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

ix

Chapter 1

Effective Administration Methods of 5-Aminolevulinic Acid as a Photosensitizer in Photodynamic Therapy for Tongue Tumor 1 Toshiyuki Ogasawara, Norio Miyoshi, Kazuo Sano, Hidetaka Kinoshita, Tetsushi Yamada, Toru Ogawa, Kazuki Miyauchi and Yoshimasa Kitagawa

Chapter 2

Relationships Between Biological and Clinicopathologic Features in Esophageal Carcinoma Takuma Nomiya, Kenji Nemoto and Shogo Yamada

Chapter 3

Prognostic Indicators in Oral Squamous Cell Carcinoma 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

11 51

95

Chapter 5

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

Chapter 6

The Role of the Percutaneous Endoscopic Gastrostomy in the Management of Head and Neck Malignancy CME Avery

155

The Biomechanical Basis for Internal Fixation of the Radial Osteocutaneous Donor Site CME Avery

183

Chapter 7

125

Chapter 8

Contents

vii

The Current Role of Prophylactic Internal Fixation of the Radial Osteocutaneous Donor Site CME Avery

195

Chapter 9

Cytologic Diagnosis of Oral Malignancies: Scope and Limitations Dilip K. Das

Chapter 10

Benign and Malignant Tumors Occurring in the Pterygopalatine Fossa and Adjacent Structures of the Pterygopalatine Fossa: Recent Advances of Diagnosis and Surgical Management Xin-Chun Jian

Chapter 11

Molecular Aspects of Oral Cancer: the Role of Phase I and II Biotransformation Enzymes in Carcinogenesis Karin Soares Gonçalves Cunha and Dennis de Carvalho Ferreira

211

229

247

Chapter 12

TP53 Mutation, c-myc Amplification and Squamous Cell Carcinoma Recurrence 263 J. Seoane, P. Varela-Centelles, M.A. Romero , A. De la Cruz, F. Barros, L. Loidi and J.L. 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 (P<0.001), particularly for hemiglossectomy (P=0.02) and maxillectomy procedures (P=0.003), following a segmental composite bone resection rather than a soft tissue resection, with or without a rim resection, (P=0.03) and finally radiotherapy alone when compared to surgery alone (P=0.004). There was no obvious relationship to age or the type of free flap. Four (2.1%) patients have a gastrostomy that is likely to be permanent. Only two patients did not use the gastrostomy. All patients with T3 and T4 oropharyngeal tumours undergoing radiotherapy or with oral tumours that required reconstruction with a free or pedicled flap were offered a PEG on the basis that nutritional support would be required for more than 2 to 4 weeks. This included T2 tumours without neck disease (stage II disease) if the site of the tumour is likely to have a significant effect on function and hence a flap reconstruction is indicated. The policy of early gastrostomy placement appears to be appropriate. However, the exact pattern of use of the gastrostomy during the various phases of treatment remains to be defined. The insertion of a PEG may be safely performed with a high degree of success and a low incidence of complications by an experienced maxillofacial surgeon. Chapter 7 - Fracture of the radial free flap osteocutaneous donor site is common and causes considerable morbidity. Most fractures are probably caused by relatively low-energy torsional forces. This complication has lead to reduced use of the flap in clinical practice. However, the incidence of fracture may be reduced by placing a bone plate, at the site of the section defect, at the time of harvesting the flap. Both anterior and posterior surgical approaches have been described. The strengthening effect of different types of plate and position were studied using the sheep tibia as a model for the radial osteocutaneous donor site. Fifty matched pairs of adult sheep tibias were tested in torsion and 4-point bending. The weakening effect of an osteotomy was first assessed by comparing an osteotomised bone with an intact bone. Then pairs of bones with an osteotomy were compared with and without

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

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

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

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

In: Oral Cancer Research Advances Editor: Alexios P. Nikolakakos, pp. 1-10

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

Chapter 1

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

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

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

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

2

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

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

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

Administration Methods of ALA in Tongue Tumors

3

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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[10] Miyoshi N, Ogasawara T, Nakano K et al. (2004). In light of recent developments, application of fluorescence spectral analysis in tumor diagnosis. Appl Spectrosc Rev 39: 437-455. [11] Fan KN, Hopper C, Speight PM et al. (1996). Photodynamic therapy using 5aminolevulinic acid for premalignant and malignant lesions of oral cavity. Cancer 78: 1374-1383. [12] Henderson BW, Vaughan L, Bellnier DA et al. (1995). Photosensitization of murine tumor, vasculature and skin by 5-aminolevulinic acid-induced porphyrin. Photochem Photobiol 62: 780-789. [13] Ma G, Ikeda H, Inokuchi T et al. (1999). Effect of photodynamic therapy using 5aminolevulinic acid on 4-nitroquinoline-1-oxide-induced premalignant and malignant lesions of mouse tongue. Oral Oncol 35: 120-124. [14] Mustajoki P, Timonen K, Gorchein A et al. (1992). Sustained high plasma 5aminolevulinic acid concentration in a volunteer: no porphyric symptoms. Euro J Clin Invest 22: 407-411. [15] Loh CS, MacRobert AJ, Bedwell J et al. (1993). Oral versus intravenous administration of 5-aminolaevulinic acid for photodynamic therapy. Br J Cancer 68: 41-51. [16] Kennedy JC, Pottier RH (1992). Endogeneous protoporphyrin IX, a clininically useful photosensitizer for photodynamic therapy. J Photochem Photobiol B 14: 275-292. [17] Szeimies RM, Sassy T, Landthaler M (1994). Penetration potency of topical applied 5aminolevulinic acid for photodynamic therapy of basal cell carcinoma. Photochem Photobiol 59: 73-76. [18] van den Akker JTHM, Lani V, Star WM et al (2000). Topical application of 5aminolevulinic acid hexyl ester and 5-aminolevulinic acid to normal nude mouse skin: differences in protoporphyrin IX fluorescence kinetics and the role of the stratum corneum. Photochem Photobiol 72: 681-689.

In: Oral Cancer Research Advances Editor: Alexios P. Nikolakakos, pp. 11-50

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

Chapter 2

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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by low Ki67 labeling index). 3) VEGF is induced by hypoxia. 4) Development of immature microvessels is induced by VEGF overexpression (as suggested by high highest-MVD). 5) These microvessels with high permeability increase the incidence of hematogenous metastasis (which seems to be one of the reasons for unfavorable prognosis).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Figure 9. Mechanism of VEGF-receptor activation. VEGF-receptors are dimerized when ligand-bound, and the intracellular domain is then activated and intracellular signaling pathways are activated. VEGF: vascular endothelial growth factor, PlGF: placenta growth factor, PI3K: phosphoinositide 3-kinase, AKT: one of protein kinases, Src: one of protein kinases.

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

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

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

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

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

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

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

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

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

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

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[218] Miura M, Sasaki T. Relationship between radiosensitivity and metallothionein content in clones from a mouse squamous cell carcinoma. Radiat Res 1990; 123(2): 171-175. [219] Nomiya T, Nemoto K, Nakata E, Miyachi H, Takai Y, Yamada S. Intrinsic radiosensitivity by metallothionein expression has no great influence on clinical radiosensitivity in esophageal carcinoma. Oncol Rep. 2004; 12(6): 1195-1199.

In: Oral Cancer Research Advances Editor: Alexios P. Nikolakakos, pp. 51-93

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

Chapter 3

PROGNOSTIC INDICATORS IN ORAL SQUAMOUS CELL CARCINOMA Márcio Diniz-Freitas1, Eva Otero-Rey1, Andrés Blanco-Carrión2, Tomás García-Caballero3, José Manuel-Gándara Rey2 and Abel García-García4 1

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

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

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

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

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

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

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

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

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

II.4. Tumor Stage (TNM Classification) The TNM system is accepted worldwide as a classification system for diverse types of neoplasm. This system evaluates the anatomic extension of malignant solid tumors, and is a key indicator of best treatment, as well as an important prognostic factor. Among the most widely used classifications in oral cancer is the staging classification proposed by the American Joint Committee on Cancer (AJCC) [37]. In this classification, stage is determined by the size of the primary lesion and by regional dissemination (to cervical lymph nodes) or distant dissemination. Cure rates for oral cancer depend on stage: the 5-year survival rate is 80% in initial stages, 40% in patients with regional involvement, and less than 20% in patients with distant metastasis [38]. Early detection significantly increases the possibility of a successful cure. At the moment of diagnosis, 36% of patients show localized disease, 43% disease with regional involvement, and 9% distant metastasis; in the remaining 12% of patients disease stage is not readily identifiable [39]. These findings suggest that early diagnosis of this disease is poor, bearing in mind that squamous cell carcinomas are formed in the superficial epithelium of the oral cavity, leading to visible changes at an early stage. Early detection of asymptomatic stages ensures not only improved survival, but also better quality of life for the patient, since aggressive treatments will be less frequently required [40,41].

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In a series of 47 patients with OSCC [42,43], we found that stages T and N independently correlated with patient survival in univariate analyses; in multivariate analyses, however, stages T and N were not statistically significant predictors of prognosis, and only the final clinical stage correlated with survival. These results indicate that combined consideration of tumor size and cervical lymph node involvement may allow more accurate assessment of prognosis in patients with oral cancer. Cervical metastasis is relatively frequent in oral cancer, and it has been suggested that this is in fact the principal indicator of prognosis [44,45]. It seems that metastatic dissemination to cervical lymph nodes is directly correlated with primary tumor size. It has been demonstrated that T1 tumors of the tongue and floor of mouth undergo cervical metastasis in 14% and 11% of cases respectively, versus 77% and 54% respectively for T4 tumors in these locations [46]. In our series of patients with OSCC, the presence of palpable cervical adenopathies, and the incidence of regional relapse, were also correlated with the size of the primary tumor. However, cervical metastases may also develop from relatively small tumors (T1 or T2, n=12), while conversely large tumors may course without cervical metastasis (T3 or T4, n = 7). Since the initial development of the AJCC TNM classification it has been revised various times. In relation to oral cancer, several modifications of the 5th edition have recently been proposed. Additionally, the German-Austrian-Swiss group for the study of tumors of the maxillofacial region (DÖSAK) has demonstrated in an extensive retrospective study of 1532 cases of primary oral cancer that modification of the TNM classification to include tumor thickness significantly improves prediction of 5-year survival rates [47]. Other studies have likewise demonstrated that tumor thickness is of prognostic value in patients with oral cancer [48,49], but nevertheless this parameter has not yet been included in the TNM system. Although it includes important prognostic factors, the TNM system does not accurately predict the biological properties of tumors. For example, the system does not explain the considerable proportion of small tumors that show poorer course than expected [50].

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

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

a

b

c

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

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III.2. The Invasive Front Pathologists have been aware for several decades that tumor cells in the most invasive areas of malignant tumors differ substantially from those in other central and surface areas. In view of this, in 1992 Bryne et al. [59] proposed a new approach to histological classification of OSCCs, considering only those cell layers showing deep invasiveness, and their interface with the host tissue (the "deep invasive front"). This approach, a simplification and modification of the multifactorial approach proposed by Anneroth et al. [60], offers highly significant additional information in patients with the same TNM stage. The approach is based on quantitative evaluation of histological parameters including degree of keratinization, nuclear polymorphism, pattern of invasion, and lymphocytic infiltration. Its prognostic value has been confirmed in diverse studies of carcinomas of the head and neck [61,62].

III.3. Pattern of Invasion Pattern of invasion, a factor common to the various classifications of the invasive front, has proved to be one of the most important parameters for predicting the course and prognosis of OSCCs. Although the scales used for classification of pattern of invasion show some differences, they mostly consider that increasing cellular dissociation is an indicator of increased tumor invasiveness, more aggressive clinical behavior, increased number of local and regional relapses, and poorer prognosis. Spiro et al. [63] have suggested a simplification of Bryne et al.'s classification of the deep invasive front (Figure 2). These authors studied pattern of invasion in squamous cell carcinomas of the tongue. They found that the pattern was associated with both patient age and TNM stage, but not with histological grade. The survival of patients with tumors showing a more invasive pattern was significantly lower than in patients with less invasive tumors. Bundgaard et al. [64] studied 8 histological parameters in 78 stage-I OSCCs, finding that pattern of invasion was the only factor with prognostic value in this patient group. We concluded that evaluation of pattern of invasion was a useful alternative to other more complex methods in which several histological parameters must be considered. Tumors with a more invasive histological pattern are typically associated with the appearance of local relapses [65], possibly because of the greater difficulty of achieving clean surgical margins without neoplastic infiltration in tumor resections. Together, these findings suggest that pattern of invasion is a criterion worth taking into account when selecting postoperative adjuvant treatments with radio- and/or chemotherapy.

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a

b

c

d

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

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

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

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

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

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

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b

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

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

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

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

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

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

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

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

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

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

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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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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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In: Oral Cancer Research Advances Editor: Alexios P. Nikolakakos, pp. 95-124

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

Chapter 4

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

ABSTRACT Despite advances in surgery, radiotherapy, and chemotherapy, the survival of patients with oral squamous cell carcinoma has not significantly improved over the past several decades. Gene therapy has the potential for the treatment of oral cancer. Cancer gene therapy is currently being met with the development of non-viral vectors, because non-viral vectors have a much lower potential for an adverse inflammatory or immune reaction, compared with viral vectors. For gene delivery, oral cancer is a particular appropriate target since it can be applied by direct injection. Also since folate and transferrin receptors are frequently overexpressed on oral tumors such as nasopharyngeal tumor and head and neck of squamous cell carcinoma, folic acid and transferrin have been utilized as a ligand for tumor-targeting gene delivery. Non-viral vectors conjugated to these ligands have been used as carriers of therap