ORAL CANCER RESEARCH ADVANCES

ORAL CANCER RESEARCH ADVANCES No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

ORAL CANCER RESEARCH ADVANCES

ALEXIOS P. NIKOLAKAKOS EDITOR

Nova Biomedical Books New York

Copyright © 2007 by Nova Science Publishers, Inc.

All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA 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 2007-11-07 616.99’491--dc22 2007026603

Published by Nova Science Publishers, Inc.

New York

CONTENTS Preface

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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 Márcio Diniz-Freitas, Eva Otero-Rey, Andrés Blanco-Carrión, Tomás García-Caballero, José Manuel-Gándara Rey and Abel GarcíaGarcía

Chapter 4

Tumor-Targeting Non-Viral Gene Therapy for the Treatment of Oral Cancer Yoshiyuki Hattori and Yoshie Maitani

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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

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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 Gonçalves Cunha and Dennis de Carvalho Ferreira

211

229

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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. López Cedrún

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

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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.

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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

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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 (P10% 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-β).

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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

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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.

a

b

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 γ2 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 α chain, a β chain and a γ 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 α3, a β3 and a γ2 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 γ2 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 α3β1, α6β1, α6β4, and α2β1 [207] (Figure 8). Results to date suggest that laminin 5 γ2 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 γ2 may serve as a marker of invasiveness in diverse types of SCC. It has been suggested that laminin 5 γ2 secreted by tumor cells stimulates cell motility and thus invasiveness [208]. Experimental studies have demonstrated that laminin 5 γ2 promotes cell dispersion when added to epithelial cell cultures [209,210].

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a

73

b

Figure 8. a) Expression of laminin 5 γ2 is limited to epithelial tissues, where it has roles in epithelial attachment and in cell motility. B) Increased laminin 5 γ2 expression is seen at the tumor invasion front, and has been associated with increased invasive potential.

The higher expression of laminin 5 γ2 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 γ2 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 γ2 in premalignant lesions that subsequently underwent transformation to OSCC or not. The authors found laminin 5 γ2 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 γ2 expression have a higher risk of undergoing malignant transformation. To date, there have been few studies of the relationship between laminin 5 γ2 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 γ2 in oral cancer. In a study of 67 SCCs of the tongue, these authors did not find any relationship between laminin 5 γ2 expression and tumor stage, cervical metastasis or depth of invasion. They did observe a relationship between laminin 5 γ2 expression and both tumor grade and mode of invasion: with increasing laminin 5 γ2 expression the tumor showed more advanced undifferentiation and a more invasive pattern. In addition, increasing laminin 5 γ2 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 γ2 expression was associated with increased tumor invasion depth and poorer prognosis. The authors also investigated possible relationships between EGFR and laminin 5 γ2 expression, finding that tumors with high EGFR expression also showed high laminin 5 γ2 expression.

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Stoltfuz et al. [220] studied laminin 5 γ2 expression in 36 T1 tumors of the tongue. They did not find any significant relationship with tumor histological grade; however, increased laminin 5 γ2 expression was correlated with increased risk of recurrence. Lim et al. [221] did not find any association between laminin 5 γ2 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 γ2 expression and survival. However, we observed increased laminin 5 γ2 expression in dysplastic epithelium adjacent to OSCCs (Figure 9) [222], suggesting that laminin 5 γ2 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 γ2 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 α, β and γ catenins. The cytoplasmic domain of E-cadherin binds directly to β and γ, and the resulting E-cadherin/β-catenin/γ-catenin is anchored to the actin cytoskeleton via catenin α [225]. E-cadherin also participates in signal transduction

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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].

a

b

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.

Bánkfalvi et al. [236] studied the role of various molecules involved in cell adhesion in the epithelium (CD44, E-cadherin, β-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 γ2, E-cadherin, βcatenin) 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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REFERENCES [1] [2]

[3] [4] [5] [6]

[7] [8] [9]

[10]

[11]

[12] [13]

[14]

[15] [16]

Scully C, Bagan JV. Recent advances in Oral Oncology. Oral Oncol 2007;43(2):10715. Ozols RF, Herbst RS, Colson YL, Gralow J, Bonner J, Curran WJ Jr, et al. Clinical cancer advances 2006: major research advances in cancer treatment, prevention, and screening--a report from the American Society of Clinical Oncology. J Clin Oncol 2007;25: 146-62. Jonson NW, Warnakulasurya KAA. Oral Cancer: Is it more common than cervical? Br Dental Res 1991; 170: 170-171. Kligerman J. Estimativas sobre a incidencia e mortalidade por cáncer no Brasil-2002. Rev Bras Cancerologia 2002; 48: 175-9. Nieto A, Ramos AR. Rising trends in oral cancer mortality in Spain, 1975-94. J Oral Pathol Med 2002; 31: 147-52 Wood NK, Sawyer DR. Cáncer Oral. En: Wood NK, Goaz PW. Diagnósticos diferenciales de las lesiones orales y maxilofaciales. Madrid: Harcourt Brace de España SA; 1998. p. 587-95 Godwin JS, Hunt WC, Janet JM. Determinants of cancer therapy in elderly patients. Cancer 1993; 72: 594-601. Reichard KW, Joseph KT, Cohen M, Greager JA. Squamous cell carcinoma of the tongue: Experience with 86 consecutive cases. J Surg Oncol 1993; 54: 239-42. El-Husseiny G, Kandil A, Jamshed A, Khafaga Y, Saleem M, Allam A, et al. Squamous cell carcinoma of the tongue: an analysis of prognostic factors. Br J Oral Maxillofac Surg 2000; 38: 193- 9. Varela-Centelles PI, Seoane J, Vazquez Fernandez E, De La Cruz A, Garcia Asenjo JA. Survival to oral cancer. A study of clinical risk markers with independent prognostic value. Bull Group Int Rech Sci Stomatol Odontol. 2002; 44: 46-51. Mackenzie J, Ah-See, Takker N, Sloan P, Maran AG, Birch J, et al. Increasing incidence of oral cancer amongst young persons: what is the aetiology? Oral Oncol 2000; 36: 387-9. Oliver RJ, Dearing J, Hindle I. Oral cancer in young adults: report of three cases and review of the literature. Br Dent J 2000; 188: 362-6. Llewellyn CD, Johnson N, Warnakulasuriya S. Factors associated with delay presentation among younger patients with oral cancer. Oral Surg Oral Pathol Oral Med Oral Endod Radiol 2004; 97: 707-13. Llewellyn CD, Linklater K, Bell J, Johnson N, Warnakulasuriya S. An analysis of risk factors for oral cancer in young people: a case-control study. Oral Oncol 2004; 304313. Sarkaria JN, Harari PM. Oral tongue cancer in young adults less than 40 years of age: rationale for aggressive therapy. Head Neck 1994; 16: 107-11. Rennie JS, McGregor AD. Intra-oral squamous cell carcinoma in patients under 40 years of age. A report of 13 cases and review of the literature. Br J Plastic Surg 1987; 40: 270-3.

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[17] Sasaki T, Moles DR, Imai Y, Speight PM. Clinico-pathological features of squamous cell carcinoma of the oral cavity in patients 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 IκBα 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

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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

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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

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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

O

+

X=

N

N

O

HO-

DC-Chol O

DODAP

O

+

X = (CH3)2NCH2CH2NHCO

O O

OH-Chol +

+ N

X = HOCH2CH2NHCH2CH2NHC

O O

DMRIE

O

+

HO

N O O

DOPE + H3N

O O

-

P O-

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-3β-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-α 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 β-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-3β-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

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(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-α and -β, are attached to the cell by a GPI-anchor, while FR-γ 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

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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-α is frequently overexpressed in tumors, including ovarian, colorectal, breast, lung, renal cell carcinomas and brain metastases derived from epithelial cancers [72,85]. FR-β is frequently overexpressed in tumors of nonepithelial cell lineages such as sarcomas and acute myeloid leukemia [86], and FR-γ 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.

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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]

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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

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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.

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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

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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 paitient’s 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-γ 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

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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 β2-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-2’deoxyuridine-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].

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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-α 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) 21–23 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

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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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Yoshiyuki Hattori and Yoshie Maitani [156] Mamot, C. and Rochlitz, C. (2006) Targeting the epidermal growth factor receptor (EGFR)--a new therapeutic option in oncology? Swiss.Med.Wkly., 136, 4-12. [157] Dias, N. and Stein, C.A. (2002) Potential roles of antisense oligonucleotides in cancer therapy. The example of Bcl-2 antisense oligonucleotides. Eur.J.Pharm.Biopharm., 54, 263-269. [158] Pai,S. I., Lin, Y.Y., Macaes, B., Meneshian, A., Hung, C.F., and Wu, T.C. (2006) Prospects of RNA interference therapy for cancer. Gene Ther., 13, 464-477. [159] McManus, M.T. and Sharp, P.A. (2002) Gene silencing in mammals by small interfering RNAs. Nat.Rev.Genet., 3, 737-747. [160] Scherr, M., Morgan, M.A., and Eder, M. (2003) Gene silencing mediated by small interfering RNAs in mammalian cells. Curr.Med.Chem., 10, 245-256. [161] Takeshita, F. and Ochiya, T. (2006) Therapeutic potential of RNA interference against cancer. Cancer Sci., 97, 689-696. [162] Brand, F.X., Ravanel, N., Gauchez, A.S., Pasquier, D., Payan, R., Fagret, D., and Mousseau, M. (2006) Prospect for anti-her2 receptor therapy in breast cancer. Anticancer Res., 26, 715-722.

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

2

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: [email protected].

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Yasuhiro Morimoto, Tatsurou Tanaka, Izumi Yoshioka et al. 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 lesion’s 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

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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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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 Waldeyer’s ring [5]. Furthermore, masses affected by dental metal streak artifact are often undetectable on X-ray CT scanning (Figure 5). It has been reported

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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 Sjögren’s 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 rotatiåons.

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 hospital’s 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 (P4 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