number/type of collateral branches are carrying
in new appliances. The artery represents the vascular axis of forehead, temporoparietal, and
parieto-occipital flap but, even if the predictability about caliber, course and terminal
subdivision is well known, Doppler examination allows to recognize those anatomical
variants capable of compromising the flap viability (10-20%) in time for changing surgical
plan even during dissection [14].

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279

When the artery becomes superficial at the tragus, it gives the following branches from
proximal to distal: transverse facial artery, anterior auricular artery, zygomatic-orbital artery,
superior auricular artery, middle temporal artery, terminal frontal and parietal arteries. All of
them can support a flap.
The transverse facial artery runs below and parallel to the zygomatic arch deep to
SMAS. It is short but large and constant. Perforators go perpendicularly through SMAS
toward the skin with almost no branching until they supply the subdermal plexus. The
dominant perforator consistently branches off between parotid gland and masseter muscle.
Whetzel and Mathes [15] have demonstrated how is statistically possible to predict the
location of the perforator within a few centimeters. This statement could promote a skin flap
drawn in the aesthetic unit of cheek and based on the subdermal vascular network, even if the
dissection of perforators is generally difficult in the face because tissues are richly
vascularized and perforators are scarcely detectable by Doppler ultrasound probe.
The zygomatic-orbital artery runs slightly upwards toward the lateral supra-orbital
region. It consists of three segments: proximal, nearly horizontal; central, vertical or oblique;
distal from eyebrow to forehead. It is inconsistent in terms of length, course and pertinent
vascular territory. This does not mean that this artery may not be used for reconstructive
purposes, it can be long enough in 35% of population. The Author, Riggio et al., [16]
illustrates a topographic diagram generated by statistical analysis of 50 Doppler
investigations, which provides a certain predictability to the anatomy of the zygomaticoorbital artery, and has successfully harvested an innovative forehead flap based on this artery,
with a lower pivot point, for total repair of the lower third of the face. Whetzel and Mathes
[15] have formerly applied biostatistics upon their study of facial perforators. The Author
infers that the zygomatic-orbital blood flow is inversely proportional to the frontal branch
flow, i.e., perfusion of the lateral forehead is kept up by both arteries in complementary way.
It is suggestive for the existence of some regulation among their flow, caliber, and height.
According to Marano [17], the frontal branch is absent in 8% of cases and the diameter is less
than 1-mm in another 8%. This means that 16% of standard foreheads flaps are potentially
jeopardized, the zygomatic-orbital artery flap may solve the problem. This point of view is
challenging and exciting for surgeons because it introduces a dynamic view of the arterial
anatomy of facial flaps in relationship with physiological and pathological variations of the
arterial circulation without taking into account the venous system [18].
Finding more options for each flap becomes possible and, moreover, consents to protect
the major artery or contiguous arteries useful for the remaining circulation of the donor site
and to preserve sources of alternative flaps as well.
The superior auricular artery runs above the root of the ear and anastomoses with the
posterior auricular artery. The retroauricular island flap is supplied by a reverse blood flow
from the frontal branch [19] or from the parietal branch (based on the contralateral
anastomoses) [20]. It can repair medium midfacial defects.
The terminal frontal artery gives off small branches, usually three: anterofrontal,
centrofrontal, posterofrontal. They can be used for distinct islands flaps in facial
reconstruction [21,22].
Anterior and posterior deep temporal arteries, branches of the internal maxillary artery,
nourish the temporalis muscle. Contribution of either the fascial vascular network or the

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muscular branch of the middle temporal artery originating from the superficial temporal
artery is often misjudged. The reverse temporalis muscle flap and related vascular anatomy
have been applied [23,24,25]. Survival is possible by a vascular plexus zone located within
1.8 cm below the superior temporal line, large 1.3 cm, and perfused by the superficial
temporal artery through the vascular network between superficial and deep temporal fascia in
the anterior two thirds and by the muscular branch of the middle temporal artery in the
posterior one third, as shown by Chen et al. [26]. For this reason the retrograde-flow flap
should include that muscular branch as well as the fascial branch that supplies the deep
temporal fascia and gives off small branches to the lower part of muscle [27]. The reverse
temporalis flap should have more applications for midfacial reconstruction, including
coverage of orbital bone grafts especially when combined to flaps based on the terminal
parietal branch. A multi-arterial axial flap of the superficial temporal artery can even
include: a) temporal fascia with/without parietal bone based on the parietal branch, b)
posterior part of the temporalis muscle based on the middle temporal artery, c) frontal skin
based on the frontal branch or zygomatic-orbital artery, d) retroauricular skin with/without
ear cartilage based on the superior auricular artery. The territory extends to superficial
temporal artery and posterior auricular angiosomes [28]. Dissection can be laborious.
Nevertheless, it is suggestive thinking about some utilization for three-dimensional composite
reconstruction of the face. The donor-site defect could be acceptable. The drainage problem
is solved by harvesting a main vein collector of the frontotemporal region.

CONCLUSION
Through research programs in different scientific fields, combination of surgical
techniques, vascular anatomy, materials, cells, growth factors, and new methodologies will be
capable of improving clinical solutions for head and neck surgery.
Experimental basic science is implementing new chances for wound healing and survival
of ischemic skin flaps. Use of growth factors, basic fibroblast growth factor (bFGF) and
vascular endothelial growth factor (VEGF), have been well-documented throughout the last
decade [29]. Gene therapy in flap survival embraces various methods to transplant plasmids
or viruses capable of coding and producing growth factors in ischemic tissue through scaffold
application over the recipient bed and in-flap or intravenous administration. The bFGF role
and interaction with hyperbaric oxygen therapy have been studied to decrease the radiationinduced damage to microcirculation in animal models [30]. Recently, the treatment with
shock waves (SW), which consists of transient pressure fluctuations with three-dimensional
spreading and apparently releasing angiogenic factors, has been compared to the gene therapy
with VEGF [31].
Tissue engineering is rapidly evolving as a therapy option. The growth of human bone
[32,33] and cartilage cells [34], pre-adipocytes [35], conjunctiva and oral mucosa equivalents
[36,37,38], and adult-derived stem cells [39] have been investigated. For cell-based bone
tissue engineering, biophysical stimulation of the host cell population around the bone defect
and autologous cell implantation are accepted for clinical proceedings. Warnke et al. [40]
report a successful reconstruction of a mandibular defect by growth of a custom bone

Recent Advances and Future Prospects Upon the Arterial Framework

281

transplant inside the latissimus dorsi muscle. Complex therapeutical options (extracorporeal
tissue engineering, stem cell use, and genetic engineering) have been tested in preclinical
investigations but have not reached clinical use until now.
Clinical investigation for new facial flaps is focused on neglected branches, anastomosis
networks, and perforators maps. Plastic surgeon must become a fine vascular anatomist. The
zygomatic artery flap by Gardetto [5], the nasolabial island perforator flap by Hofer [6], the
forehead zygomatic-orbital artery-based island flap by Riggio [16], and the ideas for flap
based on the transverse facial artery perforator, for extended submental flap or for composite
frontotemporal flaps are some of the inputs coming from recent scientific literature. In the
meanwhile, especially in case of reconstruction of the lower face after limphoadenectomy
and radiotherapy, valid options are represented by either pedicled flaps such as deltopectoral,
supraclavicular, pectoralis major, and trapezius or free flaps such as latissimus dorsi,
anterolateral thigh flap, diep, lateral brachial flap, fibula, and radial forearm.

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INDEX

A
AAV, 98
abdomen, 12, 157, 168
AC, 37, 45, 81, 89, 151, 175, 180, 225, 272, 282
access, xiv, 58, 165, 169, 198, 229, 230, 236, 237,
239, 240, 241
accuracy, xiv, 108, 134, 138, 141, 144, 145, 212,
221, 222, 236
acidi, ix, 1, 2, 9, 10, 88, 103, 105, 118, 134, 135,
149, 239, 283
acidosis, 18
ACL, 82
actinic keratosis, 212
activation, 23, 28, 29, 43, 46, 62, 66, 109, 249, 250,
251, 252, 253, 254, 260
activity level, 14, 22
acute lymphoblastic leukemia, 117
acute myeloid leukemia, 104
adaptation, 83
ADC, 133, 134
adenocarcinoma, ix, 12, 37, 47, 48, 91, 160, 170,
214, 217, 218, 222, 225, 226, 232
adenocarcinoma of the esophagus, 12
adenocarcinomas, 48, 73, 93, 233
adenoma, xiv, 146, 211, 213, 214, 216, 217, 218,
222, 225, 227, 228
adenopathy, 148
adenovirus, 108, 109, 113, 114, 115
ADH, 250
adhesion, 72, 74, 76, 92
adipocytes, 280

administrationi, ix, x, 1, 2, 3, 5, 6, 7, 8, 10, 25, 44,


99, 109, 112, 115, 134, 158, 280
adolescence, 227
adult stem cells, 283
adult tissues, 70
adults, 156, 177
aerodigestive tract, 180, 228
aetiology, 78
Africa, 212
age, xii, xv, 12, 13, 30, 52, 53, 57, 76, 78, 79, 96,
155, 159, 160, 166, 171, 172, 178, 185, 197, 200,
203, 256, 260, 263, 265, 267, 268
agent, 2, 76, 106, 114
aggregates, 100
aggressive behavior, 69
aggressive therapy, 78
aggressiveness, xi, xv, 51, 59, 63, 66, 70, 104, 123,
226, 263, 264
alanine, 265
alcohol, xv, 12, 126, 163, 171, 177, 212, 213, 247,
248, 249, 250, 255, 258, 260, 262, 270
alcohol abuse, 126, 163, 212
alcohol consumption, 248, 258
alcohol use, xv, 171, 212, 247, 270
algorithm, 157, 166
allele, 253, 255, 256, 257, 260
alternative, xiv, 57, 99, 109, 175, 196, 206, 260, 279
alternatives, 282
alters, 251
aluminium, 186
American Cancer Society, 34, 80
amines, 249, 252, 253
amino acid, 103, 120, 253
amino acids, 103

286

Index

ammonium, 4, 96, 100, 102


anastomosis, 281
anatomy, xv, 144, 233, 234, 240, 241, 246, 275, 276,
279, 280, 281, 282
anemia, 23
aneuploidy, 223
angiofibroma, 244
angiogenesis, 14, 16, 17, 18, 20, 22, 23, 25, 27, 29,
31, 38, 39, 40, 43, 45, 46, 48, 61, 71, 89, 90, 272
angiography, xi, 125, 126, 131, 136, 145, 147, 149,
234, 245
angulation, 126
anhydrase, 39
animal models, 109, 280
animals, 3, 16
anion, 103
anorexia, 166
anorexia nervosa, 166
ANOVA, 188, 190
antiangiogenic therapy, 116
anti-apoptotic, 69, 70, 108
antibiotic, 176, 179
antibody, 14, 19, 25, 29, 36, 37, 67, 107
anticancer drug, 31, 49
antigen, 14, 36, 67, 87, 88, 109, 110, 111, 123, 274
antimetabolites, 46
antioxidant, 31
antisense, 101, 106, 111, 112, 115, 116, 117, 119,
123, 124
antisense oligonucleotides, 115, 117, 124
antisense RNA, 112, 116
antitumor, 108, 109, 114, 115, 116, 121, 123
antiviral, 110
AP, 36, 41, 92, 122
apoptosis, xi, 25, 40, 43, 51, 52, 61, 62, 66, 69, 70,
71, 87, 88, 89, 90, 108, 111, 113, 115, 122, 271,
272, 274
argument, 180
arrest, 121
arteries, 136, 149, 234, 276, 277, 279
artery, xv, 137, 230, 234, 239, 240, 245, 275, 276,
277, 278, 279, 281, 282
aryl hydrocarbon receptor, 23, 42
ascites, 166
ASI, 49
Asia, 212
aspirate, 215
aspiration, xi, xiv, 126, 139, 150, 151, 164, 165, 167,
168, 171, 172, 211, 213, 214, 215, 225, 226, 227,
228, 236, 245

aspiration pneumonia, 164


assessment, xii, 35, 38, 55, 81, 82, 83, 88, 131, 139,
141, 147, 149, 150, 151, 153, 155, 190, 224, 234,
237
asymptomatic, 54, 205, 212, 214, 225
atrophy, 171
attachment, 72, 73, 187, 237
attention, xiv, 24, 34, 111, 138, 229, 230, 239, 257
Australia, 212
availability, 105, 170
averaging, 234
avoidance, 61

B
B cells, 109
BAC, 224
barriers, 99, 115
basal cell carcinoma, 10, 245
basal layer, 68, 69, 75
base pair, 111
basic fibroblast growth factor, 16, 38, 39, 90, 280,
283
Bcl-2 proteins, 70
BD, 89, 175
behavior, xi, 51, 52, 57, 61, 66, 68, 87, 92
bending, xii, xiii, 183, 184, 185, 187, 188, 189, 190,
191, 192, 196, 197, 204
benefits, 205
benign, xii, xiv, 129, 133, 141, 148, 150, 155, 172,
211, 213, 214, 216, 223, 229, 230, 231, 232, 237,
241, 246, 261
benign tumors, 129, 133, 214, 231, 246
benzene, 254
beverages, 250
bias, 166
bile, 106
binding, xv, 16, 29, 31, 39, 40, 42, 46, 62, 66, 76, 77,
103, 105, 106, 118, 119, 120, 247, 248, 253
biochemistry, 250
biologic agents, 109
biological behavior, xi, 52, 56, 61, 72
biological markers, 77
biomarkers, 228, 258
biomechanics, 192, 194, 196, 208
biophysics, 115
biopsy, xiv, 14, 17, 130, 133, 139, 148, 150, 151,
160, 193, 207, 211, 212, 213, 215, 221, 222, 223,
224, 226, 227, 228, 236, 239, 245, 268
biosynthesis, 111

Index
bladder, 8, 14, 16, 29, 31, 36, 44, 46, 47, 62, 252
blocks, 65, 187
blood, 12, 13, 15, 16, 17, 18, 23, 25, 26, 38, 42, 45,
61, 71, 106, 137, 138, 139, 150, 234, 237, 277,
279, 281, 282
blood flow, 138, 150, 279
blood plasma, 106
blood supply, 71, 281, 282
blood transfusion, 25
blood vessels, 16, 17, 18, 26, 38, 42, 61, 71, 137,
138, 234
BMI, 164
BN, 226
body weight, 165
bone cement, 193
bone grafts, 280
bone marrow, 147, 283
bone mass, 209
bone remodeling, 209
bone resorption, 131
bowel, 160
brachioradialis, 199
brain, xiv, 14, 16, 30, 36, 62, 104, 112, 137, 138,
229, 242
brain tumor, 14, 16, 30, 137, 138
branching, 218, 277, 278, 279
Brazil, 52, 79, 247, 253, 259
Breast, 39, 258
breast cancer, 14, 18, 25, 29, 30, 31, 36, 37, 40, 43,
44, 48, 90, 108, 122, 124, 227, 258
breast carcinoma, 36, 37, 39, 40, 49, 89, 258
breathing, 148
buccal mucosa, 82, 215, 218, 251, 256, 262
budding, 215
buffer, 101, 265, 266
Burkitts lymphoma, 218
bystander cells, 123
bystander effect, 110

C
cadaver, 278
cadherin, 74, 75, 76, 77, 90, 91, 92, 93
cadherins, 74, 92
cadmium, 49
calcium, 74
caliber, 278, 279
calibration, 4, 5
canals, 233, 234

287

cancer cells, 31, 70, 72, 97, 98, 104, 107, 108, 110,
111, 112, 113, 115, 117, 119, 120, 123, 141, 215,
223, 226, 260
cancer progression, 88, 111, 271
cancer treatment, ix, 1, 2, 32, 78, 139, 157, 166, 271
capillary, 71
capsule, 239
carbon, 133, 148
carcinogen, 249, 252, 255, 260
carcinogenesis, xi, xv, 51, 52, 64, 66, 67, 75, 77, 83,
89, 91, 92, 108, 228, 247, 248, 249, 250, 257,
260, 261, 262, 270, 274
carcinogens, xv, 247, 248, 249, 250, 252, 254, 255,
256, 257
carcinoma, ix, x, xi, xiv, 11, 12, 13, 14, 15, 16, 20,
21, 22, 27, 29, 32, 34, 35, 36, 37, 38, 39, 40, 41,
44, 45, 48, 49, 50, 56, 62, 63, 66, 69, 71, 76, 78,
79, 80, 81, 82, 83, 85, 89, 90, 91, 92, 93, 95, 96,
114, 122, 133, 140, 143, 147, 148, 151, 164, 169,
170, 171, 172, 177, 178, 180, 211, 212, 214, 215,
216, 217, 218, 222, 223, 225, 226, 227, 228, 231,
232, 244, 248, 253, 255, 274
carotid arteries, 234
carrier, 96, 103, 117
cartilage, 280, 283
cast, 197, 199, 200, 203, 205
catabolism, 164
catalysts, 252
catalytic activity, 29, 256
catalytic properties, 254
category a, 36
catheter, 147
Caucasians, 255, 261
CD14, 38
CD34, x, 11, 19, 21
CD44, 75, 92
CD8+, 110
cDNA, 45, 83, 97, 112, 118
CE, 46, 209
cell adhesion, 75, 92
cell culture, 72
cell cycle, 14, 17, 22, 25, 27, 65, 66, 67, 69, 86, 87,
89, 99, 108, 121, 273
cell death, 61, 65, 67, 70, 89, 110, 120, 264, 271
cell differentiation, 69, 72
cell division, 67
cell growth, 16, 39, 46, 47, 48, 90, 108, 111, 122,
264, 271
cell killing, 49

288

Index

cell line, 14, 17, 20, 22, 25, 26, 27, 28, 29, 34, 38,
41, 43, 62, 75, 76, 104, 108, 113, 114, 115, 117,
118, 121, 122, 265, 274
cell lines, 17, 20, 26, 27, 38, 41, 43, 75, 76, 108,
113, 114, 115, 118, 122
cell membranes, 100
cell surface, 100, 103, 105, 107, 110
central nervous system, 48
cerebrospinal fluid, 106
cervical cancer, 16, 17, 25, 26, 30, 40
cervix, 38, 39, 42, 44, 48, 118
chemical composition, 100
chemical reactions, 253
chemoprevention, 122, 228, 258, 261
chemoresistance, 25
chemotaxis, 46
chemotherapeutic agent, 96
chemotherapy, xi, 2, 25, 32, 33, 52, 57, 60, 63, 64,
95, 96, 108, 110, 111, 112, 114, 121, 141, 152,
157, 158, 161, 169, 174, 177, 249, 265
chicken, 120
childhood, 117, 227
children, 156
China, 229, 258, 261
Chinese, 49, 258
Chinese women, 258
chloride, 100, 101, 120
CHO cells, 49
cholesterol, 96, 100, 102, 103, 106, 117
choroid, 104, 118
chromatin, 215, 218
chromosome, 62, 65, 66, 67, 69, 74, 76, 93, 126,
253, 254, 264
chronic hypoxia, 41
cigarette smoke, 254, 256
cigarette smokers, 256
cigarette smoking, 258, 273
circulation, 279
cisplatin, 49, 96, 109, 123
classes, xv, 247, 248, 252
classification, xi, 13, 35, 51, 54, 55, 57, 80, 167, 268
cleavage, 91
clinical examination, 126, 133
clinical presentation, 79, 214, 265
clinical trials, 34, 101, 107, 110, 111, 245
clinicopathologic correlation, 273
clone, 41, 224
cloning, 47, 118
closure, 241, 277
clustering, 118

clusters, 215, 216, 217, 218


CNN, 130
coagulopathy, 166
cobalt, 43
codes, 65, 264, 271
coding, 97, 100, 110, 253, 256, 280
codon, 256, 260
cohort, 157, 225, 257
collateral, xvi, 275, 276, 277, 278
colon, 8, 30, 47, 71, 73, 90, 91, 108
colon cancer, 30, 90
colorectal cancer, 16, 36, 49, 259
combustion, 249
commissure, 278
communication, 108, 282
community, 153, 176, 179
competency, 241
competition, 70
compliance, 223
complications, xii, xiii, 52, 139, 155, 156, 157, 158,
159, 160, 163, 164, 165, 166, 167, 168, 169, 170,
171, 172, 174, 175, 176, 179, 193, 195, 196, 200,
205, 206, 207, 209, 221
components, 42, 250
compounds, 31, 249, 250, 252, 254
computed tomography, xi, xiv, 125, 144, 146, 147,
151, 152, 153, 211, 213, 236, 245
Computer simulation, 193
concentration, 3, 4, 5, 8, 10, 38, 106, 141, 190, 191,
194, 196, 204, 208, 249, 259, 265, 266
condensation, 106
confidence, 187, 188
confidence interval, 187, 188
configuration, 100, 151
confusion, 20, 213
conjugation, 105, 250, 252, 256, 261
conjunctiva, 280, 283
connective tissue, 56
consensus, 58, 271
consent, 168
consumption, 249
contaminants, 250
continuity, 277
contrast agent, 134
control, 3, 18, 26, 27, 28, 31, 35, 40, 41, 63, 66, 78,
85, 86, 89, 97, 108, 109, 112, 122, 172, 177, 239,
240, 254, 255, 256, 257, 258, 259, 260, 261, 265
control group, 172, 255, 256, 257
conversion, 31, 249

Index
correlation, x, 12, 14, 17, 19, 20, 21, 23, 24, 26, 29,
30, 32, 37, 40, 44, 45, 49, 64, 65, 70, 71, 75, 85,
89, 92, 93, 107, 112, 148, 150, 151, 221, 257,
259, 264, 271, 272
correlations, 25, 26, 37
cortex, xiii, 183, 184, 185, 186, 187, 188, 189, 190,
191, 197, 198, 205
costs, 163, 164
coverage, 278, 280
covering, 242
CpG islands, 75, 76
Croatia, 254
CT scan, 126, 127, 128, 129, 130, 131, 139, 141,
142, 144, 145
culture, 9, 283
cycles, 83, 265, 266
cyclin-dependent kinase inhibitor, 86, 87, 108, 121
cycling, 41
cyclins, 65, 88
cyclooxygenase, 113
cyclooxygenase-2, 113
cysteine-rich protein, 31
cytochrome, 23, 41, 259, 260, 261, 262
cytokines, 34
cytologic examination, 222, 227
cytology, xiv, 150, 211, 212, 213, 214, 215, 216,
218, 220, 221, 222, 223, 224, 225, 226, 227, 228,
236, 245
cytomegalovirus, 97
cytometry, xiv, 22, 212, 223
cytomorphometry, xiv, 212
cytoplasm, 23, 55, 64, 70, 103, 106, 111, 217, 218,
221
cytosine, 97, 110, 123, 253
cytoskeleton, 74, 75
cytotoxic, xi, 63, 95, 109, 111
Cytotoxic effects, 114
cytotoxicity, 49, 117

D
DD, 80, 179, 245, 272
death, xii, 60, 70, 110, 114, 123, 155, 158, 160, 162,
166, 168
deaths, xii, 52, 155, 160, 167
decision making, 172
decision-making process, 157, 166
decisions, xi, 41, 51

289

defects, xiii, xv, 100, 131, 165, 184, 190, 196, 197,
199, 203, 206, 241, 246, 275, 276, 278, 279, 281,
282, 283
deficiency, 93, 117, 255, 256
deficit, 159, 184
definition, 20, 42, 52, 53, 102, 234
deglutition, 166
degradation, 66, 71, 86, 99, 101, 108, 111, 121, 261
degradation rate, 66
delivery, xi, 41, 95, 97, 99, 100, 102, 103, 104, 105,
106, 107, 108, 111, 112, 115, 116, 117, 119, 120,
121, 122, 165
demand, 16
dementia, 166, 180
density, x, 11, 14, 15, 17, 18, 19, 20, 22, 24, 25, 26,
28, 29, 30, 31, 33, 35, 39, 40, 44, 45, 49, 67, 71,
107, 128, 129, 130, 194, 213, 234
dentists, 137
deoxyribose, 29
deposition, 38
deposits, 208
derivatives, 3, 6, 102, 103, 106, 249
destruction, 59, 231, 232
detection, xiv, 8, 54, 60, 61, 80, 87, 130, 138, 141,
147, 148, 149, 152, 153, 168, 211, 212, 215, 222,
223, 224, 227, 228, 230, 245, 265
deviation, 55
diabetes, 166
diabetes mellitus, 166
dialysis, 166
diaphyses, 209
diaphysis, 186
diet, 163, 177, 257
differential diagnosis, 129, 133, 146
differentiation, 36, 55, 56, 63, 67, 71, 72, 75, 76, 85,
118, 123, 130, 147, 264, 265, 267, 268, 271, 274
diffusion, 18, 40, 133, 134, 148
digestive tract, 80
dimerization, 29
dioxin, 42
discomfort, 164, 213
disease progression, 108
disorder, 12, 244
dispersion, 72, 213
displacement, 164, 187
dissatisfaction, 164
dissociation, 57, 58, 73, 77
distress, 168
distribution, 9, 16, 26, 41, 44, 67, 69, 141, 213, 215,
216, 233, 257, 277

Index

290

division, 230
DNA, xi, xiv, xv, 26, 33, 37, 42, 52, 65, 67, 70, 77,
89, 93, 95, 97, 98, 99, 100, 101, 102, 106, 107,
108, 110, 111, 112, 115, 116, 117, 119, 120, 123,
212, 223, 224, 225, 227, 228, 247, 248, 249, 250,
253, 256, 258, 261, 265, 266, 272, 274
DNA damage, xv, 65, 108, 247, 248, 256
DNA image cytometry, 224
DNA ploidy, 224
DNA polymerase, 110
DNA repair, 26, 33, 67, 89, 261
docetaxel, 96, 111, 115
dogs, 194, 209
doppler, 150
Doppler, 138, 139, 150, 277, 278, 279
dose-response relationship, 215
down-regulation, 108, 111, 117
drainage, 277, 278, 280, 282
drug release, 119
drug resistance, 44, 259
drugs, 63, 123, 251
dsRNA, 111
duodenum, 157
duration, xii, 67, 69, 155, 158, 159, 160, 161, 162,
163, 166, 169, 170, 171, 172, 174, 205
DWI, 134, 148
dyes, 249
dysphagia, 157, 163, 165, 166, 176
dysplasia, xiv, 63, 66, 67, 70, 86, 88, 159, 162, 211,
212, 214, 221, 222, 225, 227

E
E. coli, 24
eating, 12
E-cadherin, 72, 74, 75, 76, 77, 80, 84, 91, 92, 93
edema, 239
education, 227
effusion, 147
egg, 106
EIA, 109
elasticity, 190
elderly, 78, 160, 163, 167, 176, 180, 191, 201, 277
electrodes, 17, 40
electron, 223, 251
electron density, 251
electron microscopy, 223
electrophoresis, 265
electroporation, 100, 108, 115
embryonic development, 46

emission, 4, 5, 8, 142, 152, 153, 225, 226, 236


emulsions, 99
encapsulation, 101, 117
encoding, 43, 97, 251
endocrine, 52
endocytosis, 99, 103, 104, 105, 107, 118
endoscope, 157, 166
endoscopy, 160, 167, 170, 176, 179, 180
endothelial cells, 16, 17, 22, 27, 29, 31, 38, 39, 46,
71, 90
endothelium, 46
energy, xii, 41, 45, 183, 184, 190, 197, 250
England, 178
enlargement, 231, 234
environment, xv, 170, 247, 248, 250, 261
environmental factors, 12, 126, 257, 259
enzyme, 23, 87, 106, 108, 110, 113, 250, 253, 254,
259, 260
enzyme-linked immunoabsorbent assay, 87
enzymes, xv, 43, 110, 247, 248, 249, 250, 251, 252,
254, 255, 257, 258, 259
epidemiology, 12
epidermal growth factor, 62, 63, 83, 84, 85, 86, 91,
92, 111, 115, 116, 119, 124
epithelia, 74, 87, 113, 228
epithelial cells, 43, 55, 56, 72, 97, 213, 218, 251, 259
epithelial ovarian cancer, 47
epithelium, 36, 54, 55, 56, 60, 63, 66, 68, 72, 74, 75,
86, 92
Epstein Barr, (virus) 97, 113
equipment, 157, 169, 187
erosion, 232, 234
erythropoietin, 23, 41, 43
Escherichia coli, 24, 47
esophageal cancer, x, 11, 16, 30, 35, 38, 48
esophageal squamous cell carcinoma, x, 11, 12, 13,
19, 26, 32, 33, 34, 48, 91
esophagus, 12, 35, 37, 38, 71, 73, 93
ester, 3, 6, 9, 10
esters, 9
estimating, 21, 33, 85, 268
ethanol, 31, 101, 102, 213, 249, 250, 254
Ethanol, 249, 250, 261
ethical issues, 157, 166, 175
ethnic groups, 256
ethylene, 249, 252, 256
ethylene oxide, 249, 252, 256
etiology, 137
Euro, 10
Europe, 186, 212

Index
eustachian tube, 240
evidence, xiii, 29, 38, 41, 70, 77, 96, 109, 164, 166,
171, 176, 188, 195, 205, 206, 212, 216, 232
evolution, 77, 84, 92, 250, 265
examinations, 128, 137, 138
excision, 236, 241
excitation, 3
excretion, 252
exons, 265, 270
expertise, 165, 170
exposure, 43, 67, 99, 237, 238, 239, 241, 243, 244,
249, 250, 251, 252, 257, 277
Exposure, 258
external carotid artery, 136
external fixation, 197
extracellular matrix, 71, 72
extraction, 265

F
FAA, 274
facial nerve, 237, 238, 240, 277, 278
failure, 60, 66, 68, 166, 185, 212, 266, 272, 274
false negative, xiv, 212, 222, 224
family, xv, 18, 27, 31, 62, 70, 72, 74, 83, 85, 89,
106, 111, 247, 248, 251, 252, 253, 254, 257, 259,
264
family members, 83, 85
family studies, 253, 254
fascia, 241, 243, 280, 282
fat, 128, 131, 132, 147, 234, 241, 282
fatty acids, 251
FDA, 65
FDA approval, 65
females, 13, 204
femur, 208
fibrin, 17, 38
fibrinogen, 17
fibroblast growth factor, 65
fibroblasts, 46, 272
fibrosarcoma, 27
fibula, 163, 193, 208, 281
fidelity, 250
film, 233
fixation, xiii, 64, 190, 194, 195, 197, 198, 200, 203,
208, 209
flexor, 198, 201, 204
fluctuations, 280
fluid, 106, 137
fluorescence, ix, 1, 2, 3, 4, 5, 8, 9, 10, 224, 226

291

fluorine, 141
folate, xi, 95, 96, 97, 103, 104, 105, 106, 108, 111,
117, 118, 119, 120
folic acid, xi, 95, 97, 103, 120
foramen, 230, 232, 233, 234, 277
Ford, 153
formamide, 266
fractures, xii, xiii, 159, 183, 184, 190, 193, 194, 195,
197, 204, 205, 208, 209
fragmentation, 160
France, 257
free radical scavenger, 31
free radicals, 32, 49
frequency distribution, 257
fusion, xi, 110, 125, 133, 137, 144, 145

G
ganglion, 230, 232, 234, 240
gastrectomy, 34, 166, 167
gastric ulcer, 160
gastroenterologist, 157, 167
gastrointestinal tract, 48, 157, 164, 165
gastrostomy, xii, 155, 156, 157, 159, 165, 166, 167,
168, 169, 170, 171, 172, 174, 175, 176, 177, 178,
179, 180, 181
gel, 266
gender, 30, 166, 197, 200, 268
gene, xi, xv, 26, 36, 40, 41, 42, 43, 44, 48, 49, 60,
62, 63, 65, 66, 67, 69, 74, 76, 77, 83, 86, 87, 89,
92, 93, 95, 97, 98, 99, 100, 101, 103, 105, 106,
107, 108, 109, 110, 111, 112, 113, 114, 115, 116,
117, 119, 120, 121, 122, 123, 224, 228, 247, 248,
250, 251, 252, 253, 254, 255, 256, 258, 259, 261,
262, 264, 265, 270, 271, 272, 273, 280, 283
gene amplification, 62, 63
gene expression, 40, 42, 43, 44, 77, 97, 99, 108, 113,
122
gene promoter, 76, 113
gene silencing, 111
gene therapy, xi, 95, 97, 98, 101, 106, 107, 108, 109,
110, 112, 113, 114, 115, 116, 117, 119, 121, 123,
280, 283
gene transfer, 99, 101, 103, 105, 106, 112, 113, 115,
117, 119, 123
general surgeon, 157
generation, 17, 38, 99, 100, 102
genes, xi, 21, 23, 34, 43, 65, 66, 83, 95, 98, 107, 109,
110, 111, 122, 251, 253, 254, 255, 257, 258, 259,
261, 264, 266, 270, 271

Index

292

genetic alteration, 60, 61, 66, 99, 108, 254, 255, 256,
257
genetic disorders, 111
genetic factors, 264
genetic linkage, 45
genetic marker, 52
genome, 25, 34, 65, 99, 251
genomic instability, 83
genotype, 252, 254, 255, 256, 257, 259
germ cell tumors, 41
Germany, 101, 185, 253, 255
gingiva, ix, 218, 255
gingival, 129, 130, 132, 143, 215, 227
gland, ix, xiv, 128, 135, 211, 212, 214, 216, 218,
222, 225, 226, 228, 233, 244
glass, 213
glioma, 40, 45
glucose, 17, 18, 25, 44, 141, 142, 144, 152
glucose metabolism, 17, 18, 141, 144
glutathione, xv, 247, 248, 251, 258, 259, 261
glycogen, 215
glycol, 119
glycoprotein, 16, 62
glycoproteins, 72, 74, 106
gold, xiv, 169, 211
grading, 56, 64, 81, 224
granules, 120
graph, 134, 135
gravity, 213
groups, 20, 23, 30, 53, 58, 77, 99, 167, 172, 185,
188, 189, 190, 215, 223, 249, 252, 271
growth, ix, 14, 15, 16, 18, 22, 23, 24, 25, 27, 28, 29,
35, 38, 39, 40, 41, 44, 45, 46, 48, 49, 52, 56, 59,
61, 62, 63, 65, 67, 69, 71, 72, 75, 76, 83, 84, 89,
90, 108, 111, 113, 116, 122, 214, 216, 218, 219,
220, 265, 280, 283
growth factor, 15, 16, 28, 29, 38, 39, 40, 41, 44, 45,
46, 48, 49, 62, 63, 65, 71, 83, 84, 89, 90, 280, 283
growth factors, 45, 46, 65, 280
growth rate, 14, 22, 25
guidance, 157, 158, 169, 176, 177, 180, 236
guidelines, 13, 174

H
haplotypes, 266
harvesting, xii, 183, 208, 278, 280
hazards, 30, 266
HD, 145, 272
HE, 5, 37, 38, 43, 49, 85, 86, 88, 245, 274

head and neck cancer, 14, 17, 25, 40, 79, 108, 114,
115, 116, 121, 123, 126, 133, 146, 148, 150, 152,
153, 157, 163, 174, 175, 177, 178, 179, 180, 181,
212, 248, 252, 253, 254, 255, 257, 259, 261, 262,
264, 272, 273, 274
head trauma, 156
healing, xiii, 195, 196, 199, 204, 242
health, xiv, 53, 165, 174, 196, 205, 206, 248
health care, 205
health care system, 205
health status, 165
heat, 113
heavy drinking, 254
heavy metals, 31
height, 129, 266, 279
hemangioma, 137
hematomas, 139
hematopoietic cells, 45, 118
hematoxylin-eosin, 3
heme, 2, 23, 41, 251, 253
hemiglossectomy, xii, 155, 163, 172
hepatitis, 47
hepatocellular, 18, 29, 47, 76, 92, 93, 227, 274
hepatocellular carcinoma, 18, 29, 47, 76, 92, 93,
227, 274
hepatocyte growth factor, 35
hepatocytes, 100, 106
hepatoma, 39
hepatomegaly, 166
hernia, 160, 166
herpes, 98, 110, 114, 117, 120, 123
herpes simplex, 98, 110, 114, 117, 120, 123
heterogeneity, 17, 18, 20, 26, 34, 66, 90, 118
histologic type, ix
histological markers, xi, 52
histology, 90, 221
histone, 67, 122
histopathology, xiv, 211, 221
HIV, 115, 226
HIV-1, 115
HLA, 109, 116, 123
homeostasis, 31, 41
homogeneity, 131
Honda, 146
Hong Kong, 209
hospitals, 264
host, 12, 16, 57, 97, 99, 177, 280
host tissue, 16, 57
hot spots, 40
HPLC, 8

Index
HPV, 97, 98, 224, 272
human brain, 46
human genome, 251
human leukocyte antigen, 110
human papilloma virus, 97, 251
human papillomavirus, 112, 212, 228, 259
human subjects, 120
hybridization, 224
hydroxyl, 24
hyperbaric oxygen therapy, 280
hypermethylation, 75, 76, 93
hypothesis, 190, 256
hypoxia, 17, 18, 21, 22, 23, 24, 25, 29, 39, 40, 41,
42, 43, 44, 45
Hypoxia, 21, 23, 40, 42, 43, 44, 45
hypoxia-inducible factor, 18, 23, 24, 41, 42, 43, 44
hypoxic cells, 17, 18, 20, 26, 39, 45

I
ICD, 54
identification, xi, 51, 52, 60, 74, 131, 136, 149, 223,
239, 277
IFN, 24, 109
ilium, 142, 143
illumination, 7
image analysis, 37, 68, 223
images, xi, 125, 128, 131, 132, 133, 134, 135, 136,
137, 138, 139, 141, 142, 143, 144, 145, 147
imaging, xi, xiv, 104, 125, 126, 128, 131, 132, 133,
138, 141, 142, 144, 145, 146, 147, 148, 149, 151,
152, 153, 211, 213, 225, 229, 230, 234, 236, 237
imaging modalities, xi, 125, 126, 133, 145
imaging techniques, xiv, 145, 148, 211, 213
immune reaction, xi, 95, 97
immune response, 109, 116
immune system, 109, 163
immunity, 109
immunocytochemistry, xiv, 212
immunogenicity, 99, 105
immunoglobulin, 27
immunohistochemical markers, xi, 52, 61, 67, 76, 77
immunohistochemistry, 14, 17, 19, 32, 61, 224
immunoreactivity, 36, 63, 70, 75, 76, 77, 84, 118
immunotherapy, 64, 109, 123
implants, 205, 209, 210
implementation, 258
impurities, 250
in situ, xiv, 211, 212, 215, 222, 223, 224, 226
in situ hybridization, 224, 226

293

in vitro, 20, 23, 26, 29, 39, 49, 76, 101, 102, 105,
107, 108, 109, 110, 112, 117, 119, 121, 250, 283
in vivo, 8, 23, 27, 29, 39, 40, 41, 101, 105, 107, 108,
112, 113, 114, 115, 116, 118, 119, 122, 192, 196
incidence, xii, xiii, 21, 31, 53, 55, 71, 78, 82, 83, 96,
130, 147, 155, 156, 159, 163, 165, 166, 167, 168,
169, 170, 171, 172, 174, 183, 184, 195, 196, 197,
202, 203, 204, 205, 206, 248, 260, 270, 273, 277
inclusion, 278
India, 253, 272, 273
Indians, 258
indication, 133, 158, 277
indicators, xi, 51, 52, 69, 77, 272, 273
indices, 69
inducer, 250, 251
inducible protein, 21, 22, 23, 24, 33
induction, x, 12, 32, 39, 42, 61, 108, 109, 157
infection, 99, 152, 159, 160, 168, 205, 272
infectious disease, 111
infectious diseases, 111
inferences, 53
inflammation, 129, 141, 152
inflammatory cells, 56
inheritance, 12
inhibition, 27, 34, 45, 62, 63, 65, 66, 70, 85, 108,
111, 121, 271
inhibitor, 65, 86, 89, 115, 121, 122
initiation, 103, 248
injury, 147, 160, 179, 205, 236, 239, 278
insertion, xii, 27, 155, 156, 157, 158, 159, 166, 167,
168, 169, 171, 172, 173, 174, 175, 180, 205
instability, 131
integration, 99
integrin, 92
integrity, 72, 184, 197
intensity, ix, 2, 4, 5, 6, 7, 8, 9, 61, 65, 67, 68, 70,
133, 134
interaction, xv, 16, 99, 100, 247, 248, 254, 261, 280
interactions, 74, 100, 122, 254, 261
intercellular adhesion molecule, 72, 74, 92
interface, 56, 57
interference, 110, 124, 164
interferon, 24, 123
interferon gamma, 123
internal fixation, xiii, 183, 184, 192, 194, 195, 198,
203, 205, 206, 207, 208, 209
internalization, 111
International Classification of Diseases, 54
interphase, 67, 87
interpretation, 146, 268

Index

294

interval, 64, 68, 69, 76


intervention, 170, 258
intestine, 103, 117, 118
introns, 97
inversion, 131, 132
inversion recovery, 131, 132
ionization, 32
ionizing radiation, 49, 113
ipsilateral, 240, 278
IR, 45
iron, 106, 107, 120, 134, 148, 149
irradiation, x, 2, 9, 12, 26, 32, 33, 83, 138, 177, 180,
212
island formation, 45
isoleucine, 253
Israel, 123
Italy, 275

J
Japan, 1, 3, 4, 9, 11, 12, 13, 95, 125, 126, 127, 128,
129, 253, 255, 257
Jordan, 45, 88, 89, 272, 273
judgment, 56
justification, 166
juvenile angiofibroma, 241

K
keratin, 55, 56, 216, 223
keratinocytes, 70, 72, 251, 283
kidney, 31, 46, 104, 118
killer cells, 109
killing, 49, 123
kinase, 24, 27, 28, 43, 45, 46, 62, 64, 66, 96, 98, 110,
114, 117, 120, 121, 123
kinase activity, 45, 62
kinetics, 2, 8, 9, 10, 37, 45, 87
Korea, 253
Kuwait, 211

L
labeling, x, 11, 14, 15, 16, 20, 21, 22, 29, 30, 37
lactoferrin, 106, 120
laminin-5, 91
laparotomy, 156, 168, 174
laryngeal cancer, 260
laryngoscope, 166

larynx, 123, 165


latissimus dorsi, 163, 281
leakage, 160, 168
lesion, ix, xiv, 8, 54, 126, 139, 144, 160, 211, 212,
214, 231, 234, 236, 237, 244, 258, 265
lesions, xiv, 8, 10, 36, 54, 56, 63, 64, 66, 67, 68, 69,
70, 73, 74, 75, 81, 84, 86, 88, 90, 91, 92, 123,
126, 128, 131, 132, 134, 137, 139, 147, 150, 151,
153, 193, 208, 211, 212, 213, 214, 215, 221, 222,
223, 224, 225, 227, 228, 231, 234, 236, 237, 239,
241, 244, 245, 258, 260, 261, 267, 271, 273, 274
leukemia, 97
leukocytes, 109, 120
leukoplakia, 212, 215, 256, 258
Leukoplakia, 214, 215
lichen, 212, 224, 228
lichen planus, 212, 224, 228
life expectancy, 96, 157, 166
lifespan, 166
ligand, xi, 25, 28, 29, 45, 63, 72, 95, 97, 103, 105,
107, 108, 251
ligands, xi, 62, 95
likelihood, 70
limitation, 136, 172
linkage, 190
links, 121
lipid rafts, 118
lipids, 101, 102, 105
liposomes, 97, 99, 100, 101, 102, 104, 105, 106, 107,
108, 111, 112, 117, 119, 120
liquid chromatography, 8
literature, xiii, 53, 54, 60, 69, 77, 78, 81, 145, 157,
167, 170, 175, 195, 197, 205, 206, 220, 221, 232,
281, 283
liver, 47, 100, 221, 251, 252
liver disease, 47
local anesthetic, 223
localization, 3, 64, 84, 87, 122, 236, 251
location, 53, 54, 126, 131, 144, 218, 265, 279
locus, 29, 75
loss of appetite, 163
loss of heterozygosity, 75, 76
LPS, 24
luciferase, 113
lumen, 157
lung, 18, 31, 41, 42, 44, 45, 47, 62, 73, 91, 104, 105,
108, 111, 122, 153, 252, 258, 259, 261
lung cancer, 18, 41, 47, 258, 259, 261
lymph, xi, 52, 54, 55, 56, 58, 68, 72, 76, 80, 81, 82,
83, 90, 93, 106, 125, 126, 127, 128, 130, 131,

Index
133, 134, 138, 139, 140, 141, 142, 143, 144, 145,
146, 147, 148, 149, 150, 215, 224, 228, 256, 260,
265, 267, 271, 278
lymph node, xi, 52, 54, 55, 56, 58, 68, 72, 76, 80, 81,
82, 83, 90, 93, 125, 126, 127, 128, 130, 131, 133,
134, 138, 139, 140, 141, 142, 143, 144, 145, 146,
147, 148, 149, 150, 215, 224, 228, 256, 260, 265,
267, 271, 278
lymphadenopathy, 131, 139, 148, 150
lymphatic system, 58, 146
lymphocytes, 109, 123, 217
lymphoid, ix, 128, 141, 143, 218
lymphoid tissue, ix, 128, 141, 143
lymphoma, ix, 89, 133, 148, 212, 214, 218, 219, 226
lymphomas, xiv, 126, 211, 218
lysine, 96, 106, 213

M
mAb, 61
machinery, 111
macrophages, 17, 27, 43, 44, 48, 109
magnetic field, 131
magnetic resonance, xiv, 134, 147, 149, 151, 205,
211, 213, 234, 236, 244, 245
magnetic resonance imaging, xiv, 147, 205, 211,
213, 236, 244, 245
major histocompatibility complex, 122
Malaysia, 224
males, 13, 204, 215
malignancy, xii, xiv, xv, 12, 14, 15, 64, 70, 73, 81,
118, 128, 130, 155, 156, 157, 163, 167, 169, 171,
201, 211, 212, 215, 216, 224, 236, 248, 263, 265,
267, 268
malignant cells, 15, 19, 22, 68, 91, 215, 216, 217,
223, 226
malignant melanoma, 45, 96
malignant tumors, xiv, xv, 57, 70, 96, 129, 141, 211,
229, 230, 231, 232
malnutrition, 163, 164, 173
mammalian cells, 111, 113, 121, 123, 124
management, xii, xiii, xv, 80, 92, 112, 129, 146, 155,
156, 165, 168, 175, 177, 184, 192, 206, 208, 213,
214, 225, 226, 229, 230, 244, 261
mandible, 126, 127, 141, 197, 201, 206, 237, 238,
239, 277
manipulation, 44
mapping, 126, 133
market, 104
marrow, 147

295

masseter, 237, 238, 279


mastication, 157, 163
matrix, 17, 72, 84, 87, 91, 122, 221
matrix metalloproteinase, 84
maturation, 55, 215
maxilla, xiii, xiv, 126, 127, 196, 201, 206, 214, 229,
230, 237, 240, 243, 246
maxillary sinus, ix, xiv, 229, 230, 232, 239, 242,
243, 246, 283
MBI, 139
meals, 12
measurement, 3, 17, 25, 36, 39, 82, 131
measures, 165, 169
media, 234
median, xii, 13, 96, 155, 158, 159, 160, 161, 163,
166, 170, 171, 172
medical care, 53
medicine, 145, 246
melanoma, ix, 17, 18, 22, 25, 26, 31, 39, 40, 41, 45,
123, 214, 232
membranes, 99, 103, 104
men, 52, 53
meningioma, 223, 228, 233
messenger ribonucleic acid, 46
messenger RNA, 48
meta-analysis, 112, 167, 168, 169, 175
metabolic changes, 23
metabolic pathways, 24, 34, 253
metabolism, xv, 23, 29, 45, 47, 89, 103, 141, 142,
247, 248, 250, 251, 252, 254, 260, 262
metabolites, xv, 29, 41, 110, 247, 248, 249, 251,
252, 253, 256, 257
metabolizing, 252, 253, 254, 257, 259
metallothionein, x, 11, 31, 49, 50
metals, 31, 49
metastasis, ix, x, 9, 12, 13, 14, 21, 22, 31, 34, 40, 47,
53, 54, 55, 56, 58, 62, 65, 68, 71, 72, 73, 74, 76,
77, 79, 80, 81, 82, 83, 89, 90, 91, 93, 108, 121,
122, 130, 140, 143, 144, 145, 147, 153, 220, 224,
228, 256, 260, 264
metastatic cancer, 215, 221
metastatic disease, 148
methylation, 76, 77, 92, 93
MHC, 110, 123
mice, 3, 9, 25, 38, 41, 45, 46, 109, 111, 114, 259,
283
microarray, 34, 52
microcirculation, 280
microenvironment, 27, 43
micrometer, 59, 265

296

Index

microscope, ix, 18, 239


microscopy, 3, 56, 60, 265
migration, 16, 17, 29, 31, 47, 71, 72, 75, 91, 160,
180, 265
milk, 106
minority, 171
mitochondria, 103
mitochondrial membrane, 251
mitogen, 43
mitosis, 40, 67
mitotic, 46, 217
mixing, 103, 107
mobility, 72, 165
models, 109, 270
modulus, 190
moieties, 99
molecular biology, 86
molecular oxygen, 250, 251
molecular weight, 99, 105
molecules, 32, 46, 49, 72, 74, 75, 92, 100, 110, 111,
250
monoclonal antibodies, 64
monoclonal antibody, 17, 36, 37, 38, 87
monocytes, 27, 47
monomer, 72, 90
Moon, 207
morbidity, xii, xiii, xv, 163, 165, 167, 177, 183, 184,
192, 193, 195, 197, 204, 207, 208, 242, 275
morphology, 213, 214
mortality, xii, 52, 53, 78, 155, 159, 163, 164, 165,
167, 168, 170, 179
mortality rate, xii, 52, 53, 155, 160, 164, 168
movement, 187, 196
MRI, xi, xiv, 125, 126, 133, 134, 137, 139, 141, 146,
148, 151, 152, 211, 213, 214, 245
mRNA, 31, 66, 97, 100, 108, 111, 120, 251
mucin, 217
mucoid, 217
mucosa, ix, 8, 9, 36, 54, 59, 63, 68, 70, 75, 81, 84,
89, 118, 126, 213, 223, 225, 239, 242, 249, 273,
274, 280, 283
mucus, 217
multiple factors, 133
multivariate, 55, 60, 76, 266
muscles, 126, 131, 132, 166, 199, 201, 237, 238,
277, 278
musculoskeletal system, 194
mutant, 17, 39, 43, 108, 115, 121, 257, 260, 264
mutation, xv, 22, 29, 90, 93, 253, 263, 264, 269, 270,
271, 272, 273, 274

mutation rate, 270


mutations, xv, 60, 66, 70, 76, 85, 93, 99, 108, 117,
263, 264, 265, 268, 270, 271, 272, 273, 274

N
N-acetyltransferase (NAT), xv, 247, 248
nanoparticles, 97, 99, 100, 102, 104, 106, 107, 111,
112, 117, 120, 123
nasal cavity, ix, 242
nasogastric tube, 157, 158, 162, 176, 178
nasopharyngeal carcinoma, 113, 114, 115, 244
nasopharynx, 233, 240, 241, 242
NATO, 49
neck cancer, 108, 253
necrosis, 25, 39, 40, 130, 147, 164, 209, 215, 283
neoplasia, 66, 90, 221, 223, 264
neoplasm, xv, 54, 126, 212, 213, 214, 217, 220, 223,
226, 240, 247, 248
neoplastic cells, 59, 60, 61, 64, 65, 66, 72, 77, 217,
220
neoplastic tissue, 74, 251, 262
neovascularization, x, 11, 12, 16, 17, 18, 19, 21, 24,
27, 31, 71
nerve, 137, 138, 160, 205, 230, 234, 239, 278
nerves, 126, 230, 234
Netherlands, 253, 255
network, 40, 273, 276, 279, 281
neural tissue, 234
neuralgia, 137
neuroblastoma, 231
neurofibroma, 243
neuroimaging, 245
neurological disease, 166
neurological disorder, 156
neutral lipids, 102
New York, 80, 193, 207, 209
nickel, 236
nitric oxide, 23, 24, 44
nitric oxide synthase, 24
nitrogen, 249, 250
nitrosamines, 249, 250, 254
NK cells, 109
nodes, 130, 133, 134, 138, 139, 144, 146, 148, 150,
215
nodules, 151, 245
non-Hodgkins lymphoma, 226
normal development, 46
normal distribution, 15
North America, 12

Index
Northern Ireland, 86
notochord, 220
nuclei, 14, 55, 215, 217, 218, 220
nucleic acid, 103, 111, 123
nucleophiles, 250
nucleotide sequence, 120, 251
nucleus, 23, 55, 66, 67, 98, 111, 218, 223
nutrition, 16, 156, 157, 163, 164, 175, 177, 178, 181
nutritional supplements, 177

O
obesity, 159, 166, 167
observations, 67
obstruction, 157, 160, 163, 164, 166, 168
ODN, 96, 111
oil, 3, 8, 99
older adults, 176
olefins, 249
oligodeoxynucleotides, 111, 119
oncogene, xi, xv, 37, 84, 95, 107, 122, 263, 265,
267, 268, 272, 273
oncogenes, 66, 264
oncogenesis, 99
opacification, 232
operator, 141, 167, 169
optic nerve, 240
optimization, 97
oral cancers, ix, xi, 52, 75, 88, 96, 125, 126, 128,
131, 132, 134, 138, 141, 142, 144, 145, 166, 227,
249, 272
oral cavity, xii, xiv, 10, 53, 54, 67, 68, 69, 74, 79,
80, 81, 82, 83, 84, 85, 86, 88, 89, 90, 92, 126,
128, 133, 141, 145, 146, 155, 157, 158, 165, 181,
211, 212, 214, 215, 216, 218, 220, 221, 225, 226,
227, 228, 230, 232, 236, 248, 251, 256, 257, 259,
272, 273, 274
oral leukoplakia, 87, 255, 258, 261
orbit, 232, 237, 240, 242
organ, 14, 17, 118, 143, 251
organic chemicals, 250
organization, 41, 72
orientation, 233
oropharynx, xii, 52, 53, 54, 68, 69, 82, 85, 86, 88,
145, 146, 155, 157, 163, 166, 170, 171, 181, 232,
233, 274
osteosarcoma, 96
osteotomy, xii, xiii, 183, 184, 185, 186, 187, 188,
189, 190, 191, 192, 195, 196, 197, 198, 200, 202,
203, 205, 206, 239

297

otolaryngologist, 157, 167


ovarian cancer, 18, 29, 260
oviduct, 106
oxidation, 250, 253
oxidative damage, 32
oxidative stress, 31
oxygen, 16, 17, 18, 19, 20, 23, 25, 26, 32, 39, 40, 41,
42, 43, 44, 45
Oxygen, 17, 26, 40, 41, 49
oxygen consumption, 23
oxygen sensors, 41

P
p53, 15, 22, 24, 35, 36, 37, 60, 65, 66, 69, 76, 85, 86,
88, 89, 90, 99, 106, 108, 113, 114, 119, 121, 260,
264, 265, 267, 271, 272, 273, 274
paclitaxel, 96
pain, 239
palliative, 159, 166
palliative care, 159
palpation, 68, 139, 158, 236
pancreas, 62
pancreatic cancer, 25
parameter, 40, 55
Paris, 209
parotid, xiv, 135, 139, 146, 149, 150, 151, 216, 218,
229, 233, 237, 238, 276, 279
parotid gland, xiv, 135, 139, 146, 150, 216, 218,
229, 233, 237, 238, 279
particles, 99, 101, 102, 120, 134, 149
pathogenesis, 61, 83, 85
Pathologists, 57
pathology, xi, xii, xiv, 14, 38, 125, 138, 147, 148,
155, 167, 170, 171, 229, 230, 245
pathophysiology, 41
pathways, 24, 75, 76
patient management, 168, 170, 245
PCR, 60, 224, 251, 265, 266
pectoralis major, 281
peptides, 110
perception, 53
perforation, 157
performance, 131, 175
perfusion, 17, 18, 23, 26, 40, 205, 279
periodontal, 147
periosteum, 185, 239
peripheral vascular disease, xiv, 196, 206
peritonitis, 166
permeability, 16, 21, 22, 31, 38, 46, 48, 71, 100, 249

298

Index

permit, 8
personal, 167, 192, 204
personal communication, 204
PET, xii, 126, 131, 141, 142, 143, 144, 145, 152,
153, 213, 215
pH, 9, 106, 119, 120, 265, 266
pharmaceuticals, 104
pharynx, xiv, 79, 141, 143, 165, 211, 214
phenotype, 70, 72, 73, 75, 98, 109, 257, 271
phenotypes, 257
phosphatidylethanolamine, 96, 101, 102, 106
phosphorylase, x, 11, 29, 43, 46, 47, 48, 49
phosphorylation, 29, 65, 141
photosensitivity, 2
physiology, 41
pilot study, 93, 260
placenta, 16, 28, 39, 41, 46, 48, 90, 118
planning, 214, 278
plasma, 10, 103, 118
plasma membrane, 118
plasmid, 97, 99, 106, 107, 108, 109, 110, 111, 115,
116
Plasmids, 97
plasminogen, 17, 35
Platelet, 47, 48
platelet count, 166
plexus, 104, 118, 278, 279, 280
ploidy, 37
PM, 10, 63, 78, 79, 89, 92, 145, 146, 147, 227, 245
pneumonia, 167
point mutation, 66, 253
polarity, 75
polyacrylamide, 266
polycyclic aromatic hydrocarbon, 249
polyethylene, 119
polyethylenimine, 106
polymer, 103
polymerase, 97, 265, 266, 272
polymerase chain reaction, 272
polymers, 100, 103, 115
polymethylmethacrylate, 186
polymorphism, 57, 253, 254, 255, 256, 257, 258,
260, 261, 262
polymorphisms, 252, 253, 254, 255, 256, 257, 258,
259, 260, 261
poor, x, xiv, 2, 11, 12, 13, 14, 16, 19, 30, 32, 54, 58,
66, 68, 70, 71, 73, 75, 76, 85, 89, 90, 91, 99, 163,
164, 165, 169, 174, 180, 196, 206, 222, 223, 264,
271, 272, 274

population, 34, 45, 67, 69, 79, 166, 167, 218, 220,
248, 249, 254, 255, 256, 257, 258, 277, 279, 280
porphyrins, 9
portal hypertension, 166
Portugal, 272, 273
positive correlation, x, 11, 18, 19, 21, 29, 64
positron, xii, 43, 126, 141, 152, 153, 213, 215
positron emission tomography, xii, 43, 126, 141,
152, 153, 213, 215
posterior cortex, 204
precancerous lesions, 212, 214, 224
predictability, 278, 279
prediction, xi, 52, 55, 215, 264
predictors, 34, 55, 64, 171
preference, xiii, 167, 192, 195, 196, 206
preservative, 213
pressure, 8, 100, 199, 204, 208, 280
prevention, 78, 175, 205, 224, 258, 259
primary tumor, 34, 47, 54, 55, 60, 63, 72, 76, 77,
141, 180, 215, 224
private practice, 227
probability, 31, 105, 266
probe, 141, 279
prodrugs, 110
production, 2, 41, 97, 99, 109, 145
prognosis, x, xi, 11, 12, 13, 14, 16, 19, 21, 22, 30,
31, 32, 33, 34, 35, 36, 45, 47, 48, 51, 52, 53, 54,
55, 56, 57, 58, 60, 61, 64, 65, 66, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 80, 81, 82, 85, 86, 89, 90,
91, 108, 130, 176, 245, 264, 268, 271, 272, 274
prognostic value, xv, 37, 55, 56, 57, 60, 61, 64, 67,
70, 78, 81, 263, 264, 268
program, 70
proliferation, x, xi, 11, 14, 15, 16, 17, 20, 22, 26, 29,
33, 36, 37, 38, 41, 43, 51, 52, 62, 66, 67, 68, 69,
70, 71, 72, 87, 88, 90, 107, 108, 109, 111, 116,
273
promote, 111, 260, 279
promoter, 75, 76, 77, 93, 97, 98, 113, 116, 117
promoter region, 75, 77, 97
propane, 100, 101
prophylactic, xiii, 184, 192, 195, 198, 206, 207, 208
prophylaxis, 157, 176, 179
prostaglandins, 251
prostate, 37, 43, 62, 111, 117, 122, 218, 252, 262
prostate cancer, 37, 117, 122, 262
protein, x, 9, 12, 24, 28, 29, 31, 33, 37, 41, 42, 43,
44, 46, 62, 65, 66, 67, 69, 70, 76, 80, 84, 86, 87,
89, 91, 97, 100, 103, 108, 109, 110, 111, 118,
119, 250, 251, 253, 261, 264, 271, 272, 273

Index
protein kinase C, 91
protein kinases, 28
protein sequence, 251
protein synthesis, 43, 103
proteinase, 265
proteins, 14, 21, 23, 24, 31, 34, 61, 70, 72, 74, 75,
86, 87, 89, 99, 103, 108, 118, 251, 256, 272, 274
proteolysis, 23
protocols, 265
proto-oncogene, 69
pterygopalatine foss, xiv, xv, 229
pulse, 100, 147
pulses, 100
pyrene, 252, 256
pyrimidine, 46, 103

Q
quality of life, 54, 157, 164, 165, 166, 169, 178

R
race, 12, 268
radiation, xi, 15, 26, 32, 35, 38, 45, 53, 82, 96, 108,
119, 121, 125, 139, 152, 153, 177, 180, 215, 280,
283
Radiation, 11, 146, 215, 226, 274
radiation therapy, xi, 35, 82, 96, 125, 152, 153, 177,
180
radio, 57, 60, 152
radiography, 147, 236
radiotherapy, xi, xii, 2, 13, 16, 25, 32, 37, 40, 45, 48,
49, 52, 60, 64, 68, 80, 85, 95, 108, 114, 116, 119,
121, 134, 135, 138, 139, 141, 148, 150, 155, 156,
157, 158, 159, 161, 162, 163, 169, 170, 171, 172,
173, 174, 180, 215, 264, 271, 272, 274, 281
radius, xiii, 184, 185, 191, 192, 193, 194, 196, 197,
198, 199, 204, 205, 207, 208, 209
range, xiii, xv, 13, 15, 103, 104, 141, 159, 170, 186,
195, 197, 203, 206, 233, 263, 267, 277
reactive oxygen, xv, 247, 248, 250
reading, 187
reality, 209
receptors, xi, 27, 28, 38, 46, 48, 62, 84, 95, 97, 103,
117, 118
recognition, 97, 109, 122
recombination, 99
reconstruction, xii, xiv, xvi, 52, 127, 131, 149, 156,
158, 159, 160, 165, 169, 170, 171, 172, 173, 174,

299

185, 189, 190, 192, 193, 197, 199, 201, 203, 206,
207, 208, 209, 210, 229, 234, 241, 246, 275, 276,
277, 278, 279, 280, 281, 282
recovery, 45, 158, 163, 172, 174
rectum, 160
rectus abdominis, 242
recurrence, xv, 36, 47, 64, 68, 69, 72, 73, 74, 79, 82,
91, 133, 141, 152, 215, 224, 227, 263, 264, 265,
268, 271, 274
recycling, 104
redistribution, 26
reduction, 66, 75, 76, 169, 171, 184
redundancy, 24, 34, 43
reflection, 237, 270
reflexes, 168
refractory, x, 12, 22, 25, 96, 274
regional, xv, 44, 54, 55, 57, 58, 60, 65, 72, 76, 77,
82, 99, 146, 193, 209, 240, 275, 277
regression, xv, 108, 109, 110, 119, 121, 263, 268
regression analysis, xv, 263, 268
regulation, 23, 24, 31, 39, 41, 42, 43, 66, 69, 75, 86,
87, 89, 92, 108, 109, 111, 118, 121, 259, 279
regulators, 89, 111
rehabilitation, 165, 177, 178
reinforcement, xiii, 183, 185, 189, 190, 191, 192,
198, 199, 202, 204
rejection, 110
relapses, 57, 60
relationship, xii, 2, 16, 20, 21, 24, 25, 26, 29, 30, 31,
37, 39, 40, 60, 61, 64, 65, 66, 68, 69, 70, 71, 73,
74, 76, 89, 93, 130, 135, 136, 155, 163, 171, 172,
212, 214, 234, 254, 258, 265, 277, 279
relationships, 14, 22, 26, 31, 33, 36, 40, 64, 69, 73,
257
relatives, 248
relevance, 44, 85, 88, 90
reliability, 242, 245
remodeling, 38, 71
remodelling, 205, 209
renal cell carcinoma, 29, 47, 48, 104
repair, x, 11, 33, 65, 204, 279
replication, 65, 250
resection, xii, xiv, 52, 60, 79, 140, 144, 145, 155,
160, 161, 162, 163, 165, 169, 172, 173, 174, 175,
229, 237, 239, 241, 246, 277
residues, 62
resistance, 22, 25, 26, 32, 44, 49, 59, 97, 117, 271
resolution, 131, 144, 148, 234
respiratory, 80, 165, 168, 169
respiratory problems, 165

Index

300

responsiveness, 70
retention, 165
reticulum, 251
retinoblastoma, 65, 108
retinoic acid, 117
retroviruses, 98
RF, 78, 128
rigidity, 209
risk, xii, xv, 2, 60, 66, 68, 70, 73, 74, 77, 78, 79, 80,
82, 86, 88, 91, 155, 157, 160, 163, 164, 166, 167,
168, 169, 171, 173, 179, 180, 184, 192, 194, 198,
204, 205, 212, 215, 224, 236, 239, 247, 248, 249,
250, 252, 253, 254, 255, 256, 257, 258, 259, 260,
261, 262, 274
risk assessment, 180, 258
risk factors, xv, 78, 79, 166, 179, 194, 212, 247, 248,
261
RNA, 96, 97, 110, 111, 124
RNA processing, 110
RNAi, 96, 111

S
SA, 9, 44, 78, 86, 147, 153, 176, 179, 246, 259, 273
safety, 99
saliva, 9, 106, 134
salivary glands, xiv, 52, 53, 54, 126, 134, 149, 211,
212, 218, 222, 225, 226
salts, 32
sample, ix, 2, 3, 4, 53, 66, 79, 136, 188, 189, 213,
224, 257, 265, 267, 270
saturation, 128, 131, 132, 147
scapula, 193, 208
scatter, 90
science, 194, 280
scientific community, 264, 270
scores, 165, 169
search, xv, 275
secrete, 39
secretion, 17, 72, 97, 106
sedimentation, 213
segregation, 118
selecting, 31, 57
selectivity, 2, 112
self-assembly, 107
sensing, 43
sensitivity, xiv, 49, 61, 63, 64, 111, 123, 139, 212,
215, 221, 222, 223, 224, 236
sensitization, 26, 121
separation, 237

sequencing, 270, 272


series, xii, xiii, 53, 55, 68, 70, 71, 79, 102, 155, 157,
165, 167, 168, 170, 171, 184, 195, 197, 204, 206,
270, 271
serum, 8, 99, 108, 120, 283
severity, 63, 67, 88, 167, 205
sex, 53, 76, 265
shape, 126, 130, 193, 197, 201, 207, 215, 233
sharing, 190, 193, 207
sheep, xii, 183, 184, 185, 191, 192, 193, 196, 204,
207
shock, 280, 283
shock waves, 280
sialography, xi, 125, 126, 134, 145, 149
side effects, 8, 109
signal transduction, 46, 62, 74, 123
signaling, 28, 29, 34, 42, 45, 62, 64, 65
signaling pathway, 28, 29, 34, 42
signaling pathways, 28, 29, 34
signalling, 90
signals, 46, 61, 70, 131, 138, 265
significance level, 267
silver, 265, 274
similarity, 42, 55, 251, 255
Singapore, 244
sinus, 230, 232, 233, 239, 240, 241, 242
sinuses, 127, 128, 230, 232, 233, 240, 244, 246
sinusitis, 164
siRNA, 96, 111
sites, xiii, 46, 97, 107, 120, 123, 130, 149, 159, 162,
163, 168, 171, 184, 192, 193, 195, 196, 197, 201,
202, 203, 204, 205, 206, 210, 212, 214, 215, 221,
257, 268, 270
Sjgrens syndrome, 134
skin, 2, 8, 9, 10, 45, 199, 200, 202, 204, 236, 238,
276, 277, 278, 279, 280, 283
sleep apnea, 213, 225
smoke, 249, 260
smokers, xv, 250, 263, 267
smoking, 163, 249, 253, 254, 255, 258, 260, 270
SNP, 256
sodium, 3, 120
soft palate, 80, 213, 216, 217, 225
soft tissue tumors, 151
software, 266
solid tumors, 26, 39, 43, 44, 54, 111
solubility, 252
somatic mutations, 75, 260
Spain, 51, 52, 78, 263, 264
species, xv, 247, 248, 250, 252, 253, 260

Index
specificity, xiv, 64, 97, 99, 113, 118, 119, 139, 212,
215, 221, 223, 224, 236, 252
spectrophotometer, ix, 2, 3, 4
spectroscopy, 2
spectrum, xiv, 4, 5, 211, 214, 270
speech, 157, 164, 171
spin, xi, 125, 136, 137, 138, 149
spindle, 218, 220
splint, 208
SPSS, 267
Sri Lanka, 254, 262
St. Louis, 145, 146
stability, 97, 101, 186, 197
stabilization, 42, 99, 261
stabilizers, 97
stages, xv, 54, 55, 56, 66, 68, 75, 171, 212, 248, 263,
264, 267, 268, 271, 272
standard error, 187
Staphylococcus, 159, 180
starvation, 163
statistical analysis, 188, 279
statistical inference, 187
statistics, 112, 224
steel, 187, 190, 194, 201, 203, 209
stem cells, 280
stenosis, 12, 166
sterile, 158
steroids, 251
stock, 205, 210
stoma, 168, 170
stomach, 8, 76, 158
S-transferase (GST), xv, 247, 248
strategies, xi, 52, 63, 64, 95, 98, 107, 175, 176, 206,
258, 268
stratification, 55
strength, xiii, 98, 183, 184, 185, 187, 188, 189, 190,
191, 192, 193, 196, 197, 204, 205, 207, 208
stress, 33, 163, 186, 190, 191, 194, 196, 204, 205,
208, 209
stroke, 165, 176, 178
stroma, 17, 38
stromal cells, 283
subcutaneous tissue, 277
subgroups, 161, 163, 176
substitution, 253
substrates, 250, 251, 253
success rate, 167, 178
suicide, 98, 110, 114, 117
Sun, 178
superimposition, 136

301

supervision, 8
supply, xv, 20, 23, 233, 275, 276, 277, 279
suppression, 76, 108, 109, 110, 111, 122, 123, 131,
132, 147, 148
surface area, 45, 57
surface layer, 55
surfactant, 4
surfactants, 102
Surgeons, 168, 194
surgical resection, 79, 170, 171, 172
surveillance, 122
survival, xi, xiv, 12, 13, 26, 28, 30, 34, 36, 37, 41,
43, 45, 49, 51, 52, 53, 54, 55, 56, 57, 58, 60, 64,
65, 66, 68, 70, 71, 74, 76, 79, 80, 82, 86, 89, 95,
96, 109, 158, 163, 180, 211, 212, 265, 266, 268,
270, 271, 272, 277, 280, 282
survival rate, xi, xiv, 12, 30, 51, 52, 53, 54, 55, 58,
60, 68, 96, 211, 212
Survivin, 70, 89
susceptibility, xv, 236, 247, 248, 252, 253, 255, 256,
257, 258, 259, 260, 261, 262
suture, 160, 164, 170
SUV, 141, 144, 145
swallowing, 157, 163, 164, 166, 171, 172
Sweden, 265
swelling, 130, 215, 217, 219, 220
symptoms, 10
syndrome, 149, 168, 180, 213, 225
synthesis, 8, 23, 65, 103, 106, 107, 110
systemic circulation, 31, 107
systems, 34, 56, 77, 97, 99, 100, 101, 103, 105, 110,
116, 223

T
T cell, 122, 123
T lymphocyte, 109
Taiwan, 79, 85, 256, 258
targets, 111, 259, 261
taxanes, 96
T-cell, 109
technology, xiv, 34, 224, 225, 229, 230, 234
temperature, 266
tension, 17, 18, 19, 20, 23, 25, 26, 40, 41, 42, 44,
168, 190, 193, 208
teratoma, ix
ternary complex, 106, 107
territory, 279, 280
Tesla, 128
Texas, 27, 151

302

Index

textbooks, 53
TGF, 62, 63, 65, 116, 121
Th cells, 123
T-helper cell, 109
theory, 15, 209, 271
therapeutics, 103
therapy, ix, xi, 1, 2, 8, 9, 10, 32, 35, 44, 78, 79, 82,
89, 95, 96, 97, 98, 102, 107, 109, 110, 111, 112,
113, 115, 116, 120, 122, 124, 146, 166, 178, 234,
236, 264, 268, 280, 282, 283
thinking, 280
threshold, 66, 133, 168, 173
thymidine, x, 11, 29, 43, 47, 48, 49, 96, 98, 110, 114,
117, 120, 123
thymine, 253
thyroid, 139, 151
thyroid gland, 139, 151
tibia, xii, xiii, 183, 184, 185, 186, 190, 191, 192,
193, 196, 204, 207, 209
time, x, xii, 2, 3, 9, 55, 64, 67, 68, 129, 131, 132,
133, 134, 135, 155, 159, 168, 169, 170, 171, 174,
175, 183, 200, 203, 237, 257, 266, 267, 268, 278
timing, 8, 158, 169, 176
TIR, 131
tissue, ix, xii, xiii, xv, 1, 2, 5, 6, 7, 8, 9, 14, 15, 17,
18, 19, 20, 21, 23, 25, 26, 27, 29, 31, 39, 44, 47,
58, 59, 60, 64, 66, 67, 72, 73, 74, 77, 92, 97, 99,
100, 103, 104, 105, 113, 118, 126, 128, 129, 133,
141, 143, 149, 150, 155, 161, 162, 163, 172, 185,
195, 196, 206, 213, 218, 222, 231, 234, 236, 240,
241, 246, 251, 254, 260, 265, 275, 277, 280, 281,
283
tissue plasminogen activator, 265
titanium, 185, 186, 189, 190, 194, 201, 203, 209
TNF, 111
TNF-, 111
tobacco, xv, 85, 126, 177, 212, 247, 248, 249, 250,
252, 255, 256, 257, 261, 272, 273, 274
tobacco smoke, 249, 256, 257
tobacco smoking, 261
Tokyo, 3, 4, 46, 95, 120, 126, 127, 128, 129, 209,
259
tongue, ix, xi, xiv, xv, 1, 2, 3, 5, 6, 7, 8, 9, 10, 54,
55, 57, 58, 64, 68, 73, 74, 76, 78, 79, 80, 81, 82,
83, 84, 88, 91, 92, 93, 108, 114, 125, 135, 140,
141, 142, 143, 144, 145, 148, 149, 151, 152, 165,
171, 211, 214, 215, 216, 218, 222, 223, 226, 227,
251, 255, 263, 267, 272, 274
tonsils, 141, 143
toxicity, ix, 1, 2, 102, 104, 109

TPA, 265
tracheostomy, 159, 168, 169
training, 167
traits, 56
transcription, 23, 42, 43, 65, 77, 97, 98, 122, 254
transcription factors, 77
transcripts, 251
transducer, 141, 187
transduction, 100, 110, 113
transfection, xi, 26, 95, 97, 99, 101, 102, 105, 106,
107, 108, 109, 111, 112, 116, 117, 120, 122, 123
transferrin, xi, 95, 97, 103, 107, 120, 121
transformation, 30, 66, 67, 70, 73, 74, 86, 87, 122,
212, 258
transformations, 270
transforming growth factor, 62, 65, 84, 121
transgene, 97, 112
transgenic, 259
transition, 65, 66
translation, 97, 111
translocation, 164, 240, 241, 246
transmembrane glycoprotein, 74
transplantation, 283
transport, 97, 103, 104, 118, 213
trapezius, 281
trauma, xii, 147, 155
trend, 212
trial, 35, 114, 117, 136, 149, 157, 165, 169, 176,
177, 178, 179, 206
trigeminal nerve, 136, 137, 138, 149, 230
trigeminal neuralgia, 137, 138
triggers, 116
tumor cells, 16, 27, 32, 39, 47, 49, 57, 58, 61, 71, 72,
73, 97, 98, 100, 101, 103, 106, 107, 108, 109,
110, 119, 122, 214, 215, 217, 218, 221
tumor depth, 58
tumor growth, xi, 16, 23, 24, 25, 27, 29, 34, 38, 43,
45, 47, 52, 69, 90, 108, 109, 110, 111, 112, 115,
122
tumor invasion, 15, 58, 68, 69, 73, 77
tumor metastasis, 36, 88
tumor necrosis factor, 115
tumor progression, 44, 64, 69, 70, 72, 73, 86, 92
tumor proliferation, 37
tumor resistance, 45
tumorigenesis, 84, 108
tumors, ix, xi, xiv, xv, 1, 2, 3, 17, 20, 24, 25, 26, 31,
33, 34, 38, 39, 40, 41, 43, 44, 46, 48, 53, 54, 55,
56, 57, 58, 59, 60, 62, 63, 64, 65, 68, 71, 73, 74,
75, 76, 77, 79, 82, 95, 97, 104, 105, 108, 109,

Index
119, 120, 122, 125, 126, 129, 131, 133, 136, 137,
141, 145, 175, 177, 211, 213, 214, 216, 218, 222,
226, 227, 230, 231, 232, 236, 237, 239, 240, 244,
245, 246, 260
tumour growth, 39
tumours, xii, xv, 9, 36, 40, 41, 80, 121, 147, 150,
151, 155, 156, 161, 165, 166, 169, 170, 171, 172,
173, 174, 175, 224, 263, 264, 265, 267, 268, 271
tyrosine, 27, 45, 46, 62, 64

U
UK, 52, 155, 157, 185, 187, 201
ulcer, 216
ulceration, 13
ulna, 194, 199, 209
ultrasonography, 126, 138, 139, 140, 141, 142, 144,
151, 152, 236
ultrasound, xi, 125, 141, 149, 150, 151, 157, 169,
205, 236, 245, 279
uncertainty, 18
uniform, 9, 17, 20, 22, 26, 187, 217, 218
United Kingdom, 183, 195, 200, 205
univariate, 55, 60, 76
urea, 266
urinary tract, 166
urinary tract infection, 166
urokinase, 35
users, 256, 273
uterus, 118
UV, 9

303

VEGF, x, 11, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 38, 39, 42, 44, 45, 46,
48, 71, 90, 280
VEGF expression, x, 11, 15, 18, 19, 20, 21, 23, 24,
26, 29, 30, 31, 42, 71
vein, 277, 278, 280
vesicle, 117
vessels, xi, 17, 18, 22, 26, 45, 71, 125, 126, 131,
136, 138, 145, 234, 276, 277, 282
vimentin, 37
viral vectors, xi, 95, 97, 99, 102, 103, 109, 112
virus, 96, 97, 98, 99, 107, 110, 112, 114, 115, 117,
120, 121, 123
viruses, 83, 98, 99, 280
vision, 157
visualization, xiv, 127, 138, 213, 229, 239, 240, 244

W
weakness, 25
web, 159, 160, 167
weight gain, 165, 174
weight loss, 163, 164, 169, 171, 174, 180
WHO, 54
windows, 127
women, 52, 53, 194
World Health Organization, 212
World Health Organization (WHO), 212
wound healing, 17, 23, 38, 280, 283
wound infection, 160, 202

X
V
validity, 241
valine, 253
values, 5, 8, 15, 58, 133, 170, 191, 221, 223, 266
variability, 188
variable, 107, 167, 169, 171, 190, 205, 233, 264
variable factor, 205
variables, xv, 36, 83, 91, 263, 265, 267, 268, 270
variation, 15, 16, 53, 60, 64, 66, 141, 187, 212, 215,
221, 252, 253, 260
vascular endothelial growth factor (VEGF), x, 11,
39, 71, 280
vasculature, 10, 239
vector, 97, 99, 100, 106, 108, 110, 112, 113, 115,
123

xenobiotics, 250, 251, 252, 253


xenografts, 9, 17, 18, 20, 22, 25, 26, 27, 31, 39, 40,
47, 100, 108, 109, 110, 111, 121
xerostomia, xi, 125, 134, 145, 163

Y
yield, 170, 190, 213, 253
young adults, 78, 82

Z
zygomatic arch, 237, 238, 239, 279

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