Focus on Thyroid Cancer Research [1 ed.] 9781612097534, 9781594546266

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Copyright © 2006. Nova Science Publishers, Incorporated. All rights reserved. Milton, Carl A.. Focus on Thyroid Cancer Research, Nova Science Publishers, Incorporated, 2006. ProQuest Ebook Central,

Copyright © 2006. Nova Science Publishers, Incorporated. All rights reserved. Milton, Carl A.. Focus on Thyroid Cancer Research, Nova Science Publishers, Incorporated, 2006. ProQuest Ebook Central,

FOCUS ON THYROID CANCER RESEARCH

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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.

Milton, Carl A.. Focus on Thyroid Cancer Research, Nova Science Publishers, Incorporated, 2006. ProQuest Ebook Central,

Copyright © 2006. Nova Science Publishers, Incorporated. All rights reserved. Milton, Carl A.. Focus on Thyroid Cancer Research, Nova Science Publishers, Incorporated, 2006. ProQuest Ebook Central,

FOCUS ON THYROID CANCER RESEARCH

CARL A. MILTON

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EDITOR

Nova Biomedical Books New York Milton, Carl A.. Focus on Thyroid Cancer Research, Nova Science Publishers, Incorporated, 2006. ProQuest Ebook Central,

Copyright © 2009 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. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

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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 Available upon request. ISBN: 978-1-59454-626-6

Published by Nova Science Publishers, Inc.    New York

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CONTENTS Preface Chapter 1

Chapter 2

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Chapter 3

vii Role of Radioactive Iodine (I-131) in Management of Differentiated Thyroid Carcinoma Sin-Ming Chow Biological Characteristics and Therapeutic Strategies for Papillary Microcarcinoma of the Thyroid Yasuhiro Ito and Akira Miyauchi

43

Radioiodine in Therapy of Patients with Hürthle Cell Carcinoma of the Thyroid Nikola Besic, Barbara Vidergar-Kralj and Ivana Zagar

61

Chapter 4

Cysts of the Thyroid With Thyroid Carcinomas Jen-Der Lin, Chuen Hsueh and Tzu-Chieh Chao

Chapter 5

Value of Tumor M2-PK in Thyroid Carcinoma: A Pilot Study with Review of Actual Literature Nicole Françoise Bena-Boupda

Chapter 6

Familial Nonmedullary Thyroid Carcinoma Carl D. Malchoff and Diana M. Malchoff

Chapter 7

Recent Advances in the Treatment of Medullary Thyroid Carcinoma Levent Saydam and Mete K. Bozkurt

Chapter 8

1

Medullary Thyroid Carcinoma: Diagnostics, Treatment and Prognosis Vitaliy Zh Brzhezovskiy, Vyacheslav L Lyubaev, Tatiana T Kondratyeva, Elena A Smirnova, Faina A Amosenko and Raisa F Garkavtseva

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99 111

125

141

vi Chapter 9

Contents The Role of RT Inhibitors as a Novel Molecular Targeted Treatment in the Management of Poorly Differentiated Thyroid Tumors M. Landriscina, A. Fabiano, A. Piscazzi, C.Bagalà, S. Altamura, N. Maiorano, F. Giorgino, C. Barone and M. Cignarelli

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Index

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201

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PREFACE Thyroid cancer is cancer of the thyroid gland. These may be of many types including papillary, follicular, Hurthle cell (aka oxyphilic or oncocytic), or medullary cancers. Surgery plays an important role in treating these cancers. The thyroid concentrates iodine and so is extremely sensitive to the effects of various radioactive isotopes of iodine produced by nuclear fission. These radioactive isotopes increase the chances of developing cancer, though thyroid cancer can develop even without any exposure to radioactivity. Some evidence suggests that insufficient or excessive dietary iodine may also increase the risk for thyroid cancer. This new book presents the latest research in this field. Chapter 1 - Iodine-131 (I-131) or radioactive iodine (RAI) is an isotope used therapeutically in differentiated thyroid carcinoma (DTC) for over half a century. The selective absorption of this isotope in thyroid tissue or thyroid malignancy renders it an effective and most commonly employed isotope in the oncology field. It has been shown to be effective in reducing relapses, improving survival and in treatment of distant metastasis in DTC. However, the indications of RAI ablation in DTC is still not universally agreed. Should it be applied to all patients with DTC is debatable. From the literature, the rate of postoperative RAI ablation varies from almost none in Japan to over 90% in Germany. RAI is becoming more commonly employed in recent years, probably related to the emerging publications of its efficacy. Because of the rarity of this disease and the extremely long disease tempo, prospective randomized trials are hardly possible. Moreover, the fear of radiation exposure also impedes its application as adjuvant treatment in young adults. Acute side effects are usually well tolerated, e.g. nausea, acute sialadenitis and neck swelling. Long term effects of fertility, pregnancy outcome and secondary malignancy should be reviewed to reassure the patients of the safety of this treatment. RAI is still a mysterious tool to patients and even doctors inexperienced in the oncology field. In this article, the clinical application of RAI and data from literature will be extensively reviewed. Chapter 2 - Papillary carcinoma of the thyroid is a common malignancy originating from the endocrine organs. Usually the tumor grows very slowly with a good prognosis, although the disease frequently metastasizes to regional lymph nodes and shows multiple tumor formations in the thyroid. Recently, screening for thyroid or carotid artery lesion by

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ultrasonography (US) has been readily performed and various small thyroid lesions have frequently been detected. Furthermore, the prevalence of US-guided fine needle aspiration biopsy (FNAB) has facilitated the frequent diagnosis of papillary carcinoma measuring 1.0 cm or less in maximal diameter, which is defined as papillary microcarcinoma (PMC) in the World Health Organization monograph on histological typing of thyroid tumors. The thyroid is an organ in which latent asymptomatic carcinoma of small size is frequently detected. To date, there have been many reports from various countries describing latent thyroid carcinoma detected at autopsy, and the prevalence rate has ranged from 6.0 to 35.6 %. As US can detect thyroid lesions measuring 3 mm or larger, the size of clinically detectable PMC ranges from 3.0-10.0 mm. Previous autopsy studies showed that latent papillary carcinomas in this size range were detected in 2.3-5.2% of autopsy cases. However, a Japanese study of mass screening using US and FNAB demonstrated that papillary carcinomas measuring more than 3mm could be detected in 3.5% of otherwise healthy women aged 30 years or older, and about 84% of these lesions were 15 mm or smaller. The prevalence of US-detectable papillary carcinoma is 3500 per 100,000 females, which is not discrepant with those of previous autopsy studies. However, the prevalence of clinically apparent papillary carcinoma was only 1.9-11.7 per 100,000 females and 1.0-4.8 per 100,000 males, which is about 1000 times lower than that of US-detectable papillary carcinoma. The significant difference between these figures is notable and important in discussions of how to treat PMC. Therefore, a trial of observation without immediate surgical treatment for PMC has recently been performed with favorable results. Most PMCs do not or only slowly grow and it is concluded that observation without immediate surgical treatment can be a strategy for treatment of PMC, if the lesions are asymptomatic. In this chapter, the authors will show the outcomes of PMC patients under observation and describe their standard surgical design for patients when surgery is chosen. Furthermore, they comment on distinguishing cases that which have growth potential, and therefore, require careful treatment. Chapter 3 - Background: It is generally believed that Hürthle cell thyroid carcinoma (HCTC) does not accumulate radioiodine (RAI). The aim of their retrospective study was to find out the capacity of HCTC to accumulate RAI and the influence of RAI thyroid remnant ablation on the disease-free interval and survival. Another aim of their study was to find out if the recurrent or metastatic HCTC accumulates RAI in hypothyroid state and after the application of rhTSH. Material and methods: A total of 1612 patients with thyroid carcinoma were seen at the Institute of Oncology Ljubljana from 1972-2002 and 76 (4.7%) of them had HCTC. This study was carried out on 73 patients (55 females, 18 males; age range 31-87 years, median age 63 years) with histologically confirmed HCTC. T4 primary tumor stage, regional and distant metastases were found in 10 (=14%), 5 (=7%) and 12 (=16%) patients, respectively. After surgery, RAI ablation of thyroid remnant was performed in 56 patients. Recurrence was detected in 20 patients. Data about preoperative thyroid scintigraphy, RAI ablation of thyroid remnant after thyroid surgery and RAI uptake in distant metastases and/or locoregional recurrence were collected. The effect of RAI application on the disease-free period and survival was analyzed by univariate and chi-square statistical analysis.

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Results: The 10-year disease-free interval was 58% and 10-year survival 80%. A hot nodule was seen in 1/24 patients on preoperative thyroid pertechnetate scintigraphy. The recurrence occurred in 31% of patients with ablation and in 44% without ablation (p=0.36). The 5-year disease-free interval was 66% after ablation and 50% without ablation (p=0.40). The 10-year survival was 82% after ablation and 75% without ablation (p=0.78). Initially, metastatic (n=12) or recurrent (n=15) HCTC accumulated RAI in 16/27 (=59%) of cases. Most importantly, as seen from the X-ray, even a complete regression of lung metastases was obtained in two patients after RAI therapy. In four of eight patients with metastatic or recurrent HCTC, rhTSH-aided RAI accumulation was seen. Conclusion: The authors could not demonstrate that RAI ablation of thyroid remnant significantly decreased the recurrence or death rates. It, however, facilitates the detection of recurrent disease. The whole-body scintigraphy with RAI should be performed in HCTC. RAI may be effective for treatment of metastatic or recurrent HCTC after endogenous TSH stimulation and after application of rhTSH. Chapter 4 - Thyroid cyst is a common thyroid disease in human goiter. Prevalence of the thyroid cyst depends on definitions by ultrasonographic measurement, surgical specimens or quantity of cystic fluid. Over one third of isolated thyroid nodules are cystic, while over half exhibit cystic degeneration and 17-32% of cystic thyroid nodes are malignant. The pathology of cystic thyroid disorders may comprise clear fluid cyst, thick colloid cyst, cystic echinococcosis, hydratic cyst, intrathyroidal thyroglossal duct cyst, lymphoepithelial cyst, epithelial cyst, epidermoid cyst, thyroid abscess and various thyroid cancers. Different thyroid carcinomas may occur from the thyroid cysts or with cystic degeneration. The pathogenesis of most thyroid cysts is unknown. Possible causes include infarcts and other destructive processes like hemorrhage in the thyroid follicle, clustering of thyroid follicles followed by cystic degeneration and benign or malignant tumor necrosis. Biochemical analysis of the amylase, lactate dehydrogenase, acid phosphatase in cystic fluid show much higher levels than serum. Vascular epithelial growth factor has been mentioned raised concentration in cystic fluid of developing and recurrent thyroid nodules. Combination of ultrasound-guided aspiration near the solid part of the thyroid cysts and cytological interpretation by experienced endocrine cytopathologists constitutes the best form of preoperative diagnosis of malignant thyroid cysts. Around 14% (range 7.1% to 31.9%) of thyroid cysts were surgically proved to be malignant. Most cysts were papillary thyroid carcinoma, otherwise medullary cystc carcinoma, anaplastic thyroid carcinoma with cystic change were also reported. Observation, repeat aspirations, thyroid hormone therapy, sclerotherapy, laser photocoagulation, and surgical treatment were main methods of therapy for thyroid cysts. Percutaneous ethanol injection for treatment of thyroid cysts and solid thyroid nodules was more effective in cysts. Depending on the definition of response rate and the period of follow-up, the response rate from ethanol injection for thyroid cyst ranged from 72.1 to 93.9%. In conclusion, the pathogenesis of the thyroid cyst required further investigation. More data are needed concerning the role played by growth factors or oncogenes in the malignant transformation of these lesions. Chapter 5 - Background: Tumor M2-PK (Tu M2-PK) as the inactive dimeric form of pyruvate kinase M2 has been proven to be elevated in breast, lung, renal and gastrointestinal malignancies. The authors compared this marker in 26 patients with metastatic thyroid

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carcinoma with the established tumor marker thyroglobulin (hTg). Materials and Methods: Plasma Tu M2-PK was measured using an ELISA assay (Schebo Biotech, Giessen, Germany) and Tg was measured in serum using an electrochemiluminescence Immunoassay (Tg Elecsys systems, Roche, Germany), as a part of the routine follow-up of the patients. Results: At a cut-off of 15 U/ml, Tu M2-PK was elevated in 50% of the patients; at a cut-off of 20 U/ml (grey zone 15-20 U/ml), Tu M2-PK was elevated in 27% of the patients. The correlation coefficient between serum hTg level and Tu M2-PK was –0.093. Conclusion: Tu M2-PK is less valuable for the detection of malignant thyroid disease than hTg. Chapter 6 - Carcinomas that arise from the thyroid follicular cell include papillary thyroid carcinoma, follicular thyroid carcinoma, insular thyroid carcinoma, and anaplastic thyroid carcinoma. As a group they are referred to as nonmedullary thyroid carcinomas (NMTC). In contrast, medullary thyroid carcinoma arises from the calcitonin producing parafollicular cells (C cells) of the thyroid. NMTC usually occurs sporadically. However, several epidemiologic studies and investigations into large kindreds suggest a familial predisposition in about 5 percent of NMTC. Familial NMTC (fNMTC) can be divided into at least two clinical groups. In the first group NMTC is a relatively infrequent component of a familial tumor syndrome characterized by a predominance of non-thyroidal carcinomas. These syndromes include the Cowden syndrome, familial adenomatous polyposis, and Carney complex type 1. Multiple endocrine neoplasia type 2A and the familial paraganglioma syndromes also may be enriched in NMTC. In the second group NMTC is the predominant malignancy in affected kindreds, although other neoplasms may also occur. In this second group, NMTC is autosomal dominant with partial penetrance. As compared to sporadic NMTC, fNMTC is characterized by a younger age of onset, an increased frequency of multifocal disease, within the thyroid, an increased risk of recurrence, but no increased risk of death. Most families are relatively small, suggesting a relatively low penetrance of the susceptibility gene. An alternative explanation for this observation is that fNMTC is a polygenic disorder. If this is correct, then polygenic disease constitutes a third fNMTC group. Linkage studies suggest 3 loci for fNMTC susceptibility genes. Linkage to chromosome 1q21 has been identified in a large papillary thyroid carcinoma (PTC) kindred enriched with papillary renal neoplasia and possibly other neoplasms. Kindreds with fNMTC alone have been mapped to 2q21 and to 19p13.2. Some of the fNMTC that are linked to 19p13.2 have the pathologic finding of oxyphilia, suggesting that they are pathologically distinct from the other familial fNMTC syndromes. However, the precise susceptibility genes remain to be identified. A family history should be taken from patients with NMTC to identify kindreds with a predominance of fNMTC and kindreds that represent other familial tumor syndromes with an increased frequency of NMTC. Careful clinical evaluation is indicated for members of fNMTC kindreds, and some investigators advocate thyroid ultrasound. Prophylactic thyroidectomy is not indicated and clinically useful genetic analysis is not yet available. In summary, about 5 percent of NMTC have a familial predisposition and these can often been identified by the family history. Linkage studies have mapped at least 3 fNMTC loci, However, until the specific susceptibility genes are identified, there are no clinically useful genetic studies. Careful clinical evaluation of the members of fNMTC kindreds is indicated.

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Chapter 7 - Medullary thyroid carcinomas which arise from parafollicular or C cells of thyroid comprise about 5% to 10% of all thyroid cancers. Due to its derivative cell type medullary cancer is considered as a subset of neuroendocrine tumors. The primary hormonal product which the tumor cells secret is calcitonin. The tumor is hereditary with auotosomal dominant inheritance in approximately 25% of the cases while the rest of the cases present as in sporadic form. The hereditary forms are MEN type 2A and 2B syndromes and familial non-MEN medullary throid carcinomas which are caused by RET proto-oncogene mutations. Currently the choice of primary treatment of these tumors is surgical removal of entire gland and cervical lymph node groups having the risk of invasion by tumor cells. Radiotherapy is instituted by means of adjuvant therapy in cases with locoregional invasion or as a palliative measure in certain cases. While there is not universally accepted type of systemic treatment, currently ongoing experimental forms of treatments such as application of gene treatment procedures targeting the mutations in RET proto-oncogene, the use of somatostatin analogs or 131 I-MIBG are still under investigation. Chapter 8 - 182 patients with medullary thyroid carcinoma (MTC) were under clinical observation for this research. The pre-operative diagnostic procedures employed were ultrasound examination, fine-needle aspiration biopsy (FNAB), radioisotope examination of thyroid, calcitonin and carcinoembryonic antigen levels in the blood serum, computer tomographic scan and magneto-resonance tomography. The authors have done much work on investigation clinical and morphologic features of MTC with special interest to its diagnostics. The chapter provides discussion on the objective possibilities of cytologic method (FNAB) while making exact diagnosis of MTC and its types, with the use of their own cytograms. A number of differentially-diagnostic problems arising in practice were defined with the aim to decrease the probable errors in the distinguishing this unusually multiform thyroid cancer. Different types of MTC were studied during histologic examination of the tumors. While investigating the influence of MTC histologic type on patients’ survival, the authors failed to determine significant correlation between these parameters and prognosis. Electron-microscopic investigation of MTC demonstrated that the ratio of differentiated and un-differentiated cells in tumor is the most informative prognostic symptom. For molecular diagnostics of MTC hereditary forms, the authors performed screening of the mutations in proto-oncogene RET (in blood leukocytes) for individuals from families with MEN 2 cancer syndromes. The frequency of the activating germline mutations was 100%. In the group of patients with sporadic MTC, somatic mutations were revealed in 34.6% of tumors. Surgery is very important in the current treatment of MTC. Surgical treatment on the primary tumor lesion depends mainly on MTC form: sporadic or familial. Thyroidectomy is prescribed in hereditary form of the disease irrespective of tumor size. Preventive thyroidectomy performed for 5 asymptomatic carriers of the RET mutations (c.634) from 3 families with MEN 2A, proved to be effective in prophylaxis and/or treatment of MTC. In the case of sporadic MTC if tumor size is restricted (T1-T2), it`s possible to use organ sparing surgery. Radiation therapy has the following confined indications: 1) in doubtful radical nature of operation; 2) in inoperable forms; 3) in distant metastases with palliative and symptomatic treatment as aim. Chemotherapy in MTC is ineffective. Chapter 9 - Anaplastic thyroid carcinoma, representing only 2% of thyroid cancers, is one of the most aggressive malignancies. Indeed, the overall median survival of affected

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patients is limited to months, whereas only a minority of patients with resectable disease have demonstrated long-term survival with aggressive multimodal treatments. Unfortunately, by contrast to differentiated thyroid tumors, undifferentiated thyroid cancer cells fail to uptake iodine because of the lack of expression of the Na/I symporter (NIS) and thus their responsiveness to radio-iodine therapy is abrogated. Either undifferentiated or transformed cells express high levels of endogenous nontelomeric reverse transcriptase (RT). Pharmacological RT inhibition by two well characterized RT inhibitors (nevirapine and efavirenz) as well as the down-regulation of expression of RT-encoding LINE-1 elements by RNA interference, reversibly inhibit cell growth, promote cell differentiation and modulate gene expression in several human tumor cell lines either in vitro or in animal models. Therefore, the authors investigated, in primary cultures of human undifferentiated thyroid carcinoma as well as in anaplastic thyroid carcinoma ARO cells, whether pharmacological inhibition of RT may represent an effective tool in the treatment of poorly differentiated thyroid neoplasms. The results of the authors study demonstrated that efavirenz and nevirapine induced morphological changes resembling cell differentiation and activated the expression of thyroglobulin, a gene highly expressed by differentiated thyroid tumors. It is noteworthy that undifferentiated thyroid tumor cells exposed to RT inhibitors acquired also the ability to express NIS and pendrin, two protein involved in iodine uptake, and accumulate radioactive iodine in a TSH-dependent manner. Interestingly, the appearance of this differentiated phenotype correlated with the reversible down-regulation of either cell growth in vitro or tumor growth in vivo. Finally, the simultaneous exposure of anaplastic thyroid tumor cells to the differentiating agent all-transretinoic acid (ATRA) and nevirapine did not produce any additive effect on the inhibition of cell proliferation. Thus, the role of RT inhibitors as potential differentiating and cytostatic treatment for undifferentiated thyroid cancer is discussed.

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Chapter 1

ROLE OF RADIOACTIVE IODINE (I-131) IN MANAGEMENT OF DIFFERENTIATED THYROID CARCINOMA Sin-Ming Chow∗ Department of Clinical Oncology, Queen Elizabeth Hospital, Kowloon, Hong Kong.

ABSTRACT

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Iodine-131 (I-131) or radioactive iodine (RAI) is an isotope used therapeutically in differentiated thyroid carcinoma (DTC) for over half a century. The selective absorption of this isotope in thyroid tissue or thyroid malignancy renders it an effective and most commonly employed isotope in the oncology field. It has been shown to be effective in reducing relapses, improving survival and in treatment of distant metastasis in DTC. However, the indications of RAI ablation in DTC is still not universally agreed. Should it be applied to all patients with DTC is debatable. From the literature, the rate of postoperative RAI ablation varies from almost none in Japan to over 90% in Germany. RAI is becoming more commonly employed in recent years, probably related to the emerging publications of its efficacy. Because of the rarity of this disease and the extremely long disease tempo, prospective randomized trials are hardly possible. Moreover, the fear of radiation exposure also impedes its application as adjuvant treatment in young adults. Acute side effects are usually well tolerated, e.g. nausea, acute sialadenitis and neck swelling. Long term effects of fertility, pregnancy outcome and secondary malignancy should be reviewed to reassure the patients of the safety of this treatment. RAI is still a mysterious tool to patients and even doctors inexperienced in the oncology field. In this article, the clinical application of RAI and data from literature will be extensively reviewed. ∗

Correspondence concerning this article should be addressed to Dr. Sin-Ming Chow, Department of Clinical Oncology, Block R, Queen Elizabeth Hospital, 30 Gascoigne Road, Kowloon, Hong Kong. Tel: (852) 2958 5507; Fax: (852) 2359 4782; Email: [email protected] or [email protected].

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Keywords: differentiated thyroid carcinoma, radioactive iodine, precautions, side-effects

INTRODUCTION

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Natural History of Differentiated Thyroid Carcinoma Despite the low incidence of thyroid cancer among all cancers, it is the commonest endocrine malignancy. Papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC) are the 2 major types of differentiated thyroid carcinoma (DTC). Patients are young at presentation. The mean age at diagnosis is 44 to 46 for PTC [1-3] and 49 to 56 for FTC. [1,3,4] This disease occurs commonly in young adult women. In Hong Kong, among women of age 15 to 34 years, the incidence of thyroid malignancy is 15.7%; it ranked just second to breast cancer. [5] The prognosis is good in general. The 10-year cause-specific survival in reported series ranges from 87.7% to 96% in PTC and 69% to 87% in FTC. [1,3,6-10] The prognosis is better in PTC than FTC. [1,11-13] DTC is peculiar for its indolent course and potential for late relapses. It has been observed that 25% of first relapses occur 20 years after initial presentation. [14] The relapse rate is higher in the extremes of age. [12,15,16] The incidence of positive lymph node (LN) metastasis at diagnosis is higher in PTC: 21.8 to 56% in PTC and 2.4% to 21% in FTC. [1,3,6] At initial presentation, distant metastasis (DM) is detected in 2% to 5% of PTC [1,8,9] and in 14.9% to 21.8% of FTC. [1,3,9] The management of DTC is controversial because of the lack of prospective randomized studies. In recent years, there are emerging studies addressing the optimal surgery, postoperative management by radioactive iodine (RAI) ablation, external radiotherapy (EXT) and role of thyrotropin stimulating hormone (TSH) suppression. With the publication of reports of adjuvant RAI treatment after total or near-total thyroidectomy, role of RAI in thyroid cancer management is increasingly recognized.

Thyroid Surgery Prevalence of contralateral lobe tumor foci was 35.8 % to 61% in PTC. [17-21] The presence of multifocal disease in ipsilateral lobe [21] and LN metastasis[18] predict for cancer in contralateral lobe. However, some authors observed a low rate ( 1cm. [18] The timing of completion thyroidectomy has no impact on risk of permanent complications. [28] But the usual practice is to perform the completion thyroidectomy as early as possible. [21] Some reports that complications of total thyroidectomy, when compared with lobectomy, do not increased significantly. [29]

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Role of Radioactive Iodine (I-131)…

3

Completion thyroidectomy can be considered a safe procedure. [30] However, bilateral thyroidectomy resulted in higher rate of hypocalcemia in multivariate analyses involving large number of patients. [31,32] To avoid re-operation when incidental thyroid carcinoma is found, some surgeons advocate total thyroidectomy for multinodular goiter [33] or dominant thyroid nodules. [29] Complete thyroidectomy, apart from removing potential contralateral lobe tumors, also facilitates RAI ablation. The success of ablation depends on the dose of RAI and amount of thyroid remnant. This will be discussed in the coming sessions.

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Lymph Node Surgery Lymph node metastasis is common in PTC while rare in FTC. [13,34,35] The detection rate of LN metastasis depends on the extent of initial lymph node resection and also the pathological method of detection. Sites of LN metastasis are most commonly found in central compartment (50%), followed by mid-jugular (37%) and supraclavicular nodes (22%). [36] Detailed mapping of cervical LN metastasis reveals that in patients with positive LN metastasis, 20% had no LN metastasis at paratracheal region, 37% had no LN metastasis in midjugular region and 49% had no LN in supraclavicular region. Therefore LN metastasis can occur in several levels and skip metastasis occurs. The traditional method of LN surgery in thyroid cancer is ‘berry picking’ until recent years; a concept of central compartmental dissection (CCD) is advocated. The idea of ‘LN sampling before modified neck dissection’ is difficulty to implement because of the inherent diverse lymphatic spread in thyroid cancer. The correlation of site of LN involvement with the tumor location in thyroid is not consistent. [37] Some authors promote the use of dye or radioisotopes to detect sentinel LN. [38-40] Further studies are required to see whether this approach is worth generalized. The rate of LN metastasis, using immunohistochemical (IHC) methods for detection, is much higher than expected by H & E stain. [41] The so-called pN0 status might need to be treated more cautiously. The incidence of micrometastasis (defined as H & E stain negative but IHC positive) increases with size of primary tumor in thyroid: it is found in 26% and 66% of cervical LN when primary tumors are ≤ 1cm and > 1cm respectively. The distribution of LN metastasis follows the lymphatic drainage of the thyroid. In patients with PTC, the incidence of pathological LN metastasis ranges from 21 to 82% when there is no gross LN involvement. However, only 3-15% patients without prophylactic neck dissection developes LN relapse. [42] Will ‘inadequate LN surgery’ jeopardize survival? If LN dissection is to be performed, how do we choose the extent of LN dissection with respect to other clinicopathological factors? Can RAI be a surrogate to cervical LN dissection in areas where ‘berry picking’ is the practice? Greater local recurrence occurs with ‘berry picking’ than neck dissection. [43] CCD is promoted as part of initial surgery. However, this approach is not widely practiced in many areas. The incidence of positive LN metastasis is 60.5% to 73% when CDD is a routine practice. [36,44] CCD increases the risks of hypoparathyroidism, but not laryngeal nerve palsy. [45,46] With the observation of skip metastasis to other levels of cervical LN,

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dissection of central compartment alone, with an aim to eradicate all tumor foci, is not an adequate approach. It is difficult to decide the optimal extent of LN surgery in patients with clinically negative LN. Current approach for lymph node surgery in PTC varies inter-nationally and from centers to centers. In some Germany centers, at least CCD is practiced. [47] In Cancer Institute Hospital in Japan, CCD and hemithyroidectomy is done for tumor located in one lobe with no clinical suspicion of LN metastasis. [7] Only patients with clinically involved LN, as defined by imaging, would undergo ipsilateral modified radical neck dissection (MRND). In Noguchi Thyroid Clinic, MRND on ipsilateral side and at least lobectomy will be done for patients with tumor > 1cm. [48] The importance of LN dissection may be best demonstrated in Noguchi’s clinic because they did not treat patients with RAI ablation after curative surgery. [49] Noguchi et al reported that unilateral LN dissection resulted in 9.1% local recurrence rate compared to 17.4% of those undergone ‘node picking’ surgery. A multivariate analysis from Noguchi’s group, analyzed 1,776 patients with PTC greater than 1cm, found that modified radical neck dissection improved survival in patients with nodal metastasis. [50] This is particularly significant in a subgroup with gross nodal disease before surgery, primary tumor extension beyond thyroid capsule and women older than 60 years. Bilateral modified radical neck dissection is not favored because only 1.8% patients developed contralateral LN metastasis after a mean FU of 12.1 years. [48] Both total thyroidectomy and CCD increase the risk of hypoparathyroidism. [31,46] The complications will even be higher in inexperienced hands. In the United States, nonendocrine surgeons perform majority of endocrine operations. [51] Thyroid surgery is mostly done by general surgeons. [52] This is also a phenomenon observed in Hong Kong and probably many other countries. An increased complication rate will be expected if all patients received CCD in addition to total thyroidectomy. In Hong Kong, total thyroidectomy and berry picking is still the common practice. Our study in Queen Elizabeth Hospital showed that in patients with no LR residual disease after surgery, RAI ablation improved LR control by multivariate analysis. [27] At 10 years, the LR relapse was 7.4% after RAI ablation, compared to 19.5% of those having none. Though it cannot be ascertained that RAI can be a surrogate to CCD in patients with clinically negative LN, this result of low LR relapse is compatible with results from centers practicing primary LN surgery without RAI ablation. RAI ablation might have a role in preventing LR relapse in those with minimal LN metastasis.

THE PROCEDURE OF RADIOIODINE ABLATION Iodine-131 is an isotope with emission of both beta and gamma energies during decay. The half-life of decay was 8.02 days [53] while the median biological half-life in human body is around 14 hours, with substantial variations. [54] It is simply administered by oral route and excreted through renal system. By virtue of the specific uptake of iodine in thyroid tissue in our body, RAI will be concentrated in thyroid follicular cells or differentiated thyroid cancer cells. The isotope

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decays and emits predominantly beta rays with short treatment length of less than 1mm. A small portion of the energy is deposited as a mixture of photon emissions. Its clinical use in medical therapy is mainly in thyrotoxicosis and differentiated thyroid cancers.

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Preparation for RAI Ablation The practice of RAI preparation varies. To improve RAI uptake in thyroid tissue or cancer cells, TSH level has to be maximized by either thyroxin (T4) withdrawal for 4-6 weeks or recombinant human thyrotrophin stimulating hormone (rhTSH) without stopping T4. To reduce the side effects of prolonged hypothyroidism in T4 withdrawal, some centers replace T4 with T3 for 2 weeks and then off T3 for 2 weeks before the RAI treatment. [55] The duration of T4 withdrawal to achieve TSH level of 30mIU/l should be less than 4-6 weeks. Liel et al reported that the mean interval to reach this level of TSH was 17 days. [56] Sanchez et al found that this level can be achieved in 90% of patients at 3 weeks after discontinuing T4 suppressive therapy and in all patients after total thyroidectomy. [57] Serhal at al reported that 95% of patients, after thyroidectomy or after T4 withdrawal, achieved TSH levels of more than 30mU/l at 18 days and 22 days respectively. Therefore, the value of short-term usage of T3 in preparing the patient for RAI ablation is doubted. [58] Another important preparation is to deplete the body iodide pool. This preparation decreases iodide excretion and increases the effective half-life of RAI. There are controversial reports on the therapeutic accuracy of low iodide diet (LID). A study from United States showed that the success of ablation was not dependent on the strictness of dietary iodide content. Rather, it was RAI dose-dependent. [59] Another study from Netherlands found that a low-iodide diet for 4 days improved the efficacy of RAI ablation. [55] However, the results came from data comparison with historical control before 1992 in the same institute. The success rate, as defined by thyroglobulin (Tg) < 2ug/l and negative scintigraphy, was significantly higher in LID group. A LID was found to increase the 24-hour RAI uptake in neck scan, increased the calculated radiation dose and had higher urinary iodide excretion. In LID group, the T4 level at thyroid ablation was lower than controls. The TSH level was not affected. The optimal duration of LID is not well defined. In the procedure guideline for RAI treatment by Meier et al, they stated that ‘many experts recommend a low iodide diet for 7 to 10 days before administration’. [60] Most of the centers suggests a low iodine diet for 2 weeks before the examination. [61] The administration of RAI is usually given at 4 to 6 weeks after surgery. Patients should have sufficient renal function for excretion of the isotope. The procedure are listed in the guidelines by the Society of Nuclear Medicine, [60] British Thyroid Association and Royal College of Physicians. [62] The dose varies according to the indications: 2.75-5.55GBq (75150mCi) for thyroid remnant ablation; 5.55-7.4GBq (150-200mCi) for suspected thyroid cancer in the neck or mediastinal lymph nodes; 7.4GBq (200mCi) for distant metastases. [60]

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Ablation Efficacy The success of ablation was defined as no visual uptake at whole body scan (WBS), uptake percentage of RAI less than 0.5% to 1% (according to different institution) and Tg less than lower range of normal (< 2ng/ml). [61,63]

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Cervical Uptake of Tracer RAI The ablation efficacy is inversely correlated with 24-hour cervical uptake of RAI tracer in fixed dose of 3.5GBq or 100mCi. [61,63] Rosario et al showed that when RAI uptake percentage was more than 10%, TSH was not optimally raised (i.e. ≤ 30mIU/L) in 2 of 12 patients (16.7%). [61] When the cervical uptake percentage is less than 2%, the ablation success is high (92% to 94%). [61,64] Bulk of Thyroid Remnant The cervical uptake correlates with bulk of thyroid remnants after surgery. [65] The efficacy of RAI ablation is lower in patients having lobectomy (90%) compared to total thyroidectomy (98%). [63] Hoyes et al reported that the median pre-ablative neck uptake after total thyroidectomy was 3.3% compared to 20.1% in lobectomy. They also found that the biological half-life of RAI was markedly prolonged after lobectomy than after total thyroidectomy. Therefore, the degree of thyroid debulking is an important prerequisite for the success of ablation. Can we consider RAI ablation to replace completion thyroidectomy after an initial lobectomy? The potential advantage of this approach is the avoidance of re-operation risks and morbidity. Leung et al tried to use lesser dose of RAI (30mCi) for ablation, with high success rate of 95% after total or subtotal thyroidectomy and lower success rate of 56% in those with partial or hemithyroidectomy. [66] Randolph and Daniels used 29.9mCi of RAI as outpatient ablation in 50 patients after lobectomy. [67] They found that the mean TSH was similar to the control group of patients who underwent total or near-total thyroidectomy. The mean uptake of RAI at 24 hours was < 1% in 80% of patients. The morbidity was minimal. However, 44% of patients (22/50) required additional RAI treatments. The authors concluded that low dose RAI is a feasible alternative to completion thyroidectomy in terms of safety and cost. Hoyes et al compared the results of success after 3.5 GBq RAI ablation in patients with lobectomy and total thyroidectomy. They reported 90% success rate in a series of 60 patients who had lobectomy compared to 98% in 165 patients with total thyroidectomy. [63] The disadvantages of this approach in ablation of large thyroid remnant are the need for repeated RAI doses for ablation, [63,67] risk of radiation thyroiditis and increased whole body dose and marrow dose. The length of hospitalization may be lengthened accordingly.

Dose of RAI: High or Low Apart from the bulk of thyroid remnant, the ablation efficacy increases with dose of RAI. [68,69] In a randomized trial, a dose of < 25 mCi gave a success rate of 61.8% while dose ≥ 25mCi gave a success rate of 81.6%. [70]

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In a meta-analysis on 967 patients, the efficacy of remnant ablation following a single low dose (1,074-1,110MBq or about 29mCi) against a single high dose (2,775-3,700MBq or 75-100mCi) was compared. High dose RAI was found to be more efficient, particularly after less than total thyroidectomy. This confirms the importance of high dose and more extensive surgery as pre-requisites for success in single dose ablation. [69] The dose of RAI used in ablation varies, typically ranges from 1.1 GBq (30mCi) to 3.7 GBq (100mCi). However, optimal dose is still not well defined from the current literature. [70-72]

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Standard Fixed Dose and Alternative Practice Factors that are considered in the dose decision include prognostic factors of staging, [73] RAI uptake in neck, [74] marrow dose estimation and total volume of residual thyroid as estimated by ultrasonogram. [75] Some novel investigators try to define the heterogeneity in the thyroid tissue by I-124 PET imaging. [76,77] Other administrative factors or cost considerations also affect the dose decision. [78] The dose of RAI varies considerably in different centers. Most centers use standard fixed doses for postoperative ablation, LR recurrences and DM. [12,63,79-82] The success rate of single dose ablation after total thyroidectomy, in the dose range of 2.78 to 3.7 GBq (75mCi to 100mCi), is 92% to 98%. [27,63] Some centers determine the dose of RAI based on the 48hour uptake of iodine [83] or clinical staging [84]. In some hospitals, low dose RAI is favored because of the advantage of outpatient treatment. Logistically, lack of radiation isolation beds make this approach a favored option. [78] A fractionated RAI treatment using 30mCi in 4 weekly doses was reported by Hung et al. The success rate was only 66.7% compared to 52.6% in single 30mCi RAI dose in the same institute. This fractionated protocol has the disadvantage of much longer hypothyroid status and a higher total expense. [78]

Use of Recombinant Human TSH in RAI Treatment Use of rhTSH in ablation and therapy is not well documented. A small number of studies are published. [75,85-87] In a series of 11 patients who cannot tolerate thyroid hormone withdrawal, 108mCi RAI for ablation or recurrences under rhTSH was tried. The hormone was well tolerated and response was found in 80% of palliative treatment. [85] Another study by Robbins et al in 10 patients with low mean 24-hr uptake of 1.41% showed that all patients could be successfully ablated by mean ablative dose of 110.3mCi. [86] When rhTSH was incorporated with low dose RAI in ablation (30mCi RAI), the success rate (54%) was less than that of thyroid hormone withdrawal (84%) alone or in combination with rhTSH (78.5%). [75] Another study from 16 patients showed different conclusions. After 30mCi, the percentage of ablation success was slightly higher in patients treated with rhTSH; 81.2% vs. 75% in the group by T4 withdrawal after 42 to 57 days after surgery. [87] Their protocol involved a temporary suspension of thyroxin for 3 days, beginning at 1 day before rhTSH and

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end on the day after RAI treatment. This approach might decrease the iodine pool and resulted in less interference with rhTSH stimulation. rhTSH has the advantage of avoiding prolonged hypothyroidism and maintaining a good quality of life and productivity before treatment or scanning by RAI. Prospective studies are awaited to document its efficacy in ablation and in treating metastasis.

Hospitalisation for RAI Treatment

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The requirement for hospitalization and isolation for radiation protection differs according to local rules. According to the US Nuclear Regulatory Commission, ‘members of the public will not receive more than 1 mSv (100 mrem) in one year, and that the dose in any unrestricted area will not exceed 0.02 mSv (2 mrem) in any one hour from licensed operations. In addition, the licensee must ensure that air emissions of radioactive material to the environment will not result in a (Total effective dose equivalent) TEDE in excess of 10 millirem (0.1 mSv) per year to individual members of the public.’ [88] Then patients can be released based on certain limits of total administered dose, measured dose rate at 1m or when patient-specific calculation of the likely exposed individual is not exceeded. In conclusion, ablation efficacy depends mainly on the thyroid remnant bulk (which is reflected by the cervical uptake percent of RAI tracer) and dose of RAI. Total or near-total thyroidectomy is advantageous. Firstly, large remnants can prevent TSH rise and decrease success rate of ablation. Secondly, The acute effects of RAI may cause neck pain and transient thyrotoxicosis in those with large thyroid remnant.

Whole Body Scanning by Radioiodine In a study comparing pre-ablation scan with post-therapy scan, 13% of post-therapy scan demonstrated abnormal foci of RAI uptake not seen on pre-therapy scans and this changed the management strategy in 9% of the studied patients. [89] Post-therapy WBS was informative and changed the disease stage in 8.3% and therapeutic approach of 15% patients. It also provided clinically relevant information for 26% patients with 1 previous ablation. [90] Therefore, a post-therapy WBS rather than a diagnostic low dose WBS is favored. It is a common practice to obtain a negative whole body scan (WBS) at 6 to 12 months, in order to confirm success of ablation. This single scan provides important information. Firstly, serum Tg level under maximal endogenous TSH stimulation is measured. Secondly, the visual image of areas of RAI uptake is obtained. Thirdly, RAI uptake percentage is documented. These 3 criteria are used collaboratively to judge the success of ablation. Despite recent reports that diagnostic WBS adds little to treatment [91] and suggestions that whole body RAI scanning can be replaced by rhTSH stimulated Tg monitoring, [92,93] it is a reassuring investigation for patients who have positive anti-Tg antibodies or visible uptake in previous diagnostic scans. Thereafter, patients will be followed regularly to check the serum Tg levels and LR relapses by physical examinations and imaging where indicated. Ultrasonogram (USG) of the

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thyroid bed and cervical lymph nodes is the most sensitive imaging modality to detect neck relapses. Reported sensitivity and specificity are 90% and 82% respectively. [94] Ultrasoundguided fine-needle aspiration biopsy yielded an overall accuracy of 97.2% in patients with previously treated thyroid cancer. [95] Computerized axial tomography (CAT) scanning is good for detection of lung metastasis.

Maximal Safe Dose of Radioiodine The limitation of RAI treatment is the bone marrow or lung absorptive dose, which is very important in metastastic setting. Some guideline are set according to BM dose or whole body retention dose. [96] The requirement is that the dose to the patient’s blood would not exceed 200cGy. Other organ that might be dose limiting is the lungs when diffuse metastases is present. Dosimetry studies, e.g. by MIRDOSE3 software, can estimate the dose to BM and lungs. [97,98] The maximal tolerable dose is higher than expected, as estimated by Dorn et al, to be 38.5 GBq (1,040mCi). [98] So far, no formal dose limit of RAI treatment is published in guidelines. Probably, the cumulative dose, the sites of DM and their uptake percentage, the interval of treatment all interact. Further studies by pooling data might give insight on this important area.

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Interval of Re-Treatment by Radioiodine Practice of re-treatment interval varies from 3 to 6 months. [99-102] There is observation that short intervals of re-treatment using high dose RAI (11.1GBq or 300mCi) in 3 monthly intervals resulted in significant hematological toxicity. [100] Whether this is related to the total cumulative dose or short intervals of treatment cannot be differentiated. A study from Taiwan did not showed any benefit of weekly-fractionated RAI ablation compared with single dose (30mCi). Short interval of re-treatment requires prolonged hypothyroidism which is not favored. [78] The published guidelines did not give a clear recommended re-treatment interval. [62,103] A 1 year interval recommendation was found in a review article. [104] Probably, most centers would use a re-treatment interval of at least 6 months based on the recognition of excessive bone marrow and pulmonary toxicity in cases where high doses of RAI are repeated in short intervals of less than 6 months. (Please see discussions in sessions of ‘Risk and Precautions’)

CLINICAL USE OF RADIOIODINE IN THYROID CANCER RAI has been shown to reduce the likelihood of relapse [3,9-12,23,27,34,105,106] and to improve survival [10,12,23,27,34,106]; it is also an effective treatment for DM. [15,34,99,107-109]

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Postoperative Radioiodine Ablation of Thyroid Remnant ‘Thyroid remnant ablation’ was coined because of its use in the destruction of postoperative residual normal thyroid tissue or microscopic thyroid cancer cells. The incidence of cancer multifocality is 28% to 41% in PTC [7-9,27] and lower in FTC, from 0% to 18%. [9,27,110] Apart from eradicating microscopic foci of tumor cells in the thyroid remnant, RAI ablation facilitates detection of early relapses by serum Tg determination and RAI treatment of RAI-avid relapses. Early detection of relapses could be achieved by checking serum Tg or stimulated Tg (by endogenous TSH or rhTSH). Another possible benefit of RAI ablation is the destruction of follicular cells, which are prone to develop into malignancy at years later. Sugg et al found that rate of RET/PTC rearrangements was higher in patients with multifocal disease in PTC. [111] It is logical that the delayed appearance of relapses at decades after the primary treatment might be related to new malignancy arising from residual follicular cells rather than residual malignancy. Theoretically, eradicating all thyroid follicular cells by RAI ablation is a reasonable treatment for thyroid carcinoma of follicular cell origin. Clinical data on RAI ablation efficacy in reducing relapses and improving survival are published in recent decades. Samaan et al, in a study of 1,599 patients treated in MD Anderson Cancer Center, confirmed that RAI increased disease-free survival in DTC. [12] DeGroot et al found that more extensive surgery and RAI ablation was associated with decreased recurrence in PTC. [112] In a study of 700 patients with DTC treated in University of California, San Francisco, Loh et al observed that among patients with tumor more advanced that T1N0M0, absence of RAI ablation had a 2.1-fold greater risk of cancer recurrence. [11] Tsang et al analyzed 382 patients with DTC treated in Princess Margaret Hospital in Toronto, also confirmed the efficacy of RAI ablation in lowering LR relapse. [9] Mazzaferri and Kloos, in their review of 1,501 patients without DM at initial therapy (Ohio State University & US States Air Force), after a median of 16.6 years, found that RAI ablation decreased all cancer recurrence, DM recurrence and cancer mortality. [16] Our study on PTC in Queen Elizabeth Hospital in Hong Kong, also demonstrated a significant reduction of LR failure and DM, though the survival was not affected. [27] For FTC, RAI also improved LR control (RR 0.13) for patients without DM at presentation. [34] A French study on 172 patients with LR relapses among 3,124 patients with DTC also observed that absence of thyroid remnant ablation is an independent prognostic factor for predicting LR relapses. [105] In spite of these findings, there are inconsistent reports. Mayo Clinic, in their series of 2,512 patients with PTC, found that RAI did not significantly improve the outcome in lowrisk patients after near-total or total thyroidectomy. [8] Their results suggested that the use of bilateral thyroidectomy, instead of unilateral lobectomy, is the major step in reducing relapses and mortality in PTC. Another report from Lahey Clinic, studied the outcome of 727 patients of PTC, showed that RAI did not affect survival in any of the risk groups. [26] In view of the controversies, is it possible to do prospective randomized trials to define the role of RAI in ablation? In 1990, Wong et al combined the results of six large studies and estimated the efficacy of RAI in reducing relapses in thyroid carcinoma was 54%. [113] They also pointed out the practical difficulties in doing a prospective randomized trial to assess the

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efficacy of RAI ablation of thyroid remnants. Four thousand patients would be required in each arm. Results would be available in 35 years. Though Dragoiescu et al opined that sample size is not a problem, [114] recruiting 290 patients in each arm of study is not an easy task. So, retrospective analyses are still the most valuable source of information in guiding treatment of this rare disease. In a systematic review and metaanalysis in RAI remnant ablation for DTC, Sawka et al reviewed 1,534 English references and selected 13 cohort studies to analyze the outcome in relation to RAI ablation. The pooled data from unadjusted studies included over 8,000 patients. The pooled analyses suggested that RAI ablation reduced the 10-yr LR recurrences from 10% to 4% (RR 0.31) and distant metastasis from 4% to 2% (absolute decrease in risk 3%). [115] However, the impact on survival was not confirmed. It is still unclear about the effect of RAI ablation in low-risk patients treated by bilateral thyroidectomy and thyroid hormone suppression. Similar to all meta-analyses and retrospective studies, this study is subjected to criticisms of biases. Bearing in mind the caveats, this review gives the most valuable information on the magnitude of RAI ablation benefit on published literature. The indications for RAI ablation are inconsistent. Some routinely apply RAI ablation after surgery for all patients [116], whereas others select the high risk group for RAI treatment [8,23,106]. The recent guidelines and summary of postoperative recommended treatment are listed in table 1. The British Thyroid Association / Royal College of Physicians recommended RAI for tumors with ≥ 1cm. [62] The National Comprehensive Cancer Network (NCCN) guidelines [117] recommended RAI treatment for tumors with more than 1cm, multifocal disease, positive margins and aggressive variants. A review by Robbins and Schlumberger recommended RAI should be indicated in all patients with a DTC with size of tumor > 1.5cm, or presence of LN metastasis, extrathyroidal extension or multicentricity. Patients with incomplete surgery should have RAI ablation as well. [118] For PTC with less than 1cm, no benefit is demonstrated in preventing relapses. Studies on papillary microcarcinoma (PMC) found that multifocal disease is associated with higher LN metastasis and higher relapse rate. [79,119] The recurrence rate is low: from 3.9% to 8%. [119,120] After initial therapy, LN recurrence and DM occurred in 1.7% to 5% and 2.2% to 2.5% respectively. [79,121,122] The mortality rate is 0% to 3%. [79,121-126] Since the prognosis is so good, some authors recommend that total thyroidectomy was not necessary, [119,127] not to mention RAI ablation. However, bilateral lobar resection is a more favored option if the diagnosis is made before surgery. [79,123,124] The rationale is the high incidence of multifocal disease (28.3%) and bilateral disease (20%). [79] In Queen Elizabeth Hospital, our study showed that RAI reduced the LN recurrence rate, especially in patients with pT1N0 disease, after total thyroidectomy. [79] RAI might be considered in selected cases, e.g. multifocal disease or LN metastasis. [79] The practice of surgery affects the postoperative option of RAI ablation. As discussed in previous sessions, total thyroidectomy would be favored if RAI ablation is to be given. In centers where routine central compartmental LN dissection is not performed, RAI ablation might be employed to reduce LR relapse. All the guidelines did not separate the indications for PTC and FTC, and their variants. Aggressive PTC subtypes such as tall cell variant had higher recurrence rate. [128,129] Diffuse sclerosing variant is reported to be aggressive, but controversial reports are found.

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[130-133] For frankly invasive FTC, DM developed in 26% to 60% of patients. [134] Disease-related mortality was 30% to 50%. [134] The prognosis is much better in minimally invasive FTC. Recurrent disease was found in 4.2% to 5.8%. [34,135] Mortality was 3% to 5%. [134] Therefore, aggressive PTC variants and widely invasive FTC and should be treated with RAI irrespective of the staging.

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Radioiodine Treatment of Locoregional Relapse After primary surgery with or without adjuvant RAI or EXT, the incidence of LN relapse is 13% to 53%, [24,136,137] and LR relapse is 8.7% to 13.2%. [79,138] Recurrence in thyroid bed has worse survival compared with LN recurrence only. [105] The uptake of RAI in WBS by RAI was reported to be 66.8% in lung metastasis, 100% in bone metastasis and 33.3% in LN metastasis. [139] If surgery is feasible, it should be considered in every patient with LR relapses. [62] RAI can be tried if the LR relapse can take up RAI. RAI as the sole treatment in LR relapse is rarely reported. The role of EXT cannot be overlooked. A summary by Maxon et al showed that in patients with LN showing RAI concentration, 68% (58/85 patients) can achieve a complete resolution by scanning, x-rays or clinical assessment. [140] Robbins also observed a single dose of RAI can abolish subsequent RAI uptake in approximately 65% of patients with LR recurrence at Memorial Sloan-Kettering Hospital. In Institute Gustave-Roussy, LN relapse of > 1cm are usually just partially responsive to RAI treatment. [118] In an Italian series of 146 patients with LN relapse, 76% achieved ‘definitive cure’ after a mean cumulative dose of 123mCi. In 2.6% patients, functioning nodal metastasis lost their ability to take up RAI after a mean dose of 77mCi. [141] In patients with persistent or recurrent LN metastasis, even these LN did not take up RAI following RAI treatment, persistent disease may be present by imaging or proven by pathological examination of surgical specimen. Neither an undetectable Tg nor a negative post-therapy WBS can announce ‘cure’. [142] Therefore, surgery should remained the option of treatment for larger size LR relapse. [118] Tg monitoring revolutionized the follow-up of DTC. In patients without anti-Tg antibodies, Tg monitoring is the mainstay of monitoring after complete surgery and RAI ablation. [92,143] Impalpable LR relapse can be detected at an early stage. Nowadays, very small tumor recurrence can be confirmed by USG guided FNA. It is very important to localize the disease by imaging e.g. USG, CAT scan or MRI scans. Otherwise, re-operation of such small relapses is very difficult. EXT should be considered when surgery is not feasible, a high chance of residual disease after surgery and when the tumor is not RAI-avid. EXT is shown to be effective in LR control in patients with gross LR residual disease. Local control is improved after EXT. [9,27,144147] There is skepticism about EXT in DTC because of the significant side-effects of acute neck inflammation and subsequent fibrosis. New radiotherapy technique by intensity modulated radiotherapy provides a very good dose distribution to reduce the dose to critical organs, namely the spinal cord and lungs, and to increase dose to gross tumor in the neck. [148,149] This new technique is very promising and should be considered if patient is indicated for EXT.

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Table 1. Summary of guidelines recommendations for postoperative management of thyroid cancers. Guidelines National Comprehensive Cancer Network (NCCN) guidelines [117] 2001 AACE/AAES Medical/Surgica l Guidelines [249] 2001 Thyroid Carcinoma Task Force

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British Thyroid Association / Royal College of Physicians [62] 2002

North Cancer Network Guidelines [103] 2000

Radioiodine ≥ 1cm, positive margins, aggressive variants, multifocal disease. Tg >10ng/ml (off thyroid hormone) or RAI scan positive Dose: not mentioned. No concrete indications mentioned in the guidelines. In low-risk groups, RAI should be done by a case-by-case decision, guided by clinical judgement and experience. Dose: In the past, 75-150mCi. Now some centers use 25-30mCi. Tumours ≥ 1cm after total thyroidectomy Dose: 3.7 GBq

Tumour ≥ 1cm Dose: 3 GBq

Indications for External radiotherapy T4 disease and age > 45y

TSH suppression All patients

Poorly differentiated histology in adjuvant setting. Gross local invasion, high risk of residual disease in neck

In high risk group. The degree of suppression should be related to risk of relapse / scoring system

The main indications are: i. Unresectable tumours that do not concentrate 131I. ii. Gross evidence of local invasion at surgery, presumed to have significant macro- or microscopic residual disease, particularly if the residual tumour fails to concentrate 131I. iii. Recurrent disease in the neck which is not amenable to 131I therapy or further surgery. iv. Palliation of inoperable metastatic disease in bone, mediastinum, brain, spine or other areas. High grade tumours that do not concentrate RAI. Gross local invasion at surgery, presumed to have residual disease. Recurrent lesion in neck which is not amenable to RAI or further surgery. Palliative EXT to distant met. E.g. bone, brain. For inoperable disease. Essentially similar to BTA and RCP guidelines

All patients

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For all patients, supervised by an endocrinologist

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Radioiodine Treatment of Distant Metastasis

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At presentation, PTC is usually localized in neck, i.e. thyroid or cervical LN. Only 2% to 5% patients have DM. [1,3,8,9] The rate of DM is higher in FTC than PTC; 14.9% to 21.8% patients with FTC had DM. The pattern of distant metastasis is also different: higher rate of bone metastasis is found in FTC while in PTC, lung is the major organ for distant metastasis. The 10-year distant metastasis failure-free survival is 90.8% and 72.3% for PTC and FTC respectively. [1] The 10-year survival after DM is 25% to 65%. [150-153] The 10-year survival after diagnosis of lung metastasis is 32% to 59.6%; [152,154] that of bone metastasis is poorer, from 13% to 15%. [155,156] Multivariate analyses showed that the prognostic factors for survival after distant metastasis are age at diagnosis of DM, [151,154] site of DM, [151]and degree of extrathyroidal extension, [151] uptake of RAI in DM, [109,154,157] and morphological features of lung metastasis [109,154] and differentiation of tumor. [157] In general, patients with distant metastasis from malignant tumors are considered to be incurable and have grave prognosis. However, differentiated thyroid carcinoma is deemed a special entity because of its susceptibility to RAI treatment. Lung Metastasis RAI is effective in decreasing size of DM and prolonging survival. It is especially found to be effective in patients with lung metastasis which is not evident in chest x-rays. Positive RAI uptake is only present in 67% patients with RAI WBS. [99] A further detection of lung metastasis was found in 3.8% (15 /394 patients) after 100mCi of RAI therapy. Among patients with lung metastasis only, RAI uptake was found in 96% of those with normal CXR, 88% of those with micronodules and in 37% of those with macronodules. Overall, only 46% of patients with positive uptake achieved ‘complete remission’ or CR. With Tg monitoring, lung metastasis can be detected when chest x-ray (CXR) is normal. In Institute GustaveRoussy, only 4% patients with lung metastasis had normal CXR prior to 1977. The integration of Tg into the follow-up protocol increased the proportion of patients with normal CXR to 40%. [99] To assess response of DM to RAI is sometimes difficult. Imaging like CXR, CAT scan or MRI scan are usually employed to assess the size of DM. Effectiveness of treatment can also be evaluated by the value of Tg and WBS uptake. As discussed in session of ‘RAI in LR relapses’, Tg and WBS can be misleading because of loss of functional uptake of RAI in DM or secretary function of tumor cells. Several large studies addressing this problem showed that RAI could induce complete remission in 24.6% [158] to 33.8% [107] in DTC lung metastases. The cure rate was reported to be 45.5% in PTC and 19.5% in FTC. [141] Casara et al demonstrated that morphological, functional property of lung metastases and the presence of multiple distant metastases were important in Cox regression model. For patients with negative chest x-rays but positive RAI uptake, the complete remission rate was 78%. The CR rate dropped to 3.7% when both chest x-ray and RAI uptake were positive. There was no case of CR in patients with positive chest x-rays but no RAI uptake. [158] A recent study by Ronga et al studied 96 patients with lung metastasis. They also confirmed the importance of RAI-avidity in treatment of lung

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metastasis. Young age and positive RAI uptake was associated with better prognosis. Risk of death was increased by 5.4-fold in patients with age > 45 years. RAI treatment reduced risk of death to nearly 1/6. [154] However, Mayo Clinic found that RAI did not have s significant influence on survival, after adjusting for age and extent of metastasis involvement. [150,151] They also found that the survival rates were similar for metastasis of PTC in the years 1940-1954 and 1970-1989, suggesting that advances in diagnosis and management did not altered the average survival of patients with DM from PTC. None of the treatment variables were significant predictor of survival. With advances in diagnostic imaging, more evidence is found that RAI cannot completely eradicate all metastasis foci. Tumour load in lung metastasis is much more than what CT shows, as evidenced by histology of lung biopsies. Many lung metastases were less than 1mm in diameter. [159] After all, RAI is still the best treatment for lung metastasis from DTC, especially in the young, RAI-avid metastasis of small size. Bone Metastasis RAI is an effective treatment for bone metastasis. In multivariate analysis, independent prognostic variables for survival are: RAI uptake [99.156], cumulative dose of RAI, [155] absence of non-osseous metastasis, [155,156] bone metastasis as a revealing symptoms of thyroid carcinoma, [155] complete bone metastasis surgery in young patients. [155] RAI can prolonged survival in patients with bone metastasis which had RAI uptake. [155,156] A series of 107 patients (among 3,088 patients) with bone metastasis were treated by RAI. High RAI uptake in bone metastasis was observed in 94.2% of patients. Patients with RAI-avid bone metastasis had a mean survival of 8.9 years. Long-term remission was found in 24% and partial remission was found in 27% of patients. Patients with age < 45 and number of bone metastasis ≤ 3 had better prognosis. [101] Apart from RAI treatment, other useful treatment includes surgery (for fixation of fractures, spinal metastasis with cord compression), [160] embolization, [161,162] EXT and bisphosphonates. [163] These are good medical interventions to palliate symptoms of bone metastasis and improve quality of life. Meta-analysis showed that single dose radiotherapy was as effective as multifraction radiotherapy in relieving metastatic bone pain. [164] Spinal surgery together with EXT can give good survival and significant palliation in spinal metastasis. [165] Brain Metastasis Brain metastasis in DTC might be under diagnosed. Chiu et al found that brain metastases were only discovered at autopsy in 23% of patient. In their study of 47 cases of patients, only 3 of 18 (17%) patients had positive uptake in WBS. Role of RAI is not well defined. [166] There are case reports of acute cerebral edema shortly after RAI therapy. [167] To avoid tumor growth and swelling under continuous stimulation by endogenous TSH in case of thyroxin withdrawal, rhTSH and steroid can be tried. [166] In general, brain metastasis should be considered for surgery. Surgical removal might prolong survival. [166,168] Though there might be selection bias, those who underwent surgery had a better survival of 22 months, compared with 3.6 months in patients who had no surgery. [166] Whole brain radiotherapy with boost by stereotactic techniques might also be useful. [169]

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Radioiodine Treatment in Children and Adolescents The reported series in the literature revealed that patients with DTC in childhood and adolescence usually present with more advanced LR disease and have a high rate of DM. The incidence of LN metastasis ranged from 45% to 90%. [15,170-173] DM were found in 5.8% to 25% of patients at diagnosis. [15,170,171,174-177] Although young patients had more advanced disease at presentation and higher recurrence rate, mortality rate was low and reported to be zero in some series. [171,178-182] A report on 329 patients from 15 hospitals confirmed the good survival in patients with less than 21 years old; the mortality was 0.6%. [175] Younger age (as defined by less than 7 [179] to 15 [175]) was found to have with higher recurrence risk. [173,175,179] Schlumberger et al reported 6 cases of disease mortality among 72 patients. All of the deceased had diagnosis of DTC before the age of 10. [170] The small number of pediatric patients renders statistical analysis of RAI ablation a difficult task. The recommendations for surgery and RAI ablation should be similar to the adults. RAI ablation is recommended for pediatric DTC by a number of authors. [15,171,173,174,183] RAI is effective in treatment of DM in young patients. The reported complete response rate of lung metastasis in children or adolescents ranged from 16.7% to 55.6%. [15,184,185] RAI in young patients (age < 21) is safe in terms of second malignancy and pregnancy outcome. [15]

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Radioiodine in Patients with Raised Thyroglobulin but Negative I-131 Whole Body Scan In the follow-up of DTC, USG neck, WBS and serum Tg monitoring are considered to be complementary. [186,187] Serum Tg measurement is the single most sensitive and specific investigation for detection of relapse in DTC. The sensitivity of this test increased with TSH level. Two guidelines suggested using stimulated Tg for surveillance of low-risk patients. [92,143] Although stimulated TSH had increased sensitivity of detecting relapses, the cost of rhTSH is a real concern. In a study of 122 patients, Tg < 1ng/ml (under T4 treatment) combined with negative neck USG presented a high negative predictive value of 99.1% in low risk patients. This result doubted the recommendation of using stimulated Tg in surveillance. [188] Currently, in most of the centres, Tg measurement is performed when patients are taking T4 under replacement or suppressive dose. Understandably, this is related to its advantage of simplicity and cost-effectiveness. In the absence of anti-Tg, serum Tg is a very reliable tool to detect recurrence. The reported incidence of anti-Tg antibodies is 7.4 % to 22.6%. [80,130,189,190] Stimulated Tg, coupled with ultrasonography and other imaging, detected relapses much earlier. Therefore, it is not uncommon to encounter patients with raised Tg but negative WBS. The clinical dilemma of localization of relapse when tumour marker is elevated presents a challenge in oncology follow-up. In the past, the cut-off point of Tg under thyroid hormone replacement or suppressive dose, which necessitates further investigations, is above 5 to 10ng/ml. [186,191] With more studies, the current accepted undetectable Tg value is < 1ng/ml during T4 treatment. Whole body scan (WBS) by I-131 is part of the initial

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investigations, apart from ultrasonography and MRI of neck, CAT scan neck and thorax. For neck recurrences, ultrasonography with guided fine needle aspiration biopsy has highest specificity for detecting recurrence. [192] In case of negative WBS, some authorities promote the use of PET scan to detect sites of recurrence. In this situation, the diagnostic sensitivity and accuracy of FDG-PET were 85% to 94.6% and 87% to 90.0% respectively. [193-195] However, the specificity of FDG-PET is reported at a wide range of 25% to 90%. [193,195] It is of limited efficacy in military lung metastasis, [194] minimal disease of less than 1.5cm [192] and in patients with minimal cervical adenopathy. [196] TSH might stimulate FDG uptake. Controversial reports are found on the changes in sensitivity by TSH stimulation in hypothyroidism status. [193,197] The clinical decision of empirical RAI or so-called ‘blind’ treatment with post-therapy WBS is difficult. It involves a thorough discussion with patients for the risks and potential benefits. In view of the ‘stunning’ phenomenon observed with high diagnostic dose, high RAI dose followed by post-therapy scan is recommended. The decision of giving ‘blind’ RAI trial depends on the level of Tg, the findings from other imaging scans and surgical expertise in treating local or lymph node relapses. Pineda et al, in a series of 17 patients, found that RAI is effective because of the observation of conversion of positive scans to negative scans, decrease in mean Tg level and reduction of Tg to < 5ng/ml in about 50% of patients. [198] In a Korean study of 60 patients with raised Tg but negative DxWBS, post-therapy WBS revealed pathologic uptake in 42.9% of patients. The Tg levels were lowered after RAI therapy. Four patients even converted to negative Tg. [199] Fatourechi et al, in a series of 24 patients with raised Tg, classified the findings according to the size of tumor relapse into 3 groups: macrometastasis (>1cm), micrometastasis ( 2 ng/ml. [92] Another report from Europe recommended a cut-off point of Tg to be based on institutions. Tg can be measured during T4 suppression. [143] Further studies on rhTSH efficacy and cost-effectiveness are awaited.

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Several authors also opined that patients with detectable Tg should be treated with at least 1 dose of RAI; [202] the dose should be repeated until remission or negative uptake in 1 or 2 subsequent WBS. [203] So far, no proven benefit on survival can be shown by empirical RAI treatment in this scenario. The decision on ‘empirical’ RAI treatment should be carefully weighed.

RISK AND PRECAUTIONS OF RADIOIODINE ADMINISTRATION IN HUMAN In considering any therapy, it is very important to strike a balance between therapeutic benefit and side effects. RAI is very attractive because of its simple oral administration and mild acute side effects. The following summarized the side effects and alert the clinicians in prescription of RAI.

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Short-Term Side Effects Immediately after oral administration, acute severe side effects are rare. Mild clinical effects are nausea, acute sialadenitis, transient neck pain related to thyroiditis (especially in patients with large thyroid remnant after surgery, e.g. lobectomy), and hematological depression [204-206] Patients might be more annoyed by the symptoms of hypothyroidism which is a necessary prerequisite for RAI ablation (in centers with no available rhTSH). Another short-term inconvenience is ‘hospitalization for radiation protection’ in high dose treatment. An analysis on the symptoms immediate after RAI administration showed that 65.2% had gastrointestinal complaints, 50% had salivary gland swelling with pain, 9.8% had change in taste and 4.4% patients had headache. Multivariate analysis showed that the significant factors influencing gastrointestinal symptoms were dose per body weight and TSH values. The most common gastrointestinal symptoms were appetite loss (60.9%) and nausea (40.2%). For salivary gland swelling and pain, females had more frequent complaints. For taste changes and headache, no significant factors could be identified. [207] The most serious acute complications are acute edema or hemorrhage in tumor or metastasis. This is particularly important in cerebral metastasis [167] or those tumors situated near major airway. Transient pain was reported in bone metastasis with high uptake. [101] Transient alopecia was observed in 28.1% patients in a cohort of patients after at least 100mCi of RAI; this was not dose-dependent. [206] The use of rhTSH is now being tested in RAI treatment. The use of this hormone obviates the prolonged period of hypothyroidism and preserves normal thyroid function in patients. This is tried in metastasis setting and found to be well tolerated with high efficacy. [208] Now, rhTSH is approved in diagnostic use. More data is awaited to show its efficacy in thyroid remnant ablation and treatment of DM.

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Long-Term and Specific Side Effects The chronic side effects are decreased salivary production, radiation pneumonitis and fibrosis [209]. The effects on salivary glands, bone marrow and lung are dose-dependent. Long-term severe side effects are rare. With good precautions on the preparation for RAI treatment, RAI cumulative dose and intervals of treatment, these side effects should be minimized.

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Salivary Gland Damage Single dose of RAI of less than 5 GBq gives lower than 10% rate of sialadenitis and taste changes. [206] This study demonstrated a dose-dependent increase in frequency of sialadenitis and taste changes. For those who received 18.5 to 37 GBq, about 55% patients will experience this sialadenitis. With repeated treatment with high activity of RAI, 27% to 33% patients had salivary swelling [206,210] and 30% patients had dry mouth. [210] To avoid salivary gland damage, some centers recommend sucking of lemon candies, aiming to decrease salivary gland damage by RAI. [206] However, a recent randomized trial refuted this belief. [211] An early start of suckling lemon candy within 1 hour after RAI therapy increased the incidences of sialadenitis, taste loss and dry mouth. The author concluded that lemon candy should not be given until 24 hours after RAI therapy. Bone Marrow Toxicity and Leukaemia Bone marrow (BM) is affected by radiation dose from RAI treatment. It is important to know whether this radiation will cause any detrimental effects to the marrow. Transient leukopenia and thrombocytopenia were observed. [98,100,206] The marrow toxicity is dosedependent. [98,100] Severe leukopenia and thrombocytopenia is only seen in high dose therapy (>22.2 GBq). Will the dose cause sublethal damage to marrow cells, resulting in malignant transformation? A number of acute leukemia after RAI treatment was reported in the literature, especially found in patients with bone metastasis. [101,209,212] The bone marrow recovery after RAI treatment is lower in age older than 45. [101] The study by Petrich et al illustrated that the bone marrow suppression after RAI treatment can be analyzed according to WHO classification. Most of the blood counts alterations are grade I or II, i.e. mild and reversible. Less commonly observed was grade III (persistent severe blood count suppression) and grade IV (bone marrow aplasia or acute myeloid leukemia). In their cohort of 107 patients with bone metastasis, the blood count alterations as observed in age ≤ 45 was mild, usually grade I or II. For really high dose to bone marrow for patients with high uptake in bone metastasis, it was observed that 8 out of 107 patients died of bone marrow problems. Four patients had bone marrow aplasia (mean RAI dose 69.93 GBq) and 4 had acute myeloid leukemia (mean RAI dose 87.4 GBq). All of them were older than age 45. [101] In this center, patients were treated with total 11.1GBq of RAI when metastasis was detected (3.7 GBq followed by 7.4GBq immediately after the scan was positive for metastasis).

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Almost all cases of leukemia after RAI treatment received more than 800mCi, were more than 45 years of age and with short treatment intervals of few months. [101,209,213] Only very rarely is acute leukemia found in patients with small RAI dose of < 300mCi. [214,215] Most of the published reports indicate that the common type of leukemia is acute myeloid leukemia. Only a few cases of chronic myeloid leukemia were reported. [216,217] It is reassuring that acute leukemia is seldom found after low dose of RAI in large population of patients. A report by de Vathaire et al. on 1,497 patients who received an average of 7.2 GBq of RAI revealed no instances of leukemia at a mean follow-up of 10 years. [218] In our cohort of patients of 1,348 patients in Queen Elizabeth Hospital, we did not observe a single case of acute leukemia after a mean dose of 3.4 GBq in PTC and 4.14 GBq in FTC. We also observe no single case of second malignancy in 36 young patients of less than 21 years of age, who received a mean dose of 117 mCi (4.3 GBq), after a mean follow-up of 14 years. [15] Menzel et al also observed no case of leukemia in the nearly 2,000 patients treated in a single institution. [100] The risk of leukemia is not elevated in several large studies including patients with RAI treatment for thyrotoxicosis or diagnostic scans. [219] Wong et al [113] suggested that the risk of leukemia is so small that it does not outweigh the benefit of RAI treatment: the loss of life caused by recurrence of thyroid cancer exceeds that from leukemia by 4- to 40-fold. Secondary Malignancy Reports of second primary malignancy (SPM) after RAI treatment are controversial. A small excess of bladder cancer and leukemia was observed by Edmonds. [209] Some authors report a small excess risk of cancers in organs that concentrate RAI such as salivary gland, colon, and bladder [209,214,220]. However, most of these patients received very high RAI doses (more than 40.7 GBq or 1100 mCi). [209] A study on 7,417 patients with RAI treatment for thyrotoxicosis in England and Wales showed that the relative risk of cancer mortality was decreased after RAI treatment. [221] Incidence of cancers of the pancreas, bronchus, trachea, bladder, and lymphatic and haemopoietic systems was lowered. Mortality from cancers at all these sites was also reduced but findings were significant only for bronchus and trachea. There were significant increases in incidence and mortality for cancers of the small bowel and thyroid, although absolute risk of these cancers was small. A Swedish report by Hall et al found that organs that were estimated to have received more than 1 Gy had a significantly increased risk of a subsequent malignancy after RAI treatment. The cancer risks were studied in 834 patients with average dose of 4,551 MBq. A dose related increased risk of cancer risks was observed. For patients receiving dose of less than 1850 MBq, no significantly elevated overall risk of subsequent malignancy was observed. [219] Dottorini et al, in a study of 814 female patients, found an elevated standardized incidence for salivary gland tumors and melanoma. [222] A study by de Vatharie found that the risk of solid tumors, excluding digestive tract cancers, was not increased after RAI treatment. [218] The risk of colorectal cancer was increased and related to the total activity of RAI administered 5 years or more before its

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diagnosis (excess relative risk = 0.5 per GBq, P = 0.02). The authors suggested that this might be related to the accumulation of RAI in the colon lumen. The sites of SPM from different reports are not consistent. Among studies with large patient number on this subject, [218,219,222], SPM might be increased in colon, [218] salivary gland and melanoma. [222] Rubino et al, studied the risk of second primary malignancies in 6,841 patients who are 2-year survivors of a cohort of European thyroid cancer patients. [223] This study had high statistical power by combining data from 3 large studies of Sweden, [219] Italy [222] and French. [218] Compared to the general population of each of the 3 countries, there was an increased risk of cancer of 27%. No significant association was found between EXT and risk of SPM, except for bone and soft tissue cancers. The relative risks of cancers of bone and soft tissue (RR=4), female genital organs (RR=2.2) central nervous system (RR=2.2) and leukemia (RR=2.5) were increased after RAI exposure, after adjusting for EXT. A strong relationship was found between cumulative dose of RAI and risk of cancers of bone and soft tissue, colorectum and salivary gland. There is also an association of thyroid cancer to breast cancer, [223-226] which is not related to treatment by RAI nor EXT. [224,227] While diagnostic dose of RAI does not increase risk of thyroid cancers, [228] we should be cautious in prescription of RAI ablation in low-risk patients. Lung Toxicity As mentioned previously, RAI can induce complete remission in patients with lung metastasis, depending on the functional uptake and morphological appearance in imaging. In general, RAI for lung metastasis is safe. Studies on the capillary diffusion capacity did not showed any adverse effects of RAI. [229] Particular attention should be paid to patients with diffuse lung metastasis. Radiation pneumonitis fatality reports were found in patients with multiple fine, diffuse lung lesions. [230] Rall et al suggested that the amount of RAI delivered to patients with multiple diffuse lung metastases should not exceed 125mCi in any single dose. The interval of retreatment should be at least 6 months. Maxon and Smith, in their review of RAI induced pneumonitis, found that 6.3% (9/143) patients with lung metastasis had this complications. [140] However, it is difficult to differentiate RAI induced pneumonitis from progressive pulmonary metastases. [209] Sometimes, RAI uptake in inflammatory lung disease can mimic diffuse lung metastasis. [231] In lung metastasis, pathological examination of lung biopsies showed that small lung metastasis were found more extensive than expected. [232] However, the clinical reports of RAI induced pneumonitis, especially in recent decade, are rare in the literature. In our hospital, we did not document a single case of RAI induced pneumonitis. Bearing in mind this potential side effect, RAI is still the most effective treatment in DTC lung metastasis. Male Fertility After RAI treatment, there will be transient oligospermia, which should be reversible in few months. The damage to spermatogenesis is dose-dependent [233] with elevation of serum FSH. [233-236] FSH level subsequently fall to normal at 12 months later while the

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testosterone level did not change significantly following iodine treatment. [235] The damage is usually transient but permanent damage and infertility might be resulted after repeated high doses. [234] Hyer et al studied 122 men under the age of 40 at the time of treatment with a median follow-up of 21 years. [235] Fertility is not impaired and children fathered by male patients with history of RAI administration had no major congenital malformations. After a single ablative dose, the median estimated dose to each testis was low: 6.4 cGy following 3 GBq, 14.1 cGy following 5.5 GBq and 21.1 cGy following 9.2 GBq. RAI should be safe and infertility risk should be minimal. However, counseling and sperm banking should be considered in young males when high dose of RAI treatment is indicated. Female Fertility and Pregnancy Outcome Temporary ovarian failure was reported in 17-27% of women receiving RAI, within 10 month to 1 year after therapy. [237] There was no case of permanent ovarian failure in a series of 322 patients in United Kingdom. [238] Menstrual disturbance was found in those with greater cumulative dose of RAI [238] and older age. [237,238] RAI therapy was associated with an earlier onset of menopause. [239] The possibility of genetic or physical damage to the offspring, in terms of congenital malformation and childhood malignancies, are real concern. Several studies addressing this problem did not confirm associations of previous RAI exposure to unfavorable pregnancy outcome [222,238,240-246]. The largest reported series of 2,113 pregnancies by Schlumberger et al revealed that the miscarriage rate increased from 11% to 20% after surgery, irrespective of RAI treatment [245]. More miscarriage was observed (40%) if RAI treatment was administered within 1 year before conception or in older age group. The general consensus was to recommend pregnancy in young patients after the first post-RAI year [222,241,242,244,245]. We did a study of the gestational history of 104 patients with pregnancy after diagnosis of DTC. [246] After a mean dose of 96.6mCi of RAI, the pregnancy outcome was not adversely affected by history of RAI treatment. There were no stillbirths. The rate of miscarriage, percentage of live births, condition of neonates (sex ratio, birth weight, congenital malformation) was not different. No long-term impairment of childhood development was observed. The incidence of preterm delivery is higher in patients with history of RAI administration. Comparing the clinical data of pregnancies after RAI administration (>5mCi) with that of a territory-wide audit of hospital of 95,074 pregnancies in Hong Kong [247], the pregnancy outcome was not different. Many factors might affect pregnancy outcome after thyroidectomy in patients with DTC: the adequacy and fluctuation of thyroid hormone replacement, radiation dose to ovaries, other non-DTC related factors such as age at conception, socioeconomic class, alcohol intake and smoking. The physical half-life of iodine-131 or RAI is 8.04 days [248] while the median effective half-life of RAI is at least 14 hours [54] with substantial variations. It is reasonable to assume a rapid washout of whole body RAI after administration. Nevertheless, some guidelines recommend avoidance of pregnancy for 4 months [62] to 6 months [249] after RAI treatment

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or scanning while others recommended 1 year [103,241,242,244,245]. Observing a higher miscarriage rate [245] and some untoward pregnancy outcome like birth defects [242], Edward syndrome [241] and aplastic anemia [241] found in conceptions in the first post-RAI year, a recommendation to avoid pregnancy in the first year post-RAI treatment seems reasonable. In addition, this approach allows time for confirmation of disease remission [244] and control of thyroid hormonal status [245]. At the dose levels currently used for ablation and treatment in female patients, there is no definite evidence of an increased risk of permanent infertility or teratogenicity. [222,238,240,245,246] Education and information to patients for proper contraception and good drug compliance cannot be over emphasized. Proper procedural instructions, pregnancy testing before RAI administration and deferral of RAI administration (in case of doubt) can avoid RAI intake to a pregnant patient. During pregnancy, the thyroid function needs to be closely monitored. With all these preventive measures and precautions, RAI should be a safe treatment method in young ladies.

EPIDEMIOLOGY, PRACTICE AND GUIDELINES

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Secular Trends in Differentiated Thyroid Cancer Certain changes in DTC epidemiology was observed in some countries: increased incidence in Australia [250], Scotland, [251] Europe [252], Canada, [253] decreased size of tumour [254,255], decreased mortality rate in Switzerland, Austria, [256] Japan [254] and Hong Kong [255]. Most of the increased DTC was related to an increased frequency of PTC [252,253,255, 57] and decreased incidence of anaplastic carcinoma. [258-262] The worldwide observation of an increased frequency of PTC, less advanced tumour stages and reduction in mortality in DTC [251,254-259] was also found in our patients. [255] The possible explanations are presentation of disease at earlier stage, [254,255], change in diagnostic practice, [263] more aggressive management by bilateral surgery [8] and radiation therapy (RAI and EXT), standardized treatment protocols [251] and also early detection of disease relapse by powerful surveillance by imaging and serum Tg measurement. Epidemiology studies demonstrate a relationship of iodine-deficiency to FTC while dietary iodine supplement increases incidence of PTC but FTC remains constant [259,264]. The observed increase in incidence could be related to the change in clinical practice that the diagnosis (e.g. by USG and FNA) and management of benign thyroid problems (more surgery than observation) increased the discovery of ‘incidental’ cancers at a smaller size. The pattern of practice changed considerably during the past decades in Hong Kong: there was increase in bilateral thyroid surgery compared to unilateral resection and increased application of RAI and EXT. [255] Bilateral resection resulted in less local relapse [24,106], and improved survival in high-risk patients. [8] Improvement in pathology reporting is also observed. With more accurate histology diagnosis, description of size of primary thyroid tumor, lymph node status, resection margins, multifocality and extrathyroidal extension, the prognosis and management can be properly decided. Results from different centers can be compared.

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Despite a few negative reports of RAI on thyroid cancer management [2,26,265], the use of postoperative RAI ablation is more frequently applied, probably related to a much greater number of positive studies. [9-12,23,27,34,105] Concerning the diagnostic tools for follow-up, ultrasonography coupled with guided fine needle aspiration biopsy [94,95] and rhTSH stimulated serum Tg had highest accuracy in detecting relapses in patients with near-total thyroidectomy and RAI ablation. [189] In some centres, rhTSH stimulated Tg testing is utilized in follow-ups. Recombinant TSH should be very promising in taking up roles in diagnostic and therapy of DTC.

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RAI Usage in DTC in Different Areas It is interesting to note that the rate of postoperative RAI ablation varies significantly among countries. In a study from Yamashita et al. including 2,423 patients, postoperative RAI ablation was not employed because of the rigid regulations of use of radioisotopes in Japan. [254] RAI was only used in distant metastasis with avidity for RAI. For patients with PTC larger than 1cm, they performed thyroidectomy and ipsilateral modified radical neck dissection. [48] In Germany, 80% of patients with tumours less than 1cm and 90% patients with PTC of stages II, III and IV had postoperative RAI treatment. [266] This reflects that German physicians consider RAI an integral part of the management of DTC. RAI ablation also links to type of surgery closely. The success of ablation depends on the residual thyroid tissue bulk. In this German study, 93.8% of patients had thyroidectomy and only 2.1% had lobectomy. [266] In a pooled study of a European cohort of 6,841 thyroid cancer patients from Swedish, Italian and French, 62% received RAI. [233] In United States, RAI was only given to 50% of patients in a cohort of 5,583 patients diagnosed in 1996. [267] Lobectomy or excision was performed in 17.8% and 22.4% of PTC and FTC respectively. The application of RAI in ablation can be very low in some centers, e.g. 8.2% of FTC treated in Cleveland clinic [268] and 26% of PTC in Mayo Clinic. [8] In Hong Kong, there is an increasing trend of using RAI treatment. The rate of RAI treatment increased from 50% before 1980 to 84.3% in the years from 1991 to 2000. [255] In a report from Mexico, Herrera et al indicated that RAI ablation was administered to 84% of patients with PTC after operation. [269] In this particular disease, treatment decision is puzzled by the lack of prospective randomized trials and participation in management by different specialties (surgeons, oncologists, radiologists working in nuclear medicine, endocrinologists). Furthermore, compatibility with long-term survival even in presence of distant metastasis or local relapse complicated the picture.

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Guidelines for Management of DTC The development of consensus and guidelines is very important to maintain standard of care. Guidelines allow auditing and quality assurance. Several guidelines well serve this purpose. They give clear instructions to doctors working in different specialties: surgeons, oncologists, endocrinologists, family physicians and radiologists working with nuclear isotopes. Table 1 summarized the recommendations of postoperative management by some of the important guidelines. In general, tumor size of ≥ 1cm was an indication for total thyroidectomy and RAI ablation. External radiotherapy is indicated for locally advanced disease with high risk of residual disease after surgery. TSH suppression is recommended for all patients in western countries, with a caution that the degree of suppression should be according to the risk of relapse or scoring system.

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CONCLUSION Judging from the current literature, RAI treatment appears to be safe and effective in reducing the risk of relapse and probably in improving survival. The issue of RAI ablation in low-risk patients is still unresolved. Though the side effects are mild, cautions to procedure of administration is needed. A proper reporting of side effects and complications and pooling data would clarify the issues on secondary malignancy and leukemia. The use of rhTSH in treatment deserves further studies. This could improve the quality of life, preserving normal thyroid function and social integrity compared with thyroxin withdrawal. The improvement in outcome in DTC in recent years is probably related to a concerted effort of more radical surgery (bilateral thyroidectomy and lymph node dissection), RAI treatment in ablation and treatment of relapses, early detection of disease by imaging techniques like ultrasonography, CAT scan and serial monitoring of serum Tg.

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[99] Schlumberger, M., C. Challeton, F. De Vathaire, et al., Radioactive iodine treatment and external radiotherapy for lung and bone metastases from thyroid carcinoma. J Nucl Med, 1996. 37(4): p. 598-605. [100] Menzel, C., F. Grunwald, A. Schomburg, et al., "High-dose" radioiodine therapy in advanced differentiated thyroid carcinoma. J Nucl Med, 1996. 37(9): p. 1496-503. [101] Petrich, T., A. Widjaja, T.J. Musholt, et al., Outcome after radioiodine therapy in 107 patients with differentiated thyroid carcinoma and initial bone metastases: side-effects and influence of age. Eur J Nucl Med, 2001. 28(2): p. 203-8. [102] Hindie, E., D. Melliere, F. Lange, et al., Functioning pulmonary metastases of thyroid cancer: does radioiodine influence the prognosis? Eur J Nucl Med Mol Imaging, 2003. [103] Northern Cancer Network guidelines for management of thyroid cancer. Clin Oncol, 2000. 12(6): p. 373-91. [104] Van Nostrand, D., F. Atkins, F. Yeganeh, et al., Dosimetrically determined doses of radioiodine for the treatment of metastatic thyroid carcinoma. Thyroid, 2002. 12(2): p. 121-34. [105] Rouxel, A., G. Hejblum, M.O. Bernier, et al., Prognostic factors associated with the survival of patients developing loco-regional recurrences of differentiated thyroid carcinomas. J Clin Endocrinol Metab, 2004. 89(11): p. 5362-8. [106] Taylor, T., B. Specker, J. Robbins, et al., Outcome after treatment of high-risk papillary and non-Hurthle-cell follicular thyroid carcinoma. Ann Intern Med, 1998. 129(8): p. 622-7. [107] Samaan, N.A., P.N. Schultz, T.P. Haynie, et al., Pulmonary metastasis of differentiated thyroid carcinoma: treatment results in 101 patients. J Clin Endocrinol Metab, 1985. 60(2): p. 376-80. [108] Massin, J.P., J.C. Savoie, H. Garnier, et al., Pulmonary metastases in differentiated thyroid carcinoma. Study of 58 cases with implications for the primary tumor treatment. Cancer, 1984. 53(4): p. 982-92. [109] Casara, D., D. Rubello, G. Saladini, et al., Different features of pulmonary metastases in differentiated thyroid cancer: natural history and multivariate statistical analysis of prognostic variables. J Nucl Med, 1993. 34(10): p. 1626-31. [110] Emerick, G.T., Q.Y. Duh, A.E. Siperstein, et al., Diagnosis, treatment, and outcome of follicular thyroid carcinoma. Cancer, 1993. 72(11): p. 3287-95. [111] Sugg, S.L., S. Ezzat, I.B. Rosen, et al., Distinct multiple RET/PTC gene rearrangements in multifocal papillary thyroid neoplasia. J Clin Endocrinol Metab, 1998. 83(11): p. 4116-22. [112] DeGroot, L.J., E.L. Kaplan, F.H. Straus, et al., Does the method of management of papillary thyroid carcinoma make a difference in outcome? World J Surg, 1994. 18(1): p. 123-30. [113] Wong, J.B., M.M. Kaplan, K.B. Meyer, et al., Ablative radioactive iodine therapy for apparently localized thyroid carcinoma. A decision analytic perspective. Endocrinol Metab Clin North Am, 1990. 19(3): p. 741-60. [114] Dragoiescu, C., O.S. Hoekstra, D.J. Kuik, et al., Feasibility of a randomized trial on adjuvant radio-iodine therapy in differentiated thyroid cancer. Clin Endocrinol (Oxf), 2003. 58(4): p. 451-5.

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[115] Sawka, A.M., K. Thephamongkhol, M. Brouwers, et al., Clinical review 170: A systematic review and metaanalysis of the effectiveness of radioactive iodine remnant ablation for well-differentiated thyroid cancer. J Clin Endocrinol Metab, 2004. 89(8): p. 3668-76. [116] Lerch, H., O. Schober, T. Kuwert, et al., Survival of differentiated thyroid carcinoma studied in 500 patients. J Clin Oncol, 1997. 15(5): p. 2067-75. [117] NCCN Practice Guidelines in Oncology -v.1.2001. 2001, National Comprehensive Cancer Network via internet www.nccn.org. [118] Robbins, R.J. and M.J. Schlumberger, The evolving role of (131)I for the treatment of differentiated thyroid carcinoma. J Nucl Med, 2005. 46 Suppl 1: p. 28S-37S. [119] Baudin, E., J.P. Travagli, J. Ropers, et al., Microcarcinoma of the thyroid gland: the Gustave-Roussy Institute experience. Cancer, 1998. 83(3): p. 553-9. [120] Yamashita, H., S. Noguchi, N. Murakami, et al., Extracapsular invasion of lymph node metastasis. A good indicator of disease recurrence and poor prognosis in patients with thyroid microcarcinoma. Cancer, 1999. 86(5): p. 842-9. [121] Sugitani, I. and Y. Fujimoto, Symptomatic versus asymptomatic papillary thyroid microcarcinoma: a retrospective analysis of surgical outcome and prognostic factors. Endocr J, 1999. 46(1): p. 209-16. [122] Appetecchia, M., G. Scarcello, E. Pucci, et al., Outcome after treatment of papillary thyroid microcarcinoma. J Exp Clin Cancer Res, 2002. 21(2): p. 159-64. [123] Hay, I.D., C.S. Grant, J.A. van Heerden, et al., Papillary thyroid microcarcinoma: a study of 535 cases observed in a 50- year period. Surgery, 1992. 112(6): p. 1139-46; discussion 1146-7. [124] Furlan, J.C., Y. Bedard, and I.B. Rosen, Biologic basis for the treatment of microscopic, occult well- differentiated thyroid cancer. Surgery, 2001. 130(6): p. 10504. [125] Lin, J.D., T.C. Chao, H.F. Weng, et al., Clinical presentations and treatment for 74 occult thyroid carcinoma. Comparison with nonoccult thyroid carcinoma in Taiwan. Am J Clin Oncol, 1996. 19(5): p. 504-8. [126] Rassael, H., L.D. Thompson, and C.S. Heffess, A rationale for conservative management of microscopic papillary carcinoma of the thyroid gland: a clinicopathologic correlation of 90 cases. Eur Arch Otorhinolaryngol, 1998. 255(9): p. 462-7. [127] Noguchi, S., H. Yamashita, N. Murakami, et al., Small carcinomas of the thyroid. A long-term follow-up of 867 patients. Arch Surg, 1996. 131(2): p. 187-91. [128] Prendiville, S., K.D. Burman, M.D. Ringel, et al., Tall cell variant: an aggressive form of papillary thyroid carcinoma. Otolaryngol Head Neck Surg, 2000. 122(3): p. 352-7. [129] Terry, J.H., S.A. St John, F.J. Karkowski, et al., Tall cell papillary thyroid cancer: incidence and prognosis. Am J Surg, 1994. 168(5): p. 459-61. [130] Chow, S.M., J.K. Chan, S.C. Law, et al., Diffuse sclerosing variant of papillary thyroid carcinoma - clinical features and outcome. Eur J Surg Oncol, 2003. 29(5): p. 446-9. [131] Carcangiu, M.L. and S. Bianchi, Diffuse sclerosing variant of papillary thyroid carcinoma. Clinicopathologic study of 15 cases. Am J Surg Pathol, 1989. 13(12): p. 1041-9.

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[132] Albareda, M., M. Puig-Domingo, S. Wengrowicz, et al., Clinical forms of presentation and evolution of diffuse sclerosing variant of papillary carcinoma and insular variant of follicular carcinoma of the thyroid. Thyroid, 1998. 8(5): p. 385-91. [133] Schroder, S., Diffuse sclerosing variant of papillary thyroid carcinoma. Am J Surg Pathol, 1991. 15(5): p. 492-3. [134] Chan, J.K., Tumors of the thyroid and parathyroid glands, in Diagnostic Histopathology of Tumors, C.D.M. Fletcher, Editor. 2001, Churchill Livingstone. p. 959-1038. [135] Thompson, L.D., J.A. Wieneke, E. Paal, et al., A clinicopathologic study of minimally invasive follicular carcinoma of the thyroid gland with a review of the English literature. Cancer, 2001. 91(3): p. 505-24. [136] Coburn, M., D. Teates, and H.J. Wanebo, Recurrent thyroid cancer. Role of surgery versus radioactive iodine (I131). Ann Surg, 1994. 219(6): p. 587-93; discussion 593-5. [137] Simon, D., P.E. Goretzki, J. Witte, et al., Incidence of regional recurrence guiding radicality in differentiated thyroid carcinoma. World J Surg, 1996. 20(7): p. 860-6; discussion 866. [138] Stojadinovic, A., M. Shoup, A. Nissan, et al., Recurrent differentiated thyroid carcinoma: biological implications of age, method of detection, and site and extent of recurrence. Ann Surg Oncol, 2002. 9(8): p. 789-98. [139] Nishiyama, Y., Y. Yamamoto, Y. Ono, et al., Comparison of 99Tcm-tetrofosmin with 201Tl and 131I in the detection of differentiated thyroid cancer metastases. Nucl Med Commun, 2000. 21(10): p. 917-23. [140] Maxon, H.R., 3rd and H.S. Smith, Radioiodine-131 in the diagnosis and treatment of metastatic well differentiated thyroid cancer. Endocrinol Metab Clin North Am, 1990. 19(3): p. 685-718. [141] Pacini, F., F. Cetani, P. Miccoli, et al., Outcome of 309 patients with metastatic differentiated thyroid carcinoma treated with radioiodine. World J Surg, 1994. 18(4): p. 600-4. [142] Bachelot, A., S. Leboulleux, E. Baudin, et al., Neck recurrence from thyroid carcinoma: serum thyroglobulin and high-dose total body scan are not reliable criteria for cure after radioiodine treatment. Clin Endocrinol (Oxf), 2005. 62(3): p. 376-9. [143] Schlumberger, M., G. Berg, O. Cohen, et al., Follow-up of low-risk patients with differentiated thyroid carcinoma: a European perspective. Eur J Endocrinol, 2004. 150(2): p. 105-12. [144] O'Connell, M.E., R.P. A'Hern, and C.L. Harmer, Results of external beam radiotherapy in differentiated thyroid carcinoma: a retrospective study from the Royal Marsden Hospital. Eur J Cancer, 1994. 6: p. 733-9. [145] Farahati, J., C. Reiners, M. Stuschke, et al., Differentiated thyroid cancer. Impact of adjuvant external radiotherapy in patients with perithyroidal tumor infiltration (stage pT4). Cancer, 1996. 77(1): p. 172-80. [146] Kim, T.H., D.S. Yang, K.Y. Jung, et al., Value of external irradiation for locally advanced papillary thyroid cancer. Int J Radiat Oncol Biol Phys, 2003. 55(4): p. 100612.

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[147] Wu, X.L., Y.H. Hu, Q.H. Li, et al., Value of postoperative radiotherapy for thyroid cancer. Head Neck Surg, 1987. 10(2): p. 107-12. [148] Nutting, C.M., D.J. Convery, V.P. Cosgrove, et al., Improvements in target coverage and reduced spinal cord irradiation using intensity-modulated radiotherapy (IMRT) in patients with carcinoma of the thyroid gland. Radiother Oncol, 2001. 60(2): p. 173-80. [149] Happersett, L., M. Hunt, L. Chong, et al., Intensity modulated radiation therapy for the treatment of thyroid cancer. Proc 42nd Annual ASTRO Meeting, 2000: p. 351-2. [150] Ruegemer, J.J., I.D. Hay, E.J. Bergstralh, et al., Distant metastases in differentiated thyroid carcinoma: a multivariate analysis of prognostic variables. J Clin Endocrinol Metab, 1988. 67(3): p. 501-8. [151] Dinneen, S.F., M.J. Valimaki, E.J. Bergstralh, et al., Distant metastases in papillary thyroid carcinoma: 100 cases observed at one institution during 5 decades. J Clin Endocrinol Metab, 1995. 80(7): p. 2041-5. [152] Shoup, M., A. Stojadinovic, A. Nissan, et al., Prognostic indicators of outcomes in patients with distant metastases from differentiated thyroid carcinoma. J Am Coll Surg, 2003. 197(2): p. 191-7. [153] Lin, J.D., M.J. Huang, J.H. Juang, et al., Factors related to the survival of papillary and follicular thyroid carcinoma patients with distant metastases. Thyroid, 1999. 9(12): p. 1227-35. [154] Ronga, G., M. Filesi, T. Montesano, et al., Lung metastases from differentiated thyroid carcinoma. A 40 years' experience. Q J Nucl Med Mol Imaging, 2004. 48(1): p. 12-9. [155] Bernier, M.O., L. Leenhardt, C. Hoang, et al., Survival and therapeutic modalities in patients with bone metastases of differentiated thyroid carcinomas. J Clin Endocrinol Metab, 2001. 86(4): p. 1568-73. [156] Pittas, A.G., M. Adler, M. Fazzari, et al., Bone metastases from thyroid carcinoma: clinical characteristics and prognostic variables in one hundred forty-six patients. Thyroid, 2000. 10(3): p. 261-8. [157] Schlumberger, M., M. Tubiana, F. De Vathaire, et al., Long-term results of treatment of 283 patients with lung and bone metastases from differentiated thyroid carcinoma. J Clin Endocrinol Metab, 1986. 63(4): p. 960-7. [158] Casara, D., D. Rubello, G. Saladini, et al., Distant metastases in differentiated thyroid cancer: long-term results of radioiodine treatment and statistical analysis of prognostic factors in 214 patients. Tumori, 1991. 77(5): p. 432-6. [159] Sisson, J.C., D.A. Jamadar, E.A. Kazerooni, et al., Treatment of micronodular lung metastases of papillary thyroid cancer: are the tumors too small for effective irradiation from radioiodine? Thyroid, 1998. 8(3): p. 215-21. [160] Abdel-Wanis, M.E., N. Kawahara, A. Murata, et al., Thyroid cancer spinal metastases: report on 25 operations in 14 patients. Anticancer Res, 2002. 22(4): p. 2509-16. [161] Eustatia-Rutten, C.F., J.A. Romijn, M.J. Guijt, et al., Outcome of palliative embolization of bone metastases in differentiated thyroid carcinoma. J Clin Endocrinol Metab, 2003. 88(7): p. 3184-9. [162] Smit, J.W., G.J. Vielvoye, and B.M. Goslings, Embolization for vertebral metastases of follicular thyroid carcinoma. J Clin Endocrinol Metab, 2000. 85(3): p. 989-94.

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[163] Vitale, G., F. Fonderico, A. Martignetti, et al., Pamidronate improves the quality of life and induces clinical remission of bone metastases in patients with thyroid cancer. Br J Cancer, 2001. 84(12): p. 1586-90. [164] Sze, W.M., M.D. Shelley, I. Held, et al., Palliation of metastatic bone pain: single fraction versus multifraction radiotherapy--a systematic review of randomised trials. Clin Oncol (R Coll Radiol), 2003. 15(6): p. 345-52. [165] van der Linden, Y.M., S.P. Dijkstra, E.J. Vonk, et al., Prediction of survival in patients with metastases in the spinal column: results based on a randomized trial of radiotherapy. Cancer, 2005. 103(2): p. 320-8. [166] Chiu, A.C., E.S. Delpassand, and S.I. Sherman, Prognosis and treatment of brain metastases in thyroid carcinoma. J Clin Endocrinol Metab, 1997. 82(11): p. 3637-42. [167] Datz, F.L., Cerebral edema following iodine-131 therapy for thyroid carcinoma metastatic to the brain. J Nucl Med, 1986. 27(5): p. 637-40. [168] McWilliams, R.R., C. Giannini, I.D. Hay, et al., Management of brain metastases from thyroid carcinoma: a study of 16 pathologically confirmed cases over 25 years. Cancer, 2003. 98(2): p. 356-62. [169] Ikekubo, K., M. Hino, H. Ito, et al., [Seven cases of brain metastasis from papillary thyroid carcinoma]. Kaku Igaku, 2000. 37(4): p. 349-57. [170] Schlumberger, M., J.P. Travagli, J. Lemerle, et al., Differentiated thyroid carcinoma in childhood. Experience at Institut Gustave-Roussy, Villejuif. Acta Otorhinolaryngol Belg, 1987. 41(5): p. 804-8. [171] Dottorini, M.E., A. Vignati, L. Mazzucchelli, et al., Differentiated thyroid carcinoma in children and adolescents: a 37-year experience in 85 patients. J Nucl Med, 1997. 38(5): p. 669-75. [172] La Quaglia, M.P., T. Black, G.W. Holcomb, et al., Differentiated thyroid cancer: clinical characteristics, treatment, and outcome in patients under 21 years of age who present with distant metastases. A report from the Surgical Discipline Committee of the Children's Cancer Group. J Pediatr Surg, 2000. 35(6): p. 955-9; discussion 960. [173] Landau, D., L. Vini, R. A'Hern, et al., Thyroid cancer in children: the Royal Marsden Hospital experience. Eur J Cancer, 2000. 36(2): p. 214-20. [174] Ceccarelli, C., F. Pacini, F. Lippi, et al., Thyroid cancer in children and adolescents. Surgery, 1988. 104(6): p. 1143-8. [175] Newman, K.D., T. Black, G. Heller, et al., Differentiated thyroid cancer: determinants of disease progression in patients 15 U/ml. Patient

hTg (µg/l)

Pat. 5 Pat. 8 Pat. 9 Pat. 10 Pat. 12

816.0 1.9 3742.0 9791.0 887.0

Tu M2-PK (U/ml) 15.9 35.4 16.0 21.2 109.1

Pat. 15

5750.0

51.8

Pat. 16 Pat.18 Pat. 19 Pat. 21 Pat. 23 Pat. 25 Pat. 26

28.1 28178.0 2371.0 632.0 141.7 96.3 1611.0

15.5 25.0 18.3 16.7 32.5 17.2 26.0

Metastases Skull (histology) Diffused pulmonary (CT-scan) Lymph nodes, pulmonary and skeletal (CT-scan) Lymph nodes and pulmonary (CT scan) Sacral and pulmonary (CT-scan and bone scintigraphy) Pulmonary and tibial (CT-scan and bone scintigraphy) Cervical lymph node (histology) Pulmonary and diffuse skeletal (J131-scan) Pulmonary and cerebellar (CT-scan and histology) Skull (histology) Diffused pulmonary (CT-scan) Diffused pulmonary (CT-scan) Pulmonary (J131-scan)

RESULTS Of the 26 patients included in the study, 12 had follicular thyroid carcinoma, 9 had papillary thyroid carcinoma, 2 had combined papillary/follicular thyroid carcinoma, 1 had combined anaplastic/papillary carcinoma and 1 had hurthle cell carcinoma. In one patient who had had a recurrent goitre after thyroid resection 20 years before, the primary tumor was not found; the tumor nature was identified in the 131I whole-body-scan. We assume that the primary tumor had been resected at the time of the thyroidectomy 20 years earlier. Of the 26 patients, 25 had already had at least one radioiodine therapy. One patient was included in the study before thyroidectomy, the tumor having been detected after a cervical lymph node biopsy. In this patient, the preoperative hTg-level was 26 µg/l (normal range: < 100 µg/l) and the corresponding Tu M2-PK level was 14.6 U/ml (normal range according to the manufacturer’s instructions: ≤ 15 U/ml, “grey zone”: 15-20 U/ml). Serum Tg levels were elevated in 24 patients, ranging from 13.4 to 35,000 µg/l, with a median of 724 µg/l. In these patients, the Tu M2-PK was in the range from 6.2 to 109 U/ml, with a median of 15.1 U/ml. The patient with anaplastic/papillary carcinoma had a hTg-level of 1.9 µg/ l and an elevated Tu M2-PK level of 35.4 U/ml.

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Tu M2-PK was truly elevated (> 20 U/ml) only in 7 patients, and only in one of them significantly (109 U/ml). This patient had a papillary thyroid carcinoma, complicated by lung and skeletal metastases. Considering a cut-off value of 20 U/ml, the sensitivity for Tu M2-PK was 27%. Tu M2-PK was in the “grey zone” in 6 patients with serum hTg-levels ranging from 816 to 3,700 µg/l. Considering a cut-off value of 15 U/ml, the sensitivity for Tu M2-PK was 50%. Tu M2-PK was normal in 13 patients with serum hTg-levels ranging from 13.4 to 35,000 µg/l. Table 1 shows a synopsis of the patients with Tu M2-PK > 15 U/ml. There was no correlation between the serum hTg level and the Tu M2-PK level in the patient group studied (correlation = -0.093, see Fig. 1.).

Figure 1: Correlation between hTg and Tu M2PK.

ANALYSIS AND REVIEW OF THE CURRENT LITERATURE Considering the cut-off value recommended by the manufacturer, Tu M2-PK is elevated in about one half of the patients with metastasising thyroid carcinoma. In the other half of these patients, Tu M2-PK values were solely in the grey zone. Human thyroglobulin (hTg) is the established tumor marker for thyroid cancer; it is of high sensitivity [13] and specificity (90% in verified metastatic tumors, [12]) and the overall correlation between serum hTg and presence or absence of cancer is 96% [14]. Thus, comparing Tu M2-PK with serum hTg, the former is less reliable in the monitoring of patients with thyroid cancer. Unfortunately, our study included only one metastatic patient (anaplastic/papillary carcinoma) with a low serum hTg who had an elevated Tu M2-PK. The limitation of our study is not to have recruited

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more patients with this configuration, since it would have been interesting to observe if Tu M2-PK is more powerful in this case. On the other hand, it was a problem to gather patients who had low serum hTg levels associated with proven metastatic thyroid disease, i.e., evidence of metastases in histology, in conventional radiological imaging, in the 131I wholebody scan, in bone scintigraphy or even in the PET-scan. The patients of our collective with low hTg levels did not have metastases proven by the means cited above. Evidently, Tu M2-PK does not correlate with tumour burden in thyroid carcinoma. It may show evidence of tumour in 50% of the cases of thyroid carcinoma, but overall, it is not as reliable as the established tumour marker for thyroid cancer to date Human thyroglobulin. This pilot study could not prove that Tu M2-PK was as promising in thyroid cancer as in some others. However, in the rare cases where non-thyroglobulin-producing metastases are present, it may be worth to determine the Plasma Tu M2-PK level, as the letter was positive in the sole patient with this constellation in the study, why is not clear. A similar result was obtained by Oremek et al. [23] for haematological malignancies: They found that Tu M2-PK was not a useful indicator whether for acute nor for chronic myelocytic leukaemia and whether for acute nor for chronic lymphocytic leukaemia. They also found that in serial determinations in 3 patients with disease regression under chemotherapy, the plasma levels of Tu M2-PK was alternating, being not reliable for followup. In lung cancer, Schneider et al. demonstrated that a highly statistically significant difference of Tu M2-PK concentration was seen in patients with lung cancer as compared to healthy persons and all subjects suffering from benign lung diseases (p< 0.001), with the exception of acute inflammatory lung disorders [4]. In another publication, Schneider et al. demonstrated that the Tu M2-PK concentration correlated well with the clinical course of the disease and the tumor burden [1, 2]. Comparing Tu M2-PK with other tumor markers like CYFRA 21-1 or CEA, Schneider et al. [3] further found that Tu M2-PK had a higher sensitivity (58%) than CYFRA 21-1 (48%) or CEA (39%). In cervical carcinoma, a study-group of India [49] quantitated the plasma of 50 patients with cervical carcinoma, 10 patients with chronic cervicitis and 10 healthy controls. They found a sensitivity of the test for discrimination of malignant from non-malignant condition of 82%, the specificity was 60%. In advanced breast cancer, Lüftner et al. [6] found a sensitivity of only 52% in detecting Stage IV-disease for Tu M2-PK at a cut-off of 15 U/ml. For this purpose, they entered 121 Tu M2-PK results into a receiver operating characteristics (ROC) analysis, of which 54 samples were collected from healthy controls and 67 from patients with advanced breast cancer. They found a specificity of 85% at a cut-off of 15 U/ml. At a cut-off of 18 U/ml, the sensitivity decreased to 42% and the specificity increased to 94%. At a cut-off of 25 U/ml, the sensitivity further decreased to 28% and the specificity climbed to 96%. However, other important statistical parameters like accuracy and Youden-Index were optimal at the concentration of 15 U/ml. They compared Tu M2-PK and the tumour marker CA 27.29 and established the best cut-off level for CA 27.29 to distinguish stage IV breast cancer from healthy controls at 30 U/ml. At this level, CA 27.29 reached a sensitivity of 84% and a specificity of 91%. The authors investigated the response assessment in 45 chemotherapy blocks of 38 patients with metastatic breast cancer who could be followed-up very closely.

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The longitudinal tracking of Tu M2-PK showed that in some patients with progressive disease, there was a statistically significant increase of the baseline levels of Tu M2-PK, whereas in patients with stable disease or remission, a drastic decrease of the Tu M2-PK baseline levels was observed. However, the baseline 25/50/75 percentiles were not statistically different among the non-benefited group (progressive disease) as compared to the profiting group (stable disease or remission). The median baseline level of Tu M2-PK for patients with advanced breast cancer was 12.8 U/ml and 130 U/ml for CA 27.29. This shows that it is not only the absolute level which gives the predictive information, but also the kinetics. This was confirmed by a study of Hoopmann et al. [5]. In pancreatic cancer, Cerwenka et al. [9] demonstrated that Tu M2-PK had a sensitivity of 79% at a cut-off of 28 U/ml. This was higher than the sensitivity of CA 19-9 (65%) and CEA (22%). They found a good correlation between Tu M2-PK and the extent of the disease, but similar to our results, there was no correlation between Tu M2-PK and CA 19-9 (correlation coefficient = -0.23) or between Tu M2-PK and CEA (correlation coefficient = 0.09). A similar study was conducted by Ventrucci et al. [26]. Interestingly, they found that the two markers Tu M2-PK and CA 19-9 were statistically correlated (n = 265, r = 0.239, P < 0.001). They confirmed the higher sensitivity of Tu M2-PK (85%) in comparison to CA 19-9 (75%) in exocrine pancreatic cancer. The specificity of Tu M2-PK (41 % vs. 81%) was much lower and it was less accurate than CA 19-9 in discriminating between pancreatic cancer and acute or chronic pancreatitis. The combination of the two tests significantly increased the sensitivity to 97%, with a specificity of 38%. They reported five interesting cases where Tu M2-PK levels increased markedly within 2 weeks of surgery. In 4 of these patients, the Tu M2-PK level before surgery was lower than 15 U/ml. The reason for this increase is unknown, accelerated glycolysis in the regenerating tissue in the wound after laparatomy could play a role [30]. Tu M2-PK-plasma-levels seemed to be less affected by cholestasis than serum CA 19-9. Among the control patients, cholestasis was associated with significantly higher CA 19-9 values, while Tu M2-PK levels did not differ. This may be explained by the fact that CA 19-9 is also released by the biliary epithelium, while expression of Tu M2-PK takes place in actively proliferating cells. Of interest, furthermore, are the abnormal results the authors found for most of their patients with neuroendocrine tumors, in whom CA 19-9 is generally normal. 7 of 9 of these patients had increased Tu M2-PK levels whereas 2 patients had elevated CA 19-9 levels. Another study published by Pezzilli et al. [31] aimed to establish the diagnostic role of Tu M2-PK in patients with neuroendocrine tumors and to compare its diagnostic value with that of chromogranin A, which is well established. They examined forty-nine patients with proven neuroendocrine tumors and found Tu M2-PK to have a significantly lower specificity than chromogranin A and to be of limited value in detecting neuroendocrine tumors. However, Tu M2-PK levels correlated with the mass of the endocrine tumor, contrarily to thyroid carcinoma. In colorectal cancer, gastric cancer and oesophageal cancer, Schulze [8] found a diagnostic sensitivity for Tu M2-PK (cut-off 15 U/ml) of 50%, 67% and 59 %, respectively. Hardt et al. [7] found a sensitivity for Tu M2-PK (same cut-off) for colorectal cancer of

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76.5% and for gastric and oesophageal cancer of 60% and 45%, respectively. Interestingly, Hardt et al. also published that Tu M2-PK levels in faeces of patients with colorectal cancer patients were higher than in controls [25]. The background here is the fact that since these tumours grow intraluminally, they secrete the tumor marker in the intestinal mucosa. In healthy tissues, all isoenzymes of pyruvate kinase consist of four subunits whereby hybrids of the different forms can also occur [42,43]: M1-type in muscle, heart and brain, M2 in liver mesenchymal and bile duct epithelial cells and also in distal kidney tubules [41], L-type in the liver, the kidney and the colon and R-type in red blood cells. The epithelium of the upper gastrointestinal tract (oesophagus and stomach) reveals hybrids between M1 and M2 whereas the lower gastrointestinal tract is characterised by hybrids of L- and M2-PK [42, 43]. In gastrointestinal tumours, only subunits of the type M2 are detectable and pyruvate kinase is mainly in the dimeric form termed Tumor M2-PK (34). The increased expression of M2-PK is probably caused by mutations in the ras oncogene and p53 antioncogene, which are detected in 40-60% of colorectal cancers [44, 45, 46]. Hardt et al. [7] found a sensitivity of 73% and a specificity of 78 % of the stool-test at a cut-off of 4 U/ml Tu M2-PK in the faeces. This result was clearly superior to the sensitivity of the guaiac-based occult blood test (24%). The expression of the tumor marker by malignant cells has also been studied by Koss et al. [27]. They investigated the expression of Tu M2-PK during the metaplasia-dysplasiaadenocarcinoma sequence of Barrett’s oesophagus and found that Tu M2-PK was expressed in all cases, increased cytoplasmic expression being seen with progression along the metaplasia-dysplasia-adenocarcinoma sequence. 100% of the cases of adenocarcinoma showed 100% staining in their study. Thus, they concluded that Tu M2-PK was not specific for Barret adenocarcinoma, but may be important as a marker of transformed and highly proliferating clones during progression along the metaplasia-dysplasia-adenocarcinomasequence. In renal cell carcinoma, Oremek et al. [10] demonstrated that Tu M2 PK could discriminate very well between inflammatory renal disease and renal carcinoma. In addition, the authors found that the correlation between Tu M2-PK and Robson-stage of the tumor was highly specific. Robson-stage indirectly represents tumour size. The good discrimination between inflammatory renal disease and renal carcinoma has also been demonstrated by Wechsel et al. [28], who compared serum-levels of Tu M2-PK in 40 patients with renal cell carcinoma to serum levels of Tu M2-PK in 39 healthy persons. In their study, Roigas et al. [24] did not find a significant relationship between Tu M2PK and tumor size. They analysed Tu M2-PK in 68 patients with metastatic renal cell carcinoma after initial surgery and prior to or during chemoimmunotherapy of metastases. Of the 68 patients, 24 had metastatic disease at the time of diagnosis of the primary tumour. In this patient-group, Tu M2-PK was determined after surgery of the primary tumour and prior to chemoimmunotherapy. In the other 44 patients, metastases were diagnosed during followup after a median of 23 months. Here, Tu-M2-PK was measured when progress was revealed by radiological imaging or routinely during clinical follow-up. In 48 of the 68 patients (71%), Tu M2-PK was elevated above the cut-off of 15 U/ml, thus biochemically detecting metastatic disease. A significant difference was observed in relation to the cellular differentiation of the primary tumour when G2 and G3 carcinomas were analysed. In 34 of 50

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patients undergoing chemotherapy (68%), a positive correlation between Tu M2-PK values and response to treatment was observed. In conclusion, the relatively low sensitivity of Tu M2-PK (cut-off 15 U/ml) that we found for thyroid carcinoma is still comparable to published sensitivities of 52% for advanced breast cancer, 58% for lung cancer, 50% for colorectal cancer or 45% for oesophageal cancer. The reason for this low sensitivity is still not clear. One alteration consistently found during tumour formation is the upregulation of glycolytic enzymes. This alteration takes place at the RNA and protein level, as well as at the level of enzymatic activities [32, 33, 34, 35, 36, 37, 38]. In addition, in the case of the glycolytic enzyme pyruvate kinase, which catalyses the final step of glycolysis (phosphorylation of ADP to ATP) a loss of the tissue-specific isoenzymes and expression of the pyruvate kinase isoenzyme type M2 is described in many tumours unvestigated thus far [39, 40, 41]. All PK isozymes except M1-type are allosterically regulated and the glycolytic phosphometabolite fructose 1,6 bisphosphate (FBP) is known to act as an allosteric factor [11]. The purpose of this allosteric regulation is to coordinate the enzyme’s activity with other cellular reactions and important (signaling) pathways. As the metabolic requirements of an organism fluctuate over time and with the cell type, the organism produces related but differentially regulated forms of the same enzyme to satisfy disparate metabolic patterns. For example, PKM2 occurs in a tetrameric active and a dimeric less active form. FBP induces reassociation of the dimeric to the tetrameric form [47]. Shift of PKM2 from the active to the dimeric form (Tu M2-PK) occurs under the control of onc gene-coded kinases such as pp60vsrc kinase [29] or of the oncoprotein E7 of the human papilloma virus type 16. The pp60v-src kinase phosphorylates M2-PK to tyrosine, HPV-16 E7 directly binds to M2-PK. Result is a trigger in the dimerisation of M2-PK, a transient elevation of the cellular concentration of the glycolytic intermediates, enhancement of the glucose breakdown via the hexose monophosphate shunt and acceleration of the synthesis of nucleic acids in proliferating cells [11]. In this case of low tetramer: dimer ratio with M2-PK mainly in the nearly inactive dimeric form, energy is provided to the cells by the degradation of the amino-acid glutamine to lactate, termed glutaminolysis. A higher tetramer: dimer ratio consistent with more tetrameric form of M2-PK determines the glucose carbons to be converted into pyruvate and lactate under the production of energy. In this way, pyruvate kinase controls the ATP: ADP and GTP: GDP ratio and, in cooperation with adenylate kinase and 6-phosphofructo 1-kinase, the AMPlevels [48]. An increase in AMP levels inhibits DNA synthesis and cell proliferation by a reduction of NAD(H) as well as UTP levels and CTP synthesis [47].

CONCLUSION Going from the fact that there is a biochemical explanation for elevation of the dimeric form of M2-PK in tumour, it remains unclear why the sensitivity of the plasma-test is average in thyroid cancer, breast cancer, lung cancer, oesophageal and colorectal cancer. The reason may be that these malignant cells have a low content of M2-PK? Lower expression of Tu

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M2-PK was already observed by Yoo et al. [50] in cisplatin-resistant gastric carcinoma cells compared to parental cells. Thyroid cancer does not respond well to chemotherapy, but to assume that resistance to cytostatic substances could generally influence the expression of Tu M2-PK in such tumour cells remains a speculation at the present. On the other hand, thyroid cancer cells proliferate very slowly and this low proliferation rate may explain the limited upregulation of Tu M2-PK. This would be confirmed by the observation made by Eigenbrodt et al. 2000 [29]: The authors found that contrarily to cervix carcinomas or DS carcinosarcomas, the thyroid carcinomas neither release nor consume glutamine in great amounts. Glutamine conversion is positively linked to the rate of glucose and oxygen consumption and negatively to glucose and oxygen utilization. Pyruvate kinase is positively correlated with an increase in glucose and oxygen utilization. Less glutamine release may therefore mean less glucose and oxygen utilization, therefore less amount of pyruvate kinase, this consequently being a characteristic feature of thyroid carcinoma cells.

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REFERENCES [1] Schneider J, Neu K, Velcovsky HG, Morr H and Eigenbrodt E: Tumor M2-pyruvate kinase in the follow-up of inoperable lung cancer patients: a pilot study. Cancer Lett 193 (1): 91-8, 2003. [2] Schneider J, Neu K, Grimm H, Velcovsky HG, Weisse G and Eigenbrodt E: Tumor M2pyruvate kinase in lung cancer patients: immunohistochemical detection and disease monitoring. Anticancer Res 22: 311-318, 2002. [3] Schneider J, Velcovsky HG, Morr H, Katz N, Neu K and Eigenbrodt E: Comparison of the tumor markers tumor M2-PK, CEA, CYFRA 21-1, NSE and SCC in the diagnosis of lung cancer. Anticancer Res 20: 5053-5058, 2000. [4] Schneider J, Morr H, Velcovsky HG, Weisse G and Eigenbrodt E: Quantitative detection of Tumor M2-pyruvate kinase in plasma of patients with lung cancer in comparison to other lung diseases. Cancer Detect Prevent 24 (6): 531-535, 2000. [5] Hoopmann M, Warm M, Mallmann P, Thomas A, Gohring UJ and Schondorf T: Tumor M2 Pyruvate kinase: determination in breast cancer patients receiving trastuzumab therapy. Cancer Lett 187(1-2): 223-8, 2002. [6] Lüftner D, Mesterharm J, Akrivakis C, Geppert R, Petrides PE, Wernecke KD and Possinger K: Tumor Type M2 Pyruvate kinase expression in advanced breast cancer. Anticancer Res 20: 5077-5082, 2000. [7] Hardt PD, Ngoumou BK, Rupp J, Schnell-Kretschmer H and Kloer HU: Tumor M2Pyruvate kinase: a promising tumor marker in the diagnosis of gastro-intestinal cancer. Anticancer Res 20: 4965-4968, 2000. [8] Schulze G: The tumor marker Tumor M2-PK: an application in the diagnosis of gastrointestinal Cancer. Anticancer Res 20:4961-4964, 2000. [9] Cerwenka H, Aigner R, Bacher H, Werkgartner G, El-Shabrawi A, Quehenberger F and Mischinger HJ: Tu M2-PK (Pyruvate kinase type Tumor M2), CA 19-9 and CEA in patients with benign, malignant and metastasising pancreatic lesions. Anticancer Res 19: 849-852, 1999.

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[10] Oremek GM, Sapoutzis N, Kramer W, Bickeböller R and Jonas D: Value of Tumor M2 (Tu M2-PK) in patients with renal carcinoma. Anticancer Res 20: 5095-5098, 2000. [11] Jurica MS, Mesecar A, Heath PJ, Shi W, Nowak T and Stoddard BL: The allosteric regulation of pyruvate kinase by fructose-1,6-bisphosphate. Structure 6: 195-210, 1998. [12] Szanto J, Vincze B, Sinkovits I, Karika Z, Daubner K, Peter I, Kazatsay I and Eckhardt S: Follow up of thyroglobulin levels in patients with surgically treated, highly differentiated thyroid cancer. Orv Hetil 130(32): 1695-9, 1989. [13] Pacini F, Capezzone M, Elisei R, Ceccarelli C, Taddei D and Pinchera A: Diagnostic 131-iodine whole-body scan may be avoided in thyroid cancer patients who have undetectable stimulated serum Tg levels after initial treatment. J Clin Endocrinol Metab 87 (4): 1499-501, 2002. [14] Black EG, Sheppard MC and Hoffenberg R: Serial serum thyroglobulin measurements in the management of differentiated thyroid carcinoma. Clin Endocrinol (Oxf) 27 (1): 11520, 1987. [15] Spencer CA and Wang CC: Thyroglobulin measurement. Techniques, clinical benefits, and pitfalls. Endocrinol Metab Clin North Am 24 (4): 841-63, 1995. [16] Pellegriti G, Scollo C, Regalbuto C, Attard M, Marozzi P, Vermiglio F, Violi MA, Cianci M, Vigneri R, Pezzino V and Squatrito S: The diagnostic use of the rhTSH/thyroglobulin test in differentiated thyroid cancer patients with persistent disease and low thyroglobulin levels. Clin Endocrinol (Oxf) 58 (5): 556-61, 2003. [17] Mazurek S, Boschek CB and Eigenbrodt E: the role of Phosphometabolites in cell proliferation, energy metabolism, and tumor Therapy. J. Bioenergetics Biomembranes 29: 315-330, 1997. [18] Ibsen KH, Orlando RA, Garrat KN, Hernandez AN, Giorlando S and Nungaray G: Expression of multimolecular forms of pyruvate kinase in normal, benign and malignant human breast tissue. Cancer Res. 42: 888-892, 1982. [19] Eigenbrodt E, Reinacher M, Scheefers-Borschel U, Scheefers H and Friis R: Double role for pyruvate kinase type M2 in expansion of phosphometabolite pools found in tumor cells. Critical Rev. Oncogenesis 3: 91-115, 1992. [20] Brinck U, Eigenbrodt E, Oehmke M, Mazurek S and Fuscher G: L- and M2-pyruvate kinase expression in renal cell carcinomas and their metastases. Virch. Arch. 424: 177185, 1994. [21] Hacker HJ, Steinberg P and Bannasch P: Pyruvate kinase isoenzyme shift from L-type to M2-type is a late event in hepatocarcinogenesis induced in rats by a cholinedeficient/DL- ethionine-supplemented diet. Carcinogenesis 19: 99-107, 1998. [22] Scheefers-Borchel U, Scheefers H, Michel A, Will H, Fischer G, Basenau D, Dahlmann N, Laumen R, Mazurek S and Eigenbrodt E: Quantitative determination (ELISA) of pyruvate kinase-type tumor M2. In Klapdor R (ed): Current Tumor Diagnosis, Applications, Clinical Relevance, Resarch-trends. W. Zuckerscherdt Verlag, Muenchen 3: 365-368, 1994. [23] Oremek GM, Rox S, Mitrou P, Sapoutzis N, Sauer-Eppel H: Tumor M2-PK levels in haematological malignancies. Anticancer Research 2003 Mar-Apr; 23 (2A): 1135-8.

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[24] Roigas J, Deger S, Schroeder J, Wille A, Turk I, Brux B, Jung K, Schnorr D, Loening SA: Tumor type M2 pyruvate kinase expression in metastatic renal cell carcinoma. Urol Res. 2003 Dec 31 (6). [25] Hardt P, Mazurek S, Toepler M, Schlierbach P, Bretzel RG, Eigenbrodt E, Kloer HU: Faecal tumour M2 pyruvate kinase: a new, sensitive screening tool for colorectal cancer. Br J Cancer. 2004 Aug 31; 91 (5): 980-4. [26] Ventrucci M, Cipolla A, Racchini C, Casadei R, Simoni P, Gullo L: Tumor M2-pyruvate kinase, a new metabolic marker for pancreatic cancer. Dig Dis Sci. 2004 Aug;49(7-8): 1149-55. [27] Koss K, harrison RF, Gregory J, Darnton SJ, Anderson MR, Jankowski JA: The metabolic marker tumour pyruvate kinase type M2 (tumour M2-PK) shows increased expression along the metaplasma-dysplasia-adenocarcinoma sequenze in Barrett’s oesophagus. J Clin Pathos. 2004 Nov;57(11): 1156-9. [28] Wechsel HW, Petri E, Bichler KH and Feil G: Marker for Renal Cell Carcinoma (RCC): The dimeric form of pyruvate kinase type 2 (Tu M2-PK). Anticancer Research 19: 25832590 (1999). [29] Eigenbrodt E, Kallinowski F, Ott M, Mazurek S, Vaupel P: Pyruvate kinase and interaction of amino acid and carbohydrate metabolism in solid tumors. Anticancer Res 18: 3267-3274, 1998. [30] Oehler R, Weingartmann G, Manhart N, Salzer U, Meissner M, Schlegel W, Spittler A, Bergmann M, Kandioler D, Oismuller C, Struse HM, Roth E: Polytrauma induces increased expression of pyruvate kinase in neutrophils. Blood 95: 1086-1092, 2000. [31] Pezzilli R, Migliori M, Morselli-Labate AM, Campana D, Ventrucci M, Tomassetti P, Corinaldesi R: Diagnostic Value of tumor M2-pyruvate kinase in neuroendocrine tumors. A comparative study with chromogranin A. Anticancer Research 2003 May-Jun;23 (3C): 2969-72. [32] Durany N, Joseph J, Campo E, Molina R, Carreras J (1997): Phosphoglycerate mutase, 2,3-bisphosphoglycerate phosphatase and enolase activity and isoenzymed in lung, colon and liver carcinomas. British journal of Cancer 75(7):969-977. [33] Eigenbrodt E, Basenau D, Holthusen S, Mazurek S, Fischer G (1997): Quantification of tumor type M2 pyruvate kinase (Tu M2-PK) in human carcinomas. Anticancer Research 17: 3153-3156. [34] Mazurek S, Grimm H, Oehmke M, Weisse G, Teigelkamp S, Eigenbrodt E (2000): Tumor M2-PK and glutaminolytic enzymes in the metabolic shift of tumor cells. Anticancer Research 20: 5151-5154. [35] Hegde P, Qi R, Gaspard R, Abernathy K, Dharap S, Earle-Hughes J, Gay C, Nwokekeh NU, Chen T, Saeed AI, Sharov V, Lee NH, Yeatman J, Quackenbusch J (2001): Identification of tumor markers in models of human colorectal cancer using a 19,200element complmentatry DNA microarray. Cancer Research 61: 7792-7797. [36] Atsumi T, Chesney J, Metz C, Leng L, Donnely S, Makita Z, Mitchell R, Bucala R (2002): High expression of inducible 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase (iPFK-2;PFKFB3) in human cancers. Cancer Research 62(20): 58815887.

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[37] Birkenkamp-Demtroder K, Christensen LL, Harder Olesen S, Frederiksen CM, Lahio P, Aaltonen LA, Laurberg S, SørensenFB, Hagemann R, rntoft TF (2002): Gene expression in colorectal cancer. Cancer Research 62: 4352-4363. [38] Williams NS, Gaynor RB, Scoggin S, Verma U, Gokaslan T, Simmang C, Fleming J, Tavana D, Frenkel E, Becerra C (2003): Identification and validation of genes involved in the pathogenesis of colorectal cancer using cDNA microarrays and RNA interference. Clinical Cancer Research 9: 931-946. [39] Staal GEJ, Rijksen G (1991): Pyruvate kinase in selected human tumors. Biochemical and Molecular Aspects of Selected Cancers, Pretlow TG, Pretlow TP (eds) pp 313-317, San Diego: Academic Press. [40] Mazurek S, Boschek CB, Eigenbrodt E (1997): The role of the phosphometabolites in cell proliferation, energy metabolism, and tumor formation. J Bioenerg Biomembr 29: 315-330. [41] Steinberg P, Klingelhöffer A, Schäfer A, Wüst G, Weisse G, Oesch F, Eigenbrodt E (1999): expression of pyruvate kinase M2 in preneoplastic hepatic foci of Nnitrosomorpholine-treated rats. Virchows Arch 434 (213-220). [42] Saheki S, Harada K, Sanno Y, Tanaka T (1978): Hybrid isozymes of rat pyruvate kinase. Their subunit structure and developmental changes in the liver. Biochim Biophys Acta 526 (1): 116-128. [43] Saheki S, Saheki K, Tanaka T (1979): Changes in pyruvate kinase isoenzymes of rat small intestine during development and the synergistic effect on them of thyroid and glucocorticoid hormones. Enzyme 24 (1):8-17. [44] Rengucci C, Maiolo P, Saragoni L, Zoli W, Amadori D, Calistri D (2001): Multiple detection of genetic alterations in tumors and stool. Clin Cancer Res 7: 590-593. [45] Nishikawa T, Maemura K, Hirata I, Matsuse R, Morikawa H, Toshin K, Murano M, Hashimoto K, Nakagawa Y, Saitoh O, Uchida K, Katsu K (2002): A simple method of detecting K-ras point mutations in stool samples for colorectal cancer screening using one-step polymerase chain reaction/restriction fragment length polymorphism analysis. Clin Chim Acta 318: 107-112. [46] Smith G, Carey FA, Beattie J, Wilkie MJ, Lightfoot TJ, Coxhead J, Garner RC, Steele RJ, Wolf CR (2002): Mutations in APC, Kirsten-ras, and p53-alternative genetic pathways to colorectal cancer. Proc Natl Acad Sci USA 99: 9433-9438. [47] Mazurek S, Zwerschke W, Jansen-Dürr P and Eigenbrodt E: Metabolic cooperation between oncogenes during cell transformation: interaction between activated ras and HPV-16 E7. Oncogene (2001): 6891-6898. [48] Mazurek S, Grimm H, Wilker S, Leib S and Eigenbrodt E (1998): Metabolic characteristics of different malignant cancer cell lines. Anticancer Research 18 32753282. [49] Kaura B, Bagga R, Patel FD: Evaluation of the Pyruvate Kinase isoenzyme tumor (Tu M2-PK) as a tumor marker for cervical carcinoma. J Obstet Gynaecol Res. 2004 Jun; 30(3): 193-6. [50] Yoo BC, Ku JL, Hong SH, Shin YK, Park SY, Kim HK, Park JG: Decreased pyruvate kinase M2 activity linked to cisplatin resistance in human gastric carcinoma cell lines. Int J Cancer. 2004 Feb 10;108(4):532-9.

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Chapter 6

FAMILIAL NONMEDULLARY THYROID CARCINOMA Carl D. Malchoff∗ and Diana M. Malchoff University of Connecticut Health Center, Farmington, CT, USA.

ABSTRACT

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Carcinomas that arise from the thyroid follicular cell include papillary thyroid carcinoma, follicular thyroid carcinoma, insular thyroid carcinoma, and anaplastic thyroid carcinoma. As a group they are referred to as nonmedullary thyroid carcinomas (NMTC). In contrast, medullary thyroid carcinoma arises from the calcitonin producing parafollicular cells (C cells) of the thyroid. NMTC usually occurs sporadically. However, several epidemiologic studies and investigations into large kindreds suggest a familial predisposition in about 5 percent of NMTC. Familial NMTC (fNMTC) can be divided into at least two clinical groups. In the first group NMTC is a relatively infrequent component of a familial tumor syndrome characterized by a predominance of non-thyroidal carcinomas. These syndromes include the Cowden syndrome, familial adenomatous polyposis, and Carney complex type 1. Multiple endocrine neoplasia type 2A and the familial paraganglioma syndromes also may be enriched in NMTC. In the second group NMTC is the predominant malignancy in affected kindreds, although other neoplasms may also occur. In this second group, NMTC is autosomal dominant with partial penetrance. As compared to sporadic NMTC, fNMTC is characterized by a younger age of onset, an increased frequency of multifocal disease, within the thyroid, an increased risk of recurrence, but no increased risk of death. Most families are relatively small, suggesting a relatively low penetrance of the susceptibility gene. An alternative explanation for this observation is that fNMTC is a polygenic disorder. If this is correct, then polygenic disease constitutes a third fNMTC group.

∗

Correspondence concerning this article should be addressed to Dr. Carl D. Malchoff, Associate Professor of Medicine, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030. USA.

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Carl D. Malchoff and Diana M. Malchoff Linkage studies suggest 3 loci for fNMTC susceptibility genes. Linkage to chromosome 1q21 has been identified in a large papillary thyroid carcinoma (PTC) kindred enriched with papillary renal neoplasia and possibly other neoplasms. Kindreds with fNMTC alone have been mapped to 2q21 and to 19p13.2. Some of the fNMTC that are linked to 19p13.2 have the pathologic finding of oxyphilia, suggesting that they are pathologically distinct from the other familial fNMTC syndromes. However, the precise susceptibility genes remain to be identified. A family history should be taken from patients with NMTC to identify kindreds with a predominance of fNMTC and kindreds that represent other familial tumor syndromes with an increased frequency of NMTC. Careful clinical evaluation is indicated for members of fNMTC kindreds, and some investigators advocate thyroid ultrasound. Prophylactic thyroidectomy is not indicated and clinically useful genetic analysis is not yet available. In summary, about 5 percent of NMTC have a familial predisposition and these can often been identified by the family history. Linkage studies have mapped at least 3 fNMTC loci, However, until the specific susceptibility genes are identified, there are no clinically useful genetic studies. Careful clinical evaluation of the members of fNMTC kindreds is indicated.

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INTRODUCTION Thyroid carcinomas are divided into those malignancies derived from the thyroid epithelial cell and those derived from calcitonin producing parafollicular cells (C cells). Malignancies of the C cells are referred to as medullary thyroid carcinoma (MTC). As a group the malignancies of the epithelial cells are referred to as nonmedullary thyroid carcinoma (NMTC) and include papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), anaplastic thyroid carcinoma, and insular thyroid carcinoma. Familial tumor syndromes have been a fertile substrate for identifying molecular tumorigenic mechanisms. For example, investigation into the familial MTC has led to a description of the multiple endocrine neoplasia type 2A (MEN2A) and type 2B (MEN2B) syndromes, an understanding of the role of the RET proto-oncogene in MTC tumorigenesis, and genetic testing that improves patient care. Therefore, it is anticipated that investigations into familial NMTC (fNMTC) may uncover critical information concerning the molecular mechanisms of these neoplasms and improve the clinical care of susceptible individuals. Traditionally, NMTC have been classified as sporadic with an increased incidence following radiation exposure. A familial predisposition had not been noted. In contrast, more recent investigations suggest a familial predisposition for NMTC may occur. The evidence for this familial predisposition is found in epidemiologic studies, clinical descriptions of large kindreds, and genetic linkage studies that have mapped putative fNMTC genes. In general about 5 percent of all NMTC have a familial predisposition [1,2]. The final proof of an inherited predisposition to NMTC requires the identification of the susceptibility gene. To date no specific susceptibility gene has been uncovered. Even though the final proof is not yet available, there is enough evidence to make us highly suspicious that there is a familial predisposition to NMTC in some kindreds, and this does lead to a change in our approach to diagnosis and therapy.

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Based upon the clinical characteristics, we have chosen to divide fNMTC into two groups. In the first group fNMTC is a relatively infrequent component of a familial tumor syndrome characterized by a predominance of non-thyroidal neoplasms. Although NMTC is less common than other tumors in these syndromes, the risk of affected individuals developing NMTC appears greater than that of the general population. The syndromes within this group include familial adenomatous polyposis (FAP), the Cowden syndrome, and Carney complex, type 1 (CNC1). It may also include MEN2A and the familial paraganglioma syndromes (fPGL). In the second group, NMTC is the predominant component of the familial tumor syndrome, and other neoplasms are relatively less common or do not occur. Linkage studies suggest that at least three different genes may cause fNMTC. It is possible that fNMTC may be polygenic in kindreds, and, if this is correct, then polygenic fNMTC is a third fNMTC group. Familial benign thyroid nodules constitute a related group of disorders. Investigation into these disorders may help to uncover the molecular differences between benign and malignant thyroid neoplasms.

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EVIDENCE SUPPORTING A FAMILIAL PREDISPOSITION: EPIDEMIOLOGIC STUDIES AND FNMTC KINDREDS The initial evidence for a familial predisposition to NMTC comes from epidemiologic studies and from the identification of large kindreds with multiple subjects affected with NMTC. A general clinical description of fNMTC has been generated from review of epidemiologic studies and kindred studies. This clinical description reflects that group of disorders characterized by a predominance of fNMTC, and does not include those known familial tumor syndromes with a slight increased prevalence of NMTC. The epidemiologic studies compare the prevalence of NMTC in relatives of affected subjects to the prevalence in the general population. At least 5 such studies all suggest that the relative risk of fNMTC in first-degree relatives of affected subjects is about 5 times greater than that of normal controls [3-7]. In a 1987 study from Connecticut,159 subjects with thyroid cancer were interviewed and compared to 285 controls matched for age and sex. For first-degree relatives of PTC subjects, the relative risk of developing PTC was 5.2 times that of the control population [3]. A more recent Canadian study using patient interviews as its end point reported that first degree relatives of those affected with NMTC were about 10 times more likely than control subjects to develop NMTC [4]. These studies both relied upon patient reporting of events, which may be inaccurate. To overcome this potential bias, other studies used confirmed pathologic evidence of thyroid carcinoma. One such study was reported from the Utah Population Data Base. Again the relative risk of thyroid cancer in the first-degree relative of an affected subject was about 8 [6]. However, this study did not differentiate between the different types of thyroid cancer so that it did not exclude the known familial association of MTC. The Swedish Cancer Registry and the Swedish FamilyCancer database were used to demonstrate that subjects with NMTC were about 4 times more likely to have a parent with NMTC that would a control subject [5,7]. Although all these studies are positive and report a similar relative risk, it should be noted that they are all susceptible to bias. The family members of subjects affected with NMTC may have been

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evaluated for thyroid abnormalities more thoroughly than control subjects. In addition, it should be noted that a familial association does not prove a genetic predisposition, since a common environmental factor could predispose multiple family members to NMTC. Such an event could be considered in the Utah study in which subjects may have been exposed to ionizing radiation from nuclear testing in the area. In summary, the epidemiologic studies consistently find an increased risk of NMTC in first-degree relatives of affected subjects, but cannot prove that this is due to a familial predisposition, since alternative explanations include a common environmental factor and more vigilant clinical evaluation of the close relatives of subjects affected with NMTC. Large fNMTC kindreds are quite rare. The clustering of 2 or 3 NMTC patients within a single family may represent a concurrence of sporadic PTC. A number of these small kindreds as well as larger kindreds with 4 or more subjects have now been reported [8-17]. In a large kindred with 5 affected subjects, it was estimated that the chance of this being the concurrence of 5 sporadic PTC cases is about one in 2 billion [17]. It is these large kindreds that are most useful in performing linkage analysis to map the susceptibility gene(s). In general single large kindreds are more useful for genetic analysis than multiple small kindreds.

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CLASSIFICATIONS AND CLINICAL CHARACTERISTICS OF FNMTC If the increased familial risk of NMTC is due to a genetic predisposition, then it follows that there should be kindreds enriched in NMTC, and that other neoplasms may be present in these kindreds. Descriptions of such kindreds suggest that fNMTC can be divided into at least 2 and possibly 3 different groups. Probably about 5 percent of all NMTC belong to one of these groups. In the first group NMTC represents a relatively infrequent component of a familial tumor syndrome characterized by a predominance of nonthyroidal neoplasms. In the second group NMTC is the most common tumor in an extended kindred, although other neoplasms may also develop. Usually the concurrence of 4 or more NMTC subjects within a single kindred is felt to represent strong evidence for a predominant familial predisposition NMTC. There is potentially a third group of fNMTC, in which only 2 or 3 family members are affected with NMTC, It is not clear if these clusterings represent a familial predisposition NMTC or simply a clustering of sporadic NMTC.

NMTC as a Component of a Familial Tumor Syndrome Characterized by a Predominance of Non-Thyroidal Neoplasms Clinical descriptions indicate that kindreds affected with Cowden syndrome, FAP, CNC1, fPGL, MEN2A are enriched in NMTC. Cowden syndrome is caused by mutations of the PTEN tumor suppressor gene and also is referred to as the multiple hamartoma syndrome. It is characterized clinically not only by multiple hamartomas of the mucous membranes, breast and thyroid, but also by breast

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carcinoma and occasionally NMTC. The malignant thyroid lesions are more often FTC and less frequently PTC [18,19]. PTEN is a dual specificity phosphatase, and mutations of the PTEN gene also cause for the Bannayan-Ruvalcaba-Riley syndrome (BRRS) that is also characterized by multiple hamartomas, macrocephaly and multiple lipomas [20]. FAP is caused by mutations of the adenomatous polyposis coli gene (APC) that is another tumor suppressor gene. It has been reported that about 2 percent of genetically affected individuals develop papillary thyroid cancer [21,22]. This prevalence is about 5 to 10 times that of the normal population. In addition, there is frequently an unusual histology of these PTC that is referred to as a cribiform pattern [23,24]. The APC mutations associated with PTC are localized most frequently to exon 15 of the APC gene. In addition to PTC these specific APC mutations are associated with congenital hypertrophy of the retinal pigment epithelium [22]. APC is a tumor suppressor gene, and the normal allele is often altered in the gastrointestinal tumors of APC subjects. Therefore, it was anticipated that the normal allele would be altered in PTC of FAP subjects. Surprisingly, this was not found in a study of 6 PTC from FAP subjects [25]. Therefore, the mechanism for the apparent association of PTC with FAP is not immediately apparent, although haploinsufficiency is a possible explanation. The epidemiologic data as well as the finding of the unusual cribiform pathology make it likely that there is a true association of NMTC in FAP. Some studies have suggested an increased prevalence of PTC in MEN 2A kindreds. Although many of these neoplasms are microcarcinomas and often without clinical significance, the prevalence of 14 percent in a large group of 196 thyroid glands with MTC from MEN2 patients seems to be about twice as high as anticipated from studies of patients undergoing thyroidectomy for Graves’ disease and mutinodular goiter [26]. Activating mutations of the RET proto-oncogene do have some mitogenic activity in a cell culture line [27]. RET is not highly expressed in thyroid epithelial cells. However, illicit RET expression or increased RET expression secondary to the activation of other tumorigenic mechanisms might permit RET to become oncogenic in thyroid epithelial cells. PTC, FTC and benign thyroid nodules have been observed in CNC1 [28]. CNC1 is caused by mutations of the protein kinase A regulatory subunit 1-alpha (PRKAR1A) gene [29]. NMTC is not a common finding in either of these disorders, but it probably does occur with increased frequency as compared to the general population. Further support for a familial predisposition to thyroid neoplasia in CNC1 comes from the mouse haploinsufficiency model that develops follicular thyroid hyperplasia and adenomas [30]. PTC were reported in one clinical study of fPGL [31]. The fPGL syndromes are caused by mutations of the succinate dehydrogenase (SDH) genes: SDHB, SDHC, and SDHD [32]. The tumorigenic mechanism of these and other mitochondrial genes is under intense investigation, since it was surprising to find that genes coding for proteins of oxidative metabolism would be proto-oncogene. Surprisingly, a familial association of anaplastic thyroid carcinoma with the Li-Fraumeni syndrome has not been reported. The Li-Fraumeni syndrome is associated with inherited mutations of the p53 tumor suppressor gene [33]. In sporadic anaplastic thyroid carcinoma p53 gene mutations are relatively common [34]. Therefore, if p53 gene mutations contribute to the pathogenesis of anaplastic thyroid carcinoma, then it is predicted that germline p53 mutations would be associated with an increased frequency of anaplastic thyroid carcinoma.

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In contrast to this prediction, anaplastic thyroid cancer has not been reported as a component of the Li-Fraumeni syndrome. NMTC as the Predominant Component of a Familial Tumor Syndrome The term familial NMTC (fNMTC) refers to those disorders in which NMTC represents the most frequent tumor. Large kindreds with 4 or more NMTC subjects are quite rare. However, such kindreds are likely to represent a familial association of NMTC, and they are of particular interest, since they are likely to provide the substrate for linkage analysis that will lead to the identification of susceptibility gene(s). A small number of such kindreds have been identified and reported [9-17], and general reviews have provided insight into the clinical characteristics of fNMTC [1,2]. As a group the pathologic subtype is predominantly papillary thyroid carcinoma, rarely follicular thyroid carcinoma, and never anaplastic thyroid carcinoma. Inheritance is autosomal dominant with incomplete age-dependent penetrance, and children are rarely affected. The age of onset is younger than with sporadic NMTC, and the disease is often multifocal within the thyroid. Women are affected about twice as frequently as men, and there is an increased prevalence of benign thyroid nodules [1]. One large study from Japan demonstrated that fNMTC have an increased recurrence rate as compared to sporadic NMTC, however, life expectancy was not adversely affected by a familial predisposition [2]. Some authors suggest aggressive therapy of fNMTC [35]. Searches for associated malignancies have resulted in mixed results. Some, but not all kindreds are associated with an increased prevalence of different malignancies. The epidemiologic studies to not show a consistent relationship between NMTC and other malignancies, with the possible exception of premenopausal breast carcinoma which seems to occur more frequently in PTC subjects than would otherwise be expected [36,37]. There are at least 3 different NMTC syndromes that have been defined by genetic analysis of kindreds. Each of these is discussed below.

Small Familial NMTC Clusterings – Polygenic fNMTC or Reduced Penetrance? Familial clusterings of two or three affected subjects are more common than large kindreds, but their genetic significance is uncertain. Undoubtedly some of these clusterings represent the concurrence of sporadic NMTC. Some likely are just small kindreds caused by genes already linked to fNMTC. Others may represent novel fNMTC syndromes caused by weakly penetrant genes. Still others may represent the interaction of two or more weak susceptibility polymorphisms that occur concurrently [38]. Indeed, it has been suggested that there may be interaction of fNMTC susceptibility genes at 2q21 with the fNMTC susceptibility genes at 19p13.2 [39] in some, but not all kindreds [40].

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THREE FNMTC SYNDROMES: GENETIC, PATHOLOGIC, AND CLINICAL CHARACTERISTICS Large kindreds with a predominance of NMTC have been investigated to determine the pathologic, clinical, and genetic linkage characteristics of fNMTC. In general these studies have divided fNMTC into three different subgroups or syndromes referred to as familial papillary thyroid carcinoma enriched in papillary renal neoplasia (fPTC/PRN; OMIM %606240), thyroid carcinoma with oxyphilia (TCO; OMIM %606386), and familial nonmedullary thyroid carcinoma type 1 (fNMTC1; OMIM %606240). In each of these subgroups, linkage studies have identified chromosomal regions likely to contain the susceptibility gene. It has been suggested that a familial microcarcinoma syndrome may also exist. This is clinically different than the other syndromes, but linkage studies have not been performed to determine if it is genetically distinct. Final proof that these are familial tumor syndromes awaits identification of the specific susceptibility gene(s). The susceptibility gene for the fPTC/PRN syndrome has been mapped to chromosome 1q21 using a large kindred that includes 5 subjects affected with papillary thyroid carcinoma, 2 subjects with papillary renal neoplasia, 2 carriers without PTC or PRN, and 3 subjects with benign nodules [41]. There are no distinctive pathologic features of the PTC in fPTC/PRN. As with other fNMTC syndromes penetrance is incomplete, inheritance is autosomal dominant, and women are affected more frequently than men (4:1) [17]. However, the linkage to chromosome 1q21 and the association with papillary renal neoplasia makes this syndrome genetically and clinically distinct from other fNMTC syndromes. There were other neoplasms within this large kindred. After linkage had been established, it was determined that subjects carrying the affected allele developed premenopausal breast carcinoma (2), renal oncocytoma (1), benign thyroid nodules (3), testicular tumor (1), non-Hodgkin’s lymphoma (1), and lung adenocarcinoma (2). Some subjects developed more than one neoplasm. Therefore, the fPTC/PRN susceptibility gene may predispose to multiple malignancies. In support of this hypothesis, tumor specific loss of heterozygosity (LOH) at 1q has been observed in sporadic breast carcinoma [42,43], but gains in this region have been noted in sporadic lung adenocarcinoma [44]. The association of breast carcinoma developing after thyroid carcinoma has been observed, and it has been suggested to be related to treatment with radioactive iodine [37]. Alternatively, a susceptibility gene may predispose to both PTC and breast carcinoma. In summary, fPTC/PRN is a clinically and genetically distinct fNMTC syndrome that maps to 1q21 and is enriched in papillary renal neoplasia and possibly other tumors. The syndrome of thyroid carcinoma with oxyphilia has been mapped to 19p13.2 [45]. In this disorder the thyroid carcinomas and benign thyroid nodules demonstrate the pathologic feature of oxyphilia. Therefore, TCO is pathologically and genetically distinct from the other fNMTC syndromes. Interestingly, other families with PTC, but without oxyphilia, link to the region of 19p13 [46]. It is not yet known whether this represents a TCO variant or whether there is a second fNMTC susceptibility gene within this chromosomal region. Tumor specific LOH within this region in sporadic thyroid follicular tumors and oxyphilic thyroid tumors suggest a tumor suppressor gene in this region [40]. The identification of tumor specific LOH may help to refine the chromosomal location of this gene and aid in its identification.

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Other kindreds with fNMTC have been linked to chromosome 2q, and this putative gene is referred to as fNMTC1 [47]. There are no distinctive pathologic or clinical features of this syndrome. The presence of LOH at 2q21 in sporadic follicular tumors and sporadic oxyphilic tumors suggests a tumor suppressor gene within this region that contributes to the development of thyroid cancer [40]. Interestingly, there may be an interaction of the fNMTC1 gene (or other gene at 2q) with the TCO gene (or other gene at 19q) in some [39], but not all kindreds [40].

RELATED SYNDROMES – FAMILIAL BENIGN THYROID NODULES

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Benign thyroid nodules are often clonal thyroid neoplasms and are present in greater than expected frequency in fNMTC. Therefore, investigation into familial syndromes of benign nodules may provide clues to our understanding of malignant thyroid neoplasia. As noted previously, the CNC1 syndrome is enriched in benign thyroid nodules [28]. At least 3 loci for familial predisposition to benign nodules have been identified. The MNG syndromes have been mapped to 14q [48], Xp22 [49], and 3q26 [50]. The predisposing genes at these loci have not been identified. Inherited activating mutations of the receptor for thyroid stimulating hormone cause hyperthyroidism with thyroid nodules [51]. A kindred with FTC and goiter has been reported, but no genetic studies are available and there are no other reports of similar kindreds [52]. Inherited syndromes that result in hypothyroidism and increased TSH not unexpectedly cause thyroid nodules and thyroid growth. These will not be reviewed in this review.

CLINICAL IMPLICATIONS Although we are still accumulating information concerning fNMTC, the current information does have important clinical implications. The following are offered as suggestions to clinicians based up currently available information. It is recognized that recommendations will change in the future, and there currently is no general consensus conference that has published recommendations regarding fNMTC. It is important to recognize that patients with Cowden syndrome, FAP and CNC1 are at increased risk for the development of thyroid neoplasms. Individuals genetically affected with these disorders should receive genetic counseling and probably should undergo yearly thyroid examinations. Thyroid nodules should be appropriately evaluated, usually with fine needle aspiration biopsy. Individuals affected with MEN2A or MEN2B undergo prophylactic thyroidectomy to prevent MTC, so that they do not need to be screened for NMTC. The risk of NMTC in the PGL syndromes is not as clear, but physicians should be aware of this possible co-morbidity. For subjects with apparently sporadic NMTC, a careful family history should be taken to determine if other family members have had NMTC. As indicated from epidemiologic

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studies, about 5 percent of subjects with apparently sporadic NMTC will have a first-degree relative with NMTC, suggesting a familial predisposition. A positive family history should alert the physician to the possibility of a familial predisposition, and the fNMTC subjects should advise their family members of the potential risk for NMTC. For individuals that are members of kindreds characterized by a predominance of NMTC, careful yearly thyroid physical examinations should be considered starting at about age 20 years. NMTC usually does not occur before age 20, so that neck examinations are probably not necessary in children. The role of thyroid ultrasound remains to be determined, but will probably remain controversial. It is not know if it should be used or how often it should be performed. The results of a single screening of fNMTC kindred members has been reported. In this large investigation in Japan, thyroid ultrasound examination was performed on 149 symptom-free first and second-degree relatives of fNMTC subjects representing 53 different fNMTC kindreds. A fNMTC kindred was defined as a kindred with at least 2 known fNMTC members. At least one thyroid nodule was identified in 52 percent of the patients. Based upon thyroid aspiration results, 18 patients underwent thyroidectomy, and PTC was confirmed in 15, suggesting that the yield of PTC is about 10 percent when first-degree relatives are screened by ultrasound. In this study there were surprisingly few surgeries for benign nodules (3 of 18). Therefore, there seems to be little danger associated with screening ultrasound. It is not known how many of these nodules were identified on physical examination, nor is it known if early detection affects prognosis. Since the survival appears the same in fNMTC patients as in sporadic NMTC patients, then early screening may reduce the incidence of death from NMTC, which is already low. Therefore, there is not enough information to definitively recommend a screening thyroid ultrasound. In contrast, there are no strong contraindications to performing the ultrasound examination. Many physicians prefer to perform a thyroid ultrasound at least once for first-degree relatives of fNMTC patients. There are currently no useful genetic studies. Even in those rare large kindreds in whom linkage has been established, it seems prudent to consider all subjects potentially affected and follow them with yearly thyroid examinations. In occasional rare kindreds fNMTC appears to be more aggressive than sporadic NMTC. It may be appropriate to screen members of such kindreds with ultrasound on a routine basis. Since fNMTC is usually treatable, prophylactic thyroidectomy is not indicated as it is in the MEN2 syndromes.

CONCLUSION In about 5 percent of NMTC there is a familial predisposition. A small fraction of these will be a component of non-thyroidal familial tumor syndromes in which the relative risk of NMTC is about 10 times that of the normal population. These include FAP, Cowden syndrome, CNC1, the fPGL syndromes, and MEN2A. In the fNMTC syndromes, NMTC is the predominant neoplasm. The age of onset is younger than for sporadic PTC, the frequency of multifocality is greater, and the chance of recurrence is greater. Life expectancy and male to female ratios appear to be the same in sporadic NMTC and fNMTC.

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A careful family history should be elicited from NMTC patients. Yearly evaluation of first-degree relatives of subjects affected with fNMTC should be considered. Many endocrinologists perform a thyroid ultrasound on the first evaluation and possibly at intervals thereafter. Genetic testing is not yet available, and prophylactic thyroidectomy is not indicated.

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[30] Griffin KJ, Kirschner LS, Matyakhina L, Stergiopoulos SG, Robinson-White A, Lenherr SM, et al., A transgenic mouse bearing an antisense construct of regulatory subunit type 1A of protein kinase A develops endocrine and other tumors: comparison with Carney complex and other PRKAR1A induced lesions. J Med Genet 2004;41:923-31. [31] Parry D, LI F, Strong L, Carney J, Schottenfeld D, Reimer R, et al., Carotid body tumors in humans: genetics and epidemiology. J Natl Cancer Inst 1982;68:573-578. [32] Maher E and Eng C, The pressure rises: update on the genetics of phaeochromocytoma. Hum Mol Genet 2002;11:2347-54. [33] Levine A, p53, the cellular gatekeeper for growth and division. Cell 1997;88:323-331. [34] Fagin JA, Matsuo K, Karmarkar A, Chen DL, Tang SH, and Koeffler HP, High prevalence of mutations of the p53 gene in poorly differentiated human thyroid carcinomas. J. Clin. Invest. 1992;91:179-184. [35] Grossman RF, Tu S-H, Duh Q-Y, Siperstein AE, Novosolov F, and Clark OH, Familial nonmedullary thyroid cancer: an emerging entity that warrants aggressive treatment. Arch. Surg. 1995;130:892-899. [36] Vassilopoulou-Selin R, Palmer L, Tayler S, and Cooksley CS, Incidence of breast carcinoma in women with thyroid carcinoma. Cancer 1999;85(3):696-705. [37] Chen AY, Levy L, Goepfert H, Brown BW, Spitz MR, and Vassilopoulou-Sellin R, Development of breast carcinoma in women with thyroid carcinoma. Cancer 2001;92:225-231. [38] Links T, van Tol K, te Meerman G, and de Vries E, Differentiated thyroid carcinoma: a polygenic disease. Thyroid 2001;11:1135-1140. [39] McKay J, Thompson D, Lesueur F, Stankov K, Pastore A, Watfah C, et al., Evidence for interaction between the TCO and NMTC1 loci in familial non-medullary thyroid cancer. J Med Genet 2004;41:407-412. [40] Stankov K, Pastore A, Toschi L, McKay J, Lesueur F, Kraimps J, et al., Allelic loss on chromosomes 2q21 and 19p13.2 in oxyphilic thyroid tumors. Int J Cancer 2004;111:4637. [41] Malchoff CD, Sarfarazi MS, Tendler B, Forouhar F, Whalen G, Joshi V, et al., Papillary thyroid carcinoma associated with papillary renal neoplasia: genetic linkage analysis of a distinct heritable tumor syndrome. J Clin Endocrinol Metab 2000;85:1758-1764. [42] Gaki V, Tsopanomichalou M, Tsiftsis D, and Spandidos D, Allelic loss in chromosomal region 1q21-23 in breast cancer is associated with peritumoral angiolymphatic invasion and extensive intraductal component. Eur J Surg Oncol 2000;26:455-60. [43] Borg A, Zhang Q, Olsson H, and Wenngren E, chromosome 1 alterations in breast cancer: allelic loss on 1p and 1q is related to lymphogenic metastases and poor prognosis. Genes Chromosomes Cancer 1992;5:311-20. [44] Goeze A, Schluns K, Wolf G, thasler Z, Petersen S, and Petersen I, Chromosomal imbalances of primary and metastatic lung adenocarcinomas. J Pathol 2002;196:8-16. [45] Canzian F, Amati P, Harach R, Kraimps J-L, Lesueur F, Barbier J, et al., A gene predisposing to familial thyroid tumors with cell oxyphilia maps to chromosome 19p13.2. Am J Hum Genet 1998;63:1743-1748. [46] Bevan S, Pal T, Greenberg CR, Green H, Wixey J, Bignell G, et al., A comprehensive analysis of MNG1, TCO1, fPTC, PTEN, TSHR and TRKA in familial nonmedullary

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thyroid cancer: confirmation of linkage to TCO1. J Clin Endocrinol Metab 2001;86:3701-3704. [47] McKay JD, Lesueur F, Jonard L, Pastore A, Williamson J, Hoffman L, et al., Localization of a susceptibility gene for familial nonmedullary thyroid carcinoma to chromosome 2q21. Am J Hum Genet 2001;69:440-446. [48] Bignell GR, Canzian F, Shayeghi M, Stark M, Shugart YY, Biggs P, et al., Familial nontoxic multinodular thyroid goiter locus maps to chromosome 14q but does not account for familial nonmedullary thyroid cancer. Am J Hum Genet 1997;61:1123-1130. [49] Capon F, Tacconelli A, Giardina E, Sciacchitano S, Bruno R, Tassi V, et al., Mapping a dominant form of multinodular goiter to chromosome Xp22. Am J Hum Genet 2000;67:1004-1007. [50] Takahashi T, Nozaki J, Komatsu M, Wada Y, Utsunomiya M, Inoue K, et al., A new locus for a dominant form of multinodular goiter on 3q26.1-q26.3. Biochem. Biophys. Res. Commun. 2001;284:650-4. [51] Kopp P, Van Sande J, Parma J, Duprez L, Gerber H, Joss E, et al., Congenital hyperthyroidism caused by a mutation in the thyrotropin-receptor gene. N. Engl. J. Med. 1995;332:150-154. [52] Cooper DS, Axelrod L, DeGroot LJ, Vickery AL, and Maloof F, Congenital goiter and the development of metastatic follicular carcinoma with evidence for a leak of nonhormonal iodide: clinical, pathological, kinetic, and biochemical studies and a review of the literature. J Clin Endocrinol Metab 1981;52:294-306.

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Chapter 7

RECENT ADVANCES IN THE TREATMENT OF MEDULLARY THYROID CARCINOMA Levent Saydam∗ and Mete K. Bozkurt Department of Otolaryngology, Bayindir Hospital Ankara, Turkey.

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ABSTRACT Medullary thyroid carcinomas which arise from parafollicular or C cells of thyroid comprise about 5% to 10% of all thyroid cancers. Due to its derivative cell type medullary cancer is considered as a subset of neuroendocrine tumors. The primary hormonal product which the tumor cells secret is calcitonin. The tumor is hereditary with auotosomal dominant inheritance in approximately 25% of the cases while the rest of the cases present as in sporadic form. The hereditary forms are MEN type 2A and 2B syndromes and familial non-MEN medullary throid carcinomas which are caused by RET proto-oncogene mutations. Currently the choice of primary treatment of these tumors is surgical removal of entire gland and cervical lymph node groups having the risk of invasion by tumor cells. Radiotherapy is instituted by means of adjuvant therapy in cases with locoregional invasion or as a palliative measure in certain cases. While there is not universally accepted type of systemic treatment, currently ongoing experimental forms of treatments such as application of gene treatment procedures targeting the mutations in RET proto-oncogene, the use of somatostatin analogs or 131 IMIBG are still under investigation.

Keywords: medullary thyroid carcinoma, calcitonin, ret proto-oncogene, neuroendocrin tumors

∗

Correspondence concerning this article should be addressed to Levent Saydam, MD, Assoc. Prof. of Otolaryngology, Department of Otolaryngology, Bayindir Hospital, Sogutozu 06520, Ankara, Turkey.

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INTRODUCTION Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor arising from parafollicular or C cells of the thyroid gland. C cells produce calcitonin which is a major hormonal substance involved in calcium metabolism. MTC represents approximately 10% of all thyroid malignancies. In a major proportion (75-80%) of the cases the disease presents in its sporadic form. Hereditary forms which comprise other 20-25% of the cases can occur in two different clinical settings; as a part of multiple endocrine neoplasia syndromes: types 2A or 2B or as a single entity, familial non-MEN medullary thyroid carcinoma. These are autosomal dominant inherited syndromes which are characterized by mutations in the RET proto-oncogene. Average survival for MTC is lower than that for more common thyroid cancers, e.g., 83% 5-year survival for MTC compared with 90% to 94% 5-year survival for papillary and follicular thyroid cancer. [1,2] While medullary thyroid carcinoma presents as multifocal disease in majority (95%) of hereditary cases, only 20% of sporadic cases develop more than one focus. In parallel to its various clinical presentations, the clinical behavior of medullary thyroid cancers may range from indolent to aggressive. In this review, we will present pathologic and genetic abnormalities in medullary thyroid carcinoma. Beside the current diagnostic and therapeutic modalities will also be discussed.

C CELLS

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Embryology and Anatomy The C cells, formerly called parafollicular cells get their origin from the ultimobranchial body which is a derivative of fourth or –debatable fifth pharyngeal pouch. During embryonic life migrating cells of neural crest origin infiltrate the ultimobranchial body. As fusion of ultimobranchial body to thyroid gland develops this particular group of cells innoculates into the lateral thyroid lobes mainly at the junction of upper third and lower thirds. Ultrastructurally, C cells are located between the basal layer and follicular cells.

Function of C cells: The major substance produced by C cells is calcitonin. Also some other hormonal products such as somatostatin, vasoactive intestinal peptide, prostaglandins, serotonin, etc or carcinoembryonic antigen (CEA) which is produced by neoplastic C cells.

Calcitonin C cells’ major product calcitonin, a 32-amino acid polypeptide hormone, is involved in regulation of calcium metabolism. The gene encoding human CT (the CALC-I gene) is located on the tip of the short arm of chromosome 11 (11p15.3-15.5).It is secreted not only in

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response to calcium levels but also to gastrointestinal hormones such as glucagon. Via calcitonin receptors located in the bones and kidneys, its major actions are: 1. To lower plasma calcium and phosphate levels by inhibiting bone resorption; 2. To increase calcium excretion in urine through suppression of renal tubular reabsorption of calcium. Interestingly, calcitonin has a negligible effect on blood calcium levels in humans. Clinically low plasma Ca levels rarely occur even in patients with large amounts of circulating calcitonin from medullary carcinoma or C cell hyperplasia of the thyroid gland.

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BIOCHEMICAL SCREENING FOR MEDULLARY THYROID CARCINOMA Calcitonin measurement is indicated for the diagnosis and follow-up of patients with medullary thyroid carcinoma (MTC), the majority of which produce this hormone. Although not thoroughly specific, the elevated levels of serum calcitonin levels are found in most cases of medullary thyroid cancer or in pre-malignant C cell hyperplasia cases. In patients with normal calcitonin levels but suspected of having medullary cancer based on family history or residual and/or recurrent tumor, serum calcitonin levels should be checked by pentagastrin provocation test.[3,4] Pentagastrin is more sensitive than calcium stimulation in diagnosing early C-cell disease. Screening of first-degree relatives of MEN 2A patients with the pentagastrin test, beginning in early childhood (approximately 5 years of age) and continuing annually until 35 to 40 years of age if the intervening stimulated plasma calcitonin levels remain normal should be routinely performed. An elevated calcitonin level defines a positive result and obviates the need for a possible FNAB. Also the cases having residual disease or recurrent disease with undetectable basal CT concentrations can also be detected by this method. Despite its transient nature some adverse effects including substernal discomfort, abdominal cramping, and nausea have been reported.[4] Also the cost of screening programs, the necessity of lifelong testing for all members of a kindred, occasional false-positive results, and the difficulty of interpreting borderline pentagastrin test results make this test inappropriate as a routine application for every case.

The Technique of Pentagastrin Provocation Testing: Patient should be fasting for 10 - 12 hours prior to collection of specimen. First a basal calcitonin level is measured and pentagastrin: 0.5 ug per kilogram body weight is administered IV bolus. Consecutively at 1, 2, 5, 10 minutes serum calcitonin levels are measured. Elevated calcitonin levels increased two times or more baseline are considered positive for the diagnosis of medullary thyroid carcinoma or C cell hyperplasia.

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GENETICS OF FAMILIAL MEDULLARY THYROID CARCINOMA Hereditary medullary thyroid carcinoma is an autosomal dominant disease. Either familial non-MEN medullary carcinomas or MEN 2 syndromes both express inherited germline mutations in the RET gene which is located on chromosome region 10q11.2. The RET gene is a proto-oncogene composed of 21 exons which encodes a tyrosin-kinase receptor. It is expressed in the C-cells of the thyroid gland, in the adrenal medulla, in neurons. The extracellular domain of this receptor consists of an extracellular domain with a calciumbinding cadherin-like region and a cysteine-rich region that interacts with 1 of 4 ligands. These ligands, e.g., glial-derived neurotropic factor (GDNF), neurturin (NTN), persephin (PSF), and artemin (ATF), also interact with one of 4 coreceptors in the GDNFRa family.[5] The tyrosine kinase catalytic core is located in the intracellular domain, which causes downstream signaling events through a variety of second messenger molecules.[6] Normal tissues contain transcripts of several lengths. In MEN 2A and familial non-MEN MTC, nonconservative point mutations are found within codons specifying cysteine residues within the extracellular binding domain of the ret gene product. This results in the disruption of normal disulfide bridge formation and subsequent abnormalities in tertiary protein structure. At least 95% of families with MEN 2A or MEN2B and 85% percent of individuals with familial medullary thyroid carcinoma have a RET mutation in exon 10 or 11. [7] Mutations of codon Cys634 in exon 11 occur in about 85% of families; mutation of cysteine codons at amino acid positions 609, 611, 618, and 620 in exon 10 together account for the remainder of identifiable mutations. [7,8] In cases with codon Cys634 mutations, the cumulative risk of MTC rises linearly with age. [9] The absence of germline RET exon 10, 11, 13 or 16 mutation appears to rule out MEN 2A. In 40-60% of sporadic MTC cases express some somatic mutations. These mutations have been observed mostly in codon 918 (25-33% of the cases) and to a lesser degree in codon 618, 634, 768, 804 and 883 of the RET proto-oncogene.[9] Approximately 95% of individuals with the MEN 2B phenotype have a single point mutation in the tyrosine kinase domain of the RET gene at codon 918 in exon 16, which substitutes a threonine for methionine (Met918Thr). [10,11,12] The disease-causing point mutation in codon Met918 that causes 95% of the MEN 2B phenotype lies within the catalytic core of the tyrosine kinase and causes an alteration in substrate specificity of the normal RET. [13,14,15] In rare occasions a second mutation at codon 883 in exon 15 has been identified without a 918 mutation.[16,17,18,19] Most genotype-phenotype correlations to date have focused on the relationship between specific RET protooncogene genotypes and disease phenotype.[16,20,21] Of these the most important relations are: •

•

Mutations involving the cysteine codons 609, 618, and 620 are associated with either MEN 2A, FMTC. Mutations in these codons are detected in about 10% of families with MEN 2A and 65% of families with FMTC.[20] Any RET mutation at codon 634 in exon 11 results in higher incidence of pheochromocytomas and hyperparathyroidism. [20,22] Specifically within the 634 mutations, C634R mutation is significantly correlated with hyperparathyroidism.[20]

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Strong correlation between the presence of mutations in codon 634 and the cutaneous lichen amyloidosis has been reported.[20,21] Codon 918 and 883 mutations are associated only with MEN 2B. Somatic mutations in these codons are frequently observed in sporadic MTC.[16,23] Mutations at codons 768 or 804 may be FMTC-specific and might represent important contributors to apparently sporadic MTC.[24] Concomitant Hirschsprung’s disease is associated with mutations at codons 620 or 618.[24] Sporadic medullary thyroid carcinoma cases do not exhibit germ-line mutations but in 40-60% of the cases somatic RET mutations at codon 918 can be found. Somatic mutations of the ret protooncogene in sporadic medullary thyroid carcinoma are not restricted to exon 16 and are usually associated with tumor recurrence.[25]

CLINICAL PRESENTATIONS

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Sporadic MTC Sporadic medullary thyroid carcinoma which typically does not associate with an endocrinopathy comprises approximately 75% of all medullary thyroid cancers. In approximately 5% sporadic MTC cases reveal a germline RET proto-oncogene mutation which describes the occurrence of de novo mutations or members of previously unidentified kindreds.[9] The tumor is more frequently seen in between the fourth and sixth decades. Female to male ratio is roughly 3:2. The characteristic clinical presentation is a solitary thyroid nodule located in the upper third of a lobe with or without accompanying cervical lymphadenopathy. There is a positive correlation between palpable lymph nodes and tumor size larger than 3 cm.[26] Contrarily in a recent study reported from Institut Gustave-Roussy, the incidence of lymphatic metastases was not found to be related to thyroid tumor size.[27] Basal or stimulated calcitonin levels or both increase in all patients with sporadic medullary thyroid carcinoma, although in most cases the diagnosis of medullary thyroid carcinoma is made postoperatively rather than preoperatively. Distant metastases occur in 20% of cases involving the liver, lungs or bones in decreasing order. The family history is typically negative. Histologically, sporadic medullary thyroid carcinoma is characterized by unifocal involvement with absence of C-cell hyperplasia. Basal or stimulated calcitonin levels or both increases in all patients with sporadic medullary thyroid carcinoma.

FAMILIAL MTC SYNDROMES There are three distinctive clinical presentations: MEN 2A MEN 2B NON-MEN FAMILIAL MTC

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Multiple endocrine neoplasia type 2 (MEN 2) involves tumors of the thyroid, the adrenal medulla, and the parathyroid glands.[28] The MEN syndromes result from one or more genetic aberrations and are transmitted as autosomal dominant diseases with incomplete penetrance and variable expressivity. Because MEN II shows autosomal dominance, a child of an affected parent has a 50% chance of inheriting the causative gene. Medullary thyroid cancer in MEN2 syndromes is almost invariably multifocal and bilateral involvement is encountered in 40-50% of the cases. Genetic screening is recommended in all subjects having tumor risk before the age of 5. MEN 2A consists of medullary thyroid carcinoma, pheochromocytoma, and parathyroid hyperplasia. Pheochromocytomas are present in 10% to 50% of these patients. Hyperparathyroidism is encountered in approximately 10-25% of the MEN2A patients.[20] Less commonly cutaneous lichen amyloidosis or Hirschsprung’s disease have been observed in MEN2A families.[29] In some studies cutaneous lichen amyloidosis has been found in higher proportions (up to 36%) of MEN2A cases with RET 634 mutation.[30] In patients with MEN 2A, the MTC has a variable clinical behavior usually progressing in an indolent fashion, but it may grow rapidly in occasional cases. MEN 2B has an unusual phenotype characterized by ganglioneuromatosis, marfanoid habitus, medullary thyroid carcinoma, pheochromocytoma, and, rarely, parathyroid hyperplasia. [31] In all patients mucosal neuromas are found on the undersurface of the eyelid and throughout the gastrointestinal tractus whereas only some patients develop pheochromocytomas. These patients show a tendency to develop earlier tumors which are usually more aggressive compared to MEN2A cases. The appearence of medullary cancer foci have been reported in children 2 and 4 years of age. [32] A syndrome related to MEN 2A and MEN 2B is Familial Medullary Thyroid Carcinoma (FMTC). Patients with this disease only have MTC and express none of the extrathyroidal manifestations of the syndromes. MTC that occurs in FMTC develops later compared to Type 2B cases in life and grows slowly. The peak incidence is between the ages of 40 and 50.

TREATMENT OF MEDULLARY THYROID CARCINOMA A. Surgery Preventive Surgery for Hereditary Medullary Thyroid Carcinoma Because medullary thyroid carcinoma occur at some points throughout their life spans all patients with MEN anomalies or those in the group of non-MEN familial medullary thyroid cancers should undergo total thyroidectomy regardless of biochemical screening values. [33,34] Patients of MEN 2A with germ-line mutations and non-MEN familial medullary thyroid cancer should undergo total thyroidectomy at age 5 to 6 years. Due its aggressive nature after the confirmation of diagnosis by a RET proto-oncogen analysis all MEN 2B patients should take the same treatment in the first year of life, if possible, or as soon as the diagnosis is made. Prior the surgery in all gene carriers pheochromocytoma should be screened since it can occur practically in every case. After establishment of a specific

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medullary cancer diagnosis these patient should be kept in follow-up program to screen development of other possible endocrine anomalies. Primary Surgical Management: Since medullary thyroid carcinoma does not respond to any form of RAI treatment or thyroid suppression; surgery with the aim of removing of all neoplastic foci within the thyroid tissue or in the neck still remains the mainstay of the treatment. Preoperatively, serum calcitonin and carcinoembryogenic antigen levels should be assessed in every case. Approximately 4 weeks postoperatively these studies should be repeated in order to evaluate the completeness of surgical removal. All patients with medullary thyroid cancer should undergo total thyroidectomy with careful examination of all four parathyroid glands to exclude presence of parathyroid adenomas. In cases that no parathyroid pathology is found intraoperatively some authors still propose total parathyroidectomy with autotransplantation to ensure an adequate glandular tissue and central lymph node clearance while minimizing the chance of leaving behind a small focus of thyroid tissue which could potentially harbour medullary cancer.[35] The excised parathyroid glands are sliced to 1-3 mm fragments and autotansplanted, preferably into non-dominant forearm muscles or less frequently into the sternocleidomastoid muscle. The use of forearm muscles prevents the occurrence of hypoparathyroidism due to accidental removal of functioning glands in subsequent neck explorations. As discussed above, familial forms are uniformly multifocal and have a strong tendency for bilateral C cell disease. Sporadic MTC cases also present bilateral disease in majority (67%)of the patients.[36] These facts plus the clinical findings revealing that those patients who underwent aggressive thyroid gland removal do better than conservative surgery cases necessitates total thyroidectomy as a must for initial treatment procedure.[26] Sporadic MTC presents with palpable neck disease in up to 60% of the cases. Additionally pathologic examination reveals metastatic foci in the neck lymphatics in more than 80% of the cases.[27] Dissection of the central neck compartment (levels 6 and 7) including all nodal tissue extending from the hyoid bone superiorly to the innominate veins inferiorly is routinely done while lateral neck dissection still remains as a controversial issue. Scollo et al [27] and Cohen et al [35] based on their series consisted of 101 and 73 patients consecutively reported that lateral neck dissection (levels 2 to 5) unilaterally or bilaterally should be an integral part of initial surgical treatment. Regarding the contralateral involvement of the neck in unilateral sporadic cases, the only predictive factor is shown to be the involvement of ipsilateral neck. [27] As stated by Cohen et al [35]; ‘ For MTC, however, operative resection of all visible nodal tissue in the appropriate compartments in the neck should be the goal of treatment as there is no effective adjuvant therapy for residual tissue.’ In our clinical practice central nodal dissection as described above is routinely done and based on detailed preoperative radiological studies and careful palpation of the neck intraoperatively and depending on surgeon’s judgment a lateral neck dissection including level 2 to 5 lymph node groups is added in appropriate cases. In cases with positive lymph nodes in the central compartment dissection of the superior mediastinal lymph nodes should be performed. Sternocleidomastoid muscle, internal jugular vein and accessory nerve are not sacrificed unless overt neoplastic invasion of any of these structures is noted.

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Treatment of the Recurrent or Metastatic Disease: Surgical removal of local or regional recurrent disease with persistently elevated calcitonin levels is the mainstay of the treatment. Surprisingly the cases of high posttreatment calcitonin levels with no evidence of locoregional or distant metastatic disease do In cases of inadequate previous surgery the patient should be handled as a candidate for primary surgical treatment protocols. In the absence of distant metastasis postoperative irradiation may be instituted for further clearance of possible remnant neoplastic cells. Distant metastases are the main death-cause in MTC cases. The most frequently affected organs are liver, lungs and bones. Ten years survival after the diagnosis of metastatic disease is about 20 percent. Despite the disseminated and multiple nature of distant metastases in some patients with a single or a few metastases can be treated by means of limited resections.[37] In cases with liver metastases especially those smaller than 3 cm embolization or chemoembolization was reported to be found beneficial.[38,39] The aim of the surgical treatment in this context is to avoid occurrence of life threatening complications in critical areas such as brain or bones or to prevent metastatic pain.

NON-SURGICAL TREATMENT MODALITIES

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External Radiotherapy: External radiotherapy to the neck and upper mediastinum does appear to have a role in the treatment of locally advanced inoperable tumors. It was also found to have significant effect in reducing local relapse rate in patients with limited nodal disease.[40] But no survival advantage has been shown with radiation therapy[35] Beside this the possibility of radiation injury to the critical neck structures and lack of soft tissue plans due to postradiation fibrosis should be considered in all cases.

Chemotherapy No any established form of effective chemotherapy protocol is present among current treatment strategies for MTC.[41] Orlandi et al. [42] reported their results with combinations of dacarbazine 5-FU in five advanced or metastatic MTC cases that partial responses were observed in three patients lasting in 8,9 and 10 months. In another study, combinations of doxorubicin and streptozocin and 5-FU and dacarbazine were given alternately to 20 patients with metastatic medullary thyroid carcinoma which produced only three partial response.[43] Wu et al.[44] reported their results with combination of cyclophosphamide, vincristine and dacarbazine. While three patients remained to have progressive disease two patients had partial tumor and biochemical responses and one patient had a partial biochemical response. Use of somatostatin analogs e.i. octreotide and lanreotide is another recently employed mode of treatment for advanced cases. These agents are shown to be safe and effective treatments for tumors with somatostatin receptors such as GH-secreting pituitary adenomas, carcinoid tumors, gastrinomas, etc. The thyroid gland has also somatostatin receptors. But the use of

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somatostatin analogs and/or interferon in the thyroid gland cancer has provided contradictory results. [45] Somatostatin and its analogs fail to influence follicular thyroid function, whereas their administration in patients with medullary thyroid carcinoma induces a reduction of serum calcitonin concentrations and clinical symptoms, but fails to influence tumor size and patient survival rate.[46,47] Some authors have reported some symptomatic improvement and reduction of calcitonin levels but tumor or metastasis regression was not observed in their series.[48] The administration of octreotide and lanreotide coupled with a radioisotope with the idea of delivery of enough radiation dose to the tumor cells which specifically entrap somatostatin analogs without compromising normal tissue have indicated possible clinical potential. But the lack of somatostatin receptors due heterogeneity of tumor cells or development of resistance are current problems with this theoretically attractive treatment option.[49]

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Gene Therapy for Medullary Thyroid Carcinoma Current therapies for cancer rely on medications that kill dividing cells or block cell division. They are not tumor specific, so they not only act on tumor cells, but also affect normal proliferating cells causing increased morbidity and mortality in cancer patients. In recents years, there is a tremendous interest on genetic and immunological researches to find out a tumor specific treatment, which is more efficient and has fewer side effects. Gene therapy can be defined as the transfer of genetic material into a cell to alter the cellular phenotype. It can be done by introducing a nucleic acid or target gene (transgene) directly into cells (transfection) or transferring a transgene into a cell through a viral vector system (transduction). The goal is to administer a suitable amount of transgenes systemically to achieve a high level of tumor targeting, so that normal tissue toxicity might be avoided. Specific promoters can be used to allow targeted transcription in tumor cells. These tissuespecific, tumor-specific or inducible promoters can limit gene expression to target cells. Tissue specific promoter is the ‘thyroglobulin gene promoter’ (TG) and tumor-specific promoter is the ‘Calcitonin / Calcitonin gene-related peptide’ for the selective targeting of thyroid cells.[49,50] Medullary thyroid carcinoma (MTC) is characterized by dominant activating mutations in the RET (rearranged during transfection) proto-oncogene which has been mapped to chromosome 10. RET mutations are detected in 95% of hereditary MTC forms and up to 25% of the sporadic cases; therefore it is an attractive target for gene therapy. There are 4 main approaches in gene therapy. 1. Corrective Gene Therapy: The aim is to restore the normal function of a tumor suppressor gene or negate the effect of an oncogen. Inhibition of oncogenic activated RET is the goal of the gene therapy for MTC. Dominant-negative RET mutants delivered into tumor cells to block the oncogenic signal are found to have strong antineoplastic activity causing apoptosis induction and tumor growth retardation.[51] Zijuan et al [52] postulated that RET encodes for a tyrosine kinase

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and Src tyrosine kinases regulate MTC proliferation. They showed that interruption of tyrosine kinase signaling prevents cell growth and proliferation in MTC cells.

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2. Cytoreductive Gene Therapy (Suicide Gene Therapy): This method is based on delivering an exogenous gene that causes cell death or allows the application of cytotoxic agents. Adenovirus mediated gene transfer of herpes simplex virus type 1 thymidine kinase (HSV-tk) in tumor cells followed by application of ganciclovir (GCV) is the most common technique. GCV is phosphorylated by HSV-tk and competes with deoxyguanosine triphosphate in DNA polymerization which results in the arrest of DNA and cell death. To minimize extratumoral toxicity thyroid specific promoters have been used. Zeiger [53] was first to use the TG promoter and he reported that nearly 100% of the thyroid carcinoma cells treated with an adenovirus expressing HSV-tk under the control of the TG promoter were killed by GCV compared to only 5% of the control cells. Calcitonin gene promoter is also used by different authors. Minemura et al [54] showed that transfering a replication-defective adenovirus vector AdDCTtk, which contains a human CALC-I minigene as a promoter and HSV-tk gene, into several cell lines including human MTC-derived TT cells and rat MTC cells resulted in enhanced cell death depending on concomitant GCV concentration. A modified Calcitonin promoter has been developed by Messina et al [55] to drive expression of HSV-tk in MTC following systemic delivery by adenovirus without any evident toxicity. They created a dual specificity system, using calcitonin –specific splicing in addition to the modified calcitonin promoter, which provided enhanced selectivity for MTC. 3. Immunomodulatory Gene Therapy: Immunotherapy for tumors is aimed at augmenting the host immune response to the tumor (active immunity) or giving tumor specific antibody or T cells (passive immunity). The aim of this treatment is to induce gene expression that enhances immune responses against tumor tissue. Cytokines that include interferon-gama (IFN-γ), tumor necrosis factor alfa (TNF-α), interleukin 2 (IL-2), and interleukin 12 (IL-12) are known to have antitumor activity. However, systemic administration of recombinant IL-2 and IL-12 causes dosedependent toxicity in animals. With the technique based on direct transduction of IL expressing gene therapy vectors into the tumor tissue, this problem is solved. IL-2 has been reported in different studies for genetic immunotherapy of thyroid carcinoma. Zhang et al [56] showed tumor regression or stabilization in rat MTC after intratumoral injection of replicant-defective adenovirus mediated IL-2. IL-12 increases proliferation of natural killer cells and CD8+ T cells and activation of macrophages. Zhang et al [57] used an adenovirus carrying two subunits of the murine IL-12 gene and reported satisfactory antitumor activity after intratumoral injection in rat MTC with development of long-term antitumor immunity. DNA vaccination offers a new approach to induce immune-mediated tumor reduction in MTC. Haupt et al [58] showed that immunization against pre-procalcitonin by administration of plasmid DNA encoding pre-procalcitonin using the gene gun in mice is a potential treatment for MTC.

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Dendritic cells (DCs) also play an important role in the induction of primary immune responses. Since they are the most potent antigen presenting cells for T-cell activation, DCs are a promising option for immunization protocols. Bachleitner-Hofmann et al [59] have established an experimental tumor model for MTC to test the immunostimulatory capacity of DCs under autologous conditions. They showed that mature tumor lysate-pulsed DCs obtained from MTC patients can prime HLA class I-restricted antitumor T-cell responses against autologous tumor cells. 4. Suicide Gene Therapy + Immunotherapy: Since both suicide and immunomodulatory gene therapy alone provides promising results, combination of these approaches was suggested to further enhance the therapeutic efficacy. In a rat MTC model, Soler et al [60] showed a 52% reduction in tumor volume with HSV-tk GSV gene therapy, but when combined with IL-2, the reduction increased to 88%. Zhang et al [61] had developed an adenovirus expressing both HSV-tk and IL-2 and showed that antitumor effect in rat MTC after intratumoral injection is superior to that of each single vector.

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CONCLUSION MTC comprise a unique group of tumors with very well defined genetic characteristics which make this disease a laboratory for development of futuristic treatment options beside classical treatment procedures. Better understanding of clinical and genetic behavior of MTC cases will definitely result in longer survivals and better locoregional control rates. To achieve these goals we need much more multicentric studies based on larger patient populations and better coordination between geneticists, surgeons, radiologists, radiotherapists and medical oncologists.

REFERENCES [1] Hundahl SA, Fleming ID, Fremgen AM, et al. A National Cancer Data Base report on 53,856 cases of thyroid carcinoma treated in the U.S., 1985-1995 Cancer 1998 83 (12): 2638-48. [2] Bhattacharyya N. A population-based analysis of survival factors in differentiated and medullary thyroid carcinoma. Otolaryngol Head Neck Surg 2003128 (1): 115-23. [3] Snow KJ, Boyd AE 3d. Management of individual tumor syndromes. Medullary thyroid carcinoma and hyperparathyroidism. Endocrinol Metab Clin North Am. 1994;23:157-66. [4] McDermott MT. Calcitonin and its clinical applications. Endocrinologist. 1992;2:366-73. [5] Airaksinen MS, Saarma M. The GDNF family: signalling, biological functions and therapeutic value. Nat Rev Neurosci 2002,3 (5): 383-94. [6] Manie S, Santoro M, Fusco A, Billaud M.The RET receptor: function in development and dysfunction in congenital malformation. Trends Genet. 2001 Oct;17(10):580-9.

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[7] Eng C, Mulligan LM, Smith DP, et al. Low frequency of germline mutations in the RET proto-oncogene in patients with apparently sporadic medullary thyroid carcinoma. Clin Endocrinol (Oxf) 43 (1): 123-7, 1995. [8] Howe JR, Norton JA, Wells SA Jr.Prevalence of pheochromocytoma and hyperparathyroidism in multiple endocrine neoplasia type 2A: results of long-term follow-up. Surgery. 1993 Dec;114(6):1070-7. [9] Leboulleux S, Baudin E, Travagli JP, Schlumberger M.Medullary thyroid carcinoma Clin Endocrinol (Oxf). 2004 Sep;61(3):299-310. [10] Carlson KM, Dou S, Chi D, et al. Single missense mutation in the tyrosine kinase catalytic domain of the RET protooncogene is associated with multiple endocrine neoplasia type 2B. Proc Natl Acad Sci U S A 91 (4): 1579-83, 1994. [11] Eng C, Smith DP, Mulligan LM, et al. Point mutation within the tyrosine kinase domain of the RET proto-oncogene in multiple endocrine neoplasia type 2B and related sporadic tumours. Hum Mol Genet 3 (2): 237-41, 1994. [12] Hofstra RM, Landsvater RM, Ceccherini I, Stulp RP, Stelwagen T, Luo Y, Pasini B, Hoppener JW, van Amstel HK, Romeo G, et al.A mutation in the RET proto-oncogene associated with multiple endocrine neoplasia type 2B and sporadic medullary thyroid carcinoma. Nature. 1994 Jan 27;367(6461):375-6. [13] Chiefari E, Russo D, Giuffrida D, Zampa GA, Meringolo D, Arturi F, Chiodini I, Bianchi D, Attard M, Trischitta V, Bruno R, Giannasio P, Pontecorvi A, Filetti S.Analysis of RET proto-oncogene abnormalities in patients with MEN 2A, MEN 2B, familial or sporadic medullary thyroid carcinoma. J Endocrinol Invest. 1998 Jun;21(6):358-64. [14] Santoro M, Carlomagno F, Romano A, et al. Activation of RET as a dominant transforming gene by germline mutations of MEN2A and MEN2B. Science 267 (5196): 381-3, 1995. [15] Takahashi M, Asai N, Iwashita T, et al. Molecular mechanisms of development of multiple endocrine neoplasia 2 by RET mutations. J Intern Med 243 (6): 509-13, 1998. [16] Eng C, Mulligan LM, Healey CS, et al. Heterogeneous mutation of the RET protooncogene in subpopulations of medullary thyroid carcinoma. Cancer Res 56 (9): 216770, 1996. [17] Gimm O, Marsh DJ, Andrew SD, et al. Germline dinucleotide mutation in codon 883 of the RET proto-oncogene in multiple endocrine neoplasia type 2B without codon 918 mutation. J Clin Endocrinol Metab 82 (11): 3902-4, 1997. [18] Smith DP, Houghton C, Ponder BA Germline mutation of RET codon 883 in two cases of de novo MEN 2B. Oncogene 15 (10): 1213-7, 1997. [19] Lima J, Teixeira-Gomes J, Soares P, Maximo V, Honavar M, Williams D, SobrinhoSimoes M.Germline succinate dehydrogenase subunit D mutation segregating with familial non-RET C cell hyperplasia. J Clin Endocrinol Metab. 2003 Oct;88(10):4932-7. [20] Eng C, Clayton D, Schuffenecker I, Lenoir G, Cote G, Gagel RF, van Amstel HK, Lips CJ, Nishisho I, Takai SI, Marsh DJ, Robinson BG, Frank-Raue K, Raue F, Xue F, Noll WW, Romei C, Pacini F, Fink M, Niederle B, Zedenius J, Nordenskjold M, Komminoth P, Hendy GN, Mulligan LM, et al.The relationship between specific RET protooncogene mutations and disease phenotype in multiple endocrine neoplasia type 2. International RET mutation consortium analysis. JAMA. 1996 Nov 20;276(19):1575-9.

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[21] Yip L, Cote GJ, Shapiro SE, Ayers GD, Herzog CE, Sellin RV, Sherman SI, Gagel RF, Lee JE, Evans DB.Multiple endocrine neoplasia type 2: evaluation of the genotypephenotype relationship. Arch Surg. 2003 Apr;138(4):409-16; discussion 416. [22] Mulligan LM, Eng C, Healey CS, et al. Specific mutations of the RET proto-oncogene are related to disease phenotype in MEN 2A and FMTC. Nat Genet 6 (1): 70-4, 1994.. [23] Eng C, Smith DP, Mulligan LM, et al. Point mutation within the tyrosine kinase domain of the RET proto-oncogene in multiple endocrine neoplasia type 2B and related sporadic tumours. Hum Mol Genet 3 (2): 237-41, 1994. [24] Andreas Machens, Oliver Gimm, Raoul Hinze, Wolfgang Höppner, Bernhard O. Boehm and Henning Dralle The Journal of Clinical Endocrinology & Metabolism Vol. 86, No. 3 1104-1109 Genotype-Phenotype Correlations in Hereditary Medullary Thyroid Carcinoma: Oncological Features and Biochemical Properties. [25] Romei C, Elisei R, Pinchera A, Ceccherini I, Molinaro E, Mancusi F, Martino E, Romeo G, Pacini F. J Clin Endocrinol Metab. 1996 Apr;81(4):1619-22. [26] Clayman GL, el-Baradie TS. Medullary thyroid cancer. Otolaryngol Clin North Am. 2003 Feb;36(1):91-105. [27] Claudia Scollo, Eric Baudin, Jean-Paul Travagli, Bernard Caillou, Nicolas Bellon, Sophie Leboulleux and Martin Schlumberger Rationale for Central and Bilateral Lymph Node Dissection in Sporadic and Hereditary Medullary Thyroid Cancer. The Journal of Clinical Endocrinology & Metabolism 2003 Vol. 88, No. 5 2070-2075. [28] Sipple JH. The association of pheochromocytoma with carcinoma of the thyroid gland. Am J Med. 1961;31:163-6. [29] Cohen MS, Phay JE, Albinson C, DeBenedetti MK, Skinner MA, Lairmore TC, Doherty GM, Balfe DM, Wells SA Jr, Moley JF. Gastrointestinal manifestations of multiple endocrine neoplasia type 2. Ann Surg. 2002 May; 235(5):648-54. [30] Verga U, Fugazzola L, Cambiaghi S, Pritelli C, Alessi E, Cortelazzi D, Gangi E, BeckPeccoz P. Frequent association between MEN 2A and cutaneous lichen amyloidosis. Clin Endocrinol (Oxf). 2003 Aug;59(2):156-61. [31] Chong GC, Beahrs OH, Sizemore GW, Woolner LH. Medullary carcinoma of the thyroid gland. Cancer. 1975;35:695-704. [32] Norton JA, Froome LC, Farrell RE, Wells SA Jr Multiple endocrine neoplasia type IIb: the most aggressive form of medullary thyroid carcinoma. Surg Clin North Am. 1979 Feb;59(1):109-18. [33] Thomas PM, Gagel RF. Advances in genetic screening for multiple endocrine neoplasia type 2 and the implications for management of children at risk. Endocrinologist. 1994;4:140-6. [34] Wells SA Jr, Donis-Keller H. Current perspectives on the diagnosis and management of patients with multiple endocrine neoplasia type 2 syndromes. Endocrinol Metab Clin North Am. 1994;23:215-28. [35] Cohen MS, Moley JF. Surgical treatment of medullary thyroid carcinoma.J Intern Med. 2003 Jun;253(6):616-26. [36] Kebebew E, Ituarte PH, Siperstein AE, Duh QY, Clark OH.Medullary thyroid carcinoma: clinical characteristics, treatment, prognostic factors, and a comparison of staging systems. Cancer. 2000 Mar 1;88(5):1139-48.

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[37] Leboulleux S, Baudin E, Travagli JP, Schlumberger M. Medullary thyroid carcinoma Clin Endocrinol (Oxf). 2004 Sep;61(3):299-310. [38] Roche A, Girish BV, de Baere T, Ducreux M, Elias D, Laplanche A, Boige V, Schlumberger M, Ruffle P, Baudin E. Prognostic factors for chemoembolization in liver metastasis from endocrine tumors. Hepatogastroenterology. 2004 Nov-Dec;51(60):17516. [39] Isozaki T, Kiba T, Numata K, Saito S, Shimamura T, Kitamura T, Morita K, Tanaka K, Sekihara H. Medullary thyroid carcinoma with multiple hepatic metastases: treatment with transcatheter arterial embolization and percutaneous ethanol injection. Intern Med. 1999 Jan;38(1):17-21. [40] Fersht N, Vini L, A'Hern R, Harmer C. The role of radiotherapy in the management of elevated calcitonin after surgery for medullary thyroid cancer. Thyroid. 2001 Dec;11(12):1161-8. [41] Vitale G; Caraglia M; Ciccarelli A; Lupoli G; Abbruzzese A; Tagliaferri P; Lupoli G TI Current approaches and perspectives in the therapy of medullary thyroid carcinoma. Cancer 2001 May 1;91(9):1797-808. [42] Orlandi F, Caraci P, Mussa A, Saggiorato E, Pancani G, Angeli A. Treatment of medullary thyroid carcinoma: an update. Endocr Relat Cancer. 2001 Jun;8(2):135-47. [43] Nocera M, Baudin E, Pellegriti G, Cailleux AF, Mechelany-Corone C, Schlumberger M.Treatment of advanced medullary thyroid cancer with an alternating combination of doxorubicin-streptozocin and 5 FU-dacarbazine. Br J Cancer. 2000 Sep;83(6):715-8. [44] Wu LT, Averbuch SD, Ball DW, de Bustros A, Baylin SB, McGuire WP 3rd.Treatment of advanced medullary thyroid carcinoma with a combination of cyclophosphamide, vincristine, and dacarbazine. Cancer. 1994 Jan 15;73(2):432-6. [45] Diez JJ, Iglesia P. Somatostatin analogs in the treatment of medullary thyroid carcinoma. J Endocrinol Invest. 2002 25. 773-778. [46] Zatelli MC, degli Uberti EC. Somatostatin receptors: from basic science to clinical approach--thyroid. Dig Liver Dis. 2004 Feb;36 Suppl 1:S86-92. [47] Modigliani E, Cohen R, Joannidis S, Siame-Mourot C, Guliana JM, Charpentier G, Cassuto D, Bentata Pessayre M, Tabarin A, Roger P, et al. Results of long-term continuous subcutaneous octreotide administration in 14 patients with medullary thyroid carcinoma. Clin Endocrinol (Oxf). 1992 Feb;36(2):183-6. [48] Lupoli G, Cascone E, Arlotta F, Vitale G, Celentano L, Salvatore M, Lombardi G. Treatment of advanced medullary thyroid carcinoma with a combination of recombinant interferon alpha-2b and octreotide. Cancer. 1996 Sep 1;78(5):1114-8. [49] Kaltsas G, Rockall A, Papadogias D, Reznek R, Grossman AB.Recent advances in radiological and radionuclide imaging and therapy of neuroendocrine tumours. Eur J Endocrinol. 2004 Jul;151(1):15-27. [50] Spitzweg C Morris JC. Gene therapy for thyroid cancer: current status and future prospects. Thyroid. 2004 Jun;14(6):424-34. [51] Drosten M, Pützer BM. Gene therapeutic approaches for medullary thyroid carcinoma treatment. J Mol Med, 2003; 81:411-419.

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[52] Zijuan L, Falola J, Xudong Z, Ying G, Lawrence TK, Gearge SA, Thomas A, Fiemu EN. Antiproliferative effects of Src inhibition on medullary thyroid cancer. J Clin Encocrinol Metab, 2004; 89(7): 3503-3509. [53] Zeiger MA, Takiyama Y, Bishop JO, Ellison AR, Saji M, Levine MA. Adenoviral infection of thyroid cells: a rationale for gene therapy for metastatic thyroid carcinoma. Surgery 1996; 120(6):921-925. [54] Minemura K, Takeda T, Minemura K, Nagasawa T, Zhang R, Leopardi R, Degroot LJ. Cell-specific induction of sensitivity to ganciclovir in medullary thyroid carcinoma cells by adenovirus-mediated gene transfer of herpes simplex virus thymidine kinase. Endocrinology 2000; 141(5): 1814-1822. [55] Messina M, Yu DM, Both GW, Molloy PL, Robinson BG. Calcitonin-specific transcription and splicing targets gene-directed enzyme prodrug therapy to medullary thyroid carcinoma cells. J Clin Encocrinol Metab, 2003; 88(3): 1310-1318. [56] Zhang R, Stratus FH, Degroot LJ. Effective genetic therapy of established medullary thyroid carcinomas with murine interleukin-2: Dissemination and cytotoxicity studies in a rat model. Endocrinology, 1999; 140(5): 2152-2158. [57] Zhang R, Degroot LJ. Gene therapy of a rat follicular thyroid carcinoma model with adenoviral vectors transducing murine interleukin-12. Endocrinology, 2003; 144(4): 1393-1398. [58] Haupt K, Siegel F, Lu M, Yang D, Hilken G, Mann K, Roggendorf M, Saller B. Induction of a cellular and humoral immune response against preprocalcitonin by genetic immunization: a potential new treatment for medullary thyroid carcinoma. Endocrinology, 2000; 142(3): 1017-1023. [59] Hofmann-Bachleitner T, Stift A, Friedl J, Pfragner R, Radelbauer K, Dubsky P, Schüller G, Benkö T, Niederle B, Brostjan C, Jakesz R, Gnant M. Stimulation of autologous antitumour T-cell responses against medullary thyroid carcinoma using tumour lysatepulsed dendritic cells. J Clin Encocrinol Metab, 2002; 87(3): 1098-1104. [60] Soler MN, Milhaud G, Lekmine F, Treilhou-Lahille F, Klatzmann D, Lausson S. Treatment of medullary hyroid carcinoma by combined expression of suicide and interleukin-2 genes. Cancer Immunol Immunother, 1999; 48: 91-99. [61] Zhang R, Degroot LJ. An adenoviral vector expressing functional heterogeneous proteins herpes simplex viral thymidine kinase and human interleukin-2 has enhanced in vivo antitumour activity against medullary thyroid carcinoma. Endocrine-related cancer, 2001; 8: 315-325.

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In: Focus on Thyroid Cancer Research Editor: Carl A. Milton

ISBN: 1-59454-626-6 © 2009 Nova Science Publishers, Inc.

Chapter 8

MEDULLARY THYROID CARCINOMA: DIAGNOSTICS, TREATMENT AND PROGNOSIS Vitaliy Zh Brzhezovskiy1,∗, Vyacheslav L Lyubaev1, Tatiana T Kondratyeva1, Elena A Smirnova1, Faina A Amosenko1, 2 and Raisa F Garkavtseva1 1

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Russian NN Blokhin Cancer Research Centre, Russian Academy of Medical Sciences, Moscow, Russia. 2 Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow, Russia.

ABSTRACT 182 patients with medullary thyroid carcinoma (MTC) were under clinical observation for this research. The pre-operative diagnostic procedures employed were ultrasound examination, fine-needle aspiration biopsy (FNAB), radioisotope examination of thyroid, calcitonin and carcinoembryonic antigen levels in the blood serum, computer tomographic scan and magneto-resonance tomography. We have done much work on investigation clinical and morphologic features of MTC with special interest to its diagnostics. The chapter provides discussion on the objective possibilities of cytologic method (FNAB) while making exact diagnosis of MTC and its types, with the use of our own cytograms. A number of differentially-diagnostic problems arising in practice were defined with the aim to decrease the probable errors in the distinguishing this unusually multiform thyroid cancer. Different types of MTC were studied during histologic examination of the tumors. While investigating the influence of MTC histologic type on patients` survival, we failed to determine significant correlation between these parameters and prognosis. Electron-microscopic investigation of MTC demonstrated that the ratio of differentiated and un-differentiated cells in tumor is the most informative ∗

Correspondence concerning this article should be addressed to Dr. Vitaliy Zh Brzhezovskiy, Russian NN Blokhin Cancer Research Centre, Russian Academy of Medical Sciences, Moscow, 115478 Russia.

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Vitaliy Zh Brzhezovskiy, Vyacheslav L Lyubaev, Tatiana T Kondratyeva et al. prognostic symptom. For molecular diagnostics of MTC hereditary forms, we performed screening of the mutations in proto-oncogene RET (in blood leukocytes) for individuals from families with MEN 2 cancer syndromes. The frequency of the activating germline mutations was 100%. In the group of patients with sporadic MTC, somatic mutations were revealed in 34.6% of tumors. Surgery is very important in the current treatment of MTC. Surgical treatment on the primary tumor lesion depends mainly on MTC form: sporadic or familial. Thyroidectomy is prescribed in hereditary form of the disease irrespective of tumor size. Preventive thyroidectomy performed for 5 asymptomatic carriers of the RET mutations (c.634) from 3 families with MEN 2A, proved to be effective in prophylaxis and/or treatment of MTC. In the case of sporadic MTC if tumor size is restricted (T1-T2), it`s possible to use organ sparing surgery. Radiation therapy has the following confined indications: 1) in doubtful radical nature of operation; 2) in inoperable forms; 3) in distant metastases with palliative and symptomatic treatment as aim. Chemotherapy in MTC is ineffective.

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INTRODUCTION Medullary thyroid carcinoma (MTC) is a rare, neuroendocrine tumor accounting 5-10% of neoplasms of the organ. It arises from parafollicular or C-cells and may be multihormonal, producing calcitonin (CT), catecholamines, serotonin, prostaglandin and others bioactive substances in increased levels. This type of thyroid cancer is characterized by high frequency of regional metastatic spreading (40-55%). MTC may develop sporadically (70-80% of cases) or as a part of multiple endocrine neoplasia type 2 (MEN 2). Histologic picture of MTC is not homogenous especially regarding mixed types due its multicomponent. Currently, there is no available data in literature concerning ultrastructural criteria of C-cellular thyroid cancer, which are of great diagnostic and prognostic value. The problems associated with the interrelation between ultrastructural differentiation of MTC cells and its ability to relapsing and metastasizing studied insufficiently. The correlation between these peculiarities and clinical manifestations of the disease were not investigated. There is no sufficient evidence in literature about diagnostics of this tumor. The investigation of molecular markers of high diagnostic informativety in inherited forms of MTC is very important for confirmation of clinical diagnisis, identification of at-risk relatives, and also to select the proper method of treatment, preventive thyroidectomy being one of its forms. In order to perform a preventive thyroidectomy successfully it`s very important to choose the optimal time for the operation. However, present opinions on this problem are rather contradictory. There is a lack of information on the role of somatic RET mutations in sporadic MTC. The main method of MTC treatment is considered to be surgery. However, so far there has been no convincing evidence about the role, position and value of radiation therapy in the management of this disease. The problem regarding the volume of surgery on the primary tumor lesion remains in question.

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DIAGNOSTICS OF MTC According to the modern condition of oncology tumor diagnostics must be as early, as possible. Clinical signs don`t contribute to differentiate significantly benign and malignant thyroid cancer. On the other hand, the symptoms such as dysphonia, disphagia and neck pain were studied to occur later on as a rule, and testify to advanced cancer. For complex investigation of our patients on the preoperative stage, we used the following methods: ultrasound diagnostics, fine-needle aspiration biopsy (FNAB), radioisotopic investigation of thyroid gland, computer tomography, and magneto resonance imaging (MRI), investigation of calcitonin (CT) and carcinoembryonic antigen (CEA) levels in blood serum. Some patients had molecular-genetic analysis of specific mutations of the proto-oncogene RET, which are highly informative markers in hereditary MTC. While conducting this complex investigation for more than 1500 Russian patients with thyroid cancer treated at Russian NN Blokhin Cancer Research Centre, papillary cancer was diagnosed in 74%, – follicular in 18% – and medullary in 7%.

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Radioisotopic Investigation This investigation is used for the imaging functional tissue of thyroid gland or its nodal growths. The principle of this technique is based on the fact that I-iodine and Tc–pertechnitat are organospecific radiopharm preparations for cells of thyroid gland, e.g. infiltrate the physiologic phases of iodine exchange, repeating neoorganic phase of its exchange. This fact enabled us to state that in malignant tumors with the loss of differentiation the ability to take up organospecific radiopharm preparation decreases compared with the surrounding normal tissue. Therefore, the tumor takes on the appearance of “cold” defects of accumulation on the radionuclide imaging of thyroid gland.

a)

b)

Fig. 1. Scintigram of the MTC patient after non-radical surgery. The lesion of left lobe with expanding behind breast bone is visualize. а) scintigram with the use of 99тТс-pertechnetat. b) scintigram with the use of 99mТс-technetril. Milton, Carl A.. Focus on Thyroid Cancer Research, Nova Science Publishers, Incorporated, 2006. ProQuest Ebook Central,

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152 MTC primary patients were included into radioisotopic investigation of thyroid cancer with the use of iodine and technecium. Defect of this radiofarm preparation accumulation in thyroid cancer was revealed in 136 (89.5%) patients. In the rest of the cases the focal lesion of thyroid cancer was not detected and it may be accounted for by the insignificant tumor size. 16 patients with MTC received 99mTc-technetril as radiofarm preparation. This isotope diffuses into cellular cytoplasm passively due to negative transmembrane potentially resulting in 99mTc-technetril insertion into tumor tissue. The accumulation of this preparation, in view of “hot” and “warm” foci, was revealed in 11 (68.8%) cases (Fig. 1) that testify to relatively high specificity of technetril to tumor cells of MTC. Moreover, this isotope can detect metastatic involvement of lymphatic nodes. We noted an accumulation of radiopharm preparation in regional lymphatic nodes in 7 of 16 investigated patients with MTC. Their lymphatic involvement was supported by histologic investigation. (Fig. 2).

Fig. 2. Accumulation of 99m Tc-technetril in metastatically involved lymphatic nodes of neck in MTC.

Ultrasound Investigation Current methods of ultrasound tomography of the thyroid gland have practically 100% sensitivity in revealing focal lesions. We estimated the following parameters by means of ultrasound investigation in our practice: а) gland on the whole: 1) disposition, 2) size, 3) contour, 4) shape, 5) inner structure; b) intraorgan changes: 1) type of change (diffuse or focal), 2) disposition, 3) the number of growths, 4) focal contours, 5) size, 6) inner structure; c) interrelation of the thyroid gland with surrounding structures; d) state of regional sites of metastasizing. We have analyzed the findings of ultrasound investigations 169 both primary and repeated patients suffering from MTC. 14 out of 15 (93.3%) patients with unpalpable tumor and metastases in regional lymph nodes of the neck were found to have a tumor of the thyroid

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gland (so called occult thyroid cancer). FNAB of these nodes was done in the thyroid gland, and a malignant tumor was confirmed cytologically in 78% of cases. An ultrasound picture of MTC doesn`t provide clear specific signs. As a rule, this tumor presents hypoechogenic growth. Nodes of increased echogenicity occurred rarely (in 5% cases). Tumor contours were uneven, tuberous; it was mainly surrounded by hypoechogenic rim of irregular thickness. Microcalcinates were observed in 83% of cases. A similar ultrasound picture is typical for metastases into regional lymph nodes. However, microcalcination, a decreased echogenecity of the tumor node and irregular hypoechogenic rim may be observed in other morphologic types of thyroid cancer, particularly papillary cancer. Cystic cavities fail to occur practically in MTC compared with papillary cancer. It was noted that frequent metastasizing into regional lymph nodes of the neck was characteristic of MTC. This process is asymptomatic in a number of cases. It is very difficult to find the paratracheal area at the physical examination of a patient. Analyzing ultrasound findings of this area and comparing them with postoperative histologic investigation, metastases appeared to be confirmed histologically by ultrasound in 91% of cases. Ultrasound findings on the assessment of tumor infiltration of muscles and magistral vessels of the neck are of great value for the detection of tumor expansion. 7 cases were suspected to have tumor extension to the straight muscle of the neck. It was confirmed histologically in 5 (71%) patients. Tumor extension to the wall and lumen of the inner jugular vein was revealed by ultrasound investigation in 9 cases, and confirmed morphologically in all patients. Ultrasound investigation is very important in postoperative monitoring of patients.

Fig. 3. Computer tomography of the patient with MTC. Tumor involvement of the left lobe and isthmus of the thyroid gland. Trachea is displaced, left wall is pressed and has irregular inner contour, testifying to tumor invasion into tracheal wall.

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Computer Tomography Scanning and MRI

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In the last decades computer tomography became a routine method of diagnostics in thyroid cancer. This investigation proved to define more precisely the localization, size and interrelation between the primary tumor and metastases with surrounding organs and tissues. Computer tomography depicts a state of tracheal walls, its displacement by tumor and decrease of tracheal lumen through tumor invasion into its cartilage (Fig. 3). Computer tomography promotes the detection of tumor expansion into mediastinum and to determine its interrelation with its structures. MRI demonstrates the state of soft tissue structures in neck and mediastinum (Fig. 4). This investigation supplements computer tomography. These techniques combined, produce evidence concerning MTC localization, size and its regional metastases.

Fig. 4. MRI (frontal view) of the patient with MTC. 1,3,4 – increased metastatic nodes of neck. 2 – tumor of the right lobe of the thyroid gland.

Biochemical markers of MTC Radioimmunologic study of the content of calcitonin (CT) and carcinoembryonic antigen (CEA) in blood serum is a very important method in the diagnostics of MTC. Increased level of CT is a very specific marker in primary diagnostics of MTC. Before treatment began, CT level was determined in 164 patients with MTC. It appeared to be high in 159 patients (97%). All patients underwent radical surgery. Hemithyroidectomy, subtotal resection of thyroid gland or thyroidectomy were performed on the primary tumor lesion depending on tumor size. Neck dissection was done at the presence of regional metastases. To find out prognostic outcome for 5 years (after the operation) the CT level was observed in 56 primary patients.

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CT concentration defined in these patients preoperatively appeared to be dependent upon tumor stage. Thus, in patients with the first stage of the disease (3 patients) CT level accounted for 49.4 ± 8.3 ng/l, with the second (12 patients) – 84 ± 16.3 ng/l, and with the third stage (41 patients) – 1124 ± 209.8 ng/l. In diagnosing remote metastases of MTC, CT concentration proved to be much more increased: on average up to 4233 ng/l. CEA concentration without exceeding the standard was revealed in 10 patients (17.9%), in rest 46 patients this index was much more increased and was 82.3 ng/l on average. The results of the monitoring performed demonstrates as follows: 1. In patients having no relapse of the disease during follow-up, the CEA levels became standard for only a short period. Three years later, after the operation, these patients were found to have a gradual increase of marker level. 2. MTC patients without regional metastases at the time of detecting primary tumor have a good chance for the normalization of CT and CEA levels compared with those, who had regional metastases (table 1). 3. Higher levels of CT and CEA were observed in patients presenting distant (remote) metastases. These levels were exceeded by some times the values obtained in the involvement of regional lymph nodes in the neck or mediastinum. Table 1. The content of CT and CEA in patients with MTC. Index

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CT (ng/l) CEA (ng/ml)

Patient`s group Metastases in lymph nodes + 1751 ± 594,0 92,2 ± 38,0

Significance Metastases in lymph nodes – 67,2 ± 46 6,5 ± 3,4

Р < 0,05 Р < ,001

Tumor relapse occurred in 16 cases and different kinds of antitumor palliative treatment were performed. 6 patients were diagnosed to have relapse in the area of the neck, 3 – mediastinum, 5 patients had metastases in bones of the skeleton, 2 – in lungs. 14 patients received palliative radiotherapy and drug treatment was prescribed to 4 patients. The mean value of CT level in this group was 375 ng/l. After therapy was performed the palliative effect was noted in 4 cases. At the same time, the mean value of hormone in blood serum also decreased up to 1757 ng/l. While monitoring CEA in these patients the marker was not discovered to have a decrease of indices. Thus, monitoring CT and CEA levels in blood serum after radical MTC treatment contribute to judge the disease prognosis decrease in CT level up to standard values after surgery testifies to relatively favourable postoperative prognosis. Increased level of CT is a more unfavourable prognostic symptom. At the same time its increased value in postoperative period doesn`t always show neoplasm recurrence clinically manifested. However, insignificant MTC follow-up period (5 years) fails to confirm it confidently. Moreover, a decrease in CT level during the course of palliative treatment correlates with the degree of clinical effect from the therapy received. A comparative analysis of CT content before and after treatment is another additional test of the radical therapy administered, and in a number of cases a marker of the disease recurrence.

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Cytologic Diagnostics of MTC According to a 30-year experience of employing fine-needle aspiration biopsy (FNAB) it was established that its effectiveness mainly depends on both material taking, which was performed methodically correctly, and cytologist`s skill. Regarding sporadic MTC cytologic study of tumor punctuate, increased lymphatic nodes in the neck or other pathologic foci in occult tumor process (soft tissues, bones, lungs), it is the only morphologic method in preoperative diagnostics supporting this clinical diagnosis. Therefore, it`s very important to know objective possibilities of cytologic method in detecting this unusually variable type of thyroid cancer. In this study we analyzed tumor punctuate and its metastases in 114 patients with MTC treated and followed up at Russian NN Blokhin Oncology Research Centre RAMS for 40 years and also impressio of tumor removed during the operation (23 cases). Thus, the diagnosis of MTC in all cases was confirmed by histologic study of the operative material. Retrospective analysis on the effectiveness of cytologic study on initial stages of our investigation (1970-1980 years) revealed as follows: exact cytologic diagnosis of MTC was made in 37 cases (32.5%), cancer without noting type – in 23 cases (20.2%), malignant tumor indicating to the differentiation line which includes chemodectoma, carcinoid, paraglioma, sarcoma, melanoma along with MTC – in 48 patients (42.1%). So, at that time cytologic diagnostics of MTC presented some difficulties for a cytologist and sometimes it was impossible. It`s interesting to note that achieved ”historical” findings of specific cytologic diagnosis accuracy demonstrate clearly an uncommonness of morphologic characteristics of MTC: the epithelial nature of tumor cells was evident in only half of our cases, whereas in the other half the peculiarities of tumor cellular composition produce difficulties in specifying the histogenesis of neoplasms. Nowadays a detailed retrospective review of the same material we have performed recently –114 patients– considering our modern experience and knowledge about morphology of neuroendocrinal tumors and tumors of thyroid glands – enabled us to reveal peculiar cytomorphologic characteristics of MTC composition and to enchance the effective indices of preoperative cytologic diagnostics of this type of thyroid cancer up to 86% (98 cases). There was an error in 5 cases during cytologic diagnosis, it accounts for 4.4%, diagnosis of benign process being made only in one case by mistake. There were problems in the differentiation of MTC from follicular and papillary cancer (Fig. 5). They usually occur with the lack of amyloid in the material, with the presence of structures resembling follicles and papillae along with solid areas, and also with a small number or lack of cells with clear cytomorphologic signs of apudoma, e.g. signs showing Ccellular nature of tumor cells studied. If relatively similar cells of slightly extended form predominate in punctuate, if there are no 2-3 nuclear cells and other signs that indicate to their belonging to APUD-system, one can not make a conclusion about histological type of cancer. It is necessary to search for cells with signs of neuroendocrinal differentiation. However, a cytologist faces other diagnostic problems caused by cytomorphological characteristics compeling to suspect neuroendocrinal nature of tumor, as the term “apudom” means a number of tumors, only one of them being MTC.

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Fig. 5. Cytogram of MTC. Due to follicular disposition of tumor cells it may be difficult in making exact diagnosis according to punctuate. x400

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The main cytologic signs of MTC are variability of the process, variety of cellular types and amyloid presence. Cytologically, MTC can be presented by solid localization of tumor cells (Fig. 6) and also by presence of follicular, microfollicular, pseudopapillary and trabecular structures. The number of parenchyma and stroma in tumor varies, during the cytologic study of the material the sites of hyalinizated fibrous tissue may be revealed. All these facts combined with extended fusiform cells of MTC may produce a mistake in diagnostics of sarcoma (Fig. 7).

Fig. 6. Cytogram of MTC with solid disposition of cells and amyloid presence. x400

Detailed cytomorphologic analysis of our observations demonstrated that the diagnosis of MTC was based on the detection of one of 5 types of cells or their combinations: 1) small spherical cells often hardly distinguishing from cells of follicular epithelium; 2) extended fusiform cells; 3) bigger cells of spherically-oval and polygonal form with abundant

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cytoplasm and presence of visible granules in it; 4) giant cells with big quaint nucleus and visible micro- or macro-nucleols; 5) small undifferentiated cells. Chromatin structure may be microgranular or crumpled, rather often nuclei look like hyperchromal, nuclei with signs of nuclear membrane vagination occur. The presence of 2-3 nuclear and multinuclear cells, and also morbid mitosis is characteristic. Amyloid presented in view of lilac-violet (in staining by Leishman) amorphic mass, located intercellularly (Fig. 6). However amyloid was detected in only 24 % of our cases (27 patients). “Intranuclear vacuoles” that are typical for papillary thyroid cancer were revealed in cytogram of MTC.

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Fig. 7. Cytogram of MTC largely from fusiform cells. x400

The ratio and character of disposition of all enumerated cellular types may be different and they account for a variety of MTC pictures, and it`s necessary to differentiate it from paraganglioma, carcinoid, melanoma and other malignancies.

Fig. 8. MTC cells of high grade differentiation proved by multiple azurophil granules in cytoplasm. x1000

Having investigated the causes of errors in cytologic diagnostics, we can admit that most of them were made by incorrect interpretation of cytologic findings and, particularly, by insufficient attention to the morphologic peculiarities of the cells studied, and also by formal Milton, Carl A.. Focus on Thyroid Cancer Research, Nova Science Publishers, Incorporated, 2006. ProQuest Ebook Central,

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resemblance on the light microscopy level of colloid or stromal component of tumor (for instance, in nephrocellular cancer) with amyloid. Most authors note that dispersion disposition of cells predominate in MTC cytograms. However, one should emphasize that both in our investigations and due to publication data, about one-fourth of cases are presented by cytograms in which cells are situated in the form of groups, accumulations and different kinds of structures (follicular, microfollicular, trabecular and resembling papillary). The presence of Azurophil granules in the cytoplasm of tumor cells is a very important diagnostic sign (Fig. 8), that it is emphasized by most investigators. Granulated cytoplasm was detected most commonly (85%) in cells of the 2nd, 3rd types and more rarely in extended giant cells. Moreover, as it was shown by our study, the prevalence of undifferentiated cells (10%) and cells with sharp nuclear polymorphism occurred in cytograms of patients having unfavourable outcome. However, possible correlation of these cytologic peculiarities of MTC and the specificity of tumor cells with the differentiation grade of tumor and with the outcome of the disease remains insufficiently investigated.

Fig. 9. Cytogram of papillary cancer, columnar type. x 800

Fig. 10. Gurthle cytogram of cellular cancer. x630

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(Fig. 9), cancer from Ashkinazi cells (Fig. 10), insular cancer (Fig. 11), follicular, papillary, oncocytic types of cancer from C-cells, and also types of cancer with a low grade of cellular differentiation and slightly marked signs of organ-tissue specificity. Examples are rare types of cancer with clear cellular changes, planocellular metaplasia, with symptoms of muciparous process and anaplastic cancer (Fig. 12). One should note all above that morphologic types of cancer within the structure of thyroid cancer account for a relatively low percent (no more than 5%), but in a particular patient their uncommon occurence makes it necessary to perform an exact morphologic diagnosis (including cytologic diagnosis), determining the adequate choice of management.

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Fig. 11. Cytogram of insular type of follicular cancer. Tumor cells of low grade differentiation. x400

Fig. 12. Cytogram of undifferentiated thyroid cancer. x400

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Table 2. Indices of investigation results obtained in patients suffering from MTC. Type of investigation

Radio-nuclide diagnostics

US

Localization, size Accurence of regional metastases

+ + (only with 99mТсtechnytril) -

Tumor growing into trachea Metastases growing into magistral neck vessels volume

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Specification of morphological diagnosis

МRI

FNAB

Level of calcitonin in blood

+ +

Computer tomography + +

+ +

+

-

+

+

+

-

-

-

+

+

+

-

-

-

+

+

+

-

-

-

-

-

-

+

+

Fig. 13. Solid variant of MTC. Staining with haemotoxilin-eosin. x200

MTC Histology By histological investigation of MTC in most observations, the tumor is presented by solid sites divided with layers of connective tissues (Fig. 13). There are also tumors in which domination of sclerous stroma is marked (Fig. 14) – the tumors had thick consistency – which were presented by wide areas of hyalinous fibrinous tissue with slightly distinguished fibers and different sizes of amyloid blocks. The gigantic cells of foreign bodies are determined around the amyloid. There are also small foci of calcification. The separate tumor cells with

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distrofic changes are seen in hyalinized stroma. In tumors having solid structures (these tumors are of soft consistency) monomorphine sight of cellular composition is marked. Some tumors are presented by cells of fusiform (Fig. 19). The other tumors consist of the cells of rounded or polygonal forms (Fig.15). Some tumor cells have follicular structures (Fig. 16). Sometimes their perivascular arrangement is marked. Nuclei of tumor cells are round or elangated, light with rough chromatin blocks. Cytoplasm of these cells is eosinophilic with pronounced granulation (Fig. 18). A morphological picture corresponds to the cells differentiated according to oncocyte type (Fig. 17). Sometimes there are cells in the state of mitosis. Nuclei in these cells are elongated, polymorphic. Tumor cells often form plexiform fascicles. In some places invasion of thyroid capsule, necrosis and foci of calcification are observed.

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Fig. 14. MTC with amyloidosis of stroma. Staining with picrofuxin by Van Gizon. х 200

Fig. 15. MTC from the cells of rounded and polygonal form with well-marked amyloidosis of the stroma. Staining with hematoxylin-eosin. х 200

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Fig. 16. MTC sites of follicular structure. Staining with picrofuxin by Van-Gizon. х 450

Fig. 17. Oncocytic variant of MTC. Staining with hematoxylin-eosin. x450

In special staining (kongo-rot) the absence of amyloid content in tumor stroma was noted in 39 (21%) observations. Calcification places were revealed in 15 (8.2%) observations. In 121 (66.7%) cases the tumors consisted of polygonal cells mainly, and 25 (19.3%) predominantly consisted of fusiform cells. Polymorphic-cellular structure was noted in 25 (14%) observations. In electronic-microscopic investigation of 28 MTCs it was noted that, irrespective of histological types, two groups of tumor cells were revealed in every tumor: differentiated and non-differentiated ones.

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Fig. 18. MTC metastasis in the lymphatic node. Staining with hematoxylin-eosin. х 200

Fig. 19. Fusiform variant of MTC. Staining with hematoxylin-eosin. x 450

The first group consisted of differentiated cells in cytoplasm of which endocrine granules were found. The second group consisted of non-differentiated tumor cells without any specific ultra-structural signs. The cells of the first group were of round, oval or polygonal forms. The nuclei of most tumor cells also had a round or oval form with even or slightly festooned contours and non-deep invagination. Chromatin in the form of not so big blocks spreads over the whole karyoplasms. In some cells chromatin is condensed near the internal side of nuclear membrane. In some cells compact nucleoli are found. There are also tumor cells of elongated form with elongated nucleus and diffusive or condensated chromatin. In cytoplasm of tumor cells in this group the different quantity of specific endocrine secretory granules are defined (Fig. 20-25), cisternal of rough endoplasmic reticulum are moderately developed. Mitochondria, ribosomes, polysomes and laminar complex are found. Along with the organelles in cell cytoplasm, the lipid inclusions may be sometimes seen. In some cases,

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thin fibrillae of amyloid of thick felt-kind are also seen in the cytoplasm of tumor cells. Plasmatic membrane is smooth, and sometimes wavy. Tumor cells are mainly adjoined to each other and situated compactly. The borders between them are sometimes not clear. The cells have specialized contacts as desmosomes of different sizes (Fig. 29). The thick felt-kind accumulations of thin fibrillae of amyloid may often be seen in intercellular space.

Fig. 20. Typical variant of the MTC. Tumor cells are of rounded form with unclear cellular borders. Nuclei are with festooned nuclear membrane and condensed chromatin. In the cytoplasm, rich with organelle, mitochondria, vesicular structures, ribosomes, polysomes, lizosomes and in the moderate quantity the small, dark homogeneous endocrine granules are defined. x 12 000

Side by side with light tumor cells the darker cells with electronic-thick matrix are also found (Fig. 27, 28). The nuclear membrane of the cells were mainly tortuous with formation of the deep invaginations. In the nuclei condensated chromatin is determined. Cytoplasma of these cells, in comparison with the light cells is narrow, the organoids in it are situated compactly. There are also endocrine granules in them. Sometimes there were cells taking an intermediate situation between dark and light ones. The nuclei of these cells were similar with the nuclei of the light cells. Cytoplasma was an abundant one, organoids in it were situated more compactly in comparison with the light cells, but less compactly in comparison with the dark cells. The quantity of organoids in the cytoplasm of tumor cells shakes considerably from case to case in one observation. But sometimes there are cells in the state of apoptosis.

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Fig. 21. Solid site of MTC consists of elangated fusiform cells with moderate quantity of organelle. x 12,000

Fig. 22. Tumor cells of MTC being in close contact with blood vessel in the lumen of which erythrocytes are seen. х 12,000

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Fig. 23. Tumor cells with different degrees of ultra-structural differentiation. Cellular boundaries are seen between cells. In cytoplasm of a lowly differentiated cell there is inconsiderable quantity of usual organelle. In a highly differentiated tumor cell is a great number of endocrine granules. х 5,000

Fig. 24. This is the same as Fig. 26 with greater magnification. х 12,000

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Fig. 25. Tumor cell of low degree of ultra-structural differentiation. Round nucleus with a narrow rim of cytoplasm contains solitary organelle. х 6,000.

Fig. 26. Solid site of MTC with prevalence of the cells with a low degree of ultra-structural differentiation. х 5,000

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Fig. 27. Dark and light tumor cells of MTC. х 3,000.

Fig. 28. Dark tumor cells with moderate ultra-structural differentiation among light cells with low degree of ultra-structural differentiation. х 4,000.

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Fig. 29. On the border of tumor cells there are contacts as well-formed desmosomes. х 3,000

The second group of the cells are non-differentiated tumor cells without specific ultrastructural signs (Fig. 26-28). The cells of this group were characterized by the big round or incorrect form of nucleus with diffuse or condensated chromatin as compact conglomerates. The nuclear membrane is with deep invagination. The cytoplasma of most of these cells is scanty and contains a lot of ribosomes, polysomes and inconsiderable number of other organoids. In the cytoplasm of the cells of this group there are no endocrine secretory granules. The cells are situated compactly to each other, the cellular borders are not clear. Sometimes the short, thin desmosomes are found. Along with these cells the dark cells with electronic-thick matrix of cytoplasm are found. In different tumors the quantity correlation of the cells with the presence of specific ultrastructural signs and without them are not the same. The counting of the tumor cells with the presence of specific ultra-structural signs and without them in every neoplasm showed that in some tumors the cells of the first group (more 50%) are prevalent, in the others – the cells of the second group. In our observations in 21 (75%) cases it was marked the prevalence of ultra-structurally differentiated and in 7 (25%) cases – ultra-structurally non-differentiated tumor cells. The comparison of the degree of differentiation at histological level (on the basis of analyses histological and half-thin preparations by light microscope) and at the ultrastructural level showed that sometimes there is no complete coincidence of differentiation at the light and ultra-structural level, that is, histologically similar tumors or the same tumor in the analogous structural places may have a different degree of differentiation at the lightoptic or electronic-microscopic level. Among our observations there were medullar cancers of the high degree of differentiation at the light-optic level and low one – at the ultrastructural one, that is, the greater part of the tumor cells did not contain cytospecific signs of

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C-cellular differentiation (the presence of endocrine granules) and vice versa – histologically non-differentiated cancers had 50% more tumor cells with endocrine granules. Thus, in the result of electronic-microscopic investigations MTC it was marked that independent of histological type tumor cells were presented by two groups: differentiated with ultra-structural specific signs of C-cells (with the presence of endocrine granules in cytoplasm) and non-differentiated ones without ultra-structural specific signs of C-cells of endocrine gland (with absence of endocrine granules in cytoplasm). The correlation of these two groups of cells in different tumors varies to a considerable extent. There is also some lack of coincidence in degree of tumor cell differentiation, determined at the histological level, with differentiation degree of these cells at the ultra-structural level.

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PROTO-ONCOGENE RET MUTATIONS IN RUSSIAN PATIENTS WITH MTC: EARLIER (PRECLINICAL) DIAGNOSTICS AND PROPHYLACTIC TREATMENT MTC may develop sporadically (70-75%) or as part of the autosomal dominantly inherited syndromes: multiple endocrine neoplasia type 2 (MEN 2A, MEN 2B) and familial MTC (FMTC). These cancer syndromes vary in aggressiveness of MTC and spectrum of affected organs. The MEN 2A syndrome is characterized by MTC, pheochromocytoma in about 50% of patients and parathyroid hyperplasia in about 20-30% [1]. MEN 2B is associated with MTC, pheochromocytoma (~50%), Marfan syndrome-associated constitution, mucosal neuromas and intestinal ganglionary neurofibromatosis [1,2]. Parathyroid hyperplasia in MEN 2B is rare. In familial MTC patients, MTC is not associated with other endocrinopathies [3]. Varied expressivity and incomplete penetrance, which are characteristic for MEN 2 syndromes are the causes of significant interindividual changeability (also within one family) both according to the age at the disease starting and to the severity of its manifestation. All factors mentioned above result in certain difficulties connected with clinical diagnostics of Ccell hyperplasia and MTC on the early stage of the development. Activating germline mutations of the RET proto-oncogene have been identified as the underlying cause of MEN 2 and familial MTC [4-9]. The RET gene (REarranged during Transfection) is located in the long arm of chromosome 10 (10q11.2) [10] and encodes a receptor tyrosine kinase that plays an important role in the control of proliferation, migration and/or differentiation of neural crest cells [11,12]. There are several isoforms of the RET protein [13,14,15], which are expressed predominantly in tissues of neuroendocrine origin. MEN 2 syndrome-associated mutations convert RET into a dominant transforming oncogene whose products demonstrate constitutive tyrosine kinase activity and increased ability to phosphorylation [16,17]. The mutations common to MEN 2A and FMTC are thought to cause ligand-independent homodimerization of RET [17,18]. The MEN 2B mutations also change RET tyrosine kinase substrate specificity [17,19,20]. These RET mutations cause activation of multiple intracellular signaling pathways [21].

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Clusterization is a common feature of most RET mutations seen in inherited MTC patients. They are located in one of two domains of RET protein. The only point mutation codon 918 (cytoplasmic domain) is found in more than 95% of MEN 2B families [4,7,22]. Missense mutations in one of the five cysteine codons [609, 611, 618, 620 (exon 10) or 634 (exon 11)] in the extracellular domain of RET were identified in more than 95% of MEN 2A and in 70-80% of familial MTC [8,23,24]. About 87% of MEN 2A patients have affected codon 634 and most frequent mutation at this codon is a cysteine to arginine change (~50%) [9,25]. Point mutations, which partly coincide with MEN 2 mutations, and small deletions from exons 10-16 of the RET gene were observed in patients with sporadic MTC [26]. We have analyzed the mutations of the RET gene in 20 individuals from families with inherited cancer syndromes MEN 2 and in 26 patients with sporadic MTC applied for the help to Russian NN Blokhin Cancer Research Centre in Moscow.

Materials and Methods

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Isolation of Genomic DNA To obtain the template for PCR amplification of RET exons, genomic DNA was isolated from 1) peripheral blood lymphocytes and 2) archival material, including tumor and (in most cases) normal thyroid tissues from formalin fixed, paraffin-embedded blocks as previously described [27]. Paraffin sections (10-20μm thick) were transferred on slides. One section was stained with hematoxylin-eosin to identify the regions of tumor and normal tissues. In both cases, DNA was isolated with QIAamp blood and QIAamp tissue kits (QIAGEN) as recommended by the manufacturer. PCR Amplification, SSCP Analysis, and Sequencing Exons 11, 13, 15 and 16 of the RET proto-oncogene were PCR-amplified with specific primers (Table 3) as previously described [27]. In the case of the archival material, nested PCR was carried with primers shown in Tables 3 and 4. The primers and reaction conditions were based on the complete nucleotide sequence of the exons and introns 9-16 of the RET proto-oncogene [VN Kalinin, A Frilling, 1998; GenBank accession nos. AF082337]. The reaction mixture (100 μl) contained 67 mM Tris-Cl (pH 8.8), 1.5 mM MgCl2, 16 mM (NH4)2SO4, 10% glycerol, 200 μM each dNTP, 50 pmol each primer, 2.5 units of Taq DNA polymerase (Perkin Elmer, Langen, Germany), and 0.5 μg of DNA. Amplification included first denaturation at 950C for 2 min.; 40 cycles of 0.5 min at 950C, 0.5 min at 550C, and 1 min at 720C; and last synthesis at 720C for 5 min. Exons 11 and 16 of the RET gene were sequenced with primers shown in Table 5. PCR products were isolated using a QIAquick PCR purification kit (QIAGEN). Sequencing was carried out using BigDye dideoxy-terminators, and the ABI 377 analyzer (Applied Biosystems, Foster City, USA). Restriction enzyme analysis of the amplified products was carried out with FokI, EcoRI, HhaI, and AluI (New England BioLabs, Beverly, USA) as recommended by the manufacturer. Restriction fragments were electrophoretically separated in PAG or agarose gel and stained with ethidium bromide.

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Non-isotopic single-strand conformation polymorphism (SSCP) analysis of RET exons 11 and 16 was conducted as described previously [28]. Electrophoresis in 24-cm PAG was carried out in a Hoefer SE410 unit. Table 3. Primers used to amplify fragments of exons 11, 13, 15, and 16 of the RET proto-oncogene. Exon

11 11 13 13 15 15 16 16

Primer

Nucleotide sequence

fRET11 (RET20S) RET11R fRET13 (CRT 4F) RET13R (CRT 4E) CRT 17B CRT 17G fRET16 RET16R

5/-TGAGGCAGAGCATACGCA 5/-CCTCGTCTGCCCAGCGTTG 5/-GCAGGCCTCTCTGTCTGAACTT

PCR product size, bp

456

5/-GGAGAACAGGGCTGTATGGA 297 /

5 -GTCTCACCAGGCCGCTAC 5/-ATGGTGCACCTGGGATCCCT 5/-AGGGATAGGGCCTGGCCTTC 5/-TCTGTAACCTCCACCCCAAG

292 196

Table 4. BD primers used to amplify fragments of exons 11, 13, 15, and 16 of the RET proto-oncogene.

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Exon 11 11 13 13 15 15 16 16

Primer RET11BDF RET11BDR RET13 BDF RET13BDR RET15BDF RET15BDR RET16BDF RET16BDR

Nucleotide sequence

PCR product size, bp

/

5 -CAGTAAATGGCAGTACCCATG 5/-CACAGACTGTCCCCACACA 5/-CTCAGGGTGCTTCTTCCTCA 5/-CGTGGACTCAGCTAGACACA 5/- TATGGCTCACCACGCCCCT 5/-TAGGCGGAGTTCTAATTGGGT 5/-CTCAGGGTGCTTCTTCCTCA 5/-CGTGGACTCAGCTAGACACA

609 487 493 427

Table 5. Primers used to sequence exons 11 and 16 of the RET proto-oncogene. Exon 11 11 16 16

Primer RET11BDF-S RET11BDR-S RET16BDF-S RET16BDR-S

Nucleotide sequence

PCR product size, bp

/

5 -GGTGTTTTCAGGCCTTCCCA 5/-ACACAGCGCCCTATGGAAAT 5/-CTCCAGCCCCTTCAAAGATG 5/-CCCCTCAGTGAGGGGGTCA

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Sporadic MTC To study the somatic mutations of the RET proto-oncogene in patients with sporadic MTC, we used the archival material (paraffin –embedded tissue sections of both tumor and normal tissue) stored in NN Blokhin Cancer Research Centre for no longer than 6 years. In all cases the clinical diagnosis was verified by histological examination of the surgical material and by genealogical analysis. 26 patients with sporadic MTC are characterized in Table 6. There are 8 men and 18 women with a mean age 45.5 years (range 10 to 73) at diagnosis. About 50% of the patients have stage III, and about 31% - stage II.

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Table 6. Characterization of sporadic MTC patients examined. Patient 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Age 68 42 64 31 38 51 26 35 35 56 73 29 42 43 42 42 43 39 43 68 57 48 52 48 58 10

Sex male female female male female male female female female female female female female female female female male female female male male female male female male female

MTC stage III III III II III III III III I II II III IV I I II III II II III III III II III IV II

Molecular pathology of the studied patients is presented in Table 7.

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Таble 7. Molecular defects in patients with sporadic MTC. Patient 1 3 ∗) 3 6 6 7 7 8 11 12 ∗) 12 12*) 12 14 14 17 17 *

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**

DNA source Tumor “ Normal tissue Tumor “ Tumor Normal tissue Tumor “ “ Normal tissue Tumor Normal tissue Tumor Normal tissue Tumor Normal tissue

Exon

Codon

16 16 16 11 16 11 11 16 16 11 11 11 11 16 16 16 16

918 922 922 634 918 634 634 918 918 639 639 641 641 918 918 918 918

Substitution nucleotide ATG→ACG TCC→TTC TGC→CGC ATG→ACG TGCÆTAC TGCÆTAC ATG→ACG ATG→ACG GCA→GGA GCT→CGT ATG→ACG ATGÆACG ATGÆACG

amino acid Met→Thr Ser→Phe Cys→Arg Met→Thr CysÆTyr CysÆTyr Met→Thr Met→Thr AlaÆGly Ala→ Arg Met→Thr MetÆThr MetÆThr

Restriction site Fok I **) **)

Hha I Fok I **) **)

Fok I Fok I **) **) **) **)

Fok I Fok I FokI FokI

The mutation is new. The mutation was identified by SSCP analysis and sequencing.

Examination of RET exons 11, 16 (SSCP and restriction enzyme analyses, automatic sequencing), 13 and 15 (restriction enzyme analysis) allowed identification of the somatic mutations in 9 out 26 patients with sporadic MTC (Table 7). It is noteworthy that the mutations each occurred in heterozygote. The most common mutation was in codon 918 (exon 16). This mutation results in substitution of threonine for methionine in the cytoplasmic tyrosine kinase domain of the RET protein. This tumor specific somatic mutation was identified in 6 of 26 (23.1%) sporadic MTCs. As estimated with different samples by different authors, frequency of this mutation is 23-70% in patients with sporadic MTC from Western Europe and America [6]. In one patient (N17, Table 7) the mutation in codon 918 was observed not only in the tumor, but also in the normal thyroid tissue. Since blood of this patient was not available, we could not establish whether the mutation was germline or mosaic. A similar situation was with patient 7 with mutation in codon 634 (Table 7). We did not detect mutations in codons 768 (exon 13) and 883 (exon 15), which also affect the intracellular tyrosine kinase domain of the RET protein, and are known for patients with sporadic MTC from Western Europe, America and Japan. Mutation TGC (Cys)->CGC (Arg) in codon 634 was observed in one patient (N 7, Table 7). Another mutation of this codon, TGC (Cys)->TAC (Tyr), was also detected in one patient. One patient had two RET mutations in codons 634 and 918 (N6, Table 7). In addition, we found three new RET mutations, each occurring in heterozygous status.

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In one tumor (N3, Table 7) with abnormal SSCP variant of exon 16 sequencing, analysis revealed a heterozygotic substitution C->T at the second nucleotide of codon 922. This substitution must change the sense of this codon : TCC (Ser)->TTC (Phe). This mutation creates a new restriction endonuclease EcoRI recognition site, which was confirmed by a direct restriction analysis. The DNA isolated from unaffected (normal) part of the thyroid gland and from a blood sample of patient N3 did not show any mutation. So, it must be recognized as a somatic mutation. The restriction analysis of 100 normal chromosomes did not show any mutation at codon 922. Position 922 lies immediately adjacent to the catalytic sub-domain VIII of the RET protein [4]. The alteration of a serine residue at the position corresponding to RET codon 922, which is completely conserved in receptor tyrosine kinases [29], might be expected to alter RET function. Another SSCP abnormality in the exon 11 was observed in the thyroid tumor of patient N12 (Table 7). Direct sequencing revealed three nucleotide substitutions in two codons: a single transversion GCA (Ala)->GGA (Gly) at codon 639, and a double transversion GCT (Ala)->CGT (Arg) at codon 641 [27,30]. Again, no mutation was found in the normal part of the thyroid gland, which is a strong indication of the somatic nature of these mutations. To establish the linkage of two closely located exon 11 mutations “the artificial separation of a normal and mutant allele” was performed with allele-specific PCR as described previously [30]. Automatic sequencing of allele-specific products generated with “normal” and “mutant” codon 641-specific primers clearly demonstrates that both mutant codons belong to the same allele. Sequencing of codon 639 allele-specific products provides the same results. The mutations A639G and A641R correspond to amino acid residues of the transmembrane region [11]. The codons 639 and 641 encode the first and the last of a triad of alanine residues in the transmembrane tract. The A640G mutation, found on the same RET allele as the common C634R mutation [31], alters the second alanine residue in this tract. It is thought, that this replacement reduces the hydrophobicity of the transmembrane region and induces a conformational change in the RET protein, which might lead to increased activity [31]. Activating mutations involving amino acids of the hydrophobic transmembrane domain have been described for other receptor tyrosine kinases [32]. The combination of two mutational events in the first and in the third of a triad of alanine residues in the RET protein transmembrane tract (A639G and A641R) in our patient`s tumor may consequently result in a similar change in the activity. Thus, molecular analysis of the four exons of the RET proto-oncogene revealed six different somatic mutations (including three new ones) in 34.6% of patients with sporadic MTC. According to published data, such mutations occur in 30-70% patients [33]. The mutation-negative cases suggest the existence of additional disease alleles, or the involvement of other genes. Detection of the new defects augments the spectrum of missense mutations of the RET proto-oncogene and contributes to the knowledge of its molecular structure. The clinical significance of these mutations remains to be studied. According to Eng et al [34] somatic RET mutations are only found in parts of tumors, and therefore may not be an early event in tumorigenesis. In 26 patients with sporadic MTC (clinical data for these patients are not shown in this article) blood was the only available source of DNA for molecular analysis of the RET gene.

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To check the possibility of inherited MTC, these patients were tested for mutations in codons 634 and 918, which are most common in MEN 2A and MEN 2B, respectively. As a result, a de novo MEN 2B mutation (codon 918), causing a substitution of a threonine for a methionine was found in four patients, and a de novo MEN 2A mutation Cys634Arg was found in one patient with clinical diagnosis “sporadic MTC”. So, we included these patients in a group with inherited MTC (Tables 8 and 9). But one can`t exclude the cause of such discrepancy may be uncertain family history and incomplete penetrance.

Inherited MTC Clinical data of patients with inherited MTC are demonstrated in Table 8.

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Table 8. Characterization of inherited MTC patients examined. Patient 1 1* 21

Age 45 15

Sex male female

МТС stage IV I

Clinical diagnosis MEN 2B**** MEN 2B

32 42 53 63 73 84 94 104 115 125 136 146 156 16** 17** 18** 19** 20**

34 4 38 9 20 39 14 12 52 20 15 42 63 31 14 21 14 20

male female female female male female female male male male female female female female female female female female

III

MEN 2A**** MEN 2A MEN 2A**** MEN 2A MEN 2A MEN 2A MEN 2A MEN 2A MEN 2A**** MEN 2A MEN 2A MEN 2A MEN 2A MEN 2A MEN 2B MEN 2B MEN 2B MEN 2B

***

III *** *** ***

I III II III IV III IV II

*

Identical indices show members of one family. The patient carried a de novo mutation. *** Preventive thyroidectomy was performed. **** The patient had bilateral pheochromocytoma. **

A total of 20 consultative individuals [(6 men and 14 women); mean age 21.5 years for MEN 2B patients (range, 14-45 years) and 28.1 years (range, 4-63 years)– for MEN 2A Milton, Carl A.. Focus on Thyroid Cancer Research, Nova Science Publishers, Incorporated, 2006. ProQuest Ebook Central,

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patients] with MTC admitted to the NN Blokhin Cancer Research Centre in Moscow from January 1996 to December 2004 were enrolled in the study. This group includes patients and their asymptomatic relatives which have germline RET mutations. Identical indices show members of one family. In four families there were patients with bilateral pheochromocytoma. The data of molecular research of the patients and their asymptomatic relatives are presented in Table 9. Тable 9. Molecular defects in patients with inherited MTC.

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Patient 11* 21 32 42 53 63 73 84 94 104 115 125 136 146 156 16*** 17*** 18*** 19*** 20***

Diagnosis MEN 2B МEN 2B МEN 2A МEN 2А МEN 2А МEN 2А МEN 2А МEN 2А МEN 2А МEN 2А МEN 2А МEN 2А MEN 2A MEN 2A MEN 2A MEN 2A МEN 2B МEN 2B МEN 2B МEN 2B

Exon 16 16 11 11 11 11 11 11 11 11 11 11 11 11 11 11 16 16 16 16

Codon 918 918 634 634 634 634 634 634 634 634 634 634 634 634 634 634 918 918 918 918

Substitution nucleotide ATG→ACG ATG→ACG TCGÆCGC TGC→CGC TGC→CGC TGC→CGC TGC→CGC TGC→GGC TGC→GGC TGC→GGC TGC→TTC TGCÆTTC TCGÆCGC TCGÆCGC TCGÆCGC TCGÆCGC ATGÆACG ATGÆACG ATGÆACG ATGÆACG

Restriction site amino acid Met→Thr Met→Thr CysÆArg Cys→Arg Cys→Arg Cys→Arg Cys→Arg Cys→Gly Cys→Gly CysÆGly Cys→Phe CysÆPhe CysÆArg CysÆArg CysÆArg CysÆArg MetÆThr MetÆThr MetÆThr MetÆThr

Fok I Fok I HhaI Hha I Hha I HhaI HhaI ** ** ** ** **

Hha I Hha I Hha I Hha I Fok I Fok I Fok I Fok I

*

Identical indices show members of one family. The mutation was identified by SSCP analysis and sequencing. *** The mutation arose de novo. **

Analysis of exons 11 and 16 revealed four types of mutations in these individuals. The well-known mutation in codon 634, which results in a substitution of an arginine for a cysteine in extracellular domain of RET protein is the most common in the studied group. It was found in heterozygote in 9 of 14 individuals from MEN 2A families (64.3%). All patients with MEN 2B (6/6) carried a mutation Met918Thr. To search for other RET mutations in patients with inherited MTC we used SSCP analysis of exon 11 [27]. Results of the sequence analysis of blood DNA samples with abnormal SSCP are summarized in table 9.

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So, germline mutations in RET proto-oncogene were detected in 100% cases, which were classified as inherited MTC, and affected codon 634 or 918.

Preventive Treatment MEN 2 Patients It is well-known that patients with MEN 2A syndrome have a 100% risk of developing MTC during their lifetime. In view of the world experience and the fact that MTC commonly shows aggressive development, an early metastasis, preventive thyroidectomy was performed in five asymptomatic carriers of RET mutations from three families with genetically and clinically confirmed MEN 2A syndrome. Thyroidectomy was performed in three children and two adults at NN Blokhin Cancer Research Centre. In two families, patient`s relatives had not only MTC, but also bilateral pheochromocytoma (Table 8). And here are the pedigrees of two of these families. Pedigree 1 includes family`s members 53-73 from Table 8.

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Pedigree 1

Black symbols indicate individuals with MEN 2A. Patients II.3, III.1 and III.2 have the mutation TGC (Cys ), c. 634 (exon (Cys))->CGC (Arg (Arg), (exon 11) of the RET proto-oncogene. Patients III.1 and III.2 underwent preventive thyroidectomy. thyroidectomy.

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The arrow indicates the index patient III.2. The PCR-restriction analysis detected the mutation at codon 634 (substitution of an arginine for a cysteine) in the index patient and two of his relatives. The direct sequencing verified result of restriction analysis. The proband`s mother II.3 had undergone 5 operations for pheochromocytoma, and at the age of 36 she underwent surgery for a C-cell thyroid tumor, which was defined as MTC. The proband`s aunt II.2 died from sepsis at the age of 28 years. By autopsy she had a bilateral pheochromocytoma and thyroid enlargement. The proband`s grandmother I.2 suffered from hypertension crises and died at the age of 29 years. Autopsy revealed enlargement of both adrenals. These findings suggest that the patient had MEN 2A. So, two asymptomatic individuals from this family [patient III.2 (73) and his cousin III.1 3 (6 )] underwent total thyroidectomy. The index patient III.2 applied for help to Children Oncology Institute of NN Blokhin Cancer Research Centre complaining of nodal growth in the region of the thyroid gland and increased lymph nodes on the right side of the neck. MTC with metastases in lymph nodes of the neck on the right side was diagnosed during a complex investigation including thyroid gland scanning, ultrasound of neck, adrenal and thyroid glands, cytologic investigation of tumor punctuate, the investigation of CT level in blood (1716 ng/l). The patient underwent an operation: thyroidectomy and dissection of neck on the right. The diagnosis was confirmed histological. Besides, germline mutation at codon 634: TGC (Cys)-> GGC (Gly) in the RET proto-oncogene was revealed at DNA analysis in the patient.

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The number to the upper right of the subject symbols indicates age age in years

Black symbols indicate individuals with syndrome MEN 2A. Mutation TGC (Cys)ÆGGC (Gly), codon 634, exon 11 of the RET proto-oncogene.

Shaded symbols indicate individuals with enlarged thyroid gland. Milton, Carl A.. Focus on Thyroid Cancer Research, Nova Science Publishers, Incorporated, 2006. ProQuest Ebook Central,

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Three other members of this family: I.4, II.3 and III.3 have the same mutation. The proband`s grandmother (I.4) underwent thyroidectomy at the age of 45 (because she had an enlarged thyroid gland) before the availability of the RET testing. Today she is 72 years old. Individuals II 3 and III.3, asymptomatic carriers of the RET mutation in codon 634 with normal level of CT, underwent total thyroidectomy. The following patient received preventive thyroidectomy was a 4-year-old girl from the family with MEN 2A (42, Table 8). In three cases (patients 73, 84 and 104, Table 8) histological examinations of the surgical material revealed multifocal MTC in both lobes of the thyroid gland without metastases in lymph nodes. The result of histological analysis of one of these patients is on Fig. 19. Neither carcinoma cells nor C-cell hyperplasia were detected in two more younger patients: 4 and 9 years old (42 and 63, Table 8). Thus, detection of mutations of the RET proto-oncogene in five individuals with positive family history allowed surgical treatment of MTC at an early stage of its development in three cases. In two children, thyroidectomy will prevent MTC associated with MEN 2A. Regardless of the histological findings, all patients were recommended to consult oncologists and endocrinologists regularly. So, prophylactic thyroidectomy based on genetic testing, allows the identification and treatment of patients at an early stage of the disease and may reduce recurrence MTC. Thus, molecular genetic analysis enabled us 1) to study the spectrum of mutations of the RET proto-oncogene in Russian patients with inherited and sporadic MTCs; 2) to identify asymptomatic individuals at risk for MEN 2 cancer syndromes in early (preclinical) stage and to prevent cancer; 3) to distinguish heritable from non-heritable MTCs for the correct choice of treatment and the volume of surgery; 4) to begin the creation of a register of pathological RET gene carriers with a high (up to 100%) risk of cancer in Russia.

THYROID CANCER TREATMENT Therapy technique in cases of thyroid cancer depends on many factors. There are two different approaches concerning the volume of the operation on the thyroid gland. The first is that it`s necessary to remove all the gland, irrespective of the morphological structure, tumor size, age, sex of the patients and other factors. Some authors explain the necessity of such an approach by the following points, but in fact, they have no arguments: 1. high frequency of multiple cancer foci; 2. the decreasing of the possibility of local tumor relapses; 3. radioactive iodine therapy provided after thyroidectomy; 4. the decreasing of the possibility of recurrent interventions (interference) with high risk complications;

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Vitaliy Zh Brzhezovskiy, Vyacheslav L Lyubaev, Tatiana T Kondratyeva et al. 5. thyroid gland absence provides the conditions for the future determination of thyroglobulin level to make an early diagnosis of recurrent events and metastases of thyroid cancer.

We support the differentiated approach to the operation volume of the thyroid gland according to the primary tumor size, sex, age, familial or sporadic forms of the disease, multiple lesions of lymph nodes in neck and mediastinum. 182 patients with the primary-made diagnosis of MTC were treated in Russian NN Blokhin Cancer Research Centre; among these, 142 were followed for 5 plus years. In the determining tumor process spreading according to the TNM system the patients were distributed as following: T1N0M0 – 7, T2N0M0 – 13, T3N0M0 – 19, T0N1M0 – 4, T1N1M0 – 11, T2N1аM0 – 13, T2N1bM0 – 2, T3N1aM0 – 47, T3N1b – 20, T4N1aM0 – 9. These patients` distribution regarding the above-mentioned staging is presented in Table 10.

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Table 10. MTC patients` distribution according to the stage of the disease. The stage of the disease

I T1N0M0

II T2-4N0M0

III T0-4N1a-bM0

Number of patients %

7

32

3.8

17.6

Total number

135

IV T1-4NabM1 8

74.2

4.4

100

182

After the treatment, on the occasion of MTC previously performed in other clinics, 60 patients were hospitalized. In general, these patients were non-radically operated, in places where they lived, on the occasion of thyroid gland cancer with metastases in neck lymph nodes (39 patients) and regional metastases biopsies with subsequent radiation therapy were performed on 16 patients. The other 5 patients came for medical help following repeated operative intervention, radiation and chemotherapy. The surgical intervention volume in MTC depended on both tumor distribution in the primary focus area and regional metastases. When the tumor size in the thyroid gland was up to 4 cm, organosparing operations were conducted. When it was 4 cm or more, when we suspected multicentric growth and when we had MEN 2 syndromes, thyroidectomy was performed. Different kinds of neck dissections were performed in the presence of metastatic lesion of regional neck lymph nodes depending on the number and in a greater extent on the distribution on the surrounding tissues (sterno-cleidomastoid muscle, internal jugular vein). In three cases of cancer metastases in mediastinum lymph nodes were present, the operation approach was performed by means of sternotomy. One of the actual questions in case of MTC surgical treatments is the volume of operation on the primary cancer focus. We performed the analysis of a 5-year survival rate of radically operated patients, depending on different operation types on the thyroid gland. Hemithyroidectomy was done to the primary patients with tumor size up to 2 cm (n=20). These patients didn`t have tumor exit outside the borders of the thyroid gland capsule. In the

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subsequent observation of the patients, tumor relapses in the balance of the gland lobe were not detected. And the cause of death during the next 5 years in 3 patients (15%) was the generalization of the tumor process (cancer metastases development into the lungs and bones). In tumor size 2-4 cm in 46 patients subtotal resection of the thyroid gland was performed. In histologic testing of the removed material in 5 cases the tumor invading the thyroid gland capsule and outside (external) organ borders is noted [in 2 patients the tumor spreaded (invaded) into soft tissues; in 3 – into the trachea tissue; and due to this fact in 1 case electrocoagulation of the suspected region in the trachea area was done and in 2 – the resection of it]. The patients were followed during a period up to 5 years and the tumor recurrence reappeared in 2 patients (4.3%). In the group of patients consisting of 68 individuals with a tumor size greater than 4 cm, as well as in the group of patients with MEN 2 syndromes, thyroidectomy was done. The tumor spreading outside organ borders was noted in 18 patients. Neoplasm relapse in this zone is noted in 6 patients (8.8%) (in all cases the tumor spreads outside the borders of the thyroid gland capsule). So, the main cause of cancer relapsing in the area of the primary tumor focus is the spreading of the process outside the thyroid gland capsule. In a histological test 114 thyroid gland material after subtotal resection and thyroidectomy (20 patients with hereditary disease form of MTC and 94 – with sporadic MTC) multicentric tumor growth was found in 4 cases (4.2 %) in the group of sporadic MTC and in 100% cases – in patients of inherited MTC. Thus, the conduction of organosparing operations in cases of limited size of the primary tumor focus (Т1 – Т2) doesn`t lead to distant treatment results worsening. The patient`s prognosis in a greater extent depends on the presence, localization and spreading of the metastatic lesion. This says something about the necessity of the differentiated approach to the surgical intervention volume. The indication to thyroidectomy is believed to be the presence of one of the following features of the tumor: neoplasm size 4 cm and greater, tumor spreading on the isthmus of the thyroid gland, thyroid capsule invasion, suspected multicentric growth of the neoplasm and also MEN 2 syndromes presence. The first barrier of lymphogen metastatic process in MTC is known to be lymph nodes, localized in pre- and para-tracheal area (the 6th stage of metastatic process). 43 patients were subjected to preventive elimination of pre- and para-tracheal neck dissection (median neck dissection/in clinically undetermined metastases in this area. Thyroid tumor size, corresponding to T1 was determined in 4, T2 – in 24, T3 – in 13 and T4 – in 2 patients. Hemithyroidectomy was performed on 4 patients, subtotal resection on 19 and thyroidectomy on 21 patients. Of 43 operated patients, metastases were detected in 17 individuals (39%). So, high percentage of metastases in MTC into pre- and para-tracheal lymph nodes (39%) allows conducting such interference type to be recommended to each patient with MTC together with the operation on the primary tumor focus. Radiation therapy in treatment of the disease was used in 3 regemens: 1) in pre-operation period, 2) in post-operation period, 3) with palliative purpose in cases of inoperable tumor forms. The combined treatment method included pre-operation course of radiation therapy with the doze of 40 – 45 gr. The technique of radiation was standard: from the two lateral or one direct fields, including the area of upper mediastinum and shielding of bone marrow and

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pharynx. In 3-4 weeks surgery was performed. It is necessary to emphasize that none of the patients had the objective effect from radiation therapy course. Combined treatment with pre-operative radiation therapy was conducted in 26 patients, 7 of them were primary patients, the balance of 19 – following previous treatments performed in other hospitals (in general, regional metastases biopsies with diagnostic purpose were applied). The comparison of treatment results of the above described patient group with the patients, received only surgical treatment was done. This group consisted of 69 patients (57 were the primary patients, 12 had recurrent events). The group of patients who received post-operative radiation therapy was rather numerous. There were 65 individuals in it. 45 patients were operated on for the first time, 20 were operated on repeatedly following the surgical treatment which was performed earlier in other hospitals. We think that the combining of these patients to be justified, as tumor process spreading, both in the area of the primary focus, and in regional metastases of both groups was approximately the same. It`s necessary to say that by analyzing operation reports it is stated that a number of surgical interventions had nonradical character (tumor envasion into trachea, esophagus, intimal adhesion with neck magistral vessels). This was one of the criteria to post-operative radiation therapy appliance. Such patients were 39. Distant radiation was applied to the other 26 patients with the prophylactic purpose. 5-year and 10-year treatment results were estimated (Table 11). A rather high percent of 10-year survival in the group, with doubtful radical operation, is noteworthy. This speaks in favour of post-operative radiation therapy in such a situation. At the same time the patients’ treatment results, where radiotherapy was conducted after radical operation, were compared with the group where surgical method and combined treatment of pre-operative radiotherapy was used (the difference between them isn`t statistically significant; (p > 0.05). The group of 36 patients of whom this treatment was applied with palliative purpose was selected to estimate the effect of radiation therapy. Earlier, 27 of them had operations of different volume in NN Blokhin Cancer Research Centre or in hospitals in places where they lived; 9 patients were initially inoperable (infiltrate neoplasms, invading tracheal cartilages, spreading into mediastinum, inoperable regional metastases). Radiation therapy dose was from 40 to 70 gr. We succeeded in gaining stable palliative effect in only 4 patients (11.1%). Table 11. 5–years` and 10-years` management results in MTC patients with surgical and combined treatment methods. Treatment method

Surgical Combined with preoperation radiation therapy Combined with postoperation radiation therapy

5 – years` results Number of Of them patients alive 69 42 26 16

60.9 61.5

110 – years` results Number of Of them patients alive 58 27 19 9

26

57.7

17

15

%

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8

% 46.6 47.4

47.1

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Table 11. (Continued). Treatment method

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Non-radical operation + radiation therapy

5 – years` results Number of Of them patients alive 39 11

% 28.2

110 – years` results Number of Of them patients alive 29 5

% 17.2

Radiation therapy was also performed as a symptomatic or palliative means in distant metastases in skeletal bones. This type of treatment was applied in 10 patients. Pain lessening in metastatic process area was noted in all patients. In 4 patients when skeletal bone scanning was used, a decrease of radiopharm preparation deposition was seen following the conducted radiation therapy. Thus, to apply radiotherapy in MTC there are the following indications: 1) in the case of doubtful operation radicality; 2) in non-operable cancer forms; 3) in distant metastases with palliative and symptomatic purpose. Chemotherapy on the occasion of MTC was applied in 18 patients. All the patients had non-operable thyroid gland cancer and metastases in neck lymph nodes or distant metastases. 8 patients were given adriablasin monochemotherapy in a dose 60 mg/m2 per week for 4 weeks. Tumor process stabilization (cancer metastases into liver) was noted in 2 patients over 6 months. The other 10 patients were given drug therapy by various drug combinations including adreablastin, bleomycin, cyclophosphan and cys-platin. No less than 2 polychemotherapy courses were conducted. Tumor process stabilization was registered in 3 patients. The toxicity of the drugs employed was moderate. If we judge (to say) about palliative treatment effectiveness from the CT level, only one patient showed a decrease of this index after the performed treatment. Thus, MTC is a tumor type, showing little sensitivity to chemotherapy. It is considered the third place in treating this pathology, after surgical and radiation methods. It is necessary to gain further experience and apply new chemotherapy schemes.

MTC Treatment Results and Some Prognosis Factors of the Disease MTC treatment results depend both on a spreading of tumor process in the primary tumor focus area and, in a greater extent, on the presence and size of metastatic lesion of regional lymph nodes. The results of primary MTC patients` treatment depending on the process stage are presented in Table 12. It is known that TNM system is one of the criteria, which allows a judgment concerning the disease prognosis. Our treatment result analysis showed that the prognosis was more affected by tumor metastatic process degree into regional lymph nodes than primary tumor spreading (see Table 12), and the results worsening depending on the presence of regional metastases is statistically significant (р < 0.05).

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Stage of the disease Period of observation Total number of patients Number of survived patients %

IV

T1-4N0M0 5 years 10 years

III T1-4N1a bM0 5 years 10 years

5

27

16

102

84

8

5

5

23

12

62

41

0

100

100

85.2

75

60.8

48.8

0

I

II

Т1N0M0 5 years 10 years 5

5 years

In determining histological signs of MTC, possibly having prognostic value, neither histological tumor variant, nor tumor cells structure were found to significantly influence patients’ survival (earlier described morphological signs were seen in all observed patients groups). To find out the cause of a different prognosis in patients with MTC, having the same histological tumor variant, the same morphological characteristic and the same disease stage, electron-microscopic study of the ultra-structural features of tumor cells was done. We have examined electron-microscopic tumors’ structure to reveal the relationship between ultra-structural features of MTC, its clinical course and prognosis in 3 groups: patients who died before 5 years; patients with 5-year and 10-year survival. In each patient group, tumors were presented by different histological variants. In the group of patients who died before 5 years there were patients with I, III and IV stages of the disease; and in the group with 5- and 10-year survival there were patients with II and III stages only. A different volume of the operation was performed in all three patient groups. Metastases in regional lymph nodes were also noted in all groups. Distant metastases were only in the group of patients who died before 5 years. Characteristic for each group ultra-structural relative monomorphism, determined by ultra-structurally differentiated and non-differentiated cells ratio; dark cells presence and desmosomes development was marked in each group by electron microscopy. By using electron microscopy, the disease prognosis closely correlated with ultra-structurally differentiated cells presence and desmosomes development. The more the number of both of them, the higher the survival. The increasing number of ultrastructurally non-differentiated and dark cells worsens the prognosis.

CONCLUSION To realize the essence of the pathology presented more profoundly and to develop a more successful and effective treatment, it is of utmost importance to investigate the peculiarities of MTC clinical manifestations, histologic structure, ultrasound features of this tumor, genetic aspects of its hereditary variants, advisability and possibility of each method of investigation.

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Diagnostics of MTC must be complex and include radioisotope and ultrasound study, FNAB cytology and study of biochemical tumor markers (CT and CEA). Computed tomographic scanning and MRI must be used according to the indications. The specific mutations in the proto-oncogene RET, which are revealed in more than 95% of cases, are excellent markers in hereditary types of MTC, accounting for 20-30% of all MTCs. We have done much work on investigating clinical and morphologic features of MTC. The chapter provides a discussion on the objective possibilities of cytologic methods while making exact diagnosis of MTC and its types, with the use of our own cytograms. A number of differentially-diagnostic problems arising in practice were defined with the aim to decrease the probable errors in the distinguishing of this unusually multiform thyroid cancer. Different histologic types of MTC were studied. The majority of tumors detected in Russian patients presented a typical solid variant. In addition, we also revealed follicular, papillary, microcellular, clear cellular, oncocytic, mixed medullar-follicular and medullarpapillary types. While investigating the influence of MTC histologic type on patients` survival, we failed to determine significant correlation between these parameters and prognosis. Electron microscopic investigation of MTC demonstrated that the ratio of differentiated and undifferentiated cells in tumors is the most informative prognostic symptom. For molecular diagnostics of MTC we performed screening of mutations in the protooncogene RET (in blood leukocytes) for Russian families bearing MEN 2 (MEN 2A and MEN 2B). The frequency of activating germline mutations in RET was 100%. The preventive thyroidectomy performed for 5 asymptomatic carriers of RET mutations (codon 634). It was effective in prophylaxis and/or treatment of hereditary MTC. In the group of patients with sporadic MTC, somatic RET mutations in tumors were revealed in 34.6% of cases. The surgery is very important in the current treatment of MTC. The volume of surgical treatment on the primary tumor lesion depends mainly on MTC forms: sporadic or hereditary. Thyroidectomy is prescribed in the hereditary form of the disease (MEN 2A, MEN 2B and FMTC), irrespective of tumor size. If tumor size is restricted in the case of sporadic MTC (T1-T2), it`s possible to use organosparing surgery. Indications for thyroidectomy are as follows: tumor size corresponding to T3 symbol, neoplasm extension to the isthmus of thyroid, suspicion on the multicentric character of growth, tumor invasion of thyroid capsule and familial forms of MTC. All cases mentioned above had preventive median neck dissection (removal of pre- and para-tracheal fat) in unpalpable lymph nodes of this localization owing to the high risk of metastasizing. Concerning radiotherapy in MTC treatment, there are three main indications: 1) additional radiotherapy in case of doubtful operation radicality, assessed both macroscopically and microscopically; 2) in inoperable cancer forms; 3) in distinct metastases with palliative and symptomatic aim. At present, available chemotherapeutic drugs fail to produce significant effects on the increase of MTC patients’ survival.

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REFERENCES Amosenko FA, Brzhezovskiy VZh, Lyubchenko LN, Shabanov MA, Kozlova VM, Vanushko VE, Kazubskaya TP, Garkavtseva RF, Kalinin VN Analysis of mutations of the RET proto-oncogene in patients with medullary thyroid carcinoma. Russian Journal of Genetics 2003; 39: 706-711. Amosenko FA, Trubnikova IS, Zakharyev VM, Bannikov VM, Sazonova MA, Petrova NV, Kapranov NI, Kalinin VN TUB9 polymorphism in the CFTR gene of cystic fibrosis patients, carriers, and healthy donors from the Moscow region: SSCP and restriction analyses. Russian Journal of Genetics 1997; 33: 198-202. Asai N, Iwashita T, Matsuyama M, Takahashi M Mechanism of activation of the ret protooncogene by multiple endocrine neoplasia 2A mutations. Mol Cell Biol 1995; 15: 16131619. Bargmann CI, Hung MC, Weinberg RA Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185. Cell 1986; 45: 649-657. Carlson KM, Dou S, Chi D, Scavarda N, Toshima K, Jackson CE, Wells SA Jr, Goodfellow PJ, Donis-Keller H Single missense mutation in the tyrosine kinase domain of the RET proto-oncogene is associated with multiple endocrine neoplasia type 2B. Proc Natl Acad Sci USA 1994; 91: 1579-1583. Donis-Keller H, Dou S, Chi D, Carlson KM, Toshiba K, Lairmore TC, Howe JR, Moley JF, Goodfellow PJ, Wells SA Jr Mutations in the RET proto-oncogene are associated with MEN 2A and FMTC. Hum Mol Genet 1993; 2: 851-856. Eng C, Clayton D, Schuffenecker I, Lenoir G, Cote G, GagelRF, Ploos van Amstel HK, Lips CJM, Nishisho I, Takai SI, Marsh DJ, Robinson BG, Frank-Raue K, Raue F, Xu F, Noll WW, Romei C, Pacini F, Fink M, Niederle B, Zedenius J, Nordenskjold M, Komminoth P, Hendy G, Gharib H, Thibodeau S, Lacroix A, Frilling A, Ponder BAJ, Mulligan LM The relationship between specific RET proto-oncogene mutations and disease phenotype in multiple endocrine neoplasia type 2: International RET Mutation Consortium. JAMA 1996; 276: 1575-1579. Eng C, Mulligan LM Mutations of the RET proto-oncogene in the multiple endocrine neoplasia type 2 syndromes, related sporadic tumours and Hirschsprung disease. Hum Mutat 1997; 9: 97-109. Eng C, Mulligan LM, Healey CS, Houghton C, Frilling A, Raue F, Thomas GA, Ponder BAJ. Heterogeneous mutation of the RET proto-oncogene in subpopulations of medullary thyroid carcinoma. Cancer Res 1996; 56: 2167-2170. Eng C, Smith DP, Mulligan LM, Nagai MA, Healey CS, Ponder MA, Gardner E, Scheumann GFW, Jackson CE, Tunnacliffe A, Ponder BAJ Point mutation within the tyrosine kinase domain of the RET proto-oncogene in multiple endocrine neoplasia type 2B and related sporadic tumors. Hum Mol Genet 1994; 3: 237-241. Farndon JR, Leight GS, Dilley WG, Baylin SB, Smallridge RC, Harrison TS, Wells SA Familial medullary thyroid carcinoma without associated endocrinopathies: a distinct clinical entity. Br J Surg 1986; 73: 278-281.

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Gorlin RJ, Sedano HO, Vickers RA, Cervenka J Multiple mucosal neuromas, pheochromocytoma and medullary carcinoma of the thyroid – a syndrome. Cancer 1968; 22: 293-299. Hanks SK, Quinn AM, Hunter T The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 1998; 241: 42-52. Hofstra RMW, Landsvater RM, Ceccherini I, Stulp RP, Stelwagen T, Luo Y, Pasini B, Hoppener JWM, Pools van Amstel HK, Romeo G, Lips CJM, Buys CHCM A mutation in the RET proto-oncogene is associated with multiple endocrine neoplasia type 2B and sporadic medullary thyroid carcinoma. Nature 1994; 367: 375-376. Ishizaka Y, Itoh F, Tahira T, Ikeda I, Sugimura T, Tucker J, Fertitta A, Carrano AV, Nagao M Human ret proto-oncogene mapped to chromosome 10q11.2. Oncogene 1989; 4: 1519-1521. Iwashita T, Asai N, Murakami H, Matsuyama M, Takahashi M Identification of tyrosine residues that are essential for transforming activity of the ret proto-oncogene with MEN 2A or MEN 2B mutation. Oncogene 1996; 12: 481-487. Kalinin VN, Amosenko FA, Shabanov MA, Lubchenko LN, Hosch SB, Garkavtseva RF, Izbicki JR Three novel mutations in the RET proto-oncogene. J Mol Med 2001; 79: 609-612. Lips CJM, Landsvater RM, Hoppener JWM, Geerdink RA, Blijham G, Jansen-Schillhorn van Veen JM, van Gils APG, de Wit MJ, Zewald RA, Berends MJH, Beemer FA, Brouwers-Smalbraak J, Jansen RPM, Ploos van Amstel HK, van Vroonhoven TJMV, Vroom TM Clinical screening as compared with DNA analysis in families with multiple endocrine neoplasia type 2A. N Engl J Med 1994; 331: 828-835. Liu X, Vega QC, Decker RA, Pandey A, Worby CA, Dixon JE Oncogenic RET receptors display different autophosphorylation sites and substrate binding specificities. J Biol Chem 1996; 271: 5309-5312. Lorenzo MJ, Eng C, Mulligan LM, Stonehouse TJ, Healey CS, Ponder BAJ, Smith DP Multiple mRNA isoforms of the human RET proto-oncogene generated by alternate splicing. Oncogene 1995; 10: 1377-1383. Mulligan LM, Kwok JBJ, Healey CS, Edson MJ, Eng C, Gardner E, Love DR, Mole SE, Moore JK, Papi L, Ponder MA, Telenius H, Tunnacliffe A, Ponder BAJ Germline mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature 1993; 363: 458-460. Mulligan LM, Marsh DJ, Robinson BG, Schuffenecker I, Zedenius J, Lips CJM, Gagel RF, Takai SI, Noll WW, Fink M, Raue F, Lacroix A, Thibodeau SN, Frilling A, Ponder BAJ, Eng C Genotype-phenotype correlation in multiple endocrine neoplasia type 2B: report of the International RET Mutation Consortium. J Intern Med 1995; 238: 343346. Myers SM, Eng C, Ponder BAJ, Mulligan LM Characterisation of RET proto-oncogene 3‘splicing variants and polyadenylation sites: A novel C-terminus for RET. Oncogen 1995; 11: 2039-2045. Pasini A, Geneste O, Legrand P, Schlumberger M, Rossel M, Fournier L, Rudkin BB, Schuffenecker I, Lenoir GM, Billaud M Oncogenic activation by two distinct FMTC mutations affecting the tyrosine kinase domain. Oncogene 1997; 15: 393-402.

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Rossel M, Schuffenecker I, Schlumberger M, Bonnardel C, Modigliani E, Gardet P, Navarro J, Luo Y, Romeo G, Lenoir G, Billaud M Detection of a germline mutation at codon 918 of the RET proto-oncogene in French MEN 2B families. Hum Genet 1995; 95: 403-406. Santoro M, Carlomagno F, Romano A, Bottaro DP, Dathan NA, Grieco M, Fusco A, Vecchio G, Matoskova B, Kraus MH, di Fiore PP Activation of RET as a dominant transforming gene by germline mutations of MEN 2A and MEN 2B. Science 1995; 267: 381-383. Schimke RN Genetic aspects of multiple endocrine neoplasia. Annu Rev Med 1984; 35:25-31. Schuchardt A, D`Agati V, Larsson-Blomberg L, Costantini F, Pachnis V Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor RET. Nature 1994; 367: 380-383. Schuffenecker I, Billaud M, Calender A, Chambe B, Ginet N, Calmettes C, Modigliani E, Lenoir GM RET proto-oncogene mutations in French MEN 2A and FMTC families. Hum Mol Genet 1994; 3: 1939-1943. Segouffin-Cariou C, Billaud M Transforming ability of MEN 2A-RET requires activation of the phosphatidylinositol 3-Kinase/AKT signaling pathway. J Biol Chem 2000; 275: 3568-3576. Takahashi M, Buma Y, Hiai H Isolation of ret proto-oncogene cDNA with an amino-terminal signal sequence. Oncogene 1989; 4: 805-806. Takahashi M, Buma Y, Iwamoto T, Inaguma Y, Ikeda H, Hiai H Cloning and expression of the ret proto-oncogene encoding a tyrosine kinase with two potential transmembrane domains. Oncogene 1988; 3: 571-578. Tessitore A, Sinisi AA, Pasquali D, Cardone M, Vitale D, Bellastella A, Colantuoni V A novel case of multiple endocrine neoplasia type 2A associated with two de novo mutations of the RET proto-oncogene. J Clin Endocrinol Metab 1999; 84: 3522-3527. Wohllk N, Cote GJ, Bugalho MM, Ordonez N, Evans DB, Goepfert H, Khorana S, Schultz P, Richards CS, Gagel RF Relevance of RET proto-oncogene mutations in sporadic medullary thyroid carcinoma. J Clin Endocrinol Metab 1996; 81: 3740-3745.

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Chapter 9

THE ROLE OF RT INHIBITORS AS A NOVEL MOLECULAR TARGETED TREATMENT IN THE MANAGEMENT OF POORLY DIFFERENTIATED THYROID TUMORS M. Landriscina1,∗, A. Fabiano2, A. Piscazzi1, C.Bagalà4, S. Altamura1, N. Maiorano1, F. Giorgino3, C. Barone4 and M. Cignarelli2

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1

Cattedra di Oncologia Medica, 2Cattedra di Endocrinologia e del Metabolismo, Dipartimento di Scienze Mediche e del Lavoro, Università degli Studi di Foggia, 3 Cattedra di Endocrinologia e del Metabolismo, Università degli Studi di Bari, 4 Cattedra di Oncologia Medica, Istituto di Medicina Interna, Università Cattolica, Roma, Italy.

ABSTRACT Anaplastic thyroid carcinoma, representing only 2% of thyroid cancers, is one of the most aggressive malignancies. Indeed, the overall median survival of affected patients is limited to months, whereas only a minority of patients with resectable disease have demonstrated long-term survival with aggressive multimodal treatments. Unfortunately, by contrast to differentiated thyroid tumors, undifferentiated thyroid cancer cells fail to uptake iodine because of the lack of expression of the Na/I symporter (NIS) and thus their responsiveness to radio-iodine therapy is abrogated. Either undifferentiated or transformed cells express high levels of endogenous nontelomeric reverse transcriptase (RT). Pharmacological RT inhibition by two well characterized RT inhibitors (nevirapine and efavirenz) as well as the down-regulation of expression of RT-encoding LINE-1 elements by RNA interference, reversibly inhibit cell ∗

Correspondence concerning this article should be addressed to Dr. Matteo Landriscina, Cattedra di Oncologia Medica, Università degli Studi di Foggia, Viale Pinto, 1 - 71100 Foggia, Italy. Tel.: ++39 0881 733853; Fax: ++39 0881 736008; E-mail: [email protected].

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M. Landriscina, A. Fabiano, A. Piscazzi et al. growth, promote cell differentiation and modulate gene expression in several human tumor cell lines either in vitro or in animal models. Therefore, we investigated, in primary cultures of human undifferentiated thyroid carcinoma as well as in anaplastic thyroid carcinoma ARO cells, whether pharmacological inhibition of RT may represent an effective tool in the treatment of poorly differentiated thyroid neoplasms. The results of our study demonstrated that efavirenz and nevirapine induced morphological changes resembling cell differentiation and activated the expression of thyroglobulin, a gene highly expressed by differentiated thyroid tumors. It is noteworthy that undifferentiated thyroid tumor cells exposed to RT inhibitors acquired also the ability to express NIS and pendrin, two protein involved in iodine uptake, and accumulate radioactive iodine in a TSH-dependent manner. Interestingly, the appearance of this differentiated phenotype correlated with the reversible down-regulation of either cell growth in vitro or tumor growth in vivo. Finally, the simultaneous exposure of anaplastic thyroid tumor cells to the differentiating agent all-transretinoic acid (ATRA) and nevirapine did not produce any additive effect on the inhibition of cell proliferation. Thus, the role of RT inhibitors as potential differentiating and cytostatic treatment for undifferentiated thyroid cancer is discussed.

Keywords: nevirapine, efavirenz, thyroid tumors, differentiation, iodine uptake This study was supported by PRIN grant n. 2002063999_005 to M.C. and by PRIN grant n. 2004054004_002 to M.L.

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INTRODUCTION Most thyroid carcinomas are well differentiated and respond to surgery, radioactive iodine treatment and levothyroxine suppression therapy. However, a significant minority of thyroid cancers are poorly differentiated and often metastatic and virtually unable to concentrate radioactive iodine. These tumors, which lead to the majority of thyroid carcinoma-related death, are aggressive undifferentiated papillary or follicular thyroid cancers and anaplastic thyroid carcinomas [1]. Although, in vitro and in vivo studies, including small human trials, have showed modest effectiveness in treating anaplastic thyroid tumors with agents such as taxoids, there is no by no means a cure for this deadly disease [2]. Thus, a novel molecular targeted approach to thyroid cancer therapy may help direct novel therapies to patients who would most benefit. Retrotransposable elements are mobile DNA sequences able to retrotranspose via RNA intermediates; the latter are reverse-transcribed into cDNA copies by endogenous RT activity. The idea that these repeated sequences were mere “junk DNA” has markedly influenced the opinion of the scientific community for over two decades, during which the unquestioned assumption that the DNA of retrotransposon origin is a useless component of mammalian genomes has taken root [3]. However, in the post-genomic era this view is slowly changing. At this point, even though no clear physiological role for endogenous RT or retrotransposable elements has yet been defined, a vast body of evidences indicates that the expression of retroelements and genes harbored therein, such as the RT coding genes, is regulated in tissues

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and cell types depending on their proliferation rate and differentiation level [4]. Indeed, elevated expression levels of RT are detected in embryos [5-6], embryonic tissues [7], the male genital tract [8], germ cells [9], gametes [10-12] and tumors [13-14]. In contrast, RT is expressed at very low levels in low-proliferating and terminally differentiated cells. These observations indicate that endogenous RT activity is preferentially expressed in tissues with a high proliferation potential. It is difficult to understand whether high RT levels are causally implicated in the mechanism(s) of cell growth and differentiation or whether they become somehow up-regulated as a consequence. We have recently observed that nevirapine and efavirenz, two non-nucleosidic RT inhibitors widely employed in the therapy of AIDS [15], inhibits the endogenous RT activity in normal and transformed mammalian cells of different histological origin [16]. Moreover, nevirapine-induced RT inhibition results in a specific block of embryo development in early cleavage stages, i.e. two-cell to balstocyst and in a significant reprogramming of gene expression profiles [17]. Furthermore, either the down-regulation of the expression of RTencoding LINE-1 elements by RNA interference (RNAi) or the pharmacological inhibition of RT activity results in a reversible decrease of the rate of cell growth as well as in the induction of cell differentiation in several human tumor cell lines such as thyroid, breast, colon, lung, prostate carcinoma and melanoma cells [16,18-19]. Inhibition of RT induces a significant reprogramming of gene expression that seems to be specific for each cell type and is responsible for the commitment of cell to differentiate. E-cadherin gene, for instance, is markedly up-regulated in RT-inhibited A375 melanoma cells, while two marker genes of the differentiated prostatic epithelia, i.e. the prostate-specific antigen PSA and androgen receptor genes, are induced in response to RT inhibitors in prostate androgen-independent PC3 carcinoma cells [18]. Furthermore, in vivo inhibition of RT activity significantly antagonized tumor growth in athymic xenografts of several human tumors and cell pretreatment with RT inhibitors attenuated the tumorigenic phenotype of highly tumorigenic prostate carcinoma cells inoculated in athymic mice [18]. These data together question the traditional view that retroelements are biologically “inert” genome components, devoid of functions. Rather, the retrotransposon/retroviral machinery emerges as a dynamic genetic component with a functional role in the program of cell division and differentiation. The encouraging findings obtained with RT inhibitory drugs in tumor cells prompted us to investigate whether pharmacological modulation of the endogenous RT activity may represent a novel approach in order to inhibit tumor growth and induce differentiation in undifferentiated thyroid cancer cells. We have previously observed that anaplastic thyroid cancer cells lines treated with RT inhibitors gain morphological and molecular elements of cell differentiation and re-express several products specific of normal thyrocytes, such as TSH receptor, thyroglobulin, TPO and NIS [19]. In the present study, we extended our analysis to primary cultures obtained from human normal thyroid and poorly differentiated thyroid tumors and we report that, indeed, efavirenz and nevirapine induce cell differentiation and an extensive reprogramming of gene expression in these human-derived primary tumor cell lines. Moreover, RT inhibitors restore the TSH-dependent uptake of radioactive iodine in vitro and reversibly down-regulate cell proliferation in anaplastic thyroid tumor ARO cells either in vitro or in vivo These results suggest that endogenous RT

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may represent a novel potential target in the therapy of human undifferentiated thyroid tumors.

MATERIALS AND METHODS

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Cell Cultures Primary cultures were obtained during the surgical removal of undifferentiated papillary thyroid unifocal carcinomas. Tissue samples were collected from the tumors and from the normal non-infiltrated thyroid glands and were processed as previously reported [20]. Either these primary cultures or human thyroid carcinoma ARO cells were cultured in DMEM containing 10% fetal bovine serum (Sigma-Aldrich, Italy), glutamine (Sigma-Aldrich, Italy) and 1X Penicillin/Streptomycin (Sigma-Aldrich, Italy) [19]. Human rTSH (Sigma-Aldrich, Italy) was used at the concentration of 2 mU/ml [21]. ATRA was purchased by SigmaAldrich (Italy) and used at the concentration of 1 μM. Nevirapine and efavirenz were purified from commercially available Viramune (Boehringer-Ingelheim) and Sustiva (Bristol-Myers Squibb), dissolved in DMSO and used at concentration reported in the Results. Five-six hours after seeding, nevirapine or efavirenz, or the same DMSO volume (0,2%, controls) were added to the cultures. Incubation was carried out continuously, RT inhibitor-containing fresh medium was changed at 48 h intervals. The evaluation of the rate of cell growth, apoptosis and necrosis were performed as previously reported [18-19]. Cells were analyzed by fluorescence LEICA DM RXA2 microscope (Leica, Germany). Results of both apoptosis and necrosis are reported as percentage of total cells and represent the average (± SD) of three samples. For morphological evaluation, cells were cultured in presence of nevirapine or efavirenz for 4 days, harvested, counted and replated at high density (500.000 cells/wells) in 6-well plates and further incubated in the same medium for 2 days. Photographs have been obtained by phase contrast Leica DM IRB microscope (Leica, Germany).

Indirect Immunofluorescence (IF) and Confocal Laser Scanning Microscopy Treated and untreated cells were fixed with 4% para-formaldehyde for 30 min and permeabilized in 0.1% Triton-X 100 and 0.1% Tween 20 in PBS containing 5% Bovine Serum Albumin (Sigma-Aldrich, Italy) for 1 h (Fluorescence Blocking Solution). IF staining was performed using mouse monoclonal antibody against human thyroglobulin (Sigma, Milan, Italy) and NIS (Chemicon International, USA) and revealed by FITC-conjugated IgG secondary antibody (Sigma-Aldrich, Italy). Samples were imaged under a confocal NIKON Eclipse TE 2000-S microscope (Nikon, USA). The excitation and emission wavelengths were 488 nm and 510 nm.

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Iodine Uptake Assay Iodine uptake assay was performed as reported by Schmutzler et al. [21]. In brief, ARO cells were incubated in presence and absence of 10 μM efavirenz or 350 μM nevirapine for 10 days, harvested, counted, plated in 24-well plates and, further incubated in the same conditions in presence and absence of 2 mU/ml human rTSH for 48 h. For the assay, the medium was removed and washed with 1 ml HBSS (137 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl2, 0.4 mM MgSO4, 0.5 mM MgCl2, 0.4 mM Na2HPO4, 0.44 mM KH2PO4, 5.55 mM glucose, 10 mM Hepes, pH 7.3). Cells were overlayed with HBSS containing 10 μM NaI and carrier free Na125I to give a specific activity of 20 mCi/mmol. To control the specific uptake some of the reactions received this assay buffer supplemented with the NIS inhibitor NaClO4 (10 μM) After 30 min at 37°C in a humid atmosphere, the radioactive medium was removed, cells were washed with ice-cold HBSS and accumulated iodine was extracted at -20°C with 1 ml ethanol. Ethanol extracts were counted in Packard Cobra II auto-gamma counter (PerkinElmer, USA). In parallel cell cultures, incubated in the same conditions, cells were harvested and counted in a Burker chamber. Iodine up-take was normalized by cell numbers and expressed as percentage of the respective unstimulated control. Results represent the average (± SD) of three experiments, each repeated in quadruplicate.

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RNA Extraction and Semiquantitative RT-PCR Analysis Total RNA was extracted using the Trizol Reagent according to the manufacturer’s procedures. (Invitrogen, Italy). For the first strand synthesis of cDNA, 5 μg of RNA were used in a 20 μl reaction mixture utilizing a cDNA Superscript III (Invitrogen, Italy) according to the supplier’s instructions. A volume of 1 μl from each mixture was withdrawn and amplified using the Taq Gold DNA Polymerase kit (Applied Biosystems, Italy) in a Gene AMP PCR System 9700 Thermal Cycler (Applied Biosystems, Italy). Reaction conditions were 94° C for 10 min, followed by 35 cycles of 30 s at 94°C, 30 s at 58°C, 5 min at 72°C and 7 min at 72°C. β−actin was chosen as internal control. The following primers were utilized: Pendrin, upstream 5’-CCATTGTCGTCTGTATGGCAG-3’, downstream 5’CCTACTGACACTGCA-3’, (271 bp); β-actin, upstream 5’-GGCATCGTGATGGACTCCG3’, downstream 5’-GCTGGAAGGTGGACAGCGA-3’ (612 bp). PCR products were resolved on 1.8% agarose gels and visualized by ethidium bromide staining.

Tumors Xenografts and Animals Treatment Athymic nude mice (Harlan, Italy) four-week old, were kept in accordance with European community guidelines. Mice were inoculated sub-cutaneously in the lower back with ARO cells (1x106/mouse) suspended in PBS (100 μl). Mice were injected subcutaneously with 20 mg/kg efavirenz using a stock solution in DMSO (4 μg/μl) freshly diluted 1:1 with physiological solution, starting one day after tumor xenograft. Routinely, animals were daily injected with the RT inhibitor five days a week. Control mice were

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injected under the same conditions using a 50% DMSO solution. In order to evaluate whether the ability of efavirenz to inhibit tumor growth in vivo is reversible, in some experiments the treatment with efavirenz was discontinued 14 days after tumor injection [18]. Tumor growth was monitored by caliper measurements. Tumor volume was calculated using the formula length x width x height x 0.52 and expressed as percentage of untreated controls [18].

RESULTS RT Inhibitors Induce a Differentiated Phenotype in Primary Cultures of Poorly Differentiated Human Thyroid Papillary Tumor Cells We have previously demonstrated that the pharmacological inhibition of RT induce cell differentiation in established anaplastic thyroid tumor cell lines as well as in several other human tumor cell models [18-19]. Therefore, we used primary cultures of undifferentiated thyroid tumors to evaluate whether efavirenz and nevirapine may represent differentiating agents in human-derived tumor cells. Primary cultures of normal human thyrocytes were used as controls.

Thyroc ytes

Undiff erentiated papillary carcinoma cells

Anaplastic carcinoma ARO cells

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Control

Nevirapine

Ef avirenz

Figure 1. RT inhibitors induce the expression of thyroglobulin in primary cultures of undifferentiated papillary thyroid tumor cells. Primary cultures of normal thyrocytes and undifferentiated papillary thyroid tumor cells as well as anaplastic thyroid carcinoma ARO cells were incubated in the presence of DMSO (Control), 350 μM nevirapine or 10 μM efavirenz for 10 days, fixed, permeabilized and stained by a mouse monoclonal antibody against human thyroglobulin. Specific signal was revealed by FITC-conjugated IgG secondary antibody and imaged under a confocal NIKON microscope.

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Indeed, thyroglobulin is a gene highly expressed in normal thyroid cells and regulated by TSH receptor signaling [1,22]. It is well known that undifferentiated thyroid tumor cells are characterized by reduced or absent expression of TSH receptors as well as reduced expression of TSH-dependent genes, such as thyroglobulin [1]. Thus, primary cultures obtained for normal thyroid gland, undifferentiated thyroid tumor as well as anaplastic thyroid carcinoma ARO cells were exposed to efavirenz and nevirapine for 10 days, stained with anti-thyroglobulin antibodies and analyzed by indirect immunofluorescence. As reported in Figure 1, immunofluorescence analysis demonstrated that cells derived from normal thyroid gland exhibited detectable expression of thyroglobulin, whereas undifferentiated thyroid tumor and ARO cells expressed negligible levels of thyroglobulin. By contrast, exposure to efavirenz or nevirapine resulted in a major increase of thyroglobulin levels in both tumor cell lines, while normal thyrocytes exhibited only a minor upregulation of thyroglobulin expression. Moreover, since we previously observed morphological signs of cell differentiation in human melanoma and prostate carcinoma cells treated with RT inhibitors [18], we further investigated the induction of cell differentiation in response to RT inhibitors at morphological levels in undifferentiated thyroid tumor cells. Indeed, thyroid tumor ARO cells exhibited several morphological features representative of anaplastic tumors, such as high proliferation rate and formation of cell clusters organized into multilayer populations (Figure 2). Therefore, cells were incubated in the presence and absence of 350 μM nevirapine or 10 μM efavirenz for 4 days, harvested, replated at high density and further cultured for 2 days in the same conditions. Interestingly, exposure of anaplastic tumor cells to nevirapine or efavirenz resulted in a more flattened phenotype, an increase in cell adhesion and restoration of monolayer cell growth with a significant reduction in cluster formation (Figure 2). These results suggest that pharmacological inhibition of RT can facilitate the onset of cell differentiation in undifferentiated thyroid tumor cells.

Figure 2. RT inhibitors induce morphological differentiation in anaplastic thyroid carcinoma ARO cells. ARO cells were cultured in the presence of DMSO (Control), 350 μM nevirapine or 10 μM efavirenz for 4 days, harvested, counted and replated at high density in 6-well plates and further incubated in the same medium for 2 days. Photographs have been obtained by phase contrast microscope using 40X objectives.

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RT Inhibitors Restore the Ability to Upregulate NIS and Pendrin Expression in Poorly Differentiated Human Thyroid Papillary Tumor Cells It is well established that, while normal thyroid cells and differentiated thyroid tumors express NIS, a gene involved in iodine uptake and under the control of TSH receptor signaling [22], poorly differentiated and anaplastic thyroid tumors are devoid of NIS expression [1-2,22]. Since RT inhibitors induced cell differentiation in primary cultures obtained from human undifferentiated thyroid tumors, we further evaluated whether efavirenz and nevirapine are able to upregulate NIS expression in a TSH-dependent manner. Therefore, poorly differentiated papillary thyroid tumor cells as well as anaplastic thyroid tumor ARO cells were exposed to both RT inhibitor for 10 days, further stimulated by TSH in the presence and absence of either drug for another 2 days, stained with anti-NIS antibodies and analyzed by indirect immunofluorescence. Untreated tumor cells expressed undetectable levels of NIS (data not shown) and were not able to upregulate the NIS gene in response to TSH (Figure 3A). By contrast, the basal expression of NIS was significantly induced by TSH in nevirapine- and efavirenz-pretreated cells (Figure 3A). Recently, the characterization of Pendred syndrome, a hereditary disease characterized by neurosensorial deafness and goiter, allowed the identification of pendrin, a chloride/iodide transporter mainly expressed in the thyroid and involved in iodide transport at the apical membrane of the thyrocytes [23-24]. Indeed, modifications in the pendrin gene transcript have been demonstrated in undifferentiated thyroid tumors [25]. Therefore, we questioned whether the cell differentiation obtained by the pharmacological inhibition of RT correlated with the up-regulation of pendrin gene expression. This was evaluated by semiquantitative RT-PCR in primary cultures of poorly differentiated thyroid tumor cells and in anaplastic thyroid carcinoma ARO cells exposed to 350 μM nevirapine for 24 and 48 h. We observed that nevirapine treatment induced a significant up-regulation of pendrin gene expression (Figure 3B). These data suggest that the differentiated phenotype induced by the pharmacological inhibition of RT is characterized by an extensive reprogramming of gene expression with reactivation of TSH signaling and re-expression of several products of differentiated thyrocytes.

RT Inhibitors Restore the Ability to Accumulate Radioactive Iodine in Response to TSH in Human Undifferentiated Thyroid Tumors Normal thyroid cells are able to accumulate iodine due to the expression of NIS and pendrin, two genes under the control of TSH receptor signaling [22,24]. Differentiated thyroid tumors retain these features that are widely utilized for diagnostic and therapeutic purposes. Indeed, radio-metabolic therapy with 131I is an effective treatment for differentiated thyroid cancer. By contrast, poorly differentiated and anaplastic thyroid tumors are resistant to radioiodine therapy because of the lack of NIS expression [1]. Since efavirenz and nevirapine are able to restore the ability of TSH to induce NIS and pendrin expression in poorly differentiated thyroid tumor cells, we used ARO cells treated with RT inhibitors to study their ability to induce radioactive iodine uptake. Cells were exposed to RT inhibitors

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for 10 days, stimulated for 48 h with TSH in order to obtain the induction of the NIS gene and further incubated in the presence of radioactive iodine. ARO control cells exhibited minimal ability to accumulate iodine in response to TSH stimulation (Figure 3C). Interestingly, the pretreatment of cells with efavirenz and nevirapine elicited a strong increase in iodine uptake in response to TSH by about 20-25 times. Consistently with the observed induction of NIS expression in response to TSH, the efavirenz- and nevirapine-dependent upregulation of TSH-stimulated iodine uptake was sensitive to the NIS inhibitor, sodium perchlorate (Figure 3C). This ability of RT inhibitors to restore iodine uptake in undifferentiated thyroid tumor cells suggests that these drugs may tentatively be used to enhance the sensitivity to radiometabolic therapy in vivo.

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RT Inhibitors Reversibly Down-Regulate the Rate of Cell Growth in Vitro and in Vivo in Human Anaplastic Thyroid Tumor Cells Since RT inhibitors have been proven to induce cell differentiation in thyroid tumor cells and to exert a cytostatic activity in several human tumor cell lines [16,18], we evaluated the antiproliferative activity of efavirenz and nevirapine in anaplastic human thyroid tumor cells. Thus, ARO cells were cultured in presence and absence of 10 μM efavirenz or 350 μM nevirapine for 10 days, harvested and counted. Both untreated control and pretreated cells were replated at equal density and further incubated in the presence of efavirenz and nevirapine or in a drug-free medium, harvested every two days and counted. As reported in Figure 4A, the growth rate of ARO cells was significantly inhibited by either drugs. The withdrawal of RT inhibitors resulted in a prompt recovery of cell proliferation with a rate of cell growth similar to control cells. In parallel cell cultures ARO cells were incubated in presence and absence of 5, 10 and 20 μM efavirenz or 200, 350 and 500 μM nevirapine, harvested after 72 h and stained with Hoechst 33258 and propidium iodate to reveal the rate of apoptosis and necrosis. We did not observe any increase in the rate of apoptosis or necrosis in presence of 10 μM efavirenz or 350 μM nevirapine. By contrast, a 15-20% and 5-10% induction of, respectively, apoptosis and necrosis was induced by higher doses of either drugs (Table I). Interestingly, these non-cytotoxic and differentiating concentrations of efavirenz (10 μM) and nevirapine (350 μM) are in the therapeutic range of both drugs [19]. These findings suggest that the pharmacological inhibition of RT activity results in a significant but reversible down-regulation of the rate of cell growth in vitro in undifferentiated thyroid cancer cells. Since ARO cells are well known to be highly tumorigenic in host animals [26], we evaluated the ability of efavirenz to inhibit thyroid tumor growth in vivo, as previously demonstrated for prostate, colon and lung carcinomas as well as for melanoma cells [18]. After inoculation of ARO tumor cells in athymic mice, the animals were subjected to anti-RT treatment with efavirenz and the tumor size was determined after 28 d. The optimal dose of efavirenz (20 mg/kg body weight) was previously determined in dose-response experiments testing 4 to 40 mg/kg of the drug. Within this dose range efavirenz treatment proved to be safe for all animals and no animal death related to the treatment was observed [18]. In the present study animals were treated by subcutaneous injection with 20 mg/kg b.w efavirenz (5

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days a week), starting one day after tumor inoculation. A 50% reduction of the growth rate was recorded in efavirenz-treated mice compared to untreated tumors (Figure 4B). We also evaluated ARO cell-derived tumor growth in animals treated with efavirenz starting one day after the inoculation, but with discontinuation of the treatment after 14 days. Accordingly with the results obtained in vitro, these experiments showed that inhibition of RT-dependent tumor growth is reversible in vivo, since it is resumed in inoculated animals in which the drug treatment was interrupted (Figure 4B). These data confirm the findings from the in vitro experiments and indicate that RT inhibition reversibly antagonizes the growth of anaplastic thyroid tumor xenografts.

ATRA Does not Exert Additive Effect on the Inhibition of Cell Proliferation in Anaplastic Thyroid Carcinoma Cells Cultured with RT Inhibitors

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One of the earliest compounds tested as differentiating agent in anaplastic thyroid carcinoma cells was ATRA. Indeed, ATRA is able to inhibit cell proliferation in some thyroid tumor cell lines and to elicit the upregulation of NIS expression in ARO cells [21,27]. Therefore, we evaluated whether the simultaneous exposure of ARO cells to RT inhibitors and ATRA resulted in an additive effect on the inhibition of cell proliferation. Cells were cultured in presence and absence of 350 μM nevirapine, 1 μM ATRA or both these agents. ARO cells treated with nevirapine exhibited a strong inhibition of cell proliferation, while cells exposed to ATRA exhibited a proliferation rate similar to untreated control cells. Interestingly, the simultaneous exposure of thyroid tumor cells to nevirapine and ATRA did not elicit a response greater than that obtained by nevirapine alone. This finding suggest that ATRA is not able to synergize with the antiproliferative activity of RT inhibitors. Table 1. Analysis of cell viability in ARO cells exposed to increasing concentration of efavirenz (EFV) and nevirapine (NVR).

95.5±1.7

5 μM 10 μM EFV EFV 95.7±1.3 92.8±1.9

20 μM EFV 68.7±1.3

200 μM NVR 95.4±1.9

350 μM NVR 91.8±2.3

500 μM NVR 83.8±1.7

3.6±0.3 0.9±0.1

3.5±0.7 0.8±0.4

22.3±0.9 9.0±1.1

3.2±0.5 1.4±0.3

5.2±0.7 3.0±0.5

10.2 ±1.1 6.0±0.5

Control ARO Cells

Viable cells Apoptosis Necrosis

5.6±0.5 1.6±0.3

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Figure 3. RT inhibitors upregulate NIS and pendrin gene expression and restore iodine uptake in undifferentiated thyroid tumor cells. A. Primary cultures of undifferentiated papillary thyroid tumor cells and anaplastic thyroid carcinoma ARO cells were incubated in the presence of DMSO (Control), 350 μM nevirapine or 10 μM efavirenz for 10 days, and, then, stimulated by TSH in the presence and absence of either drug for another 2 days, stained with anti-NIS antibodies and analyzed by indirect immunofluorescence. B. Total RNA was extracted from ARO cells incubated for 24 and 48 h in the presence and absence of 350 mM nevirapine (NVR) and amplified by semiquantitative RT-PCR, using primers specific for pendrin. β−actin was used as internal standard. C. ARO cells were incubated in the presence and absence of 10 μM efavirenz or 350 μM nevirapine for 10 days, harvested, counted, plated in 24-well plates and further incubated in the same conditions in the presence and absence of 2 mU/ml human rTSH for 48 h. For the assay, the medium was removed and incubated in HBSS containing 10 μM NaI and carrier free Na125I. Some of the reactions received the assay buffer supplemented with the NIS inhibitor NaClO4, to control the specific uptake. Accumulated iodine was extracted at -20°C with ethanol and counted in a gamma counter. Iodine uptake was normalized by cell numbers and expressed as percentage of the respective unstimulated control.

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Cell Number (x1000)

A 4500

Control

4000

Efavirenz

3500

Nevirapine

3000

Efavirenz withdrawal Nevirapine withdrawal

2500 2000 1500 1000 500 0 0

2

4

6

Days

120 100 % of control

B

80 60 40 20 0 Control

C

Cell Number (x1000)

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3000 2500 2000

Nevirapine

Nevirapine 14 g

Control NVR Retinoic Ac. NVR + Retinoic Ac.

1500 1000 500 0 0

2

4

6

Days

Figure 4. RT inhibitors reversibly downregulate cell proliferation in vitro and tumor growth in vivo in anaplastic thyroid tumor cells. A. Thyroid anaplastic carcinoma ARO cells were cultured in presence and absence of 10 μM efavirenz or 350 μM nevirapine for 10 days, harvested and counted. Either control or pretreated cells were replated at equal density and further incubated in presence of efavirenz and nevirapine or in a drug-free medium, harvested every two days and counted in a Burker chamber. Data are reported as absolute cell numbers. B. ARO cells were inoculated in athymic mice and injected five days a week with 20 mg/kg efavirenz starting one day after tumor xenografts or with DMSO. In a group of animals, the treatment was discontinued 14 days after tumor injection. Tumor size was determined after 28 d by caliper measurements and reported as percentage of the volume of untreated control. C. Lack of synergism between ATRA and nevirapine in anaplastic thyroid carcinoma cells. ARO cells were cultured in presence and absence of 350 μM nevirapine, 1 μM ATRA or both drugs, harvested every two days and counted in a Burker chamber. Data are reported as absolute cell numbers.

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CONCLUSION The hypothesis of using RT inhibitors as anticancer drugs is based on several epidemiological studies which indicate that Kaposi’s sarcoma [28-29] and other AIDS-related cancers [30] have a reduced incidence in patients treated with highly active antiretroviral therapy (HAART), which is characterized by the combination of RT and protease inhibitors [30]. Although this is generally viewed as a reflection of the improved immune reaction in treated patients, it may also suggest a direct inhibitory effect of HAART on endogenous RT activity in tumor cells [30]. The results reported here support the hypothesis that endogenous RT may represent a functional “marker” of the cellular machinery associated with high proliferation and loss of differentiation; the inhibition of elevated RT levels by pharmacological means appears to be sufficient to trigger cell differentiation in vitro and reduce tumor growth in vivo. Interestingly, the reprogramming of gene expression obtained by the pharmacological inhibition of RT seems to selectively target key genes specific for each cell type. Indeed, the exposure of undifferentiated thyroid tumor cells to efavirenz and nevirapine elicited the expression of specific products of normal thyroid cells, such as thyroglobulin, NIS and pendrin. Similarly, the treatment of melanoma and prostate cancer cells with RT inhibitors or the downregulation of RT gene expression by RNAi in melanoma cells resulted in a significant reprogramming of gene expression which includes key genes involved in regulating cell proliferation and differentiation, such as androgen receptor, PSA and E-cadherin genes [18]. Moreover, the modulation of specific marker genes induced by RT inhibition seems directly correlated with the degree of morphological differentiation and the reduced rate of cell proliferation [18]. We have previously demonstrated that pharmacological inhibition of RT produced the induction of cell differentiation in established anaplastic thyroid tumor cell lines [19]. The present study extent these observations to human-derived primary cultures of undifferentiated papillary thyroid carcinoma suggesting that RT inhibitors may represent novel differentiating agents for the treatment of thyroid tumors. It is noteworthy for clinical purposes that pharmacological inhibition of RT resulted in the re-establishment of TSH signaling and in the upregulation of TSH-dependent genes responsible for thyroid-specific products, such as thyroglobulin, pendrin and NIS. Even more relevant is the observation that the reprogramming of gene expression and the differentiation process obtained by exposing anaplastic thyroid tumor cells to either efavirenz or nevirapine re-established iodine uptake in a TSH-dependent manner. Indeed, while differentiated thyroid epithelial carcinomas are characterized by the maintenance of cellular functions that are typical traits of normal thyroid follicular cells and contribute to their slow growth rate and low propensity to form distant metastases, poorly differentiated and anaplastic thyroid tumors are devoid of most of these cellular features and are characterized by aggressive biological behavior, short clinical doubling time and high metastatic potential [1]. The degree of differentiation within thyroid tumors correlates with the probability of a positive response to therapeutic options that utilize thyroid-specific functions, such as iodine uptake. Indeed, differentiated thyroid tumor cells express cell membrane receptors for TSH with functional transduction machinery capable of eliciting a second messenger cascade. These polypeptides are involved in the activation of cell cycle progression, the elaboration of thyroglobulin and the production and membrane-

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targeting of NIS. Based on these molecular features, therapeutic protocols have been designed, in which patients are transiently stimulated with recombinant TSH in order to upregulate 131I delivery to malignant cells for both diagnostic and therapeutic purposes [1]. Indeed, radiometabolic treatment is a very effective therapy that can lead to complete remission of differentiated thyroid tumors, even in the presence of distant metastases [1]. On the contrary, dedifferentiation of anaplastic thyroid cancer cell results in the diminished expression of TSH receptors and impaired signal transduction after receptor activation. The loss of thyroid-specific functions, as well as the inability to express NIS and concentrate radioactive iodine, impedes both the diagnostic and therapeutic efforts described above [1]. Therefore, the data reported in the present study strongly support the hypothesis that the reprogramming of gene expression induced in response to efavirenz and nevirapine may represent a new tool in the management of undifferentiated thyroid tumors. Interestingly, besides normal thyroid cells and differentiated thyroid tumors, several other tissues express NIS at low levels, but this appears to be insufficient to retain radioiodine within the tumor cells long enough to deliver tumoricidal radiation doses, as shown by NIS transfection studies [31-32]. Retention requires the organification of iodine, mediated by a differentiation-dependent thyroid-specific product, TPO [1]. Thyroid TPO expression is diminished by malignant transformation [33] and may account for the rapid loss of accumulated radioiodine, which is probably responsible for some treatment failures [34]. The ability of RT inhibitors to re-establish functional TSH signaling by simultaneously inducing the expression of TSH receptor, thyroglobulin, pendrin and TPO genes [19] and the ability to respond to TSH stimulation with an active transduction machinery capable of eliciting the upregulation of NIS expression and iodine uptake suggest that inhibition of RT in undifferentiated thyroid tumors may be a novel molecular-targeted differentiating treatment which may be tentatively used to restore sensitivity to radiometabolic therapy. Thus, specifically-designed clinical trials are needed to evaluate this hypothesis. Several other pharmacological agents have been tested in undifferentiated thyroid tumors with the aim of inducing differentiation, facilitating iodine uptake or inhibiting cell growth [1,35]. One of the earliest compounds advocated for this purpose was ATRA, but the results obtained are conflicting. Interestingly, ATRA elicited a strong upregulation of NIS expression in ARO cells but failed to induce iodine uptake in vitro [21]. Moreover, careful analysis of results reported aroused significant skepticism, since in vivo studies have suggested that retinoids inhibit TPO and thyroglobulin expression and do not restore iodine uptake, despite increased NIS mRNA [35]. Moreover, our results showed that while cell proliferation was significantly down-regulated in the presence of nevirapine, ATRA did not exert any cytostatic activity in ARO cells. Indeed, the simultaneous exposure of thyroid tumor cells to RT inhibitors and ATRA did not elicit any synergism of action, at least in anaplastic thyroid tumor cell lines that are devoid of retinoic acid receptor β (RARβ) which is likely responsible for the differentiating and anti-proliferative activity of ATRA [27]. Recently, the selective tyrosine kinase inhibitor STI571 (imatinib), which is remarkably effective in treating chronic myeloid leukemia and metastatic gastrointestinal stromal tumors, has been tested in anaplastic thyroid tumor cells with conflicting results. While Podtcheko et al. suggested that STI571 may induce a significant inhibition of p53 mutant anaplastic thyroid cancer cells by inducing S-G2 transition arrest [26], other authors indicated that

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imatinib has negligible antineoplastic activity against anaplastic thyroid cancer cell lines within established therapeutically useful concentrations [36]. Somatostatin and its analog octreotide have been extensively studied as antiproliferative drugs in undifferentiated thyroid tumors, but it has been established that they have no effect on the growth rate of this neoplasm either in vitro or in vivo [35]. Finally, histone deacetylase inhibitors have been recently demonstrated to induce the expression of thyroid specific genes and induce radioiodine accumulation in anaplastic thyroid tumor cells [37]. It is intriguing that both RT inhibitors and histone deacethylase inhibitors induce a similar reprogramming of gene expression in anaplastic thyroid tumor cells. Since the silencing of both TSH receptor [38] and NIS [39] genes as well as androgen receptor [40] and E-cadherin genes [41] is dependent, at least in part, on gene hypermethylation in tumor cells, it appears possible that the activity of RT inhibitors may be mediated by the redistribution of DNA methylation and chromatin remodeling. However, further studies are needed to better characterize the molecular mechanism(s) through which RT activity can instruct gene expression and cell fate.

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REFERENCES [1] Ain, KB. Management of undifferentiated thyroid cancer. Clin Endocrinol Metab, 2000 4, 615-629. [2] Ain, KB; Egorin, MJ; DeSimone, PA. Treatment of anaplastic thyroid carcinoma with paclitaxel: phase 2 trial using ninety-six-hour infusion. Collaborative Anaplastic Thyroid Cancer Health Intervention Trials (CATCHIT) Group. Thyroid, 2000 10(7), 587-594. [3] Deininger, PL; Moran, JV; Batzer, MA; Kazazian, HH. Mobile elements and mammalian genome evolution. Curr Op Gen Dev, 2003 13, 651-658. [4] Spadafora, C. Endogenous reverse transcriptase: a mediator of cell proliferation and differentiation. Cytogenet Genome Res, 2004 105, 346-350. [5] Poznanski, AA; Calarco, PG. The expression of intracisternal A particole genes in the preimplantation mouse embryo. Dev Biol, 1991 143, 271-281. [6] Packer, AI; Manova, K; Bacharova, RF. A discrete LINE-1 transcript in mouse blastocysts. Dev Biol, 1993 157, 281-283. [7] Mwenda, JM. Biochemical characterization of a reverse transcriptase activity associated with retroviral-like particles isolated from human placental villous tissue. Cell Mol Biol, 1993 39, 317-328. [8] Kiessling, AA; Crowell, R; Fox, C. Epididymis is a principal site of retrovirus expression in the mouse. Proc Acad Natl Sci USA, 1989 86, 5109-5113. [9] Branciforte, D; Martin, SL. Developmental and cell-type specificity of Line-1 expression in mouse testis - implications for transposition. Mol Cell Biol, 1994 14, 2584-2592. [10] Nilsson, BO; Kattstrom, PO; Sundstrom, P; Jaquemin, P; Larsson, E. Human oocytes express murine retroviral equivalents. Virus Genes, 1992 6, 221-227. [11] Giordano, R; Magnano, AR; Zaccagnini, G; Pittoggi, C; Moscufo, N; Lorenzini, R; Spadafora, C. Reverse transcriptase activity in mature spermatozoa of mouse. J Cell Biol, 2000 148, 1107-1113.

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[12] Sciamanna, I; Barbieri, L; Martire, A; Pittoggi, C; Beraldi, R; Giordano, R; Magnano, AR; Hogdson, C; Spadafora, C. Sperm endogenous reverse transcriptase as mediator of new genetic information. Biochem Biophys Res Commun, 2003 312, 1039-1046. [13] Deragon, JM; Sinnett, D; Labuda, D. Reverse transcriptase activity from human embryonal carcinoma cells NTera2D1. EMBO J 1990 9, 3363-3368. [14] Martin, SL. Ribonucleoprotein particles with LINE-1 RNA in mouse embryonal carcinoma cells. Mol Cell Biol 1991 11, 4804-4807. [15] Ren, J; Nichols, C; Bird, L; Chamberlain, P; Weaver, K; Short, S; Stuart, DI; Stammers, DK. Structural mechanisms of drug resistance for mutations at codons 181 and 188 in HIV-1 reverse transcriptase and the improved resilience of second generation nonnucleoside inhibitors. J Mol Biol, 2001 312, 795-805. [16] Mangiacasale, R; Pittoggi, C; Sciamanna, I; Careddu, A; Mattei, E; Lorenzini, R; Travaglini, L; Landriscina, M; Barone, C; Nervi, C; Lavia, P; Spadafora, C. Exposure of normal and transformed cells to nevirapine, a reverse transcriptase inhibitor, reduces cell growth and promotes differentiation. Oncogene, 2003 22, 2750-2761. [17] Pittoggi, C; Sciamanna, I; Mattei, E; Beraldi, R; Lobascio, AM; Mai, A; Quaglia, MG; Lorenzini, R; Spadafora, C. Role of endogenous reverse transcriptase in murine early embryo development. Mol Reprod Dev, 2003 66, 225-236. [18] Sciamanna, I; Landriscina, M; Pittoggi, C; Quirino, M; Mearelli, C; Beraldi, R; Mattei, E; Serafino, A; Cassano, A; Sinibaldi-Vallebona, P; Garaci, E; Barone, C; Spadafora, C. Inhibition of endogenous reverse transcriptase antagonizes human tumor growth. Oncogene, 2005 24, 3923-3931. [19] Landriscina, M; Fabiano, A; Altamura, S; Bagalà, C; Piscazzi, A; Cassano, A; Spadafora, C; Giorgino, F; Barone, C; Cignarelli M. Reverse transcriptase inhibitors downregulate cell proliferation in vitro and in vivo and restore TSHlaala signaling and iodine uptake in human thyroid anaplastic carcinoma. J Clin Endocrinol Metab 2005 90, 5663-5671. [20] Gianoukakis, AG; Cao, HJ; Jennings, TA; Smith, TJ. Prostaglandin endoperoxide H synthase expression in human thyroid epithelial cells. Am J Cell Physiol, 2001 280(3), C701-C708. [21] Schmutzler, C; Winzer, R; Meissner-Weigl, J; Kohrle, J. Retinoic acid increases sodium/iodide symporter mRNA levels in human thyroid cancer cell lines and suppresses expression of functional symporter in nontransformed FRTL-5 rat thyroid cells. Biochem Biophys Res Commun, 1997 240(3), 832-838. [22] Postiglione, MP; Parlato, R; Rodriguez-Mallon, A; Rosica, A; Mithbaokar, P; Maresca, M; Marians, RC; Davies, TF; Zannini, MS; De Felice, M; Di Lauro, R. Role of the thyroid-stimulating hormone receptor signaling in development and differentiation of the thyroid gland. Proc Natl Acad Sci USA, 2002 99(24), 15462-15467. [23] Everett, LA; Glaser, B; Beck, JC; Idol, JR; Buchs, A; Heyman, M et al. Pendred syndrome in caused by mutations in a putative sulphate transport gene (PDS). Nature Genetics, 1997 17, 411-422. [24] Scott, DA; Wang, R; Kreman, TM; Sheffield, VC; Karniski, P. The Pendred syndrome gene encodes a chloride-iodide transport protein. Nature Genetics, 1999 21, 440-443.

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[25] Bidart, JM; Mian, C; Lazar, V; Russo, D; Filetti, S; Caillou, B et al. Expression of pendrin and Pendred syndrome (PDS) gene in human thyroid tissues. J Clin Endocrinol Metab, 2000 85, 2028-2033. [26] Podtcheko, A; Ohtsuru, A; Tsuda, S; Namba, H; Saenko, V; Nakashima, M; Mitsutake, N; Kanda, S; Kurebayashi, J; Yamashita, S. The selective tyrosine kinase inhibitor, STI571, inhibits growth of anaplastic thyroid cancer cells. J Clin Endocrinol Metab, 2003 88(4), 1889-1896. [27] Elisei, R; Vivaldi, A; Agate, L; Ciampi, R; Molinaro, E; Piampiani, P; Romei, C; Faviana, P; Basolo, F; Miccoli, P; Capodanno, A; Collecchi, P; Pacini, F; Pinchera, A. All-trans-retinoic acid treatment inhibits the growth of RARβ mRNA expressing thyroid cancer cell lines but does not reinduce the expression of thyroid specific genes. J Clin Endocrinol Metab, 2005 90, 2403–2411. [28] Rabkin, CS. AIDS and cancer in the era of highly active antiretroviral therapy (HAART). Eur J Cancer, 2001 37(10), 1316-1319. [29] Jones, JL; Hanson, DL; Dworkin, MS; Jaffe, HW. Incidence and trends in Kaposi's sarcoma in the era of effective antiretroviral therapy. J Acquir Immune Defic Syndr, 2000 24(3), 270-274. [30] Monini, P; Sgadari, C; Toschi, E; Bacillari, G; Ensoli, B. Antitumour effects of antiretroviral therapy. Nat Rev Cancer, 2004 4(11), 861-875. [31] Shimura, H; Haraguchi, K; Miyazaki, A; Endo, T; Onaya, T. Iodide uptake and experimental 131I therapy in transplanted undifferentiated thyroid cancer cells expressing the Na+/I- symporter gene. Endocrinology, 1997 138(10), 4493-4496. [32] Lee, YJ; Chung, JK; Shin, JH; Kang, JH; Jeong, JM; Lee, DS; Lee, MC. In vitro and in vivo properties of a human anaplastic thyroid carcinoma cell line transfected with the sodium iodide symporter gene. Thyroid, 2004 14(11), 889-895. [33] Takamatsu, J; Hosoya, T; Tsuji, M; Yamada, M; Murakami, Y; Sakane, S; Kuma, K; Ohsawa, N. Peroxidase and coupling activities of thyroid peroxidase in benign and malignant thyroid tumor tissues. Thyroid, 1992 2(3), 193-196. [34] Filetti, S; Bidart, JM; Arturi, F; Caillou, B; Russo, D; Schlumberger, M. Sodium/iodide symporter: a key transport system in thyroid cancer cell metabolism. Eur J Endocrinol, 1999 141(5), 443-457. [35] Haugen, BR. Redifferentiation therapy in advanced thyroid cancer. Curr Drug Targets Immune Endocr Metabol Disord, 2004 4(3), 175-180. [36] Dziba, JM; Ain, KB. Imatinib mesylate (gleevec; STI571) monotherapy is ineffective in suppressing human anaplastic thyroid carcinoma cell growth in vitro. J Clin Endocrinol Metab, 2004 89(5), 2127-2135. [37] Furuya, F; Shimura, H; Suzuki, H; Taki, K; Ohta, K; Haraguchi, K; Onaya, T; Endo, T; Kobayashi, T. Histone deacetylase inhibitors restore radioiodide uptake and retention in poorly differentiated and anaplastic thyroid cancer cells by expression of the sodium/iodide symporter thyroperoxidase and thyroblobulin. Endocrinology, 2004 145(6), 2865-2875. [38] Xing, M; Usadel, H; Cohen, Y; Tokumaru, Y; Guo, Z; Westra, WB; Tong, BC; Tallini, G; Udelsman, R; Califano, JA; Ladenson, PW; Sidransky, D. Methylation of the thyroid-

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stimulating hormone receptor gene in epithelial thyroid tumors: a marker of malignancy and a cause of gene silencing. Cancer Res, 2003 63(9), 2316-2321. [39] Venkataraman, GM; Yatin, M; Marcinek, R; Ain, KB. Restoration of iodide uptake in dedifferentiated thyroid carcinoma: relationship to human Na+/I-symporter gene methylation status. J Clin Endocrinol Metab, 1999 84(7), 2449-2457. [40] Yamanaka, M; Watanabe, M; Yamada, Y; Takagi, A; Murata, T; Takahashi, H; Suzuki, H; Ito, H; Tsukino, H; Katoh, T; Sugimura, Y; Shiraishi, T. Altered methylation of multiple genes in carcinogenesis of the prostate. Int J Cancer, 2003 106(3), 382-387. [41] Tsutsumida, A; Hamada, J; Tada, M; Aoyama, T; Furuuchi, K; Kawai, Y; Yamamoto, Y; Sugihara, T; Moriuchi, T. Epigenetic silencing of E- and P-cadherin gene expression in human melanoma cell lines. Int J Oncol 2004 25(5), 1415-1421.

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INDEX

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A abnormalities, 113, 121, 128, 136 absorption, vii, 1 accidental, 131 accounting, 44, 49, 142, 179 accuracy, 5, 9, 17, 24, 47, 90, 103, 148 acetate, 86 acid, ix, xii, 79, 83, 86, 89, 106, 109, 126, 128, 133, 167, 168, 170, 184, 196, 198, 199 actin, 187, 193 activation, 115, 134, 135, 163, 180, 181, 182, 195 acute, vii, 1, 8, 12, 15, 18, 19, 20, 84, 103, 104 acute myeloid leukemia, 19, 20 adenocarcinoma, 105, 109, 117 adenocarcinomas, 122 adenoma, 82, 83, 84, 86, 87, 91, 93, 94, 95 adenomas, 115, 131, 132 adenopathy, 17 adenoviral vectors, 139 adenovirus, 134, 135, 139 adenylate kinase, 106 adhesion, 176, 189 administration, 5, 18, 22, 23, 25, 29, 39, 133, 134, 138 administrative, 7 adolescence, 16, 26 adolescents, 16, 36, 37, 40 ADP, 106 adult, 2, 80 adults, vii, 1, 16, 29, 36, 41, 92, 171 aerobic, 100

age, viii, x, 2, 13, 14, 15, 16, 19, 20, 22, 32, 34, 36, 39, 41, 47, 48, 56, 61, 62, 65, 68, 73, 74, 90, 111, 113, 116, 119, 127, 128, 130, 163, 166, 169, 172, 173, 174 agent, xii, 84, 184, 192 agents, 88, 89, 132, 134, 184, 188, 192, 195, 196 aggressive therapy, 116 aggressiveness, 52, 163 aid, 64, 117 AIDS, 185, 195, 199 air, 8 air emissions, 8 AKT, 182 alanine, 168 alcohol, 22 allele, 115, 117, 168 alleles, 168 allosteric, 106, 108 alopecia, 18 alpha, 86, 115, 121, 138 alternative, x, 6, 7, 29, 100, 110, 111, 114 alters, 168 Amadori, 110 amino, 106, 109, 126, 128, 167, 168, 170, 182 amino acid, 109, 126, 128, 167, 168, 170 amino acids, 168 amylase, ix, 79, 83 amyloid, 148, 149, 150, 151, 153, 155, 157 amyloidosis, 129, 130, 137, 154 analog, 197 anaplastic transformation, 48, 53 anastomoses, 85 anatomy, 126 androgen, 185, 195, 197 angiogenesis, 85, 86 animal models, xii, 184

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202 animals, 83, 134, 187, 191, 194 antibody, 37, 134, 186, 188 anticancer, 195 anticancer drug, 195 antigen, xi, 126, 131, 135, 141, 143, 146, 185 antigen presenting cells, 135 antineoplastic, 133, 197 antioxidant, 83 antisense, 121 antithyroglobulin antibody, 37 antitumor, 134, 135, 147 APC, 110, 115, 121 aplasia, 19 aplastic anemia, 23 apoptosis, 53, 85, 95, 133, 157, 186, 191 apoptotic, 53 appetite, 18 application, vii, viii, ix, xi, 1, 23, 24, 61, 62, 70, 72, 76, 81, 107, 125, 127, 134 arginine, 164, 170, 172 Armed Forces, 78 arrest, 134, 196 arteries, 95 artery, vii, 43 aspartate, 83 aspirate, 85, 91 aspiration, viii, ix, xi, 9, 17, 24, 31, 43, 44, 57, 58, 79, 80, 85, 88, 89, 90, 91, 92, 93, 95, 96, 97, 98, 118, 119, 141, 143, 148 assessment, 37, 103, 120, 145 asymptomatic, viii, xi, 33, 43, 44, 47, 58, 84, 142, 145, 170, 171, 172, 173, 179 ATF, 128 Atlas, 78 atmosphere, 187 ATP, 106 atypical, 83 auditing, 25 Australia, 23 Austria, 23 autoantibodies, 67 autolysis, 83, 90 autopsy, viii, 15, 43, 44, 45, 46, 58, 172 autosomal dominant, x, 111, 116, 117, 120, 126, 128, 130, 163 availability, 46, 52, 56, 173 avoidance, 6, 22

Index

B banking, 22 barrier, 175 basal layer, 126 bcl-2, 53 behavior, 45, 59, 78, 121, 126, 130, 135 benefits, 17, 108 benign, ix, 23, 79, 83, 84, 85, 86, 87, 88, 89, 90, 91, 93, 94, 95, 96, 103, 107, 108, 113, 115, 116, 117, 118, 119, 143, 148, 199 bias, 15, 73, 113 bile, 105 bile duct, 105 bilirubin, 83 binding, 128, 181 biological behavior, 195 biopsies, 15, 21, 58, 174, 176 biopsy, viii, xi, 9, 17, 24, 28, 31, 43, 44, 57, 80, 92, 97, 98, 101, 118, 141, 143, 148 birth, 22, 23, 40 birth weight, 22 births, 22 bladder, 20 bladder cancer, 20 blocks, 103, 153, 156, 164 blood, xi, 9, 19, 85, 86, 100, 105, 127, 141, 143, 146, 147, 153, 158, 164, 167, 168, 170, 172, 179 blood monocytes, 86 blood supply, 85 body fluid, 100 body weight, 18, 127, 191 bolus, 127 bomb, 45 bonding, 83 bone marrow, 9, 19, 175 bone resorption, 127 bone scan, 177 borderline, 127 bovine, 186 bowel, 20 brain, 13, 15, 36, 74, 100, 105, 132 breakdown, 106 breast cancer, 2, 21, 103, 106, 107, 122 breast carcinoma, 39, 114, 116, 117, 122 bronchus, 20 buffer, 187, 193

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C cadherin, 128, 200 caecum, 87 calcification, 47, 55, 90, 153 calcitonin, x, xi, 111, 112, 125, 126, 127, 129, 131, 132, 133, 134, 138, 141, 142, 143, 146, 153 calcitonin level, 127, 129, 132, 133 calcium, 87, 126, 127, 128 Canada, 23, 41, 46 cancer, vii, xi, xii, 2, 3, 5, 10, 20, 21, 30, 35, 36, 39, 40, 41, 42, 58, 80, 82, 86, 87, 90, 100, 102, 103, 104, 106, 107, 110, 113, 120, 125, 126, 127, 130, 131, 133, 139, 142, 143, 144, 145, 148, 151, 152, 163, 164, 173, 174, 175, 177, 179, 183, 184, 185, 197, 199 cancer cells, xii, 4, 5, 10, 82, 87, 107, 183, 185, 191, 195, 196, 199 cancer treatment, 39, 73, 173 capacity, viii, 21, 61, 62, 78, 135 capillary, 21, 40 capsule, 4, 88, 154, 174, 175, 179 carbohydrate, 109 carbohydrate metabolism, 109 carcinoembryonic antigen, xi, 126, 141, 143, 146 carcinogenesis, 39, 100, 200 carcinogens, 46 carcinoid tumor, 132 carcinomas, viii, x, xi, 33, 43, 45, 46, 48, 53, 57, 85, 87, 105, 107, 108, 109, 111, 112, 117, 121, 125, 128, 184, 186, 191, 195 carrier, 187, 193 cartilage, 146 CAT, 9, 12, 14, 17, 25 CAT scan, 12, 14, 17, 25 catecholamines, 142 cavities, 84, 145 CD8+, 134 cDNA, 110, 182, 184, 187 CEA, 103, 104, 107, 126, 143, 146, 147, 178 cell, vii, viii, x, xi, xii, 10, 11, 32, 33, 38, 53, 61, 62, 74, 75, 77, 78, 83, 84, 85, 86, 93, 101, 105, 106, 108, 110, 111, 112, 115, 121, 122, 125, 127, 129, 131, 133, 134, 135, 136, 139, 156, 159, 160, 163, 172, 173, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200 cell adhesion, 189 cell culture, 115, 187, 191

203 cell cycle, 195 cell death, 85, 134 cell differentiation, xii, 163, 184, 185, 188, 189, 190, 191, 195 cell division, 133, 185 cell fate, 197 cell growth, xii, 134, 184, 185, 186, 189, 191, 196, 198, 199 cell line, xii, 83, 93, 110, 134, 184, 185, 188, 189, 191, 192, 195, 196, 197, 198, 199, 200 cell lines, xii, 83, 93, 110, 134, 184, 185, 188, 189, 191, 192, 195, 196, 197, 198, 199, 200 cell metabolism, 199 central nervous system, 21 cervical carcinoma, 103, 110 cervicitis, 103 cervix, 107 CGC, 167, 170 CGT, 167, 168 chemotherapeutic drugs, 179 chemotherapy, xi, 64, 65, 67, 68, 71, 73, 74, 75, 76, 103, 106, 107, 132, 142, 174, 177 childhood, 16, 22, 26, 36, 127 children, 16, 22, 36, 37, 40, 58, 92, 116, 119, 130, 137, 171, 173 China, 45 chloride, 190, 198 chocolate, 81 cholestasis, 104 cholesterol, 83 chromatin, 90, 150, 154, 156, 157, 162, 197 chromosome, x, 111, 117, 122, 123, 126, 128, 133, 163, 181 chromosomes, 122, 168 cilia, 83, 84, 94 cilium, 94 circulation, 84 cisplatin, 107, 110 cisplatin resistance, 110 classical, 135 classification, 19, 26, 28, 59, 62, 74, 75, 78, 90, 92, 94 cleavage, 185 clinical approach, 138 clinical assessment, 12 clinical diagnosis, 148, 166, 169 clinical examination, 68, 90 clinical presentation, 41, 126, 129 clinical symptoms, 133 clinical trial, 30, 196

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204 clinical trials, 196 clinicopathologic correlation, 33 clinics, 174 clone, 100 clustering, ix, 79, 114 clusters, 189 Co, 45 coding, 115, 184 codon, 128, 129, 136, 164, 167, 168, 169, 170, 172, 173, 179, 181 codons, 128, 129, 164, 167, 168, 169, 198 cohort, 11, 18, 19, 20, 21, 24, 39, 40, 41, 42, 92 colloids, 81 Colombia, 46 colon, 20, 21, 100, 105, 109, 185, 191 colon cancer, 100 colony-stimulating factor, 97 colorectal cancer, 20, 104, 106, 109, 110 colorectum, 21 communication, 38 community, 184, 187 compatibility, 24 complete remission, 14, 21, 75, 196 compliance, 23, 89 complications, 2, 4, 18, 21, 25, 31, 132, 173 components, 90, 185 composition, 81, 94, 148, 154 compounds, 192, 196 concentrates, vii concentration, ix, 12, 64, 65, 68, 75, 79, 83, 93, 100, 103, 106, 134, 147, 186, 192 conception, 22 concrete, 13 conduction, 175 configuration, 103 confusion, 81 Connecticut, 111, 113 connective tissue, 153 consensus, 17, 22, 25, 31, 118 consent, 100 consumption, 107 control, 4, 5, 6, 10, 12, 23, 89, 104, 106, 113, 120, 134, 135, 163, 187, 190, 191, 192, 193, 194 control group, 6 conversion, 17, 107 correlation, x, xi, 3, 33, 57, 80, 91, 97, 99, 102, 104, 105, 129, 141, 142, 151, 162, 163, 179, 181 correlation coefficient, x, 99, 104

Index correlations, 97, 121, 128 cost-effective, 16, 17 counseling, 22, 118 coupling, 199 coverage, 35 covering, 78 cretinism, 83, 93 CRT, 165 CSF, 97 CT scan, 101 C-terminus, 181 cycles, 164, 187 cyclin D1, 53 cyclophosphamide, 132, 138 cyst, ix, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 cysteine, 128, 164, 170, 172 cysteine residues, 128 cystic fibrosis, 180 cysts, ix, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 cytokine, 85 cytokines, 85 cytology, 58, 80, 87, 89, 91, 97, 178 cytopathologists, ix, 80, 90 cytoplasm, 62, 144, 150, 151, 156, 157, 159, 160, 162, 163 cytotoxic, 134, 191 cytotoxic agents, 134 cytotoxicity, 139

D danger, 119 database, 113 de novo, 129, 136, 169, 170, 182 deafness, 190 death, ix, x, 15, 53, 62, 68, 76, 85, 111, 119, 132, 134, 175, 184, 191 death rate, ix, 62, 76 decay, 4, 28 defects, 23, 40, 143, 167, 168, 170 deficiency, 23, 83, 93 definition, ix, 55, 73, 80, 81, 82, 89, 92 degradation, 106 dehydrogenase, ix, 79, 83, 115, 136 delivery, 133, 134, 196 demand, 84 demographics, 37, 90 denaturation, 164

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Index dendritic cell, 139 dendritic cells, 135 density, 186, 189, 191, 194 deposition, 177 depression, 18 destruction, 10 destructive process, ix, 79, 84 detection, ix, x, 3, 9, 10, 14, 16, 23, 25, 34, 44, 47, 58, 62, 75, 76, 90, 91, 97, 99, 100, 107, 110, 119, 145, 146, 149, 173 developmental change, 110 diet, 5, 29, 65, 108 dietary, vii, 5, 23 dietary iodine, vii, 23 differential diagnosis, 82, 88, 90 differentiated cells, xi, 141, 156, 178, 185 differentiation, xii, 14, 75, 84, 91, 93, 105, 142, 143, 148, 150, 151, 152, 159, 160, 161, 162, 163, 184, 185, 188, 189, 190, 191, 195, 196, 197, 198 diffusion, 21 digestive tract, 20 dimer, 106 dimeric, ix, 99, 100, 105, 106, 109 discomfort, 127 discrimination, 103, 105 disease progression, 36, 65, 76 disease-free survival, 10, 50 diseases, 42, 48, 92, 100, 103, 130 disorder, 80, 117 dispersion, 151 displacement, 146 disposition, 144, 149, 150, 151 distribution, 3, 12, 85, 95, 174 disulfide, 128 diversity, 76 division, 122, 133, 185 DNA, 106, 109, 134, 164, 167, 168, 170, 172, 181, 184, 187, 197 DNA polymerase, 164 doctors, vii, 1, 25, 48 dominance, 130 donors, 180 dosimetry, 30, 31 down-regulation, xii, 183, 184, 185, 191 drainage, 3 drug resistance, 198 drug therapy, 177 drug treatment, 147, 192 drugs, 177, 179, 185, 191, 194, 197

205 duration, 5, 68 dust, 87 dysplasia, 105, 109 dyspnea, 75

E E-cadherin, 185, 195, 197 edema, 15, 18, 36 education, 23 efavirenz, xii, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195 elaboration, 195 electron, 84, 94, 178 electron microscopy, 84, 94, 178 ELISA, x, 99, 100, 108 email, 79 emboli, 15, 35, 132, 138 embolization, 15, 35, 132, 138 embryo, 185, 197, 198 embryos, 185 emission, 4, 37, 38, 186 encapsulated, 77 encoding, xii, 121, 126, 134, 182, 183, 185 endocrine, vii, ix, x, 2, 4, 28, 43, 44, 52, 80, 90, 104, 111, 112, 121, 126, 130, 131, 136, 137, 138, 142, 156, 157, 159, 162, 163, 180, 181, 182 endocrinologist, 13 endonuclease, 168 endoplasmic reticulum, 156 endothelial cell, 95 endothelial cells, 95 energy, 5, 100, 106, 108, 110 England, 20 enlargement, 47, 172 enolase, 109 environment, 8 enzymatic, 106 enzymes, 83, 100, 106, 109 epidemics, 84 epidemiologic studies, x, 111, 112, 113, 116, 118 epidemiology, 23, 42, 122 epidermoid cyst, ix, 79, 82, 86 epithelia, 185 epithelial cell, 83, 84, 85, 86, 94, 95, 105, 112, 115, 198 epithelial cells, 84, 86, 94, 95, 105, 112, 115, 198 epithelium, 84, 94, 104, 105, 115, 149 erythrocytes, 158

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Index

206 esophagus, 56, 105, 176 esterase, 86 ethanol, ix, 80, 88, 89, 91, 96, 98, 138, 187, 193 ethical standards, 64 ethics, 64 Europe, 17, 23, 167 evolution, 34, 197 examinations, 8, 118, 119, 173 excision, 24, 71, 75, 87 excitation, 186 excretion, 5, 127 exocrine, 104 exons, 128, 164, 165, 167, 168, 170 expertise, 17 exposure, vii, xii, 1, 21, 22, 41, 112, 184, 189, 192, 195, 196, 198 expressivity, 163 extraction, 187 eyelid, 130

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F failure, 10, 14, 22, 40 false positive, 51 familial, x, xi, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 125, 126, 128, 130, 131, 136, 142, 163, 164, 174, 179 family, x, 25, 48, 112, 113, 114, 118, 119, 127, 128, 129, 135, 163, 169, 170, 171, 172, 173, 180 family history, x, 48, 112, 118, 119, 127, 129, 169, 173 family members, 113, 114, 118 family physician, 25 fas, 85 fasting, 127 fat, 179 FDG, 17, 38 fear, vii, 1 females, viii, 18, 44, 46, 61, 62 ferritin, 93 fertility, vii, 1, 21, 22, 39, 40 fetal, 186 fibers, 153 fibrosis, 12, 19, 40, 83, 93, 132, 180 fibrous tissue, 149 fine needle aspiration, viii, 17, 24, 43, 44, 57, 58, 80, 92, 96, 98, 118 fine needle aspiration biopsy, viii, 17, 24, 43, 44, 57, 98, 118

Finland, 45, 46, 58 first degree relative, 113, 120 fission, vii FITC, 186, 188 fixation, 15 fluid, ix, 51, 79, 81, 82, 83, 85, 86, 87, 89, 90, 91, 93, 94 fluorescence, 186 fluoride, 86 FNA, 12, 23 follicle, ix, 79, 84 follicles, ix, 79, 84, 85, 86, 148 follicular, vii, x, 2, 4, 10, 25, 26, 29, 30, 32, 34, 35, 38, 42, 46, 62, 74, 77, 78, 80, 82, 84, 85, 86, 90, 93, 94, 101, 111, 112, 115, 116, 117, 120, 121, 123, 126, 133, 139, 143, 148, 149, 151, 152, 154, 155, 179, 184, 195 foramen, 87 formaldehyde, 186 fractures, 15 France, 42 free-radical, 83 fructose, 106, 108, 109 FSH, 21 FTC, 2, 3, 10, 11, 14, 20, 23, 24, 112, 114, 115, 118 fusiform, 149, 150, 154, 155, 158 fusion, 85, 126

G galectin-3, 91, 93, 98 gametes, 185 gastric, 104, 107, 110 gastrointestinal, ix, 18, 99, 105, 107, 115, 127, 130, 196 gastrointestinal tract, 105, 130 GDNF, 128, 135 GDP, 106 gel, 164 gels, 187 gender, 36, 56, 72, 90 gene, x, xi, xii, 32, 59, 106, 111, 112, 114, 115, 116, 117, 121, 122, 123, 125, 126, 128, 130, 133, 134, 135, 136, 139, 163, 164, 168, 173, 180, 182, 184, 185, 189, 190, 191, 193, 195, 197, 198, 199, 200 gene amplification, 59 gene expression, xii, 133, 134, 184, 185, 190, 193, 195, 197, 200

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Index gene promoter, 133, 134 gene silencing, 200 gene therapy, 133, 134, 135, 139 gene transfer, 134, 139 general surgeon, 4 generalization, 175 generation, 83 genes, x, 110, 111, 112, 113, 115, 116, 118, 139, 168, 184, 185, 189, 190, 195, 196, 197, 199, 200 genetic abnormalities, 126 genetic alteration, 110 genetic information, 198 genetic linkage, 112, 116, 122 genetic screening, 137 genetic testing, 112, 173 genetics, 122 genome, 185, 197 genomic, 164, 184 genotype, 121, 128, 137 genotypes, 128 germ cells, 185 Germany, vii, x, 1, 4, 24, 42, 45, 46, 99, 100, 164, 186 germline mutations, xi, 136, 142, 163, 171, 179, 182 gland, xi, 18, 19, 20, 21, 39, 121, 125, 133, 143, 144, 145, 163, 172, 173, 175, 189 glial, 128 globulin, 83 glucagon, 127 glucose, 38, 83, 106, 107, 187 glutamine, 106, 107, 186 glutathione, 83, 94 glutathione peroxidase, 83, 94 glycerol, 164 glycolysis, 100, 104, 106 goals, 135 goiter, ix, 3, 27, 48, 74, 79, 80, 84, 85, 91, 95, 115, 118, 123, 190 government, iv granules, 150, 151, 156, 157, 159, 162, 163 groups, x, xi, 10, 13, 17, 50, 84, 89, 90, 111, 112, 114, 125, 131, 151, 155, 163, 176, 178 growth, viii, ix, xii, 15, 44, 48, 56, 57, 79, 80, 81, 82, 83, 84, 85, 86, 90, 91, 93, 118, 122, 134, 145, 172, 174, 175, 179, 184, 185, 186, 187, 189, 191, 192, 194, 195, 196, 197, 198, 199 growth factor, ix, 79, 80, 83, 85, 91, 93 growth factors, ix, 80, 85, 91

207 growth rate, 191, 192, 195, 197 guidelines, 5, 9, 11, 13, 16, 22, 25, 32, 41, 78, 187

H HAART, 195, 199 half-life, 4, 5, 6, 22, 28 hamartomas, 114 hands, 4 haploinsufficiency, 115 harbour, 131 Hawaii, 46 hazards, 39 headache, 18 heart, 105 height, 188 hematological, 9, 18 hematoxylin-eosin, 154, 155, 156, 164 hemorrhage, ix, 18, 79, 84, 86 hemorrhages, 83 hepatocarcinogenesis, 108 herpes simplex, 134, 139 herpes simplex virus type 1, 134 heterogeneity, 7, 133 heterogeneous, 139 heterozygote, 167, 170 high risk, 11, 13, 23, 25, 26, 32, 47, 48, 173, 179 Hiroshima, 45 histochemical, 94 histogenesis, 148 histologic type, xi, 141, 179 histological, viii, 43, 44, 55, 58, 74, 78, 85, 90, 94, 148, 153, 155, 162, 163, 166, 172, 173, 175, 178, 185 histology, 13, 15, 23, 36, 100, 101, 103, 115 histone, 197 histone deacetylase, 199 histone deacetylase inhibitors, 197 histopathology, 63, 74 HIV, 198 HIV-1, 198 HK, 96, 110, 136, 180, 181 HLA, 135 Hoechst, 191 homogenous, 142 Hong Kong, 1, 2, 4, 10, 22, 23, 24, 26, 41 hormone, ix, 2, 5, 7, 11, 13, 16, 17, 18, 22, 29, 30, 31, 38, 65, 80, 83, 87, 88, 93, 100, 118, 126, 127, 147, 198, 200

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208 hormones, 83, 110, 127 horses, 91 hospital, 21, 22, 30, 41, 120 hospitalization, 6, 8, 18 hospitalized, 174 hospitals, 7, 16, 176 host, 134, 191 HPV, 106, 110 HR, 58 human, ix, xii, 4, 5, 7, 30, 31, 37, 38, 79, 83, 86, 94, 95, 106, 108, 109, 110, 122, 126, 134, 139, 181, 184, 185, 186, 187, 188, 189, 190, 191, 193, 195, 197, 198, 199, 200 human papilloma virus, 106 humans, 80, 122, 127 hybrids, 105 hydrochloric acid, 89 hydrophobic, 168 hydrophobicity, 168 hyoid, 131 hypermethylation, 197 hyperparathyroidism, 128, 135, 136 hyperplasia, 87, 115, 127, 129, 130, 136, 163, 173 hypertension, 172 hyperthyroidism, 39, 76, 118, 123 hypertrophy, 115 hypoparathyroidism, 3, 4, 27, 131 hypothesis, 93, 117, 195, 196 hypothyroidism, 5, 8, 9, 17, 18, 91, 118

I ice, 187 id, 9, 11, 15, 21, 47, 162 identification, 94, 112, 113, 116, 117, 142, 167, 173, 190 IFN, 134 IgG, 186, 188 IL-1, 134 IL-2, 134, 135 images, 86 imaging, 4, 7, 8, 12, 15, 16, 17, 21, 23, 25, 64, 68, 100, 103, 105, 138, 143 imaging modalities, 68 imaging techniques, 25 imbalances, 122 immune reaction, 195 immune response, 134, 135, 139 immunity, 134

immunization, 134, 135, 139 immunofluorescence, 186, 189, 190, 193 immunohistochemical, 3, 56, 59, 94, 107 immunological, 133 immunomodulatory, 135 immunostimulatory, 135 immunotherapy, 134, 135 in vitro, xii, 184, 185, 191, 192, 194, 195, 196, 197, 198, 199 in vivo, xii, 139, 184, 185, 187, 191, 194, 195, 196, 197, 198, 199 inactivation, 121 inactive, ix, 99, 100, 106 incidence, 2, 3, 10, 11, 12, 16, 20, 22, 23, 33, 39, 41, 42, 44, 45, 50, 54, 56, 90, 96, 112, 119, 128, 129, 130, 195 incurable, 14 India, 103 indication, 25, 168, 175 indicators, 35 indices, 147, 148, 169, 170 indolent, 2, 126, 130 induction, 133, 135, 139, 185, 189, 191, 195 inert, 185 infection, 139 infertility, 22, 23, 40 inflammation, 12, 83, 84 inflammatory, 21, 40, 84, 103, 105 informed consent, 100 inheritance, xi, 117, 120, 125 inherited, 112, 115, 121, 126, 128, 142, 163, 164, 169, 170, 173, 175 inhibition, xii, 139, 183, 184, 185, 188, 189, 190, 191, 192, 195, 196 inhibitor, 186, 187, 190, 191, 193, 196, 198, 199 inhibitors, vi, xii, 183, 184, 185, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 inhibitory, 185, 195 inhibitory effect, 195 initiation, 47 injection, ix, 80, 89, 96, 98, 134, 135, 138, 187, 191, 194 injury, 49, 132 innominate, 131 inoculation, 191 insertion, 144 insight, 9, 116 institutions, 17, 74 integration, 14 integrity, 25, 40

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Index intensity, 12, 35 interaction, 109, 110, 116, 117, 122 interference, xii, 8, 110, 173, 175, 183, 185 interferon, 133, 134, 138 interleukin, 97, 134, 139 interleukin-1, 139 interleukin-2, 139 interleukin-6, 97 internet, 33 interpretation, ix, 80, 86, 90, 91, 150 interval, viii, ix, 5, 9, 21, 61, 62, 65, 68, 69, 70, 72 intervention, 174, 175 interviews, 113 intracellular signaling, 163 intramuscular, 66 intramuscular injection, 66 introns, 164 invasive, 12, 34, 55, 77 iodide, 199 iodine, vii, viii, xii, 1, 2, 4, 5, 7, 8, 9, 10, 22, 23, 26, 27, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 61, 62, 65, 66, 72, 73, 74, 76, 77, 78, 83, 100, 101, 108, 117, 143, 144, 173, 183, 184, 185, 186, 187, 190, 193, 195, 196, 197, 198 iodine therapy, 32, 38, 39, 40, 72, 73, 76, 100, 101 ionizing radiation, 114 ipsilateral, 2, 4, 24, 49, 131 irradiation, 34, 35, 68, 73, 75, 76, 132 isoenzymes, 100, 105, 106, 110 isoforms, 100, 163, 181 isolation, 7, 8 isotope, vii, 1, 4, 5, 144 isotopes, vii, 25, 49 isozymes, 106, 110 Israel, 39 Italy, 21, 45, 183, 186, 187

J JAMA, 136, 180 Japan, vii, 1, 4, 23, 24, 41, 43, 45, 46, 49, 52, 56, 80, 116, 119, 167 Japanese, viii, 43, 45, 46, 58, 92 judge, 8, 147, 177 judgment, 131, 177

209

K Ki-67, 53, 56 kidney, 69, 83, 94, 100, 105, 182 kidneys, 127 kinase, ix, 99, 100, 105, 106, 107, 108, 109, 110, 115, 121, 128, 133, 134, 136, 137, 139, 163, 167, 180, 181, 182, 196, 199 kinase activity, 163 kinases, 106, 134, 168 kinetics, 104 Korean, 17

L labeling, 53, 56, 95 lactate dehydrogenase, ix, 79, 83 lamina, 156 laminar, 156 larynx, 56 laser, ix, 80, 88, 96, 186 lead, 112, 116, 168, 175, 184, 196 lesions, viii, ix, 21, 43, 44, 46, 49, 56, 57, 76, 80, 81, 83, 85, 86, 87, 89, 91, 95, 96, 97, 107, 114, 121, 144, 174 leukaemia, 19, 39, 103 leukemia, 19, 20, 21, 25, 39, 196 leukocytes, xi, 142, 179 leukopenia, 19 lichen, 129, 130, 137 life expectancy, 116 life span, 130 lifetime, 48, 171 Li-Fraumeni syndrome, 115 ligand, 163 ligands, 128 likelihood, 9 limitation, 9, 102 limitations, 49 linkage, 114, 116, 117, 119, 122, 168 links, 24 lipid, 156 liver, 83, 100, 105, 109, 110, 129, 132, 138, 177 liver metastases, 132 lobectomy, 2, 4, 6, 10, 18, 24, 27, 29, 48, 56, 75, 92 localization, 16, 53, 54, 146, 149, 175, 179 location, 3, 56, 64, 68, 117 locus, 123

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210

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London, 29 long-term, xii, 22, 24, 28, 33, 35, 38, 39, 42, 52, 75, 89, 134, 136, 138, 183 loss of heterozygosity, 117 low risk, 16 lumen, 21, 80, 86, 145, 146, 158 lung, ix, 9, 12, 14, 15, 16, 17, 19, 21, 32, 35, 40, 62, 69, 71, 73, 74, 75, 76, 78, 99, 102, 103, 106, 107, 109, 117, 122, 185, 191 lung cancer, 103, 106, 107 lung disease, 21, 40, 103, 107 lung metastases, ix, 14, 15, 21, 35, 62, 71, 73, 74, 75 lungs, 9, 12, 72, 129, 132, 147, 148, 175 lymph, vii, xi, 2, 3, 4, 5, 9, 17, 23, 25, 28, 30, 33, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 57, 58, 59, 74, 80, 82, 86, 90, 91, 97, 98, 101, 125, 129, 131, 144, 145, 147, 172, 173, 174, 175, 177, 178, 179 lymph node, vii, xi, 2, 3, 4, 5, 9, 17, 23, 25, 28, 30, 33, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 57, 58, 59, 74, 80, 82, 90, 91, 97, 98, 101, 125, 129, 131, 144, 145, 147, 172, 173, 174, 175, 177, 178, 179 lymphadenectomy, 28 lymphadenopathy, 59, 129 lymphatic, 3, 20, 129, 144, 148, 156 lymphocytes, 164 lymphoma, 117

M M.O., 32, 35, 42 M1, 63, 66, 70, 100, 105, 106 machinery, 185, 195, 196 macrophage, 97 macrophages, 86, 134 maintenance, 195 males, viii, 22, 44, 46, 61, 62 malignancy, vii, x, 1, 2, 10, 16, 20, 25, 43, 44, 47, 62, 77, 78, 80, 82, 83, 89, 90, 91, 111, 200 malignant, ix, x, 14, 19, 28, 46, 55, 59, 78, 79, 80, 83, 84, 85, 86, 90, 91, 92, 95, 97, 99, 100, 103, 105, 106, 107, 108, 110, 113, 114, 118, 127, 143, 145, 148, 196, 199 malignant cells, 105, 106, 196 malignant tumors, 14, 59, 92, 143 mammalian cell, 185 mammalian cells, 185 mammalian genomes, 184

management, vi, 1, 2, 8, 13, 15, 17, 23, 24, 25, 29, 31, 32, 33, 37, 41, 42, 57, 77, 92, 97, 108, 131, 137, 138, 142, 152, 176, 183, 196 manufacturer, 100, 101, 102, 164, 187 mapping, 3 Marfan syndrome, 163 marker genes, 185, 195 marrow, 6, 7, 19 matrix, 157, 162 measurement, ix, 16, 23, 58, 79, 88, 108, 127 measures, 23 median, viii, xi, 4, 6, 10, 17, 22, 61, 62, 65, 66, 68, 69, 71, 74, 101, 104, 105, 175, 179, 183 mediastinum, 13, 132, 146, 147, 174, 175, 176 medications, 133 medicine, 24, 61 medulla, 128, 130 medullary cancers, vii Medullary thyroid carcinomas, xi, 125 melanoma, 20, 21, 148, 150, 185, 189, 191, 195, 200 men, 22, 40, 68, 116, 117, 166, 169 menopause, 22, 40 meta-analysis, 7 metabolic, 76, 100, 106, 109 metabolic shift, 109 metabolism, 108, 109, 110, 115, 126, 199 metabolites, 100 metastases, viii, ix, xi, 5, 9, 14, 15, 21, 26, 31, 32, 34, 35, 36, 37, 40, 46, 53, 54, 61, 62, 64, 65, 68, 69, 70, 71, 72, 73, 74, 75, 76, 78, 80, 83, 86, 91, 100, 102, 103, 105, 108, 122, 129, 132, 138, 142, 144, 145, 146, 147, 148, 172, 173, 174, 175, 176, 177, 178, 179, 195 metastasis, vii, 1, 2, 3, 4, 8, 9, 11, 12, 14, 15, 16, 17, 18, 19, 21, 24, 26, 27, 28, 30, 32, 33, 36, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 69, 74, 75, 77, 78, 86, 90, 98, 132, 133, 138, 156, 171 metastasize, 45, 54 metastasizes, vii, 43, 52 metastatic, viii, ix, 13, 15, 31, 32, 34, 36, 37, 38, 39, 44, 46, 47, 50, 52, 53, 59, 61, 62, 63, 65, 66, 69, 70, 71, 72, 73, 74, 76, 82, 90, 91, 97, 98, 99, 101, 102, 103, 105, 109, 122, 123, 131, 132, 139, 142, 144, 146, 174, 175, 177, 184, 195, 196 metastatic disease, 13, 59, 71, 72, 74, 105, 132 methionine, 128, 167, 169 methylation, 197,199, 200

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Index Mexican, 42 Mexico, 24 MgSO4, 187 MIBG, xi, 125 mice, 134, 182, 185, 187, 191, 194 microarray, 109 microcirculation, 84 microenvironment, 84 micrometastasis, 3, 17, 28 microscope, 162, 186, 188, 189 microscopic investigations, 163 microscopy, 84, 94, 151, 178, 186 migration, 163 military, 17 Ministry of Education, 76 minority, xii, 183, 184 miscarriage, 22, 23 misleading, 14 mitochondria, 157 mitochondrial, 115 mitogenic, 115 mitosis, 150, 154 mitotic, 83 MND, 49, 50, 51, 52 modalities, 26, 35, 37, 65, 68, 73, 75, 77, 126 modality, 9, 76 models, xii, 109, 184, 188 modulation, 185, 195 molecular markers, 142 molecular mechanisms, 112 molecular structure, 168 molecules, 128 monoclonal antibodies, 100 monoclonal antibody, 186, 188 monocytes, 86 monograph, viii, 43, 44 monolayer, 189 monotherapy, 199 morbidity, 6, 36, 118, 133 morphological, xii, 14, 21, 78, 97, 153, 154, 173, 178, 184, 185, 186, 189, 195 morphology, 74, 86, 148 morphometric, 93 mortality, 10, 11, 12, 16, 20, 23, 26, 39, 41, 52, 133 mortality rate, 11, 16, 23 mosaic, 167 Moscow, 141, 164, 170, 180 mouse, 94, 115, 186, 187, 188, 197, 198 mouse model, 94

211 mouth, 19 MRI, 12, 14, 17, 81, 143, 146, 179 mRNA, 181, 196, 198, 199 MTC, xi, 112, 113, 115, 118, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 160, 161, 163, 164, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179 mucosa, 105 mucous membrane, 114 mucous membranes, 114 multiplicity, 44 multivariate, 3, 4, 15, 27, 32, 35, 42, 77, 78 muscle, 56, 100, 105, 131, 145, 174 muscles, 131, 145 mutant, 168, 196 mutants, 121, 133 mutation, 121, 123, 128, 129, 130, 136, 137, 164, 167, 168, 169, 170, 172, 173, 180, 181 mutations, xi, 83, 105, 110, 114, 115, 118, 121, 122, 125, 126, 128, 129, 130, 133, 136, 137, 142, 143, 163, 164, 166, 167, 168, 169, 170, 171, 173, 179, 180, 181, 182, 198 myeloid, 19, 20, 39, 196

N Na+, 199, 200 NaCl, 187 NAD, 106 natural, 32, 44, 73, 77, 134 natural killer, 134 natural killer cell, 134 nausea, vii, 1, 18, 127 neck, vii, 1, 3, 4, 5, 6, 7, 8, 9, 12, 13, 14, 16, 17, 18, 24, 28, 30, 37, 49, 58, 75, 87, 95, 119, 131, 132, 143, 144, 145, 146, 147, 148, 153, 172, 174, 175, 176, 177, 179 necrosis, ix, 79, 82, 83, 84, 85, 93, 134, 154, 186, 191 needle aspiration, xi, 9, 31, 58, 91, 92, 93, 95, 97, 141, 143, 148 needles, 47, 51 neonates, 22 neoplasia, x, 32, 85, 91, 111, 112, 115, 117, 118, 122, 126, 130, 136, 137, 142, 163, 180, 181, 182 neoplasm, 75, 82, 83, 90, 117, 119, 121, 147, 162, 175, 179, 197

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212 neoplasms, x, xii, 28, 62, 77, 78, 92, 111, 112, 113, 114, 115, 117, 118, 120, 142, 148, 176, 184 neoplastic, 75, 85, 126, 131, 132 neoplastic cells, 132 nerve, 3, 47, 49, 56, 83, 92, 94, 131 nervous system, 182 nested PCR, 164 Netherlands, 5 neural crest, 126, 163 neuroendocrine, xi, 104, 109, 125, 126, 138, 142, 163 neurons, 128 neuropathological, 94 neurturin, 128 neutrophils, 109 New England, 164 New York, 41, 58, 59, 78, 95 NIS, xii, 183, 184, 185, 186, 187, 190, 192, 193, 195, 196, 197 NIS gene, 190, 191 nodes, ix, 3, 28, 44, 45, 46, 47, 50, 51, 52, 54, 70, 79, 80, 101, 131, 144, 145, 146, 148, 172, 174 nodules, ix, 3, 27, 48, 55, 56, 57, 59, 79, 80, 81, 83, 84, 85, 86, 87, 88, 89, 90, 92, 93, 95, 96, 97, 98, 113, 115, 116, 117, 118, 119 non-Hodgkin’s lymphoma, 117 non-nucleoside inhibitor, 198 non-nucleoside inhibitors, 198 nontoxic, 123 normal, 6, 10, 14, 18, 21, 25, 58, 73, 83, 84, 85, 87, 93, 100, 101, 102, 104, 108, 113, 115, 119, 127, 128, 133, 143, 164, 166, 167, 168, 173, 185, 186, 188, 189, 190, 195, 196, 198 normalization, 147 NSE, 107 nuclear, vii, 24, 25, 90, 114, 148, 150, 151, 156, 157, 162 Nuclear Regulatory Commission, 8, 31 nuclei, 150, 156, 157 nucleic acid, 106, 133 nucleoli, 156 nucleotide sequence, 164 nucleus, 150, 156, 160, 162

O obesity, 94 observations, 78, 80, 94, 149, 153, 155, 162, 185, 195

occult blood, 105 Ohio, 10 old age, 65 oligospermia, 21 oncogen, 130, 133 oncogene, xi, 105, 110, 112, 115, 121, 125, 126, 128, 129, 133, 136, 137, 142, 143, 163, 164, 165, 166, 168, 171, 172, 173, 179, 180, 181, 182, 198 oncogenes, ix, 80, 91, 110 oncogenesis, 108 oncology, vii, 1, 16, 33, 59, 61, 62, 120, 143, 148, 172 oocytes, 197 oral, 4, 18 order statistic, 78 organ, viii, xi, 9, 14, 43, 45, 142, 152, 175 organelle, 157, 158, 159, 160 organelles, 156 organism, 106 outpatient, 6, 7 ovarian failure, 22, 40 ovaries, 22 oxidative, 115 oxygen consumption, 107

P p53, 105, 110, 115, 122, 196 paclitaxel, 197 pain, 8, 15, 18, 36, 75, 132, 143 palliate, 15 palliative, xi, 7, 35, 125, 142, 147, 175, 176, 177, 179 palpation, 44, 52, 131 Pamidronate, 36 pancreas, 20 pancreatic, 104, 107, 109, 121 pancreatic cancer, 104, 109 pancreatitis, 104 paper, 76 paraffin-embedded, 86, 164 parameter, 52, 53, 55, 100 parathormone, 88 parathyroid, 34, 86, 87, 95, 130, 131, 163 parathyroid glands, 34, 130, 131 parathyroid hormone, 87 parathyroidectomy, 131 parenchyma, 84, 149 parenchymal, 92

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Index Paris, 78 parotid, 83, 94 parotid gland, 83, 94 particles, 197, 198 passive, 134 pathogenesis, ix, 79, 83, 85, 91, 93, 110, 115 pathologist, 45 pathology, ix, 23, 58, 79, 115, 131, 166, 177, 178 pathways, 106, 110, 163 patient care, 112 PCR, 164, 165, 168, 172, 187, 190, 193 pediatric, 16, 37 pediatric patients, 16, 37 pelvis, 75 penetrance, x, 111, 116, 117, 130, 163, 169 penis, 94 peptide, 126, 133 perchlorate, 191 peripheral blood, 164 peripheral blood lymphocytes, 164 permeability, 93 permit, 115 PET, 7, 17, 30, 103 PET scan, 17 PG, 92, 95, 97, 197 pH, 89, 164, 187 phagocytosis, 87 pharmacological, xii, 184, 185, 188, 189, 190, 191, 195, 196 pharynx, 176 phenotype, xii, 121, 128, 130, 133, 136, 137, 180, 181, 184, 185, 188, 189, 190 pheochromocytoma, 130, 136, 137, 163, 169, 170, 171, 172, 180 phosphate, 127 phosphorylates, 106 phosphorylation, 106, 163 photon, 5 phylogeny, 180 physicians, 5, 11, 13, 24, 25, 29, 118, 119 physiological, 184, 187 pilot study, 100, 103, 107 pituitary, 132 placental, 197 planning, 38 plasma, 100, 103, 104, 106, 107, 127 plasma levels, 103 plasmid, 134 play, 91, 104, 135 pneumonitis, 19, 21, 40

213 point mutation, 110, 128, 164, 180 Poland, 46 polycystic kidney disease, 94 polygenic, x, 111, 113, 116, 122 polygenic disorder, x, 111 polymerase, 110 polymerase chain reaction, 110 polymerization, 134 polymorphism, 110, 151, 165, 180 polymorphisms, 116 polyp, 80 polypeptide, 126 polypeptides, 195 pools, 108 poor, 33, 51, 56, 58, 65, 73, 75, 76, 122 population, 20, 21, 25, 39, 40, 76, 113, 115, 119, 120, 135 positive correlation, 106, 129 positron, 37, 38 positron emission tomography, 37, 38 postoperative, vii, 1, 2, 7, 10, 11, 13, 24, 25, 27, 30, 35, 56, 73, 132, 145, 147 power, 21, 31 preclinical, 163, 173 prediction, 75, 115 predictors, 56 pregnancy, vii, 1, 16, 22, 23, 40, 41 pregnancy test, 23 pregnant, 23 preleukemia, 39 premenopausal, 116, 117 pressure, 84, 122 preterm delivery, 22 preventive, 23, 142, 171, 173, 175, 179 primary tumor, viii, xi, 3, 4, 32, 61, 64, 70, 74, 80, 82, 90, 101, 142, 146, 147, 174, 175, 177, 179, 185 probability, 195 proband, 172, 173 probands, 120 probe, 28 production, 19, 97, 100, 106, 195 productivity, 8 prognosis, vii, xi, 2, 11, 12, 14, 15, 23, 28, 32, 33, 37, 43, 44, 50, 52, 53, 56, 57, 58, 62, 73, 75, 77, 90, 119, 122, 141, 147, 175, 177, 178, 179 prognostic factors, 7, 14, 27, 33, 35, 42, 55, 58, 77, 137 prognostic marker, 56

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214 prognostic value, 56, 142, 178 program, 131, 185 proliferation, xii, 53, 83, 85, 86, 93, 95, 106, 107, 108, 110, 134, 163, 184, 185, 189, 191, 192, 194, 195, 196, 197, 198 proliferation potential, 185 promote, xii, 3, 17, 184 promoter, 133, 134 property, iv, 14 prophylactic, 3, 28, 49, 51, 52, 118, 119, 163, 173, 176 prophylaxis, xi, 142, 179 prostaglandin, 142 prostaglandins, 126 prostate, 73, 185, 189, 191, 195, 200 prostate cancer, 195 prostate carcinoma, 73, 185, 189 protease inhibitors, 195 protection, 8, 18 protein, xii, 59, 83, 94, 106, 115, 121, 128, 163, 164, 167, 168, 170, 180, 184, 198 protein structure, 128 proteins, 67, 83, 115, 139 protocol, 7, 14, 87, 132 protocols, 23, 132, 135, 196 protooncogene, 128, 129, 136 proto-oncogene, xi, 112, 115, 125, 126, 128, 129, 133, 136, 136, 137, 142, 143, 163, 164, 165, 166, 168, 171, 172, 173, 179, 180, 181, 182 provocation, 127 proximal, 72 PSA, 185, 195 public, 8 purification, 164 pyruvate, ix, 99, 100, 105, 106, 107, 108, 109, 110

Q quality assurance, 25 quality of life, 8, 15, 25, 36, 47

R radiation, vii, 1, 5, 6, 7, 8, 18, 19, 21, 22, 23, 26, 30, 35, 40, 58, 112, 114, 132, 133, 142, 174, 175, 176, 177, 196 radiation therapy, 23, 26, 35, 132, 142, 174, 175, 176, 177

radical, xi, 4, 24, 25, 28, 49, 75, 142, 143, 146, 147, 176, 177 radio, viii, xii, 8, 9, 10, 26, 27, 29, 30, 31, 32, 34, 35, 37, 38, 39, 40, 61, 62, 65, 66, 68, 72, 73, 76, 100, 101, 183, 190, 196, 197 radioactive iodine, v, vii, xii, 1, 2, 29, 30, 31, 32, 33, 34, 39, 40, 41, 77, 117, 173, 184, 185, 190, 196 radioactive isotopes, vii radioisotope, xi, 133, 141, 178 radiological, 17, 100, 103, 105, 131, 138 radiologists, 24, 25, 135 radiotherapy, 2, 12, 13, 15, 25, 27, 32, 34, 35, 36, 49, 65, 71, 74, 75, 76, 78, 125, 132, 138, 147, 176, 177, 179 range, viii, ix, 6, 7, 17, 43, 48, 61, 62, 65, 67, 68, 80, 81, 83, 90, 101, 126, 166, 169, 191 raphe, 94 ras, 105, 110 rat, 94, 110, 134, 135, 139, 198 rats, 108, 110 reading, 31 real time, 86 receptors, 127, 132, 138, 181, 189, 195 recognition, 9, 168 reconstruction, 95 recovery, 19, 67, 191 recruiting, 11 recurrence, viii, ix, x, 2, 3, 4, 10, 11, 12, 16, 17, 20, 28, 30, 33, 34, 36, 37, 49, 52, 53, 56, 57, 58, 61, 62, 63, 68, 70, 71, 72, 75, 76, 89, 111, 116, 119, 120, 129, 147, 173, 175 red blood cells, 83, 105 redistribution, 197 reduction, 10, 17, 23, 106, 133, 134, 135, 189, 192 reflection, 195 regional, vii, viii, 32, 34, 42, 43, 45, 52, 54, 61, 74, 132, 142, 144, 145, 146, 147, 153, 174, 176, 177, 178 registries, 41 registry, 26, 113 regression, ix, 14, 62, 65, 71, 72, 75, 103, 133, 134 regulation, xii, 106, 107, 108, 126, 183, 184, 185, 190, 191 regulations, 24 relapse, 2, 3, 4, 9, 10, 11, 12, 13, 16, 17, 23, 24, 25, 132, 147, 175

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Index relapses, vii, 1, 2, 8, 10, 11, 12, 14, 16, 17, 24, 25, 173, 175 relationship, 21, 23, 30, 53, 54, 55, 90, 91, 105, 116, 128, 136, 137, 178, 180, 200 relationships, 120 relatives, 113, 119, 120, 127, 142, 170, 171, 172 remission, 14, 15, 18, 21, 23, 36, 75, 104, 196 remodeling, 197 renal, ix, x, 4, 5, 99, 100, 105, 108, 109, 112, 117, 122, 127 renal cell carcinoma, 100, 105, 108, 109 renal disease, 105 renal function, 5 replication, 134 research, vii, xi, 141, 170 resection, 2, 3, 11, 23, 57, 75, 82, 92, 101, 131, 146, 175 residual disease, 4, 12, 13, 25, 127 residues, 30, 128, 168, 181 resilience, 198 resistance, 107, 110, 133, 198 resolution, 12, 31, 86 responsiveness, xii, 183 restriction enzyme, 167 restriction fragment length polymorphis, 110 retardation, 133 retention, 9, 199 reticulum, 156 retinal pigment epithelium, 115 retinoic acid, 196, 199 retinoic acid receptor, 196 retinoids, 196 retrovirus, 197 reverse transcriptase, xii, 183, 197, 198 ribonucleoprotein, 198 ribosomes, 156, 157, 162 risk, vii, x, xi, 2, 4, 6, 10, 11, 13, 15, 16, 17, 20, 21, 22, 23, 25, 26, 27, 31, 32, 34, 37, 40, 47, 48, 92, 111, 113, 114, 118, 119, 120, 121, 125, 128, 130, 137, 142, 171, 173, 179 risks, 3, 6, 17, 20, 21, 39, 41 RNA, xii, 106, 110, 183, 184, 185, 187, 193, 198 RNAi, 185, 195 Russian, 141, 143, 148, 163, 164, 173, 174, 179, 180 ruthenium, 101

S

215 saline, 51, 89 salivary glands, 19 sample, 11, 100, 168 sampling, 3 scanning electron microscopy, 94 scientific community, 184 scintigraphy, viii, ix, 5, 37, 41, 61, 62, 64, 68, 72, 73, 100, 101, 103 sclerosis, 96 sclerotherapy, ix, 80, 88 screening programs, 127 SDH, 115 search, 148, 170 second generation, 198 secret, xi, 125 secrete, 105 secretion, 93 seeding, 186 selecting, 82 selectivity, 134 selenium, 83, 91, 93 sensitivity, 9, 16, 17, 37, 73, 90, 102, 103, 104, 105, 106, 139, 144, 177, 191, 196 separation, 86, 168 sepsis, 172 sequencing, 164, 167, 168, 170, 172 series, 2, 6, 7, 10, 12, 15, 16, 17, 22, 45, 50, 52, 53, 55, 56, 73, 74, 75, 86, 89, 95, 96, 120, 131, 133 serine, 168 serotonin, 126, 142 serum, ix, x, xi, 8, 10, 16, 21, 23, 24, 25, 29, 31, 34, 37, 38, 51, 56, 64, 65, 67, 68, 71, 79, 83, 95, 99, 100, 102, 104, 105, 108, 127, 131, 133, 141, 143, 146, 147, 186 severity, 163 sex, 22, 113, 173, 174 sex ratio, 22 shape, 47, 144 short period, 147 short-term, 5, 18 sialadenitis, 38 side effects, vii, 1, 5, 18, 19, 25, 38, 133 sign, 75, 90, 97, 151 signal transduction, 196 signaling, 106, 128, 134, 182, 189, 190, 195, 196, 198 signaling pathway, 163, 182 signalling, 135

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216 signs, 143, 145, 148, 149, 150, 152, 156, 162, 163, 178, 189 sine, 62 Singapore, 94 sites, 9, 17, 20, 21, 144, 149, 153, 155, 181 skeleton, 147 skin, 121 Slovenia, 61, 74, 76 small intestine, 110 smoking, 22 socioeconomic, 22 sodium, 86, 191, 198, 199 sodium iodide symporter, 199 software, 9, 30 solid tumors, 20, 73, 78, 109 somatic mutations, xi, 128, 142, 166, 167, 168 somatostatin, xi, 125, 126, 132, 133, 138, 197 Spain, 45 specificity, 9, 17, 90, 102, 103, 104, 105, 114, 128, 134, 144, 151, 152, 163, 197 spectrum, 121, 163, 168, 173 speculation, 107 sperm, 22 spermatogenesis, 21 spermatozoa, 197 spinal cord, 12, 35 spine, 13, 72 spleen, 83 sporadic, x, xi, 111, 112, 114, 115, 116, 117, 118, 119, 125, 126, 128, 129, 131, 133, 136, 137, 142, 148, 164, 166, 167, 168, 173, 174, 175, 179, 180, 181, 182 SPSS, 70 stabilization, 71, 134, 177 stages, 23, 24, 44, 75, 148, 178, 185 standards, 64 statistical analysis, viii, 16, 32, 35, 61, 70 statistics, 78 stereotype, 52 sternocleidomastoid, 131 steroid, 15 stock, 187 stomach, 105 strategies, 28, 43, 81, 132 stroma, 86, 149, 153, 154, 155 stromal, 151, 196 subcutaneous injection, 191 subgroups, 70, 117 substances, 83, 107, 142 substitutes, 128

Index substitution, 100, 167, 168, 169, 170, 172 success rate, 5, 6, 7, 8 suffering, 103, 144, 153 suicide, 135, 139 sulphate, 198 summer, 84 supernatant, 100 supplements, 146 suppression, 2, 11, 13, 17, 19, 25, 127, 131, 184 suppressor, 53, 114, 115, 117, 133 surgeons, 3, 4, 24, 25, 42, 46, 48, 51, 52, 55, 56, 64, 135 surgeries, 47, 119 surgery, viii, xi, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 15, 16, 17, 18, 22, 23, 24, 25, 26, 27, 28, 33, 34, 36, 37, 42, 43, 44, 47, 48, 49, 51, 52, 56, 57, 58, 61, 63, 64, 65, 67, 68, 70, 71, 72, 75, 77, 79, 90, 92, 93, 95, 96, 97, 104, 105, 130, 131, 132, 136, 138, 139, 142, 143, 146, 147, 172, 173, 176, 179, 184 surgical, viii, ix, xi, 12, 17, 26, 27, 33, 41, 44, 45, 46, 47, 48, 50, 55, 57, 58, 63, 64, 71, 75, 77, 79, 81, 87, 88, 89, 90, 92, 125, 131, 132, 166, 173, 174, 175, 176, 177, 179, 186 surgical intervention, 174, 175, 176 surgical resection, 57 surveillance, 16, 23 survival, vii, viii, ix, xi, 1, 2, 3, 4, 9, 10, 11, 12, 14, 15, 16, 18, 23, 24, 25, 32, 35, 36, 41, 42, 50, 56, 61, 62, 68, 69, 70, 72, 73, 74, 75, 77, 78, 87, 119, 126, 132, 133, 135, 141, 174, 176, 178, 179, 183 survival rate, 15, 74, 87, 133, 174 survivors, 21 susceptibility, x, 14, 111, 112, 114, 116, 117, 123 susceptibility genes, x, 111, 112, 116 Sweden, 21, 40, 42 swelling, vii, 1, 15, 18, 19, 47, 56, 84 Switzerland, 23 symptom, xi, 119, 141, 147, 179 symptomatic treatment, xi, 75, 142 symptoms, 15, 18, 91, 133, 143, 152 syndrome, x, 23, 111, 113, 114, 115, 116, 117, 118, 119, 121, 122, 130, 163, 171, 180, 190, 198, 199 synergistic, 110 synergistic effect, 110 synthesis, 106, 164, 187 systems, x, 20, 82, 99, 137

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T T cells, 134 Taiwan, 9, 33, 45, 79 targets, 139 taste, 18, 19 TCC, 167, 168 T-cell, 135, 139 teens, 48 telomerase, 91 territory, 22 testis, 22, 94, 197 testosterone, 22 tetracycline, 88, 89, 96 TGA, 165 TGF, 56 therapeutic approaches, 76, 78, 138 therapy, ix, xi, xii, 5, 7, 8, 10, 11, 12, 13, 14, 15, 17, 18, 19, 22, 24, 26, 27, 29, 30, 31, 32, 36, 38, 39, 40, 41, 47, 49, 62, 65, 66, 67, 69, 71, 72, 73, 74, 75, 76, 77, 80, 88, 91, 96, 98, 100, 101, 112, 125, 131, 133, 135, 138, 139, 142, 147, 173, 175, 176, 177, 183, 184, 185, 190, 195, 196, 199 thoracic, 71, 72 thorax, 17 threatening, 48, 53, 132 three-dimensional, 85 three-dimensional reconstruction, 85 threonine, 128, 167, 169 thrombocytopenia, 19 thymidine, 95, 134, 139 thyroglobulin, x, xii, 5, 16, 30, 31, 34, 37, 38, 47, 51, 56, 58, 64, 80, 83, 91, 97, 99, 100, 102, 103, 108, 133, 174, 184, 185, 186, 188, 189, 195, 196 thyroid gland, vii, 28, 33, 34, 35, 42, 45, 57, 58, 77, 78, 84, 86, 87, 92, 93, 94, 96, 97, 115, 121, 126, 127, 128, 131, 132, 137, 143, 144, 145, 146, 148, 168, 172, 173, 174, 175, 177, 186, 189, 198 thyroid stimulating hormone, 83, 118 thyroiditis, 6, 18, 55, 82, 86 thyrotoxicosis, 5, 8, 20 thyrotropin, 2, 29, 31, 37, 123 thyroxin, 5, 7, 15, 17, 25, 37 tissue, vii, 1, 4, 5, 7, 10, 21, 24, 30, 56, 73, 83, 85, 86, 100, 104, 106, 108, 131, 132, 133, 134, 143, 144, 146, 149, 152, 153, 164, 166, 167, 175, 197

217 tissue homeostasis, 85 TNF, 134 TNF-α, 134 tolerance, 65 Toshiba, 180 toxicity, 9, 19, 21, 31, 133, 134, 177 TPO, 185, 196 trachea, 20, 47, 56, 153, 175, 176 tracking, 103 traits, 195 trans, 199 transcatheter, 138 transcript, 190, 197 transcriptase, xii, 183, 197, 198 transcription, 133, 139 transcripts, 128 transducer, 81, 86 transduction, 133, 134, 195, 196 transfection, 133, 196 transfer, 133, 134, 139 transformation, ix, 19, 46, 48, 53, 80, 82, 91, 110, 196 transgene, 133 transgenic, 121 transgenic mouse, 121 transition, 196 transmembrane, 144, 168, 180, 182 transmembrane region, 168 transport, 190, 198, 199 trastuzumab, 107 trastuzumab therapy, 107 treatable, 119 treatment methods, 176 trend, 24 trial, viii, 6, 10, 17, 19, 30, 32, 36, 44, 47, 58, 89, 91, 197 triglyceride, 83 triiodothyronine, 83 TSH, ix, xii, 2, 5, 6, 7, 8, 10, 13, 15, 16, 17, 18, 24, 25, 30, 38, 62, 65, 67, 72, 76, 118, 184, 185, 189, 190, 193, 195, 196, 197 tubular, 127 tumor cells, xi, xii, 10, 14, 100, 108, 109, 125, 133, 134, 135, 144, 148, 149, 151, 153, 155, 156, 157, 161, 162, 163, 178, 184, 185, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197 tumor growth, xii, 15, 80, 83, 133, 175, 184, 185, 187, 191, 194, 195, 198 tumor invasion, 145, 146, 179 tumor necrosis factor, 134

Milton, Carl A.. Focus on Thyroid Cancer Research, Nova Science Publishers, Incorporated, 2006. ProQuest Ebook Central,

Index

218 tumorigenesis, 85, 112, 168 tumorigenic, 112, 115, 185, 191 tumors, vi, viii, xi, xii, 2, 3, 11, 14, 18, 20, 35, 43, 44, 46, 47, 48, 51, 52, 53, 54, 55, 56, 57, 59, 62, 73, 76, 77, 78, 80, 85, 90, 92, 95, 102, 104, 109, 110, 113, 115, 117, 121, 122, 125, 130, 132, 134, 135, 138, 141, 143, 148, 153, 155, 162, 163, 168, 178, 179, 180, 183, 184, 185, 186, 188, 189, 190, 192, 195, 196, 200 tumour, 13, 16, 23, 103, 105, 106, 109, 139 tumours, 13, 24, 78, 95, 105, 106, 136, 137, 138, 180 Turkey, 91, 125 tyrosine, 106, 128, 133, 136, 137, 163, 167, 168, 180, 181, 182, 196, 199

Copyright © 2006. Nova Science Publishers, Incorporated. All rights reserved.

U ultrasonographic examination, 58, 80 ultrasonography, viii, 16, 17, 24, 25, 37, 43, 44, 48, 58, 80, 81, 82, 86, 88 ultrasound, ix, x, xi, 31, 37, 57, 59, 64, 68, 79, 90, 97, 98, 112, 119, 141, 143, 144, 145, 172, 178 undifferentiated cells, 150, 151, 179 uniform, 65 United Kingdom, 22 United States, 4, 5, 24, 28, 39 univariate, viii, 61, 70 uric acid, 83 urinary, 5 urine, 127 Utah, 113

virus, 106, 134, 139 visible, 8, 131, 150 voids, 81

W Wales, 20 warrants, 122 wavelengths, 186 wells, 186 western countries, 25 Western Europe, 167 WHO classification, 19, 62, 74 withdrawal, 5, 7, 15, 17, 25, 29, 30, 65, 66, 76, 85, 191 women, viii, 2, 4, 22, 39, 44, 46, 68, 117, 122, 166, 169 World Health Organization (WHO), 19, 43, 44, 62, 74, 78, 95

X xenograft, 187 xenografts, 185, 187, 192, 194 x-rays, 12, 14

Y yield, 119 young adults, vii, 1, 36

V vaccination, 134 validation, 110 values, 18, 67, 102, 104, 106, 130, 147, 152 variability, 67, 149 variable, 130, 148 variable expressivity, 130 variables, 15, 26, 32, 35, 57, 78 vascular endothelial growth factor (VEGF), VEGF, 83, 84, 86, 91, 93, 95 vasoactive intestinal peptide, 126 vector, 133, 134, 135, 139 vein, 131, 145, 174 vessels, 84, 85, 145, 153, 176

Milton, Carl A.. Focus on Thyroid Cancer Research, Nova Science Publishers, Incorporated, 2006. ProQuest Ebook Central,

Copyright © 2006. Nova Science Publishers, Incorporated. All rights reserved. Milton, Carl A.. Focus on Thyroid Cancer Research, Nova Science Publishers, Incorporated, 2006. ProQuest Ebook Central,