Papillary Thyroid Carcinoma: Methods and Protocols (Methods in Molecular Biology, 2534) [1st ed. 2022] 9781071625040, 9781071625057, 1071625047

Table of contents :
Preface
Contents
Contributors
Chapter 1: Papillary Thyroid Carcinoma: Current Position in Epidemiology, Genomics, and Classification
1 Introduction: Demographics
2 Changes in Diagnostic Criteria
3 Risk Factors for Papillary Thyroid Carcinoma
4 Radiation and Papillary Thyroid Carcinoma
5 Familial Thyroid Carcinomas
6 Genomic in Papillary Thyroid Carcinoma
7 Incidence in Papillary Thyroid Carcinoma
8 Prognosis
References
Chapter 2: Assessment of Papillary Thyroid Carcinoma with Ultrasound Examination
1 Introduction
2 Materials
3 Methods
3.1 Procedure
3.2 Interpretation
3.2.1 Recommendation for Thyroid Nodules by the American Thyroid Association (2015)
3.2.2 Recommendation for Thyroid Nodules by the American Association of Clinical Endocrinologists/the Italian Associazione Med...
3.2.3 Thyroid Imaging Reporting and Data System (TI-RADS)
3.2.4 Other Recommendations for Thyroid Nodules
3.2.5 Specific Features of Papillary Thyroid Carcinoma
3.3 Evaluation of Cervical Lymph Node Metastasis
4 Notes
References
Chapter 3: Fine-Needle Aspiration Under Guidance of Ultrasound Examination of Thyroid Lesions
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 4: Thyroidectomy for Papillary Thyroid Carcinoma
1 Introduction
2 Materials
2.1 Indications for Surgery
2.2 Preparation of Patients for Surgery
3 Methods
3.1 Choice of Approach and Incision
3.2 Anesthesia
3.3 Positioning
3.4 Operative Techniques
3.5 Postoperative Management
4 Notes
References
Chapter 5: Lymph Node Dissection for Papillary Thyroid Carcinoma
1 Introduction
2 Materials
2.1 Indications for Lymph Node Dissections
2.2 Preparation of Patients for Surgery
3 Methods
3.1 Approaches of Neck Dissection for Papillary Thyroid Carcinoma
3.1.1 Central Neck Dissection
3.1.2 Lateral Neck Dissection (Selective or Functional Neck Dissection)
3.2 Anesthesia
3.3 Positioning
3.4 Operative Techniques
3.4.1 Central Neck Dissection
3.4.2 Lateral Neck Dissection (Level II to IV ± V)
3.4.3 Removal of Other Structures When Necessary (See Note 7)
3.5 Postoperative Management
4 Notes
References
Chapter 6: Macroscopic Examination of Surgical Specimen of Papillary Thyroid Carcinoma
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 7: Histopathological Assessment for Papillary Thyroid Carcinoma
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 8: Concepts of Pathological Staging and Prognosis in Papillary Thyroid Carcinoma
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 9: Liposomal siRNA Delivery in Papillary Thyroid Carcinoma Cells
1 Introduction
2 Materials
2.1 Formulation and Characterization of siRNA-Liposomal Vesicles
2.2 Cell Culture
2.3 Liposome-Loaded siRNA Transfection
2.4 In Vitro Gene Expression Analysis
2.5 In Vivo siRNA-Loaded Liposome Delivery
3 Methods
3.1 Formulation and Characterization of siRNA-Liposomal Vesicles
3.2 Cell Culture
3.3 siRNA Transfection
3.4 In Vitro Gene Expression Analysis
3.5 In Vivo siRNA-Loaded Liposomes Delivery
4 Notes
References
Chapter 10: Long Non-Coding RNAs Profiling Using Microarray in Papillary Thyroid Carcinoma
1 Introduction
1.1 Oncogenic lncRNAs in PTC
1.2 Tumor Suppressor lncRNAs in PTC
2 Materials
2.1 Sample Collection and Preparation
2.2 RNA Extraction and First Stand cDNA Conversion
2.3 Second Strand cDNA Synthesis and Labelling
2.4 LncRNA-Microarray Hybridization
2.5 LncRNA-Microarray Washing
2.6 Data Analysis
3 Methods
3.1 Sample Collection and Preparation
3.2 RNA Extraction and First Stand cDNA Conversion
3.3 Second Strand cDNA Synthesis and Labelling
3.4 LncRNA-Microarray Hybridization
3.5 LncRNA-Microarray Washing
3.6 Data Analysis
4 Notes
References
Chapter 11: Single Nucleotide Polymorphisms in Papillary Thyroid Carcinoma: Clinical Significance and Detection by High-Resolu...
1 Introduction
2 Materials
3 Methods
3.1 Reaction Setup
3.2 High-Resolution Melting and Data Analysis
4 Notes
References
Chapter 12: BRAF Mutations in Papillary Thyroid Carcinoma: A Genomic Approach Using Probe-Based DNA Capture for Next-Generatio...
1 Introduction
2 Materials
3 Methods
3.1 DNA Fragmentation
3.1.1 Enzyme-Based DNA Fragmentation
3.1.2 Sonication-Based DNA Fragmentation
3.2 Capture
3.3 Template Amplification
4 Notes
References
Chapter 13: Application of Immunohistochemistry in Papillary Thyroid Carcinoma
1 Introduction
1.1 Confirmation of Papillary Thyroid Carcinoma in Sites Other s the Thyroid
1.2 Distinguishing Papillary Thyroid Carcinoma from Other Primary Thyroid Neoplasms in the Thyroid
1.3 Distinguish Papillary Thyroid Carcinoma from Secondary Tumors in the Thyroid
1.4 Research Studies on the Pathogenesis and Predictive Value of Markers in Papillary Thyroid Carcinoma
1.5 Principles of Working with IHC
2 Materials
2.1 Protective Measures
2.2 Equipment
2.3 Raw Materials
2.4 Reagents
3 Methods
3.1 Fixation
3.2 Sectioning
3.3 Control Tissue (Fig. 8)
3.4 Tissue Labelling
3.5 Pre-staining Sample Preparation (Dewax, Rehydration, and Antigen Retrieval)
3.6 Sample Staining
4 Notes
References
Chapter 14: Whole-Slide Imaging: Updates and Applications in Papillary Thyroid Carcinoma
1 Introduction
1.1 Research Applications of WSI
1.2 Clinical Uses of WSI
1.2.1 Limitations
1.2.2 Potential Clinical Applications
1.3 Teaching and Assessment Applications
1.4 Application Logistics
2 Materials
3 Methods
3.1 Preparation of the Slides
3.2 System Start-Up
3.3 Loading the Slides for Scanning
3.4 Scanning
3.5 Post-scanning
3.6 View and Analysis of the Image
4 Notes
References
Chapter 15: External Radiotherapy for Locoregional Control in Thyroid Carcinoma
1 Introduction
2 Materials
2.1 Indications for Adjuvant ERT After Surgery
2.1.1 Gross Residual Disease
2.1.2 High Risk for Local Recurrence
2.2 Indication for ERT for Recurrent Disease
2.3 ERT Dose and Volume
3 Methods
3.1 Patient and Simulation
3.2 Target Volumes
3.3 Organs at Risk
3.4 Monitoring During ERT
4 Notes
References
Chapter 16: Radioactive Iodine for Papillary Thyroid Carcinoma
1 Introduction
2 Materials
2.1 Indication of RAI According to Risk Stratification
2.1.1 Low-Risk Group
2.1.2 Intermediate-Risk Group
2.1.3 High-Risk Group
2.2 Contraindications of RAI
2.3 Form of Administration
3 Methods
3.1 Timing of RAI
3.2 Preparations Before RAI
3.2.1 General Preparations
3.2.2 Avoidance of Iodine Excess
3.2.3 Thyroid-Stimulating Hormone (TSH) Stimulation
3.2.4 Blood Test Before RAI: Stimulated Thyroglobulin
3.3 Administration of RAI (on the Day of Administration)
3.4 Precautions After RAI
3.5 Post-therapy Scan
3.6 Side Effects of RAI
3.6.1 Possible Early Side Effects After RAI
3.6.2 Possible Late Side Effects After RAI
3.7 Follow-Up Strategies
3.8 Dynamic Risk Stratification
4 Notes
References
Chapter 17: Radioiodine Refractory Differentiated Thyroid Cancer
1 Introduction
2 Materials
2.1 Definition of RAI-Refractory Thyroid Cancer
2.2 Diagnostic Tools for RAI-Refractory Thyroid Cancer
3 Methods
3.1 Management of RAI-Refractory Thyroid Cancer
3.1.1 Watchful Waiting Under TSH Suppression
3.1.2 Local Therapy
3.1.3 Systemic Therapies
3.2 When to Start Systemic Treatment?
3.2.1 Recommendations from International Guidelines
3.2.2 Clinical Factors Which Guide Initiation of Treatment
3.3 Targeted Agents Evident in Phase 3 Randomized Controlled Trials
3.3.1 Lenvatinib
3.3.2 Sorafenib
3.3.3 Other Targeted Agents
4 Notes
References
Index

Citation preview

Methods in Molecular Biology 2534

Alfred K. Lam Editor

Papillary Thyroid Carcinoma Methods and Protocols

METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK

For further volumes: http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-bystep fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.

Papillary Thyroid Carcinoma Methods and Protocols

Edited by

Alfred K. Lam School of Medicine and Dentistry and Menzies Health Institute Queensland, Griffith University, Gold Coast, QLD, Australia

Editor Alfred K. Lam School of Medicine and Dentistry and Menzies Health Institute Queensland Griffith University Gold Coast, QLD, Australia

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-2504-0 ISBN 978-1-0716-2505-7 (eBook) https://doi.org/10.1007/978-1-0716-2505-7 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Cover Illustration Caption: Microscopic appearance of papillary thyroid carcinoma. This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.

Preface There has been a worldwide increase in the incidence of thyroid cancer in recent years. Papillary thyroid carcinoma is the most common neoplasm in the thyroid gland and affects predominately females. The aims of the book are to present status and management protocols with the objective of identifying proper guidelines and materials for research related to the neoplasm. The different protocols provided will help to understand the pathogenesis as well as improvement of the care of patients with papillary thyroid carcinoma. The target audience of the book includes diverse groups of medical science students, medical students, academics, researchers, and multi-disciplinary clinical teams in management of thyroid cancer such as pathologists, surgeons, radiation oncologists, and endocrinologists. Chapter 1 introduces the epidemiology, genomic, and clinical characteristics of patients with papillary thyroid carcinoma. This chapter guides the readers to explore the other chapters of the book. Chapters 2, 3, 4, and 5 illustrate the diagnostic approaches (ultrasonic examination and fine needle aspiration) and initial surgical managements (thyroidectomy and neck lymph node dissection) for patients with papillary thyroid carcinoma. Chapters 6, 7, and 8 detail the guidelines for macroscopic examination, microscopic examination, and pathological staging of papillary thyroid carcinoma. Chapters 9, 10, 11, and 12 highlight different molecular approaches to the cancer which include siRNA, non-coding RNA, single nucleotide polymorphism, and detection of BRAF mutation, the most important mutation noted in papillary thyroid carcinoma. Chapters 13 and 14 identify the most used pathology laboratory approaches to study papillary thyroid carcinoma, including immunohistochemistry and whole slide imaging. Finally, it is important to be aware of the radiology oncology treatment protocols of thyroid carcinoma as presented in Chapters 15, 16, and 17. By using this book, readers will understand the most updated information on different aspects of the processes, cost, and resources available for the research and management of patients with papillary thyroid carcinoma. Increasing awareness and promoting research in this area will certainly translate to improving the management of papillary thyroid carcinoma. I thank all the authors for their invaluable contributions. Gold Coast, QLD, Australia

Alfred K. Lam

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Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 Papillary Thyroid Carcinoma: Current Position in Epidemiology, Genomics, and Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alfred K. Lam 2 Assessment of Papillary Thyroid Carcinoma with Ultrasound Examination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ichiro Abe and Alfred K. Lam 3 Fine-Needle Aspiration Under Guidance of Ultrasound Examination of Thyroid Lesions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ichiro Abe and Alfred K. Lam 4 Thyroidectomy for Papillary Thyroid Carcinoma. . . . . . . . . . . . . . . . . . . . . . . . . . . . Chung Yau Lo 5 Lymph Node Dissection for Papillary Thyroid Carcinoma . . . . . . . . . . . . . . . . . . . Chung Yau Lo 6 Macroscopic Examination of Surgical Specimen of Papillary Thyroid Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alfred K. Lam 7 Histopathological Assessment for Papillary Thyroid Carcinoma. . . . . . . . . . . . . . . Alfred K. Lam 8 Concepts of Pathological Staging and Prognosis in Papillary Thyroid Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alfred K. Lam 9 Liposomal siRNA Delivery in Papillary Thyroid Carcinoma Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Farhadul Islam, Yaoqi Zhou, and Alfred K. Lam 10 Long Non-Coding RNAs Profiling Using Microarray in Papillary Thyroid Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Farhadul Islam, Yaoqi Zhou, and Alfred K. Lam 11 Single Nucleotide Polymorphisms in Papillary Thyroid Carcinoma: Clinical Significance and Detection by High-Resolution Melting . . . . . . . . . . . . . Robert A. Smith and Alfred K. Lam 12 BRAF Mutations in Papillary Thyroid Carcinoma: A Genomic Approach Using Probe-Based DNA Capture for Next-Generation Sequencing . . . . . . . . . . Robert A. Smith and Alfred K. Lam 13 Application of Immunohistochemistry in Papillary Thyroid Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alfred K. Lam and Katherine Ting-Wei Lee

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Whole-Slide Imaging: Updates and Applications in Papillary Thyroid Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alfred K. Lam, Alfa Bai, and Melissa Leung External Radiotherapy for Locoregional Control in Thyroid Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dora L. W. Kwong and Wendy W. L. Chan Radioactive Iodine for Papillary Thyroid Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . Wendy W. L. Chan and Dora L. W. Kwong Radioiodine Refractory Differentiated Thyroid Cancer . . . . . . . . . . . . . . . . . . . . . . Wendy W. L. Chan, Sonia Chan, and Dora L. W. Kwong

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors ICHIRO ABE • Department of Endocrinology and Diabetes Mellitus, Fukuoka University Chikushi Hospital, Chikushino, Fukuoka, Japan; Cancer Molecular Pathology of School of Medicine and Dentistry, Menzies Health Institute Queensland, Griffith University, Gold Coast, QLD, Australia ALFA BAI • ACT Genomics (Hong Kong) LTD, Hong Kong Science Park, Pak Shek Kok, Hong Kong SONIA CHAN • Department of Clinical Oncology, LKS Faculty of Medicine, The University of Hong Kong, Pok Fu Lam, Hong Kong WENDY W. L. CHAN • Department of Clinical Oncology, LKS Faculty of Medicine, The University of Hong Kong, Pok Fu Lam, Hong Kong FARHADUL ISLAM • Department of Biochemistry and Molecular Biology, University of Rajshahi, Rajshahi, Bangladesh; Cancer Molecular Pathology of School of Medicine and Dentistry, Menzies Health Institute Queensland, Griffith University, Gold Coast, QLD, Australia DORA L. W. KWONG • Department of Clinical Oncology, LKS Faculty of Medicine, The University of Hong Kong, Pok Fu Lam, Hong Kong ALFRED K. LAM • Cancer Molecular Pathology of School of Medicine and Dentistry, Menzies Health Institute Queensland, Griffith University, Gold Coast, QLD, Australia; Pathology Queensland, Gold Coast University Hospital, Southport, QLD, Australia; Faculty of Medicine, University of Queensland, Herston, QLD, Australia KATHERINE TING-WEI LEE • Cancer Molecular Pathology of School of Medicine and Dentistry, Menzies Health Institute Queensland, Griffith University, Gold Coast, QLD, Australia MELISSA LEUNG • Cancer Molecular Pathology of School of Medicine and Dentistry, Menzies Health Institute Queensland, Griffith University, Gold Coast, QLD, Australia CHUNG YAU LO • Department of Surgery, The University of Hong Kong, Pok Fu Lam, Hong Kong ROBERT A. SMITH • Genomics Research Centre, Centre for Genomics and Personalised Health, Queensland University of Technology, Kelvin Grove Campus, QLD, Australia; Cancer Molecular Pathology of School of Medicine and Dentistry, Menzies Health Institute Queensland, Griffith University, Gold Coast, QLD, Australia YAOQI ZHOU • Institute for Systems and Physical Biology, Shenzhen Bay Laboratory, Shenzhen, China

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Chapter 1 Papillary Thyroid Carcinoma: Current Position in Epidemiology, Genomics, and Classification Alfred K. Lam Abstract Papillary thyroid carcinoma is the most common type of thyroid malignancy both in adults and pediatric population. Since the 1980s, there are changes in criteria in labelling thyroid lesions as “papillary thyroid carcinomas.” Radiation exposure is a well-established risk factor for papillary thyroid carcinoma. Other environmental risk factors include dietary iodine, obesity, hormones, and environmental pollutants. Papillary thyroid carcinomas could occur in familial settings, and 5% of these familial cases have well-studied driver germline mutations. In sporadic papillary thyroid carcinoma, BRAF mutation is common and is associated with clinicopathologic and prognostic markers. The mutation could aid in the clinical diagnosis of papillary thyroid carcinoma. Globally, thyroid cancer is among the top ten commonest cancer in females. In both adult and pediatric populations, there are variations of prevalence of thyroid cancer and rising incidence rates of thyroid cancer worldwide. The increase of thyroid cancer incidence was almost entirely due to the increase of papillary thyroid carcinoma. The reasons behind the increase are complex, multifactorial, and incompletely understood. The most obvious reasons are increased use of diagnostic entities, change in classification of thyroid neoplasms, as well as factors such as obesity, environmental risk factors, and radiation. The prognosis of the patients with papillary thyroid carcinoma is generally good after treatment. Nevertheless, cancer recurrence and comorbidity of second primary cancer may occur, and it is important to have awareness of the clinical, pathological, and molecular parameters of papillary thyroid carcinoma. Key words Papillary thyroid carcinoma, Incidence, Radiation, Epidemiology, Classification

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Introduction: Demographics Papillary thyroid carcinoma is the most common type of thyroid malignancy both in adults and pediatric population [1, 2]. It is a primary malignant tumor of thyroid follicular cell derivation characterized by its distinctive nuclear features. The prevalence of papillary thyroid carcinoma is approximately 80–90% of all primary thyroid neoplasms [2–4]. A unique characteristic of papillary thyroid carcinoma, when compared to many other cancers, is the occurrence predominately in females. Papillary thyroid carcinoma

Alfred K. Lam (ed.), Papillary Thyroid Carcinoma: Methods and Protocols, Methods in Molecular Biology, vol. 2534, https://doi.org/10.1007/978-1-0716-2505-7_1, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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is most commonly noted in middle-aged women, in the fifth decade; mean age is around 45 years [2, 4–7]. Approximately 5% of papillary thyroid carcinomas are noted in childhood or adolescents. In regions with high radiation exposure, many papillary thyroid carcinomas are found in this age group [8, 9]. The sporadic cases of thyroid cancer in children, adolescents, and young adults are relatively indolent and associated with good prognosis, but recurrence is frequent, and long-term follow-up is necessary [1]. It is worth noting that diffuse sclerosing subtype of papillary thyroid carcinoma and cribriform morular thyroid carcinoma occur predominately in young patients [10–13]. The female-to-male ratio of papillary thyroid carcinoma is generally between 3–4.5 and 1 [2, 4, 14]. The gender disparity of prevalence of the carcinoma is mostly confined to the detection of small (less than 20 mm) papillary thyroid carcinoma which is more common in females, with a female-to-male ratio of 4.4 to 1 [3]. When the size of the cancer increases, the ratio of detection by gender approaches 1 to 1.

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Changes in Diagnostic Criteria Starting from around the 1980s, there are numerous changes in diagnostic criteria in thyroid neoplasms that will affect the incidence of papillary thyroid carcinoma. In 1977, Chen and Rosai defined the “follicular variant of papillary thyroid carcinoma” based on nuclear features; many follicular pattern thyroid neoplasms were re-labelled to the category of “papillary thyroid carcinoma” [15]. The terminology is of widespread use in around 1980–2000 and accounts partly for the increase in incidence of papillary thyroid carcinoma in this era [16]. Nevertheless, follicular variant of papillary thyroid carcinoma is not a single entity but composed of several morphological entities with different biological behaviors [17]. Major changes in classification of thyroid neoplasms occurred in the two recent editions of the World Health Organization (WHO) classification of endocrine tumors. The basic concept of these re-classifications is to classify thyroid neoplasms according to biological aggressiveness and genomic characteristics [18, 19]. Based on genomic findings as well as prognostic findings, the previous subtype of papillary thyroid carcinoma, known as encapsulated noninvasive follicular variant, is now classified as noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP). NIFTP do not harbor the characteristic BRAF (murine sarcoma viral oncogene homolog B1) mutation of papillary thyroid carcinoma. The tumor has an extremely low risk of adverse outcomes like cancer recurrence or metastases. It is now classified

Papillary Thyroid Carcinoma: Current Position in Epidemiology, Genomics. . .

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into low-risk thyroid neoplasms, whereas papillary thyroid carcinoma is in the group of malignant neoplasms. In addition, a subtype of papillary thyroid carcinoma, invasive encapsulated follicular variant, is removed from papillary thyroid carcinoma in the WHO classification (but still classified as malignant neoplasms). There is well-known controversy between pathologists in the diagnosis of follicular variant of papillary thyroid carcinoma globally [20, 21]. This means that some cases of benign thyroid diseases were diagnosed as follicular variant of papillary thyroid carcinoma and some cases of follicular variant of papillary thyroid carcinoma were graded as benign thyroid diseases. This issue becomes not so important as most of the cases with controversy of whether they are follicular variant of papillary thyroid carcinoma were now labelled as “NIFTP” and not as “papillary thyroid carcinoma.” Before NIFTP was classified, follicular variant of thyroid carcinoma accounted for slightly less than 20% of papillary thyroid carcinoma in regional or multi-institutional international studies [2, 22]. Now, with the removal of NIFTP from the “carcinoma” category, we need to interpret the changes in incidence of thyroid cancer in this context with care. These predicted 20% decrease in incidence may be balanced by other factors which cause increase of thyroid cancer. Thus, if the global rise in incidence of thyroid cancer is due to other environmental factors, the incidence of thyroid cancer may continue to rise, remain stable, or exhibit mild decrease (decrease because of re-classification). It is worth noting that in the United States, there is a recent decline in incidence of papillary thyroid carcinoma for the first time in decades in 2015–2017 and is postulated to change in diagnostic coding (change in tumor classification) [23].

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Risk Factors for Papillary Thyroid Carcinoma The etiology of thyroid cancer is not well understood. The risk of papillary thyroid carcinoma is positively associated with high body mass index, obesity, and hormonal exposures [24–26]. High levels of dietary iodine are related to the increased risk of papillary thyroid carcinoma [27, 28]. Environmental pollutions with toxic metals (such as mercury, cadmium, etc.) in contaminated regions and volcanic area [29–32] and use of polybrominated diphenyl ethers [33] and xenobiotics [34] are associated with increased risk of papillary thyroid carcinoma. Other than these, the major risk factors for papillary thyroid carcinomas are radiation exposure and genetics.

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Radiation and Papillary Thyroid Carcinoma Exposure to radiation is the most well-known etiology factor for papillary thyroid carcinoma. Iatrogenic exposure of radiation may occur in the treatment of head and neck diseases including cancers [35]. The other exposure type is environmental related to ionizing radiation. These include radioactive fallout from testing or largescale disasters. The testing and production of nuclear weapons have been suspected of leaking out radioactive substance to environment. This could include atomic weapons testing, e.g., during the 1950s and 1960s. The first evidence of correlation of large-scale radioactive fallout and thyroid cancer was from survivors from atomic bomb dropped in Hiroshima and Nagasaki of Japan in 1945. The excess thyroid cancer risk associated with childhood exposure has persisted for >50 years after exposure [36]. Then came one of the main disasters of mankind which is the 1986 Chernobyl nuclear accident. The radioactive iodine released and the contamination of the environment lead to significant increase in thyroid carcinoma, mainly papillary thyroid carcinoma in the populations around the regions of Belarus, Ukraine, and Russia [37]. The impacts on the incidence of thyroid carcinoma and the morbidity related to carcinoma persist to date, more than 30 years after the accident [9, 38, 39]. Papillary thyroid carcinomas from these patients have different characteristics from those with sporadic papillary thyroid carcinoma. The 2011 earthquake that damaged nuclear power plants in Fukushima of Japan is another potential source of radiation. Nevertheless, studies to date did not demonstrate increase in incidence of papillary thyroid carcinoma in the region [37, 40, 41]. This may be related to the smaller doses of radiation releases in nuclear plants in Fukushima as compared with Chernobyl. Nevertheless, these findings may need to be confirmed in studies looking at the issue in a longer duration. Lastly, radiofrequency radiation from mobile phones has been classified as possibly carcinogenic to humans by the International Agency for Research on Cancer (IARC). There is a likelihood that radiofrequency radiation may be a risk factor for thyroid carcinoma, but the hypothesis of associations is weak [42, 43]. More epidemiological studies are needed in this aspect.

Papillary Thyroid Carcinoma: Current Position in Epidemiology, Genomics. . .

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Familial Thyroid Carcinomas Medullary carcinoma of the thyroid is often familial and occurs in approximately 20–25% of cases in the setting of multiple endocrine neoplasia (IIA, IIB) or familial medullary thyroid carcinoma [44]. All these syndromes are associated with aberrations of RET gene and are autosomal dominant. On the other hand, non-medullary thyroid cancer uncommonly occurs in familial settings. It is defined by the presence of the cancers in two or more first-degree relatives in the absence of other known familial syndromes. Of these familial non-medullary thyroid cancer cases, 5% have well-studied driver germline mutations [45]. Among these, Cowden syndrome (PTEN), Carney complex type I (PRKARI), Werner syndrome (WRN), and familial adenomatous polyposis/ Gardner syndrome (FAP) have the highest penetrance of thyroid cancer [46, 47]. Other syndromes in which non-medullary thyroid carcinoma could occur include DICER 1 syndrome (DICER1), Peutz-Jeghers syndrome (STK11), McCune-Albright syndrome (GNAS), Ataxia-telangiectasia (ATM), Bannayan-Riley-Ruvalcaba syndrome/PTEN-hamartoma syndrome (PTEN), Li-Fraumeni syndrome (TP53), etc. [48–50]. In the remaining 95% of familial cases of non-medullary thyroid carcinoma, the cause is mostly unknown, and the histologic type is mostly papillary thyroid carcinoma (85–91%) [45]. It is likely this group of cancer is polygenic disorder with variable penetrance. With the use of advanced genomic sequencing, some susceptibility genes have been identified (HABP2, SRGAP1, FOXE1, TITF-1/ NKX2.1, MAP 2K5, SRRM2, RTFC, DUOX2, NOP53, etc.) [46, 51], but known driving gene has not been confirmed. With the increasing incidence of papillary thyroid carcinoma in the recent years, the familial form of the disease is more common than previously reported and comprises 3–9% of all thyroid cancers [45]. This group of cancer likely occurs with relatively young age, associated with tumor multicentricity and/or bilaterally which could draw attention of the familial predisposition [52]. Nevertheless, evidence supports regular examination of individuals with a family history of thyroid cancer to prevent disease progression and to ensure early treatment [53]. Current guidelines do not recommend for or against ultrasonic screening in non-syndromic familial non-medullary thyroid cancer apart from routine physical examination [51]. Overall, meta-analysis showed that patients with familial non-medullary thyroid cancer in this setting tend to have more advanced disease at presentation and tend to receive more aggressive initial therapy [46, 54].

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Genomic in Papillary Thyroid Carcinoma Different from many cancers in which multiple genomic events occur, many papillary thyroid carcinomas harbor a main driver mutation in BRAF, accounting for around half of papillary thyroid carcinoma [55, 56]. Nearly all BRAF mutations in papillary thyroid carcinoma are in BRAF V600E [55]. The other common mutation in the cancer involves telomerase reverse transcriptase (TERT) promoter occurring in approximately 10% of papillary thyroid carcinoma [56]. Other driver alterations for the cancer include EIF1AX, PPMID, and CHEK2 [56]. The clinical roles of BRAF mutation are well studied in patients with papillary thyroid carcinoma. As BRAF mutation is common in papillary thyroid carcinoma, some centers proposed it as guidance to diagnose the presence of papillary thyroid carcinoma in fine needle aspiration biopsy [57]. In addition, BRAF protein could be demonstrated by immunohistochemistry in the tumor tissue which aims in the diagnosis of papillary thyroid carcinoma on resection specimen. Multiple studies were performed to determine the possibility of targeting BRAF mutation in the management of patients with papillary thyroid carcinoma [58–61]. In multicenter studies, BRAF mutation in papillary thyroid carcinoma is associated with increased cancer-related mortality. Because mortality of patients with papillary thyroid carcinoma is low, the association was not independent of other tumor features [56]. Also, in patients with papillary thyroid carcinoma (classical subtype) having BRAF mutation, those with lymph node metastases had increased mortality rates when compared with those without lymph node metastases [62]. In addition, male gender [63] and advanced age [64]-related increase in mortality are also documented only in patients with BRAF mutation and independent of other clinicopathologic risk factors. Furthermore, significant association between BRAF mutation and recurrence of papillary thyroid carcinoma was found in cancers with conventionally low-risk disease stage (I or II), of selected size range and within some subtypes of papillary thyroid carcinoma [65–67]. Patients with papillary thyroid carcinoma having BRAF mutation are more likely to have TERT promoter mutation. Though less common than BRAF mutation, TERT promoter mutation was an independent predictive factor for poor prognosis of patients with papillary thyroid carcinoma [68]. Patients with both BRAF and TERT promoter mutation have much higher risks of adverse outcomes compared with those with a single mutation. There are other molecular alterations in papillary thyroid carcinoma, but they are much less common [69–75]. Among these, fusions involving RET gene are commonly known in papillary thyroid carcinoma. These fusions vary among different geographic regions and are mostly related to etiology of radiation [76–78].

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Incidence in Papillary Thyroid Carcinoma According to the International Agency for Research on Cancer, thyroid cancer accounts for 3% of new cancer cases and 0.4% of new death for all cancers combined in 2020 worldwide [79]. In 2020, there is slightly more than half a million (586,000) of newly diagnosed thyroid cancer. Overall, thyroid cancer is the 11th common cancer among of all cancers. The global incidence rate in women is three to four times higher than that in men [79, 80]. In females, thyroid cancer is the fifth common cancer by incidence (excluding non-melanoma skin cancer). There is variation of prevalence of thyroid cancer worldwide. The highest incidence rates are found in Northern America (highest in Canada, the Unites States), Australia/New Zealand, Eastern Asia (highest in the Republic of Korea), and Southern Europe (highest in Cyprus, Italy, France, etc.) for both gender and in Micronesia/Polynesia and Central and South America (highest in Ecuador, Costa Rica, Brazil, etc.) for women [79]. The low-incidence regions for thyroid cancer include Africa and South-Central Asia (e.g., India). Since the 1980s, there is a continued rising incidence and prevalence rates of thyroid cancer worldwide, and the increase was almost entirely due to an increase of papillary thyroid carcinoma [81–84]. On the other hand, the mortality rates of thyroid cancer are comparatively stable, or decline has been observed in much of the world [80]. The increase in incidence of thyroid cancer is a universal phenomenon and occurs in both high-incidence and low-incidence regions in studies reported in recent years [7, 14, 82, 85–90]. This global trend in incidence of thyroid cancer in children mirrors that of increase in adult populations [83]. The reasons behind the increase incidence of thyroid cancer are complex, multifactorial, and incompletely understood. The most obvious reason behind the rapid rises in incidence rates has been largely attributed to the available and sensitive use of ultrasonography, fine-needle aspiration biopsies, and other diagnostic modalities as well as screening as surveillance in some practices. The current practices now do not recommend active surveillance and mass screening for thyroid cancer [91]. The other obvious reason is the change of classification of the thyroid neoplasms as mentioned above. The change in guidelines for screening, surgical management of thyroid nodules, and thyroid cancer classification in recent years will likely decrease the incidence of thyroid cancer, but it may take more years for the change in incidence to be apparent [92]. Other than these, attention of the potential risk factors such as increasing prevalence of obesity as well as exposure to unknown environmental risk factors such as radiation may contribute to the rising incidence of thyroid cancer.

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Prognosis Papillary thyroid carcinoma is primarily treated by removal of the thyroid gland. Lymph node dissection, radiation therapy and radioactive iodine treatment may be needed depending on the extent and nature of the carcinoma (see chapters 4, 5, 15, 16 & 17). The prognosis of patients with papillary thyroid carcinoma is generally good after appropriate treatment, especially in some patients with some histologic subtypes associated with favorable clinical and pathological parameters. It is worth noting that papillary thyroid carcinoma is characterized by more than ten different histological subtypes with diverse morphology and biological aggressiveness [18, 19]. The other clinical, pathological, and molecular parameters such as age of patients, extent of the carcinoma, presence of local and distant metastases, treatment options and responses, and genetic alterations also affect the prognosis of patients with papillary thyroid carcinoma [17]. The 10-year overall survival rate was often over 90% [5, 6, 93, 94]. Local or regional recurrence of cancer occurs in a portion of cases, in approximately 5% of patients with papillary thyroid carcinoma in large series [95, 96]. In papillary thyroid carcinoma, dedifferentiation to anaplastic thyroid carcinoma and poorly differentiated thyroid carcinoma with aggressive biological behavior may occur [97–100]. Papillary thyroid carcinoma has been reported to transform into anaplastic thyroid carcinoma over a median duration of 6 years [97]. Distant metastases are rarer and could account for 0.4–3% of papillary thyroid carcinoma in large series [96, 101]. Metastasis at multiple sites is an independent factor for mortality in patients with metastatic papillary thyroid carcinoma [101]. Some papillary thyroid carcinomas, such as papillary microcarcinoma, are clinically occult and detected at autopsy [102]. In a series, papillary microcarcinoma was noted in approximately 4% of patients with benign pathologies requiring thyroidectomy [2]. Papillary thyroid carcinoma could occur as a first or second primary cancer in the lifetime of the patient [103–105]. Awareness of the occurrence of secondary thyroid carcinoma is important and may affect the morbidity and mortality of the patients.

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for complete growth arrest in BRAF V600E mutated papillary and undifferentiated thyroid carcinomas. Endocrine 54:129–138. https://doi.org/10.1007/s12020-0160985-7. PMID: 27179656 59. Rahman MA, Salajegheh A, Smith RA, Lam AK (2015) Multiple proliferation-survival signalling pathways are simultaneously active in BRAF V600E mutated thyroid carcinomas. Exp Mol Pathol 99:492–497. https://doi. org/10.1016/j.yexmp.2015.09.006. PMID: 26403329 60. Rahman MA, Salajegheh A, Smith RA, Lam AK (2015) MicroRNA-126 suppresses proliferation of undifferentiated (BRAF(V600E) and BRAF(WT)) thyroid carcinoma through targeting PIK3R2 gene and repressing PI3KAKT proliferation-survival signalling pathway. Exp Cell Res 339:342–350. https://doi.org/ 10.1016/j.yexcr.2015.09.010. PMID: 26384552 61. Rahman MA, Salajegheh A, Smith RA, Lam AK (2014) BRAF inhibitor therapy for melanoma, thyroid and colorectal cancers: development of resistance and future prospects. Curr Cancer Drug Targets 14:128–143. h t t p s : // d o i . o r g / 1 0 . 2 1 7 4 / 1568009614666140121150930. PMID: 24446739 62. Tao Y, Wang F, Shen X, Zhu G, Liu R, Viola D, Elisei R, Puxeddu E, Fugazzola L, Colombo C, Jarzab B, Czarniecka A, Lam AK, Mian C, Vianello F, Yip L, RiescoEizaguirre G, Santisteban P, O’Neill CJ, Sywak MS, Clifton-Bligh R, Bendlova B, Sy´korová V, Zhao S, Wang Y, Xing M (2021) BRAF V600E status sharply differentiates lymph node metastasis-associated mortality risk in papillary thyroid cancer. J Clin Endocrinol Metab 106:3228–3238. h t t p s : // d o i . o r g / 1 0 . 1 2 1 0 / c l i n e m / dgab286. PMID: 34273152 63. Wang F, Zhao S, Shen X, Zhu G, Liu R, Viola D, Elisei R, Puxeddu E, Fugazzola L, Colombo C, Jarzab B, Czarniecka A, Lam AK, Mian C, Vianello F, Yip L, RiescoEizaguirre G, Santisteban P, O’Neill CJ, Sywak MS, Clifton-Bligh R, Bendlova B, Sy´korová V, Wang Y, Xing M (2018) BRAF V600E confers male sex disease-specific mortality risk in patients with papillary thyroid cancer. J Clin Oncol 36:2787–2795. https://doi.org/10.1200/JCO.2018.78. 5097. PMID: 30070937 64. Shen X, Zhu G, Liu R, Viola D, Elisei R, Puxeddu E, Fugazzola L, Colombo C, Jarzab B, Czarniecka A, Lam AK, Mian C, Vianello F, Yip L, Riesco-Eizaguirre G,

Papillary Thyroid Carcinoma: Current Position in Epidemiology, Genomics. . . Santisteban P, O’Neill CJ, Sywak MS, CliftonBligh R, Bendlova B, Sy´korová V, Xing M (2018) Patient age-associated mortality risk is differentiated by BRAF V600E status in papillary thyroid cancer. J Clin Oncol 36: 438–445. https://doi.org/10.1200/JCO. 2017.74.5497. PMID: 29240540 65. Xing M, Alzahrani AS, Carson KA, Shong YK, Kim TY, Viola D, Elisei R, Bendlová B, Yip L, Mian C, Vianello F, Tuttle RM, Robenshtok E, Fagin JA, Puxeddu E, Fugazzola L, Czarniecka A, Jarzab B, O’Neill CJ, Sywak MS, Lam AK, Riesco-Eizaguirre G, Santisteban P, Nakayama H, Clifton-Bligh R, Tallini G, Holt EH, Sy´korová V (2015) Association between BRAF V600E mutation and recurrence of papillary thyroid cancer. J Clin Oncol 33:42–50. https://doi.org/10.1200/ JCO.2014.56.8253. PMID: 25332244 66. Huang Y, Qu S, Zhu G, Wang F, Liu R, Shen X, Viola D, Elisei R, Puxeddu E, Fugazzola L, Colombo C, Jarzab B, Czarniecka A, Lam AK, Mian C, Vianello F, Yip L, Riesco-Eizaguirre G, Santisteban P, O’Neill CJ, Sywak MS, Clifton-Bligh R, Bendlova B, Sy´korová V, Xing M (2018) BRAF V600E mutation-assisted risk stratification of solitary intrathyroidal papillary thyroid cancer for precision treatment. J Natl Cancer Inst 110:362–370. https://doi.org/10. 1093/jnci/djx227. PMID: 29165667 67. Kim KJ, Kim SG, Tan J, Shen X, Viola D, Elisei R, Puxeddu E, Fugazzola L, Colombo C, Jarzab B, Czarniecka A, Lam AK, Mian C, Vianello F, Yip L, RiescoEizaguirre G, Santisteban P, O’Neill CJ, Sywak MS, Clifton-Bligh R, Bendlova B, Sy´korová V, Xing M (2020) BRAF V600E status may facilitate decision-making on active surveillance of low-risk papillary thyroid microcarcinoma. Eur J Cancer 124:161– 169. https://doi.org/10.1016/j.ejca.2019. 10.017. PMID: 31790974 68. Chen B, Shi Y, Xu Y, Zhang J (2021) The predictive value of coexisting BRAFV600E and TERT promoter mutations on poor outcomes and high tumour aggressiveness in papillary thyroid carcinoma: a systematic review and meta-analysis. Clin Endocrinol 94:731– 7 4 2 . h t t p s : // d o i . o r g / 1 0 . 1 1 1 1 / c e n . 14316. PMID: 32816325 69. Morariu EM, McCoy KL, Chiosea SI, Nikitski AV, Manroa P, Nikiforova MN, Nikiforov YE (2021) Clinicopathologic characteristics of thyroid nodules positive for the THADAIGF2BP3 fusion on preoperative molecular analysis. Thyroid 31:1212–1218. https://

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doi.org/10.1089/thy.2020.0589. PMID: 33487086 70. Panebianco F, Nikitski AV, Nikiforova MN, Kaya C, Yip L, Condello V, Wald AI, Nikiforov YE, Chiosea SI (2019) Characterization of thyroid cancer driven by known and novel ALK fusions. Endocr Relat Cancer 26:803– 814. https://doi.org/10.1530/ERC19-0325. PMID: 31539879 71. Prasad ML, Vyas M, Horne MJ, Virk RK, Morotti R, Liu Z, Tallini G, Nikiforova MN, Christison-Lagay ER, Udelsman R, Dinauer CA, Nikiforov YE (2016) NTRK fusion oncogenes in pediatric papillary thyroid carcinoma in northeast United States. Cancer 122: 1097–1107. https://doi.org/10.1002/cncr. 29887. PMID: 26784937 72. Borrelli N, Panebianco F, Condello V, Barletta JA, Kaya C, Yip L, Nikiforova MN, Nikiforov YE (2019) Characterization of activating mutations of the MEK1 gene in papillary thyroid carcinomas. Thyroid 29: 1279–1285. https://doi.org/10.1089/thy. 2019.0065. PMID: 31407636 73. Nikiforov YE (2016) Thyroid cancer in 2015: molecular landscape of thyroid cancer continues to be deciphered. Nat Rev Endocrinol 12(2):67–68. https://doi.org/10.1038/ nrendo.2015.217. PMID: 26668120 74. Yakushina VD, Lerner LV, Lavrov AV (2018) Gene fusions in thyroid cancer. Thyroid 28: 158–167. https://doi.org/10.1089/thy. 2017.0318. PMID: 29281951 75. Pekova B, Sykorova V, Mastnikova K, Vaclavikova E, Moravcova J, Vlcek P, Lastuvka P, Taudy M, Katra R, Bavor P, Kodetova D, Chovanec M, Drozenova J, Astl J, Hrabal P, Vcelak J, Bendlova B (2021) NTRK fusion genes in thyroid carcinomas: clinicopathological characteristics and their impacts on prognosis. Cancers (Basel) 13(8):1932. https://doi.org/10.3390/ cancers13081932. PMID: 33923728 76. Romei C, Elisei R (2021) A narrative review of genetic alterations in primary thyroid epithelial cancer. Int J Mol Sci 22:1726. https:// doi.org/10.3390/ijms22041726. PMID: 33572167 77. Pacini F, Elisei R, Romei C, Pinchera A (2000) RET proto-oncogene mutations in thyroid carcinomas: clinical relevance. J Endocrinol Investig 223:328–338. https://doi. org/10.1007/BF03343732. PMID: 10882153 78. Romei C, Ciampi R, Elisei R (2016) A comprehensive overview of the role of the RET

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proto-oncogene in thyroid carcinoma. Nat Rev Endocrinol 12:192–202. https://doi. org/10.1038/nrendo.2016.11. PMID: 26868437 79. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F (2021) Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 71:209–249. https://doi.org/ 10.3322/caac.21660. Epub 2021 Feb 4. PMID: 33538338 80. Lortet-Tieulent J, Franceschi S, Dal Maso L, Vaccarella S (2019) Thyroid cancer “epidemic” also occurs in low- and middleincome countries. Int J Cancer 144:2082– 2 0 8 7 . h t t p s : //d o i . o r g / 1 0 . 1 0 0 2 / i j c . 31884. PMID: 30242835 81. Olson E, Wintheiser G, Wolfe KM, Droessler J, Silberstein PT (2019) Epidemiology of thyroid cancer: a review of the National Cancer Database, 2000-2013. Cureus 11: e4127. https://doi.org/10.7759/cureus. 4127. PMID: 31049276 82. Boukheris H, Bachir Bouiadjra N (2021) Thyroid cancer incidence and trends by demographic and tumor characteristics in Oran, Algeria: 1993-2013, a populationbased analysis. Eur J Cancer Prev (in press). h t t p s : // d o i . o r g / 1 0 . 1 0 9 7 / C E J . 0000000000000699. PMID: 34519694 83. Vaccarella S, Lortet-Tieulent J, Colombet M, Davies L, Stiller CA, Schu¨z J, Togawa K, Bray F, Franceschi S, Dal Maso L, SteliarovaFoucher E, IICC-3 Contributors (2021) Global patterns and trends in incidence and mortality of thyroid cancer in children and adolescents: a population-based study. Lancet Diabetes Endocrinol 9:144–152. https://doi. org/10.1016/S2213-8587(20) 30401-0. PMID: 33482107 84. Lim H, Devesa SS, Sosa JA, Check D, Kitahara CM (2017) Trends in thyroid cancer incidence and mortality in the United States, 1974-2013. JAMA 317:1338–1348. https:// doi.org/10.1001/jama.2017.2719. PMID: 28362912 85. Loizou L, Demetriou A, Erdmann F, Borkhardt A, Brozou T, Sharp L, McNally R (2021) Increasing incidence and survival of paediatric and adolescent thyroid cancer in Cyprus 1998-2017: a population-based study from the Cyprus Pediatric Oncology Registry. Cancer Epidemiol 74:101979. https://doi.org/10.1016/j.canep.2021. 101979. PMID: 34247065

86. Li M, Pei J, Xu M, Shu T, Qin C, Hu M, Zhang Y, Jiang M, Zhu C (2021) Changing incidence and projections of thyroid cancer in mainland China, 1983-2032: evidence from Cancer Incidence in Five Continents. Cancer Causes Control 32:1095–1105. https://doi. org/10.1007/s10552-021-01458-6. PMID: 34152517 87. Park J, Park H, Kim TH, Kim SW, Jang HW, Chung JH (2021) Trends in childhood thyroid cancer incidence in Korea and its potential risk factors. Front Endocrinol (Lausanne) 12:681148. https://doi.org/10.3389/ fendo.2021.681148. PMID: 34054738 88. Oh CM, Lim J, Jung YS, Kim Y, Jung KW, Hong S, Won YJ (2021) Decreasing trends in thyroid cancer incidence in South Korea: what happened in South Korea? Cancer Med 10: 4087–4096. https://doi.org/10.1002/ cam4.3926. PMID: 33979040 89. de Morais Fernandes FCG, de Souza DLB, Curado MP, de Souza TA, de Almeida Medeiros A, Barbosa IR (2021) Incidence and mortality from thyroid cancer in Latin America. Tropical Med Int Health 26:800– 8 0 9 . h t t p s : // d o i . o r g / 1 0 . 1 1 1 1 / t m i . 13585. PMID: 33837603 90. Lee YA, Yun HR, Lee J, Moon H, Shin CH, Kim SG, Park YJ (2021) Trends in pediatric thyroid cancer incidence, treatment, and clinical course in Korea during 2004-2016: a nationwide population-based study. Thyroid 31:902–911. https://doi.org/10.1089/thy. 2020.0155. PMID: 33107409 91. Lin JS, Bowles EJA, Williams SB, Morrison CC (2017) Screening for thyroid cancer: updated evidence report and systematic review for the US Preventive Services Task Force. JAMA 317(18):1888–1903. https:// doi.org/10.1001/jama.2017.0562. PMID: 28492904 92. Kitahara CM, Sosa JA (2020) Understanding the ever-changing incidence of thyroid cancer. Nat Rev Endocrinol 16:617–618. https:// doi.org/10.1038/s41574-02000414-9. PMID: 32895503 93. Cao YM, Zhang TT, Li BY, Qu N, Zhu YX (2021) Prognostic evaluation model for papillary thyroid cancer: a retrospective study of 660 cases. Gland Surg 10:2170–2179. h t t p s : // d o i . o r g / 1 0 . 2 1 0 3 7 / g s 21-100. PMID: 34422588 94. Ito Y, Miyauchi A, Kihara M, Fukushima M, Higashiyama T, Miya A (2018) Overall survival of papillary thyroid carcinoma patients: a single-institution long-term follow-up of

Papillary Thyroid Carcinoma: Current Position in Epidemiology, Genomics. . . 5897 patients. World J Surg 42:615–622. https://doi.org/10.1007/s00268-0184479-z. PMID: 29349484 95. Kim SY, Kim YI, Kim HJ, Chang H, Kim SM, Lee YS, Kwon SS, Shin H, Chang HS, Park CS (2021) New approach of prediction of recurrence in thyroid cancer patients using machine learning. Medicine (Baltimore) 100: e27493. https://doi.org/10.1097/MD. 0000000000027493. PMID: 34678881 96. Ywata de Carvalho A, Kohler HF, Gomes CC, Vartanian JG, Kowalski LP (2021) Predictive factors for recurrence of papillary thyroid carcinoma: analysis of 4,085 patients. Acta Otorhinolaryngol Ital 41:236–242. https://doi. org/10.14639/0392-100X-N1412. PMID: 34264917 97. Yau T, Lo CY, Epstein RJ, Lam AK, Wan KY, Lang BH (2008) Treatment outcomes in anaplastic thyroid carcinoma: survival improvement in young patients with localized disease treated by combination of surgery and radiotherapy. Ann Surg Oncol 15:2500–2505. https://doi.org/10.1245/s10434-0080005-0. PMID: 18581185 98. Lam KY, Lo CY, Chan KW, Wan KY (2000) Insular and anaplastic carcinoma of the thyroid: a 45-year comparative study at a single institution and a review of the significance of p53 and p21. Ann Surg 231:329–338. https://doi.org/10.1097/00000658200003000-00005. PMID: 10714625 99. Volante M, Lam AK, Papotti M, Tallini G (2021) Molecular pathology of poorly differentiated and anaplastic thyroid cancer: what do pathologists need to know? Endocr Pathol 32:63–76. https://doi.org/10.1007/ s12022-021-09665-2. PMID: 33543394 100. Abe I, Lam AK (2021) Anaplastic thyroid carcinoma: updates on WHO classification, clinicopathological features and staging. Histol Histopathol 36:239–248. https://doi. org/10.14670/HH-18-277. PMID: 33170501 101. Nunes KS, Matos LL, Cavalheiro BG, Magnabosco FF, Tavares MR, Kulcsar MA, Hoff

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Chapter 2 Assessment of Papillary Thyroid Carcinoma with Ultrasound Examination Ichiro Abe and Alfred K. Lam Abstract Ultrasound examination of the thyroid is useful for preoperative assessment of thyroid nodules including papillary thyroid carcinoma. The examination mainly is to determine the malignant potential of thyroid nodule(s). There are different systems to predict malignant potential in the thyroid nodules and cervical lymph nodes by ultrasound. Ultrasound is used in conjunction with fine-needle aspiration to diagnosis papillary thyroid carcinoma. It is used as guidance to locate the sites to obtain the samples for diagnosis and research in papillary thyroid carcinoma. Key words Ultrasound examination, Papillary thyroid carcinoma, Lymph node metastasis

1

Introduction Ultrasound examination of the neck is useful for preoperative assessment of clinical suspicious lesion of the thyroid gland, most commonly papillary thyroid carcinoma. Besides, ultrasound examination could predict the parameters of the staging system of the American Joint Committee on Cancer, such as tumor size and extrathyroidal extension (about primary tumor (T)) (see Chapter 8) [1]. According to the American Thyroid Association Guidelines, ultrasound examinations should be performed in all patients with a suspected thyroid nodule(s) [2]. Besides, majority of cervical lymph nodes are located superficially and could be evaluated with ultrasound examination (about regional lymph nodes (N)). Considering papillary carcinoma has the frequent metastatic involvement of cervical lymph nodes, it is important to examine the neck by ultrasound in cases suspicious for papillary thyroid carcinoma. Hence, ultrasound examination is cardinal for not only diagnosis of papillary thyroid carcinoma but also its pathological staging.

Alfred K. Lam (ed.), Papillary Thyroid Carcinoma: Methods and Protocols, Methods in Molecular Biology, vol. 2534, https://doi.org/10.1007/978-1-0716-2505-7_2, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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Some systems distinguish each group of risk of malignancy about thyroid nodules or cervical lymph nodes according to the pattern of ultrasound [2–5]. According to these systems, the findings of ultrasound examination lead to the judgement of whether the fine-needle aspiration of thyroid nodules or cervical lymph nodes should be performed. Thus, ultrasound examination is a very useful guide to obtain the samples for diagnosis and research in papillary thyroid carcinoma. In this chapter, we describe the procedure and construction of findings of ultrasound examination for thyroid nodules and cervical lymph nodes, which lead to contribute the diagnosis and staging of papillary thyroid carcinoma.

2

Materials 1. Ultrasound machine (Fig. 1a). The suitable machine is with available transducer, operating mode, and system settings for scan to obtain the quality of the image (see Note 1). 2. High-frequency linear probe (Fig. 1b) (see Note 2). 3. Ultrasound transmission gels (see Note 3). 4. Operator and assistant who has good knowledge and technique of ultrasound examination of thyroid nodules as well as surrounding organs.

Fig. 1 Ultrasound machine (a) and the linear probe (b)

Assessment of Papillary Thyroid Carcinoma with Ultrasound Examination

3 3.1

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Methods Procedure

1. Preparation of the patient (a) Patients do not need to be fasted or take any pre-medications. (b) Prepare patients to lie on the bed. If impossible, prepare patients to sit on the chair. (c) Look at the clinical record to see if booking of fine-needle aspiration is included. If yes, be also aware of the precautions needed for fine-needle aspiration (see Chapter 3). 2. Physical examination to confirm the location(s) of the nodule(s). 3. Put the gel on the neck region. 4. Start scanning on the neck region. 5. Observation of the whole thyroid and surrounding organs should be performed. Most scans should be investigated in B-mode. Color flow or power Doppler examination is also required (see Note 4). 6. If nodules are detected, the operator should investigate the following characteristics: (a) The position of the nodules in the thyroid; the side as well as position in upper pole, the lower pole, the middle third of the thyroid, or the isthmus. (b) The number of nodules in the thyroid (c) The size of each nodule (d) The echogenicity of the nodules (e) The presence of calcification (f) The shape of the nodules (g) Regularity of the margins (h) The presence of halo (i) Vascularization of the nodules 7. The operator should also investigate cervical lymph nodes.

3.2

Interpretation

3.2.1 Recommendation for Thyroid Nodules by the American Thyroid Association (2015)

There are several systems for reporting the findings of thyroid lesions in ultrasonic examination. The American Thyroid Association distinguishes the following five groups of risk of malignancy about thyroid carcinoma according to the pattern of ultrasound [2] (see Note 5): 1. High suspicion 2. Intermediate suspicion 3. Low suspicion

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4. Very low suspicion 5. Benign 3.2.2 Recommendation for Thyroid Nodules by the American Association of Clinical Endocrinologists/the Italian Associazione Medici Endocrinologi (2016)

The American Association of Clinical Endocrinologists/the Italian Associazione Medici Endocrinologi distinguishes the following three groups of risk of malignancy with the pattern of ultrasound [3] (see Note 6): 1. High risk 2. Intermediate risk 3. Low risk

3.2.3 Thyroid Imaging Reporting and Data System (TI-RADS)

Thyroid imaging reporting and data system (TI-RADS) was originally described in 2009 [4]. Several societies and countries, such as in Australia, South Korea, or the United States, also adopt this system. TI-RADS is the classification system to determine the risk of malignancy for thyroid lesions based on ultrasound feature. The original TI-RADS classification distinguishes the following groups (see Note 7): TI-RADS 1: normal thyroid gland TI-RADS 2: benign conditions TI-RADS 3: probably benign nodules TI-RADS 4: suspicious nodules 4a: undetermined 4b: suspicious TI-RADS 5: probably malignant nodules TI-RADS 6: biopsy-proven malignancy

3.2.4 Other Recommendations for Thyroid Nodules

Increased intra-nodular flow and absence of halo could be the specific features indicating malignancy [6–8] (see Note 8).

3.2.5 Specific Features of Papillary Thyroid Carcinoma

Among thyroid carcinomas, papillary thyroid carcinoma commonly presents as a hypoechoic nodule, which is darker than the surrounding normal thyroid tissue in addition to the features written in Subheading 3.2.1 [9, 10]. Besides, microcalcifications are more detected in papillary thyroid carcinoma than the other thyroid carcinomas [9, 10] (Fig. 2) (see Note 9).

3.3 Evaluation of Cervical Lymph Node Metastasis

The guideline of the European Thyroid Association describes the following characteristics as suspicious for malignancy (at least one) [5] (see Note 9): 1. Microcalcifications 2. Partially cystic appearance

Fig. 2 The ultrasound imaging of papillary thyroid carcinoma. (a) The nodule is hypervascular hypoechoic with microcalcification and non-encapsulated with irregular margins. (b) The nodule has continuous calcifications at rim. The hypervascularity of the nodule is unclear. (c) The lymph node has microcalcification and cystic lesion. The lymph node is iso-hyperechoic compared with normal lymph nodes. All were diagnosed as papillary thyroid carcinomas (including lymph node metastasis) pathologically

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3. Peripheral or diffusely increased vascularization 4. Hyperechoic tissue looking like thyroid In addition, malignant lymph nodes are suspected when the height of the lymph node is equal to or greater than the width causing a more rounded appearance [11, 12] (see Note 9).

4

Notes 1. Regarding ultrasound machine, console machine should be used. Portable ultrasound machine might not be used. Recent console machine includes the computer soft which enables staff to investigate better. Besides, the image appears on an independent monitor (Fig. 1a). 2. The American Thyroid Association recommends at least 10 MHz probes, whereas the European Thyroid Association recommends at least 12 MHz probes [2, 5]. High-frequency linear probe offers the advantage of high resolution. However, if patients have obesity, high-frequency linear probe could describe only a small field and limited depth so that low-frequency linear probe is sometimes required. In addition, there are high-quality probes with at least 3.5 cm footprint. The width of each of the normal thyroid lobe is less than 2 cm. Thus, footprint probes of 3.5 cm or more could lead to better investigation of the thyroid gland, especially with large thyroid nodules, and in surrounding organs such as cervical lymph nodes [5] (Fig. 1b). 3. Standard ultrasound gels are composed of propylene glycol and water. Propylene glycol has minimal biotoxicity, is odorless, and has enough viscosity. Thus, it is suitable for thyroid and neck ultrasound as well as the other organs. 4. There are several modes of ultrasound. B mode (Brightness mode) is generally used in various clinical examinations [13]. In B mode, two-dimensional (2D) image is obtained. This mode could visualize stationary organs as well as describe moving objects such as red cells in the blood with color flow or power Doppler examination. Velocity information is displayed in the image in color flow or power Doppler examination, which could evaluate internal blood flow of thyroid nodules or cervical lymph nodes [14]. 5. The American Thyroid Association recommends thyroid nodules to be evaluated concerning risk of malignancy about thyroid carcinoma by the appearance in ultrasound [2] (Table 1).

Intermediate suspicion A solid hypoechoic nodule with smooth margins without microcalcifications, oval shape, and extrathyroidal extension

Ovoid or round shape, smooth or ill-defined margins, intra-nodular vascularity, stiffness on elastography, continuous calcifications at rim

Intermediate risk Slightly hypoechoic or isoechoic nodules with the following findings:

High suspicion High risk Nodules with at least one of the following findings: A solid hypoechoic nodule or substantial marked hypoechogenicity, speculated or microhypoechoic component of a partially cystic lobulated margins, microcalcifications, oval nodule with at least one of the following shape, and extrathyroidal growth findings: Irregular margins, microcalcifications, oval shape, extrathyroidal extension, calcifications at the rim with small extrusive soft tissue component

Recommendation for thyroid nodules by the American Thyroid Association (2015)

Recommendation for thyroid nodules by the American Association of Clinical Endocrinologists/the Italian Associazione Medici Endocrinologi (2016)

Table 1 Comparison of the three current suggestions with ultrasound findings

(continued)

TI-RADS 4b (suspicious: 10–80% malignancy) Suspicious neoplastic pattern: hyperechoic, isoechoic, or hypoechoic encapsulated with a thick capsule, hyper-vascularized, and with calcifications TI-RADS 4a (suspicious: 5–10% malignancy) Simple neoplastic pattern: solid or mixed hyperechoic, isoechoic, hypoechoic encapsulated with a thin capsule de Quervain pattern

Isoechoic or hypoechoic, non-encapsulated, hyper-vascularized, and multiple peripheral microcalcifications Mixed echogenicity or isoechoic without hyperechoic spots, non-encapsulated, and hyper-vascularized

(TI-RADS 6: biopsy-proven malignancy) TI-RADS 5 (>80% malignancy) Hypoechoic, non-encapsulated with irregular margins, and penetrating vessels

Thyroid imaging reporting and data system (TI-RADS)

Assessment of Papillary Thyroid Carcinoma with Ultrasound Examination 23

Recommendation for thyroid nodules by the American Association of Clinical Endocrinologists/the Italian Associazione Medici Endocrinologi (2016)

Benign Purely cystic nodules

Low suspicion Low risk Isoechoic or hyperechoic solid nodule, or partially Isoechoic spongiform nodules or cysts cystic nodule with eccentric solid areas without microcalcifications, oval shape, and extrathyroidal extension Very low suspicion Spongiform or partially cystic nodules without any of the sonographic features of high, intermediate, and low suspicion

Recommendation for thyroid nodules by the American Thyroid Association (2015)

Table 1 (continued)

TI-RADS 3 category (40 mm limited to thyroid

T3b

–

Tumor of any size grossly extrathyroidal extension invading only strap muscles

Tumor >20 mm but 40 mm in greatest dimension limited to thyroid

T4 T4a

Gross extrathyroidal extension invading Moderately advanced disease subcutaneous tissue, larynx, trachea, Tumor of any size extending beyond the thyroid esophagus, or recurrent laryngeal nerve capsule to invade subcutaneous soft tissues, from tumor of any size larynx, trachea, esophagus or recurrent laryngeal nerve

T4b

Very advanced disease Tumor invades prevertebral fascia or encases carotid artery or mediastinal vessels

Gross extrathyroidal extension invading prevertebral fascia encasing the carotid artery or mediastinal vessels from a tumor of any size

this new edition of AJCC. T3a is a thyroid carcinoma more than 40 mm in greatest dimension. T3b is a new category which is different in definition from T3 in the previous edition of AJCC. In this edition, minor extrathyroidal extension detected by histological examination is not included in T3 category. Gross extrathyroidal extension invading only strap muscles is included in T3b category. It is worth noting that an international study revealed that concordance was highest when pathologists (11 expert endocrine pathologists) assessed the spatial relationship of carcinoma to skeletal muscles as compared to microscopic extrathyroidal extension [38]. This supports the use of invasion of strap muscles as criterion for T staging.

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3. In the eighth edition of AJCC, lymph node metastasis carries equal weighing to prognostic staging no matter the level (location) of lymph node involvement. Nevertheless, it is recommended to document the location of involved lymph node by classifying the N into N1a and N1b. N1a is metastasis to level VI and VII lymph nodes (central lymph nodes), which can be unilateral or bilateral while N1b is metastasis to unilateral, bilateral, or contralateral lateral neck lymph nodes (levels I, II, III, IV, or V) or retropharyngeal lymph nodes (see the Chapter on macroscopic). It is worth noting that VII is included as N1b in the seventh edition of AJCC but is included as N1a in the eighth edition of AJCC. In addition, the number of involved lymph nodes, number of lymph nodes sampled, size of largest involved lymph node, size of the largest involved lymph node, size of metastatic foci within involved lymph nodes, and extranodal extension could be useful to be documented as recommended by AJCC. These parameters are also useful for (ATA) risk predication system [27]. 4. The presence of distant metastases (M1) will put the papillary thyroid carcinoma in most advanced pathological stage grouping as distant metastasis is associated with poor prognosis in patients with papillary thyroid carcinoma. Multi-organ metastasis and brain involvement are associated with lower survival rates in papillary thyroid carcinoma [39]. Papillary thyroid carcinoma does not often develop distant metastases. Biological aggressive subtype, such as diffuse sclerosing subtype, could have higher rates of distant metastases [40, 41]. Overall, large populational studies reveal that 2% of papillary thyroid carcinoma had distant metastases at initial diagnosis and slightly over one fourth presented with multiorgan disease [39]. The most common site of distant metastasis is the lung, followed by bone, liver, and brain. 5. In the current edition, the major change is the cut-off age which has been moved from 45 to 55 to better reflect the prognostic differences between the different age groups. This is based on an international multi-institutional validation (from 9,494 patients from ten institutions) of age 55 as a cut-off for risk stratification in the AJCC/UICC staging system for welldifferentiated thyroid cancer [42]. 6. The revised AJCC staging for papillary thyroid carcinoma leads to the down staging of many patients with papillary thyroid carcinoma to avoid unnecessary treatment of patients with indolent thyroid carcinoma. The changes in the prognostic stage grouping are summarized in Table 3. The prognostic stage grouping for younger patients remains the same apart from changes form cut-off age from 45 to 55. All these

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Table 3 Comparison of the two prognostic staging grouping for papillary thyroid carcinoma Seventh edition

Eighth edition

Under 45

Under 55

Stage I

Any T, any N, M0

Any T, any N, M0

Stage II

Any T, any N, M1

Any T, any N, M1

45 and above

55 and above

T1, N0, M0

T1, N0/Nx, M0

Stage I

T2, N0/Nx, M0 Stage II

T2, N0, M0 T1, N1, M0 T2, N1, M0 T3a/T3b, any N, M0

Stage III

T1, N1a, M0 T2, N1a, M0 T3, N0, M0 T3, N1a, M0 T4a, any N, M0

Stage IVA

T4a, N0, M0 T4a, N1a, M0 T4a, N1b, M0 T1, N1b, M0 T2, N1b, M0 T3, N1b, M0 T4b, any N, M0

Stage IVB

T4b, any N, M0 Any T, any N, M1

Stage IVC

Any T, any N, M1

relatively young patients with papillary thyroid carcinoma are classified in either Stage I (without distant metastases) or Stage II (with distant metastases). On the other hand, for patients with age 55 and above, many patients with papillary thyroid carcinoma are downstaged. Patients with T2, N0/x, M0 carcinoma were previously in Stage II, and now groups with T1, N0/x carcinoma are in Stage I. Those carcinomas previously in Stage III (T1/T2, N1, M0 or T3 any N, M0) are now in Stage

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II. It means that in older patients (55 or above), patients with positive lymph nodes are downstaged to Stage II. Stage III carcinomas only comprise T4a, any N M0 carcinomas (in the previous edition, these cancers are Stage IVA). In this new prognostic stage grouping, Stage IV, only two subgroups IVA and IVB are used (instead of three subgroups in the seventh edition). Stage IVA carcinomas are T4b any N M0 (previously Stage IVB) carcinomas while Stage IVB carcinomas are carcinomas with distant metastases (M1) (previously Stage IVC). References 1. Lam KY, Lo CY, Chan KW, Wan KY (2000) Insular and anaplastic carcinoma of the thyroid: a 45-year comparative study at a single institution and a review of the significance of p53 and p21. Ann Surg 231:329–338 2. Nies M, Vassilopoulou-Sellin R, Bassett RL, Yedururi S, Zafereo ME, Cabanillas ME, Sherman SI, Links TP, Waguespack SG (2001) Distant metastases from childhood differentiated thyroid carcinoma: clinical course and mutational landscape. J Clin Endocrinol Metab 106:e1683–e1697 3. Beahrs OH, Myers MH (1977) Thyroid gland. In: Beahrs OH, Myers MH (eds) American Joint Committee on Cancer: manual for staging of cancer, 2nd edn. J. B. Lippincott Company, Philadelphia, pp 55–57 4. Lang B, Lo CY, Chan WF, Lam KY, Wan KY (2007) Restaging of differentiated thyroid carcinoma by the sixth edition AJCC/UICC TNM staging system: stage migration and predictability. Ann Surg Oncol 14:1551–1559 5. Vrachimis A, Gerss J, Stoyke M, Wittekind C, Maier T, Wenning C, Rahbar K, Schober O, Riemann B (2014) No significant difference in the prognostic value of the 5th and 7th editions of AJCC staging for differentiated thyroid cancer. Clin Endocrinol 80:911–917 6. Michael Tuttle R, Morris LF, Haugen BR, Shah JP, Sosa JA, Rohren E, Subramaniam RM, Hunt JL, Perrier ND (2017) Thyroiddifferentiated and anaplastic carcinoma. In: Amin MB, Edge S, Greene F, Byrd DR, Brookland RK, Washington MK, Gershenwald JE, Compton CC, Hess KR, Sullivan DC, Jessup JM, Brierley JD, Gaspar LE, Schilsky RL, Balch CM, Winchester DP, Asare EA, Madera M, Gress DM, Meyer LR (eds) AJCC cancer staging manual, 8th edn. Springer, New York, pp 873–890 7. Cavalheiro BG, de Matos LL, Leite AKN, Kulcsar MAV, Cernea CR, Kowalski LP (2021) Survival in differentiated thyroid

carcinoma: comparison between the 7th and 8th editions of the AJCC/UICC TNM staging system and the ATA initial risk stratification system. Head Neck 43:2913–2922 8. Alzahrani AS, Albalawi L, Mazi S, Mukhtar N, Aljamei H, Moria Y, Elsayed T, Amer L, Alanazi F, Alnasser L, Alqarni B, Fadel R, AlMatar A, Alqahtani A, Tuttle RM (2021) How does the AJCC/TNM staging system Eighth Edition perform in thyroid cancer at a major Middle Eastern medical center? Endocr Pract 27:607–613 9. Thewjitcharoen Y, Chatchomchuan W, Karndumri K, Porramatikul S, Krittiyawong S, Wanothayaroj E, Butadej S, Nakasatien S, Veerasomboonsin V, Kanchanapituk A, Rajatanavin R, Himathongkam T (2021) Impacts of the American Joint Committee on Cancer (AJCC) 8 edition tumor, node, metastasis (TNM) staging system on outcomes of differentiated thyroid cancer in Thai patients. Heliyon 7:e06624 10. Kim K, Kim JK, Lee CR, Kang SW, Lee J, Jeong JJ, Nam KH, Chung WY (2020) Comparison of long-term prognosis for differentiated thyroid cancer according to the 7th and 8th editions of the AJCC/UICC TNM staging system. Ther Adv Endocrinol Metab 11. h t t p s : // d o i . o r g / 1 0 . 1 1 7 7 / 2042018820921019 11. Zhi J, Wu Y, Hu L, Zhao J, Liu H, Ruan X, Hou X, Zhang J, Zheng X, Gao M (2020) Assessment of the prognostic value and N1b changes of the eighth TNM/AJCC staging system for differentiated thyroid carcinoma. Int J Clin Oncol 25:59–66 12. Dwamena S, Patel N, Egan R, Stechman M, Scott-Coombes D (2019) Impact of the change from the seventh to eighth edition of the AJCC TNM classification of malignant tumours and comparison with the MACIS prognostic scoring system in non-medullary thyroid cancer. BJS Open 3:623–628

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13. Chereau N, Oyekunle TO, ZambeliLjepovic´ A, Kazaure HS, Roman SA, Menegaux F, Sosa JA (2019) Predicting recurrence of papillary thyroid cancer using the eighth edition of the AJCC/UICC staging system. Br J Surg 106:889–897 14. Gan T, Huang B, Chen Q, Sinner HF, Lee CY, Sloan DA, Randle RW (2019) Risk of recurrence in differentiated thyroid cancer: a population-based comparison of the 7th and 8th Editions of the American Joint Committee on Cancer Staging Systems. Ann Surg Oncol 26:2703–2710 15. Hulse K, Williamson A, Gibb FW, Conn B, Nixon IJ (2019) Evaluating the predicted impact of changes to the AJCC/TMN staging system for differentiated thyroid cancer (DTC): a prospective observational study of patients in South East Scotland. Clin Otolaryngol 44:330–335 16. Shaha AR, Migliacci JC, Nixon IJ, Wang LY, Wong RJ, Morris LGT, Patel SG, Shah JP, Tuttle RM, Ganly I (2019) Stage migration with the new American Joint Committee on Cancer (AJCC) staging system (8th edition) for differentiated thyroid cancer. Surgery 165: 6–11 17. Nam SH, Bae MR, Roh JL, Gong G, Cho KJ, Choi SH, Nam SY, Kim SY (2018) A comparison of the 7th and 8th editions of the AJCC staging system in terms of predicting recurrence and survival in patients with papillary thyroid carcinoma. Oral Oncol 87:158–164 18. Tam S, Boonsripitayanon M, Amit M, Fellman BM, Li Y, Busaidy NL, Cabanillas ME, Dadu R, Sherman S, Waguespack SG, Williams MD, Goepfert RP, Gross ND, Perrier ND, Sturgis EM, Zafereo ME (2018) Survival in differentiated thyroid cancer: comparing the AJCC Cancer Staging Seventh and Eighth Editions. Thyroid 28:1301–1310 19. van Velsen EFS, Stegenga MT, van Kemenade FJ, Kam BLR, van Ginhoven TM, Visser WE, Peeters RP (2018) Comparing the prognostic value of the Eighth Edition of the American Joint Committee on Cancer/Tumor Node Metastasis Staging System between papillary and follicular thyroid cancer. Thyroid 28: 976–981 20. Tran B, Roshan D, Abraham E, Wang L, Garibotto N, Wykes J, Campbell P, Ebrahimi A (2018) The prognostic impact of tumor size in papillary thyroid carcinoma is modified by age. Thyroid 28:991–996 21. Verburg FA, M€ader U, Luster M, Reiners C (2018) The effects of the Union for International Cancer Control/American Joint Committee on Cancer Tumour, Node, Metastasis

system version 8 on staging of differentiated thyroid cancer: a comparison to version 7. Clin Endocrinol 88:950–956 22. Shteinshnaider M, Muallem Kalmovich L, Koren S, Or K, Cantrell D, Benbassat C (2018) Reassessment of differentiated thyroid cancer patients using the Eighth TNM/AJCC classification system: a comparative study. Thyroid 28:201–209 23. Lamartina L, Grani G, Arvat E, Nervo A, Zatelli MC, Rossi R, Puxeddu E, Morelli S, Torlontano M, Massa M, Bellantone R, Pontecorvi A, Montesano T, Pagano L, Daniele L, Fugazzola L, Ceresini G, Bruno R, Rossetto R, Tumino S, Centanni M, Meringolo D, Castagna MG, Salvatore D, Nicolucci A, Lucisano G, Filetti S, Durante C (2018) 8th edition of the AJCC/TNM staging system of thyroid cancer: what to expect (ITCO#2). Endocr Relat Cancer 25:L7–L11 24. Kim TH, Kim YN, Kim HI, Park SY, Choe JH, Kim JH, Kim JS, Oh YL, Hahn SY, Shin JH, Kim K, Jeong JG, Kim SW, Chung JH (2017) Prognostic value of the eighth edition AJCC TNM classification for differentiated thyroid carcinoma. Oral Oncol 71:81–86 25. van Velsen EFS, Visser WE, Stegenga MT, M€ader U, Reiners C, van Kemenade FJ, van Ginhoven TM, Verburg FA, Peeters RP (2021) Finding the optimal age cutoff for the UICC/AJCC TNM staging system in patients with papillary or follicular thyroid cancer. Thyroid 31:1041–1049 26. Park SY, Kim HI, Kim JH, Kim JS, Oh YL, Kim SW, Chung JH, Jang HW, Kim TH (2018) Prognostic significance of gross extrathyroidal extension invading only strap muscles in differentiated thyroid carcinoma. Br J Surg 105: 1155–1162 27. Cipriani NA (2019) Prognostic parameters in differentiated thyroid carcinomas. Surg Pathol Clin 12:883–900 28. Park J, Lee S, Park J, Park H, Ki CS, Oh YL, Shin JH, Kim JS, Kim SW, Chung JH, Kim K, Kim TH (2021) Proposal of a new prognostic model for differentiated thyroid cancer with TERT promoter mutations. Cancers (Basel) 13:2943 29. Tao Y, Wang F, Shen X, Zhu G, Liu R, Viola D, Elisei R, Puxeddu E, Fugazzola L, Colombo C, Jarzab B, Czarniecka A, Lam AK, Mian C, Vianello F, Yip L, Riesco-Eizaguirre G, Santisteban P, O’Neill CJ, Sywak MS, CliftonBligh R, Bendlova B, Sy´korova´ V, Zhao S, Wang Y, Xing M (2021) BRAF V600E status sharply differentiates lymph node metastasisassociated mortality risk in papillary thyroid

Pathological Staging and Papillary Thyroid Carcinoma cancer. J Clin Endocrinol Metab 106(11): 3228–3238 30. Kim KJ, Kim SG, Tan J, Shen X, Viola D, Elisei R, Puxeddu E, Fugazzola L, Colombo C, Jarzab B, Czarniecka A, Lam AK, Mian C, Vianello F, Yip L, RiescoEizaguirre G, Santisteban P, O’Neill CJ, Sywak MS, Clifton-Bligh R, Bendlova B, Sy´korova´ V, Xing M (2020) BRAF V600E status may facilitate decision-making on active surveillance of low-risk papillary thyroid microcarcinoma. Eur J Cancer 124:161–169 31. Wang F, Zhao S, Shen X, Zhu G, Liu R, Viola D, Elisei R, Puxeddu E, Fugazzola L, Colombo C, Jarzab B, Czarniecka A, Lam AK, Mian C, Vianello F, Yip L, RiescoEizaguirre G, Santisteban P, O’Neill CJ, Sywak MS, Clifton-Bligh R, Bendlova B, Sy´korova´ V, Wang Y, Xing M (2018) BRAF V600E confers male sex disease-specific mortality risk in patients with papillary thyroid cancer. J Clin Oncol 36:2787–2795 32. Shen X, Zhu G, Liu R, Viola D, Elisei R, Puxeddu E, Fugazzola L, Colombo C, Jarzab B, Czarniecka A, Lam AK, Mian C, Vianello F, Yip L, Riesco-Eizaguirre G, Santisteban P, O’Neill CJ, Sywak MS, CliftonBligh R, Bendlova B, Sy´korova´ V, Xing M (2018) Patient age-associated mortality risk is differentiated by BRAF V600E status in papillary thyroid cancer. J Clin Oncol 36:438–445 33. Huang Y, Qu S, Zhu G, Wang F, Liu R, Shen X, Viola D, Elisei R, Puxeddu E, Fugazzola L, Colombo C, Jarzab B, Czarniecka A, Lam AK, Mian C, Vianello F, Yip L, Riesco-Eizaguirre G, Santisteban P, O’Neill CJ, Sywak MS, Clifton-Bligh R, Bendlova B, Sy´korova´ V, Xing M (2018) BRAF V600E mutation-assisted risk stratification of solitary intrathyroidal papillary thyroid cancer for precision treatment. J Natl Cancer Inst 110:362–370 34. Xing M, Alzahrani AS, Carson KA, Shong YK, Kim TY, Viola D, Elisei R, Bendlova´ B, Yip L, Mian C, Vianello F, Tuttle RM, Robenshtok E, Fagin JA, Puxeddu E, Fugazzola L, Czarniecka A, Jarzab B, O’Neill CJ, Sywak MS, Lam AK, Riesco-Eizaguirre G, Santisteban P, Nakayama H, Clifton-Bligh R, Tallini G, Holt EH, Sy´korova´ V (2015) Association between BRAF V600E mutation and recurrence of papillary thyroid cancer. J Clin Oncol 33:42–50 35. Xing M, Alzahrani AS, Carson KA, Viola D, Elisei R, Bendlova B, Yip L, Mian C, Vianello F, Tuttle RM, Robenshtok E, Fagin JA, Puxeddu E, Fugazzola L, Czarniecka A, Jarzab B, O’Neill CJ, Sywak MS, Lam AK,

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Riesco-Eizaguirre G, Santisteban P, Nakayama H, Tufano RP, Pai SI, Zeiger MA, Westra WH, Clark DP, Clifton-Bligh R, Sidransky D, Ladenson PW, Sykorova V (2013) Association between BRAF V600E mutation and mortality in patients with papillary thyroid cancer. JAMA 309:1493–1501 36. Smith RA, Salajegheh A, Weinstein S, Nassiri M, Lam AK (2011) Correlation between BRAF mutation and the clinicopathological parameters in papillary thyroid carcinoma with particular reference to follicular variant. Hum Pathol 42:500–506 37. Abe I, Lam AK (2021) Anaplastic thyroid carcinoma: updates on WHO classification, clinicopathological features and staging. Histol Histopathol 36:239–248 38. Turk AT, Asa SL, Baloch ZW, Faquin WC, Fellegara G, Ghossein RA, Giordano TJ, LiVolsi VA, Lloyd R, Mete O, Rosai J, Suster S, Thompson LDR, Wenig BM (2019) Interobserver variability in the histopathologic assessment of extrathyroidal extension of well differentiated thyroid carcinoma supports the New American Joint Committee on Cancer Eighth Edition criteria for tumor staging. Thyroid 29:619–624 39. Toraih EA, Hussein MH, Zerfaoui M, Attia AS, Marzouk Ellythy A, Mostafa A, Ruiz EML, Shama MA, Russell JO, Randolph GW, Kandil E (2021) Site-specific metastasis and survival in papillary thyroid cancer: the importance of brain and multi-organ disease. Cancers (Basel) 13:1625 40. Lam AK, Lo CY (2006) Diffuse sclerosing variant of papillary carcinoma of the thyroid: a 35-year comparative study at a single institution. Ann Surg Oncol 13:176–181 41. Pillai S, Gopalan V, Smith RA, Lam AK (2015) Diffuse sclerosing variant of papillary thyroid carcinoma--an update of its clinicopathological features and molecular biology. Crit Rev Oncol Hematol 94:64–73 42. Nixon IJ, Wang LY, Migliacci JC, Eskander A, Campbell MJ, Aniss A, Morris L, Vaisman F, Corbo R, Momesso D, Vaisman M, Carvalho A, Learoyd D, Leslie WD, Nason RW, Kuk D, Wreesmann V, Morris L, Palmer FL, Ganly I, Patel SG, Singh B, Tuttle RM, Shaha AR, Go¨nen M, Pathak KA, Shen WT, Sywak M, Kowalski L, Freeman J, Perrier N, Shah JP (2016) An international multiinstitutional validation of age 55 years as a cutoff for risk stratification in the AJCC/ UICC Staging System for well-differentiated thyroid cancer. Thyroid 26:373–380

Chapter 9 Liposomal siRNA Delivery in Papillary Thyroid Carcinoma Cells Farhadul Islam, Yaoqi Zhou, and Alfred K. Lam Abstract The discovery of RNA interference (RNAi) has opened a new strategy in cancer therapy, especially by silencing target genes. Pharmacologically it can be achieved by introducing of small (19–21 base pairs) dsRNA molecules known as small interfering RNA (siRNA) targeting interested genes. siRNA mediated gene has been widely investigated for its utility in treating various diseases including cancer. However, the systemic delivery of interested siRNA via non-viral methods remains a major challenge with large numbers of polymeric and liposomal systems being tested. The most effective methods involving cationic liposomes delivery to cells. Nonetheless, systemic delivery of siRNA via cationic lipid particles is often poor due to rapid uptake by reticuloendothelial organs, resulting in decreased delivery of these particles to the site of interest. Polyethylene glycol (PEG) has been used in siRNA-liposomes formulation to minimize reticuloendothelial uptake. Also, PEGylation permits the accumulation of the liposomes-loaded siRNA at the tumor sites with defective vasculatures such as enhanced permeability and retention phenomena. Thus, a simple method to prepare stable PEGylated siRNA-loaded lipid particles could provide better systemic delivery system in treating various cancers, including papillary thyroid carcinoma. Here we illustrate a simple protocol for the formulation of siRNA-loaded lipid particles by hydration of freeze-dried matrix (HFDM) method for effective delivery of target specific siRNA to papillary thyroid carcinoma cells. Key words RNAi, siRNA, Liposome, Systemic delivery, Papillary thyroid carcinoma

1

Introduction Small interfering RNA (siRNA), also known as short interfering RNA or silencing RNA, are a class of double stranded non-coding RNA molecules of 19–25 nucleotides in length and operating within the RNA interference (RNAi) pathway [1, 2]. They work by interacting with the complementary mRNA sequence of target gene and induces mRNA degradation, thereby preventing the translation followed by loss of protein expression [3]. Thus, the delivery of siRNA of interested gene have widely been investigated for their effective utility in the treatment of various diseases, including cancer, neurodegenerative and infectious diseases [4–6].

Alfred K. Lam (ed.), Papillary Thyroid Carcinoma: Methods and Protocols, Methods in Molecular Biology, vol. 2534, https://doi.org/10.1007/978-1-0716-2505-7_9, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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Several systems have been developed to deliver the siRNA to the target sites, however, the size, charges, and instability of these molecules limit their effective delivery, especially in vivo [7]. Most of these systems are cationic particles, which facilitate the interaction of negatively charged siRNA molecules and induce their cellular entry. The systemic delivery of siRNA by cationic particles is poor due to the rapid uptake by reticuloendothelial organs, resulting in insufficient delivery of these particles to the target site such as in cancer tissue [8–10]. The development of liposomal carriers using polyethylene glycol (PEG) enhances the systemic stability of the formulations [11]. Also, PEGylation decreases reticuloendothelial system uptake and increased the accumulation of the particles at tumor site where defective vasculature present due to enhanced permeability and retention effect [12–14]. Thus, PEGylated liposome entrapped drugs exhibited a great advantage of therapeutic efficacy over the naked drugs against several cancers [15, 16]. For example, PEGylated liposomal doxorubicin (DOXIL) had shown reduced uptake by reticuloendothelial system and showed extended circulation time with a reduced distribution in patients with AIDS-related Kaposi’s sarcoma, ovarian, breast, and prostate carcinomas, thereby significantly improve the therapeutic index of doxorubicin [15]. siRNA-loaded PEGylated liposome particles have been formulated using various methods, including post insertion, reverse phase evaporation, detergent dialysis, ethanol dialysis, etc. [17– 21]. These formulations are complicated and required lengthy procedures with end particles suspended in an aqueous state, which lead long term storage problems including aggregation and/or fusion of the particles, hydrolysis of lipids, degradation siRNA nucleotides [22, 23]. Also, these particles could be affected by agitation and temperature fluctuation during transport, along with these methods required significant effort for large-scale production, which limit their adoption of the use of siRNA-loaded liposomes in the clinical uses [23]. To overcome these limitations, Li and Deng developed hydration of a freeze-dried matrix (HFDM) method for liposomes preparation using lipid and sugar [22]. This formulation is easy, simple, and effective for PEGylated siRNAloaded liposomes and provides long term storage capacity to the particles. Most importantly, HFDM-mediated siRNA-loaded liposomes protected siRNA from nuclease degradation and permit accumulation of the particles at the tumor site after intravenous injection [24]. These siRNA-loaded liposomes can induce silencing of target gene in tumors. For example, siRNA-loaded liposomes targeting E6/7 oncogenes expression exhibited around 50% reduction of expression in cervical cancer, resulting in significant reduction of tumor size [24].

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In a recent study, we have illustrated HFDM-formulated liposomal delivery of microRNA-34b-5p in thyroid cancer cells [25]. These liposomes effectively delivered microRNA-34b-5p to anaplastic and papillary thyroid carcinoma cells both in in vitro and in vivo and suppress cell proliferation, migration, wound healing, and tumor growth by targeting the expression of vascular endothelial growth factor A [25]. Another study reported that liposomal delivery of microRNA-791 induces reduced proliferation and cell cycle progression of papillary thyroid carcinoma cells by targeting Cyclin D1, CDK4, and CDK6 expression [26]. Thus, liposomal delivery of target gene or gene segment could be a promising strategy for effective therapy in papillary thyroid carcinomas. In this chapter, we discuss the simple and easy method of HFDM-mediated siRNA-loaded liposomes formulation followed by their delivery to papillary thyroid carcinoma cells both in vitro and in vivo. The siRNA entrapment efficacy of liposomal particles and expression of the target gene followed by transfection will also be described.

2

Materials All chemicals and solvents are of at least analytical and or molecular biology grade as required. Use ultrapure or Milli-Q water to prepare all the reagents and solutions. The materials are kept at room temperature unless otherwise indicated. In addition, use appropriate personal protective equipment (PPE) during experiments and follow the guidelines of waste disposal for disposing the waste materials.

2.1 Formulation and Characterization of siRNA-Liposomal Vesicles

1. Dioleoyl trimethylammonium propane (DOTAP). 2. Dioleoylphosphatidylethanolamine (DOPE). 3. Cholesterol. 4. PEG2000-C16 ceramide: N-palmitoyl-sphingosine-1{succinyl [methoxy (polyethylene glycol)2000]}. 5. siRNA silencer sequence for the target gene. 6. Scramble siRNA oligonucleotides. 7. Sucrose. 8. Tert-butanol (tertiary alcohol). 9. Nitrogen/Phosphate. 10. Freeze-dryer. 11. Zetasizer Nano ZS (molecular/particle size and zeta potential analyzer). 12. Distilled water.

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13. PicoGreen reagent (DNA assay reagent). 14. 10 mM Tris HCl, 1 mM EDTA (TE buffer; pH 7.5). 15. pH meter. 16. 96-well black bottom plate. 17. Fluorescence plate reader. 18. 0.5% Triton-X 100. 2.2

Cell Culture

1. Papillary thyroid carcinoma cells storage in liquid nitrogen. 2. Cell culture media: Roswell Park Memorial Institute (RPMI) 1640, L-glutamine, pH 7.2. Take 900 mL water to a glass beaker and add 10.4 g RPMI1640 powder media to the water with gentle stir (see Note 1). Dissolve the powder completely and do not heat the media (see Note 2). Add 2.0 g of sodium bicarbonate to the media and stir for complete dissolving. Adjust the pH with 1N HCl (hydrogen chloride) and NaOH (sodium hydroxide). Finally, add water up to mark 1 L, sterilize with 0.22-micron membrane filter and aseptically transfer media into sterile container. Store at 4
C. 3. 10% fetal bovine serum (FBS) in sterile media (see Note 3). Store at 4
C. 4. 1% penicillin and streptomycin. Store at 4
C (see Note 4). 5. Cell washing buffer: Phosphate buffer saline (PBS), pH 7.4. Take 8.0 g sodium chloride (NaCl), 0.2 g potassium chloride (KCl), 1.42 g disodium hydrogen phosphate (Na2HPO4), and 0.24 g potassium dihydrogen phosphate (KH2PO4) in a glass beaker and dissolve all the salts in 800 mL water. Adjust the pH with 1N HCl, add water to a total volume of 1 L. Sterilize the buffer by autoclaving at 121
C for 30 min. Cool it down and store at 4
C. 6. Cell dissociation solution: 0.25% Trypsin- ethylenediaminetetraacetic acid (EDTA), pH 7.2–8. Stored at 20
C (see Note 5). 7. Cell culture flasks: Polystyrene 25 cm2 and 75 cm2 flasks. 8. Falcon 15 mL and 50 mL conical centrifuge tubes. 9. Microscope and bench top centrifuge machines. 10. 80% ethanol in water (v/v).

2.3 LiposomeLoaded siRNA Transfection

1. Tissue culture plates (6-well, 12-well, 96-well plates). 2. 0.4% trypan blue. 3. Cell counter. 4. Hemocytometer.

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1. Total RNA extraction kit. 2. Phosphate-buffered saline (PBS). 3. Deoxyribonuclease (DNase). 4. First strand cDNA Synthesis Kit. 5. SYBR green (asymmetrical cyanine dye used as nucleic acid stain). 6. Primers for target gene (e.g., Endothelial PAS domaincontaining protein 1 [EPAS1]) and internal control genes (β-actin, 18s, glyceraldehyde 3-phophaste dehydrogenase [GAPDH], etc.) 7. RNase free water. 8. Nano drop/spectrophotometer. 9. Standard thermal cycler. 10. Real-time polymerase chain reaction system. 11. Centrifuge with plate holders. 12. Micro-centrifuge. 13. Vortex. 14. Polypropylene tube. 15. Reagent tubes with caps. 16. 96-well optical reaction plates. 17. Optical adhesive covers.

2.5 In Vivo siRNALoaded Liposome Delivery

1. Experimental animal (e.g., female NU/NU nude mouse). 2. Cell counter (e.g., hemocytometer or automated cell counter). 3. PBS. 4. 1 mL syringe. 5. 25-gauge needle. 6. Slide caliper. 7. Weighing scale. 8. siRNA-loaded liposomes, control siRNA and empty liposomes. 9. Formalin. 10. Plastic container for animal organs.

3

Methods

3.1 Formulation and Characterization of siRNA-Liposomal Vesicles

Hydration of a freeze-dried matrix (HFDM) method was used for the process of preparing siRNA-loaded lipid particles (liposome). A flow diagram for this liposome formation method is outlined in the Fig. 1. The steps are given below.

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Fig. 1 Schematic flow diagram of siRNA-loaded liposomes formulation using hydration of freeze-dried matrix (HFDM) method

1. Mix DOTAP, cholesterol, DOPE, and PEG2000-C16 ceramide at a molar ratio of 50:35:5:10 with interested siRNA at nitrogen: phosphate (N:P) ratio of 4:1 in sucrose containing water/ tert-butanol (1:1 v/v) co-solvent system (see Note 6). 2. Snap-freeze and freeze-dried the resultant formulation overnight at a condensing temperature of 80
C and pressure less than 0.1 mbar. 3. Add sterile water for hydration of freeze-dried lyophilized product with gentle shaking. 4. Characterize the liposomes by measuring size, polydispersity index, and zeta potential using a Zetasizer Nano ZS at room temperature. 5. Assay the siRNA entrapment efficiency of the liposomes using PicoGreen reagent. For this, dilute the liposomes in Tris-HClEDTA (TE) buffer (pH 7.5) to the concentration that fall the fluorescence reading of linear range of standard curve (1 ng/mL to 1.0 μg/mL). 6. Add 100 μL of diluted (1:200) PicoGreen reagent to 100 μL of diluted liposome sample in a 96-well black bottom plate and incubate for 2–5 min at dark. 7. Measure the fluorescence intensity using a plate reader at 485 nm excitation and 520 nm emission wavelength.

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Fig. 2 Standard curve for PicoGreenR dsDNA assay. Known concentration of DNA was added to the plate containing PicoGreenR dsDNA reagent diluted in 10 mM Tris-HCl, 1 mM EDTA at pH 7.5 (TE). The samples were excited at 480 nm and the fluorescence emission intensity was measured at 520 nm using a plate reader and fluorescence emission intensity was plotted versus DNA concentration

8. Measure the fluorescence intensity after treating the liposome samples with 0.5% Triton-X 100, which allows the release of entrapped siRNA. 9. Generate a standard curve using 1 ng/mL to 1.0 μg/mL siRNA (Fig. 2). 10. Determine the entrapment efficiency of the liposomes using the formulae: ([siRNA] with Triton-X-100[siRNA] without Triton-X-100)/[siRNA] with Triton-X-100. 3.2

Cell Culture

1. Take the cryovial containing papillary thyroid carcinoma cells from liquid nitrogen facility and transfer them into a 37
C water bath. 2. Quickly thaw the cells by gently swirling the vial in water bath at 37
C (see Note 7). 3. Transfer the vial into a biosafety cabinet and wipe out site of the vial with 80% ethanol. 4. Add pre-warmed complete growth media (2 mL) in a 15 mL centrifuge tube and add the thaw cells dropwise into the tube. 5. Centrifuge the cell suspension at approximately 400g for 3–5 min. 6. Check the cell pellet after centrifugation and aseptically decant the supernatant without disrupting the cells pellet. 7. Add 2 mL PBS and re-suspend the cells pellet with slow pipetting. 8. Centrifuge the suspension at 400g for 3–5 min and discard the supernatant.

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9. Resuspend the cell pellets in complete growth media (containing FBS) and transfer them into an appropriate cell culture flask containing recommended growth media and incubate in the carbon dioxide (CO2) incubator at appropriate conditions (see Note 8). 3.3 siRNA Transfection

1. Seed the papillary thyroid carcinoma cells in 6-well plate at the density of 2  104 cells and grow them 24-h in complete media. 2. Remove the media and gently wash the cells with PBS after 24 h of initial seeding and prepare the liposome complex for transfection. 3. Add 4 μL of PEGylated liposomes (40 μg siRNA 300 μL) in 1 mL RPMI media supplemented with 10% FBS and 1% penicillin/streptomycin, resulting in 40 nM final siRNA concentration. 4. Add 2 mL 5 nM siRNA-liposomes complex in each well and incubate at 37
C for 48 h. 5. Perform the same protocol for liposome loaded-scramble siRNA (non-targeted control siRNA) and empty liposome transfections.

3.4 In Vitro Gene Expression Analysis

Quantitative real-time polymerase chain reaction (qRT-PCR) is used to analyze the expression of the siRNA targeted gene followed by total RNA extraction. The protocol involves the following steps. 1. Wash the cells with PBS and extract total RNA from siRNAloaded liposomes treated and control cells using RNA extraction kit by following the manufacturer guidelines (see Note 9). 2. Check the purity spectrophotometer.

of

the

extracted

RNA

using

3. Then convert the RNA into first stand using cDNA-first stand synthesis kit according to the suppliers’ instructions. 4. Quantify the expression of the interested gene and internal control gene (e.g., 18s, β-actin, GAPDH, etc.) in siRNAloaded liposomes treated and control cells using qRT-PCR as follows (see Note 10). 5. Label your PCR-strips or tubes or 96-well plate for polymerase chain reaction. 6. Add 10 μL 2 Universal PCR Master Mix (SYBR green) to 7.67 μL Nuclease-free water for a total of 17.67 μL on ice into a polypropylene tube labelled PCR reaction (see Note 11). 7. Mix and centrifuge with bench-top centrifuge machine for 15 s and add 1.0 μL (or alternative according to the kit manual) of 20 SYBR Green assay mix into the corresponding PCR reaction tube.

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8. Add this into corresponding wells and finally add 1.33 μL of cDNA first stand each well of the PCR plate and seal with optical adhesive cover. 9. Mix by tapping the plate/tubes and centrifuge with bench-top machine for 15 s. 10. Place the PCR plate/tubes in real-time PCR system and run real-time PCR at standard conditions: 1 cycle of 10 min at 95
C for enzyme activation and 40 cycles of PCR (15 s at 95
C, followed by 60 s at 60
C) (see Note 12). 11. After completion of PCR reactions, do the analysis and transfer the results to Excel or an alternative spreadsheet for analysis the expression of siRNA targeted gene. 12. Expression of the siRNA targeted gene can be presented as the ratio of expression (CT values of target gene/CT values of internal control gene. The 2-DDct method can be used to calculate the fold changes of siRNA targeted gens’ expression in each sample group (Fig. 3).

Fig. 3 Relative expression of target gene. The expression of target gene significantly ( p < 0.001) downregulated in siRNA-loaded liposome treated cells when compared to that of scramble control and empty liposome treated groups

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3.5 In Vivo siRNALoaded Liposomes Delivery

Subcutaneous tumor model can be used to examine the effects of liposomal mediated siRNA delivery in tumors. Effective delivery of siRNA suppresses the expression of the target gene in mouse xenoplants, thereby reduced growth and progression should be noted in mice receiving interested liposomal siRNA (see Note 13). 1. Culture the papillary thyroid carcinoma cells as described in Subheading 3.2 and collect the experimental animal (e.g., female NU/NU nude mouse). Mouse should be 6–8 weeks of age and should be acclimatized to condition for several days. 2. Count the cells using a cell counter (e.g., hemocytometer or automated cell counter) and adjusted the cell number as two million cells in 100 μL of cell in sterile PBS. 3. Inject the cells subcutaneously into right and left sides of the abdomen of each mouse. 4. Monitor the tumor growth daily by palpation (see Note 14) and allow the tumor to grow till progressive growth establish. 5. Record the tumor weight and size using caliper and scale from the day when tumors first detect by palpation until the end of experiment. 6. After progressive tumor establish, inject the siRNA-loaded liposomes, control siRNA and empty liposomes intravenously on days according to your experimental design. 7. After treatment, monitor and record the tumors weight and size until the end of experiment. 8. Collect the tumors and asses by measurement with calipers using the following formula: tumor volume ¼ (length _ width _ height)/0.5236. 9. Collect all the major organs and kept in formalin for further analysis.

4

Notes 1. Powdered media are highly hygroscopic and should be protected from atmospheric moistures. Preparing a concentrated media solution may form precipitation. 2. pH needs to be reduced to 4.0 with 1N HCl to dissolve the media completely. After that, pH can be raised up to 7.2 with 1N NaOH prior to add sodium bicarbonate. 3. We noted that 10% FBS is good for the growth and maintenance of the papillary thyroid carcinoma cells. 4. The combined antibiotics, penicillin and streptomycin, were used to prevent bacterial contamination of cultured papillary thyroid carcinoma cells. This concentration is to maintain and keep the cells free from bacterial contaminations.

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5. Avoid repeated freeze thawing and warm up of trypsin. Multiple freezing, thawing, and warming may cause the reduced enzymatic activity of trypsin. 6. The amount of siRNA depends on how much siRNA concentration is desired at final solution. Forty micrograms of siRNA can be added to obtain a solution of 40 microgram siRNA in 300 μL isotonic sucrose solution. Lipid molar ratio and sucrose concentration can be optimized for better liposome formation. 7. Thawing procedures is stressful for the frozen cells. Using the good techniques and fast thawing at 37
C ensure high proportion of the cells to survive the procedures. The technique includes dilute the frozen cells with pre-warmed complete media and mix with slow pipette up and down. 8. Flask size depends on the number of frozen cells present in the cryovial and culture conditions vary based on cell type and media used. Generally, cells seeded at 10,000 cells/cm2, in 5% CO2 at 37
C used for routine culture and passages. 9. Most of the RNA extraction kits uses Trizol (also known as TRI REAGENT) for the isolation of total RNA. Trizol is a mixture of guanidine thiocyanate and phenol, which effectively dissolves DNA, RNA and protein on homogenization or lysis of cells and tissue sample. After adding chloroform and centrifuging, the mixture separates into 3 phases with the upper clear aqueous phase containing the RNA. For RNA expression analysis, it is highly recommended to treat the sample with DNase, an enzyme that digests DNA. Most of the commercial kits that enable simple RNA extractions, use a column that binds the RNA, included a DNase digestion step in their protocol. As Trizol are very toxic, always use fume hood during usages. 10. Design the primers for siRNA target gene and internal control genes. Also, needs to optimize the annealing temperature of the primers. 11. Adjust these volumes for the appropriate number of reverse transcription reactions and run all the sample in triplicate at least. 12. Can generate melt-curve to see the specificity of amplicon and make sure that the assays are running correctly. 13. Get the animal ethic for using animal and follow guidelines in accordance with the ethical standards of the institution as well as 1964 Helsinki declaration and its later amendments comparable to the ethical standards. Some institution could take long time to approve your application, so follow your institutional guidelines in advance to prepare the application. 14. Usually, tumors can be detected after 1 week of transplantation and grow the tumor according to your experimental plan.

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References 1. Islam F, Gopalan V, Lam AK (2018) RNA interference-mediated gene silencing in esophageal adenocarcinoma. Methods Mol Biol 1756:269–279 2. Das PK, Asha SY, Abe I, Islam F, Lam AK (2020) Roles of non-coding RNAs on anaplastic thyroid carcinomas. Cancers (Basel) 12: 3159 3. Lagana` A, Veneziano D, Russo F, Pulvirenti A, Giugno R, Croce CM, Ferro A (2015) Computational design of artificial RNA molecules for gene regulation. Methods Mol Biol 1269:393–412 4. Landen CN Jr, Chavez-Reyes A, Bucana C, Schmandt R, Deavers MT, Lopez-Berestein G, Sood AK (2005) Therapeutic EphA2 gene targeting in vivo using neutral liposomal small interfering RNA delivery. Cancer Res 65: 6910–6918 5. Dı´az-Hernández M, Torres-Peraza J, SalvatoriAbarca A, Morán MA, Go´mez-Ramos P, Alberch J, Lucas JJ (2005) Full motor recovery despite striatal neuron loss and formation of irreversible amyloid-like inclusions in a conditional mouse model of Huntington’s disease. J Neurosci 25:9773–9781 6. Giladi H, Ketzinel-Gilad M, Rivkin L, Felig Y, Nussbaum O, Galun E (2003) Small interfering RNA inhibits hepatitis B virus replication in mice. Mol Ther 8:769–776 7. Ewert KK, Ahmad A, Bouxsein NF, Evans HM, Safinya CR (2008) Non-viral gene delivery with cationic liposome-DNA complexes. Methods Mol Biol 433:159–175 8. Zamboni WC (2005) Liposomal, nanoparticle, and conjugated formulations of anticancer agents. Clin Cancer Res 11:8230–8234 9. Kim JK, Choi SH, Kim CO, Park JS, Ahn WS, Kim CK (2003) Enhancement of polyethylene glycol (PEG)-modified cationic liposomemediated gene deliveries: effects on serum stability and transfection efficiency. J Pharm Pharmacol 55:453–460 10. Opanasopit P, Nishikawa M, Hashida M (2002) Factors affecting drug and gene delivery: effects of interaction with blood components. Crit Rev Ther Drug Carrier Syst 19: 191–233 11. Gjetting T, Andresen TL, Christensen CL, Cramer F, Poulsen TT, Poulsen HS (2011) A simple protocol for preparation of a liposomal vesicle with encapsulated plasmid DNA that mediate high accumulation and reporter gene activity in tumor tissue. Results Pharma Sci 1: 49–56

12. Li W, Szoka FC Jr (2007) Lipid-based nanoparticles for nucleic acid delivery. Pharm Res 24:438–449 13. Sapra P, Tyagi P, Allen TM (2005) Ligandtargeted liposomes for cancer treatment. Curr Drug Deliv 2:369–381 14. Wagner E, Kircheis R, Walker GF (2004) Targeted nucleic acid delivery into tumors: new avenues for cancer therapy. Biomed Pharmacother 58:152–161 15. Gabizon A, Shmeeda H, Barenholz Y (2003) Pharmacokinetics of pegylated liposomal doxorubicin: review of animal and human studies. Clin Pharmacokinet 42:419–436 16. Gabizon A, Catane R, Uziely B, Kaufman B, Safra T, Cohen R, Martin F, Huang A, Barenholz Y (1994) Prolonged circulation time and enhanced accumulation in malignant exudates of doxorubicin encapsulated in polyethyleneglycol coated liposomes. Cancer Res 54:987– 992 17. Li SD, Huang L (2006) Targeted delivery of antisense oligodeoxynucleotide and small interference RNA into lung cancer cells. Mol Pharm 3:579–588 18. Stuart DD, Allen TM (2000) A new liposomal formulation for antisense oligodeoxynucleotides with small size, high incorporation efficiency and good stability. Biochim Biophys Acta 1463:219–229 19. Wheeler JJ, Palmer L, Ossanlou M, MacLachlan I, Graham RW, Zhang YP, Hope MJ, Scherrer P, Cullis PR (1999) Stabilized plasmid-lipid particles: construction and characterization. Gene Ther 6:271–281 20. Maurer N, Wong KF, Stark H, Louie L, McIntosh D, Wong T, Scherrer P, Semple SC, Cullis PR (2001) Spontaneous entrapment of polynucleotides upon electrostatic interaction with ethanol-destabilized cationic liposomes. Biophys J 80:2310–2326 21. Jeffs LB, Palmer LR, Ambegia EG, Giesbrecht C, Ewanick S, MacLachlan I (2005) A scalable, extrusion-free method for efficient liposomal encapsulation of plasmid DNA. Pharm Res 22:362–372 22. Li C, Deng Y (2004) A novel method for the preparation of liposomes: freeze drying of monophase solutions. J Pharm Sci 93:1403– 1414 23. Anchordoquy TJ, Carpenter JF, Kroll DJ (1997) Maintenance of transfection rates and physical characterization of lipid/DNA complexes after freeze-drying and rehydration. Arch Biochem Biophys 348:199–206

siRNA Delivery in Papillary Thyroid Carcinoma 24. Wu SY, Putral LN, Liang M, Chang HI, Davies NM, McMillan NA (2009) Development of a novel method for formulating stable siRNAloaded lipid particles for in vivo use. Pharm Res 26:512–522 25. Maroof H, Islam F, Dong L, Ajjikuttira P, Gopalan V, McMillan NAJ, Lam AK (2018) Liposomal delivery of miR-34b-5p induced

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Chapter 10 Long Non-Coding RNAs Profiling Using Microarray in Papillary Thyroid Carcinoma Farhadul Islam, Yaoqi Zhou, and Alfred K. Lam Abstract Long non-coding RNAs (lncRNAs) have been implicated in various cancers, including papillary thyroid carcinomas (PTCs). Genome-wide analysis (GWAS) of lncRNAs expression in PTC samples exhibited up and down regulation of lncRNAs, thus, acting as tumor promoting oncogenes or tumor suppressors in the pathogenesis of PTC by interacting with target genes. For example, lncRNAs such as HOTAIR, NEAT1, MALAT1, FAL1, HOXD-AS1, etc. are overexpressed in PTC in comparison to that of non-cancerous thyroid tissues, which stimulate the pathogenesis of PTC. On the other hand, lncRNAs such as MEG3, CASC2, PANDAR, LINC00271, NAMA, PTCSC3, etc. are down regulated in PTC tissues when compared to that of non-cancerous thyroid samples, suppressing formation of PTC. Also, several lncRNAs such as BANCR acts as oncogenic or tumor suppressor in PTC formation depending on which they are interacting with. In addition, lncRNAs expression in patients with PTC associated with clinicopathological parameters such as distance metastasis, lymph node metastasis, tumor size, pathological stage, and response to therapy. Thus, lncRNAs profiles could have the potential to be used as prognostic or predictive biomarker in patients with PTC. Therefore, we describe the microarray method to examine lncRNAs expression in PTC tissue samples, which could facilitate better management of patients with PTC. Furthermore, this method could be fabricated to examine lncRNAs expression in other biological and/or clinical samples. Key words LncRNA, Non-coding RNAs, Microarray, Papillary thyroid carcinoma, Cancer biomarkers

1

Introduction More than 90% of the human genome is transcribe, while only 2% of the genome is translated subsequently to protein products [1]. The rest of the transcribed RNAs do not have protein coding capacity, thus, remains as untranslated and hence called non-coding RNAs (ncRNAs). These ncRNAs categorized into two groups, i.e., short ncRNAs including microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), small interacting RNAs (siRNAs), transfer RNAs (tRNAs), some ribosomal RNAs (rRNAs) and long ncRNAs such as

Alfred K. Lam (ed.), Papillary Thyroid Carcinoma: Methods and Protocols, Methods in Molecular Biology, vol. 2534, https://doi.org/10.1007/978-1-0716-2505-7_10, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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long intergenic ncRNAs (lincRNAs), intronic long ncRNAs (ilncRNAs), promoter-upstream transcript (PROMPT), promoterassociated long ncRNAs (paRNAs), repetitive element-associated long ncRNAs, pseudogene long ncRNAs, enhancer-associated long ncRNAs, etc. based on their size [2]. Long ncRNAs (lncRNAs) are 200 base-pair to 100 kilopase-pair in length and it was noted that approximately 80% of the human genome is transcribed to 14,880 lncRNAs from 9277 loci and play crucial roles in epigenetic regulation of genes [3]. They regulate the expression of various genes at different levels, including chromatin, splicing, transcriptional and post-transcriptional stages [4]. LncRNAs mediated modulation of genes involved in vital biological and cellular process such as proliferation, survival, apoptosis, invasion & migration, differentiation, autophagy in cancer cells. Accumulating information suggested that deregulation of lncRNAs have been implicated in many cancers including papillary thyroid carcinomas (PTCs) [5–7]. Several studies identified that the genome-wide analysis (GWAS) of lncRNAs expression in PTC samples had shown up and down regulation of various lncRNAs when compared to that of non-cancerous tissues [5, 7–11]. For example, microarray analysis of 62 PTC tissues exhibited significantly differentially expressed thousands of lncRNAs and mRNA in comparison to that of paired non-cancerous tissues [5]. Also, they noted 1805 deregulated lncRNAs have associated with cis or trans target genes [5]. Among the cis target genes, 463 were differentially expressed in PTC tissues, thus, implied the lncRNAs mediated regulation of them during PTC carcinogenesis [5]. Another lncRNAs microarray analysis of three PTC identified abnormal expression of 675 lncRNAs when compared to that of paired noncancerous thyroid tissues [7]. Expression of lncRNAs by RNA-sequencing and qRT-PCR analysis detected 188 differentially expressed lncRNAs in PTCs (n ¼ 12) in comparison to that of matched non-cancerous thyroid tissues [8]. The differentially expressed lncRNAs associated with lymph node metastasis (NONHSAT076747 and NONHSAT122730) and tumor size (NONHSAG051968) in patients with PTC [8]. In addition, a total of 777 lncRNAs were differentially expressed in PTC (n ¼ 22) samples relative to the matched non-cancerous thyroid tissues [10]. Among the deregulated lncRNAs, 325 were upregulated and 452 were downregulated in PTC tissues [10]. The differentially expression of lncRNAs in PTCs indicating that like protein-coding genes, lncRNAs might have functional roles in carcinogenesis as oncogenes or as tumor suppressor genes by regulating the expression of target genes [12]. 1.1 Oncogenic lncRNAs in PTC

LncRNAs can induce cell cycle progression, cell proliferation, invasion & migration, and metastasis while inhibiting apoptosis, acting as oncogenes in cancer pathogenesis [13]. In PTCs, several

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Table 1 Oncogenic lncRNAs in pathogenesis of papillary thyroid carcinoma

lncRNAs

Sample size

Functions

References

HOTAIR

Promote cell growth and invasion, associated with 35 poor survival of patients with PTC 496 (TCGA)

[27, 28]

NONHSAT076754

72

Associated with LNM in patients with PTC

[29]

NEAT1

87

Promote tumor progression and overexpression associated with tumor size in PTC

[14]

n340790

85

Promote cell growth, invasion, metastasis and inhibit [30] apoptosis in PTC

ENSG00000273132.1 59 ENSG00000230498.1

Overexpression associated with BRAF (V600E) mutation in PTC

[31]

CTD-3193013 AC007255.8 HOXD-AS1 RP11-40216.1

45

Overexpression associated with tumor size, LNM, clinical stages of patients with PTC

[32]

HIT000218960

55

Associated with TMN stage, LNM, multifocality in PTC

[11]

MALAT1

195

Modulates proliferation, migration and EMT via TGF-β in PTC

[33]

NR_036575.1

83

Promotes proliferation and migration of PTC cells

[34]

XLOC_051122 XLOC_006074

12

Associated with LNM and BRAF (V600E) mutation, [9] promotes tumor progression and metastasis in PTC

LOC100507661

64

Stimulates proliferation, migration, and invasion of PTC cells

[35]

FAL1

100

Correlated with multifocality in PTC

[36]

ENST00000537266 ENST00000426615

46

Regulate proliferation, migration, cell cycle and apoptosis in PTC

[10]

PTC papillary thyroid carcinoma, LNM lymph node metastases, EMT epithelial-mesenchymal transition

lncRNAs promotes carcinogenesis by modulating various target genes, thereby regulating key cellular process (Table 1). For example, nuclear-enriched abundant transcript 1 & 2 (NEAT 1 & 2) has played important roles in PTC pathogenesis by modulating the expression of oncogene ATAD2 (ATPase family AAA-domain containing protein 2) via suppression of miR-106-5p expression [14]. NEAT1 &2 are overexpressed in PTC tissues and cells, and suppression of them resulted in growth arrest and reduced metastasis of PTC cells (K1 and TPC1) when compared to non-neoplastic control cells [14]. Also, NEAT1 & 2 overexpression

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was associated with advanced TMN stage and larger tumor size in patients with PTC [14]. HOX transcript of antisense RNA (HOTAIR), another lncRNA significantly overexpressed in PTC tissues when compared to non-cancerous tissues. Also, HOTAIR promotes proliferation and survival of PTC (BCPAP) cells via modulating Wnt/β-catenin signaling pathways [15]. Suppression of HOTAIR with antisense RNA induced growth arrest of PTC cells in vitro and tumor formation in vivo, while overexpression of HOTAIR increased cyclin D2 expression via modulating miR-1, resulting in development and progression of PTC (TPC1) cells [16]. Another lncRNA known as H19 significantly overexpressed in PTC and associated with the poor prognosis of patients with PTC [17]. Silencing of H19 inhibited proliferation and tumor progression via decreasing ERβ (estrogen receptor beta) expression in PTC stem cells [17]. A list of the oncogenic lncRNAs involved in the PTC pathogenesis is summarized in Table 1. 1.2 Tumor Suppressor lncRNAs in PTC

Downregulation of tumor suppressive lncRNAs play critical roles in cancer initiation, development, and progression by modulating target genes involved in cellular proliferation, survival, cell cycle progression, apoptosis, invasion, migration, and metastasis [18]. It was demonstrated that several lncRNAs was downregulated in PTC tissues and cells in comparison to that of noncancerous tissues and cells (Table 2). Also, their ectopic overexpression associated reduced tumor formation properties, thereby acting as the tumor suppressive lncRNAs in PTCs [19–23]. For instance, NAMA (noncoding RNA associated with MAK kinase pathway and growth arrest) lncRNA downregulated in PTCs with BRAF (V600E). Thus, it is involved in the regulation of MAPK pathway in PTC pathogenesis [19]. Furthermore, induction of NAMA in thyroid carcinoma cells (NPA87) results in G1 cell cycle arrest and apoptosis followed by modulating MAPK pathway [19]. Papillary thyroid carcinoma susceptibility candidate 3 (PTSCS3), another lncRNA was significantly downregulated in PTCs when compared with noncancerous samples [20]. Transient overexpression of PTSCS3 in PTC cells (TPC1 and BCPAP) induced reduced cellular growth and decreased the expression of genes associated with DNA replication, recombination, motility, and apoptosis of cells [20]. Maternally expressed 3 (MEG3), a lncRNAs, was significantly downregulated in PTC tissues and low expression of MEG3 was associated with lymph node metastasis (LNM) in patients with PTC [22]. In vitro overexpression of MEG3 in thyroid carcinoma (TPC1 and HTH83) cells induced significant inhibition of cellular migration and invasion via suppressing tumor promotor Rac1 [22]. Thus, these lncRNAs exhibited protective roles in PTC pathogenesis, thereby act as a tumor suppressor. Table 2 summarizes the lncRNAs acts as tumor suppressor in papillary thyroid carcinoma.

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Table 2 Tumor suppressor lncRNAs in PTC pathogenesis of papillary thyroid carcinoma

lncRNAs

Sample size

MEG3

16

RP5-1024C24.1

45 Negatively associated with advanced clinical stage of [32] 18 (GWAS) patients with PTCs

CASC2

86

Downregulation associated with multifocality and advanced TNM of PTC

PANDAR

64

Inhibits cell proliferation, cell cycle progression and [38] promote apoptosis in PTC

Functions

References

Reduced invasion of PTC cells and associated with LNM

[22]

[37]

ENSG00000235070.3 59 ENSG00000255020.1

Associated with BRAF (V600E) mutation in PTC

[31]

GAS8-AS1

402 97

Inhibits cell proliferation and downregulation associated with LNM in patients with PTCs

[39, 40]

NONHSAG051968 NONHSAG018271 NONHSAG007951

12

Inhibits cell growth and downregulation associated [8] with increase tumor size of PTC

LINC00271

Associated with extra-thyroidal invasion, LNM, [23] 50 advanced tumor stage and recurrence in patients 471 with PTCs (TCGA) 185 (FUSCC)

NONHSAT037832

87

Correlates with LNM and tumor size of PTC

[41]

PTCSC2

65

Prone genetically to the development of PTC

[42]

PTCSC3

46 73

Inhibits cell growth and invasion of PTC

[20]

PTCSC1

26

Susceptible candidate gene for PTC

[43]

NAMA

40

Targets MAPK signaling pathway in PTC

[19]

PTC papillary thyroid carcinoma, LNM lymph node metastases

Additionally, several lncRNAs acts as oncogenic or tumor suppressor in PTC pathogenesis depending on the target they intact with [24]. For example, BRAF-activated long non-coding RNA (BANCR) can acts as oncogene or tumor suppressor gene in PTC pathogenesis [25, 26]. Zheng et al. reported that BANCR significantly upregulated in PTC (n ¼ 40) tissues when compared to that of adjacent noncancerous tissues. Also, silencing of BANCR in PTC cells caused reduced expression of cyclin D1, resulting in inhibition of cell proliferation and colony formation in comparison to that of control cells [25]. BANCR was significantly downregulated in PTC (n ¼ 92) tissues when compared to that of matched normal thyroid

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epithelial tissues [26]. Also, downregulation of BANCR in PTC tissues was associated with large tumor size, presence of multifocal lesions and advanced PTC stage. Moreover, overexpression of BANCR was associated with reduced proliferation, metastasis, and increased apoptosis in vitro and reduced tumor formation in vivo via modulating ERK1/2 and p38 signaling axis of PTC cells [26]. The accumulating information suggested that lncRNAs involved in the tumorigenesis of PTC by acting as oncogene and/or tumor suppressor, which implied their clinical applications such as the prognostic marker or therapeutic target for better management of patients with PTC. Therefore, we aim to describe here the microarray-based method for lncRNAs profiling using PTC biopsy sample. The method has four major steps including (i) sample collection and preparation, (ii) RNA extraction and first stand cDNA conversion, (iii) Second stand cDNA synthesis and labelling (iv) lncRNA microarray hybridization, (v) lncRNAmicroarray washing, and (iv) data analysis. An overview of lncRNA microarray experiment is shown in Fig. 1.

Fig. 1 Overview of lncRNA microarray experiment

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Materials

2.1 Sample Collection and Preparation

1. Papillary thyroid carcinoma and non-cancerous thyroid tissues (see Note 1). 2. Microtome/cryostat. 3. 80% ethanol. 4. Positively charged slides (see Note 2). 5. 1.5 mL tubes.

2.2 RNA Extraction and First Stand cDNA Conversion

1. Trizol total RNA extraction reagent. 2. 0.8 M sodium citrate, 1.2 M NaCl. 3. Isopropanol. 4. Chloroform. 5. 70% ethanol. 6. RNase free water. 7. RNase inhibitor. 8. 50 mL sterile centrifuge tube. 9. Nano drop. 10. cDNA synthesis kit/(Oligo-dT, Superscript buffer, Superscript II RT, 0.1 M DTT, 10 mM dNTPs). 11. RNase A/H. 12. PCR Thermal cycler.

2.3 Second Strand cDNA Synthesis and Labelling

1. RNA/cDNA labelling kit/Cy3-dUTP or Cy5-dUTP (see Note 3). 2. Random primer. 3. Klenow buffer. 4. 10 deoxynucleotide triphosphates (dNTPs), the substrates for DNA polymerizing enzymes.

2.4 LncRNAMicroarray Hybridization

1. LncRNA expression microarray kit (see Note 4). 2. Hybridization station/chamber/oven. 3. Hybridization gasket slide kit. 4. Slide staining dishes. 5. Nuclease free water. 6. Slide racks.

2.5 LncRNAMicroarray Washing

1. Wash buffer: 2 Saline-Sodium Citrate (SSC), 0.03% Sodium Dodecyl Sulphate (SDS); 20 SSC (3 M, pH 7). Take 800 mL water in a container, add 175.3 g of sodium chloride (NaCl) to the water, add 77.4 g of sodium Citrate to the solution. Adjust pH with HCl (14 N). Add water up to 1 L.

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2. Vacuum concentrator/speed vac. 3. Slide washing dishes. 4. Glass slide holders. 5. Forceps. 6. Microarray slide scanner. 7. Working wash buffer: wash buffer A (2 SSC, 0.03% SDS), wash buffer B (1 SSC), wash buffer C (0.05 SSC). 2.6

3

Data Analysis

1. Data extraction and analysis software packages.

Methods

3.1 Sample Collection and Preparation

1. Collect papillary thyroid non-cancerous tissues.

carcinoma

and

matched

2. Snap freeze the tissues with liquid nitrogen and stored in 80  C for further uses. 3. Cut the fresh frozen tissue sections (5 μM slices) for histological examination using cryostat. Microtome needs to be used in the case of formalin fixed paraffin embedded tissues (see Note 2). 4. Take 1 g (5–7 μM slices) of the tissues in 1.5 mL centrifuge tubes for RNA extraction. 5. Disinfect the cryostat or microtome using 80% ethanol.

3.2 RNA Extraction and First Stand cDNA Conversion

1. Transfer the tissues to 50 mL centrifuge tube using forceps. 2. Add 15 mL Trizol reagent and incubate at 60  C for 5 min, then homogenize the tissues for 15 s. Repeat the homogenization step (see Note 5). 3. Centrifuge the tube at 12,000g for 10 min at 4  C and take the supernatant into a new sterile 50 mL tube. 4. Add 3 mL chloroform to the supernatant under the hood, vortex the tubes vigorously for 15 s. Keep the tube at room temperature for 2–3 min. 5. Centrifuge the tube at 10,000g for 15 min at 4  C. Take the aqueous phase into a new tube carefully, discard the interphase and lower phase into waste container. 6. Add isopropanol and 0.8 M sodium citrate, 1.2 M NaCl, half volume of the aqueous phase each to precipitate the RNA. Let the tube sit for 10 min at room temperature. 7. Spin the tube at 10,000g for 10 min at 4  C, discard the supernatant. Wash the pellet with 20 mL of 75% ethanol by vortexing.

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8. Centrifuge the tube at 10,000g for 10 min at 4  C and discard the supernatant. 9. Dry the pellet briefly in air (5–10 min, no longer), add 250 μL RNase free water to the pellet and resuspend the RNA pellet by pipetting up and down for several times (see Note 6). 10. Add 1 μL RNase inhibitor to the RNA solution and transfer the sample to 1.5 mL and determine the RNA concentration and quality using a nano drop spectrophotometer (see Note 7). RNA sample (~1 μg)

 μL

RNase free water

24- μL

Oligo-dT

0.5 μL

Total Volume

24.5

11. For cDNA synthesis, mix the following materials in a 0.2 mL PCR tube. 12. Incubate at 70  C for 10 min and then transfer to ice. 13. After the incubation, add the following reagents: 5 superscript buffer

8 μL

0.1 M DTT

4 μL

10 mM dNTPs

2 μL

Superscript II RT

1.5 μL

Total volume

40 μL

14. Mix thoroughly by tapping the tubes and incubate at 42  C (in a PCR machine) for 1 h. 15. Add 0.25 μL RNase A/H and incubate at 37  C for 30 min followed by mixing. 3.3 Second Strand cDNA Synthesis and Labelling

1. Mix the following materials: First-stand cDNA product

28 μL

Klenow buffer

4 μL

Random primer

1 μL

Total volume

33 μL

2. Incubate the mixture at 100  C for 2 min and then keep the tubes at room temperature for 5 min.

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3. Add the following reagents to the tubes: 10 dNTPs

4 μL

Cy3-dUTP or Cy5-dUTP or labelling kit

1 μL

Klenow buffer

2 μL

Final volume

40 μL

4. Incubate at 37  C for 3 h in a PCR machine (see Note 8). 3.4 LncRNAMicroarray Hybridization

1. Mix the following materials: Labelled cDNA

18 μL

Blocking buffer

5 μL

Nuclease free water

2 μL

Total volume

25 μL

2. Incubate the mixture at 60  C for 30 min. 3. Add 25 μL 2 hybridization buffer (supplied with LncRNA Microarray kit). 4. Centrifuge at 13,000g for 1 min at room temperature. 5. Put the sample on ice and place them on the array surface quickly. 6. Put a 24  30 mm cover slip on top of the slide (see Note 9). 7. Clamp the microarray/backing gasket slide sandwich into the hybridization station/chamber. 8. Rotate the slide assembly to wet the surface and ensure the air pockets are not stuck in position and can be switch easily. 9. Incubate the assembly at 65  C for 17 h in a hybridization oven with gentle rotation. 3.5 LncRNAMicroarray Washing

1. Take out the hybridization assembly from chamber and disassemble quickly (see Note 10). 2. Place the slides onto the slide holder and place the slides into the washing buffer A quickly to avoid indiscriminate hybridization. 3. Remove the coverslip by a forceps and decant the washing buffer. 4. Transfer the slides in wash buffer B and wash for 2 min by up and down of the slides in a continuous motion. 5. Then transfer the slides in wash buffer C and wash for another 2 min.

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6. Place the slide rack with the slides to the metal spin-rack and put in speed vac for drying. 7. Spin with vacuum, no heat for 5–10 min. Place the slides into a light-proof slide box for analysis. 8. Scan the slides as soon as possible, otherwise stored them at 80  C for 1–2 weeks. 3.6

4

Data Analysis

Analysis of microarray data required special software packages for data extraction, analysis, and presentation. GeneSpring GX (https://www.agilent.com/search/?Ntt¼GeneSpring%20GX; Agilent, USA) software package for microarray data analysis provides extensive features, graphic user interface-guided analyses on raw or processed data. Also, there are several open-source or free of cost software available for microarray data analysis. For example, GenePatter (www.broadinstitute.org/cancer/software/gen epattern), MultiExperiment Viewer MeV (www.tm4.org/mev. html), Chipster (chipster.csc.fi), and R/Bioconductor packages (www.bioconductor.org) for microarray data analysis.

Notes 1. The samples need to be collected with proper informed consent from the patients with papillary thyroid carcinoma. The non-cancerous tissues must be free from cancer cells and cancer tissue section should have significant volume (>70%) of cancer cells. Also, ethics approval from the authority needs to be obtained beforehand for using the human samples. 2. Histological examination of the tissue sections needs to be performed by expert pathologist followed by Hematoxylin H & eosin (H & E) staining. The protocol for H & E protocol is as follows: (a) Place slides in quick dip fixation for at least 30 s, (b) Rinse in clean dH2O, 10 dips, (c) Stain in hematoxylin for 30 s, (d) Rinse in clean dH2O, 10 dips, (e) Place the slides in Scott’s blues solution for 10 s, (f) Rinse in clean dH2O, 10 dips, (g) Rinse in 70% ethanol, 5 dips, (h) Stain with Eosin for 20 s and rinse with 100% ethanol, 10 dips (3 times), (i) Rinse with xylene, 10 dips (2 times) and finally mount the slides. 3. Some RNA labelling kit contained reverse transcriptase, oligo (dT) and random primers. Thus, it can convert the RNA to double stand cDNA and amplified the template using T7 promoter along with labelling with fluorophore. In that case, researchers do not need extra cDNA conversion step. 4. Different lncRNA expression Microarrays for human, mouse and rat species are available. As the lncRNA conservation

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among the species is low, thus, lncRNA microarray designed for one species cannot be used for other species. 5. Insufficient or incomplete homogenization may lead to significant reduction in RNA yield. TissueLyser and rotor-stator mediated homogenization could provide higher RNA yields. 6. If the RNA pellet does not resuspend by pipetting, then incubate the sample at 55–60  C for 10 min. 7. Approximately 2 μg of total RNA is required for a microarray experiment and lower amount may causes significant constraints on reliable handling, storage, sample quality control, repetition of experiment, success of the experiment, quality of the data, etc. Also, an aliquot of the RNA should be kept for confirming the microarray results using qPCR. The quality/integrity of RNA are very important for microarray experiment, thus, check the RNA quality and integrity of RNA by measuring A260/A280 and A260/A230 ratios (1.9–2.1), which indicates the presence of impurities of the RNA. Also, the integrity of RNA can be checked by a Bioanalyzer or by denaturing gel electrophoresis. In gel electrophoresis, sharp and intense bands of 28S and 18S rRNA with a 28S:18S ratio of 2:1 indicate good quality RNA. 8. The number of dyes depends on experimental design. Two dyes are used to label different samples for comparison analysis. 9. Do not trap air bubbles. Carefully place the coverslip on top of the slide to avoid air bubble formation. 10. Keep the array surface up and wipe off the excess water from the chamber before opening it up. Also, avoid dust by minimizing the exposure of the slide to the air. References 1. Pertea M (2012) The human transcriptome: an unfinished story. Genes (Basel) 3:344–360 2. Hombach S, Kretz M (2016) Non-coding RNAs: classification, biology and functioning. Adv Exp Med Biol 937:3–17 3. Derrien T, Johnson R, Bussotti G, Tanzer A, Djebali S et al (2012) The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression. Genome Res 22:1775–1789 4. Huarte M (2015) The emerging role of lncRNAs in cancer. Nat Med 21:1253–1261 5. Lan X, Zhang H, Wang Z, Dong W, Sun W, Shao L, Zhang T, Zhang D (2015) Genomewide analysis of long noncoding RNA expression profile in papillary thyroid carcinoma. Gene 569:109–117

6. Liao D, Lv G, Wang T, Min J, Wang Y, Liu S (2018) Prognostic value of long non-coding RNA BLACAT1 in patients with papillary thyroid carcinoma. Cancer Cell Int 18:47 7. Yang M, Tian J, Guo X, Yang Y, Guan R, Qiu M, Li Y, Sun X, Zhen Y, Zhang Y, Chen C, Li Y, Fang H (2016) Long noncoding RNA are aberrantly expressed in human papillary thyroid carcinoma. Oncol Lett 12:544– 552 8. Wang Q, Yang H, Wu L, Yao J, Meng X, Jiang H, Xiao C, Wu F (2016) Identification of specific long non-coding RNA expression: profile and analysis of association with clinicopathologic characteristics and BRAF mutation in papillary thyroid cancer. Thyroid 26:1719– 1732

Long Non-Coding RNAs and Papillary Thyroid Carcinoma 9. Liyanarachchi S, Li W, Yan P, Bundschuh R, Brock P, Senter L, Ringel MD, de la Chapelle A, He H (2016) Genome-wide expression screening discloses long noncoding RNAs involved in thyroid carcinogenesis. J Clin Endocrinol Metab 101:4005–4013 10. Xu B, Shao Q, Xie K, Zhang Y, Dong T, Xia Y, Tang W (2016) The long non-coding RNA ENST00000537266 and ENST00000426615 influence papillary thyroid cancer cell proliferation and motility. Cell Physiol Biochem 38:368–378 11. Li T, Yang XD, Ye CX, Shen ZL, Yang Y, Wang B, Guo P, Gao ZD, Ye YJ, Jiang KW, Wang S (2017) Long noncoding RNA HIT000218960 promotes papillary thyroid cancer oncogenesis and tumor progression by upregulating the expression of high mobility group AT-hook 2 (HMGA2) gene. Cell Cycle 16:224–231 12. Braconi C, Kogure T, Valeri N, Huang N, Nuovo G, Costinean S, Negrini M, Miotto E, Croce CM, Patel T (2011) microRNA-29 can regulate expression of the long non-coding RNA gene MEG3 in hepatocellular cancer. Oncogene 30:4750–4756 13. Do H, Kim W (2018) Roles of oncogenic long non-coding RNAs in cancer development. Genomics Inform 16:e18 14. Sun W, Lan X, Zhang H, Wang Z, Dong W, He L, Zhang T, Zhang P, Liu J, Qin Y (2018) NEAT1_2 functions as a competing endogenous RNA to regulate ATAD2 expression by sponging microRNA-106b-5p in papillary thyroid cancer. Cell Death Dis 9:380 15. Zhu H, Lv Z, An C, Shi M, Pan W, Zhou L, Yang W, Yang M (2016) Onco-lncRNA HOTAIR and its functional genetic variants in papillary thyroid carcinoma. Sci Rep 6:31969 16. Di W, Li Q, Shen W, Guo H, Zhao S (2017) The long non-coding RNA HOTAIR promotes thyroid cancer cell growth, invasion and migration through the miR-1-CCND2 axis. Am J Cancer Res 7:1298–1309 17. Chu R, van Hasselt A, Vlantis AC, Ng EK, Liu SY, Fan MD, Ng SK, Chan AB, Liu Z, Li XY, Chen GG (2014) The cross-talk between estrogen receptor and peroxisome proliferatoractivated receptor gamma in thyroid cancer. Cancer 120:142–153 18. Guzel E, Okyay TM, Yalcinkaya B, Karacaoglu S, Gocmen M, Akcakuyu MH (2019) Tumor suppressor and oncogenic role of long non-coding RNAs in cancer. North Clin Istanb 7:81–86 19. Yoon H, He H, Nagy R, Davuluri R, Suster S, Schoenberg D, Pellegata N, Chapelle Ade L

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(2007) Identification of a novel noncoding RNA gene, NAMA, that is downregulated in papillary thyroid carcinoma with BRAF mutation and associated with growth arrest. Int J Cancer 121:767–775 20. Jendrzejewski J, He H, Radomska HS, Li W, Tomsic J, Liyanarachchi S, Davuluri RV, Nagy R, de la Chapelle A (2012) The polymorphism rs944289 predisposes to papillary thyroid carcinoma through a large intergenic noncoding RNA gene of tumor suppressor type. Proc Natl Acad Sci U S A 109:8646– 8651 21. Wang Y, He H, Li W, Phay J, Shen R, Yu L, Hancioglu B, de la Chapelle A (2017) MYH9 binds to lncRNA gene PTCSC2 and regulates FOXE1 in the 9q22 thyroid cancer risk locus. Proc Natl Acad Sci U S A 114:474–479 22. Wang C, Yan G, Zhang Y, Jia X, Bu P (2015) Long non-coding RNA MEG3 suppresses migration and invasion of thyroid carcinoma by targeting of Rac1. Neoplasma 62:541–549 23. Ma B, Liao T, Wen D, Dong C, Zhou L, Yang S, Wang Y, Ji Q (2016) Long intergenic non-coding RNA 271 is predictive of a poorer prognosis of papillary thyroid cancer. Sci Rep 6: 36973 24. Murugan AK, Munirajan AK, Alzahrani AS (2018) Long noncoding RNAs: emerging players in thyroid cancer pathogenesis. Endocr Relat Cancer 25:R59–R82 25. Zheng H, Wang M, Jiang L, Chu H, Hu J, Ning J, Li B, Wang D, Xu J (2016) BRAFactivated long noncoding RNA modulates papillary thyroid carcinoma cell proliferation through regulating thyroid stimulating hormone receptor. Cancer Res Treat 48:698–707 26. Liao T, Qu N, Shi RL, Guo K, Ma B, Cao YM, Xiang J, Lu ZW, Zhu YX, Li DS, Ji QH (2017) BRAF-activated LncRNA functions as a tumor suppressor in papillary thyroid cancer. Oncotarget 8:238–247 27. Zhang Y, Yu S, Jiang L, Wang X, Song X (2017) HOTAIR is a promising novel biomarker in patients with thyroid cancer. Exp Ther Med 13:2274–2278 28. Li HM, Yang H, Wen DY, Luo YH, Liang CY, Pan DH, Ma W, Chen G, He Y, Chen JQ (2017) Overexpression of LncRNA HOTAIR is associated with poor prognosis in thyroid carcinoma: a study based on TCGA and GEO Data. Horm Metab Res 49:388–399 29. Xia S, Wang C, Ni X, Ni Z, Dong Y, Zhan W (2017) NONHSAT076754 aids ultrasonography in predicting lymph node metastasis and promotes migration and invasion of papillary thyroid cancer cells. Oncotarget 8:2293–2306

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30. Li Q, Shen W, Li X, Zhang L, Jin X (2017) The lncRNA n340790 accelerates carcinogenesis of thyroid cancer by regulating miR-1254. Am J Transl Res 9:2181–2194 31. Goedert L, Plac¸a JR, Fuziwara CS, Machado MCR, Plac¸a DR, Almeida PP, Sanches TP, Santos JFD, Corveloni AC, Pereira IEG, de Castro MM, Kimura ET, Silva WA Jr, Espreafico EM (2017) Identification of long noncoding RNAs deregulated in papillary thyroid cancer and correlated with BRAFV600E Mutation by bioinformatics integrative analysis. Sci Rep 7:1662 32. Du Y, Xia W, Zhang J, Wan D, Yang Z, Li X (2017) Comprehensive analysis of long noncoding RNA-mRNA co-expression patterns in thyroid cancer. Mol BioSyst 13:2107–2115 33. Zhang R, Hardin H, Huang W, Chen J, Asioli S, Righi A, Maletta F, Sapino A, Lloyd RV (2017) MALAT1 long non-coding RNA expression in thyroid tissues: analysis by in situ hybridization and real-time PCR. Endocr Pathol 28:7–12 34. Sun W, Lan X, Wang Z, Dong W, He L, Zhang T, Zhang H (2016) Overexpression of long non-coding RNA NR_036575.1 contributes to the proliferation and migration of papillary thyroid cancer. Med Oncol 33:102 35. Kim D, Lee WK, Jeong S, Seol MY, Kim H, Kim KS, Lee EJ, Lee J, Jo YS (2016) Upregulation of long noncoding RNA LOC100507661 promotes tumor aggressiveness in thyroid cancer. Mol Cell Endocrinol 431:36–45 36. Jeong S, Lee J, Kim D, Seol MY, Lee WK, Jeong JJ, Nam KH, Jung SG, Shin DY, Lee EJ, Chung WY, Jo YS (2016) Relationship of focally amplified long noncoding on chromosome 1 (FAL1) lncRNA with E2F transcription factors in thyroid cancer. Medicine (Baltimore) 95:e2592

37. Xiong X, Zhu H, Chen X (2017) Low expression of long noncoding RNA CASC2 indicates a poor prognosis and promotes tumorigenesis in thyroid carcinoma. Biomed Pharmacother 93:391–397 38. Li Z, Gao B, Hao S, Tian W, Chen Y, Wang L, Zhang X, Luo D (2017) Knockdown of lncRNA-PANDAR suppresses the proliferation, cell cycle and promotes apoptosis in thyroid cancer cells. EXCLI J 16:354–362 39. Zhang D, Liu X, Wei B, Qiao G, Jiang T, Chen Z (2017) Plasma lncRNA GAS8-AS1 as a potential biomarker of papillary thyroid carcinoma in Chinese patients. Int J Endocrinol 2017:2645904 40. Pan W, Zhou L, Ge M, Zhang B, Yang X, Xiong X, Fu G, Zhang J, Nie X, Li H, Tang X, Wei J, Shao M, Zheng J, Yuan Q, Tan W, Wu C, Yang M, Lin D (2016) Whole exome sequencing identifies lncRNA GAS8AS1 and LPAR4 as novel papillary thyroid carcinoma driver alternations. Hum Mol Genet 25:1875–1884 41. Lan X, Sun W, Zhang P, He L, Dong W, Wang Z, Liu S, Zhang H (2016) Downregulation of long noncoding RNA NONHSAT037832 in papillary thyroid carcinoma and its clinical significance. Tumour Biol 37: 6117–6123 42. He H, Li W, Liyanarachchi S, Jendrzejewski J, Srinivas M, Davuluri RV, Nagy R, de la Chapelle A (2015) Genetic predisposition to papillary thyroid carcinoma: involvement of FOXE1, TSHR, and a novel lincRNA gene, PTCSC2. J Clin Endocrinol Metab 100: E164–E172 43. He H, Nagy R, Liyanarachchi S, Jiao H, Li W, Suster S, Kere J, de la Chapelle A (2009) A susceptibility locus for papillary thyroid carcinoma on chromosome 8q24. Cancer Res 69: 625–631

Chapter 11 Single Nucleotide Polymorphisms in Papillary Thyroid Carcinoma: Clinical Significance and Detection by High-Resolution Melting Robert A. Smith and Alfred K. Lam Abstract Single nucleotide polymorphisms (SNPs) can have a variety of implications for the progression and development of papillary thyroid carcinomas (PTCs). Identification of SNPs, either as germline variants or mutations occurring in tumor tissue, can thus have useful implications for patient management. There are many potential methods that can be used to identify a specific SNP or other genetic variant, and among these is high-resolution melting (HRM). HRM can be used to detect the presence of a genetic variant in a single sealed tube, involving undertaking a polymerase chain reaction (PCR) in the presence of a saturating intercalating dye. Once PCR is complete, the amplicons produced can be melted through incremental raising of the temperature and the genotype of individual samples determined by changes in the change in fluorescence as the fluorescent dye is released by the melting DNA. In this chapter, we detail a method for the genotyping of DNA samples using HRM. Key words High-resolution melting, HRM, Polymerase chain reaction, PCR, Single nucleotide polymorphisms, SNP, Cancer management, Mutations

1

Introduction Thyroid cancer is a relatively common malignancy, with age-standardized rates of >8 cases per 100,000 population in many nations [1]. Much of the thyroid cancer incidence is the result of the most common subtype, papillary thyroid cancer (PTC), the incidence of which has been rising and rose significantly in between 2014 and 2017 [2–4]. This raises an interesting clinical problem, since PTCs overall have excellent prognosis and are quite indolent, and active treatment may not be necessary in many cases, resulting in a burden of overtreatment [3]. By contrast, some PTCs can be lethal and require aggressive treatment [3, 4]. Thus, it is becoming more important to identify biomarkers that can differentiate between the less aggressive forms of PTC and those which are

Alfred K. Lam (ed.), Papillary Thyroid Carcinoma: Methods and Protocols, Methods in Molecular Biology, vol. 2534, https://doi.org/10.1007/978-1-0716-2505-7_11, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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already life-threatening or are likely to become so in the future. One possible avenue for such clinical differentiation is with genetic biomarkers including single nucleotide polymorphisms (SNPs). Single nucleotide polymorphisms are distinct from mutations as biomarkers in that they already exist within the genetic makeup of the patient prior to the initiation of the cancer and are not themselves the cause of the malignancy. SNPs may, however, influence the susceptibility of the individual to cancer development and may influence the course taken by the disease once it develops. Alternately, associations between SNPs and various aspects of PTC biology may occur due to linkage between SNPs assayed in research and other genetic variations nearby, including genetic variations in promoters, enhancers, repressors, and other genetic sequences that control gene expression. Regarding PTC, several SNPs have been identified that are associated with several characteristics of the disease. At the basic level, there have been SNPs which have been found to increase the risk of PTC development. These include the FAS c.642T>C SNP (rs2234978), where the frequency of the T allele is elevated in PTC samples, and the DICER1 c.*3473A>G SNP (rs3742330), where the frequency of the G allele is higher in controls [5, 6]. More complex relationships to PTC development have also been identified, where haplotypes of multiple SNPs combine to signal changes in PTC risk. These include the TAS2R3/4 CC haplotype (for rs2270009 and rs2234001) which shows lower PTC risk; the ATM haplotype rs373759 (C), rs664143(G), and rs4585 (T) showing lower PTC risk; the TITF1/TITF2 rs944289, rs965513, and rs1443434 haplotypes T-G-G and T-G-T showing increased risks of PTC; the MDM2 rs2279744 (G) rs3730485 (deletion) haplotype associated with increased PTC incidence; and haplotypes of multiple SNPs in BRCA1 including rs1799950, rs799917, rs16941, rs16942, rs1060915, and rs1799966 affecting PTC risk [7–11]. The influence of SNPs may also include altering the course of disease progression, and SNPs have been associated with specific characteristics of PTCs in multiple studies. For example, the distribution of the VEGF-A polymorphisms 141 A>C, +405 C>G, and +936 C>T, where alleles A, G, and T, respectively, are significantly more common in malignant thyroid tissue compared to benign lesions [12]. Similarly, the T allele of the MMP9 1562 C>T polymorphism was found to be associated with increased MMP9 expression, extrathyroidal extensions, and higher TNM stage, with increased expression also associating with extrathyroidal invasion, lymph node metastasis, larger tumor size, and advanced stage [13]. Jendrzejewski and colleagues also determined that rs965513, located near the Forkhead Box E1 (FOXE1) gene and the non-coding RNA Papillary Thyroid Carcinoma Susceptibility Candidate 2 (PTCSC2) and potentially regulating both, is

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associated with larger tumor size and extrathyroidal expansion [14]. In the same paper, they also determined that rs2439302, located in Neuregulin (NRG1), was associated with lymph node metastasis and multifocality of the tumor [14]. Thus, there are reasons that identifying the presence of one or more SNPs may be potentially useful for patient management, and there are several ways to ascertain the status of SNPs in a patient. In this chapter, we will discuss a basic method for ascertaining SNPs, high-resolution melting (HRM). High-resolution melting is a single-tube, polymerase chain reaction (PCR)-based method for the identification of DNA sequence changes. HRM utilizes a saturating intercalating fluorescent dye, which binds to double-stranded DNA, and a PCR machine equipped with a fluorescent camera, such as those used to perform qPCR [15]. HRM allows discrimination of genetic variants through subtle alterations to the melting temperature caused by differences between the hydrogen bonds and orientation of base pairs of the available alleles [15]. The process involves slow, stepwise raising of the temperature of a PCR product while at each step assessing the amount of fluorescence present, which will drop as the amplicon melts and the dye is released. Homozygous samples will show a simple S curve (Fig. 1) as the amplicon melts, with each homozygous condition showing a slight shift to the right or left, depending on the relative binding strength of the amplicon. Heterozygous samples show a distorted curve, and the melting of the different amplicons in the solution is assessed. HRM is limited in that it does not precisely indicate the identity of the genetic variants present, only that there is a deviation from the homozygous genotype used as a control. This means that if working with DNA where mutations may be likely to occur (e.g., micro-dissected tumor tissue), alternative methods may be preferable. Conversely, HRM can detect genetic variants regardless of their position in the amplicon, or whether their identity is known beforehand, and can detect multiple genetic variants in the same amplicon, as each different sequence has its own melting pattern. This means that it may be suitable as a higher throughput and less expensive first-line test to detect the presence of mutation from the wild type, with specific variant identity later determined using Sanger sequencing. When performing HRM, it is advisable to include positive control genotypes, preferably at least one of each genotype being sought, as well as performing sample replicates (triplicate or greater) to monitor for deviations caused by impurities or temperature variation in the PCR block, if using a block-based instrument. With careful use of control genotypes and sample replicates, HRM can provide a wealth of useful information about sequence changes in an amplicon.

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Fig. 1 HRM curve differences. This figure shows data from an HRM experiment. Homozygous samples, whether mutant or wild type, show as a simple S curve, with each genotype separated by a distance determined by the relative binding strength of the amplicon. In this case, separation is approximately 0.25  C. Heterozygotes show a distorted curve shape, typically displaying a “kink” in the curve as shown here. This curve may be separate from the homozygotes as in this example, or on top of them. Vertical axis is relative sample fluorescence. Horizontal axis is sample temperature in  C

2

Materials Prior to commencing the method, you will need the following: 1. PCR box, or biocontainment hood with (UV) sterilization (optional, but desirable).

ultraviolet

2. HRM capable PCR machine, with fluorescent camera matching the emission spectrum of the dye you intend to use (Fig. 2). 3. HRM analysis software suitable for your instrument. 4. Pipettes suitable for batch sizes you wish to use. 5. Pipette tips suitable for PCR, preferably barrier tips. 6. Sterile PCR tubes or PCR plate suitable for your instrument of choice. 7. PCR loading rack for holding tubes or plate. 8. Sterile capped tube for master mix. Volume of tube as required, typically 1 mL.

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Fig. 2 HRM instrument samples loaded for HRM within a Rotor-Gene Q instrument. The Rotor-Gene Q undertakes PCR and melting in a single temperaturecontrolled chamber, which limits temperature differences between samples that can occur between the edges and center of Peltier cooling plates

9. Ice bucket or tray. 10. Crushed ice. 11. Aluminum foil. 12. Magnesium chloride (MgCl) solution, 25 mM. 13. PCR buffer 10 (e.g., 200 mM Tris HCl (pH 8.4), 500 mM potassium chloride (KCl), but any commercial buffer will work). 14. Forward and reverse primers for your PCR, 5 mM. 15. Nucleotides (dNTPs). 10 mM each of dATP, dTTP, dCTP, and dGTP. 16. Saturating intercalating dye. For example, SYTO9 at 5 mM (see Note 1). 17. DNA polymerase, for example, Taq polymerase, at 5 U/μL. 18. Sterile, molecular biology grade water. 19. Template DNA at 20 ng/μL.

3

Methods The setup and running of a HRM is generally like other PCR applications, and care should be taken to prevent contamination of samples, as this can lead to erroneous results, especially if contamination occurs only in a few samples or tubes. This is another reason for the inclusion of replicates and positive controls (where possible), as deviations from the expected pattern of melting curves for different genotypes caused by contamination can be identified through these means. It is also advisable to ensure that the

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Table 1 PCR initial setup Reagent

Stock concentration

Concentration for individual PCR solution

MgCl

25 mM

2.5 mM

PCR buffer

10

1

Primers (each)

5 mM

0.25 mM

dNTPs

10 mM (each)

0.5 mM (each)

SYTO-9

5 mM

0.25 mM

Taq polymerase

5 U/μL

1 U/rxn

Sterile water

~

~

DNA

20 ng/μL

2 ng/μL

intercalating dye used is protected from light as much as possible to maintain fluorescence, so in addition to protecting stock tubes, mastermix tubes and reaction tubes should be protected with foil or tight, opaque racks. 3.1

Reaction Setup

1. Prepare a location to set up the PCR (see Note 2). 2. Place reagents on ice for the duration of PCR setup; only permit sufficient thawing to allow pipetting. 3. Ensure intercalating dye used is covered by foil to protect it from light. 4. Pipette reagents for all reactions, excepting template DNA, into a sterile capped tube, in sufficient quantities to produce a mastermix that will result in the desired final concentration in individual PCR tubes. For an example on starting concentration, see Table 1 (see Notes 3 and 4). 5. Ensure enough mastermix is produced to allow for all reactions to be pipetted, plus a safety margin of additional reactions to account for pipette calibration error (see Note 5). 6. Seal mastermix tube, cover in foil, and place on ice until later. 7. Set up rack for PCR tubes or plates as desired, and place tubes or plate within the rack. 8. Pipette template DNA and negative controls into PCR tubes/ rack (see Note 6). 9. Seal PCR tubes/rack. Ensure all reagents are at the bottom of each reaction tube. 10. Place sealed tube in HRM capable PCR machine, and commence the thermal amplification steps. See Table 2 for an example on thermal protocol (see Note 7).

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Table 2 Thermal conditions initial setup Stage Initial denaturation

Time 1 min

Temperature a

Number of repeats



1



95 C

Denaturation Primer annealing Extension

30 s 1 min 30 sb

95 C 60  Cc 72  C

30

Final extension

5 min

72  C

1

a

Initial denaturation may need to be 5 min, if heat activated polymerase is used This step can be eliminated for some efficient PCRs, as extension can take place during the annealing step as well. Optimize as required c Temperature for primer annealing will depend on the melting temperature of primer pairs and the need to avoid non-specific PCR. Adjust as required b

3.2 High-Resolution Melting and Data Analysis

Analysis of an HRM run is a relatively simple process. The basic success of a run can be determined if a fluorescence acquisition step is included in the PCR stage by acquiring data in the extension phase of each cycle. This is not strictly necessary, but it can be useful to see the rate of amplification for each sample and identify potential problems in the amplification stage. Once the amplification step is completed, you will need to melt the amplicons by slowly raising the temperature of the completed reaction and, at each temperature increase, observing the amount of fluorescence present. The more extreme the change in binding forces caused by any genetic variation present, the more different the data for genotypes will be. Practically speaking, this means that if you are looking for a known mutation with a large change in binding forces, such as an adenine to cytosine change or small insertion, the change in temperature at each step of the melting process can be relatively large. By contrast, when dealing with small changes in binding forces, such as a cytosine to guanine change, smaller increments in temperature will help improve the resolution of the melting curve obtained. 1. Once thermal amplification steps are completed, melting steps can be performed. For an example on melting protocol, see Table 3 (see Note 8). 2. Obtained fluorescence data from each reaction tube should now be normalized using the analysis software for your instrument. 3. Data ranges for normalization should be selected from the regions before and after the full S phase of the melting curve. For an example on regions to use, see Fig. 3. 4. Genotype samples with reference to known positive controls included in the experiment (see Note 9).

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Table 3 HRM conditions initial setup Lowest data-acquisition temperature

80  C

Highest data-acquisition temperature

90  C

Size of temperature increment per step

0.1  C

Time for data acquisition

2s

Fig. 3 HRM data normalization. This figure shows data from an HRM experiment before and after normalization using different parameters. (a) Data prior to normalization with small regions (red boxes) selected for normalization; (b) data prior to normalization with larger region (red boxes) selected for normalization; (c) data post-normalization with small normalization regions; (d) data post-normalization with large normalization regions. Vertical axis is sample fluorescence (absolute for panels a and b and relative for panels c and d). Horizontal axis is sample temperature

4

Notes 1. Setup for an HRM reaction is essentially identical to a basic PCR, with only the addition of the saturating fluorescent intercalating dye, for example, SYTO-9. Basic SYBR Green does not saturate nucleic acid as effectively and can produce inferior results, though it is still possible to use it in a properly optimized reaction.

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2. The location used for PCR setup should be free of potential contamination by extraneous DNA or PCR amplicons from previous reactions. Ideally, this should be in a dedicated PCR box or a biocontainment hood with UV sterilization. 3. As with all PCR, appropriate optimization for reagent concentrations and thermal conditions should be undertaken until the PCR performs to requirements. In the case of an HRM, ensuring that the signal from the intercalating dye is sufficient is important, as well as that the production of amplicon is effective. When setting up signal acquisition parameters for assessment of the PCR phase, signal from the dye should be examined by assessing fluorescence at the end of each elongation step, or primer annealing if elongation steps are not used. It is also important in an HRM that no non-specific bands are produced, as these have a high potential for distorting the results of any downstream analysis. Likewise, excessive primer dimer can cause interference in signal assessment, and PCRs should be run for as many cycles as needed to deplete any leftover primers. Thus, PCR product should be checked on gels during optimization to ensure amplicons are clean. Finally, the subtle differences in melting temperature between different genotypes can be difficult to differentiate in larger amplicons, so amplicons should only be as large as necessary in your assay. 4. Premixed solutions are available from several commercial providers and may simplify the process of setting up the reaction by eliminating all components other than the specific primers and template DNA. Optimization will still be needed, however. 5. Additional reactions should be assumed when creating a mastermix for a batch, to avoid there being insufficient mastermix available for all intended reactions due to pipetting error and instrument calibration variation. The number of additional reactions to make up will depend on personal comfort, budget, and prior experience of mastermix shortfall, but somewhere between two and five additional reactions per batch is useful as a starting point. 6. When undertaking a HRM analysis, it is a good idea to perform HRM on each target DNA sample in triplicate to allow crossverification of sample genotypes. This is most important when the variant involves a change to or from an adenine/thymine or a cytosine/guanine, as the difference in melting temperature can be extremely subtle between different alleles. This may also be important when utilizing a block-based PCR machine, as subtle temperature differences for samples on the edges of the heating block may lead to small changes in the apparent melting curve.

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7. Table 2 is an example starting point for PCR thermal conditions. The time taken for each step in the reaction can be shortened or lengthened as needed. Generally, increasing the time for any step will increase the amount of overall amplification, but since this also applies to any possible non-specific amplification, shorter times for certain steps may be required. The elongation step is not always necessary, and a sufficiently long primer annealing step may allow sufficient time and temperature of the DNA polymerase to complete the amplicon. Primer annealing temperature will depend on melting temperature of your primers, and increasing the temperature will increase specificity, but decrease amplification, and reducing temperature will increase amplification, but also decrease specificity. If you are assessing fluorescence during the PCR stage of the process, data acquisition should always be set at the end of the elongation steps, or the primer annealing steps if the elongation steps are not being used. 8. Once the initial PCR is optimized, the actual HRM step will need to be assessed and optimized. The HRM step consists of gradually raising the temperature of the completed PCR solution and pausing to assess the amount of remaining fluorescence. Table 3 is an example starting point. The parameters that are most likely to be altered are the size of the temperature increment for each temperature step, as well as the range of temperatures to acquire data from. Smaller increments of temperature produce finer-grained data, enabling discrimination of alleles with very close melting temperatures, while a wider or narrower range of melting temperatures may be needed to allow for sufficient maximum and minimum fluorescence values to be obtained for data normalization. 9. During analysis, samples should be compared to the positive controls for each genotype included in the reaction. Triplication is useful here in being able to see individual tubes where melting characteristics have been affected by instrument error or contamination and avoid mis-genotyping the sample. Direct comparison of experimental samples to the positive controls is most useful where there are subtle differences between the homozygous melting curves, and some curves may drift into an ambiguous area between them. Heterozygotes can also benefit from reference to the positive control if there are multiple possible variants in the amplicon, to allow differentiation of samples that are heterozygotic at different positions.

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References 1. Ferlay J, Ervik M, Lam F, Colombet M, ˜ eros Mery L, Pin M, Znaor A, Soerjomataram I, Bray F (2020) Global cancer observatory: cancer today. International Agency for Research on Cancer, Lyon. Available from: https://gco.iarc.fr/today. Accessed 30 Apr 2021. 2. Abdullah MI, Junit SM, Ng KL, Jayapalan JJ, Karikalan B, Hashim OH (2019) Papillary thyroid cancer: genetic alterations and molecular biomarker investigations. Int J Med Sci 16(3): 450–460. https://doi.org/10.7150/ijms. 29935 3. Roman BR, Morris LG, Davies L (2017) The thyroid cancer epidemic, 2017 perspective. Curr Opin Endocrinol Diabetes Obes 24(5): 332–336. https://doi.org/10.1097/MED. 0000000000000359 4. Prete A, Borges de Souza P, Censi S, Muzza NN, Sponziello M (2020) Update on fundamental mechanisms of thyroid cancer. Front Endocrinol (Lausanne) 11:102. https://doi. org/10.3389/fendo.2020.00102 5. Basolo F, Giannini R, Faviana P, Fontanini G, Patricelli Malizia A, Ugolini C, Elisei R, Miccoli P, Toniolo A (2004) Thyroid papillary carcinoma: preliminary evidence for a germline single nucleotide polymorphism in the Fas gene. J Endocrinol 182(3):479–484. https://doi.org/10.1677/joe.0.1820479 6. Mohammadpour-Gharehbagh A, Heidari Z, Eskandari M, Aryan A, Salimi S (2020) Association between genetic polymorphisms in microrna machinery genes and risk of papillary thyroid carcinoma. Pathol Oncol Res 26(2): 1235–1241. https://doi.org/10.1007/ s12253-019-00688-z 7. Choi JH, Lee J, Yang S, Lee EK, Hwangbo Y, Kim J (2018) Genetic variations in TAS2R3 and TAS2R4 bitterness receptors modify papillary carcinoma risk and thyroid function in Korean females. Sci Rep 8(1):15004. https:// doi.org/10.1038/s41598-018-33338-6 8. Song CM, Kwon TK, Park BL, Ji YB, Tae K (2015) Single nucleotide polymorphisms of ataxia telangiectasia mutated and the risk of papillary thyroid carcinoma. Environ Mol Mutagen 56(1):70–76. https://doi.org/10. 1002/em.21898

9. Zhang X, Gu Y, Li Y, Cui H, Liu X, Sun H, Yu Q, Yu Y, Liu Y, Zhan S, Cheng Y (2020) Association of rs944289, rs965513, and rs1443434 in TITF1/TITF2 with risks of papillary thyroid carcinoma and with nodular goiter in Northern Chinese Han populations. Int J Endocrinol 2020:4539747. https://doi.org/ 10.1155/2020/4539747 10. Maruei-Milan R, Heidari Z, Salimi S (2019) Role of MDM2 309T>G (rs2279744) and I/D (rs3730485) polymorphisms and haplotypes in risk of papillary thyroid carcinoma, tumor stage, tumor size, and early onset of tumor: a case control study. J Cell Physiol 234(8):12934–12940. https://doi.org/10. 1002/jcp.27960 11. Xu L, Doan PC, Wei Q, Liu Y, Li G, Sturgis EM (2012) Association of BRCA1 functional single nucleotide polymorphisms with risk of differentiated thyroid carcinoma. Thyroid 22(1):35–43. https://doi.org/10.1089/thy. 2011.0117 12. Salajegheh A, Smith RA, Kasem K, Gopalan V, Nassiri MR, William R, Lam AK (2011) Single nucleotide polymorphisms and mRNA expression of VEGF-A in papillary thyroid carcinoma: potential markers for aggressive phenotypes. Eur J Surg Oncol 37(1):93–99. https://doi. org/10.1016/j.ejso.2010.10.010 13. Roncevic J, Djoric I, Selemetjev S, Jankovic J, Dencic TI, Bozic V, Cvejic D (2019) MMP-91562 C/T single nucleotide polymorphism associates with increased MMP-9 level and activity during papillary thyroid carcinoma progression. Pathology 51(1):55–61. https://doi. org/10.1016/j.pathol.2018.10.008 14. Jendrzejewski J, Liyanarachchi S, Nagy R, Senter L, Wakely PE, Thomas A, Nabhan F, He H, Li W, Sworczak K, Ringel MD, Kirschner LS, de la Chapelle A (2016) Papillary thyroid carcinoma: association between germline DNA variant markers and clinical parameters. Thyroid 26(9):1276–1284. https://doi.org/ 10.1089/thy.2015.0665 15. Er TK, Chang JG (2012) High-resolution melting: applications in genetic disorders. Clin Chim Acta 414:197–201. https://doi.org/10. 1016/j.cca.2012.09.012

Chapter 12 BRAF Mutations in Papillary Thyroid Carcinoma: A Genomic Approach Using Probe-Based DNA Capture for Next-Generation Sequencing Robert A. Smith and Alfred K. Lam Abstract The BRAF V600E mutation in papillary thyroid carcinoma is the major mutation in classical subtype of papillary thyroid carcinoma and other cancers. It is the most studied predictor of clinical and pathological characteristics as well as molecular targets for cancer therapy. On the other hand, there is potential for many more forms of activating mutation in BRAF that are not detectable by simple assays to detect V600E, or even simple polymerase chain reaction (PCR)-based sequencing for full-length BRAF. Such activating mutations could arise from larger-scale rearrangements which may apparently leave no sequence change to BRAF while causing increased expression or activation by unusual means, such as gene fusion. Detection of these kinds of changes can take place using a variety of methods, though capture-based sequencing can identify the existence of such forms of mutant BRAF without needing foreknowledge of the loci involved in these kinds of mutation. In this chapter, we detail a method for capture of specific DNA sequences and their amplification to prepare for massively parallel sequencing. Key words BRAF, Probe capture, Degenerate oligonucleotide PCR, DOP PCR, Papillary thyroid carcinoma

1

Introduction BRAF V600E mutation is the classical pathogenic mutation in papillary thyroid carcinoma and other cancers [1, 2]. It is the most common mutation in papillary thyroid carcinoma, particularly the classical subtype [3], and uncommon or not being detected in follicular or diffuse sclerosing subtype [4]. BRAF mutations also in collaboration with TERT promoter mutations are associated with aggressive clinicopathological characteristics of papillary thyroid carcinoma [5]. Because of this, inhibitors for BRAF are actively investigated for treatment of papillary thyroid carcinoma and the de-differentiated follicular-type carcinoma, anaplastic thyroid carcinoma [6–9].

Alfred K. Lam (ed.), Papillary Thyroid Carcinoma: Methods and Protocols, Methods in Molecular Biology, vol. 2534, https://doi.org/10.1007/978-1-0716-2505-7_12, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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Among the different mutations noted in papillary thyroid carcinoma, BRAF V600E mutation is the most studied mutation as a predictor of behavior of the carcinoma. In multicenter studies, BRAF mutation is associated with increased mortality and cancer recurrence in patients with papillary thyroid carcinoma [10, 11]. In these studies, the mutation was also shown to be a strong predictor of aggressive clinical behavior in certain clinical subgroups of patients with papillary thyroid carcinoma, such as in advanced age [12], male sex [13], with lymph node metastases [14], and with certain tumor sizes [15, 16]. It is worth noting that BRAF V600E mutation is not the only potential mutation that can enhance BRAF signalling. Other point mutations as well as insertions and fusions have been observed as potential driver mutations in cancer [17]. This means that while a simple screen for the BRAF V600E will identify a high proportion of mutation carriers, such an approach will fail to detect many individuals who might respond to forms of BRAF inhibition not specific to V600E. As a result, undertaking a next-generation sequencing (NGS) approach to the identification of BRAF mutations, using genomic or semi-genomic strategies for library generation, may prove useful for prognostic or treatment selection purposes. Additionally, NGS approaches typically allow more robust detection of mutations existing in relatively small proportions of biopsied tumor tissue than Sanger sequencing [17]. Such detection is important for potential escape from treatments not targeting BRAF signalling and can take place in initial identification of the tumor, or in later surveillance testing of patient blood. The ability of an assay to detect a variety of different mutation types will depend on the method of generation used for the library used in sequencing [18]. Different NGS technologies have different capabilities and requirements for their libraries, and as a result, in most cases, the manufacturers provide proprietary kits for library generation, which we will not cover in this chapter. Selection of the library type and preparation of the target nucleic acid are important, however, and that is what we will be discussing. Broadly speaking, NGS libraries will derive from a targeted or un-targeted sequence selection process. The specific mechanisms involved may be broadly similar, though targeted libraries will obviously have some additional mechanism to select the specific sequences desired. The most direct form of this kind of selection is a polymerase chain reaction (PCR) of the desired regions, which can enable identification of mutations across an entire gene or several selected genes or loci. PCR-based approaches can also be used for exome and genome-based sequencing, and though these seem effectively untargeted, it is important to remember that set primer sequences will produce set amplicons, and mutations that significantly disrupt an amplicon’s size or change its location such as insertions, deletions, and translocations may be missed by the assay.

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An alternative to PCR-based targeting is sequence capture enrichment. In this method, the target nucleic acid is fragmented through use of restriction enzymes or sonication, and a complimentary sequence to the desired region or regions is incubated with the sample nucleic acid [19, 20]. This capture sequence is chemically modified to carry a biotin group or similar bondable tag, allowing the hybridized sequence to be drawn out of solution through binding of a magnetic bead and exposure to a magnet [20, 21]. Following capture of the target sequences, the captured nucleic acid is subject to non-specific amplification. This method allows for the identification of translocations, large insertions/ deletions close to the loci of interest, and other such changes that may not be detected by PCR-based approaches. As such, sequence capture enrichment may be of interest in cancer patients to identify and characterize amplifications, deletions, or translocations to genes of interest, including BRAF. Another alternative to PCR-based targeting is immunocoprecipitation, whereby an antibody is used to target proteins of interest that may be bound to a sequence of interest. Target proteins may include histones with specific modifications, transcription factors, or zinc-finger proteins. As in sequence capture enrichment, the target nucleic acid is fragmented prior to incubation of biotinlabelled antibodies, which can then be bound to magnetic beads, and the target proteins and any nucleic acid bound to them can be recovered. Such an approach may be of use to identify genes activated in a specific pathway in cancer tissue, or the strength of activation for a specific pathway, as determined by the amount of a particular sequence appearing in the resulting sequencing. This may be of interest when attempting to identify patients who may benefit from interventions, by detecting activation of specific signalling pathways. Untargeted libraries will be generated by use of all available nucleic acid for sequencing. Use of untargeted sequencing is generally a trade-off between the depth of sequencing possible on the platform of interest and the ability to detect unusual or unexpected genetic variants, such as mutations in regulatory regions and largerscale alterations. It is important to be aware that even whole genome sequencing has limitations, and there are regions that even untargeted sequencing will not be able to interrogate, often due to unusual structure or complexity in a region that limits analysis of obtained reads [18]. As such, selection of the right library preparation approach is important before commencing work so that the appropriate data will be available. In this method, we will cover the fragmentation and probebased recovery of targeted DNA sequences, followed by degenerate oligonucleotide amplification for further sequencing through massively parallel means. The process is shown schematically in Fig. 1.

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Fig. 1 Schematic method. This figure shows the schematic process to generate targeted DNA for inclusion in a massively parallel sequencing library. Genomic DNA from the source is first fragmented by sonication or restriction enzymes and biotinylated probes for the target DNA hybridized to the fragments. The biotinylated probes are further hybridized to streptavidin conjugated magnetic beads and target DNA separated by use of a magnet. Target DNA is purified by denaturation followed by removal and retention of supernatant. After purification, target DNA is amplified by degenerate oligonucleotide primed PCR, producing double-stranded DNA that is ready to be tagged with appropriate sequencing adaptors for massively parallel sequencing

2

Materials Prior to commencing the method, you will need the following: 1. PCR box, or biocontainment hood with UV sterilization for handling DNA and reactions (optional, but desirable). 2. Pipettes suitable for batch sizes you wish to use. 3. Pipette tips suitable for pipettes in use, preferably barrier tips. 4. Sterile sample tubes suitable for your instrument(s) of choice. 5. Rack for holding tubes. 6. Sterile, molecular biology-grade water. 7. PCR thermal cycler. 8. Vortex mixer. 9. Template DNA at 20 ng/μL. 10. Molecular biology-grade water. 11. Crushed ice. 12. Ice bucket.

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Fig. 2 Sonicator. A sonicator unit with a dedicated waterbath, in addition to a pump system for constant refreshment of chilled water during sonication. Where possible, use of a unit with dedicated systems to refresh the water is desirable to prevent excessive damage to DNA and encourage consistency between samples

13. [For enzymatic fragmentation] Sterile capped tube for master mix. Volume of tube as required, typically 1 mL. 14. [For enzymatic fragmentation] Restriction Enzyme (e.g., EcoRI) 15. [For enzymatic fragmentation] Buffer for restriction enzyme (e.g., for EcoRI, 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 μg/ml bovine serum albumin (BSA), pH 7.9 at 25  C). 16. [For sonication] Ice bucket or tray. 17. [For sonication] Crushed ice. 18. [For sonication] Low TE buffer (10 mM Tris-HCl 0.1 mM EDTA at pH 7.5–8.0 @25  C). 19. [For sonication] Sonication device, with separate or attached waterbath (Fig. 2). 20. [For sonication] Sterile, deionized water @ 4  C. 21. [Optional for purification] Ethanol 100%, chilled at

20  C.

22. [Optional for purification] 3M sodium acetate. 23. [Optional for purification] Liquid nitrogen. 24. [Optional for purification] Insulated vessel for freezing samples. 25. [Optional for purification] Tongs/forceps for freezing/ retrieving samples.

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26. [Optional for purification] Centrifuge capable of 16000 RCF. 27. [Optional for purification] Waterbath or incubator capable of heating to 37  C. 28. [For capture] Biotinylated probes for desired region(s) at 50 μM. 29. [For capture] Streptavidin conjugated magnetic beads. 30. [For capture] 10x saline sodium citrate (SSC) Buffer (1.5M NaCl, 0.15M Na3C6H5O7, pH 7.0). 31. [For capture] Magnetic separation rack. 32. [For amplification] PCR buffer 10X (e.g., 200 mM Tris HCl (pH 8.4), 500 mM KCl, but any commercial buffer will work). 33. [For amplification] 25 mM MgCl2. 34. [For amplification] Universal degenerate oligonucleotide primers (see Note 1), 5 mM. 35. [For amplification] Nucleotides (dNTPs). 10 mM each of dATP, dTTP, dCTP, and dGTP. 36. [For amplification] DNA polymerase, for example, Taq Polymerase, at 5 U/μL.

3

Methods

3.1 DNA Fragmentation

The choice of precise method for fragmentation will depend upon what you wish to accomplish with your method and your intended sequencing assay. If you wish to identify potentially large insertions, deletions, or translocations, you will wish to use a fragmentation method that produces long DNA sequences, to allow for potentially large distances between your target sequence and the mutation point. If you wish to sequence very specific loci or an entire genome, smaller fragments to suit the library size of your sequencing instrument would be best. Fragmentation can be achieved by enzymatic digestion, or sonication. Both methods, or multiple rounds of the same method, may be used if further fragmentation after the initial fragmentation is desired, for example, when capturing large fragments to find translocation points followed by sonication of selected large fragments to obtain an appropriate size for the sequencer library creation kits. When using restriction enzymes for fragmentation, selection of an appropriate enzyme will depend upon the size of fragment desired [22]. In general, restriction enzymes with smaller recognition sequences will have more cutting sites in a target genome and produce smaller fragments, while larger recognition sites will produce larger fragments. Selection of restriction enzyme should also take into consideration any star activity, altered cutting of methylated DNA, capacity for heat inactivation, and the kinds of cut it

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Fig. 3 Gel tanks. Multiple sizes of tanks for agarose gel electrophoresis. Electrophoresis provides the capacity to visualize size differentiated DNA fragments. While not an explicit part of this protocol, electrophoresis can be useful to optimize the amount of fragmentation required for your input DNA by comparing the effects of different enzymes and/or sonication protocols

performs. Blunt and overhanging cuts require different strategies for attaching library adaptors if you are doing that directly after fragmentation, so bear this in mind. When performing sonication, the precise number of cycles will determine the size of fragments produced, with more cycles producing smaller fragments, ranging from kilobases down to ~150 bp. The exact number of cycles used will need to be determined for your instrument, its power settings (if any), the sonication tubes used, and volume of sample, which can be accomplished by using identical aliquots, removing them at staged points and checking the output DNA using an agarose gel [23] (Fig. 3). 3.1.1 Enzyme-Based DNA Fragmentation

1. Place reagents on ice for the duration of reaction setup; only permit sufficient thawing to allow pipetting. 2. Pipette reagents for all reactions, excepting template DNA, into a sterile capped tube, in sufficient quantities to produce a mastermix that will result in the desired final concentration in individual reaction tubes. For an example on starting concentration, see Table 1. 3. Ensure enough mastermix is produced to allow for all reactions to be pipetted, plus a safety margin of additional reactions to account for pipette calibration error. (see Note 2) 4. Seal mastermix tube, and place on ice until later. 5. Setup rack for reaction tubes, either 0.5 mL or 0.2 mL PCR tubes as desired, and place tubes or plate within the rack. 6. Pipette template DNA and into reaction tubes. 7. Seal reaction tubes, and vortex and spin briefly. Ensure all reagents are at the bottom of each reaction tube.

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Table 1 Digestion initial setup Reagent

Stock concentration Concentration for individual PCR solution Example volume 20 U/rxn

1 μL

Digestion buffer 10X

1X

1 μL

DNA

16 ng/μL

8 μL

EcoRI

a

20,000 U/mLa

20 ng/μL

a

Example enzyme only

Table 2 Thermal conditions initial setup Stage

Time

Temperature

Digestion

8–16 h

37 ºCa

Heat inactivation

20 min

65 ºCa

a

For EcoRI as example enzyme

8. Place sealed tubes in a waterbath or PCR machine, and set the instrument for the restriction temperature of the chosen enzyme. See Table 2 for an example on thermal protocol. 9. Once sufficient time for digestion has occurred, heat inactivate the enzyme to prevent interference with downstream steps (Table 2). 10. If restriction enzyme might interfere with later library preparation steps, ethanol precipitation as in Subheading 3.1.1, step 2 can be performed to purify sample. Optional Ethanol Precipitation 1. To the entire volume of digested DNA, add 2 volumes of 100% ethanol, chilled to 20  C. 2. Add 3M sodium acetate to 3% of the total volume. 3. Invert several times to mix. 4. Freeze in liquid nitrogen, dipping the containing tube into the liquid nitrogen vertically, so that sample freezes at the bottom of the tube. 5. Remove sample from liquid nitrogen, and centrifuge at 14–16,000 RCF for 15 min (see Note 3). 6. Remove supernatant by gently pipetting on the tube wall opposite from the DNA pellet. 7. Resuspend pellet in equivalent volume of water to initial sample size. 8. Incubate overnight in a 37  C waterbath or incubator. 9. DNA can now be quantitated for later steps.

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1. Prepare a ~100 μL volume of starting DNA at ~20 ng/μL in Low TE buffer. (see Note 4). 2. Add the DNA to a sonication tube suitable for your instrument. 3. Place DNA on ice for at least 10 min. 4. Ensure sterile, deionized water in sonication bath is at ~4  C. Ensure enough water is on hand to replace water in bath every few cycles (if no active cooling on instrument). 5. Load samples into sonicator, or place sonication probe into sample, and submerge sample container in waterbath (see Note 5). 6. Subject sample to cycles of 15 s or 30s of sonication, followed by 30s of rest in between. Ensure water in the waterbath remains cold, changing as necessary. 7. Total number of cycles needed will depend on the size of fragment desired and must be determined for concentration and volume of sample used (see Note 6).

3.2

Capture

1. Take 100 ng of fragmented DNA, and mix with 100 pmol of the desired biotinylated probe(s) along with 60 μL of SSC buffer in a total volume of 100 μL, diluting with water as needed. 2. Heat reaction mix at 95  C for 10 min. 3. Allow reaction to cool to hybridization temperature for probes (see Note 7). 4. Incubate reaction for 1 h at the hybridization temperature. 5. In a separate tube, aliquot 100 μg of streptavidin conjugated magnetic beads. Add 100 μl of 6x SSC buffer and vortex. 6. Apply the tube containing the beads to the magnetic separation rack for 30 s to 1 min. Remove and discard the supernatant while the tube is still in the rack. 7. Repeat steps 5 and 6 twice more (three washes total). 8. Resuspend the magnetic beads in 100 μl of 6x SSC buffer and vortex. 9. Add suspended beads to Probe/DNA hybridised tube. 10. Incubate at room temperature for at least 10 min with occasional gentle mixing. 11. Apply the tube to the magnetic separation rack for 30 s to 1 min. Remove and discard the supernatant while the tube is still in the rack. 12. Add 200 μl of 4x SSC buffer and mix gently. 13. Apply the tube to the magnetic separation rack for 30 s to 1 min. Remove and discard the supernatant while the tube is still in the rack.

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14. Repeat steps 12 and 13. 15. Add 200 μl of 2x SSC buffer and mix gently. 16. Apply the tube containing the beads to the magnetic separation rack for 30 s to 1 min. Remove and discard the supernatant while the tube is still in the rack. 17. Repeat steps 15 and 16. 18. Add 50 μl of water to resuspend beads/DNA and mix gently. 19. Incubate tubes at the melting temperature of the probe(s) for 5–10 min (see Note 8). 20. Without allowing tubes to cool, apply the tube to the magnetic separation rack for 30 s. 21. Remove supernatant containing unbound target DNA, and add it to a new tube for retention. 22. DNA is now ready for amplification. 3.3 Template Amplification

Following capture, target DNA will consist of single-stranded fragments which need to be rendered double stranded and amplified for use in most sequencing libraries. With that said, from here, you may apply alternate steps to generate your library if your chosen system can work directly with single-stranded templates of low concentration. Following this amplification step, your PCR products may have overhangs that need to be removed for the generation of your sequencing libraries. Consult your library kit instructions to determine whether this is the case or not. 1. Prepare a location to set up the PCR. 2. Place reagents on ice for the duration of PCR setup; only permit sufficient thawing to allow pipetting. 3. Pipette reagents for all reactions, excepting template DNA, into a sterile capped tube, in sufficient quantities to produce a mastermix that will result in the desired final concentration in individual PCR tubes. For starting concentrations, see Table 3 (see Note 9). 4. Ensure enough mastermix is produced to allow for all reactions to be pipetted, plus a safety margin of additional reactions to account for pipette calibration error (see Note 10). 5. Seal mastermix tube, and place on ice until later. 6. Set up rack for PCR tubes or plates as desired, and place tubes or plate within the rack. 7. Pipette template captured DNA and negative controls into PCR tubes/rack. 8. Pipette mastermix captured DNA.

into

tubes

containing

template

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Table 3 PCR initial setup Reagent

Stock concentration

Concentration for individual PCR solution

MgCl

25 mM

3 mM

PCR buffer

10X

1X

Universal primer 5 mM

0.4 μM

dNTPs

10 mM (each)

0.5 mM (each)

Taq polymerase

5 U/μL

2.5 U/rxn

Sterile water

~

~

DNA

Variable depending on capture efficiency ~10 ng

Table 4 Thermal conditions initial setup Stage

Time

Temperature

Number of repeats

Initial denaturation

1 min

95  C

1x

Denaturation Primer annealing Ramping Extension

1 min 1 min 0.3  C/second 3 min

95  C 30  C 30  C–72  C 72  C

6x

Denaturation Primer annealing Extension

30 s 1 min 2 min

95  C 56 ºCb 72  C

35x

Final extension

5 min

72  C

1x

a

a

Initial denaturation may need to be 5 min, if heat activated polymerase is used Optimal temperature for universal primer annealing in the second phase will depend on the melting temperature of the 5’ sequence used. Value given is for the primer used by Telenius et al. Adjust as required b

9. Seal PCR tubes/rack. Ensure all reagents are at the bottom of each reaction tube. 10. Place sealed tubes/rack in PCR machine, and commence the thermal amplification steps. See Table 4 for an example on thermal protocol.

4

Notes 1. The universal degenerate oligonucleotide primer consists of sequence tagged on the 5’ and 3’ends of random hexamers. The original form used by Telenius et al. is CCGACTC GAGNNNNNNATGTGG , but alterations to the sequence, such as the GTGAGTGATGGTAGTGTGGAGNNNNN NATGTGG used by Blagodatskikh et al., are possible

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[24, 25]. The 3’ sequence used is fairly standard, but the 5’ sequence can be altered for downstream uses such as appending your sequencing method’s adaptor key sequences to eliminate the need to ligate them on later or introducing restriction sites for later insertion into in plasmids. 2. Additional reactions should be assumed when creating a mastermix for a batch, to avoid there being insufficient mastermix available for all intended reactions due to pipetting error and instrument calibration variation. The number of additional reactions to make up will depend on personal comfort, budget, and prior experience of mastermix shortfall, but somewhere between two and five additional reactions per batch is useful as a starting point. 3. When loading samples for centrifugation, it is a good idea to orient all tubes in the same manner, for example, lid hinge facing outward, or label facing outward. This will ensure that the DNA pellet precipitates in the same place in all tubes and help minimize any DNA loss if the pellet is not visible while pipetting away the supernatant. 4. Different starting volumes of DNA will shear at different rates. Larger volumes will tend to shear more slowly. Thus, starting volumes should be consistent to ensure replicable results. 5. Sonicators may require the insertion of a probe into the sample, while others utilize sealed, special vials. Sealed vial-type sonicators are greatly preferred for molecular biology to limit contamination, but if a direct probe instrument must be used, extreme care to decontaminate probes should be taken between samples. 6. Generally, 5 cycles at 30s will produce fragments in the 400–600 bp range. 7. Hybridization temperature should be ~5  C colder than the calculated melting temperature for the probe used. If using multiple probes, the temperature used should be as close to suitable for as many probes as possible, so designing probes to have similar melting temperatures is advisable. 8. When melting probes from DNA, ensure that the temperature used is at least as high as the hottest melting temperature of all the probes used. Increasing this temperature by 1–2  C will help ensure better separation of probe bound beads from target DNA. 9. Increasing the availability of polymerase, universal primer, and dNTPs in the reaction can allow amplification of potentially rare sequences in the captured DNA. You may need to adjust the initial protocol depending on the potential for rare mutations in your pool of cells.

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10. Additional reactions should be assumed when creating a mastermix for a batch, to allow for error in pipetting and instrument calibration. Failure to include spare capacity can leave a shortfall in final reaction volumes in one or more reactions, which may affect outcomes. References 1. Pakneshan S, Salajegheh A, Smith RA, Lam AK (2013) Clinicopathological relevance of BRAF mutations in human cancer. Pathology 45: 346–356. https://doi.org/10.1097/PAT. 0b013e328360b61d. PMID: 23594689 2. Ng JY, Lu CT, Lam AK (2019) BRAF mutation: current and future clinical pathological applications in colorectal carcinoma. Histol Histopathol 34:469–477. https://doi.org/ 10.14670/HH-18-079. Epub 2018 Dec 28. PMID: 30592501 3. Smith RA, Salajegheh A, Weinstein S, Nassiri M, Lam AK (2011) Correlation between BRAF mutation and the clinicopathological parameters in papillary thyroid carcinoma with particular reference to follicular variant. Hum Pathol 42:500–506. https:// doi.org/10.1016/j.humpath.2009.09. 023. PMID:21167555 4. Pillai S, Gopalan V, Smith RA, Lam AK (2015) Diffuse sclerosing variant of papillary thyroid carcinoma--an update of its clinicopathological features and molecular biology. Crit Rev Oncol Hematol 94:64–73. https://doi.org/10. 1016/j.critrevonc.2014.12.001. PMID: 25577570 5. Liu X, Qu S, Liu R, Sheng C, Shi X, Zhu G, Murugan AK, Guan H, Yu H, Wang Y, Sun H, Shan Z, Teng W, Xing M (2014) TERT promoter mutations and their association with BRAF V600E mutation and aggressive clinicopathological characteristics of thyroid cancer. J Clin Endocrinol Metab 99:E1130–E1136. h t t p s : // d o i . o r g / 1 0 . 1 2 1 0 / j c . 2013-4048. PMID: 24617711 6. Rahman MA, Salajegheh A, Smith RA, Lam AK (2014) BRAF inhibitor therapy for melanoma, thyroid and colorectal cancers: development of resistance and future prospects. Curr Cancer Drug Targets 14:128–143. https://doi.org/ 1 0 . 2 1 7 4 / 1568009614666140121150930. PMID: 24446739 7. Rahman MA, Salajegheh A, Smith RA, Lam AK (2015) Multiple proliferation-survival signalling pathways are simultaneously active in BRAF V600E mutated thyroid carcinomas. Exp Mol Pathol 99:492–497. https://doi.

org/10.1016/j.yexmp.2015.09.006. PMID: 26403329 8. Rahman MA, Salajegheh A, Smith RA, Lam AK (2015) MicroRNA-126 suppresses proliferation of undifferentiated (BRAF(V600E) and BRAF(WT)) thyroid carcinoma through targeting PIK3R2 gene and repressing PI3KAKT proliferation-survival signalling pathway. Exp Cell Res 339:342–350. https://doi.org/ 10.1016/j.yexcr.2015.09.010. PMID: 26384552 9. Rahman MA, Salajegheh A, Smith RA, Lam AK (2016) Inhibition of BRAF kinase suppresses cellular proliferation, but not enough for complete growth arrest in BRAF V600E mutated papillary and undifferentiated thyroid carcinomas. Endocrine 54:129–138. https://doi.org/ 1 0 . 1 0 0 7 / s 1 2 0 2 0 - 0 1 6 0985-7. PMID:27179656 10. Xing M, Alzahrani AS, Carson KA, Viola D, Elisei R, Bendlova B, Yip L, Mian C, Vianello F, Tuttle RM, Robenshtok E, Fagin JA, Puxeddu E, Fugazzola L, Czarniecka A, Jarzab B, O’Neill CJ, Sywak MS, Lam AK, Riesco-Eizaguirre G, Santisteban P, Nakayama H, Tufano RP, Pai SI, Zeiger MA, Westra WH, Clark DP, Clifton-Bligh R, Sidransky D, Ladenson PW, Sykorova V (2013) Association between BRAF V600E mutation and mortality in patients with papillary thyroid cancer. JAMA 309:1493–1501. h t t p s : // d o i . o r g / 1 0 . 1 0 0 1 / j a m a . 2 0 1 3 . 3190. PMID: 23571588 11. Xing M, Alzahrani AS, Carson KA, Shong YK, Kim TY, Viola D, Elisei R, Bendlová B, Yip L, Mian C, Vianello F, Tuttle RM, Robenshtok E, Fagin JA, Puxeddu E, Fugazzola L, Czarniecka A, Jarzab B, O’Neill CJ, Sywak MS, Lam AK, Riesco-Eizaguirre G, Santisteban P, Nakayama H, Clifton-Bligh R, Tallini G, Holt EH, Sy´korová V (2015) Association between BRAF V600E mutation and recurrence of papillary thyroid cancer. J Clin Oncol 33:42–50. https://doi.org/10.1200/ JCO.2014.56.8253. Epub 2014 Oct 20. PMID: 25332244 12. Shen X, Zhu G, Liu R, Viola D, Elisei R, Puxeddu E, Fugazzola L, Colombo C,

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Jarzab B, Czarniecka A, Lam AK, Mian C, Vianello F, Yip L, Riesco-Eizaguirre G, Santisteban P, O’Neill CJ, Sywak MS, CliftonBligh R, Bendlova B, Sy´korová V, Xing M (2018) Patient age-associated mortality risk is differentiated by BRAF V600E status in papillary thyroid cancer. J Clin Oncol 36(5): 438–445. https://doi.org/10.1200/JCO. 2017.74.5497. Epub 2017 Dec 14. PMID: 29240540 13. Wang F, Zhao S, Shen X, Zhu G, Liu R, Viola D, Elisei R, Puxeddu E, Fugazzola L, Colombo C, Jarzab B, Czarniecka A, Lam AK, Mian C, Vianello F, Yip L, RiescoEizaguirre G, Santisteban P, O’Neill CJ, Sywak MS, Clifton-Bligh R, Bendlova B, Sy´korová V, Wang Y, Xing M (2018) BRAF V600E confers male sex disease-specific mortality risk in patients with papillary thyroid cancer. J Clin Oncol 36:2787–2795. https://doi. org/10.1200/JCO.2018.78.5097. PMID: 30070937 14. Tao Y, Wang F, Shen X, Zhu G, Liu R, Viola D, Elisei R, Puxeddu E, Fugazzola L, Colombo C, Jarzab B, Czarniecka A, Lam AK, Mian C, Vianello F, Yip L, Riesco-Eizaguirre G, Santisteban P, O’Neill CJ, Sywak MS, CliftonBligh R, Bendlova B, Sy´korová V, Zhao S, Wang Y, Xing M (2021) BRAF V600E status sharply differentiates lymph node metastasisassociated mortality risk in papillary thyroid cancer. J Clin Endocrinol Metab 106:3228– 3238. https://doi.org/10.1210/clinem/ dgab286. PMID: 34273152 15. Huang Y, Qu S, Zhu G, Wang F, Liu R, Shen X, Viola D, Elisei R, Puxeddu E, Fugazzola L, Colombo C, Jarzab B, Czarniecka A, Lam AK, Mian C, Vianello F, Yip L, Riesco-Eizaguirre G, Santisteban P, O’Neill CJ, Sywak MS, Clifton-Bligh R, Bendlova B, Sy´korová V, Xing M (2018) BRAF V600E mutation-assisted risk stratification of solitary intrathyroidal papillary thyroid cancer for precision treatment. J Natl Cancer Inst 110(4):362–370. https://doi.org/10. 1093/jnci/djx227. PMID: 29165667 16. Kim KJ, Kim SG, Tan J, Shen X, Viola D, Elisei R, Puxeddu E, Fugazzola L, Colombo C, Jarzab B, Czarniecka A, Lam AK, Mian C, Vianello F, Yip L, RiescoEizaguirre G, Santisteban P, O’Neill CJ, Sywak MS, Clifton-Bligh R, Bendlova B, Sy´korová V, Xing M (2020) BRAF V600E status may facilitate decision-making on active surveillance of low-risk papillary thyroid microcarcinoma. Eur J Cancer 124:161–169. https://doi.org/10.1016/j.ejca.2019.10. 017. PMID:31790974

17. Frisone D, Friedlaender A, Malapelle U, Banna G, Addeo A (2020) A BRAF new world. Crit Rev Oncol Hematol 152:103008. https://doi.org/10.1016/j.critrevonc.2020. 103008. PMID: 32485528 18. Barbitoff YA, Polev DE, Glotov AS, Serebryakova EA, Shcherbakova IV, Kiselev AM, Kostareva AA, Glotov OS, Predeus AV (2020) Systematic dissection of biases in wholeexome and whole-genome sequencing reveals major determinants of coding sequence coverage. Sci Rep 10:2057. https://doi.org/10. 1038/s41598-020-59026-y. PMID: 32029882 19. Jensen MR, Sigsgaard EE, Liu S, Manica A, Bach SS, Hansen MM, Møller PR, Thomsen PF (2021) Genome-scale target capture of mitochondrial and nuclear environmental DNA from water samples. Mol Ecol Resour 21:690–702. https://doi.org/10.1111/ 1755-0998.13293 20. Demidov VV, Bukanov NO, FrankKamenetskii D (2000) Duplex DNA capture. Curr Issues Mol Biol 2:31–35. PMID: 11464918 21. St John J, Quinn TW (2008) Rapid capture of DNA targets. BioTechniques 44:259–264. h t t p s : // d o i . o r g / 1 0 . 2 1 4 4 / 000112633. PMID: 18330355 22. Zhang X, Garnerone S, Simonetti M, Harbers L, Nicos´ M, Mirzazadeh R, Venesio T, Sapino A, Hartman J, Marchio` C, Bienko M, Crosetto N (2019) CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples. Nat Commun 10:4732. https://doi.org/ 10.1038/s41467-019-12570-2. PMID: 31628304 23. Sambrook J, Russell DW (2006) Fragmentation of DNA by sonication. CSH Protoc 2006: pdb.prot4538. https://doi.org/10.1101/ pdb.prot4538. PMID: 22485919 24. Telenius H, Carter NP, Bebb CE, Nordenskjo¨ld M, Ponder BA, Tunnacliffe A (1992) Degenerate oligonucleotide-primedPCR: general amplification of target DNA by a single degenerate primer. Genomics 13:718– 725. https://doi.org/10.1016/0888-7543 (92)90147-k. PMID: 1639399 25. Blagodatskikh KA, Kramarov VM, Barsova EV, Garkovenko AV, Shcherbo DS, Shelenkov AA, Ustinova VV, Tokarenko MR, Baker SC, Kramarova TV, Ignatov KB (2017) Improved DOP-PCR (iDOP-PCR): a robust and simple WGA method for efficient amplification of low copy number genomic DNA. PLoS One 12: e0184507. https://doi.org/10.1371/journal. pone.0184507. PMID: 28892497

Chapter 13 Application of Immunohistochemistry in Papillary Thyroid Carcinoma Alfred K. Lam and Katherine Ting-Wei Lee Abstract Immunohistochemistry (IHC) is an economic and precise method to localize the presence of specific protein at cellular level in tissue. Although many papillary thyroid carcinomas do not require IHC to render a diagnosis, there are certain scenarios in which IHC are important. The major diagnostic applications of IHC include confirmation of papillary thyroid carcinoma in sites other than the thyroid, distinguish papillary thyroid carcinoma from other primary thyroid neoplasms in thyroid, and identify papillary thyroid carcinoma from secondary tumors to the thyroid. At research level, IHC could help identify prognostic information, identify underlying genetic alterations, and predict response to treatment in papillary thyroid carcinoma. The understanding of principle and recent advances in IHC will improve the diagnosis and management of patients with thyroid lesions including papillary thyroid carcinoma. Key words Immunohistochemistry, Papillary thyroid carcinoma, Diagnosis, Review, Differential diagnoses

1

Introduction Papillary thyroid carcinoma often shows classic histological features in excision specimen. However, in some instances, immunohistochemistry (IHC) is essential for diagnosis, patients’ management, and research purposes. IHC, different from other more expansive molecular tests, is cheaper and easy to work on most clinical samples and has an advantage of being able to locate the target protein at the cellular location in carcinoma. The major applications of IHC in papillary thyroid carcinoma and related thyroid lesions are summarized below.

Alfred K. Lam and Katherine Ting-Wei Lee contributed equally with all other contributors. Alfred K. Lam (ed.), Papillary Thyroid Carcinoma: Methods and Protocols, Methods in Molecular Biology, vol. 2534, https://doi.org/10.1007/978-1-0716-2505-7_13, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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1.1 Confirmation of Papillary Thyroid Carcinoma in Sites Other s the Thyroid

One of the most common applications of IHC is to identify the origin of a metastatic cancer. It is important especially in small biopsy specimen in which the classical morphological features of different tumors may not be obvious. There are a few IHC makers, thyroglobulin, TTF-1, and PAX-8, frequently being employed in this context for identifying thyroid cancer as the origin of metastatic carcinoma or thyroid carcinoma from ectopic thyroid tissue. Thyroglobulin in the most specific marker for carcinomas originates from the thyroid gland. It is the ideal marker for identifying thyroidal origin of a metastatic carcinoma [1]. However, it shows cytoplasmic staining, not so sensitive and sometimes loss in highgrade thyroid carcinomas and necrotic lesions [2]. Thyroid transcription factor (TTF-1) is used more often in pathology practice for identifying primary differentiated thyroid carcinomas as well as lung carcinomas [3]. It has the advantage of a being a sensitive (strong) and easily located nuclear staining (Fig. 1). Nevertheless, TTF-1 also express, sometimes with focal staining, in many carcinomas [4]. Thus, positivity to TTF-1 stain should be interpreted in appropriate clinicopathological context.

Fig. 1 Papillary thyroid carcinoma metastasizes to lymph node (top). TTF-1 nuclear stain highlights the carcinoma (bottom). Lymphoid tissue in the lymph node is negative (blue on counterstain) for TTF-1

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Paired-box gene 8 (PAX-8) is a paired-box gene that plays an important role in thyroid, kidney, and Mu¨llerian tract development [5]. Nuclear positivity to PAX-8 could be used to support the origin of cancer from thyroid. However, renal cell tumors, Mullerian (female genital tract) tumors, and thymic tumors are also positive for PAX-8. In addition, a minority of lung carcinomas show low expression of PAX-8 [6]. PAX-8 could be positive in some highgrade follicular-derived thyroid carcinoma when reactive to thyroglobulin and TTF-1 are lost. Overall, combination of markers is important as sometimes unusual expression pattern may occur. A papillary thyroid carcinoma metastasis in the bone has been labelled as lung carcinoma because of positivity to cytokeratin 7, napsin-A, and TTF-1 [7]. It is worth noting that although napsin A (functional aspartic proteinase that is often positive in lung adenocarcinoma) expression is common in lung adenocarcinoma, it could also be expressed in close to one fourth of metastatic thyroid carcinoma [8]. Further stain with thyroglobulin and PAX-8 could confirm the origin from the thyroid. Papillary thyroid carcinoma may occur in thyroid tissue in locations other than the thyroid gland such as in struma ovarii [9]. In usual site, identification of papillary thyroid carcinoma with thyroglobulin, TTF-1, and cytokeratin 19 (CK19) is useful [9, 10]. 1.2 Distinguishing Papillary Thyroid Carcinoma from Other Primary Thyroid Neoplasms in the Thyroid

Although papillary thyroid carcinoma is the most common malignant neoplasm in the thyroid, there are other benign and malignant thyroid lesions in the thyroid which may sometimes mimic papillary thyroid carcinoma and need to be differentiated by IHC. Strong positivity to CK19 and HBME1 (marker of mesothelial cells but positive in thyroid carcinomas) and loss of CD56 are commonly employed panel of markers to distinguish follicular variant of papillary thyroid carcinoma from mimics like follicular adenoma and adenomatous nodule [11–13] (Figs. 2 and 3). CK19 is sensitive, and taking into consideration the intensity and proportion of positive staining in association with morphology is useful in differential diagnosis of benign follicular lesions from follicular tumors with papillary thyroid carcinoma nuclear features [14, 15]. With the changes in the World Health Organization (WHO) classification of thyroid tumors [16], these stains are mainly used to detect noninvasive follicular thyroid neoplasm with papillary-like features (NIFTP) (previously labelled as encapsulated follicular variant of papillary thyroid carcinoma) or invasive encapsulated follicular variant papillary carcinoma from benign follicular thyroid lesions [17]. Galectin 3 (belong to a family of carbohydrate-binding proteins with multiple physiological functions) and TROP-2 (tumor-associated calcium signal transducer 2) are less commonly used but also noted to have high specificity to papillary thyroid carcinoma [18–

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Fig. 2 Papillary thyroid carcinoma on hematoxylin-eosin stain (right). The carcinoma region is highlighted by strong brown cytoplasmic staining of HBME1 and CK19. Note that only carcinoma is positive for HBME1, whereas the non-neoplastic thyroid follicles are negative for the stain (blue on counterstain). For CK19, papillary thyroid carcinoma is strongly positive, whereas non-neoplastic thyroid follicles are negative or weakly positive

Fig. 3 Papillary thyroid carcinoma on hematoxylin-eosin stain (right). The carcinoma region is highlighted by loss of staining to CD56 (blue on counterstain) and strong brown cytoplasmic staining of CK19. Note that the non-neoplastic thyroid follicles show strong positive brown cytoplasmic staining to CD56

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Fig. 4 Positive cytoplasmic staining (brown) of BRAF protein in papillary thyroid carcinoma. The non-neoplastic thyroid follicles are negative (blue on counterstain)

20]. Co-expressions of galactin-3, HBME1, and TROP-2 are consistent in differentiating the invasive follicular papillary thyroid carcinoma from benign follicular lesions or NIFTP [21]. In addition, in core needle biopsy [22], galectin-3 and CK19 showed higher specificity and expressed mainly in classical papillary carcinoma, while HBME-1 showed higher sensitivity for the diagnosis of carcinoma with expression in both conventional type and follicular variant papillary thyroid carcinoma. BRAF staining occurs mostly in classical subtype papillary thyroid carcinoma (Fig. 4) and is useful to exclude the diagnosis of NIFTP in cases with questionable papillae or other features that are more frequently seen in classical papillary thyroid carcinoma such as pseudo-inclusions or high-grade nuclear features. Medullary thyroid carcinoma (carcinoma of C-cell differentiation in the thyroid as distinguished from other thyroid carcinomas often of follicular cell differentiation) could mimic follicularderived thyroid carcinoma such as papillary thyroid carcinoma. Rarely, it can occur in combination with papillary thyroid carcinoma in the same patient [23]. Like papillary thyroid carcinoma, it expresses TTF-1 and PAX-8. Also, medullary carcinoma could stain up (non-specificity) for thyroglobulin [1]. For any tumor located within the thyroid, false positivity can occur because of tissue contamination by thyroglobulin-rich thyroid colloid from adjacent thyroid normal tissue [1]. On the other hand, it is positive for calcitonin (hormone secreted by C-cells in thyroid) (Fig. 5) and neuroendocrine marker such as chromogranin. Awareness of the possibility of the diagnosis and correct use of IHC could identify the carcinoma with ease. Papillary thyroid carcinoma could dedifferentiate to anaplastic thyroid carcinoma [24, 25]. IHC could help identify foci of transformation to anaplastic thyroid carcinoma by high Ki-67 index, p53 overexpression, as well as low or loss of expression of keratin,

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Fig. 5 Positive cytoplasmic staining (brown) of calcitonin in medullary thyroid carcinoma. The non-neoplastic thyroid follicles are negative (blue on counterstain)

PAX-8, and TTF-1 [26]. There is also increased expression of smooth muscle actin in spindle (sarcomatoid) area and abnormal expression of p16 [27]. Papillary thyroid carcinoma could have squamous metaplasia which can be highlighted by p63, p40, and CK5/6. It is worth noting that anaplastic thyroid carcinoma could be predominately squamous differentiation, labelled previously as squamous cell carcinoma [28, 29]. The anaplastic differentiation could be detected by morphological examination as well as confirmed by high proliferative index, as detected by Ki-67 on IHC. Cribriform-morular thyroid carcinoma, formally a variant of papillary thyroid carcinoma and now classified as a type of tumor of uncertain histogenesis, is a tumor associated with familial adenomatosis polyposis and occurs exclusively in young women [30]. The carcinoma is characterized by nuclear and cytoplasmic staining of beta-catenin which is different from weak membranous staining in papillary thyroid carcinoma or hyalinizing trabecular tumor (histological mimicker of cribriform-morular carcinoma) [31]. Secretory carcinoma of the thyroid gland is a salivary glandtype carcinoma which is rare and with specific arrangement of ETV6 gene [32]. The carcinoma has follicular-like microcystic pattern with colloid-like secretions, and papillary carcinoma could mimic papillary thyroid carcinoma. Positive staining for S100 and mammaglobin as well as negative for TTF-1 and thyroglobulin should be able to confirm the diagnosis [33]. 1.3 Distinguish Papillary Thyroid Carcinoma from Secondary Tumors in the Thyroid

Common tumors which metastasize to the thyroid include renal cell carcinoma, lung carcinoma, breast carcinoma, gastric carcinoma, and melanoma as well as direct infiltration from head/neck and esophageal carcinomas [34–36]. It is often difficult to distinguish papillary thyroid carcinoma from these secondary metastases

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on core biopsy or fine-needle aspiration biopsy. Most often, the diagnosis is based on clinical suspicious and confirmation or exclusion with IHC. In renal cell carcinoma (the most common clear cell subtype), the clear cell morphology and the vascular networks are very characteristics and could be easy to be identified by pathologist even at metastatic sites. IHC findings of positivity to makers often used to identify renal cell carcinoma such as CD10, carbonic anhydrase IX (CAIX), and negativity to thyroid follicular marker (e.g., TTF-1) will confirm the diagnosis of metastatic renal cell carcinoma. Head/neck carcinomas and some esophageal carcinomas are often squamous cell carcinoma. The squamous differentiation could be identified by positivity to p63, p40, and CK5/6 as well as the typical squamous morphology on histology. It is worth noting that papillary thyroid carcinoma could show squamous metaplasia as well as dedifferentiation to anaplastic thyroid carcinoma with squamous differentiation. In these circumstances, PAX-8 and TTF-1 which could be positive in anaplastic squamous cell carcinoma and often negative in head and neck (including esophageal) squamous cell carcinoma could be employed [28]. Metastatic melanoma is a known mimicker of other cancers on histopathology examination. On the other hand, melanoma is easily identified by melanocytic makers like positive to SOX-10 (nuclear transcription factor in the differentiation of neural crest progenitor cells to melanocytes) and Melan A (also known as MART-1, melanoma-specific antigen which a transmembrane protein). Gastric/ esophageal adenocarcinoma [37] and breast carcinoma [38] are the other adenocarcinomas that could be distinguished from papillary thyroid carcinoma by IHC. Gastric/esophageal adenocarcinoma are often positive for CDX2 (intestine-specific transcription factor expressed in the nuclei of epithelial cells throughout the intestine), whereas breast carcinomas are positive for GATA-3 (GATA binding protein 3, a transcription factor important in the differentiation of breast epithelia, urothelial, and subsets of lymphocytes). Lung carcinomas, especially adenocarcinoma, are one of the commonest types of cancer metastasis to the thyroid. It is worth noting the TTF-1 and napsin A could be positive in lung adenocarcinoma and thyroid carcinoma. Thus, correlations with clinical features and further stains such as PAX-8 and thyroglobulin to exclude papillary thyroid carcinoma in the setting are required. In fine-needle aspiration specimen, there may be some instances where differentiation of a thyroid lesion from parathyroid lesion such as parathyroid adenoma is needed. If cell block is available, parathyroid adenoma is positive for GATA-3 and parathyroid hormone (PTH), whereas thyroid lesions are positive for TTF-1 and thyroglobulin [39].

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1.4 Research Studies on the Pathogenesis and Predictive Value of Markers in Papillary Thyroid Carcinoma

Some immunohistochemical markers, such as BRAF, galectin, and TROP-2, currently used for pathological diagnosis of papillary thyroid carcinoma could have additional applications. BRAF mutation is the key player in the pathogenesis of papillary thyroid carcinoma. The mutation is also related to clinicopathological parameters [40, 41] as well as having predictive values in cancer recurrence and patient mortality [42–46]. In recent years, BRAF protein could be detected by immunohistochemistry and appears to be an adjunct tool for detection of BRAF mutation in papillary thyroid carcinoma [47–49]. The sensitivity and specificity as detected by immunohistochemical method is comparable to those by direct sequencing or polymerase chain reaction [50, 51]. IHC for BRAF protein, which is cheaper to use than PCR or sequencing, could be used as a first-line predictive marker in the management of patients with papillary thyroid carcinoma. Galectin 3 could be detected by IHC in papillary thyroid carcinoma, especially those carcinomas with lymph node metastases [18]. It could be detected in the serum of patients with papillary thyroid carcinoma and could be a marker for molecular imaging and progression in cancer [52]. TROP-2 has prognostic value in many cancers, in particular breast cancer and gynecologic cancers, as well as in papillary thyroid carcinoma [53]. P53 overexpression is uncommon in classical papillary thyroid carcinoma but noted in carcinoma with aggressive phenotype [54] and as predictors of regional lymph node recurrence [55]. Vascular endothelial growth factor (VEGF) is an indicator of tumor angiogenesis and is a target for angiogenesis inhibitors in cancer therapy. In papillary thyroid carcinoma, VEGF is shown to be involved in the pathogenesis, interactions with other pathogenetic mechanisms and aggressive biological behaviors, and potential target for therapy [56–62]. Expressions of VEGF proteins (VEGFA, VEGF-C) were used in the analysis [62–64].

1.5 Principles of Working with IHC

The principle of IHC is to detect the protein of interest (antigen) by antibody to the protein using immunological and chemical reaction. The processes involve preparation of the tissue for optimization of IHC. These include fixation and embedding the tissue, cutting and mounting of section, deparaffinizing and rehydrating the section, retrieval of the antigen, incubation of antibody and antigen, amplification step (secondary antibody), and staining by chromogens, for visual identification [65]. Nowadays, all these steps are done automatically by machine (autoimmunostainer). The use of autoimmunostainer reduces the manpower required in doing IHC and allows multiple antibodies to be done at the same time. In research situations working on only a few tissue sections, it may be simpler to use the traditional manual approach. IHC is mostly done on sections from paraffin-embedded histological

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sections. In some instances, IHC is required on cells in solution. These often occur in clinical situations for thyroid lesions; many of the specimens come from fine-needle aspiration. Also, cancer cells in solution are used in research, such as in cell culture or from blood (circulating tumor cells). In these situations, it is important to convert the cells of interest in solution to cell block. IHC could be done on these cell blocks [66]. IHC is a routine test done in pathology laboratory. In pathology practice, IHC proficiency programs in quality assurance programs are popular and provide pathology laboratories with an international performance analysis and continued professional development. In this chapter, the step-by-step manual approach of working on immunohistochemical staining is presented. The option of doing some steps with autoimmunostainer is highlighted. In addition, the protocols for making cell blocks from cells in solution for immunohistochemistry in additional to traditional paraffin blocks are also presented.

2

Materials

2.1 Protective Measures

2.2

Equipment

Appropriate personal protective equipment must be worn during experimental procedures as a safety measure against potential laboratory hazards. Follow all rules and regulations for waste disposal. 1. Microtome machine, tweezers (preferably with a curved tip for handing sections), small paintbrush, microtome blades (Caution! Blades are extremely sharp; handle with care), block of paraffin-embedded tissue, water bath (at 40–45  C), cold plate (at 20  C), positively charged coated adhesion glass slides, slide rack, and pencils are required for this section. 2. Centrifuge with centrifuge tubes and lens paper. 3. Tissue processing machine for making paraffin blocks of cancer tissue. 4. Autoimmunostainer (optional).

2.3

Raw Materials

Thyroid carcinoma in tissues or cells in solution.

2.4

Reagents

Dissolve all reagents in distilled water unless otherwise specified. If using autoimmunostainer, some of the regents are in the package and no need to obtain in separate. 1. Buffers – can be commercially obtained but may require dilution before use. These include: • 10% buffered formalin. • Phosphate-buffered saline (PBS) buffer. • Tween 20 (Polysorbate 20).

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• Tris-buffered saline, 0.1% Tween® 20 Detergent (TBST) buffer. • Copper sulfate or Scott’s solution (buffered reagent for the conversion of red hematoxylin-stained nuclei to blue, a solution which contains magnesium sulfate and sodium bicarbonate in optimal proportions in dilated in water). 2. Paraffin. 3. Plasma and thrombin (for precipitation of cells in solution). 4. Epitope retrieval solution (range of options or provided by commercial companies). 5. Xylene. 6. Ethanol (100%, 95%) diluted with double distilled water. 7. Desired primary antibody for thyroid tissue (e.g., TTF-1). 8. Secondary antibody (HRP – horseradish peroxidase) or polymers (for use in polymer-based detection method in autoimmunostainer). 9. DAB (3,3-diaminobenzidine) (as chromogen) and other buffers. 10. Mayer’s hematoxylin (as counterstain).

3 3.1

Methods Fixation

A. Tissue specimen (Fig. 6) 1. Immerse fresh thyroid specimen with carcinoma tissue into 10% buffered formalin for 18–24 hours (see Note 1) before embedding in paraffin at 58 to 60  C (see Note 2). B. Cell culture or cytology specimen 1. Resuspend thyroid carcinoma cells in PBS in a 15 ml tube to wash them. 2. Centrifuge at 100 g for 5 minutes to pellet the cells, and remove the PBS solution. Add a few drops of plasma (ensure the pellet of cells is thoroughly covered), and add equal drops of thrombin (i.e., if four drops of plasma were added, add four drops of thrombin). 3. Mix by tapping gently on the counter. 4. Allow mixture to stand until clot. 5. Flip the tube to pour the clot onto the lens paper. 6. Wrap the sample inside the lens paper, and process it as tissue specimen to embed it in paraffin (see Note 2).

3.2

Sectioning

1. Chill the paraffin-embedded tissue blocks on the cold plate for at least half an hour.

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Fig. 6 IHC of papillary thyroid carcinoma. (a) Paraffin block containing thyroid tissue with a papillary thyroid carcinoma (arrow). (b) Hematoxylin and eosinstained section showing the papillary thyroid carcinoma cut from block in (a) (arrow). (c) Section of paraffin block in (a) by IHC which shows papillary thyroid carcinoma (arrow) is positive (brown color) for BRAF protein

2. In the meantime, set the water bath containing distilled water to 40–45  C. 3. Place the blade in the holder of the microtome and secure it. Set the clearance angle (see Note 3). 4. Then, carefully approach the block with the blade and cut a few thin sections (approximately 3–4 μm) to ensure the positioning is correct. Adjust the block orientation as required. 5. Trim the block to expose the tissue surface to a level where a representative section can be sliced. During this stage, slicing can be done at a thickness of 10–30 μm. 6. Once tissue surface exposure level is satisfactory, slice the section with a thickness of about 3–4 μm (Fig. 7). 7. Using tweezers, pick up the ribbons of sections, and float them on the surface of the water in the hot water bath to allow them to flatten out (see Note 4). 8. Pick the sections out of the water bath using microscope slides, and store it upright in a slide rack until ready for immunohistochemical analysis. 3.3 Control Tissue (Fig. 8)

1. Multiple tissue control blocks are usually constructed. 2. Select the appropriate control(s) based on the antibody used (see Note 5).

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Fig. 7 Cut sections (with labels) of cancer tissues from the paraffin block on tray for IHC (ready to be inserted into autoimmunostainer for immunohistochemical reactions). Note that each section has control tissues sections (arrows) besides the cancer tissues for IHC (see Fig. 8)

Fig. 8 Strong TTF-1 staining (brown color) in both thyroid cancer and non-neoplastic thyroid tissue. Control tissues were shown on the left side which is composed of cocktails of tissues from different organs (appendix, liver, lung, and small intestine – from top to bottom). The lung tissue shows focal strong brown staining as positive control for TTF-1

3. Cut the tissue control blocks sections as in step 3.2, and put side by side with the papillary thyroid carcinoma tissue or cell block tissue on the same glass slide. 3.4

Tissue Labelling

1. Enter the case identification information on each slide with cut section of tissue and control tissue. This can be done through pre-entering the details (patient last name, case identification number, slide number, and number of slides) onto the computer system linked to autoimmunostainer which is then saved onto a hard disc and printed onto slide labels. Alternatively, in small research projects, enter the identification information manually on the slide.

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2. Apply the slide labels onto appropriate slides. 3. Clip the labelled slides onto the slide racks, and keep the slides in order (Fig. 7). 3.5 Pre-staining Sample Preparation (Dewax, Rehydration, and Antigen Retrieval)

1. Heat slides at 60–65  C for at least 2 hours to fix tissue onto slide. 2. Dewax slides by washing it in xylene 3 times, 5 minutes each (see Note 6). 3. Rehydrate slides by washing it with 100% ethanol twice, 5 minutes each, followed by 95% ethanol twice, 5 minutes each, and distilled water twice, 5 minutes each. 4. Dilute the epitope retrieval buffer by adding 540 ml of distilled water with 60 ml of buffer solution (10%). 5. Add 2 drops of Tween 20 to buffer and mix well. 6. Add slides into epitope retrieval buffer (see Note 7) for 10 to 40 minutes (depending on the antibody and needs to be optimized), and cover with cling film and microwave on full power for 3 minutes. Solution will turn cloudy. 7. Place sections in buffer. Ensure they are completely covered and heat at 95  C for 15 minutes. 8. Allow sections to cool for 20 to 30 minutes.

3.6

Sample Staining

1. Rinse sections thoroughly in TBST for 5 minutes. 2. Incubate slides in peroxidase blocking reagent for 5 minutes to block endogenous peroxidases. 3. Wash slides 3 times, 5 minutes each. 4. Dilute primary antibody to desired concentration using the diluent solution (see Note 8). 5. Incubate slides with primary antibody (the incubation time varies and could be 15 minutes, an hour or longer, depending on optimization results) at 4  C overnight in humidity chamber. 6. Wash slides with TBST three times, 5 minutes each. 7. Incubate with secondary antibody (HRP – horseradish peroxidase) for 30 minutes at room temperature (different chemicals if using polymer-based detection method in the autoimmune stainer system). 8. Wash slides with TBST three times, 5 minutes each. 9. Prepare DAB (3,3-diaminobenzidine) by adding 1 ml DAB buffer + 1 drop DAB, according to the manufacturer’s guideline (see Note 9). 10. Incubate the slides with DAB for approximately 3 minutes (see Note 10).

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11. Wash slides in distilled water 2 times, 2 minutes each, and counterstain in hematoxylin for 2 minutes (see Note 11). 12. Wash the slides by dipping it up and down in distilled water for 2 minutes (see Note 12). 13. Stain with Scott’s solution for 30 seconds or Copper sulfate for one minute (see Note 13). 14. Wash the slides by dipping it up and down in distilled water for 2 minutes. 15. Dehydrate with 100% ethanol for 2 minutes. 16. Clear with xylene for 2 minutes, twice. 17. Mount slides with mounting media and cover the tissue section with a coverslip (see Note 14).

4

Notes 1. Fixation in buffered formalin allows for immobilization of antigens and maintenance of cellular and subcellular structure integrity. However, the antigenicity of some cellular components may be reduced as the crosslinking can obstruct antibody binding. The ideal fixation time depends on the size and type of tissue with a general fixation time between 18 and 24 hours. Over-fixation can mask epitope of which additional antigen retrieval techniques may be used to help overcome the masking effect. Under-fixation on the other hand can result in strong staining at the edges of the section and minimal to no signal in the center portion of the tissue. In addition, prolonged fixation may result in loss of signal even after antigen retrieval. 2. To embed the tissue in a paraffin block, rinse the formalin fixed tissue under running tap water for 1 hour before dehydrating it through 70%, 80%, and 95% ethanol for 45 min each and then in 100% ethanol for 1 hour, three times. Next, soak the tissue in 100% xylene twice (using fresh xylene each time), 1 hour each. Then, move the cassette into 58–60  C paraffin for 1 hour, three times (using fresh paraffin each time). In the meantime, coat the bottom layer of the mold with melted paraffin. Transfer the tissue sample onto the top of the base wax, and cover it with more paraffin. Next, place the pathology cassette on top of the mold and completely fill the mold. Allow mold to cool on the embedding station (approximately 10 to 15 minutes) before removing embedded sample from the mold. The paraffin embedded sample can be stored in room temperature until ready to section. 3. Set the clearance angle according to the manufacturer’s instruction, ensuring that there is no contact between the

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knife facet and the face of the block. Insert the paraffin block, and orientate it to be parallel to the blade to allow smooth slicing across the block. (Caution! Ensure that blade protector is on when doing this.) 4. Try to avoid creases or folding to the paraffin ribbons. 5. The presence of controls is essential for accurate interpretation. An example of a multi-tissue control block consists of several different types of tissue samples (Figs. 7 and 8). An immunohistochemical assay must consist of controls; otherwise, it cannot be validly interpreted. The absence of positive and negative controls that work as expected can result in erroneous scientific conclusions and clinical misdiagnoses. 6. Automated systems are available for the following steps. Steps 3.5 and 3.6 sample could be done with an autoimmunostainer with minor alterations of reagents and conditions (Fig. 9). Steps like autoimmunostainer are highly recommended for bulk sample to ensure consistent staining quality. The time and step (peroxide blocking) involved may have modifications depending on the antibody and the system used. Wherever

Fig. 9 Autoimmunostainer: (a) IHC could be done in the machine with slides, one slide (left, see also Fig. 7) with reagents and antibodies trays on the other slide (right). The regents and antibodies are suctioned and delivered to the slides. (b) The tray with reagents. (c) The tray with different antibodies to apply on the sections

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mentioned, optimization should be done to allow for best staining results. Peroxide blocking step (Step 2) could be after the polymer step (Step 7) in using the autoimmunostainer. 7. Slides are treated with epitope retrieval buffer to unmask antigenic sites. The masking is caused by crosslinking of disulfide bonds (methylene bridges) due to formalin fixation. Antigen retrieval can be heat-induced with sodium citrate or Tris/ethylenediaminetetraacetic acid (EDTA) or enzymatic with trypsin, pepsin, or other proteases. The heating method could be microwave, pressure cooker, vegetable steamer, or the heating system in autoimmunostainer. 8. Different antibodies and different supplies may have different starting concentrations. A serial dilution of antibodies concentration may need to be done according to the manufacturer’s suggested dilution range to obtain the optimal antibody concentration for satisfactory staining. 9. DAB is to visualize the positive antigen-antibody complex in IHC and will appear in brown color if positive. DAB is very sensitive to light. Make sure that while preparing the DAB cocktail, the tube used to hold DAB is either an ambient tube or has been wrapped with aluminum foil. Always make the DAB cocktail fresh right before it is needed. 10. DAB is a hazardous substance and should be disposed of appropriately. The common disposal method is to dilute DAB by 100x and then flushing it down the sink with constant running water. 11. During this step, check the slide regularly. Remove slide from stain earlier if stain appears dense to avoid overstaining. 12. Change a fresh batch of distilled water, and continue washing by dipping up and down until adequate staining is acquired. 13. During this stage, please check the slide regularly, and stop staining once the desired density of blue color is achieved. Use of Scott’s solution is optional. It can be replaced by copper sulfate for 1 minute for enhancement of weak DAB color. 14. Take care to avoid bubble formation between the coverslip and the slide. This can be done by slanting the coverslip at a 30 angle and slowly dropping it down to the slide. References 1. Steurer S, Schneider J, Bu¨scheck F, Luebke AM, Kluth M, Hube-Magg C, Hinsch A, Ho¨flmayer D, Weidemann S, Fraune C, Mo¨ller K, Menz A, Bernreuther C, Lebok P, Sauter G, Simon R, Jacobsen F, Uhlig R,

Wilczak W, Minner S, Burandt E, Krech RH, Dum D, Krech T, Marx AH, Clauditz TS (2021) Immunohistochemically detectable thyroglobulin expression in extrathyroidal cancer is 100% specific for thyroidal tumor origin.

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17-year-old nulliparous patient: a case report. Diagnostics (Basel) 10:45. https://doi.org/ 10.3390/diagnostics10010045. PMID: 31952290 11. Abouhashem NS, Talaat SM (2017) Diagnostic utility of CK19 and CD56 in the differentiation of thyroid papillary carcinoma from its mimics. Pathol Res Pract 213:509–517. https://doi.org/10.1016/j.prp.2017.01. 017. PMID: 28214214 12. Tastekin E, Keskin E, Can N, Canberk S, Mut A, Erdogan E, Asa N, Gu¨ldiken S, Sezer A, Azatcam M (2019) CD56, CD57, HBME1, CK19, Galectin-3 and p63 immunohistochemical stains in differentiating diagnosis of thyroid benign/malign lesions and NIFTP. Pol J Pathol 70:286–294. https://doi.org/10. 5114/pjp.2019.93131. PMID: 32146798 13. Zargari N, Mokhtari M (2019) Evaluation of diagnostic utility of immunohistochemistry markers of TROP-2 and HBME-1 in the diagnosis of thyroid carcinoma. Eur Thyroid J 8:1– 6 . h t t p s : // d o i . o r g / 1 0 . 1 1 5 9 / 000494430. PMID: 30800635 14. Lam KY, Lui MC, Lo CY (2001) Cytokeratin expression profiles in thyroid carcinomas. Eur J Surg Oncol 27:631–635. https://doi.org/10. 1053/ejso.2001.1203. PMID:11669590 15. Wa Kammal WS, Yahaya A, Shah SA, Abdullah Suhaimi SN, Mahasin M, Mustangin M, Md Isa N (2019) The diagnostic utility of cytokeratin 19 in differentiating malignant from benign thyroid lesions. Malays J Pathol 41: 293–301. PMID:31901914 16. Lam AK (2017) Pathology of endocrine tumours update: World Health Organization new classification 2017-other thyroid tumours. AJSP: Rev Rep 22:209–216 17. Sadiq Q, Sekhri R, Dibaba DT, Zhao Q, Agarwal S (2021) HBME1 and CK19 expression in non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) vs other follicular patterned thyroid lesions. World J Surg Oncol 19:143. https://doi.org/ 10.1186/s12957-021-02258-7. PMID: 33964951 18. Salajegheh A, Dolan-Evans E, Sullivan E, Irani S, Rahman MA, Vosgha H, Gopalan V, Smith RA, Lam AK (2014) The expression profiles of the galectin gene family in primary and metastatic papillary thyroid carcinoma with particular emphasis on galectin-1 and galectin3 expression. Exp Mol Pathol 96:212–218. https://doi.org/10.1016/j.yexmp.2014.02. 003. PMID: 24530443 19. Saffar H, Jahanbin B, Ameli F, Farhang F, Tavangar SM, Saffar H (2021) Diagnostic value of TROP2 expression in papillary thyroid

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carcinoma. Appl Immunohistochem Mol Morphol 29:218–222. https://doi.org/10.1097/ PAI.0000000000000886. PMID:33264108 20. Abdou AG, Shabaan M, Abdallha R, Nabil N (2019) Diagnostic value of TROP-2 and CK19 expression in papillary thyroid carcinoma in both surgical and cytological specimens. Clin Pathol 12:2632010X19863047. https://doi. org/10.1177/2632010X19863047. PMID: 31384753 21. Chuang HW, Wang JS, Tsai JW, Hsu CT, Lin KJ (2021) Immunohistochemistry helps to distinguish noninvasive follicular thyroid neoplasm with papillary-like nuclear features/ noninvasive encapsulated follicular variant of papillary thyroid carcinoma with other follicular thyroid lesions. Medicina (Kaunas) 57: 1 2 4 6 . h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 / medicina57111246. PMID: 34833464 22. Song S, Kim H, Ahn SH (2019) Role of immunohistochemistry in fine needle aspiration and core needle biopsy of thyroid nodules. Clin Exp Otorhinolaryngol 12:224–230. https:// doi.org/10.21053/ceo.2018.01011. PMID: 30531651 23. Samarasinghe S, Yuksel S, Mehrotra S (2020) Intermixed medullary and papillary thyroid cancer in a patient with renal cell carcinoma. Endocrinol Diabetes Metab Case Rep 2020: 20–0025. https://doi.org/10.1530/EDM20-0025. PMID: 32538376 24. Volante M, Lam AK, Papotti M, Tallini G (2021) Molecular pathology of poorly differentiated and anaplastic thyroid cancer: what do pathologists need to know? Endocr Pathol 32: 63–76. https://doi.org/10.1007/s12022021-09665-2. PMID: 33543394 25. Abe I, Lam AK (2021) Anaplastic thyroid carcinoma: updates on WHO classification, clinicopathological features and staging. Histol Histopathol 36:239–248. https://doi.org/ 10.14670/HH-18-277. PMID: 33170501 26. Lam KY, Lo CY, Chan KW, Wan KY (2000) Insular and anaplastic carcinoma of the thyroid: a 45-year comparative study at a single institution and a review of the significance of p53 and p21. Ann Surg 231:329–338. https://doi. o r g / 1 0 . 1 0 9 7 / 0 0 0 0 0 6 5 8 200003000-00005. PMID: 10714625 27. Ragazzi M, Torricelli F, Donati B, Ciarrocchi A, de Biase D, Tallini G, Zanetti E, Bisagni A, Kuhn E, Giordano D, Frasoldati A, Piana S (2021) Coexisting well-differentiated and anaplastic thyroid carcinoma in the same primary resection specimen: immunophenotypic and genetic comparison of the two components in a consecutive series of 13 cases and a review of the literature. Virchows Arch 478:

265–281. https://doi.org/10.1007/s00428020-02891-9. PMID: 32683537 28. Lam AK (2020) Squamous cell carcinoma of thyroid: a unique type of cancer in World Health Organization classification. Endocr Relat Cancer 27:R177–R192. https://doi. org/10.1530/ERC-20-0045. PMID: 32252028 29. Lam KY, Lo CY, Liu MC (2001) Primary squamous cell carcinoma of the thyroid gland: an entity with aggressive clinical behaviour and distinctive cytokeratin expression profiles. Histopathology 39:279–286. https://doi.org/10. 1046/j.1365-2559.2001.01207.x. PMID: 11532039 30. Lam AK, Saremi N (2017) Cribriform-morular variant of papillary thyroid carcinoma: a distinctive type of thyroid cancer. Endocr Relat Cancer 24:R109–R121. https://doi.org/10. 1530/ERC-17-0014. PMID: 28314770 31. Lam AK, Fridman M (2018) Characteristics of cribriform morular variant of papillary thyroid carcinoma in post-Chernobyl affected region. Hum Pathol 74:170–177. https://doi.org/ 10.1016/j.humpath.2018.01. 006. PMID:29320754 32. Dettloff J, Seethala RR, Stevens TM, Brandwein-Gensler M, Centeno BA, Otto K, Bridge JA, Bishop JA, Leon ME (2017) Mammary analog secretory carcinoma (MASC) involving the thyroid gland: a report of the first 3 cases. Head Neck Pathol 11:124–130. https://doi.org/10.1007/s12105-0160741-6. PMID:27400918 33. Dogan S, Wang L, Ptashkin RN, Dawson RR, Shah JP, Sherman EJ, Michael Tuttle R, Fagin JA, Klimstra DS, Katabi N, Ghossein RA (2016) Mammary analog secretory carcinoma of the thyroid gland: a primary thyroid adenocarcinoma harboring ETV6-NTRK3 fusion. Mod Pathol 29:985–995. https://doi.org/ 10.1038/modpathol.2016.115. PMID: 27282352 34. Stergianos S, Juhlin CC, Zedenius J, Calissendorff J, Falhammar H (2021) Metastasis to the thyroid gland: characterization and survival of an institutional series spanning 28 years. Eur J Surg Oncol 47:1364–1369. https://doi.org/10.1016/j.ejso.2021.02. 018. PMID: 33642087 35. Lam KY, Lo CY (1998) Metastatic tumors of the thyroid gland: a study of 79 cases in Chinese patients. Arch Pathol Lab Med 122:37– 41. PMID: 9448014 36. Ghossein CA, Khimraj A, Dogan S, Xu B (2021) Metastasis to the thyroid gland: a single-institution 16-year experience.

Immunohistochemistry in Thyroid Carcinoma Histopathology 78:508–519. https://doi. org/10.1111/his.14246. PMID: 32897542 37. Wu Z, Guo T, Li Q, Cheng L, Hu X, Xu A (2021) Rare metastasis of gastric cancer to the thyroid gland: a case report and review of literature. Front Surg 8:731673. https://doi.org/ 10.3389/fsurg.2021.731673. PMID: 34692762 38. Bourcier K, Fermeaux V, Leobon S, Deluche E (2018) Lobular breast carcinoma metastasis to the thyroid gland: case report and literature review. J Breast Cancer 21:463–467. https:// doi.org/10.4048/jbc.2018.21.e55. PMID: 30607169 39. Shi Y, Brandler TC, Yee-Chang M, Cangiarella J, Wei XJ, Leung A, Szeto O, Deng FM, Liu CZ, Simsir A, Sun W (2020) Application of GATA 3 and TTF-1 in differentiating parathyroid and thyroid nodules on cytology specimens. Diagn Cytopathol 48: 128–137. https://doi.org/10.1002/dc. 24338 40. Smith RA, Salajegheh A, Weinstein S, Nassiri M, Lam AK (2011) Correlation between BRAF mutation and the clinicopathological parameters in papillary thyroid carcinoma with particular reference to follicular variant. Hum Pathol 42:500–506. https:// doi.org/10.1016/j.humpath.2009.09. 023. PMID:21167555 41. Shen X, Zhu G, Liu R, Viola D, Elisei R, Puxeddu E, Fugazzola L, Colombo C, Jarzab B, Czarniecka A, Lam AK, Mian C, Vianello F, Yip L, Riesco-Eizaguirre G, Santisteban P, O’Neill CJ, Sywak MS, CliftonBligh R, Bendlova B, Sy´korová V, Xing M (2018) Patient age-associated mortality risk is differentiated by BRAF V600E status in papillary thyroid cancer. J Clin Oncol 36:438–445. https://doi.org/10.1200/JCO.2017.74. 5497. PMID: 29240540 42. Kim KJ, Kim SG, Tan J, Shen X, Viola D, Elisei R, Puxeddu E, Fugazzola L, Colombo C, Jarzab B, Czarniecka A, Lam AK, Mian C, Vianello F, Yip L, RiescoEizaguirre G, Santisteban P, O’Neill CJ, Sywak MS, Clifton-Bligh R, Bendlova B, Sy´korová V, Xing M (2020) BRAF V600E status may facilitate decision-making on active surveillance of low-risk papillary thyroid microcarcinoma. Eur J Cancer 124:161–169. https://doi.org/10.1016/j.ejca.2019.10. 017. PMID: 31790974 43. Xing M, Alzahrani AS, Carson KA, Viola D, Elisei R, Bendlova B, Yip L, Mian C, Vianello F, Tuttle RM, Robenshtok E, Fagin JA, Puxeddu E, Fugazzola L, Czarniecka A, Jarzab B, O’Neill CJ, Sywak MS, Lam AK,

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Riesco-Eizaguirre G, Santisteban P, Nakayama H, Tufano RP, Pai SI, Zeiger MA, Westra WH, Clark DP, Clifton-Bligh R, Sidransky D, Ladenson PW, Sykorova V (2013) Association between BRAF V600E mutation and mortality in patients with papillary thyroid cancer. JAMA 309:1493–1501. h t t p s : // d o i . o r g / 1 0 . 1 0 0 1 / j a m a . 2 0 1 3 . 3190. PMID: 23571588 44. Wang F, Zhao S, Shen X, Zhu G, Liu R, Viola D, Elisei R, Puxeddu E, Fugazzola L, Colombo C, Jarzab B, Czarniecka A, Lam AK, Mian C, Vianello F, Yip L, RiescoEizaguirre G, Santisteban P, O’Neill CJ, Sywak MS, Clifton-Bligh R, Bendlova B, Sy´korová V, Wang Y, Xing M (2018) BRAF V600E confers male sex disease-specific mortality risk in patients with papillary thyroid cancer. J Clin Oncol 36:2787–2795. https://doi. org/10.1200/JCO.2018.78. 5097. PMID:30070937 45. Tao Y, Wang F, Shen X, Zhu G, Liu R, Viola D, Elisei R, Puxeddu E, Fugazzola L, Colombo C, Jarzab B, Czarniecka A, Lam AK, Mian C, Vianello F, Yip L, Riesco-Eizaguirre G, Santisteban P, O’Neill CJ, Sywak MS, CliftonBligh R, Bendlova B, Sy´korová V, Zhao S, Wang Y, Xing M (2021) BRAF V600E status sharply differentiates lymph node metastasisassociated mortality risk in papillary thyroid cancer. J Clin Endocrinol Metab 106:3228– 3238. https://doi.org/10.1210/clinem/ dgab286. PMID: 34273152 46. Xing M, Alzahrani AS, Carson KA, Shong YK, Kim TY, Viola D, Elisei R, Bendlová B, Yip L, Mian C, Vianello F, Tuttle RM, Robenshtok E, Fagin JA, Puxeddu E, Fugazzola L, Czarniecka A, Jarzab B, O’Neill CJ, Sywak MS, Lam AK, Riesco-Eizaguirre G, Santisteban P, Nakayama H, Clifton-Bligh R, Tallini G, Holt EH, Sy´korová V (2015) Association between BRAF V600E mutation and recurrence of papillary thyroid cancer. J Clin Oncol 33(1):42–50. https://doi.org/10. 1200/JCO.2014.56.8253. PMID: 25332244 47. Rashid FA, Tabassum S, Khan MS, Ansari HR, Asif M, Sheikh AK, Sameer Aga S (2021) VE1 immunohistochemistry is an adjunct tool for detection of BRAF V600E mutation: validation in thyroid cancer patients. J Clin Lab Anal 35:e23628. https://doi.org/10.1002/ jcla.23628. PMID: 33305405 48. Choden S, Keelawat S, Jung CK, Bychkov A (2020) VE1 immunohistochemistry improves the limit of genotyping for detecting BRAF V600E mutation in papillary thyroid cancer. Cancers (Basel) 12:596. https://doi.org/10. 3390/cancers12030596. PMID: 32150939

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49. Zhang Y, Liu L, Liu Y, Cao N, Wang L, Xing C (2021) Clinical significance of immunohistochemistry to detect BRAF V600E mutant protein in thyroid tissues. Medicine (Baltimore) 100:e25566. https://doi.org/10.1097/MD. 0000000000025566. PMID: 33879712 50. Parker KG, White MG, Cipriani NA (2020) Comparison of molecular methods and BRAF immunohistochemistry (VE1 Clone) for the detection of BRAF V600E mutation in papillary thyroid carcinoma: a meta-analysis. Head Neck Pathol 14:1067–1079. https://doi.org/ 1 0 . 1 0 0 7 / s 1 2 1 0 5 - 0 2 0 01166-8. PMID:32358715 51. Singarayer R, Mete O, Perrier L, Thabane L, Asa SL, Van Uum S, Ezzat S, Goldstein DP, Sawka AM (2019) A systematic review and meta-analysis of the diagnostic performance of BRAF V600E immunohistochemistry in thyroid histopathology. Endocr Pathol 30:201– 218. https://doi.org/10.1007/s12022-01909585-2. PMID: 31300997 52. Li J, Vasilyeva E, Wiseman SM (2019) Beyond immunohistochemistry and immunocytochemistry: a current perspective on galectin-3 and thyroid cancer. Expert Rev Anticancer Ther 19:1017–1027. https://doi.org/10. 1080/14737140.2019.1693270. PMID: 31757172 53. Sun H, Chen Q, Liu W, Liu Y, Ruan S, Zhu C, Ruan Y, Ying S, Lin P (2021) TROP2 modulates the progression in papillary thyroid carcinoma. J Cancer 12:6883–6893. https://doi. org/10.7150/jca.62461. PMID: 34659576 54. Asioli S, Erickson LA, Sebo TJ, Zhang J, Jin L, Thompson GB, Lloyd RV (2010) Papillary thyroid carcinoma with prominent hobnail features: a new aggressive variant of moderately differentiated papillary carcinoma. A clinicopathologic, immunohistochemical, and molecular study of eight cases. Am J Surg Pathol 34: 44–52. https://doi.org/10.1097/PAS. 0b013e3181c46677. PMID: 19956062 55. Ali KM, Awny S, Ibrahim DA, Metwally IH, Hamdy O, Refky B, Abdallah A, Abdelwahab K (2019) Role of P53, E-cadherin and BRAF as predictors of regional nodal recurrence for papillary thyroid cancer. Ann Diagn Pathol 40:59– 65. https://doi.org/10.1016/j.anndiagpath. 2019.04.005. PMID: 31031216 56. Salajegheh A, Vosgha H, Rahman MA, Amin M, Smith RA, Lam AK (2016) Interactive role of miR-126 on VEGF-A and progression of papillary and undifferentiated thyroid carcinoma. Hum Pathol 51:75–85. https:// doi.org/10.1016/j.humpath.2015.12. 018. PMID: 27067785

57. Salajegheh A, Smith RA, Kasem K, Gopalan V, Nassiri MR, William R, Lam AK (2011) Single nucleotide polymorphisms and mRNA expression of VEGF-A in papillary thyroid carcinoma: potential markers for aggressive phenotypes. Eur J Surg Oncol 37:93–99. https://doi.org/ 10.1016/j.ejso.2010.10.010. PMID: 21093207 58. Maroof H, Irani S, Arianna A, Vider J, Gopalan V, Lam AK (2019) Interactions of vascular endothelial growth factor and p53 with miR-195 in thyroid carcinoma: possible therapeutic targets in aggressive thyroid cancers. Curr Cancer Drug Targets 19:561–570. https://doi.org/10. 2174/1568009618666180628154727. PMID:29956628 59. Maroof H, Islam F, Ariana A, Gopalan V, Lam AK (2017) The roles of microRNA-34b-5p in angiogenesis of thyroid carcinoma. Endocrine 58:153–166. https://doi.org/10.1007/ s12020-017-1393-3. PMID: 28840508 60. Salajegheh A, Vosgha H, Md Rahman A, Amin M, Smith RA, Lam AK (2015) Modulatory role of miR-205 in angiogenesis and progression of thyroid cancer. J Mol Endocrinol 55:183–196. https://doi.org/10.1530/JME15-0182. PMID: 26342107 61. Maroof H, Islam F, Dong L, Ajjikuttira P, Gopalan V, McMillan NAJ, Lam AK (2018) Liposomal delivery of miR-34b-5p induced cancer cell death in thyroid carcinoma. Cell 7: 2 6 5 . h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 / cells7120265. PMID: 30544959 62. Yu XM, Lo CY, Chan WF, Lam KY, Leung P, Luk JM (2005) Increased expression of vascular endothelial growth factor C in papillary thyroid carcinoma correlates with cervical lymph node metastases. Clin Cancer Res 11:8063– 8069. https://doi.org/10.1158/1078-0432. CCR-05-0646. PMID: 16299237 63. Salajegheh A, Pakneshan S, Rahman A, DolanEvans E, Zhang S, Kwong E, Gopalan V, Lo CY, Smith RA, Lam AK (2013) Co-regulatory potential of vascular endothelial growth factorA and vascular endothelial growth factor-C in thyroid carcinoma. Hum Pathol 44:2204– 2212. https://doi.org/10.1016/j.humpath. 2013.04.014. PMID: 23845470 64. Mohamad Pakarul Razy NH, Wan Abdul Rahman WF, Win TT (2019) Expression of vascular endothelial growth factor and its receptors in thyroid nodular hyperplasia and papillary thyroid carcinoma: a tertiary health care centre based study. Asian Pac J Cancer Prev 20:277– 282. https://doi.org/10.31557/APJCP. 2019.20.1.277. PMID: 30678450

Immunohistochemistry in Thyroid Carcinoma 65. Ramos-Vara JA (2017) Principles and methods of immunohistochemistry. Methods Mol Biol 1641:115–128. https://doi.org/10.1007/ 978-1-4939-7172-5_5. PMID: 28748460 66. Abram M, Huhtamella R, Kalfert D, HaksoMa¨kinen H, Ludvı´ková M, Kholová I (2021)

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Chapter 14 Whole-Slide Imaging: Updates and Applications in Papillary Thyroid Carcinoma Alfred K. Lam, Alfa Bai, and Melissa Leung Abstract Whole-slide imaging (WSI) has wide spectrum of application in histopathology, especially in the study of cancer including papillary thyroid carcinoma. The main applications of WSI system include research, teaching, and assessment and recently pathology practices. The other major advantages of WSI over histological sections on glass slides are easier storage and sharing of information as well as adaptation of use in artificial intelligence. The applications of WSI depend on factors such as volume of services requiring WSI, physical factors (computer server, bandwidth limitation of networks, storages requirements for data), adaption of the WSI images with the laboratory workflow, personnel (IT expert, pathologist, technicians) adaptation to the WSI workflow, validation studies, ethics, and cost efficiency of the application(s). Key words Whole-slide imaging, Pathology, Artificial intelligence, Teaching, Thyroid

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Introduction Whole-slide imaging (WSI) system is a major milestone for digitization of histopathology images which has tremendous potential of applications in different fields. Fading of color and eventually complete loss of color occur in all both hematoxylin and eosin (H&E) and immunochemical stained sections. On the other hand, WSI system allows permanent storage of the digital visual information. In terms of storage of information, histological sections on glass slides require a lot of physical space, whereas WSI files need to consider different digital storage solutions. In addition, WSI allows easy sharing of digital images over the Internet and facilitates the development and application of artificial intelligent (AI) in histopathology analysis. “Machine learning” (ML) is a subgroup of AI

Alfred K. Lam and Alfa Bai contributed equally with all other contributors. Alfred K. Lam (ed.), Papillary Thyroid Carcinoma: Methods and Protocols, Methods in Molecular Biology, vol. 2534, https://doi.org/10.1007/978-1-0716-2505-7_14, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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Table 1 Main applications of WSI in Pathology Research – tissue microarray sections, facilities collaborations, standardization of assessment criteria, assessment of histological and cytological features Clinical – small/subset diagnostic services, assessment of histopathology parameter(s), frozen section assessment for remote sites, sending consultations, recording of received consult cases Teaching and assessment – undergraduate teaching, postgraduate teaching, conference presentation, assessment, quality assurance

application which is broadly divided into supervised and unsupervised ML [1]. Both forms need large datasets to train the ML algorithm. For assessment of histological parameters by AI, capturing of many images of the representative fields of H&E is needed. WSI images, in comparison to digital images manually taken by pathologist by usual digital camera, are more precise in focusing, of high resolution, and less time-consuming in obtaining and have acquired more information. In 2000, whole-slide scanners were available for commercial use. WSI or virtual microscopy has wide spectrum of applications in histopathology, especially in the study of cancer including papillary thyroid carcinoma. The technology is mainly used in research and teaching, but it is not until recently, around two decades later, that the technology is beginning to be of routine use in clinical diagnostic field. Table 1 summarizes the major potential applications of WSI. 1.1 Research Applications of WSI

In papillary thyroid carcinoma or other cancers, one of the most common applications of WSI system is to assess immunohistochemical staining or in situ hybridization (ISH) of markers in tissue microarray (TMA) sections. TMA sections are used for studies of biochemical pathways as well as pharmaceutical research for biomarkers particularly in oncology. Assessment of TMA sections with almost approximately 100 stained histological sections on a microscopic slide is almost impossible for pathologists to interpret and score without enlarging digitized sections on the computer screen. In papillary thyroid carcinoma, WSI has helped score and verify the use of markers such as TROP-2, galaectin-1, galaectin-3, endothelin 1, and endothelin receptor A on larger number of cases in papillary thyroid carcinoma on TMA sections [2–4]. WSI enhances research collaborations and flow of works in research. For instance, after the scanning of slides of papillary thyroid carcinoma by WSI scanner, WSI images could be sent via the Internet to pathologist(s), in different locations, for assessment of histological features. This is of particular importance in research

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which requires contributions of pathologists in different regions in reaching conclusion of standardization of diagnostic criteria and assessing observation variations in diagnostic parameters in tumors, including thyroid tumors [5–7]. WSI allows validation of different image analysis systems as well as AI projects to be performed on histological parameters. Most commonly in thyroid neoplasms, nuclear features of thyroid follicular cells in fine-needle aspiration are evaluated by application of machine learning algorithm on WSI images to determine the likelihood of carcinoma [8–12]. The impact of the application of WSI in thyroid carcinoma cytology has been reviewed recently, and the results look promising in clinical use on condition of resolution of technical issues for adaption of use and after validation studies [13]. In thyroid carcinomas, histological parameters like capsular invasion, vascular invasion, and volume of nodal metastases are important parameters for management of patients [14] and could potentially be detected and assessed by WSI and AI [15]. In addition, election of region(s) of interest could be done by pathologist (s) remotely to extract DNA or RNA for molecular studies. 1.2 Clinical Uses of WSI 1.2.1 Limitations

Recently, commercial market for WSI scanners has been extended to histopathology diagnostic service. Approval of the US Food and Drug Administration (FDA) to a WSI system for use in primary histopathology diagnosis has made the possibility of WSI in routine practice a wider acceptance [16]. However, why not yet for universal histopathological practice using WSI system? One of the most important factors to consider for use of WSI in clinical diagnosis is the additional time (added by using WSI) adding on turnaround time in pathology services. The additional time is related to the speed of scanning of the slides (scanning time) plus other time in logistics required for scanning. The scanning time depends on maximum magnification needed to be achieved, the volume of services, size, and nature of the tissue. In pathology assessment, the slide should be scanned at 40 which takes much longer scanning time when compared to laboratory scanning the slide for documentation at 20. For tertiary university hospital-based pathology, there are a large number of complex cases in which a case may have more than ten hematoxylin and eosin (H&E)-stained slides of area around 4 mm2 (2 mm  2 mm). For example, a typical total thyroidectomy, sampling of papillary thyroid carcinoma, and representative tissues from right lobe, left lobe, and isthmus normally require more than ten paraffin blocks with corresponding H&E sections (Chapter 6). For additional procedures such as neck dissection, scanning of more than 40 slides is required. Currently, the average time in scanning (including loading time) for a standard histopathology section (2.5 mm  2.0 mm) is approximately 12 min per slide. It would tremulously decrease

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the turnaround time for reporting of pathological diagnosis if large number of slides must be scanned before giving to the pathologist. Nevertheless, there are a few scanners in the commercial market accredited for pathology diagnostic purposes tailor-made for application of whole-slide scanning in histopathology diagnostic services. The scanners concentrate on improvement of functional capacity to scan large number of H&E-stained histologic slides (more than 400) in a run, and a few logistics have been set up in different laboratories to improve the workflow for practical applications in diagnostic histopathology. Another limitation of clinical use of WSI system is because it is only best developed for H&E-stained histologic sections. In cytology, such as in fine-needle aspiration of papillary thyroid carcinoma, the thickness of smear and cell clusters makes WSI images difficult to interpret. Also, for some special stains to find organisms (such as acid-fast bacilli), the resolution is not adequate to identify the organisms. Furthermore, the assessment of polarization effects (e.g., amyloid) cannot be performed on WSI and necessitates evaluation from glass slides. 1.2.2 Potential Clinical Applications

There is a good potential for clinical use of WSI in some pathology laboratories, reporting mainly on small biopsy-type biopsies (such as skin biopsies and endoscopic gastrointestinal biopsies). In this biopsy samples, only one or few stained slides per patient were produced and the size of the tissue in each slide is small. The scanning time will be much decreased in proportion to the size of the histologic section. With the newer models, the scanning time of small biopsies could decrease to 1 min. No time is needed to spend on selecting the focus points. In addition, in large laboratories, many WSI scanners are available so that many slides could be captured simultaneously. In many big laboratories, the current approach is to scan small portion of certain subsets of slides. These may include those slides borrowed for consultation in which the laboratory wants to keep a record after reporting and returning of the slides. It may be the other way around: a laboratory would like to send slides for expert opinion. It is quicker to scan the slide and send the WSI images to the expert to avoid long-time waiting in sending postages and the cost of the postages. An important application of WSI images in histopathology is counting of immunohistochemical-stained Ki-67 proliferative index which is important in clinical management of many tumors such as breast carcinoma, neuroendocrine tumors, adrenal tumors, etc. [17–20]. There are many complementary image analysis tools available to use for this purpose [21, 22]. WSI is also important for urgent procedures such as getting preliminary diagnosis at frozen sectioning of tissue at the time of operation in operation theatre without an on-site pathologist. The sections produced could be scanned by a technician and send to the

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pathologist(s) offsite on duty or for expert opinion in laboratories operating in many locations. This is one of the best uses of telemedicine and especially in the time of the coronavirus (COVID) pandemic when many staff could temporarily work at home. An unexpected boost of use of WSI system has been reported in the pandemic [23–25]. In addition, AI diagnostic system has been tested in helping detect carcinoma at frozen section [26, 27]. WSI is a solution for remote laboratories with limited volume of services (slides/day) and no diagnostic pathologist on site. In this situation, WSI files could be sent to pathologists in main diagnostic laboratories. WSI images may also delivered via smartphones [28]. Lastly, the adaption of new diagnostic platform and psychological resistance of pathologists trained for assessment of histology under light microscope needs to be addressed [23, 29]. Many pathologists require additional glass slide evaluation before signing out the case on WSI. Overall, advance on technology and wellplanned strategies will allow WSI to be of more use in clinical diagnostic services. 1.3 Teaching and Assessment Applications

WSI images are used for teaching in undergraduate and postgraduate education assessment for a long time. The digital images provided by WSI are often of better quality for presentation and can easily be annotated as well as posting up on social media. The application in the setting, unlike clinical application, has much less medical-legal issues to be consideration as accuracy for patient management is not the consideration. The WSI are well received for postgraduate pathology trainees in cytopathology in additional to histopathology [30]. For undergraduate teaching, WSI images are being used in teaching of histological features of cancers (such as papillary thyroid carcinoma) and other diseases in practical demonstrations. This has replaced the use of glass slides and microscope and concentrated teaching on the computer screen. This has the advantage of easier integration with other teaching materials like PowerPoint presentation sharing on the same computer screen. The students’ learning experience in WSI and computer appears to be higher than using slides and microscope [31, 32]. In clinical practice, as discussed above, it is not practical to do WSI for all the slides. Most often, the pathologist will scan some of the slides with histological features of teaching value such as for teaching of medical students and pathologist in training as well as use in multidisciplinary team meeting for management of cancer. In many parts of assessments (examinations) for pathologist in training, the assessments are now based on the skills of diagnosis of WSI scanned histology sections rather than histology sections on glass slides. This saves the time and expenses in cutting multiple sections for candidates, checking multiple sections for the presence

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of identifiable histological features. Also, using WSI for assessments avoids complaints that some histological features may not be prominent in some sections compared to others. With good selection and manipulation, digital cytology could also be used successfully for assessment in examination involving immunohistochemistry, cytology, and special stains in addition to histopathology sections. For continued professional education, WSI files are being circulated to all pathologists involved in the quality assurance programs [33]. For example, in programs of Royal College of Pathologists of Australasia, WSI files are used in laboratories in Australia and New Zealand as well as to pathology laboratories over 80 countries including countries in Europe, Asia (the United Arab Emirates, India, Malaysia, Taiwan, etc.), and South Africa. In addition, WSI files of the cases presented in national or international conferences are often available for pre-review before attending the conferences. These WSI also make web conferencing for pathologists and pathologists-in-training feasible in the midst of the pandemic (COVID-19). Also, the digital pathology application could be used to engage pathology workforce in developing countries. 1.4 Application Logistics

Many scanners are available for purchase in the market though the cost of the machine should be considered. The choice of type and number of scanners used depend on the requirements of individual laboratory use. For clinical use, large numbers of scanners and with capacity of loading hundreds of slides at one run are important for smooth and fast workflow of the laboratory. For research and teaching use, it is unlikely to be of large volume of histopathology slides to be scanned in a day. The choice may be a small, movable scanner on working desk with capacity to scan one or few slides at a time. It is worth noting that if the research slides are for TMA sections, the time of scanning per TMA slide is much longer and may require half an hour for each TMA section. It is important to align the scanner in the workflow for the purpose of using the WSI images by considering various physical and human factors. The first group of factors is mainly physical which includes the computer server, Internet provider (bandwidth limitation of networks), and storages for data. The image files may need to upload to the big server to be assessed by pathologists on the web or directly copy out to hard drive to work for manual or AI manipulations. A safe and large capacity storage system is needed for these WSI images. The second group of factors includes the adaption of the WSI images with the laboratory workflow such as pathologists’ workstation, software system to use, laboratory information system, data management system, data archiving system, etc. Lastly are the human factors like need for information technology (IT) expert, pathologist, and technicians to adapt to the WSI workflow and conduction of validation studies (e.g., applications of

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AI). In addition, guidelines on ethics in using the WSI images for different purposes also needed to be developed. At the end of the day, the cost efficiency of using WSI must be considered. There are only few studies available, but the data unlikely could be generalized [23]. It is important to know the economic benefits of WSI system such as easy storage, avoidance of transport cost, education and research applications, and flexibility in labor could outbalance the additional time and cost investigate for the system. In addition, occupational health issues (computer vision syndrome) may occur in a small proportion of users, and more studies may need to be conducted in the area [23]. In this chapter, the details of scanning histologic sections of papillary thyroid carcinoma are discussed. In addition, the application of WSI images to select region on the carcinoma for use of extraction of DNA is demonstrated.

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Materials 1. A well-prepared glass slide holding histologic section of papillary thyroid carcinoma tissue with clean coverslip is the key for smooth scanning progress and getting high-quality slide images. 2. Cleaning applications for the slides (ethanol, xylene, alcoholbased hand sanitizer, cotton cloth or Kimwipes, tissue towel, and scalpel blades). 3. Whole-slide scanner (see Note 1). 4. Computer with whole-slide scanner control software installed (linked to the scanner) (Fig. 1). 5. External hard disc for image backup (need high storage capacity as the approximate size of one scanned image is 1 GB) (optional). 6. Additional desktop computer/server tower with imagesharing software (digital slide server, DSS) installed, to view the scanned images and as well as analyze the images locally or remotely (optional). 7. Handheld tablet and (optional) (Fig. 2).

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3.1 Preparation of the Slides

1. Prepare the barcode label of the slides (see Note 2). 2. Arrange the slides in order and record the order of the label number (as well as other information, e.g., type of stain) of the slides to be scanned.

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Fig. 1 Whole-slide scanner linked to a desktop computer

Fig. 2 Digital image server software provided a webpage-based image viewing and annotation marking interface which could be easily operated via a computer or a tablet

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3. Clean the slides, and make sure all the slides for scanning have coverslips and are dry and not sticky and the labels are not sticky or overhanging, too (see Note 3). 4. With excessive glass coverslip on slide, it can be resolved by trimming the slide edge with razor blade. 3.2

System Start-Up

1. Switch on the computer which is linked to the scanner, server and other optional devices. 2. Start up the scanner. 3. Log in to the computer system with password (if there is a pre-set password).

3.3 Loading the Slides for Scanning

1. Load the slide to the slide adaptor tray (Fig. 3a). 2. Adjust the clips to ensure the slide is securely fixed on the tray (Fig. 3b). 3. Open the lid of the tray loading box on the side of the scanner (right-hand side), and put the adaptor tray in the box (Fig. 3c).

Fig. 3 Loading slide to the adaptor tray. (a) Load the slide at an angle of about 30 . (b) Adjust the clips (green circles) at the side and bottom of the slide to secure it. (c) Adaptor tray at the correct position of the tray loading box – the white dot at the bottom and three arrows at the right side of the adaptor are aligned to the “Go” position and the three arrows, respectively (red circles)

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Fig. 4 Scanner control software (a) before putting in an adaptor tray. (b) Preview mode of an adaptor tray; barcode (not showing) and scanning area (green circle) are automatically detected, adjusting the red rectangle to change the desired scanning area if necessary. (c) The adaptor tray moves to the unload tray on the lefthand side after scanning finished and “Unload” button clicked

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Fig. 4 (continued)

3.4

Scanning

1. Activate the scanner control software and start scanning progress. 2. Select the location of the adaptor tray loaded with slide, and click “Preview” to check if the slide will be correctly scanned (Fig. 4a). Select the scanning magnification that will be used, and further crop or adjust the scan area to make sure the barcode information is correctly read (Fig. 4b). Click “Scan” to start the full scanning progress (see Note 4).

3.5

Post-scanning

1. After scanning, click “Unload Tray.” The adapter tray moves to the unload box on the left-hand side of the scanner (Fig. 4c). 2. Take the adaptor tray out and remove the slide from it.

3.6 View and Analysis of the Image

1. After the scanning finishes, the image file could be uploaded to DSS automatically or manually. 2. Connect to the DSS with an Internet browser using the assigned web address. Log into the system; the image thumbnails are listed here (Fig. 5a); double click the image thumbnail to view the image, and the analysis tools are listed at left side of the image (Fig. 5b). 3. Selection of area of interest for identification and further studies (Fig. 6).

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Fig. 5 Digital slide server (DSS) for two slides from a patient with papillary thyroid carcinoma. (a) Thumbnail list view after login. (b) Whole-slide view of a scanned image; analysis tools on the left-hand side which allow the pathologist to draw a region of interest (ROI), measure the distance and the area, give comment to the ROI, and take snapshot of the current view and then export to a new image file. (c) Optical zoom to 40 of the image of papillary thyroid carcinoma in lymph node. (d) Digital zoom to 80 of the image in (c)

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Fig. 5 (continued)

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Notes 1. Different models of whole-slide scanners are available in the market. Factors like, the optical quality and resolution, scanning speed, operation interface and integration of Internet elements are important for considerations when setting up in individual’s laboratory. Different end users, such as clinical service laboratory and academic research units, might have different concerns on throughputs, add-on functions (such as fluorescence signal detection, z-stacking layering scanning, etc.), instinct collaboration mode via the Internet, etc. The scanner showed in this demonstration (Fig. 1) is a low- to mid-throughput (six slides per run) whole-slide scanner, which comes with a digital image server software for easier collaboration and sharing image information with collaborators at different locations on the planet (Fig. 2). It is a good choice for small research laboratory that work on research involving histology sections in which the scanned images could be distributed electronically nationally or internationally for analysis. 2. The barcode label could be scanned and recognized by the scanner control software. The information hidden in the barcode could be automatically applied to the filename of the image and showed on the screen during browsing. As such, if there is a huge information stored in the barcode, such as a QR code with a patient’s personal information and medical information, make sure that the label is covered before scanning.

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Fig. 6 The primary papillary thyroid carcinoma (top) and metastatic papillary thyroid carcinoma in lymph node (bottom) are examined by a pathologist using a software to select the area of interest for further processing

3. The surface of the slide should be kept in clean and good condition. There must not be any dirt, fingerprints, excessive adhesion glue, or scratches on it. If there is felt pen or ink marking on the glass slide, clean with Kimwipes and alcoholbased solvents. When there is mounting glue on the coverslip, gently remove with Kimwipes and xylene. After wiping with organic solvent, the slide shall be dried by cotton cloth or tissue towel, or allow it to air-dry completely before starting scanning. 4. Some pre-set or default setting could be prepared that fit to the different section types such as tissue microarray (TMA) (Fig. 7), serial section, cytology, blood smear, very faintly

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Fig. 7 Tissue microarray section of papillary thyroid carcinoma. (a) Multiple small tissue samples of papillary thyroid carcinoma from different patients were in the same section for testing of immunohistochemical stain, galectin 3. (b) Higher magnification of the two pieces of tissue sections in A showing the staining in cytoplasm of the carcinoma

stained section, in situ hybridization, etc. Meanwhile, cropping and adjusting the scan area correctly might significantly reduce scanning time if more complex settings were applied. However, a high magnification-powered scanning is suggested to have clearer optical zoom in effect during analysis (Fig. 5c and d). References 1. Abels E, Pantanowitz L, Aeffner F, Zarella MD, van der Laak J, Bui MM, Vemuri VN, Parwani AV, Gibbs J, Agosto-Arroyo E, Beck AH, Kozlowski C (2019) Computational pathology definitions, best practices, and recommendations for regulatory guidance: a white paper from the Digital Pathology Association. J Pathol 249:286–294. https://doi.org/ 10.1002/path.5331. PMID: 31355445 2. Bychkov A, Sampatanukul P, Shuangshoti S, Keelawat S (2016) TROP-2immunohistochemistry: a highly accurate method in the differential diagnosis of papillary

thyroid carcinoma. Pathology 48:425–433. https://doi.org/10.1016/j.pathol.2016.04. 002. PMID: 27311870. 3. Salajegheh A, Dolan-Evans E, Sullivan E, Irani S, Rahman MA, Vosgha H, Gopalan V, Smith RA, Lam AK (2014) The expression profiles of the galectin gene family in primary and metastatic papillary thyroid carcinoma with particular emphasis on galectin-1 and galectin3 expression. Exp Mol Pathol 96:212–218. https://doi.org/10.1016/j.yexmp.2014.02. 003. PMID: 24530443.

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4. Irani S, Salajegheh A, Gopalan V, Smith RA, Lam AK (2014) Expression profile of endothelin 1 and its receptor endothelin receptor A in papillary thyroid carcinoma and their correlations with clinicopathologic characteristics. Ann Diagn Pathol 18:43–48. https://doi. org/10.1016/j.anndiagpath.2013.11. 001. PMID: 24332749. 5. Williams TA, Gomez-Sanchez CE, Rainey WE, Giordano TJ, Lam AK, Marker A, Mete O, Yamazaki Y, Zerbini MCN, Beuschlein F, Satoh F, Burrello J, Schneider H, Lenders JWM, Mulatero P, Castellano I, Kno¨sel T, Papotti M, Saeger W, Sasano H, Reincke M (2021) International histopathology consensus for unilateral primary aldosteronism. J Clin Endocrinol Metab 106:42–54. https://doi. org/10.1210/clinem/dgaa484. PMID: 32717746 6. Aloqaily A, Polonia A, Campelos S, Alrefae N, Vale J, Caramelo A, Eloy C (2021) Digital versus optical diagnosis of follicular patterned thyroid lesions. Head Neck Pathol 15:537– 543. https://doi.org/10.1007/s12105-02001243-y. PMID: 33128731. 7. Jung CK, Bychkov A, Song DE, Kim JH, Zhu Y, Liu Z, Keelawat S, Lai CR, Hirokawa M, Kameyama K, Kakudo K (2021) Molecular correlates and nuclear features of encapsulated follicular-patterned thyroid neoplasms. Endocrinol Metab 36:123–133. h t t p s : //d o i . o rg / 1 0 . 3 8 0 3 / E n M . 2 0 2 0 . 860. PMID: 33677934 8. Collins BT, Collins LE (2013) Assessment of malignancy for atypia of undetermined significance in thyroid fine-needle aspiration biopsy evaluated by whole-slide image analysis. Am J Clin Pathol 139:736–745. https://doi.org/ 10.1309/AJCPQU29GHXYSZRR. PMID: 23690115. 9. Gerhard R, Teixeira S, Gaspar da Rocha A, Schmitt F (2013) Thyroid fine-needle aspiration cytology: is there a place to virtual cytology? Diagn Cytopathol 41:793–798. https:// doi.org/10.1002/dc.22958. PMID: 23441010. 10. Chain K, Legesse T, Heath JE, Staats PN (2019) Digital image-assisted quantitative nuclear analysis improves diagnostic accuracy of thyroid fine-needle aspiration cytology. Cancer Cytopathol 127:501–513. https://doi. org/10.1002/cncy.22120. PMID: 31150162. 11. Elliott Range DD, Dov D, Kovalsky SZ, Henao R, Carin L, Cohen J (2020) Application of a machine learning algorithm to predict malignancy in thyroid cytopathology. Cancer Cytopathol 128:287–295. https://doi.org/ 10.1002/cncy.22238. PMID: 32012493.

12. Lin YJ, Chao TK, Khalil MA, Lee YC, Hong DZ, Wu JJ, Wang CW (2021) Deep learning fast screening approach on cytological whole slides for thyroid cancer diagnosis. Cancers (Basel) 13:3891. https://doi.org/10.3390/ cancers13153891. PMID: 34359792 13. Girolami I, Marletta S, Pantanowitz L, Torresani E, Ghimenton C, Barbareschi M, Scarpa A, Brunelli M, Barresi V, Trimboli P, Eccher A (2020) Impact of image analysis and artificial intelligence in thyroid pathology, with particular reference to cytological aspects. Cytopathology 31:432–444. https://doi.org/ 10.1111/cyt.12828. PMID: 3224858 14. Ghossein R, Barletta JA, Bullock M, Johnson SJ, Kakudo K, Lam AK, Moonim MT, Poller DN, Tallini G, Tuttle RM, Xu B, Gill AJ (2021) Data set for reporting carcinoma of the thyroid: recommendations from the International Collaboration on Cancer Reporting. Hum Pathol 110:62–72. https://doi.org/10. 1016/j.humpath.2020.08.009. PMID: 32920035 15. Xu B, Teplov A, Ibrahim K, Inoue T, Stueben B, Katabi N, Hameed M, Yagi Y, Ghossein R (2020) Detection and assessment of capsular invasion, vascular invasion and lymph node metastasis volume in thyroid carcinoma using microCT scanning of paraffin tissue blocks (3D whole block imaging): a proof of concept. Mod Pathol 33:2449–2457. https://doi.org/10.1038/s41379-0200605-1. PMID: 32616872 16. Kumar N, Gupta R, Gupta S (2020) Whole slide imaging (WSI) in pathology: current perspectives and future directions. J Digit Imaging 33:1034–1040. https://doi.org/10.1007/ s10278-020-00351-z. PMID: 32468487. 17. Feng M, Deng Y, Yang L, Jing Q, Zhang Z, Xu L, Wei X, Zhou Y, Wu D, Xiang F, Wang Y, Bao J, Bu H (2020) Automated quantitative analysis of Ki-67 staining and HE images recognition and registration based on whole tissue sections in breast carcinoma. Diagn Pathol 15: 65. https://doi.org/10.1186/s13000-02000957-5. PMID: 32471471 18. Satturwar SP, Pantanowitz JL, Manko CD, Seigh L, Monaco SE, Pantanowitz L (2020) Ki-67 proliferation index in neuroendocrine tumors: can augmented reality microscopy with image analysis improve scoring? Cancer Cytopathol 128:535–544. https://doi.org/ 10.1002/cncy.22272. PMID: 32401429. 19. Lam AK, Ishida H (2021) Pancreatic neuroendocrine neoplasms: clinicopathological features and pathological staging. Histol Histopathol 36:367–382. https://doi.org/10.14670/ HH-18-288. PMID: 33305819.

Whole-Slide Imaging in Papillary Thyroid Carcinoma 20. Lam AK (2017) Update on adrenal tumours in 2017 World Health Organization (WHO) of endocrine tumours. Endocr Pathol 28:213– 227. https://doi.org/10.1007/s12022-0179484-5. PMID: 28477311. 21. Aeffner F, Zarella MD, Buchbinder N, Bui MM, Goodman MR, Hartman DJ, Lujan GM, Molani MA, Parwani AV, Lillard K, Turner OC, Vemuri VNP, Yuil-Valdes AG, Bowman D (2019) Introduction to digital image analysis in whole-slide imaging: a white paper from the Digital Pathology Association. J Pathol Inform 10:9. https://doi.org/10. 4103/jpi.jpi_82_18. PMID: 30984469 22. Dzaparidze G, Kazachonok D, Laht K, Taelma H, Minajeva A (2020) Pathadin – the essential set of tools to start with whole slide analysis. Acta Histochem 122:151619. https://doi.org/10.1016/j.acthis.2020. 151619. PMID:33066841. 23. Jahn SW, Plass M, Moinfar F (2020) Digital pathology: advantages, limitations and emerging perspectives. J Clin Med 9:3697. h t t p s : // d o i . o r g / 1 0 . 3 3 9 0 / jcm9113697. PMID:33217963 24. Koelzer VH, Grobholz R, Zlobec I, Janowczyk A, Swiss Digital Pathology Consortium (SDiPath) (2021) Update on the current opinion, status and future development of digital pathology in Switzerland in light of COVID-19. J Clin Pathol. https://doi.org/ 10.1136/jclinpath-2021-207768. PMID: 34518361 25. Lujan GM, Savage J, Shana’ah A, Yearsley M, Thomas D, Allenby P, Otero J, Limbach AL, Cui X, Scarl RT, Hardy T, Sheldon J, Plaza JA, Whitaker B, Frankel W, Parwani AV, Li Z (2021) Digital pathology initiatives and experience of a large academic institution during the coronavirus disease 2019 (COVID-19) pandemic. Arch Pathol Lab Med 145:1051– 1061. https://doi.org/10.5858/arpa.20200715-SA. PMID: 33946103. 26. Li Y, Chen P, Li Z, Su H, Yang L, Zhong D (2020) Rule-based automatic diagnosis of thyroid nodules from intraoperative frozen sections using deep learning. Artif Intell Med

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108:101918. https://doi.org/10.1016/j. artmed.2020.101918. PMID: 32972671. 27. Chen P, Shi X, Liang Y, Li Y, Yang L, Gader PD (2020) Interactive thyroid whole slide image diagnostic system using deep representation. Comput Methods Prog Biomed 195:105630. https://doi.org/10.1016/j.cmpb.2020. 105630. PMID: 32634647. 28. Yu H, Gao F, Jiang L, Ma S (2017) Development of a whole slide imaging system on smartphones and evaluation with frozen section samples. JMIR Mhealth Uhealth 5:e132. h t t p s : // d o i . o r g / 1 0 . 2 1 9 6 / m h e a l t h . 8242. PMID: 28916508. 29. Unternaehrer J, Grobholz R, Janowczyk A, Zlobec I, Swiss Digital Pathology Consortium (SDiPath) (2020) Current opinion, status and future development of digital pathology in Switzerland. J Clin Pathol 73:341–346. h t t p s : // d o i . o r g / 1 0 . 1 1 3 6 / j c l i n p a t h 2019-206155. PMID: 31857377. 30. Van Es SL, Kumar RK, Pryor WM, Salisbury EL, Velan GM (2015) Cytopathology whole slide images and adaptive tutorials for postgraduate pathology trainees: a randomized crossover trial. Hum Pathol 46:1297–1305. https://doi.org/10.1016/j.humpath.2015. 05.009. PMID: 26093936. 31. Van Es SL, Kumar RK, Pryor WM, Salisbury EL, Velan GM (2016) Cytopathology whole slide images and adaptive tutorials for senior medical students: a randomized crossover trial. Diagn Pathol 11:1. https://doi.org/10. 1186/s13000-016-0452-z. PMID: 26746436 32. Gopalan V, Kasem K, Pillai S, Olveda D, Ariana A, Leung M, Lam AKY (2018) Evaluation of multidisciplinary strategies and traditional approaches in teaching pathology in medical students. Pathol Int 68:459–466. https://doi.org/10.1111/pin.12706. PMID: 30043440 33. Niazi MKK, Parwani AV, Gurcan MN (2019) Digital pathology and artificial intelligence. Lancet Oncol 20:e253–e261. https://doi. org/10.1016/S1470-2045(19) 30154-8. PMID: 31044723.

Chapter 15 External Radiotherapy for Locoregional Control in Thyroid Carcinoma Dora L. W. Kwong and Wendy W. L. Chan Abstract Most patients with well-differentiated thyroid cancers (WDTC) are adequately treated with surgery, radioactive iodine, and TSH suppression by thyroxine. External radiotherapy (ERT) is reserved for selected cases and for older patients. Some of the indications for ERT to neck include adjuvant treatment for gross or microscopic disease after surgery, palliation of locally advanced unresectable tumor, or as salvage for recurrent disease which is not amenable to surgery or does not uptake radioactive iodine. High radiation dose of at least 60Gy is required for locoregional control of gross or microscopic residual disease. As even patients with recurrent or metastatic disease can have long survival, it is important to minimize late radiation-induced morbidity without compromising local control. Modern ERT technique like intensitymodulated radiotherapy allows high radiation dose to be delivered to the large, complex target volume while protecting the adjacent critical normal structures like the trachea, larynx, esophagus, and cervical spinal cord. Key words External radiotherapy, Locoregional control, Intensity-modulated radiotherapy

1

Introduction Well-differentiated thyroid cancers (WDTC) include papillary, follicular, and mixed papillary-follicular histological types. Typically, the neoplastic tissue of WDTC can concentrate iodine, and radioactive iodine (RAI) is the main stay of radiotherapy. External radiotherapy (ERT) is infrequently used. Most patients have resectable disease who are adequately treated with thyroidectomy, radioactive iodine (RAI), and thyroid stimulating hormone (TSH) suppression, with excellent prognosis. The use of ERT is mainly limited to patients with gross residual tumor or with microscopic disease at high risk of recurrence after resection, those with neck recurrence not amenable to resection, and tumors that do not uptake RAI. Most of the evidence supporting benefit of ERT in locoregional control of WDTC comes from retrospective series from single center as there is no randomized trial comparing standard therapy

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with and without the addition of ERT. Because patients with WDTC have long survival, these retrospective series vary highly in the eras of treatment, radiotherapy techniques, and inclusion criteria for ERT treatment. Thus, the role of ERT is not well delineated, and there is no uniform approach or recommendations regarding patient selection or practice of ERT in WDTC. Some of the more accepted indications for ERT are discussed below.

2

Materials

2.1 Indications for Adjuvant ERT After Surgery 2.1.1 Gross Residual Disease

2.1.2 High Risk for Local Recurrence

The main indication for ERT is incomplete surgery. In patients with T4 disease, where there is tumor involvement of the larynx, trachea, esophagus, blood vessels, or mediastinum, gross residual tumor may remain after surgery. Tubiana et al. reported 97 patients treated by ERT after an incomplete surgical excision; the local recurrence was 11% at 15 years versus 23% for patients treated by surgery alone, although patients with ERT had larger and more extensive tumors [1]. O’Connell et al. reported 49 patients treated with ERT for gross residual disease (with either follicular or papillary CA thyroid). Complete regression was obtained in 37.5% of patients, partial regression in 25%, and no regression in 37.5% with an overall 5-year survival of 27% [2]. In a retrospective review by Chow et al. of 1297 patients with papillary thyroid carcinoma (PTC) treated in Hong Kong, 217 patients had gross postoperative locoregional disease, of which 59.5% had ERT. ERT not only improved locoregional control but also failure-free survival and overall survival. With addition of ERT to treatment, the locoregional recurrencefree survival increased from 24% to 63.4% at 10 years, while the cause-specific survival improved from 49.7% to 74.1% [3]. This demonstrates the efficacy of ERT (see Note 1). Residual disease is expected if the tumor is shaved off the trachea, recurrent laryngeal nerve, or blood vessels in surgery. Pathologic features including widespread/multifocal extrathyroidal extension, pT4, positive margin, involvement of tracheal perichondrium or esophageal muscularis, and/or multiple positive nodes indicate that there may be microscopic residual disease and high risk of locoregional recurrence after surgery (Fig. 1). In the study by Chow et al., ERT improved local failure-free survival in patients with pathologically confirmed positive resection margins and reduced local failures in patients with T4 disease. ERT also improved the 10-year lymph node failure-free survival in patients with N1b disease and patients with lymph node metastases larger than 2 cm [3]. In another review of 729 patients with WDTC reported by Brierley et al., ERT improved the 10-year locoregional failure-free survival (86.4% vs 65.7%) and cause-specific survival (81% vs 64.6%) among patients over 60 years old, with extrathyroidal extension but

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Fig. 1 A 65-year-old man presented with hoarseness and dysphagia. CT scan (a axial, b coronal view) showed large thyroid mass with erosion of the thyroid cartilage and cricoid cartilage. The left common carotid artery was partially encased by the mass and left internal jugular vein was obliterated with tumor thrombosis. He underwent total thyroidectomy and total laryngectomy and neck node dissection. Pathology showed widely invasive follicular carcinoma, oncocytic (Hurthle cell) variant from left lobe of thyroid, and papillary microcarcinoma in isthmus. There was extensive extrathyroidal extension and angioinvasion by the follicular carcinoma. There was no lymph node metastasis. He had adjuvant ERT to thyroid bed according to the preoperative tumor extent. (c) Coronal view showing target volumes: preoperative tumor extent in blue, CTV in yellow line, and PTV in red line. (d) IMRT dose in color wash (dose scale on left, 60gy in red and 40Gy in blue). The PTV received 60Gy in 30 fractions. (e) Sagittal view showing spinal cord dose from IMRT was kept below 40Gy

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no gross residual disease (n ¼ 70) [4]. In 44 patients with WDTC with extracapsular spread, Chen et al. found that none of the 11 patients treated with adjuvant ERT experienced local recurrence, while local recurrences were seen in nine out of 33 patients treated without ERT [5]. Tam et al. reviewed adjuvant treatment of 88 patients with T4a WDTC in MD Anderson Cancer Center, 44 patients underwent RAI alone, and 44 patients underwent RAI with ERT after surgery [6]. Patients treated with RAI and ERT had significant tumor invasion into soft tissue and visceral structures, while patients who had RAI alone had invasion into the recurrent laryngeal nerve only, or minimal invasion into the tracheal perichondrium and/or esophageal muscularis requiring shave resection only. Five-year disease-free survival was 43% in those undergoing RAI alone, compared with 57% in those undergoing RAI and ERT. Patients undergoing RAI alone had an increased rate of locoregional failure. Age and esophageal invasion were independent predictors of worse disease-free survival. In a systemic review of 16 published studies, including a pooled population of 5114 patients, Fussey et al. found that ERT decreased the mean rate of locoregional recurrence from 20.1% to 13.2% [7] (see Note 1). They concluded that ERT improved locoregional control when used in patients over the age of 45 at high risk for locoregional recurrence. The benefit of ERT in high-risk patients should be balanced against the morbidity of radiation (see Note 2). Age is an important prognostic factor in WDTC, and young patients have excellent prognosis if the tumor uptakes RAI. RAI is a more targeted delivery of high-dose radiation and can treat microscopic disease. In addition, there is concern of radiation-induced second malignancy with ERT. Thus, ERT is generally avoided in young patients. Thyroid carcinomas with adverse histologic features like Hurthle cells, poorly differentiated cells, tall cell, or clear cell have worse patient outcomes. These tumors are less iodine avid and would benefit from ERT also. In a statement of the American Head and Neck Society (2016), there were four recommendations for ERT for locoregional control in WDTC [8] (see Note 3): (a) ERT is recommended for patients with gross residual or unresectable locoregional disease, except for patients 45 years old with high likelihood of microscopic residual disease and low likelihood of responding to RAI. (d) Cervical lymph node involvement alone should not be an indication for adjuvant RAI.

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This statement preceded the release of the American Joint Committee on Cancer (AJCC)American Joint Committee on Cancer (AJCC) eight edition that uses 55 years as the cutoff age for poor prognosis.Prognosis. Thus, 55 years may be a more appropriate age cutoff. The American Thyroid Association guideline is also widely adopted for treatment of thyroid diseases. In the 2015 guideline, it recommended that for WDTC that invades the upper aerodigestive tract, surgery combined with additional therapy such as RAI and/or ERT is generally advised [9]. 2.2 Indication for ERT for Recurrent Disease

Surgery is the standard treatment for local or neck node recurrence. Additional RAI should be considered after surgery and TSH suppression continued. The indications for ERT after surgery are like that as discussed above for adjuvant ERT. ERT can be considered as salvage treatment for recurrence that are not resectable or do not uptake RAI. Beckham et al. reported treatment with definitiveintent ERT using intensity-modulated radiotherapy (IMRT) for 88 patients with unresectable or gross residual disease with or without concurrent chemotherapy (weekly doxorubicin, 10 mg/ m2). At 4 years, the local progression-free survival was 77.3%, and overall survival was 56.3% for all patients. Patients receiving concurrent chemotherapy with IMRT had better local progression-free survival (85.8% vs. 68.8%) and overall survival (68% vs 47%) compared with those treated with IMRT alone [10].

2.3 ERT Dose and Volume

The dose used in older series was usually lower because older radiotherapy methods could not deliver an adequate dose of radiation to the thyroid bed without exceeding the tolerance of the spinal cord. In the modern series, the recommended doses for gross residual or inoperable disease ranged from 60Gy to 70Gy with the higher dose preferentially used for unresectable disease, and doses for microscopic residual disease range from 50Gy to 70Gy with a median of 60Gy. Modern radiotherapy should be conformal, and IMRT will allow high dose to be delivered to the neck while limiting dose to normal structures like the larynx, esophagus, and spinal cord in the neck. High-dose ERT is associated with significant acute and late morbidity. Schwartz et al. reported treatment with conformal ERT for 131 patients, including recurrent disease in 76 patients and gross residual disease in 15 patients [11]. Of these, 57 patients were treated with IMRT. The median dose was 60Gy. IMRT did not impact survival but was associated with less frequent severe late morbidity (12% vs. 2%). Late morbidities included six patients with esophageal stricture requiring dilatation, one patient had subglottic laryngeal stenosis requiring dilatation, one patient had subglottic laryngeal edema requiring tracheostomy, and one patient remained feeding tube dependent. In the series reported by Beckham et al., 88 patients

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were treated with IMRT (45 patients with concurrent chemotherapy) to median dose of 70Gy in median 33 fractions [10]. Severe acute toxicities occurred in 23.9% of patients, mostly dermatitis and mucositis. In addition, 17.1% of patients required a percutaneous endoscopic gastrostomy (PEG) tube during or shortly after completion of ERT, and 10.1% of patients required a PEG for more than 12 months after ERT. The volume to be included in ERT is controversial. There are large variations in the irradiated volume adopted by different centers. Radiation can be limited to the thyroid bed only or also include the bilateral lateral neck nodes. The superior extent can start from the tip of the mastoid process or start from the upper level of the thyroid bed. Inferiorly, it can extend into the upper mediastinum or just including the supraclavicular fossa. These large differences can partially explain the differences in the observed locoregional control and treatment-related toxicities. There is no uniform guideline for target delineation. In deciding the target volume for irradiation, one should consider the toxicities of ERT, and it is reasonable to only include the area(s) at highest risk of recurrence in the target volume.

3

Methods

3.1 Patient and Simulation

Patient will lie in supine position, with head at rest and arms down by the sides. A thermoplastic mask is made for immobilization and covers from the head to the chest. Planning CT scan will be taken with patient in immobilization and in treatment position and extending from vertex to below diaphragm to include the whole lungs. CT with contrast is preferred for better delineation of residual disease and normal structures. However, residual thyroid tissue or tumor will concentrate iodine from the CT contrast, and this will preclude treatment with RAI for 2 months. This should be taken into consideration when scheduling the sequence of ERT and RAI. For intensity-modulated radiotherapy (IMRT), slice thickness of 2–3 mm is required.

3.2 Target Volumes [12, 13]

The thyroid bed extends from the hyoid to just below the suprasternal notch. Image fusion with preoperative imaging and planning CT helps in delineation of the original extent of tumor and defines the at-risk areas. Unlike other head and neck cancers, the first echelon of nodal drainage of thyroid cancer is to level VI which is at midline and then to lateral neck (levels III, IV, and II and may be part of level V) [14]. Typically, level I or retropharyngeal nodes are not involved except in the setting of recurrence. ERT target volumes should be custom designed for each patient according to their risks for local and regional recurrence (Case 1 and Case 2 for illustration).

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The gross tumor volume (GTV) will include the gross disease identified clinically or on imaging. The high-risk clinical target volume (CTV) should include GTV and areas of positive margin or shave excision with margin (at least 5 mm). This high-risk CTV (h-CTV) should be treated to 60–70Gy. The intermediate-risk CTV (i-CTV) is defined to cover the thyroid bed, tracheoesophageal groove, and level VI. The i-CTV can be treated to 56–60Gy. A low-risk CTV (l-CTV) may be considered for uninvolved nodal level II–V and VII if there are multiple nodal metastases and treated to 50–56Gy. Target volumes should be adjusted according to the surgical and pathology findings. Extensive nodal irradiation may not be necessary unless there is residual disease after neck dissection or extensive nodal extracapsular extension (see Note 4). The planning target volume (PTV) is created by adding margin to CTV to account for setup errors and typically is 3–5 mm for IMRT. IMRT allows for dose painting and concomitant treatment of all volumes in 30–35 fractions over 6–7 weeks (Fig. 2) (see Note 5). Fractional dose should not be larger than 2Gy per fraction. 3.3

Organs at Risk

3.4 Monitoring During ERT

4

Organs at risk (OARs) include the parotid glands (if level II irradiated), larynx, lungs, esophagus, brachial plexus, and spinal cord, and these structures should be delineated in the planning CT. Dose constraints are employed in IMRT planning to limit dose to OARs to avoid excessive toxicities. Table 1 shows the usual dose constraints. Priority should be given to critical OARs like the spinal cord, brachial plexus, and lungs. Esophagus dose should be kept as low as possible without compromising target dose. The parotid glands may be irradiated if the upper cervical nodes are involved. To avoid xerostomia after ERT, mean parotid dose should be kept below 26Gy, but a higher dose is acceptable since the parotid glands are not critical organs. Thyroxine is continued during ERT. Patients are monitored for acute toxicities. Skin reaction includes skin erythema, dry desquamation, and maybe moist desquamation if high dose employed. Mucositis of the esophagus, trachea, and larynx may require a soft diet, analgesics, and possibly enteral feeding, especially toward the end of ERT. Body weight should be monitored during ERT. Skin reaction and mucositis usually subside within 2–4 weeks after the end of ERT, and it is important to keep up with the nutrition of the patient during this period.

Notes 1. Retrospective data showed efficacy of ERT in improving locoregional control of WDTC.

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Fig. 2 A 41-year-old female had thyroidectomy and excision of central neck lymph nodes for papillary thyroid carcinoma. Pathology showed 6.5 cm primary in left lobe and three lymph nodes positive for metastases. Postoperative RAI was given. Thyroglobulin remained high after RAI: 28-44 μg/L on thyroxine and 1429 μg/L

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2. Because of significant toxicities associated with ERT, its use should be limited to cases where there is significant risk of locoregional failure despite other effective treatment including surgery, RAI, and TSH suppression. Discretion is required in patient selection so that the benefit of ERT will outweigh the morbidity of treatment. 3. Main indications for ERT for WDTC are for control of advanced locally advanced disease including gross residual or high risk of microscopic residual disease after surgery or unresectable recurrence which do not uptake RAI. Young patients have good prognosis, and ERT is not preferred. 4. ERT volume should be customized to limit radiation to highrisk areas. Extensive neck irradiation is not necessary. 5. Dose painting with IMRT will allow multiple dose levels to be prescribed to multiple target volumes at different risks. High dose can be delivered to targets without exceeding tolerance of critical stricture like the spinal cord. Table 1 Dose constraint for organs at risk (OAR) OAR

Goal

Spinal cord

Dmax 45Gy

Brachial plexus

Dmax 60Gy

Larynx

Mean dose 3 cm, incomplete tumor resection, or distant metastasis. Distant metastasis is not unusual, which may be diagnosed on presentation,

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Table 2 The ATA risk stratification system can be used to guide the postoperative radioactive iodine and dose ATA risk group

Use of RAI

Dose of RAI

Low

Consider or no

1.1 GBq

Intermediate

Consider

3.7 GBq

High

Recommended

3.7–5.5 GBq

especially those with aggressive histological subtypes (e.g., tall, hobnail, solid, diffuse sclerosing, and columnar cell variants; vascular invasion and multiple locoregional lymph nodes), or during follow-up which can happened over decades after the first diagnosis. The most common sites of metastasis are the lungs and bones (involved in 49% and 25% of all cases, respectively) [10]. RAI is the first choice of treatment in DTC with distant metastases. The dose of RAI ranges from 3.7 GBq to 5.5 GBq (Table 2). A few studies have demonstrated that RAI treatment can reduce specific diseasespecific mortality. In the study by Dania and colleagues, which included 138 patients with DTC and distant metastasis, with a mean follow-up period of 8.2 years, 24.6% of patients had complete resolution, and 31.6% had improvement/stable disease after RAI [11]. In a study by Slook et al. which included 64 patients with DTC with bone metastases, with a mean follow-up period of 11  9.6 years, 7.8% had complete resolution, 7.8% had structural improvement, and 23.4% had stable disease. Factors associated with overall mortality and disease-specific mortality included older age at diagnosis, T3/T4 disease, extra-thyroidal extension, lymph node metastases, co-presence of distant metastases at sites other than the bone, and spinal metastases [12]. 2.2 Contraindications of RAI

1. Pregnancy: The RAI can pass through the placenta and destroy the fetal thyroid tissue and pose irreversible mental retardation to the developing fetus. 2. Breastfeeding: Estrogenized breast tissue has increased sodium iodide symporter activity. The lactating breast concentrates a substantial amount of iodide. Breastfeeding must be stopped at least 4 weeks prior to RAI therapy to avoid breast irradiation and to prevent RAI in the milk from passing to the infant. 3. Carcinoma with no iodine uptake.

2.3 Form of Administration

RAI (Iodine-131) is available as oral capsule, oral drinking solution, or intravenous injections. RAI for thyroid cancer is mostly in oral capsule form.

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Methods Timing of RAI

The time interval between total thyroidectomy and RAI administration is usually between 3 and 4 weeks to 3 months. However, the impact of the timing of post-thyroidectomy RAI therapy as a prognostic factor is still a matter of debate. In a retrospective analysis of 198 high-risk DTC patients, the risk of death was 4.22 times higher in patients who received RAI more than 180 days after total thyroidectomy than those who received RAI within 180 days [13]. On the other hand, in another study analyzing the National Cancer Database (NCDB) with 9706 patients in the high-risk group, the OS was not significantly different between those who received early RAI (3 months) and those who received delayed RAI (between 3 and 12 months after thyroidectomy) [14]. Similarly, another study from Korea which included 526 patients having PTC of low-risk PTC found no significant difference in OS or DFS between the groups with early ( 22.2 GBq (600 mCi). – The fifth scenario should not be translated into a universal cutoff value for RAI activity. For instance, as is acknowledged in the ATA guidelines, if a patient has stable disease after multiple therapies with RAI, this should not be regarded as RAI-refractory disease [4]. – The above criteria only reflect the likelihood of a tumor being RAI-refractory, but do not indicate if systemic treatment needs to be started. – It is important to define and recognize RAI-refractory DTC as these patients will no longer benefit from RAI, and unnecessary treatments and adverse events can be avoided.

2.2 Diagnostic Tools for RAI-Refractory Thyroid Cancer

– Iodine whole-body scan is needed to detect RAI-refractory thyroid cancer foci which are not iodine-avid on the scan or show disease progression compared with the previous scan done within 12–14 months [6]. – Serial imaging with whole-body CT at 6–12 months intervals and applying RECIST 1.1 criteria to determine disease status is

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useful for assessing tumor growth [7]. PET-CT may yield additional information on tumor biology with a negative prognostic impact for lesions with increased FDG uptake [8]. – Serum thyroglobulin (Tg) is checked regularly, usually with 3-monthly interval, to detect the thyroglobulin doubling time which is associated with the pace of disease progression [9]. – Tumor biology including histological features and molecular features of RAI-refractory thyroid cancer is important to determine tumor growth and ability to concentrate iodine [10]. Tall cell, columnar cell, diffuse sclerosing, solid/trabecular, and insular variants of well-differentiated papillary thyroid cancer are all potentially more aggressive than conventional papillary thyroid cancer. For molecular pathology, in particular screening for BRAF mutations and ALK, NTRK and RET rearrangement may have a therapeutic impact for considering druggable targets.

3

Methods

3.1 Management of RAI-Refractory Thyroid Cancer

3.1.1 Watchful Waiting Under TSH Suppression

The ATA guidelines and the European Thyroid Association guidelines specified four basic principles for managing RAI-refractory thyroid cancers, including watchful waiting, local therapy for localized or symptomatic lesions, systemic treatment with multikinase inhibitors, and clinical trials [4, 11] (see Fig. 1). 1. RAI-refractory DTC can be asymptomatic for several years. Active surveillance and watchful waiting with TSH suppression can be considered for patients with asymptomatic and indolent disease; low tumor burden, or tumor size less than 1 cm; low likelihood of developing rapidly progressive disease; and no adverse impact from disease burden [11]. 2. According to the 2015 ATA guidelines, TSH suppression should be considered below 0.1 mIU/L in patients with RAI-refractory thyroid disease [4]. 3. Careful monitoring of disease activity is crucial when watchful waiting approach is selected in order not to miss the optimal timing to start multikinase inhibitors. 4. Follow-up assessment should be in interval of every 3–6 months and includes checking for potential symptoms, imaging studies for disease evaluation, and blood tests with thyroid function test and thyroglobulin level.

3.1.2 Local Therapy

– Local therapies indicated for RAI-refractory thyroid cancer is determined by the location of the disease (e.g. cervical lymph nodes, brain, bones, aerodigestive tract, lung, etc.), number of

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Fig. 1 Suggested flowchart for management of RAI-refractory thyroid carcinoma

lesions, tumor burden, and technical feasibility. Main local therapies include surgery, external beam radiotherapy (EBRT), radiofrequency ablation, ethanol ablation, and laser ablation [12]. – For example, for locoregional recurrence, surgery with central or lateral neck dissection is the first choice of treatment. In patients with lung metastases, laser ablation, metastasectomy, or EBRT can be considered to palliate symptoms like cough or bleeding. In patients with bone metastases, particularly those with isolated progressive or symptomatic metastases impending fracture, surgery with external beam radiotherapy can be used to relieve pain and maintain functional status [13]. Bonemodifying agents like denosumab or bisphosphonate are recommended to delay time of occurrence of skeletal-related events, including fracture, pain, and neurological complications. In patients with brain metastasis, radiotherapy (stereotactic radiosurgery or whole-brain radiotherapy) with or without prior surgery is often used, and the decision depends on the size, number, and location of the brain metastasis [14].

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– For patients with RAI-refractory DTC, systemic treatment with multikinase inhibitors is a standard option when local treatment options have been exhausted. Two multikinase inhibitors (sorafenib, lenvatinib) have been approved for use in RAI-refractory DTC based on their significant improvement in progression-free survival (PFS) in phase III randomized controlled trials (DECISION and SELECT trials) [15, 16]. – Despite the prognostic significance in PFS, the overall survival (OS) benefit has not clearly been proven compared with placebo for either sorafenib or lenvatinib. Moreover, both multikinase inhibitors are accompanied by side effects compromising patients’ quality of life. The risks and benefits of the multikinase inhibitors should be weighted before their introduction.

3.2 When to Start Systemic Treatment? 3.2.1 Recommendations from International Guidelines

– Several guidelines have suggested parameters to determine when to initiate TKI treatment [4, 11, 17, 18]. Common themes in these guidelines include symptomatic patients with progressive disease or asymptomatic patients with considerable tumor load or with progressive disease which might cause complications soon if systemic treatment is not given. Details about the recommendations of different guidelines are shown in Table 1.

Table 1 Recommendation from different international guidelines on initiation of systemic treatment in radioactive refractory differentiated thyroid cancer Guideline

Recommendation

National Comprehensive Cancer Network (NCCN)

Patients with rapidly growing lesions or symptomatic disease

American Thyroid Association (ATA)

Patients with metastatic, rapidly progressive, symptomatic, and/or imminently threatening disease not otherwise amenable to local control using other approaches

European Society for Medical Oncology (ESMO)

Patients with symptomatic disease with multiple lesions or asymptomatic progressive disease with multiple lesions

European Thyroid Association

Patients with progressive RAI-refractory disease, with considerable tumor load, and when refraining from treatment would lead to considerable harm/ clinical complications within the near future Patient-related medical factors (age, health status, comorbidities, and contraindications) and patient preferences should be considered with respect to treatment goals and values and acceptance of adverse effects

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Table 2 Checklist of factors needed to consider for initiation of multikinase inhibitors for RAI-refractory thyroid cancer Factors

Favor starting multikinase inhibitor

Favor watchful waiting

Tumor-related symptoms

Symptomatic

Asymptomatic

Tumor progression

Within 12 months

>12 months

Tumor location

Close to major vessels, bronchi, esophagus

Metastatic site

Metastasis to the bone, liver, and brain

Tumor load

Lung metastasis
10 mm Sum of dimensions of all measurable target lesions
42 mm

Lung metastasis
10 mm Sum of dimensions of all measurable target lesions 65 years, the median overall survival (OS) was significantly longer in the lenvatinib arm than the placebo arm (HR 0.53, 95% CI ¼ 0.31–0.91, p ¼ 0.02) [24]. The incidence of grade
3 adverse events of lenvatinib was significantly higher in older patients than in younger patients (88.7% vs. 67.1%; p < 0.001). Older patients also had higher incidences of dose reductions, dose interruptions, and treatment discontinuations. This indicated that careful management of adverse events should be practiced when starting lenvatinib in older patients. (c) Thyroglobulin and thyroglobulin doubling time Baseline thyroglobulin is not a marker for starting multikinase inhibitor but is a significant prognostic factor for RAI-refractory thyroid cancer [25]. Retrospective studies demonstrated that a shorter doubling time of Tg (Tg-DT) is associated with more rapidly growing tumor [9, 26, 27]. Patients with Tg-DT less than 1 year had a significantly shorter survival compared with patients with Tg-DT over 1 year. Tg-DT less than 1 year may be a possible indicator for starting systemic treatment in asymptomatic patients with progressive disease. (d) Tumor load Previous studies showed that tumor size was a prognostic factor for RAI-refractory thyroid cancer [28]. Larger tumor volume is associated with poor blood supply and elevated interstitial pressure, leading to a hypoxic microenvironment in the tumor. Tumor hypoxia may be involved in drug resistance to anti-VEGF therapy including multikinase inhibitors. In a post hoc analysis of the SELECT trial, for patients with lung metastasis
10 mm, median survival was longer in the lenvatinib arm compared with the placebo arm (44.7 months vs. 33.1 months, HR 0.63, 95% CI 0.37–0.85, p ¼ 0.025) [29]. Another retrospective study also demonstrated that patients with baseline tumor size (defined as the sum of the longest dimensions of all measurable target lesions) less than 42 mm had a better outcome in terms of PFS and OS on lenvatinib compared with those with larger tumor size (
42 mm) [30]. These studies suggested that treatment may need to be started with the lung metastases progress up to > 10 mm or when the sum of the target lesions is up to approximately 40 mm.

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(e) Site of metastasis Different tumor locations reflect different needs for TKI therapy. Tumors located near major vessels, such as the carotid artery, pulmonary artery, and aortic arch, are at a higher risk of tumor invasion if the watchful waiting approach were to be adopted. Tumors near other organs such as the esophagus, bronchi, and skin will face an increased risk of treatment-related fistula if TKI were not initiated. In addition, there is no definite relationship identified between metastatic sites and efficacy of multikinase inhibitors, but bone, liver, and brain metastases can be less responsive to treatment and shorter duration of response [31]. Patients with metastasis to these sites often have worse prognosis and might be considered for earlier systemic treatment. 2. Novel treatment targeting BRAF – BRAF V600E is one of the most common mutations in papillary thyroid carcinoma [32]. Although papillary thyroid cancer overall has a low risk of death from the cancer and is usually slow growing, the presence of the BRAF V600E mutation in the cancer predicts a faster rate of growth and spread and a higher risk of death. BRAF inhibitors such as vemurafenib or dabrafenib have been studied in phase I and II studies for the management of PTC. In an open-label, non-randomized, phase 2 trial which included 51 patients with histologically confirmed recurrent or metastatic papillary thyroid cancer refractory to radioactive iodine and positive for the BRAF V600E mutation, vemurafenib demonstrated an overall response rate of 38.5% [33]. Grade 3 or grade 4 adverse events were recorded in 65% of patients, and the most common grade 3 and 4 adverse events were squamous cell carcinoma of the skin (20–27%), lymphopenia, (8%), and increased γ-glutamyl transferase (4–12%). – Dabrafenib is a reversible and potent adenosine triphosphate (ATP)-competitive inhibitor that selectively inhibits the BRAF V600E kinase. In another phase 1 study with subgroup analysis on 14 patients with BRAF V600E mutant thyroid carcinoma, dabrafenib showed a response rate of 29%, and the median PFS was 11.3 months [34]. 3. Novel treatment targeting NTRK – NTRK fusions have been reported in PTC, Hu¨rthle cell thyroid carcinoma, poorly differentiated thyroid carcinoma, and anaplastic thyroid cancer, and the prevalence ranges from 2.3% to 3.4% of thyroid carcinomas. Recently, there has been a growing interest in testing and characterizing

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NTRK fusion genes because they are therapeutically targetable. NTRK fusion-positive tumors are sensitive to TRK inhibitors, such as larotrectinib and entrectinib, which appear to be well tolerated and effective. – Larotrectinib is a selective inhibitor of tropomyosin receptor kinase (TRKA, TRKB, and TRKC) which has been approved by the Food and Drug Administration (FDA) in the USA for treatment of solid tumors with NTRK fusion. In phase I and II clinical trials, larotrectinib showed marked and durable antitumor activity in patients with NTRK-positive tumors. The overall response rate was unremarkably high (75%) [35]. At a median follow-up of 9.4 months, 86% of the patients with a response (38 of 44 patients) were continuing treatment or had undergone surgery that was intended to be curative. Most of the adverse events reported in the primary analysis were grade 1 and grade 2, with the most common being elevated liver enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (42%), fatigue (36%), vomiting (33%), dizziness (31%), nausea (31%), diarrhea (29%), and anemia (29%). – Entrectinib is another selective inhibitor of TRKA, TRKB, and TRKC that also inhibits ALK and ROS1 tyrosine kinases. In phase 1 and 2 clinical trials which involved 54 adults with advanced or metastatic NTRK fusionpositive solid tumors, entrectinib attained an objective response rate of 57% (complete response 7% and partial response 50%) and median duration of response of 12.9 months [36]. The most common adverse events included dysgeusia (43%), dizziness (33%), constipation (33%), diarrhea (28%), and weight increase (26%). 4. Novel treatment targeting RET – RET kinase fusions occur in