Liver Fluke, Opisthorchis viverrini Related Cholangiocarcinoma: Liver Fluke Related Cholangiocarcinoma (Recent Results in Cancer Research, 219) 3031351657, 9783031351655

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Recent Results in Cancer Research Series Editor: Heike Allgayer

Narong Khuntikeo Ross H. Andrews Trevor N. Petney Shahid A. Khan   Editors

Liver Fluke, Opisthorchis viverrini Related Cholangiocarcinoma Liver Fluke Related Cholangiocarcinoma

Recent Results in Cancer Research Volume 219 Series Editor Heike Allgayer, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Baden-Württemberg, Germany

This book series presents comprehensive, high-quality updates on areas of current interest in basic, clinical, and translational cancer research. The scope of the series is broad, encompassing epidemiology, etiology, pathophysiology, prevention, diagnosis, and treatment. Each volume is devoted to a specific topic with the aim of providing readers with a thorough overview by acclaimed experts. While advances in understanding of the cellular, genetic, and molecular mechanisms of cancer and progress toward personalized cancer care are a particular focus, subjects such as the lifestyle, psychological, and social aspects of cancer and public policy are also covered. Recent Results in Cancer Research is accordingly of interest to a wide spectrum of researchers, clinicians, other health care professionals, and stakeholders. The series is listed in PubMed/Index Medicus.

Narong Khuntikeo · Ross H. Andrews · Trevor N. Petney · Shahid A. Khan Editors

Liver Fluke, Opisthorchis viverrini Related Cholangiocarcinoma Liver Fluke Related Cholangiocarcinoma

Editors Narong Khuntikeo Department of Surgery Khon Kaen University Khon Kaen, Thailand Trevor N. Petney Karlsruh Erbprinzenstr State Museum of Natural History Karlsruhe Karlsruhe, Baden-Württemberg, Germany

Ross H. Andrews Faculty of Medicine Cholangiocarcinoma Research Institute Khon Kaen University Khon Kaen, Thailand Shahid A. Khan Division of Digestive Diseases Faculty of Medicine Imperial College London London, UK

ISSN 0080-0015 ISSN 2197-6767 (electronic) Recent Results in Cancer Research ISBN 978-3-031-35165-5 ISBN 978-3-031-35166-2 (eBook) https://doi.org/10.1007/978-3-031-35166-2 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 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. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Contents

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2

3

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Narong Khuntikeo, Ross H. Andrews, Trevor N. Petney, and Shahid A. Khan References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Opisthorchis viverrini Life Cycle, Distribution, Systematics, and Population Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Weerachai Saijuntha, Ross H. Andrews, Paiboon Sithithaworn, and Trevor N. Petney 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1 Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2 Life Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3 Source of Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Systematics and Population Genetics . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1 The Liver Fluke O. viverrini . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2 The Bithynia Snail Host . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Biological and Morphological Variations . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epidemiology and Control of Opisthorchis viverrini Infection: Implications for Cholangiocarcinoma Prevention . . . . . . . . . . . . . . . . . . . Narong Khuntikeo, Bandit Thinkhamrop, Thomas Crellen, Chatanun Eamudomkarn, Trevor N. Petney, Ross H. Andrews, and Paiboon Sithithaworn 3.1 Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Tools for Screening, Surveillance, and Control Program . . . . . . 3.3.1 Isan Cohort . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2 New Urine Assay for OV Screening . . . . . . . . . . . . . . . . . 3.3.3 Teleconsultation Ultrasonography . . . . . . . . . . . . . . . . . . . . 3.4 Intervention and Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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8 9 9 10 11 12 16 16 18 20 21 27

28 29 30 31 31 33 33

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3.4.1

The Primary Prevention Program: Food Safety, School-Based Health Education, and Screening of O. viverrini . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.2 Secondary Prevention Program . . . . . . . . . . . . . . . . . . . . . . 3.4.3 Tertiary Patient Care Program: Confirmation and Management of Suspected CCA . . . . . . . . . . . . . . . . . 3.5 Past and Current Control Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1 Education . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2 Medication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.3 Sanitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.4 Comprehensive Control Program . . . . . . . . . . . . . . . . . . . . 3.6 Modeling for Control of O. viverrini Related Pathology and Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 Progress Toward Control of OV and CCA . . . . . . . . . . . . . . . . . . . . 3.8 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

The Hallmarks of Liver Fluke Related Cholangiocarcinoma: Insight into Drug Target Possibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Watcharin Loilome, Nisana Namwat, Apinya Jusakul, Anchalee Techasen, Poramate Klanrit, Jutarop Phetcharaburanin, and Arporn Wangwiwatsin 4.1 Host–Parasite Interactions: Chronic Infection-Induced Inflammation-Related CCA Development . . . . . . . . . . . . . . . . . . . . 4.2 Hallmarks of Ov-Related CCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Sustaining Proliferative Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Evading Growth Suppressors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 Resisting Cell Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6 Enabling Replicative Immortality . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7 Inducing Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.8 Activation of Invasion and Metastasis . . . . . . . . . . . . . . . . . . . . . . . . 4.9 Enabling Characteristics and Emerging Hallmarks . . . . . . . . . . . . 4.9.1 Genome Instability and Mutation . . . . . . . . . . . . . . . . . . . . 4.9.2 Tumor-Promoting Inflammation . . . . . . . . . . . . . . . . . . . . . . 4.9.3 Reprogramming Energy Metabolism . . . . . . . . . . . . . . . . . 4.9.4 Evading Immune Destruction . . . . . . . . . . . . . . . . . . . . . . . . 4.9.5 The Tumor Microenvironment . . . . . . . . . . . . . . . . . . . . . . . 4.10 Molecular Heterogeneity and Therapeutic Opportunities . . . . . . 4.10.1 Genetic Profiling of iCCA and Its Clinical Implication in Targeted Therapy . . . . . . . . . . . . . . . . . . . . . 4.10.2 Genetic Profiling of eCCA and Its Clinical Implication in Targeted Therapy . . . . . . . . . . . . . . . . . . . . . 4.11 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

33 35 35 37 37 38 38 39 39 43 45 45 53

54 55 55 58 59 61 61 63 66 66 67 68 69 70 72 72 74 76 79

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Pathology of Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Supinda Koonmee, Prakasit Sa-ngiamwibool, Chaiwat Aphivatanasiri, Waritta Kunprom, Piyapharom Intarawichian, Walailak Bamrungkit, Sakkarn Sangkhamanon, and Malinee Thanee 5.1 Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Spreading Pattern of Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . 5.3 Surgery and Residual Tumor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6 Intrahepatic Cholangiocarcinoma; iCCA . . . . . . . . . . . . . . . . . . . . . . 5.7 Macroscopic Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.8 Histopathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.9 Cancer Staging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.10 Extrahepatic Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.11 Macroscopic Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.12 Histopathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.13 Cancer Staging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.14 Molecular Pathology and Specimen Handling . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . New Imaging Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nittaya Chamadol, Richard Syms, Vallop Laopaiboon, Julaluck Promsorn, and Kulyada Eurboonyanun 6.1 Imaging of Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.1 The Role of Imaging in CCA Diagnosis . . . . . . . . . . . . . 6.1.2 Anatomical and Morphological Classification . . . . . . . . 6.1.3 Ultrasound Screening of CCA . . . . . . . . . . . . . . . . . . . . . . . 6.1.4 Ultrasound Findings of MF-CCA . . . . . . . . . . . . . . . . . . . . 6.1.5 Ultrasound Findings of PI-CCA . . . . . . . . . . . . . . . . . . . . . 6.1.6 Ultrasound Findings of ID-CCA . . . . . . . . . . . . . . . . . . . . . 6.1.7 Use of CT in CCA Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . 6.1.8 Imaging Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.9 Diagnosis of Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . 6.1.10 Perihilar Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . 6.1.11 Intrahepatic Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . 6.1.12 Distal Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.13 Staging and Treatment Planning . . . . . . . . . . . . . . . . . . . . . 6.1.14 CT Volumetry and Estimation of Future Liver Remnant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.15 Post-treatment Follow-Up and Surveillance . . . . . . . . . . 6.1.16 MRI Imaging of Cholangiocarcinoma . . . . . . . . . . . . . . . . 6.1.17 Positron Emission Tomography (PET)/CT of Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.18 Magnetic Resonance Imaging and Relaxometry . . . . . .

93 94 94 95 95 96 96 97 99 101 102 102 103 106 106 109

110 110 110 111 114 115 116 117 120 120 121 122 124 125 126 127 127 132 132

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6.1.19 Limitations to Contrast and Resolution . . . . . . . . . . . . . . . 6.1.20 Internal Receivers for CCA Imaging . . . . . . . . . . . . . . . . . 6.1.21 In Vitro Imaging of CCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

135 135 137 140

Surgery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Narong Khuntikeo, Ake Pugkhem, Tharatip Srisuk, Vor Luvira, Attapol Titapun, Theerawee Tipwaratorn, Vasin Thanasukarn, Vivian Klungboonkrong, and Jitraporn Wongwiwatchai 7.1 Part I: Surgical Anatomy of the Liver and Biliary Tract . . . . . . . 7.1.1 Liver Anatomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.2 Pancreas Anatomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Part II Pre-operative Treatment for CCA . . . . . . . . . . . . . . . . . . . . . 7.2.1 Clinical Presentation for Each Type in Different Symptoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.2 Liver Function Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.3 Volume Assessment Method . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.4 Pre-operative Biliary Drainage . . . . . . . . . . . . . . . . . . . . . . . 7.2.5 Portal Vein Embolization . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3 Part III: Surgery for Intrahepatic Cholangiocarcinoma . . . . . . . . 7.3.1 General Consideration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2 Patient Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.3 Margin of Resection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.4 Role of Lymph Node Dissection . . . . . . . . . . . . . . . . . . . . . 7.3.5 Vascular Resection for iCCA . . . . . . . . . . . . . . . . . . . . . . . . 7.3.6 Resection After Neoadjuvant Treatment . . . . . . . . . . . . . . 7.3.7 Resection of Tumor Recurrence . . . . . . . . . . . . . . . . . . . . . 7.3.8 Special Consideration for Intraductal Growth Subtype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4 Part IV: Surgery for Perihilar Cholangiocarcinoma . . . . . . . . . . . . 7.4.1 Definition of Perihilar Cholangiocarcinoma . . . . . . . . . . 7.4.2 Assessment of Curative Resectability . . . . . . . . . . . . . . . . 7.4.3 Surgical Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4.4 Left and Right Trisectionectomy . . . . . . . . . . . . . . . . . . . . . 7.4.5 Hepatopancreatoduodenectomy (HPD) . . . . . . . . . . . . . . . 7.5 Part V: Surgical Treatment in Distal Cholangiocarcinoma . . . . . 7.5.1 Assessment of Resectability . . . . . . . . . . . . . . . . . . . . . . . . . 7.5.2 Arterial Approach Pancreaticoduodenectomy . . . . . . . . . 7.5.3 Extended Lymphadenectomy . . . . . . . . . . . . . . . . . . . . . . . . 7.5.4 Resection Part . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5.5 Reconstruction Part . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6 Part VI: Minimally Invasive Surgery in Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6.1 Intrahepatic Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . 7.6.2 Distal Bile Duct Cholangiocarcinoma . . . . . . . . . . . . . . . .

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Part VII: Role of Liver Transplantation in Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7.2 Hilar Cholangiocarcinoma and Liver Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7.3 Liver Transplantation Versus Liver Resection . . . . . . . . 7.7.4 Intrahepatic Cholangiocarcinoma and Liver Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Systemic Treatment for Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . Aumkhae Sookprasert, Kosin Wirasorn, Jarin Chindaprasirt, Piyakarn Watcharenwong, Thanachai Sanlung, and Siraphong Putraveephong 8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2 Neoadjuvant Treatment in Cholangiocarcinoma . . . . . . . . . . . . . . . 8.2.1 Resectability Criteria and Rationale of Neoadjuvant Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.2 Clinical Data and Ongoing Data . . . . . . . . . . . . . . . . . . . . . 8.2.3 Ongoing Clinical Trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.4 Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3 Adjuvant Systemic Treatment in Cholangiocarcinoma . . . . . . . . 8.4 Systemic Treatment for Advanced or Unresectable Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4.1 Chemotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4.2 Second-Line Chemotherapy . . . . . . . . . . . . . . . . . . . . . . . . . 8.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Palliative Care in Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Attakorn Raksasataya, Anucha Ahooja, Vivian Krangbunkrong, Apiwat Jareanrat, Attapol Titapun, and Narong Khuntikeo 9.1 General Principles in Palliative Care . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.1 Definition of Palliative Care . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.2 Model of Care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.3 Palliative Care Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.4 Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.5 Symptom Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.6 Pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.7 Dyspnea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.8 Nausea and Vomiting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.9 Communication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.10 Bereavement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.11 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2 Palliative Biliary Drainage for Advance Stage Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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9.2.1 9.2.2 9.2.3

Percutaneous Palliative Biliary Drainage . . . . . . . . . . . . . Palliative PTBD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Complication of Percutaneous Transhepatic Biliary Drainage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.4 Hemorrhage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.5 Pericatheter Leakage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.6 Palliative PTBS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.7 Planning and Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.8 Indications for PTBS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.9 Contraindication for PTBS . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.10 Instruments and Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.11 Complications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.12 Endoscopic Biliary Stenting . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.13 Type of Metallic Biliary Stent . . . . . . . . . . . . . . . . . . . . . . . 9.2.14 Fully Covered Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.15 Partly Covered Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.16 Uncovered Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.17 Outcome of SEMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.18 Surgical Biloenteric Bypass . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.19 Operative Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.20 Right Sided Hepaticojejunostomy . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Digital Innovations (Isan Cohort) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bandit Thinkhamrop, Kavin Thinkhamrop, Chaiwat Tawarungrueng, and Panuwat Prathumkham 10.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3 OV-CCA Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4 Tele-Radiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5 Pathology Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6 Surgery Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.7 Palliative Care Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.8 Randomized Controlled Trial (RCT) Database . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 RAW ATTITUDES: Socio-Cultures, Altered Landscapes, and Changing Perceptions of an Underestimated Disease . . . . . . . . . . . Carl Grundy-Warr, Ross H. Andrews, Narong Khuntikeo, and Trevor N. Petney 11.1 An Ecologically Embedded and Socially Entwined Life Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 An Underestimated and Neglected Parasite in World Public Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3 Socio-Economic Dimensions of Disease . . . . . . . . . . . . . . . . . . . . . . 11.4 Developmental Landscapes and Anthropogenic Ecologies . . . . .

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11.4.1 Aquaculture and Irrigation . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.4.2 Dams and Reservoirs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.4.3 Roads and Rivers: Migration and Mobility . . . . . . . . . . . 11.5 Uneven Regional Knowledge About the Extent of Opisthorchis viverrini and Opisthorchiasis . . . . . . . . . . . . . . . . . 11.6 Public Health Programs Tackling OV and CCA in Thailand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.7 Raw Attitudes in Isan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.8 Raw Attitudes in Lao PDR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.9 Raw Attitudes in Vietnam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.10 Raw Attitudes in Cambodia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.11 Altering Attitudes and Public Health Approaches . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Community Awareness and Education: In the West and Southeast Asia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Helen Morement and Narong Khuntikeo 12.1 Southeast Asia—Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2 The UK and Europe—Background . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3 How Can the CCA Challenges in Southeast Asia and the West Be Overcome? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3.1 Raising Awareness and Understanding in Southeast Asia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.4 Combatting Cholangiocarcinoma in the Lao People’s Democratic Republic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.5 The Challenges Faced by Healthcare Professionals in Southeast Asia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.6 The Challenges Faced by Cholangiocarcinoma Patients in Southeast Asia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.7 Raising Awareness and Understanding in the West . . . . . . . . . . . 12.8 The Challenges Faced by Healthcare Professionals in the West . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.9 The Challenges Faced by CCA Patients in the West . . . . . . . . . . 12.10 Molecular Profiling and Targeted Therapies . . . . . . . . . . . . . . . . . . 12.11 How Can the CCA Challenges Be Overcome in Southeast Asia and the West? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.12 In Southeast Asia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.13 In the West . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.14 National and International Bodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.15 Looking to the Future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.16 In the West . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.17 Final Thoughts … . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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13 Synopsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361 Narong Khuntikeo, Ross H. Andrews, Trevor N. Petney, and Shahid A. Khan

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Introduction Narong Khuntikeo, Ross H. Andrews, Trevor N. Petney, and Shahid A. Khan

Cholangiocarcinoma (CCA) is a lethal cancer arising in the bile ducts within and just outside the liver. It occurs worldwide and falls into two etiologically defined groups, one related to chronic liver fluke infection and the other not. Liver fluke related CCA is found in continental Southeast Asia (caused by Opisthorchis viverrini with infection leading to opisthorchiasis), East Asia (Clonorchis sinensis), and Eastern Europe and Russia (Opisthorchis felineus). Both O. viverrini and C.

Dedication This book is dedicated to the millions of people worldwide who have died and are continuing to die from cholangiocarcinoma. This book is also dedicated to the late Professor Narong Khuntikeo a pioneer in the treatment of cholangiocarcinoma, an outstanding surgeon, inspirational leader and mentor who will be sadly missed. N. Khuntikeo (B) Department of Surgery, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand e-mail: [email protected] N. Khuntikeo · R. H. Andrews Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen 40002, Thailand e-mail: [email protected] R. H. Andrews Department of Surgery and Cancer, Faculty of Medicine, Imperial College, London, UK T. N. Petney Departments of Zoology and Paleontology and Evolution, State Museum of Natural History Karlsruhe, Erbprinzenstrasse 13, 76133 Karlsruhe, Germany e-mail: [email protected] S. A. Khan Division of Digestive Diseases, Faculty of Medicine, Imperial College, London, UK e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. Khuntikeo et al. (eds.), Liver Fluke, Opisthorchis viverrini Related Cholangiocarcinoma, Recent Results in Cancer Research 219, https://doi.org/10.1007/978-3-031-35166-2_1

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sinensis are classified as group one carcinogens, while recent data from O. felineus suggest the same. The following estimates of the number of people infected and/or at risk of infection with O. viverrini and developing CCA are most certainly substantially too low as hard data are only available from Thailand. In this context, it has been estimated that in SE Asia 67.3 million people are at risk of infection by O. viverrini and subsequently developing CCA. When the other two liver fluke species are considered, an estimated 700 million people are at risk of infection and developing CCA globally. The northeast of Thailand (Isan) is the world’s hot spot of liver fluke infection and CCA. Currently, available data show that upwards of 20,000 people die of CCA each year, i.e., 56 people/day. In Thailand, 10 million people are infected with O. viverrini, the majority of whom will develop CCA, and once diagnosed will die within three months. Early detection, diagnosis, and surgical intervention/ curative treatment of CCA are critical to increase life expectancy and quality of life of people in the region and globally. Despite concentrated recent efforts focusing on a multidisciplinary approach to understand the ecology, epidemiology, biology, and public health and social significance of infection by cancer-causing liver flukes, it remains an underestimated and under-resourced public health problem. In addition, it is still believed to be a regional problem without global significance—this is not the case. This book focuses on O. viverrini as the main causative agent of CCA in Southeast Asia, but many aspects detailed in the following chapters also relate to the two other liver fluke species. Our aim is to produce a holistic framework including the basic biology of O. viverrini and its relation to the epidemiology of the disease through diagnosis to treatment, including palliative methods, pathology, and control [1–7]. It is for the above reasons that we enlisted the assistance of specialists with many years of multidisciplinary expertise to provide the most up-to-date and comprehensive examination of the liver fluke, O. viverrini related CCA in the following chapters. In Chap. 2, Saijuntha et al. discuss the field biology of O. viverrini including its morphology, life cycle, source of infection, and distribution. They also examine in detail the systematics, population genetics, and population structure of O. viverrini as well as the first intermediate host Bithynia snails, which are the critical amplifying point within the life cycle. Additionally, biological and morphological differences detected in O. viverrini across its distribution are examined. In Chap. 3, Khuntikeo et al. examine the epidemiology and control of O. viverrini infection and its implications for cholangiocarcinoma prevention. Topics covered include the prevalence of infection and incidence of CCA, tools for screening, surveillance and control programs, intervention and solutions, and progress toward control of O. viverrini infection and CCA. In Chap. 4, Loilome et al. discuss the hallmarks of liver fluke related CCA and explore insights into drug target possibilities. Topics discussed include host–parasite interactions, sustaining proliferative signaling, evading growth suppressors,

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resisting cell death, enabling replicative immortality, inducing angiogenesis, activation of invasion and metastasis, processes of epithelial mesenchymal transition, genome instability and mutation, tumor-promoting inflammation, reprogramming energy metabolism, evading immune destruction, the tumor microenvironment, and molecular heterogeneity and therapeutic opportunities. In Chap. 5, Koonmee et al. examine the pathology of CCA in the biliary tract, with topics including surgery and residual tumor, prognosis, biomarkers, intrahepatic and extrahepatic cholangiocarcinoma, and molecular pathology and specimen handling. In Chap. 6, Chamadol et al. consider and discuss the advancement of new imaging techniques, the role of imaging in CCA diagnosis, anatomical and morphological classification, ultrasound screening of CCA, ultrasound findings of MF-CCA, PI-CCA, ID-CCA, the use of CT in CCA diagnosis, staging, and treatment planning, CT volumetry and estimation of future liver remnant, posttreatment follow-up and surveillance, MRI imaging, Positron emission tomography (PET)/CT, limitations to contrast studies and resolution, internal receivers for CCA imaging, and in vitro imaging of CCA. In Chap. 7, Khuntikeo et al. examine in detail the surgical aspects of CCA by considering, surgical anatomy of the liver and biliary tract, preoperative treatments for CCA, the clinical presentation for each type of CCA in different symptoms, surgical procedures for intrahepatic CCA, surgery for perihilar CCA, surgical treatment in distal CCA, minimally invasive surgery in CCA, and the role of liver transplantation in CCA. In Chap. 8, Sookprasert et al. examine systemic treatments for CCA including neoadjuvant treatment, adjuvant systemic therapy, systemic treatment for advanced or unresectable CCA, first- and second-line chemotherapy, targeted therapy, tumoragnostic therapy, and immunotherapy. In Chap. 9, the role of palliative care in CCA is discussed by Raksasataya et al. in which they detail the general principles of palliative care, models of care, palliative care criteria, assessment, symptom management, communication, palliative biliary drainage for advanced CCA, endoscopic biliary stenting, and surgical bilioenteric bypass. In Chap. 10, innovations in digital procedures are discussed and highlighted by Thinkhamrop et al. who provide comprehensive background and an overview of the Cholangiocarcinoma Screening and Care Program (CASCAP), and the innovative database known as the Isan cohort, including the O. viverrini—CCA module, tele-radiology, databases for pathology, surgery, palliative care, and a randomized controlled trial database. In Chap. 11, Grundy-Warr et al. examine “raw attitudes” and sociocultures, altered landscapes, and the changing perceptions of an underestimated disease, opisthorchiasis, and CCA. They explore topics such as an ecologically embedded and socially entwined life cycle, an underestimated and neglected parasite in world public health, socio-economic dimensions of disease, developmental landscapes and anthropogenic ecologies, aquaculture and irrigation, dams and reservoirs, roads and rivers, migration and mobility, uneven regional knowledge about the

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extent of O. viverrini infection and opisthorchiasis, public health programs tackling O. viverrini and CCA in Thailand, raw attitudes in Isan, Lao PDR, Vietnam and in Cambodia, and altering attitudes and public health approaches. In Chap. 12, Morement and Khuntikeo examine community awareness and education of CCA in the West (spontaneous CCA in the UK and Europe) and Southeast Asia (parasite related CCA). They provide background of how the challenges in Southeast Asia and the West are being overcome by raising the awareness of CCA and education programs instigated by the CCA Foundation and CASCAP in Thailand and the UK CCA Foundation (the Alan Morement Memorial Foundation, AMMF). Other topics that are considered are the challenges faced by patients and healthcare professionals in the West and Southeast Asia, molecular profiling and targeted therapies, newly formed national and international bodies raising CCA awareness and future possibilities and directions. In Chap. 13, the Synopsis by Khuntikeo et al. highlights advances and future directions in the areas covered by the chapters in this book. These chapters provide up-to-date detailed interrelated multidisciplinary information which is providing the basis and tools necessary to increase life expectancy and the quality of life of millions of people worldwide. Acknowledgements The editors wish to acknowledge the numerous people who have been instrumental to our work and contributed over many years to instigate and foster collaborative biological, medical, and social research into liver fluke O. viverrini related cholangiocarcinoma, as well as associated public health/education programs, and the National Research Council of Thailand (NRCT) for their ongoing support for CCA research and innovation. In particular, we wish to thank and acknowledge two outstanding colleagues. Firstly, we wish to acknowledge Professor Simon Taylor-Robinson, Imperial College, London (ICL), who specializes in diseases of the liver, in particular liver cancer and cholangiocarcinoma (CCA), and who prompted and motivated us to enlist and edit the numerous contributions from colleagues for this unique and critical book which we believe provides the foundation for a comprehensive knowledge base to combat an insidious disease affecting millions of people worldwide. Over a decade ago, as Dean of the Faculty of Medicine at ICL, Simon was the catalyst to initiate and foster many international public health programs in research and training between (ICL) and Khon Kaen University (KKU), Thailand. These programs have been implemented and focus on liver fluke, Opisthorchis viverrini, and related CCA. Importantly, the reciprocal undergraduate, postgraduate, and postdoctoral exchange programs Simon helped establish together with the Cholangiocarcinoma Screening and Care Program (CASCAP) and the Cholangiocarcinoma Research Institute (CARI) have provided the basis for a greater understanding and awareness globally of the enormous public health problems that O. viverrini related CCA causes. He is the instrumental driving force that underpinned the transfer and implementation from Thailand of the CASCAP to Lao PDR, which is proving very successful in the early screening, diagnosis, treatment, specialist radiology and surgery training and care for CCA patients, thereby enhancing patient survival and quality of life in the region. He has also been instrumental in fostering global collaboration between the CASCAP/CARI, and the Thailand Cholangiocarcinoma Foundation with the UK Cholangiocarcinoma Foundation the Alan Morement Memorial Foundation (AMMF), as well as collaboration with the Global Cholangiocarcinoma Alliance (GCA). It is through such reciprocal collaborations that Professor Simon Taylor-Robinson continues to implement and actively champion that progress has been made in increasing the life expectancy and the quality of life of millions of people who are at risk of infection by liver flukes and resultant CCA, as well as increasing the awareness of the severity and impact that CCA has globally.

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Secondly, we wish to thank and acknowledge the late Professor Puangrat Yongvanit, Khon Kaen University, Thailand, who spent a lifetime of work to help the poor communities in Thailand and elsewhere in the Southeast Asian region to gain knowledge and understanding of opisthorchiasis and CCA, as well as their risk factors. She was singularly focused on increasing the life expectancy and quality of life of predominately the poor people of Thailand and the Mekong Region. Her legacy remains perpetual in the thousands of people she contacted and influenced, from the local poor people in villages and communities in Isan to throughout Thailand and the Mekong Region, as well as inspiring the next generation of young researchers and health professionals. She was a fierce champion for an in-depth holistic understanding of the biochemistry of carcinogenesis, specifically CCA. Equally, she was a fierce and singularly focused champion and pioneer whose lifetime aim was to help her beloved people of the northeast of Thailand, her Isan people and their communities. She was a catalyst for the evolution of ongoing research collaboration between, Khon Kaen University and many other institutions extending regionally and internationally, for instance with ICL, with the AMMF, and with the Global Cholangiocarcinoma Alliance (GCA).

References 1. Saijuntha W, Sithithaworn P, Wongkham S, Laha T, Piptgool V, Tesana S, Chilton NB, Petney TN, Andrews RH (2007) Evidence of a species complex within the food borne trematode Opisthorchis viverrini and possible coevolution with their first intermediate host. Int J Parasitol 37:695–703 2. Andrews RH, Sithithaworn P, Petney TN (2008) Opisthorchis viverrini: an underestimated parasite in world health. Trends Parasitol 24:497–501 3. Grundy-Warr C, Andrews RH, Sithithaworn P, Petney TN, Sripa B, Lathavewat L, Zeigler AD (2012) Raw attitudes, wetland cultures, life-cycles: socio-cultural dynamics relating to Opisthorchis viverrini in the Mekong Basin. Parasitol Int 61:65–70 4. Khuntikeo N, Chamadol N, Yongvanit P, Loilome W, Namwat N, Sithithaworn P, Andrews RH, Petney TN, Promthet S, Thinkhamrop K, Tawarungruang C, Thinkhamrop B (2015) Cohort profile: cholangiocarcinoma screening and care program (CASCAP). BMC Cancer 15:1475–1487 5. Khuntikeo N, Titapun A, Loilome W, Yongvanit P, Thinkhamrop B, Chamadol N, Boonmars T, Nethanomsak T, Andrews RH, Petney TN, Sithithaworn P (2018) Current perspectives on opisthorchiasis control and cholangiocarnoma detection in Southeast Asia. Front Med 5:117 6. Khuntikeo N, Thinkhamrop B, Bundhamcharoen K, Andrews RH, Grundy-Warr C, Yongvanit P, Loilome W, Chamadol N, Kosuwan W, Sithithaworn P, Petney TN (2018) The Socioeconomic burden of cholangiocarnoma associated with Opisthorchis viverrini sensu lato infection in Northeast Thailand: a preliminary analysis. Adv Parasitol 102:141–163 7. Saijuntha W, Sithithaworn P, Kiatsopit N, Andrews RH, Petney TN (2014) Liver flukes: Clonorchis and Opisthorchis. In: Toledo R, Fried B (ed) Digenetic trematodes, Chap 6. Springer, pp 153–199

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Opisthorchis viverrini Life Cycle, Distribution, Systematics, and Population Genetics Weerachai Saijuntha, Ross H. Andrews, Paiboon Sithithaworn, and Trevor N. Petney

Contents 2.1 2.2

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1 Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2 Life Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3 Source of Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Systematics and Population Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1 The Liver Fluke O. viverrini . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2 The Bithynia Snail Host . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Biological and Morphological Variations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8 9 9 10 11 12 16 16 18 20 21

W. Saijuntha (B) Faculty of Medicine, Mahasarakham University, Maha Sarakham 44000, Thailand e-mail: [email protected] R. H. Andrews · P. Sithithaworn Cholangiocarcinoma Research Institute, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand P. Sithithaworn Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand R. H. Andrews Department of Surgery and Cancer, Faculty of Medicine, Imperial College, London, UK T. N. Petney Departments of Zoology and Paleontology and Evolution, State Museum of Natural History Karlsruhe, Erbprinzenstrasse 13, 76133 Karlsruhe, Germany © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. Khuntikeo et al. (eds.), Liver Fluke, Opisthorchis viverrini Related Cholangiocarcinoma, Recent Results in Cancer Research 219, https://doi.org/10.1007/978-3-031-35166-2_2

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Introduction

Opisthorchis viverrini is a medically important foodborne trematode (FBT) causing a major public health issue in many developing countries affecting the health of more than 10 million people in Southeast Asia, particularly Thailand and Lao PDR [3]. It has been classified as a group 1 carcinogen (i.e., a carcinogenic agent) in people causing bile duct cancer, cholangiocarcinoma (CCA) [75]. Although precise prevalence figures are not currently available, it is also known to occur in Cambodia and southern Vietnam [13, 33, 62], with several new foci having been reported in Myanmar [4, 57]. The history of human infection with O. viverrini started with two publications [20, 34] dealing with the same data set describing the infection in prisoners in a jail in Chiang Mai in the north of Thailand [40]. Thereafter, a series of reports began to appear, widely scattered in the literature, extending the known range of the infection to the northeast of Thailand [44] and Lao PDR [5]. The first major, coordinated research project on O. viverrini infection, starting in 1951, was carried out between the Thai Ministry of Public Health in cooperation with the United States Special Technical and Economic Mission to Thailand. In 1955, the project leader, Elvio Sadun, published the first comprehensive paper on the epidemiology of O. viverrini infection in Thailand [48]. The infections of O. viverrini are associated with the consumption of raw or partially cooked cyprinid fish, which are infected with the parasites metacercariae [15, 62] due to intensive control programs [18], in the past 20 years the prevalence has remained at similar levels [41]. Infection rates of O. viverrini in their snail and fish intermediate hosts are still being reported [53]. The habit of eating raw or partially cooked freshwater fish in the northeast region of Thailand has not changed [15, 54, 64]. At present, up to 20,000 people die of Opisthorchis-associated liver cancer (CCA) every year in the northeast of Thailand alone [21]. The liver fluke O. viverrini has a complex life cycle which requires three hosts to complete [55]. The freshwater snails in the genus Bithynia and cyprinid fish act as the first and second intermediate hosts, respectively. Humans are the definitive hosts, whereas rodents, canines, and felines are reservoir hosts [55]. These multiple hosts have been shown to be important factors that drive the biological and morphological variation including genetic variations in parasitic trematodes, including opisthorchiidae [41]. Studies of the systematics and population genetics of the liver fluke O. viverrini and their snail hosts are important to understand the evolutionary processes of their complex life cycle [45]. Thus, biological and morphological variation as well as genetic investigations of O. viverrini alone may not be sufficient to provide answers to the evolutionary processes of this liver fluke. Despite this, investigations of its intermediate hosts (both snails and cyprinid fish) could provide more understanding of the adaptations and evolutionary processes between O. viverrini and its hosts, including humans. Thus, this chapter will focus on the basic biological and ecological information available for the carcinogenic liver fluke O. viverrini including its life cycle, sources of infection and distribution, systematics and population genetics. This

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information can be integrated into control programs for the parasite feeding onto strategies for the prevention of opisthorchiasis.

2.2

Biology

2.2.1 Morphology Opisthorchis viverrini is a hermaphroditic trematode containing both female and male sexual organs, i.e., ovary and testes. Thus, both cross- and self-fertilization can occur. Their body size ranges from 5.5 to 9.55 mm (mean=7.44 mm) in length, by 0.77 to 1.65 mm (mean= 1.47) in width [58, 76]. The body is transparent and flattened with an oral sucker situated anteriorly and a ventral sucker at midbody. The ventral sucker is larger than the oral sucker. Two extra-caecal chains of vitelline glands run between the ventral sucker and the anterior or posterior testes. The ovary is located in the posterior third of the body. The uterus, packed with oval-shaped eggs, loops irregularly between the ventral sucker and the ovary. The three-lobed ovary is located slightly sub-median or median in the posterior third of the body. The seminal receptacle is voluminous, sited posteriorly to the ovary. The testes are large and located one after the other (tandem) with their extremities deeply lobed. They occupy almost the entire posterior body’s width, overlapping both caeca laterally, and extending between the ventral sucker and the testis. The caeca end blinds in the posterior part of the body. Eggs are a yellowish-brown color with an ovoid shape. The size, length, and width are on average 26.80±1.5 and 14.9±0.7 µm. They have an operculum, shoulder, knob, and thickened eggshell. The operculum is large and fits into a broad rim of the eggshell. When passed, the egg contains a well-developed miracidium that is rather asymmetrical in its internal organization [16, 19, 69]. It has been estimated that a single mature worm can produce approximately 50–200 eggs per gram feces (epg) in humans [14, 59]. After a period of intra-molluscan development, free-swimming cercariae are released from the snail in an elongated form with a long tail bearing prominent fin folds and obliquely striated muscles. The oral sucker is well-developed, protractible, and located at the anterior end, whereas the ventral sucker is faintly developed. Proteinaceous tegumental spines, which are arranged transversally, and different kinds of presumed sensory structures cover the cercarial body surface. Two well-pigmented eye spots with rhabdomeric photoreceptors are located near the oral sucker in the anterior part of the body. An extended protonephridial system is connected to a voluminous excretory bladder. The excretory tube opens at the end of the body and does not lead into the tail [1]. Metacercariae are an infective stage that usually has an oval shape contained in a double-walled cyst surrounded by a thick layer of tissue, 201 × 167 µm in average size. The body of a metacercaria is folded within the cyst and frequently appears to be C-shaped. The mature larva moves vigorously at room temperature. The excretory bladder appears as an oval area composed of masses of dark

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granules. The oral and ventral suckers are clearly visible [73, 76]. The definitive or reservoir host ingests the metacercaria by eating undercooked cyprinid fishes which they infect. The metacercaria excysts in the hosts’ duodenum by the action of the gastric juices and trypsin after which it migrates through the ampulla of Vater into the common bile duct, cystic duct, gall bladder, and intrahepatic ducts [39] before finally reaching the distal bile ducts where it attaches to biliary epithelium by using its oral and ventral suckers. The metacercariae then develop into mature worm within 30 days [76].

2.2.2 Life Cycle The life cycle includes snails as primary intermediate hosts, namely Bithynia siamensis sensu lato (i.e., B. s. siamensis and B. s. goniomphalos) and Bithynia funiculata (Fig. 2.1). Hatching of the miracidium occurs only after a suitable snail ingests the egg. The miracidium transforms into a sporocyst and redia in the wall of the snail’s intestine or in other organs. The cercaria that then develops has a pair of eyespots and is packed with delicate bristles and tiny spines. The tail has dorsal and ventral fins (pleurocercous cercaria) [58]. Once shed, the cercaria hangs upside down in the water and slowly sinks to the bottom. When contacting any object, it rapidly swims upward toward the surface and again begins to sink. Even a slight current of water will cause this reaction. Thus, when a fish swims by the cercaria is stimulated to react in a way favoring its contact with its next (second intermediate) host. On touching the epithelium of the fish, the cercaria attaches with its suckers, casts off its tail and bores through the skin, coming to rest and encysting under a scale or in the muscle becoming a metacercaria. Freshwater fish, mostly Cyprinidae, for example Cyclocheilichthys, Barbodes, Henicorhynchus, Puntioplites, and Hampala act as the second intermediate hosts of O. viverrini. Most cyprinid fish in wetland systems have been found

Fig. 2.1 Bithynia snails first intermediate host of the liver fluke Opisthorchis viverrini, Bithynia funiculata (A), and Bithynia siamensis sensu lato (B)

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to be infected with the metacercariae of O. viverrini but some fish species are more susceptible than others [75]. Mammals other than humans that have been found to be infected with adult O. viverrini include dogs and cats. Experimentally, rabbits, hamsters, and guinea pigs are highly susceptible to infection. Perhaps any fish-eating mammals can become infected. Dogs and cats undoubtedly are important reservoir hosts [75]. However, to date their roles in the epidemiology of the liver fluke infection particularly in Thailand are unclear. Humans are infected by eating uncooked, infected fish. Immature flukes excyst in the duodenum and the normal route of juvenile to the liver appears to be by the way of the bile duct. Immature flukes have been found in the liver 10–40 hours after the infection of experimental animals [58]. The worms mature and begin producing eggs in about a month after infection. Adult worms can live several years in humans but the mean life span is probably a few years [61]. Completion of the life cycle occurs when definitive hosts (carnivores and people) are infected by eating raw, fermented, or partially cooked fish containing infective metacercariae. After migrating up the bile ducts they reach sexual maturity (after several weeks) in the bile ducts where they commence laying eggs [47]. An extensive zoonotic definitive host spectrum which includes domestic animals such as cats and dogs, stock animals such as pigs, human followers such as the brown rat (Rattus norvegicus), as well as a wide range of wild fish-eating carnivores [40]. The zoonotic cycle of O. viverrini, however, involves few carnivores, predominately domestic cats and dogs [40]. A complex life cycle of O. viverrini is shown in Fig. 2.2.

2.2.3 Source of Infections Metacercariae are the infective stages of O. viverrini found in the second intermediate hosts (Fig. 2.3). Transmission to people and animal hosts occurs when infected fish are eaten raw, fermented, or partially cooked [; see Chap. 11]. In Southeast Asia, particularly Thailand and Lao PDR, raw, fermented, or partially cooked fish dishes (Fig. 152.4), the source of the liver fluke infection, can be grouped into three categories, i.e., raw fish, fermented fish, and partial cooked or undercooked fish. Fresh raw fish dishes “koi pla,” “soi pla,” and “lap pla” pose a high risk of infection. Quickly fermented dishes (1–2 days) known as “pla som,” which are usually produced by using medium to large size of fish with a marinating medium, and “pla jom” (some local people call this dish as “som pla noi”), usually using small fish for fermentation, pose a moderate risk of infection. There are also fermented fish dishes (pla ra) which normally require long-term fermentation. Short-term and variable ingredients, however, may provide favorable environments for metacercarial survival. “Pla ra” in Thailand or “pla dak” (as it is known in Lao PDR and northeast Thailand) is a common ingredient in many dishes in Southeast Asia, for example in papaya salad (“som tum”) [15, 54]. Another risk dish is grilled fish (ping pla), which has a chance of accidentally being only partially

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Fig. 2.2 Life cycle of O. viverrini

cooked, leading to the presence of viable metacercariae and subsequent of infection [55]. In other Southeast Asian countries, for instance Cambodia, shorted-term fermented fish are prepared as “pla hoc” which is similar to “pla som,” and “goi ca mai” (raw fish salad), slices of raw silver carp in Vietnam may serve as a source of infection.

2.3

Distribution

Opisthorchiasis caused by the O. viverrini is currently being reported in several countries in Southeast Asia, namely Thailand, Lao PDR, Cambodia, Vietnam, and Myanmar, with greater than 200 million people currently at risk of infection, and

Fig. 2.3 Four common genera of cyprinid fish commonly found infected with O. viverrini, (A) genus Henicorhynchus; (B) genus Puntioplites; (C) genus Barbodes; (D) genus Hampala. Photos courtesy by Komgrit Wongpakam

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Fig. 2.4 Sources of human infection by eating raw, partially cooked, and fermented cyprinid fish. Photos by Weerachai Saijuntha

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at least 12.39 million persons are currently estimated to be infected by O. viverrini. The highest prevalence of O. viverrini was in Thailand with an estimated 6.71 million people known to be infected, most of whom live in the northeastern region [66]. There are a further 2.45, 2.07, and 1.00 million people who are estimated to be infected in Lao PDR, Vietnam, and Cambodia, respectively [56], with no information available from Myanmar [77]. A high prevalence of opisthorchiasis was predominantly found in northeast Thailand, Lao PDR, Cambodia, central-southern Vietnam where raw fish or undercooked fish dishes are frequently consumed [15, 53–55, 76 Studies on the epidemiology of opisthorchiasis in northeast Thailand in the last 20 years ago have revealed that the prevalence was 18.57% with the highest prevalence found in Nakhon Phanom Province (56.25%). The lowest prevalence rate was found in Nakhon Ratchasima Province (5.2%), but one-third of prevalences were higher than 20% even after a successful medical prevention strategy [17]. More recently [65] indicates that O. viverrini infection rates range between 2.1 and 71.0% (mean 25%) in different districts within Khon Kaen Province. A current survey of opisthorchiasis in Nakhon Phanom Province has found the prevalence of O. viverrini infection at an average of 12.9% [11]. In the northern region, the prevalence of O. viverrini infection was 23.3% in Lampang Province [72]. Studies conducted during 2002 to 2009 in a rural community of Sanamchaikaet District, Chachoengsao Province in the central region of Thailand have shown that the prevalence of Opisthorchis-like eggs was from 17.4 to 21.3% [6]. Low prevalence of O. viverrini infection has been found in Bithynia snails, but the prevalence in infected fish is very high. Typically, a low prevalence (< 1%) of infection in snail intermediate hosts was found. Such a very low prevalence of O. viverrini cercariae in Bithynia siamensis sensu lato snails from the canal network system in the Bangkok Metropolitan Region of Thailand has also been reported [46]. Recently, [27] have also reported that a low prevalence of O. viverrini infection (0.71%) exists in B. s. goniomphalos in northeast Thailand. However, there were exceptions in some areas where high infection rates of O. viverrini in Bithynia snails were detected, e.g., the highest prevalence levels per locality were 6.93% and 8.37% in Thailand and Lao PDR, respectively [23]. Examination of the emergence patterns of O. viverrini cercariae in B. s. goniomphalos from Lao PDR revealed that peak cercarial emergence was not consistent in different seasons, occurring between 08.00 and 10.00 h during the hot-dry season, and between 13.00 and 14.00 h during the rainy and cool-dry seasons. The cercarial output was highest in the hot-dry season. The prevalence of infection and the emergence of cercariae were strongly dependent on snail size [25]. There are at least 40 species belonged to 18 genera of cyprinid fish that have been recognized as second intermediate hosts of O. viverrini [53]. The most common species of cyprinid fish are Babonimus, Cyclocheilichthys, Henicorhynchus, and Hampala (Fig. 2.3) [32, 53, 75]. The intensity of O. viverrini infection in fish varies by season, type of water body, species, and individual of fish [60, 70, 74]. Additionally, the metacercarial burden peaks in winter between October to February and dips during the rainy and summer seasons [37, 60]. The

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infection was associated with fish body size and was predominantly found in Hampala dispar (86.5%), Cyclocheilichthys armatus (73.2%), and Puntius brevis (42.7%) in Thailand [37]. More recently, five cyprinid species of fish were examined in northeast Thailand, all species were positive for O. viverrini metacercariae, for instance, Henicorhynchus siamensis (13.7%), Cyclocheilichthys spp. (12.7%), Hampala spp. (8.1%), Systomus spp. (6.9%), and Barbonymus gonionotus (5.0%) [10]. A survey of O. viverrini metacercariae infection in 18 species of cyprinid fish in Khammouane Province, Lao PDR found that eight species were infected, namely Cyclocheilichthys repasson (58.5%), C. armatus (43.1%), C. enoplos (10.0%), Dangila lineata (69.6%), Henicorhynchus lineatus (42.9%), Hampala dispar (44.4%), Puntioplites proctzysron (26.8%), and Osteochilus waandersii (30.5%) [35]. Interestingly, 17 different species of cyprinid fish collected in Kandal Province, Cambodia, were found to be infected with metacercariae of O. viverrini, ranging from a minimum of 5.6% in Crossocheilus reticulatus to 100% in Amblyrhynchichthys truncates [70]. The findings that seasonality, sampling locality, fish size, and species of fish play roles in the risk of O. viverrini infection imply that these host and environmental factors are important for transmission dynamics and the control of O. viverrini.

2.4

Systematics and Population Genetics

2.4.1 The Liver Fluke O. viverrini Evidence of genetic variation of populations of O. viverrini was initially recorded by Sueblinvong et al. [67]. Adult worms of O. viverrini which were collected from an autopsy and CCA patients in Srinagarind Hospital, Khon Kaen, northeast Thailand, were examined by the multilocus enzyme electrophoresis (MEE) technique. Three enzyme markers, glucose-6-phosphate dehydrogenase (G6PD), glucose phosphate isomerase (GPI), and phosphoglucomutase (PGM) exhibited 16 banding patterns among 109 individual worms. However, genetic variation of O. viverrini was not examined again until 2001, following advances in molecular biology, and at this time, it was based on DNA markers [2]. Genetic variation of adult O. viverrini from different geographical localities including an autopsy in northeast Thailand was examined at the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene and internal transcribed spacer 2 (ITS2). Intra- and interspecific genetic variations of the CO1 sequence were detected, and five haplotypes were defined based on four variable nucleotide sites. Analyses of the ITS2 sequences on the other hand were found to be identical among all samples [2]. Following these initial studies, the systematics and population genetic variation of O. viverrini have been examined progressively with initial evidence being provided by Saijuntha et al. [49] that O. viverrini is not a single species but consists of a species complex. For instance, defined allozyme markers provided by Saijuntha et al. [49] have been used to characterize a geographical isolate from Savannakhet Province, Lao PDR [22]. The Savannakhet isolate from Lao PDR clustered closely

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with an O. viverrini population from Vientiane Province, Lao PDR. Interestingly, the Savannakhet isolate also clustered with populations from the Songkhram River, Thailand, which is across from the Mekong River. Data from these analyses provides support for the initial data provided by Saijuntha et al. [49] that a O. viverrini is indeed species complex. Other studies have consecutively utilized independent DNA markers and mitochondrial CO1 and ND1 sequences to analyze the genetic variation of O. viverrini between 14 different geographical isolates in Thailand and Lao PDR [50]. Nucleotide variation was observed to be low and varied between 0 and 0.3% among O. viverrini isolates, and hence, they were not suitable to reveal the genetic structure of O. viverrini related to isolates from geographical localities. Nevertheless, a single marker, a partial sequence of ND1, was suitable and used to examine the genetic structure of six populations of O. viverrini from Thailand, Lao PDR, and Cambodia [71]. Results showed no significant differences among populations analyzed by AMOVA. This implied the “rejection” of previous studies that O. viverrini contained a species complex. It is important to note that mitochondrial DNA appears to be unsuitable and unreliable for the systematic and population genetic studies of O. viverrini. This is primarily due to the observed low genetic variation that has been detected in a number of independent studies examining different geographical isolates [2, 51, 71]. Three polymorphic enzyme loci were used to study the population genetics of O. viverrini from Ban Phai District, Khon Kaen Province [51]. A lack of heterozygosity at the phosphoglucomutase (PGM) locus was detected providing evidence that O. viverrini has a high rate of self-fertilization or non-random mating. The genetic variation has been reported for O. viverrini populations based on temporal factors as well as different species of fish secondary intermediate hosts using three polymorphic enzyme loci [52]. Heterozygote deficiency was detected in O. viverrini collected at different times (temporal populations) and in different species of fish host. No significant genetic differences, however, were found among O. viverrini populations on a temporal basis or from different fish species. Later studies of the population genetics of O. viverrini from different endemic foci in Vientiane, Lao PDR using polymorphic enzyme loci found similar results based on spatial, temporal, and fish host species [26], which provided evidence that self-fertilization and/or a clonal distribution of O. viverrini occurs in Lao PDR. The role of different species of fish host and temporal factors appears to have little effect on the levels of genetic differentiation. Interestingly, spatially related population genetic differentiation is possibly occurring between O. viverrini populations located in the either the upper and lower areas of Nam Ngum Dam. This hypothesis requires further comprehensive population genetic studies, for example using microsatellite markers which provide the basis for finer resolution at the population genetic level than the more evolutionary conserved enzyme/allozyme markers. Microsatellite DNA has provided highly polymorphic genetic markers which have been developed and used to successfully examine the population genetic structure of O. viverrini from Thailand and Lao PDR [30, 31]. Results showed significant heterozygote deficiency which confirmed previous studies supporting predominant self-fertilization of O. viverrini. Furthermore, comparisons of O.

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viverrini populations uncovered genetic differentiation with high F ST values providing independent evidence supporting previous studies of the sub-structuring of O. viverrini populations based on wetland systems. Several studies have been subsequently undertaken supporting the hypothesis that O. viverrini is a species complex using other effective/polymorphic molecular markers, such as microsatellite DNA [31, 38, 42]. Population structure and genetic diversity of four populations located closely to each other within Khon Kaen Province have been examined using microsatellite DNA markers. Unique alleles were detected in each population which also showed significant genetic differentiation, as well as population sub-structuring between these four O. viverrini population localities. These data highlight the versatility of microsatellite markers to undertake studies to examine the genetic structure of O. viverrini populations at a microscale [31]. For instance, a recent population genetics study examined O. viverrini populations in different species of cyprinid fish using microsatellite DNA. The study provided evidence for the first time that second intermediate species of fish host contribute to the genetic diversity of O. viverrini. An important and significant result from this study was that in O. viverrini endemic areas there are significant differences in the genetics of O. viverrini found infecting different species of fish within and between different geographical localities [43]. A study of the systematics of O. viverrini using four independent DNA markers, namely two mitochondrial genes, CO1 and ND1, and two nuclear genes (i.e., Paramyosin and Cathepsin F) from eight geographical localities in Thailand and Lao PDR uncovered a new cryptic population/species from the Pangkon District, Sakon Nakhon Province. This cryptic species of O. viverrini was clearly genetically distinct from all the other isolates examined [43]. Namsanor et al. [38] in a more recent study using microsatellite DNA, as well as nuclear and mitochondrial DNA markers confirmed the discovery of a cryptic species from Pangkon District, Sakon Nakhon Province. Furthermore, and of particular significance, has been that preliminary data from nuclear intron sequence analyses have shown that O. viverrini from Pangkon District was the most genetically distinct from all others examined (Saijuntha et al. unpublished). Comprehensive investigations on the genetics, morphology, biology, and ecology of this novel genetic group and/or potential cryptic species should be conducted to accurately assess its systematics status [56, 63].

2.4.2 The Bithynia Snail Host Systematics and population genetics investigations using enzyme markers of Bithynia snails, the first intermediate hosts of O. viverrini, have revealed that B. s. goniomphalos is not a single subspecies of Bithynia but it is indeed a species complex. The B. s. goniomphalos species complex contains at least nine cryptic morphologically similar but genetically very distinct, hence, cryptic species that have specific associations with defined wetlands in Thailand and Lao PDR

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[24]. Furthermore, results showed that cryptic species of B. s. goniomphalos were directly associated with defined genetic distinct groups of O. viverrini from the same wetlands in Thailand and Lao PDR. This provides evidence supporting previous independent studies of the systematics of O. viverrini uncovering the existence of cryptic species and co-evolution between O. viverrini and its snail first intermediate host [49] and critical amplifying point in the life cycle of cryptic species within the O. viverrini species complex. Subsequently, [28] used the mitochondrial CO1 sequence to successfully examine genetic differentiation and genetic diversity of the genus Bithynia among the 10 species/subspecies in the family Bithyniidae. Surprisingly, these molecular analyses revealed the existence of B. s. siamensis in the south and northeast of Thailand. The distribution of B. s. siamensis has previously been believed to be restricted to the central region of Thailand [28]. A more recent study of B. s. goniomphalos which was intensively collected from a wider geographical range which included 33 localities in six different wetland (catchment) systems of the Lower Mekong basin in Thailand, Lao PDR, and Cambodia was examined by 16S rDNA and mitochondrial CO1 sequences [68]. Three major lineages (I–III) of B. s. goniomphalos were detected and classified corresponding to specific water catchment systems. Lineage I was made up of B. s. goniomphalos from the vast majority of catchment systems in Thailand and Lao PDR, namely the Kok, Wang, Yom, Nan, and Pasak catchments of the northern Mekong, the Chi, Mun, Songkram, Huai Bagn Koi, Huai Ma Hiao, Nam Kam and Nam Loei catchments of the northeast region, the Prachin Buri and Bang Pakong catchments in the eastern region, the Chao Phraya catchment in central Thailand, and the Nam Ngum catchment in Lao PDR. On the other hand, Lineage III was found to contain snails from the Mekong and Sea Bang Heang catchments in Thailand and Lao PDR, respectively. All remaining populations from the Tonle Sap catchment clustered within lineage II. These genetically distinct groups in defined lineages, therefore, should undergo detailed investigations of their morphological/taxonomic characteristics, biology, ecology, and molecular genetics to re-assess their systematics and unique taxonomic status. Data to date provides strong support that B. s. goniomphalos in Thailand is a species complex, namely B. s. goniomphalos sensu lato [68]. The intron regions of arginine kinase (AkInt) are potentially co-dominant genetic markers which can be used to determine heterozygosity in freshwater mollusks, including Bithynia snails [7]. Four regions of AkInt have been recently characterized for B. s. siamensis, B. s. goniomphalos, and B. funiculata [7]. Of these four regions, intron 1 (AkInt1) was assessed as an appropriate genetic marker to examine the population genetics of B. s. goniomphalos, B. s. siamensis, and B. funiculata. Results showed that AkInt1 could genetically differentiate the three Bithynia species and subspecies of snail [7] and can be used in future studies as a genetic marker to resolve and define the systematics and evolutionary relationships of Bithynia snails, as well as determine hybridization levels within and between populations of Bithynia snails.

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As detailed above, mitochondrial DNA sequences have been successfully used for the molecular analyses of the distribution of Bithynia snails covering a substantial geographical range in the north, central, and west of Thailand [8]. The genetic structure of B. s. goniomphalos as well as that of B. s. siamensis was directly related to defined water catchment systems. Furthermore, the genetic structure of a closely related species, Hydrobioides nassa was found to be similar and sub-structuring detected was also related to different water catchment systems [9]. Surprisingly, data from [9] also showed that different species and subspecies of Bithynia examined were not clearly separated by regions in Thailand as has previously been recognized. Results have shown that the three taxa of Bithynia, B. funiculata, B. s. siamensis, and B. s. goniomphalos are found in the northern region of Thailand, whereas B. s. siamensis and B. s. goniomphalos coexist in the central region of Thailand (Fig. 2.2). These results strongly suggest that the current taxonomically defined subspecies B. s. siamensis and B. s. goniomphalos should be included within the species complex of “Bithynia siamensis sensu lato” [8].

2.5

Biological and Morphological Variations

Based on the wide O. viverrini distribution covering different localities and wetland systems in Thailand and Southeast Asia, the biological and morphological variation among different isolates would provide important basic information for better understanding of the systematic and taxonomic status of this liver fluke. A comprehensive comparison of the biological variation among O. viverrini sensu lato populations was initially conducted in a Ph.D. thesis by Saijuntha [49] and subsequently published by Laoprom et al. [29]. Previous molecular genetic analyses indicate that O. viverrini is a species complex containing at least two cryptic species divided into six genetic groups, which correlate with different wetland systems in Thailand and Lao PDR [49]. A comparison of the infectivity, growth, fecundity, and body size (12 characteristics) of O. viverrini from these different wetland systems, namely four wetlands from Thailand; Chi River, Mun River, Wang River, Songkhram River wetlands, and one wetland from Lao PDR; Nam Ngum River wetland was conducted. Worm recovery was lowest in the Songkhram River, which differed significantly from all other wetland systems. The Chi River differed significantly from Nam Ngum River populations. A similar pattern was found for fecundity. The biological and morphological data support the previous genetic data indicating a subdivision of O. viverrini populations on the basis of wetland systems and indicate the existence of cryptic species in Thailand and Lao PDR [29]. Later a novel genotypic group of O. viverrini from a new isolate of Phang Kon District, Sakon Nakhon Province (SPk), Thailand, detected by nuclear and mitochondrial sequences analyses, was found [43]. This finding raises many questions related to the systematic and taxonomic status of O. viverrini distributed in Thailand. Most recently, the Ph.D. thesis of Namsanor [36] measured the body size (17 characteristics) including fecundity, growth, and infectivity comparing between a

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new cryptic isolate SPk and the other isolates from different wetlands in Thailand and Lao PDR. The overall biological and morphological comparisons among SPk and other isolates were not significantly different. This finding is not strong enough to support the evidence of the biological and molecular data [29, 43] which hypothesized that SPk is a cryptic species of O. viverrini. Thus, increasing the sample size from SPk for further morphological studies of adult worms, cercaria, metacercaria, and eggs should be made. Although the morphological comparison between SPk and O. viverrini-like flukes recovered from domestic ducks in Vietnam revealed very similar characteristics, both showed significant differences from Opisthorchis lobatus [36]. This finding supports the suggestion that O. viverrini from SPk and the O. viverrinilike fluke found in ducks from Vietnam are cryptic species within the O. viverrini complex. Interestingly, the discovery of an O. viverrini-like flukes in domestic ducks and birds in an O. viverrini endemic area in Central Vietnam revealed a complex situation with respect to the epidemiology of opisthorchiasis in Vietnam due to the coexistence of two related genotypes. Therefore, morphological comparison of adult O. viverrini recovered from different hosts, i.e., humans, cats, and hamsters, and an O. viverrini-like flukes collected from ducks was reported [12]. The flukes recovered from ducks revealed morphometric differences from the flukes isolated from cats, humans, and hamsters. Overall morphometric data demonstrated that the flukes found in ducks is larger than O. viverrini found in mammal hosts, except for the body length [12]. The testes are two to four times larger, as is the size of the oral and ventral suckers. These morphological variations may fall into the intraspecific variability of O. viverrini in different hosts. However, information on the biological and morphological variation among Bithynia siamensis sensu lato populations is still scarce. According to a comprehensive genetic investigation, there is evidence that Bithynia siamensis is a species complex containing at least four distinct genetic groups, i.e., three genetic groups of B. s. goniomphalos including one genetic group of B. s. siamensis [8, 68]. Future research on the biological and morphological variation within the B. siamensis species complex needs to be conducted. Probably at least two new species exist in the B. siamensis sensu lato populations in the Lower Mekong Subregion.

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Epidemiology and Control of Opisthorchis viverrini Infection: Implications for Cholangiocarcinoma Prevention Narong Khuntikeo, Bandit Thinkhamrop, Thomas Crellen, Chatanun Eamudomkarn, Trevor N. Petney, Ross H. Andrews, and Paiboon Sithithaworn

Contents 3.1

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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N. Khuntikeo Department of Surgery, Faculty of Medicine, Khon Kaen University and Cholangiocarcinoma Research Institute, Khon Kaen, Thailand B. Thinkhamrop Faculty of Public Health, Khon Kaen University and Cholangiocarcinoma Research Institute, Khon Kaen, Thailand T. Crellen School of Biodiversity, One Health and Veterinary Medicine, University of Glasgow, G12 8QQ Glasgow, United Kingdom e-mail: [email protected] C. Eamudomkarn Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand T. N. Petney Evolution and Paleontology, State Museum of Natural History Karlsruhe, Erbprinzenstrasse 13, 76133 Karlsruhe, Germany R. H. Andrews Department of Surgery and Cancer, Faculty of Medicine, Imperial College, London, UK P. Sithithaworn (B) Department of Parasitology, Faculty of Medicine, Khon Kaen University and Cholangiocarcinoma Research Institute, Khon Kaen, Thailand e-mail: [email protected] T. Crellen Big Data Institute, Nuffield Department of Medicine, University of Oxford, Old Road Campus, Oxford OX3 7LF, London, UK © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. Khuntikeo et al. (eds.), Liver Fluke, Opisthorchis viverrini Related Cholangiocarcinoma, Recent Results in Cancer Research 219, https://doi.org/10.1007/978-3-031-35166-2_3

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3.2 3.3

Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tools for Screening, Surveillance, and Control Program . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1 Isan Cohort . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2 New Urine Assay for OV Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.3 Teleconsultation Ultrasonography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Intervention and Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1 The Primary Prevention Program: Food Safety, School-Based Health Education, and Screening of O. viverrini . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.2 Secondary Prevention Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.3 Tertiary Patient Care Program: Confirmation and Management of Suspected CCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Past and Current Control Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1 Education . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2 Medication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.3 Sanitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.4 Comprehensive Control Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 Modeling for Control of O. viverrini Related Pathology and Carcinogenesis . . . . . . . . 3.7 Progress Toward Control of OV and CCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3.1

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Preface

Infection with Opisthorchis viverrini (OV) is the most significant risk factor for the development of cholangiocarcinoma (CCA) in the countries bordering the Mekong River in Southeast Asia, where this cancer has its highest incidence worldwide. Opisthorchis viverrini infection occurs via consumption of raw or insufficiently heated cyprinid fish, with humans as the dominant definitive hosts. Prevention of OV infection should contribute to a substantial reduction or elimination of O. viverrini-induced CCA. Although education programs and curative treatment using praziquantel have had some effect on reducing the prevalence of infection, this is not yet visible in a reduction of cases of CCA, probably due to the lag phase between the time of infection and the induction of cholangiocarcinogenesis, which may take many years. To-date, the cholangiocarcinoma research center at Khon Kaen University, Thailand, have generated three innovative tools for the campaign toward a fluke-free Thailand. Firstly, the Isan cohort database system for data storage and management of the high-risk population. The second tool is the establishment of a point-of-care test via an OV Rapid Diagnostic test (OV-RDT) for screening of opisthorchiasis by urine assay. Thirdly, ultrasound telecommunication for screening of the at-risk population to find individuals with early-stage, curable CCA, thus reducing the burden of the disease on the families and communities affected. The comprehensive control program being implemented in Thailand was designed and divided into primary prevention which dealt with school health education, food safety, and screening of OV by urine assay. The secondary prevention covered ultrasound screening and care of early CCA cases. The tertiary patient care program included confirmation and management of early CCA cases. This program

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has the potential to be expanded into other endemic countries and due to the ecoepidemiology of opisthorchiasis-induced CCA, the long-term anticipated impacts and outcomes include the reduction of CCA incidence throughout the region. However, for these aims to be achieved, concerted efforts to raise social awareness and participating action by the general public, non-government organizations, and government are required.

3.2

Background

Recent estimates indicate that 10 million people are infected with O. viverrini in Thailand, Lao PDR with limited information from Cambodia, southern Vietnam, and Myanmar [82, 92]. Prevalences of O. viverrini sensu lato (see Chap. 2) infection within this geographic range shows high variability by locality [15, 23, 76, 84, 102, 117]. Most estimates of the parasite prevalence are based on conventional diagnosis by fecal examination. The availability of new and more sensitive diagnostic techniques, such as the urine antigen assay and molecular diagnostics, has suggested that these figures could underestimate the true prevalence of infection [74, 115]. As O. viverrini is linked causally to both the precancerous changes to the biliary duct epithelium and to cholangiocarcinogenesis [83, 90, 94, 118], a reduction in the incidence of CCA should be achieved by reducing the number of people infected with the parasite. There are, however, complicating factors. Evidence suggests that CCA can develop years after infection with O. viverrini, and that only a relatively small proportion of individuals infected develop CCA. There will, therefore, be a lag between the infection and the development of CCA: measures taken today will only have an effect after some years have passed. Repeated infection is also common and without prior cure leads to severe disease in the hamster animal model [62]. Evidence in animal models and epidemiological investigations in humans has suggested that opisthorchiasis can also induce kidney abnormalities [10, 26]. With the advent of praziquantel as a pharmacological agent against O. viverrini infection, it is possible to treat individuals infected with the parasite and this can subsequently reduce liver pathology. Pinlaor [61] showed that praziquantel successfully reduced nitrative and oxidative DNA damage to biliary tissue [61]. Although it did not kill all of the parasites, it did dramatically reduce histopathological changes. However, as no immunity develops, reinfection is possible, and indeed common, with the use of the drug tending to the belief that eating infected partially cooked or fermented fish is then not health damaging [71, 106]. Although CCA is a relatively rare cancer in most countries, it shows very high incidences in those Southeast Asian countries bordering the Mekong River [11]. As symptoms are poorly defined and usually only become apparent at an advanced stage of the disease, prognosis is poor with palliative care often being the only option [44] In Thailand, an estimated 89% of liver cancers are due to CCA [87, 91]. This is the most common cancer in males and the third most common cancer in females [95, 96]. Liver cancer is also the third-highest cause of death in males

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aged 50–74 and the sixth-most common cause of death in females within this age group [64]. It is therefore a major cause of death, with diagnosis usually occurring at the late stage of the disease leading to a very poor prognosis with five-year survival being highly unlikely [41, 104]. In particular, data from the northeast of Thailand indicate that mortality from liver cancer is very high and affects mostly adults aged 40 and above, causing a major socioeconomic burden on the usually poor families of the victims [43]. Although accurate data are lacking, there is also a consensus among medical specialists that CCA represents a major medical problem in Lao PDR [46, 85, 86]. The limited data available from Cambodia suggest that primary liver cancer is substantially more common than CCA [20]. Information is not currently available from Myanmar or Vietnam. The high incidences of CCA in these Southeast Asian countries are associated with the high prevalence of infection with the liver fluke O. viverrini. This species has been classified by the International Agency for Research on Cancer as a group 1 carcinogen based on a wide variety of in vivo, in vitro and statistical studies that range in direction from the detection of precancerous changes to the epithelial lining of the biliary tract, through cholangiocarcinogenesis to progression and postmortem pathology [19, 31]. Although other risk factors, such as alcohol consumption and smoking [98], may be of relevance to the development of CCA, the strong geographical correlation between areas with high O. viverrini prevalence and high CCA incidence and the lack of correlation outside of these areas are very strong indicators of the significance of this parasitic infection for CCA development. Human infection occurs through eating traditional fish dishes that have not been sufficiently heated to kill the metacercariae (see Chap. 11). These include dishes made with raw fish (e.g., koi pla) or with freshly prepared, lightly fermented fish (e.g. pla-ra and pla-som) [25, 55, 56, 65, 119]. The presence of social food networks may also play a role in maintaining the pattern of transmission in local communities [45, 59, 72]. Hence, food safety is an important issue in control of opisthorchiasis.

3.3

Tools for Screening, Surveillance, and Control Program

In order to set up a comprehensive program for control of CCA, the Cholangiocarcinoma Screening and Care Program (CASCAP) was established at the Faculty of Medicine, Khon Kaen University in cooperation with the Cholangiocarcinoma Foundation, Thailand. The aim was to represent the most detailed and comprehensive study of the application and optimization of screening methods for the liver fluke infection and early diagnosis of CCA combined with treatment and followup studies. There were three innovative tools derived from the research outcomes of the Cholangiocarcinoma Research Institute to be used for the control program.

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3.3.1 Isan Cohort The Isan Cohort is a web application located at www.cascap. in.th. (see Chap. 10) This database facility uses the CASCAP protocol in an internet platform so that healthcare institutes across Thailand can use it in a systematic, unified, single approach. The name “Isan” refers to the northeast region of Thailand, which is known to have the highest rate of CCA worldwide. The protocol started with the enrollment of at-risk individuals. This created two initial cohorts: the OV Screening Cohort and the CCA Screening Cohort. Cohort members undergo the OV test followed by hepatobiliary US. Baseline demographic information and risk factors were recorded during enrollment, and screening results were recorded following US. If positive findings were detected, the OV Screening Cohort received praziquantel, while the CCA Screening group patients were transferred for advanced diagnosis using CT/MRI. If confirmed, subjects are moved to a Patient Cohort. Information collected during these processes was recorded on case report forms designed by CASCAP. To date as of March 2022, >3.5 million individuals have registered with the health information required by CASCAP archived within the Isan Cohort. Approximately, two-fifths were diagnosed with fatty liver disease and periductal fibrosis, and approximately, 1.0% of individuals were diagnosed with suspected CCA and referred for further investigation. The Isan Cohort includes a sophisticated data collection and analysis system (see Chap. 10) that can be used to determine government policy for the treatment and control of CCA, not only within Thailand but also in other Mekong countries. CASCAP has multiple aims in addition to developing a strategy for diagnosing early-stage CCA. The program increases awareness of CCA in the at-risk population and by doing so reduces the costs of screening. It further reduces the incidence of CCA by determining which individuals are at high risk based on precancerous pathology and following them up. As CASCAP data are collected both longitudinally as well as cross-sectionally, progressive changes in the bile duct and liver of individuals can be monitored. Once diagnosed, patients will receive the most appropriate and best available treatment. They will then be followed-up and provided with the best supportive care and clinical assessment until the end of life (see Chap. 9).

3.3.2 New Urine Assay for OV Screening The conventional diagnostic method for human helminths, including OV, is searching for eggs in fecal samples. Several methods have been used successfully in the past, such as the modified formalin ether concentration technique [22], the modified thick Kato smear [32], and Stoll’s dilution egg count technique [109]. Currently these techniques have become unreliable and are not sensitive. In fecal egg negative cases, an autopsy study revealed many infected individuals with worms in the liver leading to under diagnosis by as much as 20% [81]. Another diagnostic problem is the concurrent infection with fishborne zoonotic trematodes

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referred to as minute intestinal flukes (MIF) which have a similar size and shape to the liver fluke eggs causing false-positive diagnoses [14], 107]. Molecular diagnosis of opisthorchiasis by PCR targeting a repeat DNA element has offered high specificity but variable sensitivity [4, 5, 38, 99, 112]. Loop-mediated isothermal amplification (LAMP) has been established for the detection of both OV and C. sinensis with a higher sensitivity than conventional PCR [4, 5, 49]. Species-specific PCRs are now also available to distinguish between different liver fluke species: OV, Opisthorchis felineus, and C. sinensis [12, 38, 99]. Molecular methods discussed earlier (see Chap. 2) will contribute significantly toward a more effective and accurate diagnosis of trematode infections but improvement and simplification of the tests regarding cost-effectiveness under various socioeconomic scenarios are needed. Alternatively, several serological antibody tests for opisthorchiasis and clonorchiasis have been developed as diagnostic assays with greater sensitivity and specificity than fecal examination [74, 113]. These include the intradermal test, immunoelectrophoresis, indirect hemagglutination assay, indirect fluorescent antibody test and indirect enzyme-linked immunosorbent assay (indirect ELISA) [90, 94, 113] as well as a rapid test format [67, 68]. Indirect ELISA is preferred for the detection of antibodies using different types of antigen including crude somatic extracts of adult worms [63, 111] and excretory secretory antigens [47, 101]. These are superior to fecal examination. The detection of parasite-specific antibodies in other clinical samples, such as urine and saliva, is possible and offers the potential for the serodiagnosis of opisthorchiasis, and these antibodies could act as markers for associated morbidities [16, 73, 77]. To increase diagnostic performance and reduce the cross-reactivity of parasite proteins, several recombinant antigens from eggs and worms were produced and tested [56–59]. However, our inability to discriminate current and past infections poses the main problem for serological antibody diagnosis [83]. To avoid the drawback of antibody-based detection, secretory products from adult worms could be used to indicate a current infection [1, 78, 79, 110, 114]. In this regard, monoclonal antibody-based systems offer increased diagnostic sensitivity, as they discover infections when eggs are not detectable in fecal samples, as corroborated in an autopsy study [81]. Currently, both copro- and urine antigen detection are possible for opisthorchiasis, with the advantage of antigen detection when fecal examination for eggs is negative [110, 114]. The antigen concentration measured is also correlated with the intensity of infection. Due to its simplicity and the noninvasive nature of sample collection, the urine antigen assay provides a better alternative diagnostic method to conventional fecal examination and has revolutionized the diagnostic approach of opisthorchiasis. The rapid diagnostic test for diagnosis of O. viverrini (OV-RDT) aimed for pointof-care use has been produced and an initial study showed that it had similar performance to that by ELISA (unpublished data). The urine assay is currently being applied for large-scale population screening in four provinces in northeast Thailand. Based on its diagnostic potential, the OV-RDT test can facilitate the control program of opisthorchiasis aimed toward the reduction of CCA.

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3.3.3 Teleconsultation Ultrasonography In order to assess the massive number of radiological images, a teleconsultation system was set-up to allow ultrasound images to be viewed by an expert radiologist who then provides a provisional diagnosis, recommending more advanced diagnostic techniques (CT and MRI) for suspected cases [18; see Chap. 6]. This innovative information transfer procedure will also be made available to Lao PDR, Cambodia, and Vietnam, where O. viverrini infection is also common. Screening by US is a suitable method for the initial diagnosis of CCA, as well as for periductal fibrosis, which is a potential precursor of this cancer. The screening program started with the recruitment of at-risk individuals who had previously registered at a subdistrict health clinic. US examination takes place at local medical facilities (clinics and hospitals) or through the CASCAP mobile unit, which can examine 500 individuals a day.

3.4

Intervention and Solution

The Cholangiocarcinoma Screening and Care Program (CASCAP) The CASCAP is a package with different levels of activities. It consists of a primary control program targeting the prevention and control of opisthorchiasis. The ultrasound screening (US) for hepatobiliary disease including CCA serves as a secondary control program in which at-risk population is systematically recruited for screening. Suspected patients are then sent for confirmatory diagnosis, treatment, and care in a tertiary control program.

3.4.1 The Primary Prevention Program: Food Safety, School-Based Health Education, and Screening of O. viverrini Previous control programs and activities for opisthorchiasis have been initiated since 1950 and continued until 1992 with varying scale and intensity (Table 3.1). The primary prevention program involves a campaign for food safety education and an improved strategy for opisthorchiasis screening and treatment based on the urine assay. For the long-term vision, school-based curricula dealing with OV and CCA were initiated and expanded to all education levels. Because cyprinid fish are the source of infection, the primary preventive effort is to stop raw or insufficiently cooked fish consumption (see Chap. 11). Food safety is the key issue for the control of opisthorchiasis, but it has proven difficult to reduce or stop the consumption of infective fish products as these have a longstanding tradition as key food items in the affected areas [25]. Reports indicated that OV is still prevalent in different forms of fermented fish dishes in northeast Thailand [55, 56] and “Pla som,” a short-term fermented cyprinid fish dish (3–4 days at ambient temperature) can contain viable metacercariae [55, 56]. This recent finding

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Table 3.1 Major programs for control of opisthorchiasis in Thailand Date

Area

Method and activity

Organization

1950–1958

Nakorn Ratchasima, Udon Thani, Sakhon Nakorn, Ubon Ratchathani, Songkhla

Education, diagnosis, treatment

Thai Department of Health supported by the US government [69] lists the spring of 1953 as the start of the program directed against O. viverrini infection)

1967–1974

Sakhon Nakorn

Community health education

Liver fluke control unit, Thai government, MOPH [33]

1983–1987

Khon Kaen, Roi Et Sakhon Nakorn, Ubon Ratchathani

Mass screening and selective treatment with praziquantel

Communicable disease control department

1989–1992

Northeast, north, and some central provinces

Mass screening and selective treatment with praziquantel

National Public Health Development Plan, Thai Department of Communicable Diseases Partial supported by the Federal Republic of Germany [33]

1992–1994

Nationwide (42 Provinces)

Regular government activities

MOPH [33]

2014–2023

Central, north, and throughout northeast Thailand

Comprehensive program for screening of opisthorchiasis and early-stage CCA

Cholangiocarcinoma Screening and Care Program (CASCAP) [40–42]

supports the hypothesis that Pla som is likely to be an important source of infection (see Chap. 11). School-based health education has been advocated for the prevention of many infectious diseases [116] as well as for OV and CCA [120]. Based on our initial trial [48], a school-based curricula program was established through the Faculty of Education, Khon Kaen University, and included primary to secondary school courses as well as vocational study. Selected schools were located in active OV transmission localities in the Chi, Mun, and Songkhram River wetlands, and the program spanned 3 years with formal educational evaluation as well as the impact on OV infection. The CASCAP screening program is being operated as a Thai Ministry of Public Health (MOPH) national program, from 2015 to 2026, as part of the national policy to control of opisthorchiasis and CCA. In 2015, the targeted population screened by fecal examination (Kato thick smear) involved 76,000 people in 84 subdistricts in 27 provinces in the north, northeast, and central Thailand. The urine assay based on our previous work (64) was applied in opisthorchiasis endemic regions nationwide to evaluate the current OV prevalence. Pending detailed analysis, the initial

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prevalence of opisthorchiasis was estimated by urine assay with an average prevalence of 40% with the highest prevalence of 60%. Importantly, reinfection and new infections can be evaluated. Thus, the screening and treatment of opisthorchiasis can be planned for parasite control and eventually eliminate the parasite in the community.

3.4.2 Secondary Prevention Program Most patients presenting at a hospital have late-stage CCA with palliative care being the most common treatment option. Thus, there is a second, undetected group that has early-stage CCA without being aware of it [40]. If, however, these individuals with early-stage disease can be accurately identified, then curative surgery is possible [104]. To detect and identify these individuals, the CASCAP was initiated [40, 41]. Given that an estimated six million people in Thailand fall within a risk group (over 40 years old, with a history of OV infection, eating raw or fermented fish, and/or having relatives with CCA), the screening program is a massive undertaking. Screening by US is a suitable method for the initial diagnosis of CCA, as well as for periductal fibrosis, which is a potential precursor of this cancer [19]. The screening program started with the recruitment of at-risk individuals who had previously registered at a subdistrict health clinic. Examination by US takes place at local medical facilities (clinics and hospitals) or through the CASCAP mobile unit, which can examine 500 individuals a day. Screening divides the patients into four groups: (1) without liver pathology, (2) with liver pathology not directly related to CCA (e.g., fatty liver or cirrhosis), (3) with periductal fibrosis as a potential precursor of CCA, and (4) with suspected CCA (liver mass and bile duct dilatation). Patients with no pathology are asked to return for control in about a year, and people with significant pathology not related to CCA are referred to a local hospital. People with periductal fibrosis are asked to return after 6 months for a control examination, and those with suspected CCA are referred for further diagnostic tests. A teleconsultation system, via the CASCAP Cloud software, is coupled with the US examination, allowing diagnostic confirmation by a specialized radiologist at a tertiary hospital [18, 40]. The cohort studies form a sound basis for the discovery of biomarkers for CCA and potentially speed up the process of screening.

3.4.3 Tertiary Patient Care Program: Confirmation and Management of Suspected CCA Ultrasound screening has had a major impact on active, early-stage CCA detection and the subsequent treatment of patients, saving many lives. The initial results can be seen among the cohort that underwent screening, with a confirmed diagnosis of CCA. Although more data confirmation is still required, there is a trend from diagnosing predominantly late-stage to increasingly identifying early-stage CCA,

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and this is a positive sign toward the eventual curative treatment and reduction of CCA. The CCA Center of Excellence was established as a national, regional, and international hub at Srinagarind Hospital, KKU. This contains five dedicated facilities: (1) a CCA ward (19 beds), (2) 1 operating theater, (3) an intensive care unit of 2 beds, (4) a biobank, and (5) research laboratories. This forms the basis for establishing five dedicated CCA functions, namely for tertiary referral, training, research, networking, and a national, regional, and as an international focal point. Currently, all five functions have reached their goals and are operating successfully. Furthermore, a multidisciplinary CCA patient care team has been established to improve the quality of CCA care, which includes nurses, medical oncologists, hepatobiliary pancreatic (HBP) surgeons, radiologists, pathologists, and radiotherapists. This provides avenues for a better understanding and effective dissemination of information, as well as for acquiring specialist skills at all levels to combat CCA. To strengthen both screening and surgery, capacity building and networking to cope with the increasing number of CCA patients are needed. Two training programs were established to enhance screening and care. A Training Program in Cholangiocarcinoma US Screening for Radiologists and General Practitioners was initiated to provide radiologists and general practitioners with the knowledge, skills, and expertise to diagnose CCA using US. This training program operates every 2 months. More than 60 radiologists have passed the training program and can further train GPs and consult with GPs via teleconsultation [18]. In Thailand, there are an estimated 20,000 CCA cases/year. Considering a single hospital, for instance, Srinagarind Hospital in northeast Thailand, unfortunately only about 10% of those affected (ca. 200 of 2000) finally reached the hospital where they received appropriate treatment and palliative care [51]. To increase the capability of the healthcare system, particularly the provincial hospitals, improvement, and expansion of facilities and personnel are inevitable. In particular, HBP surgeon training is a priority. Thus, a Training Program in Hepatobiliary and Pancreatic Surgery was initiated and is currently offered by the Department of Surgery, Faculty of Medicine, Khon Khan University. The objective is to provide trained surgeons with knowledge, skills, and expertise in the field of surgical diseases of the liver, biliary tract, and pancreas. This is a one-year course, and up to eight HBP surgeons can be trained for work in north and northeast Thailand. The partner hospitals for surgical training included Khon Kaen, Sunpasitthiprasong, Surin Hospitals, and National Cancer Institute in Bangkok. In addition to the HBP training, special financial support from NGOs and charity organizations has been implemented to help increase the number of surgical treatments, i.e., 500 cases/year, within these network hospitals.

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Past and Current Control Programs

Although O. viverrini infection in humans was recorded as early as 1915 by Leiper, with scattered additions in the following years [9, 39, 66], it was only with the comprehensive survey published by Sadun [69] which was carried out under the sponsorship of the Thai Ministry of Public Health, and supported by the United States government, that an initial indication into the relationship between O. viverrini infection and CCA became apparent. Sadun reported “carcinomatous changes of the liver” and that “carcinoma of the bile ducts was often observed in the northeast,” while leaving a causal relationship open [69]. Nevertheless, he did suggest the necessity of control programs based on education to limit or prevent eating infected fish and changes in the sanitary situation, table, and toilet [58], to prevent eggs from contaminating freshwater bodies containing the first and second intermediate hosts, both preferred methods today. He also discarded as impractical the control of reservoir hosts such as cats and dogs, and of the Bithynia first intermediate hosts. At the time he wrote in the 1950s, effective anthelminthic therapy was not available (see Chap. 2).

3.5.1 Education Based on Sadun’s work, the first, local, health education program aimed at preventing O. viverrini and other helminth infections was initiated in 1953 in various provinces predominantly in the northeast of Thailand. The program included the display of posters explaining the dangers of eating infected fish at schools and on trees by bodies of freshwater, brochures given to village headmen and lectures at Buddhist temples [69]. When support from the USA ended in 1958 the control activities continued even though the helminth control units ceased to exist [34]. Education has continued to be an important component in the drive to reduce the prevalence of O. viverrini infection. The effectiveness of this approach has been estimated in various independent studies: [48] developed an education program on the dangers of O. viverrini infection for schools. This program was presented in certain schools with others acting as controls [48]. Although knowledge increased after the program, there was also a basic knowledge in the control schools where the program was not taught, suggesting local community transfer of information. Khuntikeo [41, 42] showed that a major decrease in the prevalence of O. viverrini infection had occurred if the periods 1955–1983 and 1994–2013 were compared [42]. In a recent study, [97] Srithongklang and colleagues showed that educational intervention based on a “health belief model” was effective in increasing the knowledge, perceived susceptibility, perceived severity, perceived benefits, perceived barriers, cues to action, self-efficacy, and CCA preventive behaviors compared to a control group [97]. These studies suggest that control programs, especially those including education and medication, have had a significant, positive effect on disseminating knowledge of the association between O. viverrini

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infection and CCA. This is particularly true for school-age children where prevalences have dropped from up to 90% or more to under 10% [42]. Forrer [23] showed that there was a higher risk of infection with a higher level of schooling but suggested that this result was confounded by these individuals having occupations more likely to be associated with infection [23]. Chaiputcha et al. [15] showed that higher education was protective against O. viverrini infection [15]. In contrast, [100] found that there was no change in prevalence in a central Thai village (21.6% in 2002–2004 and 21.4% in 2007–2009) after the promotion of hygienic defecation and health education focusing on avoiding raw fish consumption, as well as case diagnosis and treatment [100]. One study of ten randomly selected villages in Saravane District, Southern Lao PDR showed a low awareness of the problems associated with O. viverrini (and other helminth) infection [60].

3.5.2 Medication The introduction of praziquantel radically changed the potential for curing and thus controlling opisthorchiasis and is undoubtedly responsible for a significant component of the reduction in the prevalence of O. viverrini infection between 1984 and 2001 [34]. Treatment by praziquantel showed a protective effect against DNA damage and against carcinogenesis. Saowakontha et al. [75] found that praziquantel treatment combined with education and improved sanitation effectively reduced the prevalence and intensity of O. viverrini infection in two experimental villages in northeast Thailand compared to a control village [21, 75]. Unfortunately, reinfection did occur over a 6-month period. There are, however, possible negative effects of praziquantel use as it is seen as a repeatable therapy allowing the continual consumption of the traditional raw or mildly fermented fish [70]. Individuals with a frequent history of PZQ use who still continued raw fish consumption showed high levels of repeated reinfection with O. viverrini. They were infected, treated, and reinfected repeatedly. This is a particular problem in highly endemic areas for O. viverrini. Thus, the introduction of this drug may have increased the cycle of infection-cure-reinfection [103]. The repeated use of praziquantel does not relate with an increased risk of CCA [36, 50] nor reinfection after treatment [115].

3.5.3 Sanitation Sanitary practices in many of the areas where opisthorchiasis is prevalent remain basic, with most fecal material introduced into the environment where it is deposited or washed into freshwater bodies containing the snail and fish, first and second intermediate hosts, respectively [58]. The elimination of such practices, for example by using modern sanitary techniques, would break the cycle of infection with the exception of fecal input from reservoir hosts, which is likely to be of limited significance [13]. Forrer et al. [23], for example, found that safe sanitation

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significantly reduced the risk of infection with O. viverrini in Lao PDR [23]. The disposal of toilet sewage was recognized as a potential source of environmental contamination and the hygienic sewage disposal plant, and a by-product of fertilizer production has been initiated in some provinces in northeast Thailand. But the investment cost and suitable construction sites required local acceptance and cooperation of the local population. In addition, the availability of budget allocation from local and central governments is an essential issue. Unfortunately, to date there has been little progress regarding this issue highlighting the urgent need for concerted efforts.

3.5.4 Comprehensive Control Program A pilot project in villages around Lawa Lake in Khon Kaen province, northeast Thailand, based on chemotherapy and intensive education at schools and within the community, was successful in reducing O. viverrini infection by more than two-thirds [93]. Whether such intensive input is logistically feasible for the larger area and on a long term remains to be seen. A broader approach has been made by the Fluke-free Thailand initiative which includes 11 interrelated components covering education and sanitation (see Chap. 10). This approach also includes research on reservoir hosts, data collection, collation and analysis, new diagnostic methods, screening for precancerous changes to the biliary tract and CCA, in particular early-stage curable disease run by the Cholangiocarcinoma Screening and Care Program, CASCAP, [41], clinical trials, socioeconomic consequences [43], and health/government policy advice.

3.6

Modeling for Control of O. viverrini Related Pathology and Carcinogenesis

Infection with O. viverrini has been repeatedly found in cross-sectional studies to be a risk factor for the development of liver pathology and CCA, with higher infection intensities producing stronger associations with chronic disease. Parkin et al. [57] reported that antibodies against O. viverrini gave an increased odds ratio of 5.0 for the development of CCA in a hospital-based case-control study [57]. Population surveys of CCA have found similar results, with [28] reporting elevated odds ratios of 1.67, 3.23, and 14.08 for individuals with 1–1500, 1501–6000, and >6000 O. viverrini eggs per gram of stool respectively in Khon Kaen province, Thailand [28]. While the categorization of egg count intensities varies by study, making direct comparisons difficult, subsequent surveys have reported similar strengths of association [30, 102]. The liver fluke is thought to induce pathology in a number of ways, including through mechanical damage, autoimmune inflammatory responses, and secreted products [90, 94]. Some of the strongest evidence for parasite-mediated pathology relates to the production of granulin, a wound-healing protein. When gene expression of granulin is knocked down in O. viverrini, this

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results in significantly less morbidity in infected hamster models [8]. Liver pathology in humans living in O. viverrini endemic settings is more prevalent among older age groups (>40 years), while infection with liver fluke can occur early in childhood [27], indicating a lag between the start of infection and the onset of liver disease. Taking these points together, that i) heavier worm burdens give an increased risk of developing CCA, ii) proteins secreted by the parasite directly increase liver pathology; and iii) pathology follows after potentially decades of infection, it may be assumed that chronic damage to the bile ducts accumulates primarily as a function of the length of parasite exposure and intensity of infection [89]. Mathematical modeling can provide a framework for phenomenological or quantitative assessment of pathogen transmission and disease progression [29]. A number of modeling frameworks have already considered the chronic pathology that results from infection with macroparasites [54, 105, 108]. Here, we consider a simple dynamic model, applied initially by [53], which we parameterize for O. viverrini [53], We have applied these models in order to stimulate discussion on the development of quantitative methods that link exposure to liver flukes with the progression of CCA, and how best to optimize methods of control to reduce morbidity. The number of worms () acquired at age a (the force of infection) is given by (a) = aαe(−aβ) ,

(3.1)

where α and β are parameters that determine the infection rate by age. We have set β=1/40, which assumes that the rate of parasite acquisition is greatest at forty years of age [27], and α=1/2, which gives an infection rate of approximately five worms per year [106]. The mean parasite burden at age a, M(a) is given by the differential equation dM = (a) − μM. da

(3.2)

The parasite death rate, μ, is set to 1/5, so that around half of parasites are dead after five years and 90% after 10 years [80]. The age-specific force of infection and mean worm burdens produced by the models are shown in Figure 3.1.

We consider damage, or chronic morbidity, resulting from infection as i) a linear accumulation of the parasite burden (area under the curve M at age a), and ii) with a rate of resolution (γ ) that decreases exponentially (σ ) with increasing morbidity. If Dd (a) is the damage at age a with the latter form of resolution, then the rate of change is given by dDd = M − γ e−σ Dd Dd da

(3.3)

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Fig. 3.1 The mean Opisthorchis viverrini worm burden (M) as a function of age is shown in the absence of treatment (solid line), and with anthelmintic treatment every decade from ages ten to sixty at 95% efficacy (dashed line). The rate of infection establishment per year, or force of infection (), is shown as a dot-dashed line. Models are given in Eqs. 3.1 and 3.2, which have been parameterized for O. viverrini

The latter function accounts for the observation that more severe hepatobiliary damage may not resolve with time or following anthelmintic treatment [52]. Introducing control measures into this framework, we can reduce the rate of infection (a) i) directly through health education to lower the feeding rate [3], ii) indirectly by sanitation measures to reduce human egg output into water sources with Bithynia spp. snails. Anthelmintic treatment, typically with praziquantel, directly impacts the worm burden (M) at the point in time when treatment is given. Any of these interventions reduce the accumulated worm burden, and thus  the cumulative damage D(a) to a lower value D (a). The benefit of the interventions can therefore be expressed by the ratio of the areas under the curves D(a)  and D (a). In a scenario where we introduce five treatments with praziquantel over the life course of an individual at every decade from ten until sixty years of age, with a 95% treatment efficacy against adult worms (Fig. 3.1), this results in a i) 37% reduction in the total damage accumulated by seventy years of age; if damage accumulates linearly with worm burden, or ii) 95% reduction in the total damage accumulate by seventy years of age; if morbidity can resolve however the rate of resolution declines exponentially with increased damage (Fig. 3.2). A qualitative conclusion from this modeling is, therefore, that the nature of the

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Fig. 3.2 Accumulated damage resulting from chronic infection with Opisthorchis viverrini. We consider both a linear accumulation of damage, given by the area under the curve of Eq. 3.2 (solid lines), and a function (Eq. 3.3) where morbidity initially resolves; however, the rate of resolution decays exponentially as damage accumulates (dashed lines). Both models are shown i) in the absence of treatment, where the damage shown is a proportion of the maximum absolute value and the curves reach unity; and ii) with five deworming treatments given each decade from ages ten to sixty. In the latter case, the curves are shown as a proportion of the maximum untreated values.

“damage function” impacts substantially on our assessment of interventions, such as anthelmintic treatment with praziquantel, on hepatobiliary morbidity. The modeling framework we have adopted thus far is limited by our inability to relate an unscaled measure of parasite-induced “damage” with observed clinical outcomes such as periductal fibrosis, bile duct dilation, and CCA [17]. A separate approach would be to estimate the rate of CCA in the population as a function of age using a stochastic model fitted to age distributions of cancer incidence or fatalities [37], which has been a commonplace approach in the epidemiological literature for some time [6, 7]. While it has been argued that rates of CCA have risen or remained stable in Thailand while the prevalence of O. viverrini has decreased [41], this phenomenon could be attributable to i) a lag between reduction of infection intensity and the reduction in incidence in CCA, as hepatobiliary damage acquired early in life may still lead to a higher risk of carcinogenesis in later years even with a cessation of transmission; and ii) increases in the reporting rate of CCA due to public health programs in Thailand such as CASCAP. As awareness of O. viverrini and CCA is raised among the general population, enhanced parasite, and disease control may coincide with a higher proportion of

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CCA cases being reported. Here we explore these forces by use of a simple simulated example. If the equilibrium mean worm burden in the population at baseline is given by M(0), following a complete cessation in transmission the mean worm burden at time t is given by M(t) = M(0)e−μt .

(3.4)

The mean worm burden therefore declines exponentially as worms die at a rate of μ [2]. If we consider then the number of CCA cases in the population per 100,000 in year t, yt , to be a realization of a Poisson process yt ∼ Poisson(λt )

(3.5)

λt = M(t − n)ρ(t)

(3.6)

The time varying incidence rate (λt ) is a product of the average worm burden at some earlier time point (t−n, where n>0), a time-dependent reporting rate, ρ(t), and a scaling parameter ε. A stochastic realization of a model run is shown in Fig. 3.3 for illustrative purposes. The reported CCA incidence initially rises while worm burdens decline, due to both the time lag and the rise in reporting rates after the start of interventions. This simple example serves to illustrate some of the basic epidemiological patterns underpinning observed data. A more complete schema for assessing how the risk of CCA relates to infection with O. viverrini infection would incorporate information on mutation rates and the presence and timing of driver mutations [35]. Cancer genomic studies are increasingly able to time the occurrence of mutational events, with cancers that arise as a result of exogenous mutagens, such as tobacco smoking in lung adenocarcinoma and ultraviolet light exposure in melanomas, showing early mutation events [24]. Combining molecular data from host tissue with longitudinal assessments of O. viverrini infection intensity would therefore provide a strong evidence-base for understanding the link between parasite exposure and carcinogenesis in human populations.

3.7

Progress Toward Control of OV and CCA

Although the CASCAP is healthcare oriented, the data collected represent a comprehensive research resource. For instance, a database for scientific monitoring and evaluation and in particular the assessment of long-term changes in the liver and the bile duct, the rate of early detection, and long-term clinical outcome of the patients in response to various medical interventions. The procedures defined by the CASCAP are ultimately aimed at being adopted as part of routine healthcare practice. All core data items are part of the routine data collection at all cancer hospitals in the northeastern region of Thailand and later throughout the Mekong region. CASCAP serves as an innovative and comprehensive approach to combat CCA in the region (see Chap. 10).

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Fig. 3.3 Shows simulated model output from Eqs. 3.4–3.6. The reported cholangiocarcinoma (CCA) incidence per 100,000 is given by the solid line, while the mean O. viverrini worm burden is given by the dot-dashed line. We consider a scenario where O. viverrini transmission ceases immediately at t = 5 (vertical dashed line) due to stringent control measures; that the reporting rate of CCA rises from ρ=1/2 prior to the onset of control to ρ=2/3 thereafter; that the initial equilibrium worm burden M(0)=30, and that a five-year lag exists between the worm burden and it’s impact on rates of CCA; and the rate of CCA is initially 71 per 100,000 population [88]

On 21st June 2016, the Thai Ministry Of Public Health (MOPH) announced a decade long policy to eliminate OV and reduce CCA. The vision is for Thai citizens to be safe from OV and CCA and to have a better quality of life and life expectancy. It encourages preventive behavior, and the risk groups for CCA are provided with adequate and holistic medical care until cure or final stage disease. A set of strategic plans was laid down to cover activity related to: (1) intensive surveillance of OV and CCA, (2) strengthening preventive strategies in Thailand and neighboring countries, (3) enhancing screening, care, and referral systems, (4) supporting and facilitating community and local authority participation in the prevention and management of OV and CCA, and (5) strengthening research and development for efficient comprehensive database management. As a result of these plans, the cumulative targets over the next 10 years for OV screening and treatment are >4 million, US screening >5 million, and up to 15,000 cases for surgical treatment. Based on the number of CCA victims/ year, the cumulative numbers for CCA surgery and treatment are clearly less than the annual number of CCA cases. However, the initiative is expected to create

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an environment stimulating a more concerted effort and willingness of all parties concerned to move forward in a desirable direction. Nevertheless, the current global community is involved in population movement and migration, including borderless economic communities such as ASEAN, leading to trans-border disease movement. The success of OV and CCA control within a single country, Thailand, can never be ensured without the close collaboration of the neighboring countries where OV is endemic, namely Lao PDR, Cambodia, Vietnam, and Myanmar. The CASCAP model can be modified be extended to these countries.

3.8

Summary

The elimination of O. viverrini infection would largely remove the risk of developing CCA in and along the Lower Mekong Region once the long-term precancerous pathology caused by the infection has been eliminated from the population, which could take decades. Although education has been partially effective, currently, in reducing the prevalence of infection, eating traditional dishes of raw or fermented infected fish remains common, especially within the rural populations, indicating that education alone is unlikely to solve the problem of parasite transmission. The use of praziquantel has not reduced the likelihood of reinfection, and to date sanitary improvements, although occurring, have not effectively broken the chain of transmission. What is required is a multilevel, interdisciplinary action, such as that found in the fluke-free Thailand concept, to eliminate the threat of CCA not only within Thailand, but also throughout the Mekong region. The strategic plans developed for this program can be expanded for use in other endemic areas as well as being a model for use in other chronic diseases. Due to the eco-epidemiology of opisthorchiasis-induced CCA, the anticipated impacts and outcomes of the program include short-, medium-, and the long-term goals for the reduction of CCA incidence. To achieve long-term sustainable impacts, concerted efforts to raise social awareness and participating action by general public, non-government organizations, and government agencies are necessary.

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The Hallmarks of Liver Fluke Related Cholangiocarcinoma: Insight into Drug Target Possibility Watcharin Loilome, Nisana Namwat, Apinya Jusakul, Anchalee Techasen, Poramate Klanrit, Jutarop Phetcharaburanin, and Arporn Wangwiwatsin

Contents 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8

Host–Parasite Interactions: Chronic Infection-Induced Inflammation-Related CCA Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hallmarks of Ov-Related CCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sustaining Proliferative Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evading Growth Suppressors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Resisting Cell Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enabling Replicative Immortality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inducing Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Activation of Invasion and Metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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W. Loilome (B) · N. Namwat · P. Klanrit · J. Phetcharaburanin · A. Wangwiwatsin Department of System Biosciences and Computational Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand e-mail: [email protected] N. Namwat e-mail: [email protected] P. Klanrit e-mail: [email protected] J. Phetcharaburanin e-mail: [email protected] A. Wangwiwatsin e-mail: [email protected] A. Jusakul · A. Techasen Faculty of Associated Medical Science, Khon Kaen University, Khon Kaen 40002, Thailand W. Loilome · N. Namwat · A. Jusakul · A. Techasen · P. Klanrit · J. Phetcharaburanin · A. Wangwiwatsin Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen 40002, Thailand © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. Khuntikeo et al. (eds.), Liver Fluke, Opisthorchis viverrini Related Cholangiocarcinoma, Recent Results in Cancer Research 219, https://doi.org/10.1007/978-3-031-35166-2_4

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4.9

Enabling Characteristics and Emerging Hallmarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.9.1 Genome Instability and Mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.9.2 Tumor-Promoting Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.9.3 Reprogramming Energy Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.9.4 Evading Immune Destruction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.9.5 The Tumor Microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.10 Molecular Heterogeneity and Therapeutic Opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.10.1 Genetic Profiling of iCCA and Its Clinical Implication in Targeted Therapy . . 4.10.2 Genetic Profiling of eCCA and Its Clinical Implication in Targeted Therapy . 4.11 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Cholangiocarcinoma (CCA) is a malignant tumor of the biliary tree that is classified into three groups based on its anatomic location: intrahepatic (iCCA), perihilar (pCCA), and distal (dCCA). Perihilar CCA is the most common type and accounts for 50–60% of CCA cases. It is followed by distal CCA and then intrahepatic CCA that account for 20–30% and 10–20% of cases, respectively [1]. CCA represents the second most common hepatobiliary malignancy and accounts for 10–20% of primary liver cancers worldwide after hepatocellular carcinoma (HCC) [2]. It also accounts for an estimated 3% of gastrointestinal malignancies [1]. Globally, the incidence of CCA varies in different regions and is closely associated with the prevalence of the risk factors in each region [3]. Cholangiocarcinogenesis has multiple risk factors some of which are associated with both iCCA and extrahepatic (eCCA), while other risk factors are specific to iCCA or eCCA [4]. The highest incidence of CCA is found in Asian countries, especially in Thailand where the highest incidence worldwide has been reported, particularly in the north-eastern region where liver fluke, Opisthorchis viverrini (Ov), infection is the major risk factor for its development [5].

4.1

Host–Parasite Interactions: Chronic Infection-Induced Inflammation-Related CCA Development

Chronic inflammation is defined as a risk factor of many human cancers. Two of the mechanisms that link cancer and inflammation are reactive oxygen species (ROS) and reactive nitrogen species (RNS) which are generated by leukocytes that infiltrate the area of inflammation. Oxidative/nitrative stress can cause damage to many cellular biomolecules including proteins, lipids, or DNA. The chronic inflammation induced by Ov infection is also involved in generating of nitric oxide (NO) and other reactive oxygen and nitrogen species (ROS & RNS) [6]. Production of endogenous NO is catalyzed by inducible nitric oxide synthase (iNOS), the enzyme mainly produced by inflammatory cells, especially macrophages that are induced by inflammatory cytokines [7]. There is the evidence which has been addressed that iNOS activation in response to inflammatory cytokines caused over-production of NO, resulting in DNA damage and inactivation of the enzyme that is involved in the DNA repair process [8]. In addition, Pinlaor and

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colleagues found that Ov infection can cause oxidative and nitrative DNA damage in hamster liver tissues, which was demonstrated by the presence of 8-oxo-7, 8-dihydro-2’-deoxyguanosine (8-oxodG), and 8-nitroguanine, biomarkers for the damage of DNA. The nitrative, oxidative DNA damage, and the expression of iNOS were induced via the infection of Ov and may participate in CCA carcinogenesis [9]. Furthermore, high level of 8-oxodG was seen in the liver tissues of CCA patients and the level of 8-oxodG which detected in urine and leukocytes of Ov-infected patients were higher than in healthy subjects [10]. Finally, genotoxic events caused by DNA damage can lead to a DNA mismatched repair mechanism. However, if the damage is beyond repair, or cell death occurs through apoptosis, then these mutated cells can survive and transform into malignant cells. CCA causes the alteration of several genes and expression of proteins [11, 12], and these alterations lead to CCA progression. The next section describes the progression of CCA according to the alteration of genes and/or proteins.

4.2

Hallmarks of Ov-Related CCA

The “hallmarks” are an organizing principle for understanding the biology and complexities of cancer. In 2000, Hanahan and Weinberg demonstrated six biological capabilities during the multistep process of the development of tumors to explain the progression of cancers [13]. Recently, the next-generation hallmarks of cancer were developed and two emerging hallmarks were added, namely evading immune destruction and reprogramming of energy metabolism [14]. In this section, eleven hallmarks of cancer were used to describe tumor development and progression in Ov related CCA.

4.3

Sustaining Proliferative Signaling

Normal cells require growth signaling to control cell proliferation. The components that drive growth signaling are composed of growth factor ligands and the cognate receptors or cytosolic signaling molecules. When a growth factor ligand binds to its specific cell surface receptor, which typically contains an intracellular tyrosine kinase domain, cell growth signaling will be generated. The signal is subsequently transferred through intracellular signaling cascades that control cell growth and the cell cycle. Under biological condition, growth signaling is tightly regulated to proliferate in a controlled fashion and maintain tissue homeostasis. Conversely, this mechanism is deregulated in cancer cells. The capability of cancer cell to promote sustained proliferative signaling can occur via several alternative pathways [15, 16]. 1) Cancer cells may produce growth factor ligands by themselves that respond by the expression of the cognate receptors. This activation manner is called autocrine stimulation.

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2) Cancer cells may send growth signals to stimulate normal cells within the tumor stromal area and then reciprocally activate cancer cells with several growth factors. This is called paracrine activation. 3) Overexpression of cell surface receptors results in a hyper-response even if there are limited amounts of growth factor ligands. 4) Autoactivation caused by mutation at the active site of the cell surface receptor leads to the activation of growth signaling without binding between the growth factor ligand and the cognate receptor. An important characteristic of CCA involves the production of growth-promoting signals. The important growth factor ligand that drives carcinogenesis and the progression of Ov-associated CCA is IL-6. This is an inflammatory cytokine that is secreted from inflammatory cells and plays roles in inflammation, the immune response, and cell development. The elevation of IL-6 in the plasma of Ov-infected patients who have advanced periductal fibrosis defines the high-risk group for CCA [17, 18]. Moreover, secretion of IL-6 has been detected in LPS-induced human macrophage-conditioned media mediating CCA cell proliferation through the activation of STAT3 [19]. These results show that IL-6 is a signaling ligand which plays a role in the inflammatory process that drives CCA carcinogenesis and growth. Additional to the elevation of the growth signal ligand, overexpression and/ or overactivation of the cell membrane receptor is a mechanism that promotes the continuing proliferative signaling of Ov-associated CCA. Dokduang and co-workers reported the activation of receptor tyrosine kinases (RTKs) in Ovassociated CCA. They surveyed the activation profiles of protein kinases in both human CCA cells, as well as in human CCA patients’ tissues, using human phospho-RTKs array analysis. The results revealed the activation of several RTKs. Commonly activated RTKs that have been identified in CCA cell lines and tissues include EGFR, MPSR, and Ephrin B2, and an RTK EGFR that has been identified and may be involved in sustained growth signaling in CCA [11]. Epidermal growth factor receptors (EGFR), also called the HER or ErbB receptor family, are receptor protein kinases that belong to the HER receptor family. This is composed of four members, EGFR (HER1), HER2, HER3, and HER4. EGFR can be activated by different ligands, such as epidermal growth factor (EGF), heparin-binding factor (HB-EGF), and amphiregulin. Activation of EGFR initiates an intracellular signaling cascade that increases the potential for cell proliferation, angiogenesis, and resistance to apoptosis [20]. Overexpression of EGFR in Ov- associated CCA patient tissues has recently been elucidated. High expression of EGFR was found in >80% of CCA tissue cases and was associated with a poor prognosis and recurrence status in CCA patients [21]. Therefore, EGFR was suspected as an important molecule that plays a significant role in CCA progression and in the molecular mechanism(s) underlying EGFR-mediated Ov-associated CCA growth. These results have been supported by the study of Dokduang et al. which revealed that high expression of EGFR and HER2 was also detected in CCA cell lines. By using a pan-HER inhibitor, they showed that varlinib inhibited

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CCA cell growth and induced cell death both in vitro and in an animal model [22]. These results show that the overexpression of membrane receptors is involved in CCA development. Apart from the membrane receptor, deregulation of downstream signaling molecules is an important component that drives the continuous proliferation signal in CCA. Loilome et al. reported the upregulation of the protein kinase A regulatory 1 subunit alpha gene (PRKAR1A) in an Ov-induced hamster CCA carcinogenesis model. They also revealed the overexpression of PRKAR1A mRNA and proteins in human CCA tissue and cell lines [23, 24]. PRKAR1A is a regulatory subunit of protein kinase A (PKA), which is the enzyme that regulates the metabolism of cellular carbohydrates, glycogens, and lipids. Silencing PRKAR1A expression induces growth inhibition and apoptosis of CCA cells. This is associated with a decrease in MAPK, PI3K/Akt, JAK/STAT, and Wnt/b-catenin pathway signaling. Moreover, inhibiting PRKAR1A by specific PKA inhibitors led to growth inhibition and apoptosis induction in CCA cells [23]. This suggests an important role for PRKAR1A in the control of CCA cell growth, and the overexpression of PRKAR1A is implicated in the sustained growth signaling in Ov-associated CCA. As we mentioned above, chronic inflammation caused by Ov infection is the main risk factor that contributes to carcinogenesis in CCA. This can alter the expression of several genes including kinase genes during cholangiocarcinogenesis. The study of Dokduang et al. revealed the activation of the signal transducer and activator of transcription (STAT) protein family in CCA. The STAT family plays roles in the immune response, inflammation, and cell development. From this study, the activation of STAT3 and STAT5b was found to be correlated with CCA patient survival. The activation of STAT3 and STAT5b was strongly increased in the chronic inflammatory phase of CCA carcinogenesis. STAT3 activation was found in bile duct epithelial cells, while STAT5b activation was detected in the surrounding inflammatory cells. Moreover, high activation of STAT3 was observed in CCA cell lines, whereas STAT5b activation was found in macrophages. Interestingly, stimulating CCA cell lines by macrophage-conditioned media resulted in an increasing of STAT3 activation [19]. Moreover, an anti-inflammatory agent, xanthohumol, can inhibit STAT3 activity in CCA cell lines leading to CCA cell growth inhibition and apoptosis [25]. This supports the hypothesis that STAT3 and STAT5b act as mediators between inflammation that drives Ov-associated CCA carcinogenesis and cell proliferation. Wnt-β-catenin is another activated kinase that is implicated in CCA cell proliferation. The study of Loilome and co-workers demonstrated the upregulated mRNA and protein expression of Wnt3a, Wnt5a, and Wnt7b in CCA tissues. Wnt3 was prominently found in inflammatory cells. High expression of Wnt5a was significantly correlated with the poor survival of CCA patients. The accumulation of cytoplasmic β-catenin in bile duct tumor cells was also observed in this study. Interestingly, the increase of Wnt3 mRNA and protein expression was found in the macrophage cell line when induced with lipopolysaccharide (LPS). When CCA cell lines were stimulated with LPS-induced macrophage-conditioned media, β-catenin accumulation in CCA cells was determined. Furthermore, suppression of

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β-catenin protein reduced CCA cell growth as well as cyclin D1 protein expression [26]. The other factors that promote the continuous proliferative signaling in Ovassociated CCA are growth mediators that are produced and secreted by Ov such as granulin [27], as well as the mutation of oncogenes that cause the constitutive activation of growth signaling in CCA, such as c-ski and Ras signaling [28, 29]. In addition, a recent study from our research group revealed the crucial role of fatty acid metabolism enzymes on CCA cell proliferation. This study demonstrated that fatty acid synthase enzyme (FASN), a crucial enzyme in de novo fatty acid synthesis, was found to be overexpressed in human CCA tissues and CCA cell lines. High expression of FASN was associated with late-stage CCA and poor survival of CCA patients. Suppression of FASN expression by using shRNA knockdown in CCA cell lines leads to CCA cell growth inhibition, cell cycle arrest, and induced cell apoptosis. Interestingly, metabolomic analysis in FASN knockdown CCA cells demonstrated FASN knockdown-associated metabolic changes in purine metabolism of CCA cells. Purine is a molecule that is required for DNA synthesis, especially in a rapidly proliferative cell. Results from this study indicate the involvement of lipid metabolism enzyme, FASN, that sustains the proliferative capacity of CCA [30].

4.4

Evading Growth Suppressors

An additional hallmark capability of sustaining response to the growth-stimulating signal is the evasion of the cell growth suppressor, leading to the promotion of carcinogenesis and progression of the cancer. Tumor suppressors, including retinoblastoma (RB) and P53, are well-known regulatory proteins that govern cell apoptosis, proliferation, and senescence in Ov-associated CCA. The RB gene encodes RB protein that acts as the critical gatekeeper in the cell cycle, preventing excessive cell growth by inhibiting cell cycle progression. RB controls the cell’s ability to replicate DNA, which prevents cell cycle progression from the G1 to the S phase by binding and inhibiting E2 promoter-binding–proteindimerization partner (E2F-DP) dimers. These are transcription factors of the E2F family that drive the cell to the S phase [31]. TP53 encodes P53 protein that acts as a negative regulator of cell proliferation and a positive regulator of apoptosis in response to DNA damaging agents. P53 activates DNA repair proteins when DNA has sustained damage. It can arrest the cell cycle at the G1/S regulation point on DNA damage recognition until the damaged DNA has been repaired [32]. The studies in the Ov-induced hamster CCA carcinogenesis model revealed the mutation of RB and TP53 during the CCA tumorigenesis process. Boonmars and colleagues identified the kinetic expression of genes in the RB pathway. Their results demonstrated that the expression of RB1 and p16INK4 was down-regulated during the development of CCA induced by Ov infection plus N-nitrosodimethylamine treatment in a time-dependent manner. Conversely, the

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expression of cyclin D1 and CDK4 was upregulated. These findings are consistent with the study results in human CCA tissue samples, indicating the involvement of the RB gene in the carcinogenesis of OV-associated CCA [33]. In addition to RB gene downregulation, a point mutation of TP53 gene has been reported in CCA carcinogenesis in the hamster model. Tungkawattana et al. found that a point mutation in exon 6 of TP53 may be involved in the cholangiocarcinogenesis process [34]. Exome sequencing in human CCA tissue samples was also used to explore the genetics of CCA. The results of this study revealed different mutation patterns in liver fluke related and non-infection-related CCA. Mutation of TP53 was significantly more frequent in Ov related tumor tissue samples when compared to non-Ov related CCA. Recently, Jusakul et al. demonstrated the genetic clustering of CCA tissues samples with a different etiology from ten countries by integrated whole-genome and epigenomic analysis. This study revealed the integrative clustering of Ov-associated CCA clusters which were enriched in the TP53 mutation as well as ERBB2 amplification [35]. The TP53 mutation was found in approximately 50% of cancers including CCA [36]. It causes conventional chemotherapeutic resistance. A recent study in Ov related CCA discovered that the anti-cancer drug PRIMA-1 MET can restore P53 activity in CCA cell lines, leading to cell senescence induction and resulting in CCA cell growth inhibition [37]. This finding indicates the role of the TP53 mutation in CCA progression and chemotherapy resistance and therefore can be a candidate target to improve CCA treatment. In addition to the downregulation and mutation mechanisms that lead to tumor suppressor gene dysfunction in Ov related CCA, loss of function in tumor suppressor gene by phosphorylated inactivation has been reported. Yothaisong and co-workers identified an alteration of the PI3K/Akt pathway in Ov-associated CCA human tissues. They found an overactivation of protein components in the PI3K/ Akt signaling cascade which is resulted from the dysfunction of the tumor suppressor proteins phosphatase and tensin homolog (PTEN). PTEN was suppressed by loss of expression or inactivation by phosphorylation, dysfunction of PTEN was observed in the majority of Ov related CCA patient tissues [38]. Moreover, a similar result was found in Ov related CCA carcinogenesis hamster tissues [39]. Taken together, the above evidences indicate that evading growth suppressors capacity of Ov related CCA involves with a tumor suppressor loss of function in the carcinogenesis and progression.

4.5

Resisting Cell Death

The machinery of apoptosis depends on the regulators and downstream effector components. The regulators consist of two major pathways, namely the receiving and processing of extracellular death signals (extrinsic pathway such as the Fas ligand and Fas receptor), and the program that senses and integrates a variety of signals of intracellular origin (the intrinsic pathway). Both pathways converge

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in the activation of proteolytic cleavage proteins called caspases that efficiently control the execution phase of apoptosis. Moreover, apoptosis is controlled by counterbalancing pro- and antiapoptotic members of the Bcl-2 family of regulatory proteins that convey signals between the regulators and effectors [40, 41]. In cancer, homeostasis between cell proliferation and cell death is disturbed. In bile duct cancer cells, increasing expression of the anti-apoptotic proteins Bcl-2, BclXL, and in myeloid cell leukemia-1 (Mcl-1) were identified in benign, dysplastic, and malignant biliary epithelium. Moreover, IL-6, which is a critical signaling molecule in the pathogenesis of Ov related CCA [18], can induce the expression of Mcl-1 which leads to CCA cell survival [42]. In addition, cancer cells can survive by increased activation of the anti-apoptotic pathway. The PI3K/Akt signaling pathway has been recognized as a survival pathway for cancer cells. PI3K/Akt plays roles in the activation of cell cycle progression, as well as cell survival. In terms of anti-apoptosis, Akt can modulate proapoptotic protein Bad activity by phosphorylating at S112. Bad induces apoptosis by inhibiting antiapoptotic Bcl-2-family members (Bcl-x and Bcl-2); phosphorylation of Bad by Akt results in the dissociation of Bad from Bcl-2 and is associated with cell survival [43]. In Ov related CCA, the activation of protein components in the PI3K/Akt signaling pathway has been documented. Dokduang et al. demonstrated the activation of the PI3K/Akt pathway in CCA patient tissues and CCA cell lines [11]. The expression and roles of the PI3K/Akt pathway in CCA were further identified by Yothaisong and co-workers. This study found increased PI3K/Akt signaling activation in CCA tissues. Interestingly, overactivation of the PI3K/Akt signaling cascade in CCA resulted from PTEN inactivation by either loss of expression or phosphorylation. These results were also observed in liver tissue in the Ov-induced cholangiocarcinogenesis hamster model, indicating the involvement of PI3K/Akt activity in CCA carcinogenesis and progression [38]. Apart from apoptosis, the cellular homeostasis mechanism, implicated in both pro-survival and pro-death processes, is an autophagic mechanism. Autophagy is a cellular adaptive response to stress conditions, such as nutrient deprivation, hypoxia, as well as anti-cancer agents that enable cells to break down cellular organelles or dysfunctional components allowing the resulting catabolites to recycle as cellular components used for biosynthesis and energy metabolism [44, 45]. There is an intersection between autophagy, apoptosis, and cell homeostasis. The survival signal that stimulates PI3K/Akt/mTOR to block apoptosis, similarly, inhibits autophagy; when the survival signals are insufficient and PI3K/Akt/mTOR signaling is downregulated, autophagy may be induced [46]. In Ov related CCA, the study by Yothaisong et al. showed that inhibiting PI3K/Akt/mTOR signaling by using a dual PI3K/mTOR inhibitor, NVP-BEZ235, resulted in CCA cell growth and movement inhibition, while lacking an effect on apoptotic induction. Conversely, they found that NVP-BEZ235 induced autophagy in CCA cells [38]. Autophagy is implicated in both pro-survival and pro-death processes; however, it is also defined as a mechanism that may enable tumor cells to survive antineoplastic therapy. If an anti-cancer agent has a potent enough effect, this will drive

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the damaged cells to undergo programmed cell death by autophagy. On the other hand, cells can survive and evade apoptosis by using the energy which is produced from the digested biomolecules via autophagic mechanisms when the anti-cancer agent is insufficient to contribute to cell death by autophagy [47]. Moreover, a recent study reported that the expression of hypoxic responsive proteins (HIF-1α and BNIP3) and a key regulator of autophagy (PI3KC3) was associated with poor survival of CCA patients and the aggressiveness of CCA [48]. The above results suggest that there is a cell death evading property in CCA. A critically important issue that requires comprehensive examination and clarification is the cell biological conditions that dictate when and how autophagy enables cancer cells to survive or enhance cell death.

4.6

Enabling Replicative Immortality

Unlimited replication in cancer cells generates macroscopic tumors. This capability contrasts with normal cell lineages in the body that have a limited number of cell growth and division cycles. Growth limitation in normal cells is controlled by the shortening of the telomeres at the end of chromosomes. Normally, telomeres of non-immortalized cells shorten with each cell division until complete uncapping leads to growth arrest called the “hayfrick limit,” which is the normal aging process for cells [49]. The unlimited proliferation capability of tumor cells involves the telomerase (TERT) enzyme [50]. Telomerase is a specialized DNA polymerase enzyme that maintains chromosome integrity and genomic stability by adding “TTAGGG” repeats at the end of chromosomes. Telomerase activity was detected in 85%–100% of cancer patients, whereas normal somatic cells have low or undetectable levels [51]. Likewise, upregulation of TERT has been detected in bile duct cancer. Ozaki et al. found that the expression of TERT mRNA, including human telomerase RNA (hTR) and telomerase protein components (TP1) mRNA, is highly expressed in carcinoma as well as the pre-neoplastic lesions of intrahepatic CCA tissues. Conversely, TERT expression was not detected in non-tumor or normal liver areas. This indicates the roles of TERT in the malignant progression of CCA [52]. In Ov-associated CCA, expression of TERT in CCA tissues has been reported [53]. Moreover, TERT mRNA can be detected in patient serum and can be used as a tumor marker for CCA patients [54]. However, there is limited evidence about the telomere and telomerase status on Ov-inducing CCA.

4.7

Inducing Angiogenesis

Like normal tissues, tumor tissues require nutrients and oxygen to supply their growth requirements tumors cannot grow over 2–3 mm3 nor can they metastasize to the other sites without the formation of new blood vessels [55]. Therefore, angiogenesis is a crucial process that promotes tumor progression. Angiogenesis

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is the process that forms new blood vessels through the division, movement, and assembly of endothelial cells from pre-existing vessels [56]. Regulation of the angiogenic switch is controlled by pro- and anti-angiogenic factors that either induce or inhibit angiogenesis. Some of the pro-angiogenic regulators are signaling proteins that stimulate surface receptors expressed on vascular endothelial cells [57, 58]. A well-known angiogenesis inducer is vascular endothelial growth factor-A (VEGF-A), which is the ligand that activates VEGF signaling via receptor tyrosine kinase, VEGFR-1-3 [59]. For Ov related CCA, angiogenesis is generally implicated in the progression of this tumor type. Dokduang and co-workers reported the activation of receptor tyrosine kinases in CCA tissues and cell lines. They found that VEGFR3 was activated in CCA tissues but was, however, undetectable in CCA cell lines [11]. This implies the involvement of the tumor microenvironment in the angiogenesis of CCA. VEGF signaling is controlled by hypoxia-inducible factors (HIFs), which are the transcription factors that respond to the decrease of available oxygen in the cellular environment, or hypoxia, in order to generate new blood vessels to supply oxygen for the growing tumor [60, 61]. In Ov related CCA, the expression of hypoxic responsive proteins including HIF-1α and BNIP3 is related to poor survival of CCA patients, as well as the development of metastatic stages [48]. Additional to the surface receptors that play a role in CCA angiogenesis, the downstream effector kinase that is associated with angiogenesis is also activated in Ov related CCA. Endothelial nitric oxide synthase (eNOS) is an isoform of the enzyme nitric oxide synthase (NOS) and is constitutively expressed in endothelial cells where it plays important roles in vasodilation [62, 63]. The activity of eNOS can be modulated by VEGFR3 and EphrinA3 via the PI3K/Akt signaling pathway [64]. Interestingly, eNOS and the upstream regulators are highly activated in CCA [11]. A study by Suksawat et al. revealed that eNOS, phosphorylated-eNOS (p-eNOS), and their upstream regulators including VEGFR3, VEGF-C (VEGFR3 specific ligand), EphrinA3, and ephrin A1 (EphrinA3 specific ligand) increased during CCA genesis. Moreover, an increase in eNOS and p-eNOS was found to be significantly associated with a high micro-vessel level in human CCA tissues [65], supporting tumor angiogenesis as an enabling hallmark of liver fluke-associated CCA.

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Activation of Invasion and Metastasis

The difference between benign and malignant tumors is the capability of cancer cells to leave a primary tumor to a distant tissue via blood circulation where they form a secondary tumor, i.e., invasion and metastasis. This is another important hallmark of cancer, which is generally composed of five steps termed the metastatic cascade [66]. 1) Invasion and migration: The detachment process of individual cells from the primary tumor is to invade to adjacent or healthy tissue. Actin polymerization is the rearrangement of the actin cytoskeleton to extend motile structures, and the modulation of cell–cell and cell–extracellular matrix adhesions. These can promote cell invasion and migration through molecular coordination and signaling orchestrated by both cancer cells and the stromal cells in the tumor microenvironment. During this process, a secretion of several lytic enzymes, e.g., matrix metalloproteases (MMPs), degrades the extracellular matrix (ECM) under the basement membrane to contribute to invasion and migration [66]. Our studies on Ov related liver injury revealed a high expression of hydroxyproline (precursor of collagen synthesis), collagen I, MMP-2, -9, -13, -14 in serum and liver tissue during tissue remodeling and periductal fibrosis [67, 68]. In addition, human CCA serum associated with liver fluke infection showed that hydroxyproline, collagen I, and MMP-7 were higher than in healthy controls [69]. Moreover, Subimerb and co-workers found that macrophage MAC387-positive cells could express MMP-9 [70]. 2) Intravasation: After cancer invasion and migration, the cancer cells enter into the blood and lymphatic vessels mainly via attachment to the endothelial cells using cell adhesion molecules (CAMs) such as intercellular cell adhesion molecule-1 (ICAM-1), vascular endothelial cell adhesion molecule-1 (VCAM1), E-selectin, and P-selectin [71]. The secretion of proteolytic enzymes by neoplastic cells also enables them to intrude into blood vessels [66]. In CCA, serum ICAM-1 levels are significantly higher than in healthy controls [72]. 3) Circulation: The aberrant cells travel via the bloodstream and have to withstand the conditions present in the blood. These are toxic for cancer cells due to the high concentration of oxygen and cytotoxic lymphocytes. Thus, a selection for particularly resistant and aggressive tumor cells takes place. 4) Extravasation: The cells often get stuck in the capillaries of an organ and leave the bloodstream by penetrating the endothelium through proliferation and/or proteolytic enzymes. 5) Colonization: Proliferation and angiogenesis: The neoplastic cell settles at a distant tissue site and builds a secondary tumor. The latter proliferates and induces neoangiogenesis in order to ensure sufficient vascularization.

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Processes of epithelial-mesenchymal transition (EMT) Epithelial-mesenchymal transition (EMT) is the process of conversion from an epithelial to a mesenchymallike phenotype. It plays a crucial role for many steps of cancer invasion and metastasis. In general, cancer cells use the same genetic programs as healthy cells, which are induced by transcription factors, but are activated at the wrong time and in the wrong cells. They normally control crucial steps in early embryogenesis. The molecular and cell biological principles of invasion and metastasis are highly complex but essential for a profound understanding of carcinogenesis. Various genetic, anatomical, and physiological conditions affect metastasis, including components of the extracellular matrix, integrins, cadherins, cell–cell adhesion molecules (CAMs), cellular adhesion molecules, factors involved in cellular movement (motility and chemotaxis), metastasis genes and metastasis suppressors, and several proteinases (plasminogen activators, cathepsins, matrix metalloproteinases) as well as their inhibitors. Additionally, the nature and localization of the primary tumor as well as angiogenic factors and the immune system should be considered. CCA is a malignant tumor derived from the epithelial tissues, which is connected to the ECM by the basement membrane and mainly expresses epithelial cadherin (E-cadherin). E-cadherin, a cell adhesion molecule, is frequently downregulated and has been proposed as an important mediator in epithelialmesenchymal transition (EMT) in tumors. Apart from E-cadherin, there are other molecules, N-cadherin and vimentin, which are expressed by mesenchymal cells (in particular fibroblasts). N-cadherin is often upregulated in cancer cells, whereas E-cadherin is frequently found to be down-regulated. Phenomena like this not only occur in the context of a tumor EMT, but can also be observed during gastrulation [66]. Our group demonstrated a statistically significant association between positive metastasis status with low E-cadherin protein expression in human CCA tissues. Suppressing the E-cadherin expression caused the upregulation of vimentin, a mesenchymal marker, resulting in increasing migration and invasion abilities. These findings suggest that loss of E-cadherin contributes to CCA progression by attenuating the strength of cellular adhesion, affects motility as well as regulating the expression of EMT-related genes during CCA invasion and metastasis [73]. In addition, we have shown that there is an upregulation of Twist transcription factor and N-cadherin mesenchymal marker in CCA tissues. The high expression of Twist was significantly associated with a poor prognosis of CCA patients. Moreover, CCA samples with Twist nuclear expression were significantly correlated with the upregulation of N-cadherin [74]. Moreover, qRTPCR analysis revealed that Snail expression significantly increased in CCA, and was correlated with tumor metastasis. Interestingly, the expression of Snail was inversely associated with E-cadherin [75]. A previous study has also shown that the activation of vimentin could promote the metastatic potential of CCA cells. Suppression of vimentin significantly decreased migration and invasion capabilities [76].

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Chemokines and cytokines contribute to the EMT process. The activation of EMT can be induced by various types of inflammatory cytokines including transforming growth factor β (TGF-β) and tumor necrosis factor-α (TNF- α). Inflammatory cytokines are mainly produced by autocrine with cancer as well as paracrine with stromal cells including tumor-associated macrophages (TAMs). Previously, we have demonstrated that various cytokines, such as IL4, IL-6, IL10, TNF-α, and TGF-β1, secreted by LPS-activated macrophages could induce EMT by the reduction of epithelial markers (E-cadherin and CK-19), and the induction of mesenchymal markers S100A4 and MMP-9 resulting in the modification of EMT in CCA [77]. It is well known that the activation of EMT can be induced by various types of inflammatory cytokines including transforming growth factor β (TGF-β), whereas bone morphogenetic protein-7 (BMP-7) can inhibit this process. In CCA, the inflammatory mediator TGF-β induces CCA cell migration, one of the metastatic processes, possibly via stimulation of Twist, N-cadherin, and vimentin expression. Additionally, BMP-7 inhibits TGF-β-induced CCA cell migration through the inhibition of TGF-β-mediated Twist and N-cadherin expressions. These data reinforce the rationale to use BMP-7 as an EMT inhibitor to suppress the progression of CCA. This might be a valuable therapeutic approach to improve the efficiency of CCA treatment [74]. With respect to TNF-α, its stimulation enhances migration behavior and significantly induces the expression of Snail in CCA cell lines. However, expression of E-cadherin and CK-19 (the epithelial marker) was found to be reduced. Immunofluorescence analyses have revealed that TNF-α-treated CCA cell lines increased nuclear translocation of Snail, whereas E-cadherin was dramatically decreased. These findings suggest that changes in the expression of Snail or E-cadherin might regulate EMT development in CCA resulting in the promotion of tumor progression [75]. We have recently reported that TNF-α modulates the epithelial-mesenchymal transition mediators ZEB2 and S100A4 to promote CCA progression. Results showed that ZEB2 and S100A4 mRNA levels were higher in CCA tissues, and that high expression was significantly associated with CCA metastasis. In addition, TNF-α-induced CCA cell migration by the induction of TGF-β resulting in ZEB2 and S100A4 mRNA and protein activation. These results showed that TNF-α plays a crucial role in the progression of CCA by activating TGF-β signaling, and the induction of ZEB2 and S100A4 EMT-related proteins expression [75]. In addition, this research supported the hypothesis that migration and metastasis are involved with inflammatory lipid mediators, namely PGE2 [78] and leukotrienes [79]. Two enzymes which produce inflammatory lipid mediators are cyclooxygenases (COX-2) are suppressed by meloxicam [80] and 5-lipoxygenase (5-LOX). Moreover, zileuton-inhibited 5-LOX could reverse the epithelial mesenchymal transition to the mesenchymal-epithelial transition phenotype [79]. Additionally, the secretion of cancer-associated fibroblasts (CAFs) powerfully induced IL-6 mediated motility in CCA cells, while CAFs pre-treated with Resveratrol completely halted cancer cell motility and reverted the N- to E-cadherin switch in migrating cells [81].

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Other factors are associated with CCA metastasis and invasion via EMT, such as HIF-1 [48, 82], progranulin [83], EGFR [84], and eNOS activation via the upregulation of phosphorylated vasodilator-stimulated protein (p-VASP) [85], Ephb2 [86], CD44 [87], TCTP [88]. To summarize, an activation of CCA invasion and metastasis occurs via EMT processes due to the stimulation of inflammatory cytokine production, such as TNF-α and TGF-β, as well as inflammatory lipid mediators. Based on previous findings, loss of E-cadherin is regulated by Snail via TGF-β activation. Conversely, upregulation of N-cadherin and vimentin is delimited by Twist transcription factor via stimulating TNF-α. During this process, several protease enzymes, e.g., hydroxyproline, collagen I, and MMP-9, have been detected in both human and hamster CCA.

4.9

Enabling Characteristics and Emerging Hallmarks

4.9.1 Genome Instability and Mutation The alteration of the genome is an important point to turn normal cells into cancer cells. Several reports have demonstrated that a succession of clonal expansions, triggered by the chance of an enabling mutant genotype, e.g., inactivation of tumor suppressor gene, can be acquired through epigenetic mechanisms, such as DNA methylation and histone modifications [14]. Chronic inflammation and liver fluke infection caused by CCA, with an over-production of free radicals, leading to the adaptive response of the genome protection mechanism in cells. Aberrant epigenetic regulation, such as promoter hypermethylation in CpG-islands, has been shown in numerous important cancer-associated genes (OPCML, SFRP1, HIC1, PTEN, and DcR1) in CCA [89]. These findings suggested that the aberrant hypermethylation of certain loci is a common event in Ov related CCA and may potentially contribute to CCA genesis. Interestingly, microsatellite instability (MSI)-high was present in 69% of patients and was associated with poor prognosis [90], whereas MSI-low was present in 31 of patients. Additionally, a large CCA cohort study (n=102) linked with Ov infection showed a promoter of hypermethylation in a handful of target genes compared to adjacent tissue [89]. Whole-exome and targeted sequencing revealed frequent mutations of TP53 (44%), KRAS (16.7%), and SMAD4(16.7%), and novel CCA-related genes to be associated with chromatin remodeling [BAP1(2.8%), ARID1A (17.6%), MLL3 (13%), and IDH1/ 2 (2.8%)], WNT signaling [ RNF43(9.3%) and PEG3 (5.6%)] as well as KRAS/G protein signaling GNAS(9.3%) and ROBO2 (9.3%)]. Interestingly, there is a significant difference in the frequency of mutated genes between Ov related CCA and non Ov related CCA, such as TP53 and IDH1/2, reflecting the different pathways of pathogenesis. Altered DNA methylation and transcriptional profiles associated with the xenobiotic metabolism and pro-inflammatory responses were also found in Ov related CCA [12]. Recently, there has been a considerable effort to elucidate the different patterns of whole-genome, targeted/exome, copy-number, gene

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expression, and DNA methylation information from different parts of the world. Integrative clustering defined 4 CCA clusters-fluke-positive CCAs (clusters 1/2) which are enriched in ERBB2 amplifications and TP53 mutations. Conversely, fluke-negative CCAs (clusters 3/4) exhibit high copy-number alterations and PD-1/ PD-L2 expression, or epigenetic mutations (IDH1/2, BAP1) and FGFR/PRKArelated gene rearrangements. Whole-genome analysis has highlighted the FGFR2 3’ untranslated region deletion as a mechanism of FGFR2 upregulation. Integration of noncoding promoter mutations with protein-DNA binding profiles demonstrates pervasive modulation of H3K27me3-associated sites in CCA. Clusters 1 and 4 exhibit distinct DNA hypermethylation patterns targeting either CpG-islands or shores-mutation signature. Sub-clonality analyses suggest that these reflect different mutational pathways. Integrated whole-genome and epigenomic analysis of CCA on an international scale identified new CCA driver genes, noncoding promoter mutations, and structural variants. CCA molecular landscapes differ radically by etiology, underscoring how distinct cancer subtypes in the same organ may arise through different extrinsic and intrinsic carcinogenic processes [35].

4.9.2 Tumor-Promoting Inflammation Histological data of carcinogenesis in an animal model as well as human CCA tissues clearly demonstrates a high population of immune cells [70, 91–95]. In 1978, Thamavit and co-worker were the first to report CCA induction with Ov infection and NDMA administration using the hamster animal model. They found that early pathological changes consisted of an acute inflammatory reaction involving the bile ducts of the second order and the portal connective tissue, especially the large veins. As the flukes developed into adults, they induced hyperplasia of the bile duct epithelium and adenomatous formation. Granubomatous responses to the adult worms and ova were present [91]. In addition, a recent study suggests that Ov antigen can trigger the inflammatory response of macrophages (RAW cell line) through the TLR2-mediated pathway leading to the NF-kappa-B-mediated expression of iNOS and COX-2 [95]. In 2011, the links between parasitism by the carcinogenic Ov and this co-regulator using both an Mta12/2 mouse model of infection and a tissue microarray of Ov-associated human CCA was investigated and demonstrated that master co-regulator (MTA1) status plays an important role in conferring an optimal cytokine response in mice following infection with Ov and is a major player in parasite related CCA in humans [96]. This finding indicates that inflammatory cytokines might play a vital role in CCA genesis. Thanee and co-workers revealed that a high density of macrophages was associated with carcinogenesis in the hamster animal model [94]. Similarly, our research on human CCA tissues shows high densities of tumor-associated macrophages (MAC387positive cells) in CCA tissues and blood circulating CD14(+) CD16(+) monocytes were significantly associated with a poor prognosis [70, 93]. With respect to tumor heterogeneity, tumor tissues are composed of not only of cancer cells, but also have

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a microenvironment that is called the tumor microenvironment. Tumor-associated macrophages (TAMs) are divided into two types depending on T cell activation and their cytokines. M1 can inhibit cancer promotion, and M2 macrophages can promote cancer development. Our group demonstrated that a high density of M2 macrophages was present in CCA patients with poor prognosis. Moreover, we found that some of the CCA cells could act like the macrophage phenotype and express macrophage markers (CD163). These correlated with metastasis [94]. The details are described in the section below on the tumor microenvironment.

4.9.3 Reprogramming Energy Metabolism Emerging evidence indicates that cancer is primarily a metabolic disease involving disturbances in energy production through respiration and fermentation. The genomic instability observed in tumor cells and all other recognized hallmarks of cancer are considered downstream epiphenomena of the initial disturbance of cellular energy metabolism. The disturbances in tumor cell energy metabolism can be linked to abnormalities in the structure and function of the mitochondria. When viewed as a mitochondrial metabolic disease, the evolutionary theory of Lamarck can better explain cancer progression than can the evolutionary theory of Darwin. Cancer growth and progression can be man-aged following a wholebody transition from fermentable metabolites, primarily glucose, and glutamine, to respiratory metabolites, primarily ketone bodies. As each individual is a unique metabolic entity, personalization of metabolic therapy as a broad-based cancer treatment strategy will require fine-tuning to match the therapy to an individual’s unique physiology [97]. Altered energy metabolism in cancer cells was discovered over 80 years ago: Otto Warburg discovered that cancer cells predominantly produce energy (adenosine triphosphate, ATP) through the glycolytic pathway rather than through the tricarboxylic acid (TCA) cycle, even in the presence of adequate oxygen. This altered energy dependency is known as “aerobic glycolysis” or the “Warburg effect.” Under aerobic conditions, normal cells process glucose, first to pyruvate via glycolysis in the cytosol and thereafter to carbon dioxide in the mitochondria; under anaerobic conditions, glycolysis is favored and relatively little pyruvate is dispatched to the oxygen-consuming mitochondria [98, 99]. A key metabolic enzyme of anaerobic glycolysis in the final step is lactate dehydrogenase A (LDHA). An increase in LDHA expression is associated with the progression of CCA genesis and CCA progression, supporting the aberrant energy metabolism of CCA [100]. In addition, hexokinase 2 (HKII) an enzyme involved in rate-limiting step of glycolysis pathway was found to be overexpressed in CCA tissues and targeting HKII could suppress CCA progression [101]. Extracellular glucose level was shown to alter the progression of CCA cells and evidence supports that high glucose conditions affect CCA cell proliferation, migration, and invasion [102, 103]. Recently, Padthaisong reported that patients with cancer recurrence have different metabolic profiles compared with those without recurrence. Patients with

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cancer recurrence represent high activity of glycolysis and pyruvate metabolism while the activity of the TCA cycle is low [104]. Apart from glycolysis, the metabolism of amino acids is also important. Cancer cells undergo unlimited growth and multiplication, causing them to require massive amounts of amino acid to support their continuous metabolism. Among the amino acid transporters expressed on the plasma membrane, l-type amino acid transporter-1, a Na+ -independent neutral amino acid transporter, is highly expressed in many types of human cancer, including CCA. Previously, we have reported that l-type amino acid transporter-1 and its co-functional protein CD98 were highly expressed and implicated in CCA progression and carcinogenesis. Moreover, this report showed that the suppression of l-type amino acid transporter1 activity using JPH203 inhibits CCA progression in vitro and in vivo [105, 106]. In addition, lipids are also important in terms of energy storage, signal transduction as well as cell structure. The upregulation of lipid biosynthesis enzymes, such as fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), and HMG-CoA reductase (HMGCR), have been found in various cancers. In CCA, high expression of lipid biosynthesis enzymes, FASN and HMGCR were correlated with low survival of patients. In addition, differences in metabolic profile were found in CCA cell lines with and without FASN knockdown. The FASN knockdown cells showed low activity in purine metabolism which is important in the production of DNA components which are required for CCA cell proliferation [30]. Recently, Thanee demonstrated that an interruption of CCA metabolism pathway using sulfasalazine, well known as an xCT inhibitor, could inhibit CCA cell progression in vitro and in vivo [107].

4.9.4 Evading Immune Destruction The immune system plays a role in resisting or eliminating the formation and progression of incipient neoplasia, late-stage tumors, and micrometastases. Tumors somehow escape detection by the immune system, thereby evading suppression as in the case of the increase in immunocompromised individuals with certain cancers [108]. Mice genetically engineered to be deficient for various components of the immune system were assessed for the development of carcinogen-induced tumors. The results showed that tumors arose more frequently and grew more rapidly in immunodeficient mice compared to immunocompetent controls. In particular, deficiencies were found in the development or function of CD8+ cytotoxic T lymphocytes (CTLs), CD4 + Th1 helper T cells, or natural killer (NK) cells, each of which led to demonstrable increases in tumor incidence. Furthermore, mice with combined immunodeficiencies of both T cells and NK cells were more susceptible to cancer development [109, 110].

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In CCA, there is an enhanced cytotoxic activity of effector T cells through RNA- and protein-pulsed dendritic cells. A study has revealed that activation of anti-tumor effector T cells against CCA by RNA-pulsed dendritic cells is more effective than that of protein lysate-pulsed dendritic cells. In addition, pulsing dendritic cells with pooled messenger RNA from multiple cell lines enhanced the efficacy of a cellular immune response against CCA [111]. In recent years, the animal model of CCA induction with Ov infection and N-nitrosodimethylamine (NDMA) administration revealed significantly higher numbers of IL-17-producing CD4+ T cells (Th17) and CD4+ Foxp3+ T cells (Treg) during carcinogenesis, indicating their involvement in the early stages of CCA genesis Infiltration of IL-17 + inflammatory cells and Foxp3+ cells, as well as increases in their transcription expression levels, was significantly lower in the melatonintreated group In contrast, increased CD4+ cell infiltration and TNF-α expression were also observed through melatonin treatment Melatonin exerts an immunomodulatory effect, suppressing eosinophils and Th17 cells, and expression of Foxp3, but enhancing CD + cells and TNF-α [112].

4.9.5 The Tumor Microenvironment Tumors have progressively been recognized as organs, and the explanation of tumor biology can be understood by studying the individual specialized cell types that surround the neoplasm and influence the course of multistep tumorigenesis, termed “tumor microenvironment” [14]. The tumor microenvironment (TME) includes numerous non-neoplastic cells such as leukocytes (e.g., macrophages, neutrophils, dendritic cells) and stromal cells (e.g., fibroblasts, endothelial cells). Unlike hepatocellular cacinoma (HCC), CCA is characterized by a noticeable desmoplastic tumor microenvironment (TME) composed of various cell types, especially tumor-associated macrophages, TAMs, and cancer-associated fibroblasts (CAFs) that support and promote the development of CCA [70, 81, 93–95, 113, 114]. Recently, evidence of cancer stem cells (CSC) in CCA has been increasingly reported [87, 115, 116]. Cancer cells and cancer stem cells Traditionally, the cancer cells within a tumor have been portrayed as being comprised of reasonably homogeneous cell populations until relatively late in the course of tumor progression, when hyperproliferation combined with increased genetic instability spawn distinct clonal subpopulations. Such clonal heterogeneity points to the existence of a new dimension in intratumor heterogeneity and a hitherto-unappreciated subclass of neoplastic CSC cells within tumors [14]. The vital property of CSCs is their ability of self-renewal, which dysregulated from redox regulation assists in CSCs to resist general chemotherapy. This may be one of the causes for drug resistance and recurrence [117]. CSC markers, namely CD44 and its variants [87, 118], CD133, and Oct3/4 [116] are expressed in CCA tissues and CCA cell lines. Their high expression is linked with a poor prognosis in CCA patients. Importantly, our group found that EBF1 downregulation induces

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stem cell properties via oxidative stress leading to CCA genesis with aggressive clinical outcomes [115]. Similarly, the accumulation of CD44v9 has been recorded in transforming bile duct cells and is related to the redox status regulation of CCA cells via the contribution of the xCT function. Sulfasalazine, an xCT inhibitor, could inhibit CCA cell growth and activate cell death of the CD44v9-subpopulation. This suggests that an xCT-targeting drug may improve CCA therapy by sensitization to the available drug (e.g., gemcitabine) by blocking the mechanism of the cell’s ROS defensive system [87]. Recently, the association of the existing of CSC and CCA recurrence was also reported [104]. However, the interaction of CCA cells and CSCs is still unclear. Immune inflammatory cells Over the past decade, an early transformation of bile duct epithelial cells in the animal model induced by Ov infection and NDMA administration was observed along with an acute inflammatory response [91]. Obviously, Ov antigen can trigger an inflammatory response by macrophages (RAW cell line) through the TLR2-mediated pathway leading to NF-kappa-B-mediated expression of iNOS and COX-2 [95]. The accumulation of tumor-associated macrophages was observed during the development of CCA in the animal model [94]. Similar to the animal model, high densities of tumor-associated macrophages (MAC387-positive cells) in CCA tissues and blood circulating CD14(+) CD16(+) monocytes were found to be significantly associated with a poor prognosis [70, 93]. Tumor-associated macrophages (TAMs) are divided into two types depending on T cell activation and their cytokines. M1 macrophages can be stimulated by LPS and can inhibit cancer promotion [119]. Conversely, M2 macrophages are activated by IL4 or IL-13 and can promote cancer progression [119]. We have currently demonstrated a high density of M2 macrophage in CCA patients which is associated with a poor prognosis [94]. Interestingly, M2 macrophages can trigger the migration of CCA cells through the EMT process [94]. Cancer-associated fibroblasts Cancer-associated fibroblasts (CAFs) have been proposed to play a role in promoting carcinogenesis and tumor progression. Evidence concerning CAFs in the genesis of cholangiocarcinoma (CCA) has previously been reported [94]. Results from CCA patients indicate a statistically significant correlation between the high expression of CAF markers (alpha-SMA or FSP-1) and aggressive CCA progression [94, 113]. CCA-derived CAFs have proliferative effects which may directly affect CCA progression [113]. The microRNA (miRNA) profile found miRNA-15a downregulated in CAFs when compared with normal skin fibroblasts using microarrays. Moreover, miR-15a of CAFs can suppress the migration of CCA cells [120]. Interestingly, Thongchot demonstrated that the condition media collected from CAFs could induce the CCA cell migration [121]. The conditioned medium from CCA-derived CAFs further stimulated the secretion of IL-6, and to a lesser extent of IL-8, by CCA cells. More importantly, they showed that Resveratrol has the potential to abrogate the secretion of IL-6 by CAFs and inhibit the capability of CCA migration via the switching of N-to E-cadherin. This finding indicates that CAFs secretory products directly revert the malignant phenotype of cancer cells [81].

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Molecular Heterogeneity and Therapeutic Opportunities

Genomic alterations play a role in the development and progression of cancer and thus have important clinical implications, from diagnosis to therapy. In recent years, advances in genomic profiling techniques have enabled comprehensive mutational profiling of CCA tumors, identifying novel treatment targets, and providing new insights into the genetic basis of cholangiocarcinogenesis [35, 122– 128]. Studies on different population cohorts characterized different chromosomal, expressional, and aberrant signaling in intrahepatic (iCCA), perihilar (pCCA), and distal (dCCA). These data highlighted the molecular subgroups associated with the motor genes and a patient’s prognosis.

4.10.1 Genetic Profiling of iCCA and Its Clinical Implication in Targeted Therapy Genetic alterations vary across different subtypes of CCA [129, 130]. Mutations in common oncogenes and tumor suppressors previously have been found in iCCA including KRAS, BRAF, EGFR, PI3CA, PTEN, TP53. Interestingly, 25–50% of iCCAs exhibited genetic alterations involving at least one of the chromatin remodeling genes [131–134]. ARID1A, PBRM1, and BAP1 functioned as tumor suppressors and were enriched in inactivating mutations in iCCA. CCA patients with mutations in any one of these genes tended to have poorer prognosis [133]. These mutations may provide additional therapies for iCCA, as drugs targeting chromatin remodeling like histone deacetylase (HDAC) inhibitors may have a therapeutic benefit [135]. It should be noted that the main exploitable genetic aberrations in iCCA were IDH1/2 and FGFR alteration. The IDH1/2 mutations were found in 10–25% of iCCA tumors and rarely in extrahepatic CCA (eCCA) and gall bladder cancer (GBC) [122, 125, 136]. In contrast, they are virtually absent in pCCA and dCCA [137]. Among the three isoforms of IDH, IDH1 mutations are more frequent than IDH2 mutations and tend to appear in non-OV-related CCA [128]. The most common IDH1-2 variants are the “hotspot” IDH1/2 activating mutations (R132 and R172). These somatic gain-of-function mutations result in the production of oncometabolite 2-hydroxyglutarate (2-HG) and are involved in tumor development. Currently, several IDH-selective inhibitors that are highly specific to the IDH-mutant alleles and block the function of mutant IDH1 or IDH2 leading to reduced 2-HG levels have been developed. The inhibitors of IDH1 (AG120) and IDH2 (AG221), ivosidenib and enasidenib (NCT02273739), respectively, are currently under investigation in several trials. A recent phase III trial in 185 iCCA patients with IDH1 mutations (R132C/L/G/H/S mutation variants) demonstrated a significant risk reduction of disease progression with ivosidenib [138]. Additionally, through a high-throughput drug screen of a large panel of cancer cell lines, including 17 biliary tract cancers (BTCs), demonstrated that CCA cell lines

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harboring IDH mutations exhibited a striking response to the multi-TKI, dasatinib [139]. In addition, dasatinib-treated IDH-mutant xenografts demonstrated pronounced apoptosis and tumor regression. Recently (2021), the FDA approved ivosidenib, an oral small-molecule IDH1 inhibitor used for the treatment of adults with previously treated locally advanced or metastatic IDH1-mutated CCA. Data from the phase 3 ClarIDHy (NCT02989857) clinical trial revealed that ivosidenib achieved a 63% reduction in the risk of progression or death in patients with IDH1mutanted CCA compared with placebo. Several other strategies for the exploitation of IDH mutations are also being investigated [140]. Firstly, increasing 2-HG production has been shown to reduce NAPDH production and increase levels of ROS during carcinogenesis, thus increasing susceptibility to ionizing radiation and PARP inhibitors [141, 142]. Secondly, IDH-mutant CCA cells have been shown to be dependent on Src activity and are sensitive to the kinase inhibitor dasatinib in vitro [139], suggesting a rationale for phase II evaluation in IDH1/2-mutant iCCA (NCT02428855). Additionally, it has also been evidence to indicate that tumors with IDH mutations have been associated with hypermethylation in CCA [35, 128]. Most studies have identified that fibroblast growth factor receptor (FGFR) aberrations are enriched in iCCA. FGFRs are tyrosine kinase receptors involved in many biological processes. Aberrant FGFR activity caused by genetic alterations including gene rearrangement, activating mutations, and amplifications can initiate malignant transformation. Alterations in FGFR identified in CCA are mostly located in FGFR2; moreover, there is a predominance of rearrangements or fusions (3.5%) over amplifications (2.6%), with few mutation events (0.9%) [130, 143]. FGFR2 rearrangement (fusion and translocation) is the most common type of FGFR alteration in iCCA with a prevalence of 14%–23%, and rarely in Ovassociated CCA [136, 144, 145]. Common FGFR2 fusions observed in CCA are FGFR2-AHCYL1, FGFR2-BICC1, FGFR2-PARK2, FGFR2-MGEA5, FGFR2TACC3, and FGFR2-KIAA1598 [144, 146–148]. The study from the Mayo Clinic showed that the survival of CCA patients with FGFR2 fusions was significantly higher than those without FGFR2 fusions, suggesting the potential utility of FGFR2 fusion identification as a prognostic marker. Kongpetch and colleagues demonstrated that in fluke-associated iCCA, the presence of rare FGFR2 fusions indicated a trend toward better overall survival compared with that of fusionnegative tumors, although the difference was not statistically significant [145]. Interestingly, the FGFR2 fusions were found in CCA patients with non-advanced, early stages of the disease. This suggests that FGFR2 fusion may be an early oncogenic event that serves as a driver of tumorigenesis [149, 150]. Interestingly, FGFR2 fusions are more relevant to treatment with FGFR inhibitors than mutations. In vitro and in vivo studies demonstrated that the oncogenic ability of FGFR2 fusion proteins can be suppressed by treatment with FGFR kinase inhibitors [147, 148, 151]. Several candidate drugs targeting the FGFR pathway are currently being developed, particularly non-selective and selective FGFR tyrosine

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kinase inhibitors (TKIs) [152]. FGFR2 inhibitors, such as pemigatinib, derazantinib (NCT03230318), and infigratinib (BGJ394), have shown promising results in clinical trials. With these positive results, pemigatinib was recently approved by the FDA for previously treated, unresectable locally advanced or metastatic CCA, with a FGFR2 fusion or other rearrangement. Note, that for the majority of FGFR inhibitors under development responses have been absent for patients with other FGFR aberrations [130, 153]. Apart from FGFR2 fusions, c-ros oncogene 1 receptor tyrosine kinase (ROS1) fusions have been identified in 8.7% of iCCAs [154]. ROS1 encodes an orphan receptor tyrosine kinase related to anaplastic lymphoma kinase (ALK) which can be continuously activated by chromosomal rearrangement. FIG-ROS identified in iCCA patients was validated as a potent oncogene in an orthotopic allograft mouse iCCA model that could accelerate tumor onset forming an aggressive and metastatic subtype with cooperation of KRAS G12D and mutant p53 [155]. Crizotinib, a small-molecule tyrosine kinase inhibitor of ALK, ROS1, and a hepatocyte growth factor receptor (HGFR, also known as c-MET), have shown marked antitumor activity in patients with advanced ROS1-rearranged non-small-cell lung cancer regardless of the type of ROS1 rearrangement [156]. Furthermore, an integrative genomic analysis has identified two classes of iCCA with distinct characteristics, activated genes, and clinical outcome [124]. The proliferation class (62%) is characterized by the activation of oncogenic signaling pathway (i.e., RAS, MAPK, and MET) mutations in KRAS and BRAF. While the inflammation class (38%) exhibited activated inflammatory signaling pathways, with overexpression of cytokines, and STAT3 activation. The genomic heterogeneity of iCCA between Asian and Western populations was also investigated [157]. The Asian iCCA patients (72%) had ≥1 actionable genetic alteration, with a significantly higher frequency in KMT2C, BRCA1/2, and DDR2 compared with Western patients. While 60.9% of Western patients had ≥1 actionable genetic alteration and higher frequency of genetic alterations in CDKN2A/B and IDH1/2. Additionally, DNA repair genes and higher mutational burdens occurred more frequently in Asian patients with defects in nuclear factor kappa B pathway regulators.

4.10.2 Genetic Profiling of eCCA and Its Clinical Implication in Targeted Therapy The prognosis of eCCA has remained generally poor with neither molecular targeted therapies nor immunotherapies recommended for this subtype of CCA [158]. To date, no targeted therapies have demonstrated survival benefits in eCCA, which may result from the limited understanding of the biological mechanisms of action [129]. Previous constraints in conducting a molecular characterization of eCCA have included the low number of samples analyzed, and the inclusion of heterogeneous samples from different biliary tract cancer (BTC) subtypes, particularly iCCA and gall bladder cancer (GBC) [35, 122, 127].

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Recent integrative molecular analyses of eCCA have revealed a better understanding of the molecular landscape with the identification of novel therapeutic targets for eCCA [126]. The study included an international multicenter dataset of 189 Western patients with eCCA. Mutations of KRAS (36.7%), TP53 (34.7%), ARID1A (14.0%), and SMAD4 (10.7%) were the most prevalent mutations. Recurrent chromosomal amplifications were observed in YEATS4 (6.0%), MDM2 (4.7%), CCNE1 (2.7%), CDK4 (1.3%), and ERBB2 (1.3%). Mutations of these genes were classified into four main oncogenic signaling pathways: RTKRAS-PI3K (53% of tumors), TP53-RB (47%), histone modification (22%), and transforming growth factor-β (TGFβ, 18%). In contrast to iCCA, IDH1/2 mutations were identified in only 4.7% of cases of eCCA. Moreover, unsupervised clustering of whole-genome expression data from 182 eCCA revealed four distinct molecular classes of eCCA with potentially targetable genomic alterations, which can be classified as follows: metabolic class (18.7%), proliferation class (22.5%), mesenchymal class (47.3%), and immune class (11.5%). The metabolic class demonstrated overexpression in hepatocyte markers and enrichment in gene signatures linked to the deregulated metabolism of bile acids. The proliferation class presented overexpression of MYC targets, HER2/neu aberrations, and enrichment of oncogenic AKT/mTOR and Ras/MAPK pathways. The mesenchymal class showed aberrant TGF-β and TNF-α and poorer prognosis. Lastly, the immune class exhibited several immune-related features, comprising overexpression of PD-1/PD-L1 and higher lymphocyte infiltration. Notably, actionable genomic alterations, which have potential prognostic and therapeutic implications, were identified in 25% of eCCA. Moreover, Nakamura and colleagues showed for the first time genetic aberrations in eCCA, namely ATP1B-PRKACA and ATP1B-PRKACB fusions, which were observed only in pCCA and dCCA patients [122]. It should be noted that while few studies of specific therapies in eCCA have been completed to date, clinical, and molecular data continue to accumulate. Until now, the main targetable findings of eCCA rely on the HER gene family. Regarding HER2, its alterations seem to be more frequent in eCCA (15%) compared to iCCA [159–161]. Despite promising preclinical results, available data on HER2-targeted therapies in BTCs are controversial and are limited to case reports and case series evaluating monoclonal antibodies in HER2-positive patients with metastatic disease [162, 163]. A report of retrospective series of 14 BTC including GBC and CCA patients harboring HER2 aberrations who received lapatinib, pertuzumab, or trastuzumab described the response rate (50%) and duration of response (median of 40 weeks). Moreover, disease control was achieved in eight out of 14 patients, including a complete response in a GBC patient. However, this benefit seems limited to GBC since no responses were detected in other BTC groups [164].

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KRAS mutations occur in high frequency in eCCA, with KRAS aberrations being associated with poorer prognosis and more advanced stage at diagnosis [165, 166]. Previous efforts have focused on the inhibition of the KRAS downstream targets, MEK and BRAF. A Phase II trial assessing selumetinib, a MEK inhibitor as first- or later-line treatment, showed interesting activity in 25 advanced BTC patients, with a median PFS of 3.7 months and a median OS of 9.8 months [167]. Another MEK inhibitor, trametinib has been evaluated in a trial on 20 previously treated BTC patients including six cases of eCCA and reported that the median PFS occurred in 10.6 weeks, with a 1-year OS rate of 20% [168]. Interestingly, VEGF-A overexpression has been reported in approximately 60% of eCCAs [169]. The overexpression of VEGF-A has been associated with poorer survival and a more aggressive clinical course in advanced BTC [170]. Combination strategies using VEGF antibodies or tyrosine kinase inhibitors (TKIs) with systemic chemotherapy or immunotherapy in advanced BTC have been tested. For example, cediranib-a multi-kinase inhibitor targeting VEGFR, PDGFR, and c-KIT has been evaluated in the phase II trial which includes 48 eCCAs. Unfortunately, the addition of cediranib to the CisGem plus did not improve PFS in any BTC patient or in selected subgroups [171]. Recently, NTRK gene fusions have been identified in a wide number of solid tumors, including BTCs [172]. A study presented at ESMO World Congress on Gastrointestinal Cancer 2020 attempted to determine the incidence of NTRK gene fusions in biliopancreatic malignancies, including pancreatic cancer and BTCs [173]. The presence of NTRK gene fusions was observed in only 0.67% of BTC malignancies. Larotrectinib is under investigation as monotherapy in a Phase II basket trial of NTRK-positive solid tumors, including eCCAs (NAVIGATE, NCT02576431).

4.11

Conclusion

This chapter provides a useful conceptual framework for understanding the complexity of CCA carcinogenesis and biology. Chronic inflammation caused by liver fluke, Opisthorchis viverrini (Ov) infection leads to the over-production of the reactive oxygen species (ROS) and reactive nitrogen species (RNS). This can cause damage to many cellular biomolecules including protein, lipid or DNA and has been defined as a risk factor of CCA in the endemic region. In addition, the six capabilities are a basic requirement for cancer development and can be termed the hallmarks of CCA: sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, induction of angiogenesis, and promoting invasion and metastasis. These characteristics are mainly associated with the kinase pathways, such as PI3K/Akt/mTOR signaling, Wnt/β-catenin signaling, STAT signaling, EGFR signaling, Ephrin signaling, HIF-1α signaling,

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eNOS signaling, and VEGF-C signaling. In particular, an invasion and metastasis of CCA have revealed that these processes are related to inflammatory cytokine-mediated EMT regulation. Currently, an emerging hallmark is composed of two enabling characteristics, genomic instability and tumor microenvironment. Therefore, the emerging hallmarks of CCA consist of genome instability and mutation, tumor-promoting inflammation, reprogramming energy metabolism, and evading immune destruction. The data suggest that the genomic instability and mutation of several genes in CCA occur in various patterns, such as hypermethylation (i.e., OPCML, SERP1, HIC1, PTEN, DcR1), microsatellite instability, and mutation (i.e., TP53, KRAS, SMAD4, BAP1, MLL3, IDH1/2, WNT, ARID1A, KRAS/G, PEG3). Interestingly, CCA genesis and progression are regulated by the tumor microenvironment, especially TAMs and CAFs, via the secretion of cytokines. In addition, the new technology for metabolomics is being developed indicating that the reprogramming of energy metabolism might act as a player in the motivation for cellular function in several processes. Several years ago, alteration of CCA metabolism was rarely discussed in detail due to limitation of research methodology. However, the new technology for investigating metabolic profile, also known as metabolomics, has recently been used in CCA research to investigate the phenotypic features in various aspects including cancer biomarker, and drug metabolism and advancing our understanding of its molecular mechanism. Hence, metabolomics could be helpful in elucidating the phenotype of CCA, an interaction of TME and CCA, as well as the individual drug responses. It could be useful in understanding the molecular mechanisms of carcinogenesis and cancer development, resulting in the improvement of targeted CCA therapy in the future. The mechanisms and the hallmarks of abnormal genes, proteins that associated with the liver fluke infection-related CCA genesis and progression were summarized in Fig. 4.1.

Fig. 4.1 This figure encompasses mechanisms and the hallmarks of abnormal genes, proteins, which contribute to the liver fluke infection-related CCA genesis and progression

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Incyte (Inst), Puma Biotechnology (Inst), Adaptimmune (Inst), Merck Serono (Inst), RedHill Biopharma (Inst), Basilea (Inst) Travel, Accommodations, Expenses: ArQule, Celgene, AstraZenecaKai WangEmployment: OrigiMed Leadership: OrigiMedMilind JavleConsulting or Advisory Role: Qed, Oncosil, Incyte, Mundi Other Relationship: Rafael Pharmaceuticals, Incyte, Pieris Pharmaceuticals, Merck, Merck Serono, Novartis, Seattle Genetics, BeiGene, QED Therapeutics, Bayer No other potential conflicts of interest were reported. Rizvi S, Khan SA, Hallemeier CL, Kelley RK, Gores GJ. Cholangiocarcinoma - evolving concepts and therapeutic strategies. Nat Rev Clin Oncol. 2018 Feb;15(2):95–111. PubMed PMID: 28994423. Pubmed Central PMCID: PMC5819599. Morizane C, Ueno M, Ikeda M, Okusaka T, Ishii H, Furuse J (2018) New developments in systemic therapy for advanced biliary tract cancer. Jpn J Clin Oncol 48(8):703–11. PubMed PMID: 29893894 Galdy S, Lamarca A, McNamara MG, Hubner RA, Cella CA, Fazio N, et al. HER2/HER3 pathway in biliary tract malignancies; systematic review and meta-analysis: a potential therapeutic target? Cancer Metastasis Rev. 2017 Mar;36(1):141–57. PubMed PMID: 27981460. Pubmed Central PMCID: PMC5385197. Merla A, Liu KG, Rajdev L. Targeted Therapy in Biliary Tract Cancers. Curr Treat Options Oncol. 2015 Oct;16(10):48. PubMed PMID: 26266637. Pubmed Central PMCID: PMC4534500. Czink E, Heining C, Weber TF, Lasitschka F, Schemmer P, Schirmacher P, et al. Durable remission under dual HER2 blockade with Trastuzumab and Pertuzumab in a patient with metastatic gallbladder cancer. Z Gastroenterol. 2016 May;54(5):426–30. PubMed PMID: 27171333. Dauerhafte Remission unter dualer HER2-Blockade mit Trastuzumab und Pertuzumab bei metastasiertem Gallenblasenkarzinom. Sorscher S. Marked radiographic response of a HER-2-overexpressing biliary cancer to trastuzumab. Cancer Manag Res. 2013;9:1–3. PubMed PMID: 24376362. Pubmed Central PMCID: PMC3864990. Javle M, Churi C, Kang HC, Shroff R, Janku F, Surapaneni R, et al. HER2/neu-directed therapy for biliary tract cancer. J Hematol Oncol. 2015 May 29;8:58. PubMed PMID: 26022204. Pubmed Central PMCID: PMC4469402. Voss JS, Holtegaard LM, Kerr SE, Fritcher EG, Roberts LR, Gores GJ et al (2013) Molecular profiling of cholangiocarcinoma shows potential for targeted therapy treatment decisions. Hum Pathol 44(7):1216–22. PubMed PMID: 23391413 Ross JS, Wang K, Gay L, Al-Rohil R, Rand JV, Jones DM, et al. New routes to targeted therapy of intrahepatic cholangiocarcinomas revealed by next-generation sequencing. Oncologist. 2014 Mar;19(3):235–42. PubMed PMID: 24563076. Pubmed Central PMCID: PMC3958461. Bekaii-Saab T, Phelps MA, Li X, Saji M, Goff L, Kauh JS, et al. Multi-institutional phase II study of selumetinib in patients with metastatic biliary cancers. J Clin Oncol. 2011 Jun 10;29(17):2357–63. PubMed PMID: 21519026. Pubmed Central PMCID: PMC3107751. Ikeda M, Ioka T, Fukutomi A, Morizane C, Kasuga A, Takahashi H, et al. Efficacy and safety of trametinib in Japanese patients with advanced biliary tract cancers refractory to gemcitabine. Cancer Sci. 2018 Jan;109(1):215–24. PubMed PMID: 29121415. Pubmed Central PMCID: PMC5765304. Yoshikawa D, Ojima H, Iwasaki M, Hiraoka N, Kosuge T, Kasai S, et al. Clinicopathological and prognostic significance of EGFR, VEGF, and HER2 expression in cholangiocarcinoma. Br J Cancer. 2008 Jan 29;98(2):418–25. PubMed PMID: 18087285. Pubmed Central PMCID: PMC2361442. Miyamoto M, Ojima H, Iwasaki M, Shimizu H, Kokubu A, Hiraoka N, et al. Prognostic significance of overexpression of c-Met oncoprotein in cholangiocarcinoma. Br J Cancer. 2011 Jun 28;105(1):131–8. PubMed PMID: 21673683. Pubmed Central PMCID: PMC3137414.

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Pathology of Cholangiocarcinoma Supinda Koonmee, Prakasit Sa-ngiamwibool, Chaiwat Aphivatanasiri, Waritta Kunprom, Piyapharom Intarawichian, Walailak Bamrungkit, Sakkarn Sangkhamanon, and Malinee Thanee

Contents 5.1 5.2 5.3 5.4 5.5 5.6 5.7

Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Spreading Pattern of Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Surgery and Residual Tumor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Intrahepatic Cholangiocarcinoma; iCCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Macroscopic Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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S. Koonmee (B) · P. Sa-ngiamwibool · C. Aphivatanasiri · W. Kunprom · P. Intarawichian · W. Bamrungkit · S. Sangkhamanon · M. Thanee Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand e-mail: [email protected] P. Sa-ngiamwibool e-mail: [email protected] C. Aphivatanasiri e-mail: [email protected] W. Kunprom e-mail: [email protected] P. Intarawichian e-mail: [email protected] W. Bamrungkit e-mail: [email protected] S. Sangkhamanon e-mail: [email protected] M. Thanee e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. Khuntikeo et al. (eds.), Liver Fluke, Opisthorchis viverrini Related Cholangiocarcinoma, Recent Results in Cancer Research 219, https://doi.org/10.1007/978-3-031-35166-2_5

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5.8 Histopathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.9 Cancer Staging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.10 Extrahepatic Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.11 Macroscopic Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.12 Histopathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.13 Cancer Staging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.14 Molecular Pathology and Specimen Handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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The biliary tract is the pathway for bile transportation. To begin with, the bile at the bile canaliculi flows into the bile ductule and continues down to the interlobular and intrahepatic bile duct and the right and left hepatic ducts. Afterward, these leave the liver and join to form a large tube-like structure, the main bile duct. When this joins with the cystic duct, it is called the common bile duct and connects with the pancreatic duct to transport bile and digestive juices from the pancreas to the duodenal ampulla (Fig. 5.1). Cholangiocarcinoma (CCA) is a primary tumor arising from the progenitor cells of the epithelial cells. It can occur in any area of the bile duct (Fig. 5.2), for example, in the intrahepatic bile or the extrahepatic bile duct. Tumors that arise in any part of the hepatic bile duct are intrahepatic (Fig. 5.2A), while tumors that arise outside the liver are called extrahepatic. The tumors occurring in the area where the bile duct separates from the liver are further divided into perihilar cholangiocarcinoma (perihilar CCA, Fig. 5.2B) and distal cholangiocarcinoma (distal CCA, Fig. 5.1 Anatomy of the biliary tract. Bile is transported from the bile ducts within the liver into the right and left large bile ducts located outside of the liver. These combine into the large bile duct at the perihilar area, which then flows from the cystic duct into the area called the common hepatic duct. Afterward, this joins the pancreatic duct and enters the major duodenal ampulla (Adapted from AJCC Cancer Staging Manual, 8th edition)

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Fig. 5.2 Characteristics of cholangiocarcinoma by anatomical location (A) Shows the characteristics of intrahepatic CCA. The boundaries are unclear. The tumor color is gray-white, and there are multiple nodules. The tumor grows in the bile ducts and then spreads to the nearby liver tissues. (B) Shows the characteristics of perihilar CCA. There is a gray-white mass with an infiltrative boundary. The bifurcation of the common hepatic duct is distended. (C) Shows the characteristics of distal CCA that invades nearby organs

Fig. 5.2C). In addition, CCA that arises in the junctional area of the right and left hepatic duct to the common bile duct may be called Klatskin tumors [1]. Each type of cholangiocarcinoma is classified according to the biliary duct’s anatomical characteristics and present different clinical symptoms requiring specific surgical techniques. According to a study based on the anatomical location of cholangiocarcinoma, the patients with cholangiocarcinoma at Srinagarind Hospital and in the northeastern region of Thailand have the highest prevalence rate in the world. The most frequently occurring types of cholangiocarcinoma found in this area are perihilar CCA (52.9%), intrahepatic CCA (37.1%), distal CCA (4.1%), and combined intrahepatic and extrahepatic CCA (5.83%). The incidence seems different from other populations across the globe. Tumors with multifocal lesions may be found in intrahepatic CCA. The tumor mass is most often associated with a small or large bile duct. Tumors with skip lesions may be present in extrahepatic CCA. As mentioned earlier, these pathological characteristics can be found both in intrahepatic and extrahepatic CCA.

5.1

Pathology

The pathological characteristics of most CCA found during surgery are often at an advanced stage when the disease has spread. According to a cross-sectional picture of the tumor, the gross pathology of intrahepatic CCA shows a gray-white mass with an infiltrative border. It may also be accompanied by the appearance of

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mucus secretion from the tumor; a single mass or several multiple masses may be present on one side or both sides of the liver. Such pathological characteristics may be misdiagnosed as metastasis. In addition, proximal bile duct dilatation may be present. The nearby liver tissues are often non-cirrhotic. This characteristic is different from the nature of hepatocellular carcinoma (HCC), yet it can also be accompanied by cirrhosis and may be seen in some patients with chronic hepatitis B or C infection [2]. Extrahepatic CCA is present in the upper bile duct (arising at two levels of both hepatic ducts and the common hepatic duct or hilar CCA). It accounts for 60% of the cases. The tumor arising in the middle third from the distal common bile duct, cystic duct, and its confluence to the proximal common bile duct accounts for 20% of cases. The lower third from the distal common bile duct to the periampullary region, distal CCA, accounts for the remaining 20%. In addition, a crucial pathological characteristic that occurs in approximately 5.8% of cases is combined intrahepatic and extrahepatic CCA.

5.2

Spreading Pattern of Cholangiocarcinoma

Cholangiocarcinoma is a group of cancers with slow growth. However, the disease progression and the spread of cancer cells are faster-growing than some other types of cancers when spreading out of the bile duct, invading the liver, and encroaching on the common hepatic duct. The cancer cells can spread to other organs as they come from small tumors, especially spreading to regional lymph nodes. They also spread to nerve tissue (perineural invasion). Other organs to which cancer cells most often spread are the lungs, bones, adrenal glands, kidneys, spleen, pancreas, and brain. Skin and subcutaneous tissue are less prone to invasion. It is noteworthy that this type of cancer often spreads to the lymph nodes faster than to the blood vessels.

5.3

Surgery and Residual Tumor

Curative resection, which includes both removing part or all of the cancerous tissue, is a strong predictor of prognosis. In addition, a pathological examination can help the doctor diagnose the presence of residual tumor in the resected region. The status of residual tumor can be divided into three types: R0 R1 R2

No residual tumor found at the resected margin Microscopic residual tumor found in the resected region Residual tumor found in the resected region by visual examination

For instance, according to a study on perihilar CCA, patients who underwent curative resection without residual tumor at the resected margin (R0) showed a survival

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rate of 49.7% over three years, while those with microscopic residual tumor in the resected site (R1) had an 8.9% chance of survival [3].

5.4

Prognosis

Cholangiocarcinoma has been reported as a poor prognosis disease because patients are often admitted in advanced stages, with locally advanced or metastatic disease. Therefore, prognostic factors are needed to predict and plan for the curative treatment. Nowadays, many prognostic factors can be used to predict and relate to poor outcomes of CCA patients, such as growth pattern, tumor size, and surgical margin. These factors are the preliminary predictors for the prognosis of the progression and aggressiveness of CCA, which is correlated with the poor survival rate of CCA patients. Growth pattern is one of the main prognostic factors for CCA patients, and in particular, mass-forming and periductal infiltrating types are correlated with poor survival [4–6]. In contrast, the intraductal growth type is more likely to have a good outcome. Tumor size is also a good prognostic factor for predicting the progression of CCA, and previous research has shown that large tumors are associated with tumor grade, staging, positive regional lymph nodes and more frequent vascularization resulting in poor survival. In addition, a positive surgical margin is usually related to lymph node metastasis and tumor recurrence, leading to a poor treatment, outcome and consequently short survival due to cancer metastasis [7, 8].

5.5

Biomarkers

Typically, serum biomarkers are helpful for an early diagnosis of the disease. Carcinoembryonic antigen (CEA) and the carbohydrate antigens 19-9 (CA 19-9) and 125 (CA 125) are biomarkers used as screening tests for CCA in clinical practice. Even though they do not have sufficient sensitivity and specificity for detection, they differentiate CCA from other entities such as hepatocellular carcinoma and combined hepatocellular-cholangio-carcinoma. The other benefits of serum markers are for predicting recurrent risk and prognostic outcomes. CA 19-9 may aid in predicting tumor resectability in which the patients with unresectable tumors have significantly higher CA 19-9 levels than patients with resectable tumors. High preoperative serum CA 19-9 levels are also associated with poor survival rates after surgery. However, high serum CA 19-9 can increase the likelihood of other conditions such as obstruction and cholangitis, which can be a coincidence in a patient with CCA.

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Intrahepatic Cholangiocarcinoma; iCCA

Intrahepatic CCA originates from cells of the bile duct lining within the liver. It is found in male patients over 60 years of age more often than in female patients. In particular, people in the northeastern region of Thailand have a higher incidence than those from the other regions of Thailand. Also, the northeastern region of Thailand has the most iCCA cases globally (88 males per 100,000 population and 37 females per 100,000 population). In addition, intrahepatic CCA accounts for 37% of all cholangiocarcinomas. It is associated with liver fluke infections with Opisthorchis viverrini (Thailand and other Southeast Asian countries) and Clonorchis sinensis (China and Korea) [9–11]. The exact causes of cholangiocarcinoma are unclear. Experts postulate that it relates to chronic inflammatory biliary disease, primary sclerosing cholangitis, hepatolithiasis, parasitic biliary infestation, biliary malformation (Caroli disease or choledochal cysts), and chronic hepatitis C virus infection. Nevertheless, studies have shown that liver fluke infection in combination with exogenous nitrosamine compounds is the main cause of cholangiocarcinoma in Thailand (Fig. 5.5), which contributes to chronic inflammation of the biliary tract.

5.7

Macroscopic Features

The macroscopic features on visual examination of cancer tissues can be divided into three types as follows (Fig. 5.3) [6, 12]:

Fig. 5.3 Macroscopic features of cholangiocarcinoma. A. Mass-forming type (MF): The boundaries of the nodules are unclear and gray-white. It may have yellowish necrotic spots. B. Periductal infiltrating type (PI): The bile duct thickens due to the spread of cancer cells around it. C. Intraductal growth type (IG): A white papilla is found within the bile duct

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1. Mass-forming type; MF The cancerous tissue looks like a hard mass to the naked eye. The boundaries of the nodules are unclear; they may be found as a single mass (Fig. 5.3A) or multiple masses. They may be found to diffuse into neighboring tissues. The nodules usually appear gray-white. The original bile duct may not be found because the cancerous mass has destroyed it. The survival rate from this malignant mass-forming CCA within five years is about 39%. 2. Periductal infiltrating type; PI The cancerous tissue spreads throughout the bile duct and causes periductal fibrosis (Fig. 5.3B); it might be accompanied by narrowing of the bile duct in the same area. In addition, the distal bile duct distends due to the obstruction of the proximal bile duct leading to cholangitis. It is found that patients with PI have the lowest chance of survival because the cancer cells often spread to the lymph nodes and nerves. In particular, the survival is very low if the patients have both MF and PI. 3. Intraductal growth type; IG The cancerous tissue looks like a papilla or protruding nodule. It arises within the bile ducts that are distended, protuberate, or bulge out as a cyst with a papilla inside (Fig. 5.3C). This kind of tumor develops and becomes cancer deriving from the pathological characteristics of the intraductal papillary neoplasm of the bile duct (IPNB). The survival rate of IG within five years is 69%; this is relatively good compared to the other types [3, 6]. According to a study, the proportion of IG found in Thailand is higher than in other countries. When classified on macroscopic appearance, these three types of cancer cells can also be found together, starting from the IG (invading into the liver tissue) to the MF (forming a mass) and to the PI (proliferating throughout the bile duct).

5.8

Histopathology

Intrahepatic CCA can be the non-invasive intraductal type (IPNB, Fig. 5.4). The invasive CCA has two main histological subtypes, the large duct type and small duct type [13–15]. Other rare variant subtypes have also been reported. Large duct type The large duct-type histology of iCCA is similar to that of perihilar and extrahepatic CCA. A desmoplastic invasive tubular adenocarcinoma infiltrates the portal connective tissue, the surrounding bile ducts and the hepatic parenchyma. The tumor tissue usually becomes sclerotic or obliterates the large bile channels from which it emerged. Invasion of the perineural and lymphatic systems, as well as lymph nodes, may be seen (Fig. 5.5).

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Fig. 5.4 Characteristics of intraductal growth-type cholangiocarcinoma shows cancer cells arranged in a papillary structure within the bile duct. A: low power view (50x), B: high power view (400x). Red and black arrows indicate the bile duct surface and tumor region, respectively

Fig. 5.5 Characteristics of well-differentiated adenocarcinoma. The cancer cells appear cuboidal and are arranged in a complex tubular shape. A: low power view (50x), B: high power view (400x)

Small duct type Small duct iCCA shows tubular formations with distinct lumina formed by cuboidal to low columnar tumor cells with scant cytoplasm, or small tubular, cord-like, or spindle cell formations with a slit-like lumen. The components can be blended in a variety of ways. Replacement development of tumor cells in the hepatic lobules or regenerative nodules is seen in small duct iCCA. Early stage, small-scale iCCA may cause complications.

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Fig. 5.6 Mucinous type of intrahepatic cholangiocarcinoma. Histopathologically, the cancer cells are arranged in clusters or show a cuboidal epithelium floating in the mucin lake. A: low power view (50x), B: high power view (400x). Black arrows and asterisks represent cancer cells and mucin, respectively

Other types There are several other types of intrahepatic CCA. Combined hepatocellular-CCA is a rare type showing a histomorphology of overlapping hepatocellular carcinoma and CCA. Intraductal papillary neoplasm with an associated invasive carcinoma showing an intraductal papillary growth component resembles extrahepatic counterparts with an area of stromal invasion. Mucinous cystic neoplasm with an associated invasive carcinoma displays a cyst-forming epithelial neoplasm with variably mucin-producing epithelium associated with ovarian-type subepithelial stroma with associated invasive carcinoma (Fig. 5.6). Undifferentiated carcinoma lacks standard morphological and immunohistochemical features of specific carcinoma differentiation, including HCC and CCA.

5.9

Cancer Staging

According to the AJCC Cancer Staging Manual eighth edition, this TNM staging system divides the classification of CCA into intrahepatic and extrahepatic types. For primary carcinomas of the intrahepatic bile ducts, this staging system can be used for any type of intrahepatic CCA, including mass-forming tumors, periductal infiltrating tumors, and mixed tumor growth patterns. It classifies carcinoma in situ as biliary intraepithelial neoplasia grade 3 (BilIN-3), high-grade dysplasia in an intraductal papillary lesion, or mucinous cystic lesion as ‘Tis.’ The T classification of invasive intrahepatic CCA consists of the tumor focality, tumor size, vascular invasion and tumor perforation. T1 is defined as a solitary tumor without vascular invasion, and it is divided into T1a if the tumor size is less than or 5 cm

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Table 5.1 Classification of intrahepatic CCA staging (AJCC 8th ed., 2017)

Stage

Tumor

Node

Metastasis

0

Tis

N0

M0

IA

T1a

N0

M0

IB

T1b

N0

M0

II

T2

N0

M0

IIIA

T3

N0

M0

IIIB

T3

N0

M0

IIIC

Any T

N1

M0

IV

Any T

Any N

M1

and T1b if the tumor is larger than 5 cm. A tumor with vascular invasion and multiple intrahepatic tumors are classified as T2. Tumors perforating the visceral peritoneum are categorized as T3, and a tumor directly invading local extrahepatic structures is defined as T4. The N category of the intrahepatic CCA classifies by metastasis in the followings group of lymph nodes: The right lobe of the liver (segments 5–8): hilar, periduodenal, and peripancreatic; the left lobe of the liver: hilar, inferior phrenic, and gastrohepatic. Intrahepatic CCA can be staged as in Table 5.1 according to the TNM staging of the AJCC, 8th edition. The details in each section are as follows: Primary tumor (T) Tx T0 Tis T1 T1a T1b T2 T3

Primary tumor cannot be assessed No evidence of primary tumor Carcinoma in situ (Intraductal tumor) Solitary tumor without vascular invasion, Size ≤ 5 cm or > 5 cm Solitary tumor without vascular invasion, size ≤ 5 cm Solitary tumor without vascular invasion, size > 5 cm Solitary tumor with intrahepatic vascular invasion or multiple tumors, with or without vascular invasion Tumor perforating the visceral peritoneum

Regional lymph nodes (N) N0 N1

No regional lymph nodes metastasis Regional lymph nodes metastasis

Distant metastasis (M) Mx

Distant metastasis cannot be assessed

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M0 M1

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No distant metastasis Distant metastasis

Extrahepatic Cholangiocarcinoma

This type of CCA is usually found in patients between the ages of 60 and 70 years. On most occasions, they visit a doctor when they have jaundice or yellow eyes as the cancer mass becomes so large that it obstructs the bile duct, causing obstructive jaundice. In addition, abdominal pain in the right ribcage area can also be found. Obstructive jaundice can gradually grow concomitantly with the spread of the cancer to adjacent organs, nerves (Fig. 5.7), and lymph nodes surrounding the bile duct. The cause of extrahepatic CCA in this area may be similarly associated with intrahepatic CCAs, such as liver fluke infection [11] by O. viverrini or C. sinensis, and choledochal cysts. Furthermore, this cancer has more potential to develop in patients with primary sclerosing cholangitis, ulcerative colitis, and patients with a choledocho-pancreatic junction. As mentioned earlier, most extrahepatic CCAs are present in the biliary tract in the porta hepatis area. Since cancer often arises on either side of the liver lobes (the right or left hepatic duct) or common hepatic bile duct around the porta hepatis area, it is called hilar or perihilar CCA. If the cancer is found in the distal region, it is called distal CCA. Patients with this type of cancer often have jaundice, yellowing of the eyes without abdominal pain. As the distal bile duct is very small and becomes clogged easily, jaundice can be found faster. Moreover, the cancer cells often spread to nearby organs such as the pancreas, duodenum, stomach, or omentum. However, ampullary carcinoma is not classified as distal CCA. Fig. 5.7 Neural invasion of perihilar CCA. The arrow shows the part of the nerve and the arrowheads show cancer cells

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Macroscopic Features

There are three different growth patterns of extrahepatic cholangiocarcinoma. These are similar to intrahepatic cholangiocarcinoma that arises in the large duct and may be observed by visual examination: 1. Papillary Papillary tumors are frequently found growing in the bile duct. They are soft and become easily flaky and spread along the bile ducts without biliary papillomatosis. Extrahepatic CCA shows a better prognosis compared to intrahepatic CCA. The characteristics of papillary tumors are comparable to those of the intraductal growth type in intrahepatic CCA. 2. Nodular type Nodular tumors are found as small masses which spread outside the bile duct and are concomitantly found with the sclerosing type. They are comparable with those in the mass-forming type in intrahepatic CCA. 3. Sclerosing or scirrhous constriction Sclerosing tumors are frequently found. The prognosis is relatively poor as they spread to nearby organs and lymph nodes faster than the first two growth patterns. Cancer cells spread outside the bile duct. When comparing with intrahepatic CCA, their characteristics are comparable to those of the periductal infiltrating type.

5.12

Histopathology

Extrahepatic CCAs, including the perihilar and distal types, have a similar histology. They form tubular or glandular structures as for adenocarcinoma with high cellular proliferation in the bile duct. According to its pathogenesis, the premalignant lesions can be classified as biliary intraepithelial neoplasia (BilliNs) and intraductal papillary neoplasia of the bile duct (IPNB). The invasive adenocarcinoma can be classified by its histology: pancreatobiliary-type, intestinal-type, foveolar-type, or mucinous type. Pancreatobiliary-type adenocarcinoma Pancreatobiliary-type adenocarcinomas are characterized by widely spaced and well-formed irregular glands and small cell tumor clusters. The tumor is usually associated with a sclerotic desmoplastic stroma, often with perineural infiltration and lymphovascular invasion.

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Intestinal type These tumors are composed of absorptive columnar cells that resemble colonic adenocarcinoma with intestinal metaplasia in the adjacent dysplastic bile duct. They show an intraluminal spread along the dilated intrahepatic ducts with minimal ductal stromal invasion. Foveolar type The tumor glandular cells are lined by a single layer of malignant cuboidal-tocolumnar epithelial cells with basally located bland nuclear features and abundant, mucin-secreting cytoplasm that resembles the gastric foveolar-type epithelium. Mucinous type Mucinous adenocarcinoma is a tumor with extracellular mucinous components occupying more than 50% of the total tumor volume. The tumor cells have almost no glandular pattern with marked mucin production. CCA with a mucinous component has more aggressive biological behaviors and a poor prognosis when compared with the conventional type.

5.13

Cancer Staging

The cancer stage in perihilar and distal CCA is identifiable. Although they are seen as extrahepatic CCAs, these two types are distinct. To illustrate, distal CCA mainly invades outside of the bile duct. According to the TNM system (AJCC 8th edition) [16], the cancer stages can be summarized as in Table 5.2. The T category of extrahepatic CCA classifies by the depth of the tumor invading into the bile duct wall. T1 is defined as the tumor invading the bile duct wall less than 5 mm in depth, tumor invasion 5 to 12 mm is T2 and tumor invasion over 12 mm in depth is T3. A tumor involving the celiac axis, superior mesenteric artery, and/or common hepatic artery is categorized as T4. The N category of the extrahepatic cholangiocarcinoma is classified by metastasis in the following groups of lymph nodes: common bile duct, hepatic artery, Table 5.2 Classification of extrahepatic CCA staging (AJCC 8th ed., 2017)

Stage

Tumor

Node

Metastasis

0

Tis

N0

M0

I

T1

N0

M0

II

T2a–b

N0

M0

IIIA

T3

N0

M0

IIIB

T4

N0

M0

IIIC

Any T

N1

M0

IVA

Any T

N2

M0

IVB

Any T

Any N

M1

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posterior and anterior pancreaticoduodenal nodes, nodes along the right lateral wall of the superior mesenteric artery, and hilar node of the gallbladder. Details of each section are given as follows: Primary tumor (T) Tx T0 Tis T1 T2 T2a T2b T3 T4

Primary tumor cannot be assessed No evidence of primary tumor Carcinoma in situ/ High-grade dysplasia Tumor confined to the bile duct with extension up to the muscular layer or fibrous tissue Tumor invades beyond the wall of the bile duct to surrounding adipose tissue or tumor-adjacent hepatic parenchyma Tumor invades beyond the wall of the bile duct to surrounding adipose tissue Tumor invades adjacent hepatic parenchyma Tumor invades unilateral branches of the portal vein or hepatic artery Tumor invades the main portal vein or its branches bilaterally, or the common hepatic artery, or unilateral second-order biliary radicals with contralateral portal vein or hepatic artery involvement.

Regional lymph nodes (N) Nx No N1

N2

Regional lymph nodes cannot be assessed No regional lymph node metastasis One to three positive lymph nodes typically involved: the hilar, cystic duct, common bile duct, hepatic artery, posterior pancreaticoduodenal, and portal vein lymph nodes Four or more positive lymph nodes from N1 site.

Distant metastasis (M) Mx M0 M1

Distant metastasis cannot be assessed No distant metastasis Distant metastasis.

Distal cholangiocarcinoma can be staged according to the TNM system (AJCC 8th edition): The cancer stage is summarized in Table 5.3. Details of each section are given as follows: Primary tumor (T) Tx T0 Tis T1

Primary tumor cannot be assessed No evidence of primary tumor Carcinoma in situ/high-grade dysplasia Tumor invades the bile duct with a depth less than 5 mm

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Table 5.3 Classification of distal CCA staging (AJCC 8th ed., 2017)

T2 T3 T4

Tumor invades the bile duct with a depth less than 5–12 mm Tumor invades the bile duct with a depth greater than 12 mm Tumor involves the celiac axis, superior mesenteric artery and/or common hepatic artery

Regional lymph nodes (N) Nx No N1 N2

Regional lymph nodes cannot be assessed No regional lymph node metastasis Metastasis in one to three positive lymph nodes Metastasis in four or more positive lymph nodes

Distant metastasis (M) Mx M0 M1

Distant metastasis cannot be assessed No distant metastasis Distant metastasis

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Molecular Pathology and Specimen Handling

Over the past several years, there has been increasing evidence that the progression of malignant biliary epithelial cells is associated with their molecular and genetic aspects. An individual molecular background of CCA patients affects the CCA drug response. Moreover, targeted therapy has recently been used as an alternative for effective treatment [17]. The classification of molecular pathology, diagnosis and therapeutic targets plays a crucial role in CCA treatment [18]. Therefore, molecular techniques (immunohistochemistry; IHC, DNA sequencing, real-time PCR, fluorescence in situ hybridization; FISH) are increasingly applied. Generally, IHC is a simple technique and is commonly used in surgical pathology and cancer diagnostics [19, 20]. This technique involves the reaction of antibody binding to a specific antigen. The quality of specimen preparation in the pre-analytic phase is critical to prevent biomolecular protein degradation, DNA degradation, and structural distortion, which strongly influence the IHC result. The CCA specimen is mostly more than 10 cm in diameter. The surgical specimens should be fixed in 10% neutral buffered formalin; cold ischemic time within 60 minutes [21]; these specimens should be fixed in formalin with a fixative duration of between 24 and 72 hours [22, 23]. The fixative duration generally is not an issue compared to the cold ischemic time, which is not well monitored and managed. Our experience is that the cold ischemic time (control cold ischemic time less than 60 minutes) is associated with good immunohistochemical staining of CK19, reduces artifact deposition, and improves cellular structure as well as nuclear and cytoplasmic morphologies. The importance of controlling cold ischemic time in cholangiocarcinoma specimens will be critical in the future.

References 1. Nagtegaal ID, Odze RD, Klimstra D, Paradis V, Rugge M, Schirmacher P, Washington KM, Carneiro F, Cree IA (2020) Board WHOCoTE: the 2019 WHO classification of tumours of the digestive system. Histopathology 76(2):182–188 2. Lee CH, Chang CJ, Lin YJ, Yeh CN, Chen MF, Hsieh SY (2009) Viral hepatitis-associated intrahepatic cholangiocarcinoma shares common disease processes with hepatocellular carcinoma. Br J Cancer 100(11):1765–1770 3. Luvira V, Pugkhem A, Bhudhisawasdi V, Pairojkul C, Sathitkarnmanee E, Luvira V, KamsaArd S (2017) Long-term outcome of surgical resection for intraductal papillary neoplasm of the bile duct. J Gastroenterol Hepatol 32(2):527–533 4. Suzuki S, Sakaguchi T, Yokoi Y, Okamoto K, Kurachi K, Tsuchiya Y, Okumura T, Konno H, Baba S, Nakamura S (2002) Clinicopathological prognostic factors and impact of surgical treatment of mass-forming intrahepatic cholangiocarcinoma. World J Surg 26(6):687–693 5. Yamamoto M, Takasaki K, Yoshikawa T, Ueno K, Nakano M (1998) Does gross appearance indicate prognosis in intrahepatic cholangiocarcinoma? J Surg Oncol 69(3):162–167 6. Tawarungruang C, Khuntikeo N, Chamadol N, Laopaiboon V, Thuanman J, Thinkhamrop K, Kelly M, Thinkhamrop B (2021) Survival after surgery among patients with cholangiocarcinoma in northeast Thailand according to anatomical and morphological classification. BMC Cancer 21(1):497

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7. Choi SB, Kim KS, Choi JY, Park SW, Choi JS, Lee WJ, Chung JB (2009) The prognosis and survival outcome of intrahepatic cholangiocarcinoma following surgical resection: association of lymph node metastasis and lymph node dissection with survival. Ann Surg Oncol 16(11):3048–3056 8. Yeh CN, Hsieh FJ, Chiang KC, Chen JS, Yeh TS, Jan YY, Chen MF (2015) Clinical effect of a positive surgical margin after hepatectomy on survival of patients with intrahepatic cholangiocarcinoma. Drug Des Devel Ther 9:163–174 9. Parkin DM, Srivatanakul P, Khlat M, Chenvidhya D, Chotiwan P, Insiripong S, L’Abbe KA, Wild CP (1991) Liver cancer in Thailand. I. A case-control study of cholangiocarcinoma. Int J Cancer 48(3):323–328 10. Sripa B, Pairojkul C (2008) Cholangiocarcinoma: lessons from Thailand. Curr Opin Gastroenterol 24(3):349–356 11. Haswell-Elkins MR, Satarug S, Elkins DB (1992) Opisthorchis viverrini infection in northeast Thailand and its relationship to cholangiocarcinoma. J Gastroenterol Hepatol 7(5):538–548 12. Yamasaki S (2003) Intrahepatic cholangiocarcinoma: macroscopic type and stage classification. J Hepatobiliary Pancreat Surg 10(4):288–291 13. Nakanuma Y, Sato Y, Harada K, Sasaki M, Xu J, Ikeda H (2010) Pathological classification of intrahepatic cholangiocarcinoma based on a new concept. World J Hepatol 2(12):419–427 14. Aishima S, Oda Y (2015) Pathogenesis and classification of intrahepatic cholangiocarcinoma: different characters of perihilar large duct type versus peripheral small duct type. J Hepatobiliary Pancreat Sci 22(2):94–100 15. Nakanuma Y, Harada K, Ishikawa A, Zen Y, Sasaki M (2003) Anatomic and molecular pathology of intrahepatic cholangiocarcinoma. J Hepatobiliary Pancreat Surg 10(4):265–281 16. Amin MB, Greene FL, Edge SB, Compton CC, Gershenwald JE, Brookland RK, Meyer L, Gress DM, Byrd DR, Winchester DP (2017) The eighth edition AJCC cancer staging manual: continuing to build a bridge from a population-based to a more “personalized” approach to cancer staging. CA Cancer J Clin 67(2):93–99 17. Rogers JE, Law L, Nguyen VD, Qiao W, Javle MM, Kaseb A, Shroff RT (2014) Second-line systemic treatment for advanced cholangiocarcinoma. J Gastrointest Oncol 5(6):408–413 18. Montal R, Sia D, Montironi C, Leow WQ, Esteban-Fabro R, Pinyol R, Torres-Martin M, Bassaganyas L, Moeini A, Peix J et al (2020) Molecular classification and therapeutic targets in extrahepatic cholangiocarcinoma. J Hepatol 73(2):315–327 19. Bonacho T, Rodrigues F, Liberal J (2020) Immunohistochemistry for diagnosis and prognosis of breast cancer: a review. Biotech Histochem 95(2):71–91 20. Thunnissen E, Allen TC, Adam J, Aisner DL, Beasley MB, Borczuk AC, Cagle PT, Capelozzi VL, Cooper W, Hariri LP et al (2018) Immunohistochemistry of pulmonary biomarkers: a perspective from members of the pulmonary pathology society. Arch Pathol Lab Med 142(3):408– 419 21. Yildiz-Aktas IZ, Dabbs DJ, Bhargava R (2012) The effect of cold ischemic time on the immunohistochemical evaluation of estrogen receptor, progesterone receptor, and HER2 expression in invasive breast carcinoma. Mod Pathol 25(8):1098–1105 22. van Seijen M, Brcic L, Gonzales AN, Sansano I, Bendek M, Brcic I, Lissenberg-Witte B, Korkmaz HI, Geiger T, Kammler R et al (2019) Impact of delayed and prolonged fixation on the evaluation of immunohistochemical staining on lung carcinoma resection specimen. Virchows Arch 475(2):191–199 23. Khoury T (2012) Delay to formalin fixation alters morphology and immunohistochemistry for breast carcinoma. Appl Immunohistochem Mol Morphol 20(6):531–542

6

New Imaging Techniques Nittaya Chamadol, Richard Syms, Vallop Laopaiboon, Julaluck Promsorn, and Kulyada Eurboonyanun

Contents 6.1

Imaging of Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.1 The Role of Imaging in CCA Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.2 Anatomical and Morphological Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.3 Ultrasound Screening of CCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.4 Ultrasound Findings of MF-CCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.5 Ultrasound Findings of PI-CCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.6 Ultrasound Findings of ID-CCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.7 Use of CT in CCA Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.8 Imaging Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.9 Diagnosis of Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.10 Perihilar Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.11 Intrahepatic Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.12 Distal Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.13 Staging and Treatment Planning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.14 CT Volumetry and Estimation of Future Liver Remnant . . . . . . . . . . . . . . . . . . . . 6.1.15 Post-treatment Follow-Up and Surveillance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.16 MRI Imaging of Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.17 Positron Emission Tomography (PET)/CT of Cholangiocarcinoma . . . . . . . . . . 6.1.18 Magnetic Resonance Imaging and Relaxometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.19 Limitations to Contrast and Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.20 Internal Receivers for CCA Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

110 110 110 111 114 115 116 117 120 120 121 122 124 125 126 127 127 132 132 135 135

N. Chamadol (B) · V. Laopaiboon · J. Promsorn · K. Eurboonyanun Department of Radiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand e-mail: [email protected] R. Syms Department of Electrical and Electronic Engineering, Imperial College London, Exhibition Road, London SW7 2AZ, UK e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. Khuntikeo et al. (eds.), Liver Fluke, Opisthorchis viverrini Related Cholangiocarcinoma, Recent Results in Cancer Research 219, https://doi.org/10.1007/978-3-031-35166-2_6

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6.1.21 In Vitro Imaging of CCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

6.1

Imaging of Cholangiocarcinoma

6.1.1 The Role of Imaging in CCA Diagnosis As stated in previous chapters, cholangiocarcinoma (CCA) is the most common primary malignant tumor of the biliary tract, arising from the biliary epithelium [1]. It is the second most common primary liver tumor after hepatocellular carcinoma (HCC). CCA is relatively rare worldwide, but it is one of the most important disease problems of the biliary tract throughout southeast Asia, with the highest global incidence in northeast Thailand. The predominant risk factor is the liver fluke, O. viverrini, a group 1 carcinogen [2]. The medical imaging modalities available include ultrasonography (US), computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), PET-CT, and endoscopic cholangiography. The different modalities and their advantages for diagnosis have seen considerable development in the last decade. This advance has increased diagnostic accuracy, providing reliable information that can allow appropriate treatment and management programs to be selected for each patient. The main roles of imaging are detection, characterization, differential diagnosis, staging, and follow-up after treatment. Selection of the most suitable modality is critical for detection of CCA in its early stage. Early stage detection is fundamental to the assessment of surgical resectability and the outcome of disease management, which in turn is reflected by survival time after treatment. In the past, early stage detection in patients with non-specific symptoms was difficult, primarily due to most patients (80%) presenting at an advanced stage of the disease. The general result was a poor prognosis and a low five-year survival rate [3]. Accurate detection of early stage cases suitable for curative surgery is therefore crucial to improving survival time. The most important role for imaging is screening or surveillance imaging of the at-risk population. Because of its relatively low cost and portability, ultrasound screening with teleconsultation may easily be used in regional clinics or villages and consequently has proven highly effective for early stage detection [4–6].

6.1.2 Anatomical and Morphological Classification Classification of CCA from anatomical location and tumor morphology has been based on a variety of imaging features in both early and advanced stages of the disease [7]. Because of the considerable range of both anatomical and morphological classification, this tumor can present a widely varying appearance in each of the common imaging modalities (US, CT, and MRI) [8]. Currently, CCA is classified

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by anatomical location into intrahepatic (I-CCA), perihilar and distal CCA, and by tumor morphology into mass-forming (MF-CCA), periductal-infiltrating (PI-CCA) and intraductal (ID-CCA) types [9].

6.1.3 Ultrasound Screening of CCA The accepted and widely used primary imaging modality of the hepatobiliary system is ultrasound. US imaging of MF-CCA shows mass lesions with or without bile duct dilatation. For PI-CCA, the most common ultrasound observation is dilatation of bile ducts depending on the anatomical location of the tumor. For ID-CCA, the most common observation is dilatation of the bile ducts, which may occur with intraductal mass. These findings are easily detected in advanced CCA cases, providing baseline information for further characterization by CT or MRI. Ultrasound imaging of early stage cases is still very challenging. Despite this, US is the currently acceptable screening modality, because it has high sensitivity for detection of liver mass and bile duct dilation, the most important disease indicators in the risk group. In O. viverrini associated CCA, chronic inflammation of bile ducts has been documented as periductal fibrosis (PDF), using ultrasound imaging of patients with CCA. PDF can occur along both intrahepatic and extrahepatic ducts. In normal ultrasound of the liver, the hepatic artery and intrahepatic bile ducts are not visible due to their small size. The hepatic vein and portal vein are both well demonstrated in different echo patterns and their anatomical location. The hepatic vein has a thin anechoic wall and runs intersegmentally toward the inferior vena cava. The portal vein has an echoic wall with a periportal echo that represents the periportal space and runs intrasegmentally at the central part of the liver. Bile ducts running in the periportal space accompany the hepatic artery, autonomic nerve, lymphatic channel and potential space. The periportal space runs parallel with the portal vein in both intrahepatic and extrahepatic regions. Consequently, the echo patterns of normal bile ducts are normally apparent within the periportal echo. Figures 6.1, 6.2, 6.3 and 6.4 show example ultrasound images obtained at 3.5 MHz frequency using a convex probe and highlighting the most important observations. Figure 6.1 shows a normal liver, with an echo pattern containing relatively thin echoic periportal echo, in each case indicated by arrows. Figure 6.2 shows a case of periductal fibrosis type 1 (PDF1), which demonstrates an increase in the thickness of the periportal echo in both the so-called starry sky pattern (which consists of bright echogenic rings or dots in a background of decreased echogenicity corresponding to liver parenchyma) for transverse sections of portal triads and in the corresponding linear segments obtained for more parallel sections. Figure 6.3 shows a case of periductal fibrosis type 2 (PDF2), which demonstrates increased thickness of the periportal echo along the segmental branch of the portal vein in a so-called pipe-stem pattern in the left liver lobe. Figure 6.4 shows a case of periductal fibrosis type 3 (PDF3), which demonstrates increased periportal echo along branches of the portal vein along the liver hilum as indicated by the arrows.

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Fig. 6.1 US image through right and left lobe of normal liver in right subcostal scan (A), right intercostal scan (B), left subcostal scan (C), and left sagittal scan (D) showing homogeneous parenchymal echo thin periportal echo in right (arrows in A and B) and left (arrows in C and D) liver lobe

Identification of these different characteristics represented a major advance in tumor surveillance through ultrasound imaging [10–12]. It has been hypothesized that carcinogenesis of O. viverrini associated CCA occurs as the combination of PDF with other risk factors such as nitrosamine and genetics. Results of ultrasound screening have shown that: 1. PDF detection is important for close follow-up of patients by ultrasound to detect early cases of O. viverrini associated CCA. 2. Liver mass, bile duct dilatation, or mass with bile duct dilatation are important for further characterization by CT or MRI.

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Fig. 6.2 US image through right and left lobe of liver with PDF1 in right subcostal scan (A, B), left sagittal scan (C), and left subcostal scan (D), showing increased echo in starry sky pattern (arrows in A and B) and increased echo pattern in linear pattern (arrows in C and D)

Fig. 6.3 US image through right and left liver lobe with PDF2 in left subcostal scan (A) and left sagittal scan (B) showing increased periportal echo along segmental branch of portal vein in pipestem pattern in left liver lobe (arrows)

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Fig. 6.4 US image through right and left liver lobe with PDF3 in right subcostal scan (A) and right intercostal scan (B) showing increased periportal echo along branches of the portal vein along the liver hilum (arrows)

6.1.4 Ultrasound Findings of MF-CCA The most common ultrasound imaging result has been found to be liver mass. The echogenicity of the mass can be hypoechoic, isoechoic, hyperechoic, or heteroechoic, with or without a hypoechoic rim. In most of the advanced cases of CCA, ultrasound findings have shown there is accompanying bile duct dilatation [13]. For example, Fig. 6.5 shows ultrasound images of mass-forming CCA (MF-CCA). Figure 6.5A shows a heteroechoic mass in the right liver lobe (arrow 1), together with associated bile duct dilatation in the right liver lobe (arrow 2). Figure 6.5B shows a less distinct echoic mass in the right liver lobe (arrow 1), again with associated bile duct dilatation in the right liver lobe (arrow 2).

Fig. 6.5 Ultrasonography of MF-CCA. A) Heteroechoic mass in right lobe liver (arrow 1) with associated bile duct dilatation in right liver lobe (arrow 2). B) Echoic mass in right liver lobe (arrow 1) with associated bile duct dilatation (arrow 2)

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Fig. 6.6 Ultrasonography of MF-CCA. A) Echoic mass in right liver lobe without hypo-echoic rim (arrow). B) Hypoechoic mass in right liver lobe (arrow)

In early cases of O. viverrini associated CCA, most of the lesions are hyperechoic or hypoechoic with or without hypoechoic rim, with some cases exhibiting dilatation of adjacent bile ducts. For example, Fig. 6.6A shows an echoic mass in the right liver. There is no hypoechoic rim, and the extent of the mass is indicated by the annotation. Figure 6.6B shows a hypoechoic mass in the right liver lobe, whose extent is clearly now self-evident. Figures 6.7 and 6.8 show further examples of ultrasonography of MF-CCA. Figure 6.7A shows an echoic mass with a hypo-echoic rim in the right liver lobe, and Fig. 6.7B shows a similar mass in the left liver lobe. The extent of the mass is again indicated by the annotation where necessary. Figure 6.8A and B both show an echoic mass in left liver lobe with associated bile duct dilatation. In each case, the arrows 1 and 2 indicate the mass and bile duct dilatation, respectively.

6.1.5 Ultrasound Findings of PI-CCA The most common US imaging result that has been documented is bile duct dilatation without liver mass. For most of the advanced cases, this has been associated with atrophic change of the related liver lobe, and discontinuation of bile duct dilatation at the porta hepatis. For example, Fig. 6.9A shows US images from PICCA cases. The arrows show dilatation of the intrahepatic duct in the right and left liver lobes, while the circle in Fig. 6.9B indicates a set of crowded, dilated ducts that may represent atrophic change of right liver lobe. In early cases of O. viverrini associated CCA, the most common finding is segmental dilatation of the bile ducts without an accompanying liver mass. Figures 6.10 and 6.11 show different examples of dilatation of intrahepatic ducts and the common bile duct, in each case indicated by the arrows.

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Fig. 6.7 Ultrasonography of MF-CCA. A) Echoic mass with hypo-echoic rim in right liver lobe. B) Echoic mass with hypo-echoic rim in left liver lobe

Fig. 6.8 Ultrasonography of MF-CCA. A) 2 cm in size echoic mass in left liver lobe (arrow 1) with associated bile duct dilatation (arrow 2). B) 4 cm in size echoic mass in left liver lobe (arrow 1) with associated bile duct dilatation (arrow 2)

6.1.6 Ultrasound Findings of ID-CCA The most common ultrasound findings of ID-CCA are bile duct dilatation with or without an intraductal/parenchymal mass. In most of the advanced cases, the findings are dilatation of the bile ducts with intraductal and parenchymal mass (Fig. 6.12). For early cases of CCA, the most common ultrasound finding is segmental dilatation of the bile ducts, some with small intraductal mass (Figs. 6.13 and 6.14). In some cases, the tumor may present as a liver cyst with intramural echoic nodules or septation (Fig. 6.15).

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Fig. 6.9 Ultrasonography of PI-CCA. A) Dilatation of intrahepatic duct in right (arrow 1) and left liver lobe (arrow 2) with discontinuation of right and left lobe bile ducts. B) Group of dilatations of the intrahepatic duct in the right liver lobe (circle)

Fig. 6.10 Ultrasonography of PI-CCA. A) Dilatation of intrahepatic bile duct in left liver lobe without parenchymal mass (arrows). B) Dilatation of intrahepatic bile duct in right liver lobe without parenchymal mass (arrow)

6.1.7 Use of CT in CCA Diagnosis Despite the advantage it offers in screening of large risk groups, the field-of-view, resolution, and range of soft tissue contrast mechanisms of US are all limited. CT, PET, and MRI allow improvements to all aspects, but using significantly more expensive and bulky equipment that is necessarily based in major hospitals. All three imaging modalities are therefore more suitable for confirmatory diagnosis and staging rather than initial disease detection. Due to its wider availability, lower

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Fig. 6.11 Ultrasonography of PI-CCA. A) Dilatation of intrahepatic bile duct in right liver lobe (arrow) without parenchymal mass. B) Liver hilum shows dilatation of the common bile duct (arrow) Fig. 6.12 Ultrasonography of ID-CCA. A) Dilatation of intrahepatic duct in right (arrow 1) with echoic mass in right liver lobe (arrow 2); B and C) Dilatation of intrahepatic duct in right liver lobe (arrows)

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Fig. 6.13 Ultrasonography of ID-CCA. Dilatation of single intrahepatic duct (arrow 1) with intraluminal echoic nodule (arrow 2) in right liver lobe

Fig. 6.14 Ultrasonography of ID-CCA. Dilatation of group of intrahepatic ducts (arrow 1) with intraluminal echoic nodule (arrow 2) in right liver lobe

Fig. 6.15 Ultrasonography of ID-CCA. There is cystic mass (arrow 1) with intramural nodule (arrow 2) in right liver lobe

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Table 6.1 CT protocol Protocol Name: CT of the upper abdomen Scanner: Siemens Definition Flash 128-Slice (Dual Source) FOV: From above the dome of diaphragm to below the lower pole of the kidneys Section width/Increment: 2 mm/1 mm Intravenous contrast: Yes, hypo- or isoosmolar iodinated contrast Rate of i.v. contrast administration: 3 ml/sec using power injector Bolus tracking: Yes, marker placed in the upper abdominal aortic lumen Oral contrast: None Phases of study – Pre-contrast – Arterial phase: 25 seconds after bolus tracking – Venous phase: 70 seconds after bolus tracking – Delayed phase (additional): 5 minutes after bolus tracking

cost, and higher patient throughput, CT is currently more widely used than PET and MRI [14]. The role of CT in CCA management includes: 1. Detection and diagnosis. 2. Tumor staging and treatment planning. 3. Post-treatment follow-up and surveillance.

6.1.8 Imaging Protocols Multiphasic multi-detector CT is required for a comprehensive assessment of CCA. Pre-contrast CT is helpful in the identification and differentiation between intraductal stones and intraductal tumors. Post-contrast imaging, which allows for the assessment of tumoral enhancement characteristics, is usually performed in arterial and portal venous phases (approximately 30 seconds and 60–70 seconds after contrast administration, respectively). The sample CT protocol and parameters used in our institution can be found in Table 6.1. The arterial and portal venous phases also help delineate vascular anatomy and vascular involvement by the tumor. The additional delayed phase (approximately 5 minutes after contrast administration) can help differentiate mass-forming cholangiocarcinoma from hepatocellular carcinoma [15, 16]. Imaging should be undertaken before any drainage treatment, because such a procedure might alter bile duct walls and lead to a false interpretation of tumor extent [17].

6.1.9 Diagnosis of Cholangiocarcinoma For an abnormal lesion in a patient who has presented with abnormal liver function, biliary obstruction, or elevated tumor marker, CT can demonstrate

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Fig. 6.16 Schematic illustration of CCA subtype according to the growth pattern; (a) mass-forming, (b) periductal-infiltrating, and (c) intraductal-growth

and validate tumors and their anatomical relation. As discussed above, cholangiocarcinomas are commonly classified into intrahepatic and extrahepatic CCA. Extrahepatic CCA can be divided into perihilar and distal CCA [18]. Cholangiocarcinomas can also be classified according to their morphologic growth pattern, namely into mass-forming, periductal-infiltrating, and intraductal-growth types. Each subtype reflects different tumor behavior and has a different treatment outcome [9]. The morphologic subtypes can be recognized based on their characteristic imaging features as illustrated in Fig. 6.16 [19].

6.1.10 Perihilar Cholangiocarcinoma Perihilar cholangiocarcinoma (pCCA) is a term for tumors located above the common bile duct and below the secondary branch of the right or left hepatic ducts. It is the most common type of tumor, and accounts for 50–60% of all CCAs [20]. The majority of perihilar CCAs have periductal-infiltrating growth pattern [16, 21]. Most pCCAs can be accurately detected and localized using CT. Perihilar CCA usually manifests as irregular thickening of the ductal wall, causing luminal narrowing and upstream intrahepatic duct dilatation (Fig. 6.17). A discrete mass

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Fig. 6.17 Perihilar CCA; the CT images show enhancing thickening wall of the common hepatic duct (arrow in b) with upstream dilatation of intrahepatic bile ducts (Fig. 6.17a)

might not be visible. Delayed enhancement of the thickened ductal wall is usually present. The tumor may extend beyond the bile duct into the periductal space and invade the surrounding liver parenchyma or adjacent vessels [16, 21, 22]. Perihilar CCAs are commonly subclassified according to the Bismuth-Corlette classification. Coronal reformatted images can help determine the longitudinal extent of the tumor.

6.1.11 Intrahepatic Cholangiocarcinoma Intrahepatic cholangiocarcinoma (iCCA) is a tumor that arises proximal to the second-order bile ducts. It is the second most common type of CCA and found in approximately 20% of all cases [9, 15]. Recently, a classification of iCCA based on histological features has been proposed. This new classification subclassifies iCCA into four categories: conventional type (small and large bile duct type), bile ductular type, intraductal type, and rare variant. Each histologic subtype exhibits a different morphological growth pattern [23]. Most iCCAs are of a mass-forming growth subtype. Patients with iCCA tend to present with a more advanced stage at the time of diagnosis compared to patients with pCCA or dCCA [24]. The typical imaging characteristics of the mass-forming iCCA are a welldefined lobulated mass that is centrally hypoattenuating in the arterial phase and shows progressive enhancement in the portal venous and delayed phases. The progressively enhanced portion of the tumor reflects the varying degree of desmoplastic changes. Rim enhancement may be observed in the arterial phase (Fig. 6.18) [25]. Some mass-forming iCCAs may show diffuse hyperenhancement in the arterial phase. This so-called hypervascular iCCA can be found in 15 to 40% of patients,

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Fig. 6.18 Mass-forming iCCA; CT images of a large mass-forming CCA at the right hepatic lobe. The mass appears hypodense in the pre-contrast image (a) and shows peripheral enhancement in the arterial phase (b) with progressive enhancement in the venous phase (c)

with increasing incidence in cirrhotic patients. Hypervascular iCCA may also manifest with early arterial enhancement with portal venous or delayed washout, which further complicates the diagnosis of HCC in the cirrhotic population [26–28]. The intraductal type encompasses the intraductal papillary neoplasm of bile duct (IPNB), intraductal tubular neoplasm of bile duct (ITNB), and superficial spreading iCCA. The imaging manifestation of the intraductal-type tumors can be a papillary lesion projecting into the duct lumen, or plaque-like lesions that spread along the inner wall of the bile ducts. IPNB often presents with varying degrees of diffuse or localized bile duct dilatation due to abundant mucin secretion (Fig. 6.19). Rarely, cystic dilatation of the bile duct is observed. On the contrary, mucin secretion is usually absent in ITNB. Thus, the imaging manifestation is usually of a cast in a slightly dilated or non-dilated bile duct. Histologically, IPNB and ITNB both show a spectrum from premalignant lesion to invasive carcinoma. Multi-focal lesions are common, and the tumors can involve either intrahepatic or extrahepatic bile ducts or both. For the superficial spreading type, bile duct dilatation without wall thickening, mass, or visible intraductal lesion is typical [23, 29–31].

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Fig. 6.19 Intraductal-growth type intrahepatic CCA; CT images show diffuse bile duct dilatation (a) with a small intraductal nodule (arrow in b) and a larger papillary mass in the common bile duct (asterisk in c)

6.1.12 Distal Cholangiocarcinoma Cholangiocarcinomas that arise in the common bile duct below the cystic duct opening are called distal cholangiocarcinomas (dCCA). They are the least common type, found in less than 10% of cases, but offer the most favorable prognosis [24]. Patients usually present with symptoms caused by obstructive jaundice. Distal CCA is one of the differential diagnoses of periampullary cancers; the others are pancreatic ductal adenocarcinoma, duodenal adenocarcinoma, and carcinoma of the ampulla of Vater. Distal CCA and other periampullary cancers share similar imaging characteristics: gallbladder distention and intrahepatic and extrahepatic bile duct dilatation. A focal mass lesion may or may not be visible. Unlike pancreatic adenocarcinoma, concurrent pancreatic duct and CBD dilatation, also known as the double-duct sign, are rarely present in dCCA (Fig. 6.20) [32].

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Fig. 6.20 Distal CCA; CT images show a mass at the distal CBD (a) causing upstream dilatation of the CBD and IHDs (arrows in b). Concomitant upstream dilatation of the main pancreatic duct (arrows in c), uncommon in distal CCA, is also present in this patient

6.1.13 Staging and Treatment Planning Surgery remains the only curative treatment for patients with CCA. The surgical procedures vary according to the extent and location of the tumors, ranging from bile duct resection to major hepaticopancreaticoduodenectomy. A thorough assessment of tumor staging and resectability is necessary for the proper management of each patient. Computed tomography is an essential tool for the assessment of tumor staging and resectability. It also demonstrates vascular anatomy and provides an anatomical roadmap for the definitive surgery or drainage procedure [17]. The current edition of the American Joint Committee on Cancer (AJCC) TNM classification (8th edition) has a separate staging system for iCCA, pCCA, and dCCA. This staging system may not reflect the resectability of cholangiocarcinomas, particularly the perihilar type. Additional classification systems have been proposed to assess resectability. The Bismuth-Corlette and the Memorial Sloan Kettering Cancer Center (MSKCC) classifications are the most common [16, 33– 35]. Regardless of the staging system, computed tomography can provide the information needed for local tumor staging and resectability assessment, such as

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the longitudinal and radial extension of the tumor, tumor size and multiplicity, vascular involvement, and associated lobar atrophy. One meta-analysis showed pooled sensitivity and specificity of CT for the resectability of CCA were 95% and 69%, respectively. The sensitivity of CT was comparable to MRI and significantly higher than PET/CT, while the specificity of CT, MRI, and PET/CT was not statistically significant [14]. Lymph node metastasis is one of the important prognostic markers. It correlates with the higher risk of irresectability. Moreover, it has been shown that the number of metastatic lymph nodes affected the overall survival. Patients with N1 disease also have an increased risk of death compared with those with N0 diseases [14, 36]. Even in patients with curative resection (R0 resection), the presence of lymph node involvement was significantly associated with early recurrence and poorer long-term outcome [37–39]. Size enlargement is the most reliable sign for CT assessment of lymph node metastasis. According to the RECIST 1.1 guidelines, the criteria for diagnosis of pathologic lymph nodes include a short axis of at least 10 mm [40]. Further imaging features such as a loss of fatty hilum, spherical shape, strong or heterogeneous contrast enhancement, central necrosis, threshold growth, and distribution pattern are also helpful in determining a pathologic lymph node, especially in smallersized nodes [40, 41]. Even so, with a reported accuracy ranging from 60 to 85%, the ability of CT to diagnose a metastatic lymph node is still far from perfect [42]. As the lymph nodes which are larger than 10 mm have a higher chance for malignancy, most of the enlarged lymph nodes are still found to be benign. Moreover, a normal-sized lymph node does not preclude metastasis. In one study, two-thirds of the metastatic lymph nodes were smaller than 10 mm in the short axis [42]. Distant metastasis is another important factor affecting the prognosis of tumors. The presence of a distant metastasis would render a patient ineligible for surgery. Cholangiocarcinomas commonly metastasize to the lung, peritoneum, distant lymph nodes, and bone. For extrahepatic CCA, however, metastasis to the liver is much more common than other sites [43, 44]. It is thought that MRI is superior to CT in detecting occult liver metastasis, and PET/CT is superior in detecting unsuspected distant metastasis. However, a recent study showed that the performance of CT in the detection of distant metastasis is not significantly different from that of MRI or PET/CT [45].

6.1.14 CT Volumetry and Estimation of Future Liver Remnant Liver remnant after hepatic resection is a significant predicting factor for postoperative liver failure [46]. Thus, in a patient planned for a major hepatic resection, an estimate of the future liver remnant (FLR) should be made to ensure that the patient would have an adequate amount of functioning liver after surgery. Volumetric assessment with computed tomography is a well-recognized and accurate method that can estimate the FLR in both normal and cirrhotic livers [47, 48].

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In a patient deemed to have small FLR, procedures such as portal vein embolization or Associating Liver Partition and Portal vein ligation for Staged hepatectomy (ALPPS) might be performed. These procedures can induce hypertrophy of the liver portion that will remain after surgery, thus increasing FLR. CT volumetry is often the best tool to monitor liver volume changes after such interventions [47–50].

6.1.15 Post-treatment Follow-Up and Surveillance As with other malignancies, computed tomography is the mainstay imaging modality to follow-up patients who underwent surgical or systemic treatment. Even after curative resection, tumor recurrence is frequent and can occur in approximately one-third of patients [39]. The recurrence can occur locoregionally, in the liver, lymph nodes, or other distant sites. The number and the location of recurrent sites may affect the management and overall survival of patients, and the role of CT is to identify recurrent lesions as early as possible [39, 51–53].

6.1.16 MRI Imaging of Cholangiocarcinoma MR imaging with dynamic contrast enhancement and MR cholangiography has been accepted as the comprehensive imaging modality for diagnosis and characterization of CCA. MR imaging and MR cholangiography can provide tumor characterization of cholangiocarcinoma depending on tumor cell components, namely fibrosis, tumor necrosis, intratumoral bleeding, calcification, and especially tumor vascularity. Cholangiocarcinoma can be divided into subtypes according to tumor location, for instance, as intrahepatic cholangiocarcinoma, perihilar cholangiocarcinoma, and distal cholangiocarcinoma. Intrahepatic cholangiocarcinoma is located beyond the second-order branches of the biliary tree, which are located in the liver and found in 40% of cholangiocarcinoma patients [54, 55]. Perihilar CCA predominately arises in the perihilar region, including the common hepatic duct, hilar, right hepatic duct, and left hepatic duct which occurs in 50% of cases. It is the most common location of CCA, while distal CCA tumor is raised in the distal common bile duct below the level of the common hepatic duct and represented 10% of CCA cases [54, 55] (Fig. 6.21). Infrequently CCA can be found in multifocal tumors within the liver, perihilar, or even combined distal CCA. However, in these cases, CCA can be classified regarding to tumor morphology, including mass-forming type which is the most common type of the tumor, as well as periductal and intraductal types. We usually find a combination of tumor types which may be periductal with intraductal, or intraductal with mass-forming type, or mass-forming with periductal type. Recently, some research studies have demonstrated that intrahepatic CCA and perihilar CCA may have different pathogenesis. Intrahepatic CCA may originate

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Fig. 6.21 Underlying case of choledochal cyst type IV A involving both intrahepatic and extrahepatic bile duct contains a polypoid intraluminal mass within CHD down to distal CBD showing intraluminal filling defect on coronal MIP MRCP (A). The tumor shows heterogeneous enhancement of the intraluminal mass (B) and heterogeneous mix low and high signal intensity on T2WI (C)

from a progenetic cell in the canals of Hering, while the perihilar large duct type may have a different origin, for instance, from peribiliary glands which further develop from the biliary intraepithelial neoplasm (BilIN) or intraductal neoplasm of the bile duct (IPNB) [25] (Fig. 6.22). These different cell origins of two different locations of CCA provide the basis for different enhancements by MR imaging. In general, intrahepatic mass-forming CCA is hypo- to iso-intense on T1-weighted MR images compared to normal liver parenchyma and heterogeneous intense on T2-weighted MR images, which are depended on the tumor component of fibrous tissue, hemorrhage, calcification, and necrosis. These are usually heterogeneous with high SI on T2-weighted MR image (Fig. 6.22). There are also specific ancillary features of mass-forming intrahepatic CCA, namely capsular retraction, satellite nodules, and peripheral bile duct dilatation [25]. These imaging characteristic features of intrahepatic mass-forming CCA

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Fig. 6.22 Perihilar intraductal cholangiocarcinoma with some foci of IPNB. The tumor shows heterogeneous slightly high signal intensity polypoid mass in the left hepatic duct and hilar on T2WI (A). There is a heterogeneous enhancement of the intraluminal polypoid mass on postgadolinium-enhanced MR imaging (B)

are useful to differentiate intrahepatic cholangiocarcinoma from hepatocellular carcinoma (HCC) [25] (Fig. 6.23). With regards to diffusion-weighted MR image (DWI), CCA shows restriction diffusion due to the high cellularity of the tumor like other malignant liver tumors. DWI is a quantitative imaging method using apparent diffusion coefficient (ADC) values to differentiate between benign and malignant liver tumors. Many studies have reported that malignant liver tumors have higher ADC values compared to the benign liver tumors. The ADC of intrahepatic CCA is 1.15×10–3 which is slightly higher than hepatocellular carcinoma and liver metastasis which have ADC values

Fig. 6.23 Intrahepatic mass-forming cholangiocarcinoma at segment 7 of liver demonstrated heterogeneous low signal intensity on T1WI (A) with adjacent smaller low signal intensity nodule nearby the tumor (white arrow) and retraction of an adjacent liver capsule (white curved left arrow). On T2WI the mass also shows heterogeneous high signal intensity (B) with peripheral irregular intrahepatic bile duct dilatation (white curved arrow)

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Fig. 6.24 Intrahepatic mass-forming cholangiocarcinoma showing peripheral restriction diffusion on peripheral of the tumor compared to darker signal intensity on B value 800 of DWI (A) with also a restriction diffusion of a daughter nodule beside the mother tumor (white arrow) and showing peripheral low signal intensity on ADC map with high signal intensity on the central part of the tumor (B)

of 1.02×10–3 and 0.95×10–3 , respectively [56]. CCAs with higher ADC values are also associated with microvascular invasion as well as with lymph node metastases which are related to poor disease outcomes/survival [57, 58] (Fig. 6.24). In relation to DWI, intrahepatic mass-forming CCA may have more characteristic features which demonstrate peripheral restriction diffusion with centrally non-restriction diffusion stimulating a target-like lesion. This finding represents central stromal tumor fibrosis or even central necrosis with some inflammation along with some fibrosis. This target-like lesion of intrahepatic mass-forming CCA can provide differential diagnosis of this tumor with respect to hepatocellular carcinoma, which has a more homogeneous diffusion restriction on DWI (Fig. 6.25). For dynamic post-gadolinium-base-enhanced MR images, CCA usually shows heterogeneous arterial enhancement in the peripheral zone of the tumor, with some gradual centripetal enhancement on the portovenous phase and delay phase due to the fibrous component of the tumor (Figs. 6.26 and 6.27). Hepatocyte-specific contrast has been used to investigate and characterize intrahepatic CCA. Hepatobiliary-specific contrast, such as gadoxetate disodium or gadobenate dimeglumine-enhanced MR imaging, can detect specific characteristics and provide a prognosis tool for intrahepatic mass-forming CCA. For 20 minutes delay of the hepatobiliary phase of gadoxetate disodium-enhanced MR imaging, intrahepatic mass-forming CCAs exhibit a target-like lesion (targetoid) which represents a centrally fibrous core stroma, and a central necrosis with desmoplastic change (Fig. 6.26). However, not all the intrahepatic mass-forming CCAs exhibit target-like lesions on the hepatobiliary phase of post-hepatocyte-specific contrast-enhanced MR imaging.

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Fig. 6.25 Hepatocellular carcinoma always has homogeneous restriction diffusion which could be a differential diagnosis from mass-forming intrahepatic cholangiocarcinoma

Fig. 6.26 Mass-forming intrahepatic cholangiocarcinoma shows low signal intensity with a target-like appearance on 20 minutes delayed gadoxetate disodium-enhanced MR imaging (white arrow)

There have been some previous studies which have used MR elastography to evaluate liver masses which have reported some higher stiffness value of the malignant tumor and CCA has been documented to certainly have a higher tumor stiffness value than hepatocellular carcinoma [59]. In general, CCA is a fibrous-containing tumor that leads to a higher stiffness value (Fig. 6.27). Cholangiocarcinoma which contains more amounts of fibrous tissue within the tumor may a have less favorable disease prognosis compared to tumors which have less fibrous components.

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Fig. 6.27 Mass-forming intrahepatic cholangiocarcinoma at segment 7 of liver on T1WI post gadobenate dimerglumine-enhanced MR imaging showing heterogeneous enhancement (white arrow) (A). The mean stiffness of the tumor is 14.6 kPa and 3.3 kPa of liver parenchyma (white curved arrow) (B)

6.1.17 Positron Emission Tomography (PET)/CT of Cholangiocarcinoma 2-[fluorine-18] fluoro-2-deoxy-d-glucose (18F-FDG) positron emission tomography (PET)/CT has been widely used to evaluate malignant tumors and can be useful to detect small, infiltrating tumors or distant metastasis of malignant tumors as well. Some cholangiocarcinoma can uptake 18F-FDG PET (Fig. 6.28); however, some mucinous type CCA may be difficult to demonstrate optimal 18F-FDG PET uptake [60, 61]. Moreover, there are some other inflammatory processes or infections, such as abscesses, granulomatous infection of liver, or biliary system that could mimic the malignancy process on 18F-FDG PET, such as demonstrating vivid 18F-FDG PET uptake which is the same for malignant tumors. Further investigations of CCA with 18F-FDG PET/CT should certainly be undertaken in the future.

6.1.18 Magnetic Resonance Imaging and Relaxometry The following outlines recent investigations of possible new methods for improving the resolution of magnetic resonance imaging of CCA. Conventional MRI [62] is carried out with the patient lying in the bore of a superconducting magnet, which provides a static B0 field. In equilibrium, nuclear magnetic dipoles in the body (primarily, of protons) process closely around the B0 direction at a frequency set by the field strength and the gyromagnetic ratio. Imaging is carried out in a two-step process. In the first step, excitation, a radio frequency source generates an additional rotating B1 magnetic field. A specialized RF coil (the body coil [63]) embedded in the magnet tunnel ensures this field is uniform. Application of the

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Fig. 6.28 Clinical case distal cholangiocarcinoma. 18F-FDG PET shows vivid tumor uptake of 18F-FDG at tumor at distal CBD with SUV Max 3.27 SUV-bw (A, B). A CT demonstrating pulmonary metastasis at the left lower lobe with uptake 18F-FDG (white arrow C, D)

B1 field in a sequence rotates the processing dipoles out of alignment with the B0 field. In the second step, detection, the signals emitted as the dipoles relax back to equilibrium are detected by induction. For abdominal imaging, the receiver is an array of coils covering the torso [64]. Relaxation of the longitudinal and transverse components of magnetization follows two different time constants T1 and T2. Although other factors are important, design of the sequence for T1- or T2weighting allows the signal to depend mainly on one time constant [65]. Contrast is obtained by sampling after a fixed time, the echo time (TE), when signals from different tissues have relaxed by different amounts. There is a trade-off between contrast and signal-to-noise ratio (SNR). Figure 6.29a shows representative decay curves for two tissues during T2-weighted imaging. A short TE yields a high SNR but limited contrast, while a long TE gives increased contrast but a reduced SNR that may fall below a useful threshold. Image formation is achieved through gradient magnetic fields, controlled by the sequences, which localize signals to individual volumetric elements or voxels. Fast spin echo (FSE) sequences allow rapid imaging of large volumes [66].

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Images are acquired in a stack of slices, each containing a two-dimensional array of voxels, and each array is used to create a gray-scale image. Images can contain several types of artifact [67], due to material with nonzero magnetic susceptibility (which locally distorts B0 ), non-uniform detection sensitivity (which causes a spatial variation in image brightness), direct coupling between coils (which induces local over-excitation), diffusion (which acts as a further relaxation mechanism) and respiratory motion (which blurs the image [68]). Artifacts are suppressed by careful choice of material, optimized receiver design and detuning the receiver during excitation [69]. Diffusion can be compensated [70] or exploited in diffusion-weighted imaging [71]. Respiratory motion is managed by breath-holding, respiratory gating [72], or sequences that effectively track motion [73, 74]. An alternative modality, relaxometry [75], uses different sequences to obtain the time constants directly. Multiple signal samples are acquired at regularly spaced TE. A nonlinear least-squares algorithm is then used to estimate the relaxation process, for example by fitting an exponential to a T2-weighted response [76]. Figure 6.29b shows the process for similar data to Fig. 6.29a. A colored plot of time constants is then known as a T2 map. Relaxometry eliminates the effects of non-uniform reception, but its drawbacks have led to an overwhelming preference for imaging. Even though fast sequencies are known [77], acquisition takes longer, increasing the cost of imaging. Time constants vary with B0 [78], and even from scanner to scanner [79]. If the voxel size is too large, the presence of multiple tissues may lead to the appearance of multi-exponential decay [80]. As a result, time constants may not be diagnostic in vivo [81–83], and alter with time after

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resection for in vitro specimens [82]. Despite this, the result is a high-resolution presentation that may be obtained for any coil orientation without correction.

6.1.19 Limitations to Contrast and Resolution The contrast provided by intrinsic time constant differences is limited [65]. In cancer imaging, contrast is enhanced and targeted through careful choice of T1- or T2-weighting combined with the injection of chemical agents into the bloodstream [84]. The agents accumulate in tumors after a short delay due to their increased microvasculature. Often, they are based on gadolinium [85], which shortens T1 so that tumors appear bright in T1-weighted images. Designing an imaging protocol and interpreting the resulting images are highly skilled tasks, and the varied etiology of CCA poses special difficulties even for an experienced radiologist. The procedure, magnetic resonance cholangiopancreatography (MRCP), is well established [86]. However, although mass-forming tumors may be clearly visible, the extent of periductal and intraductal disease (which affects smaller tissue volumes) is not. Early stage disease, which involves small changes to duct walls, may be indistinct, and affected ducts are often simply described as thickened or blurred [87–89]. Often, disease is inferred from cholestasis, due to the presence of bile (which has a large T2 [90]) in blocked ducts. As a result, staging may be imprecise, and patients can be re-assessed as unresectable at surgery. Consequently, there is a need to improve image resolution as well as contrast. Resolution is determined by SNR, which sets a lower limit to the useful voxel size [91]. Signal is maximized by placing the detector close to the tissue of interest. SNR is then optimized by reducing noise as far as possible. Since the main source of noise in human MRI is thermal noise from the body itself [92], the use of an external RF detector such as a torso array may be suboptimal: each coil is relatively distant from the ductal system and detects noise from a large volume. Resolution in MRI is therefore currently limited to around 1 mm3 . There is therefore a case for the use of internal coils, which can have a smaller field-of-view (FOV) for noise by virtue of their non-uniform reception [93], and hence may offer increased SNR for nearby tissue. Noise also impacts on relaxometry, where its effect is to broaden time constant values into a distribution [94]. Improved SNR also allows smaller voxels, reducing multi-exponential effects.

6.1.20 Internal Receivers for CCA Imaging Any internal coil requires safe introduction into and removal from the body. Many catheter coils have been developed for arterial imaging [95, 96], and intrabiliary imaging has also been carried out via drainage tubes [97]. A smaller number of endoscope coils have been constructed for imaging the gastro-intestinal tract [98, 99]. Duodenoscopes and catheters are used to access the biliary ductal system during endoscopic retrograde cholangiopancreatography (ERCP) [100]. However,

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adaptation to MRCP is non-trivial. Existing instruments may not generally be used, since these often contain magnetic materials [101], and any receiver must avoid interference with steering, optical imaging, and catheter deflection. Cathetermounted receivers must be flexible and devoid of protrusions to allow passage through the biopsy channel and ideally be disposable. However, to accommodate a guide wire—used to cannulate blocked ducts—catheter receivers must have a hollow bore. To avoid heating of adjacent tissue by coupling to the B1 field or its associated electric field [102], open loops and long continuous wires must be avoided, although loops are required for detection and wires for output coupling. We have developed a solution in the form of flexible thin-film circuits based on the magneto-inductive (MI) waveguide, a linear array of magnetically coupled resonant loops [103]. The inductors and capacitors needed for resonance are formed by patterning and etching copper layers on either side of a thin Kapton substrate, and the mutual inductance needed for coupling between loops is obtained by overlay. The circuits have no protrusions and have been batch-fabricated in lengths up to 2 m. Individual receivers can be separated from a panel with a scalpel and mounted on a tubular scaffold using heat shrink tubing. Depending on the loop size, the circuits may be used as catheter [104] or endoscope [105] receivers, and the fabrication process is cheap enough for disposability. When tuned to the Larmor frequency, each resonant loop may detect MRI signals. However, an enhanced signal may be detected in a resonant loop at the distal end. Signals propagate by induction to the proximal end, where the waveguide is impedance matched to the scanner electronics. The use of figure-of-eight shaped loops avoids direct coupling to the B1 field, and the use of an elements shorter than the length of a standing wave resonance avoids coupling to electrical fields [106]. Detection sensitivity is highly non-uniform [107]. In the axial direction, the discrete coil construction leads to image segmentation. This effect cannot easily be compensated but may be unimportant. Within each segment, the conductor layout approximates a parallel-wire receiver. Figure 6.30a shows the sensitivity pattern on a two-dimensional plane, which leads to a variation with radial distance r that reduces as ~1/r2 [95]. This limits the FOV for noise. However, the SNR must also vary with radius, and will typically match that of an external coil at a multiple of the conductor separation. Figure 6.30b compares typical variations in SNR for external and internal coils. Outside the shaded region, there is no advantage to the internal coil. The useful detection volume therefore consists of a set of co-axial lobes; the first may be used for imaging and the remainder for tracking. The radial sensitivity variation generates images that are over-bright near the coil, especially its conductors. Compensation may be achieved by an r2 correction, but only if the coil is approximately parallel to the magnet bore and the correction center is known [95]. Since neither is likely in biliary imaging, we have used relaxometry, plotting T2 maps rather than gray-scale images. Prototype endoscope and catheter coils with diameters of ~13 mm and ~3 mm have been demonstrated using 1.5T and 3T clinical scanners in the UK and Thailand. Experiments with phantoms have confirmed the local SNR advantage [105, 108], and monitoring of extended imaging with fiber-optic thermometers has shown no signs of RF heating [106].

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Fig. 6.30 a) Transverse variation of detection sensitivity for two-conductor coil mounted on a catheter, b) spatial variation of SNR for external and internal coils

6.1.21 In Vitro Imaging of CCA Tissue imaging experiments have been limited to in vitro work, carried out on freshly resected [109] or formalin fixed [110] specimens obtained from Thai patients with CCA. In all cases, patient permission was obtained using appropriately worded consent forms. Fresh specimens allowed cannulation but required careful synchronization between surgery, radiology, and pathology. Few such specimens were available, and imaging sessions were limited to a few hours. Fixed specimens were generally incomplete, but available in larger numbers. Histopathology was used to establish tissue type and distinguish disease. Tissue imaging experiments were performed with an Achieva™ 3T clinical whole-body system (Philips Healthcare, Best, Netherlands) at Khon Kaen University Hospital. Imaging was carried out using FSE sequences and T2 mapping with FSE and mGRASE [77] sequences. Because of the lack of perfusion, contrast agents could not be used, and imaging contrast relied on intrinsic differences in

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T2. The body coil was used for excitation, and results were compared for signal reception by the body coil, a torso coil and experimental endoscope and catheter coils. Axial images obtained using internal coils were compensated (where possible) using a MATLAB algorithm. T2 maps were generated using manufacturer’s software or using MATLAB. In each case, mono-exponential relaxation models were fitted to image data obtained at different echo times. Similar results were obtained by least-squares fitting to log-domain data. The results confirmed the increase in SNR offered by the torso coil compared with the body coil and a local increase in SNR for both endoscope and catheter coils compared with the torso coil. However, while the image obtained using the torso coil was highly uniform, internal coil images were restricted to a set of bright lobes in the axial direction, which decayed rapidly in the radial direction. Despite this, T2 mapping confirmed the potential advantage of internal coils. The local increase SNR reduced the component of time constant spread caused by body noise, enhancing the sharpness of T2 maps. Cross-sectional maps showed new detail of diseased ducts, including thickened, irregular walls often reported simply as ‘blur’ in MRI. Figure 6.31a shows a freshly resected specimen with polypoid intraductal tumor in Segments 2 and 3 being cannulated with a catheter receiver in an intrahepatic duct [109]. Figure 6.31b shows the arrangement for imaging, with the receiver approximately parallel to the magnet bore and the specimen mounted on a tank phantom to assist with localization.

Fig. 6.31 In vitro specimen: a) cannulation with catheter receiver and b) arrangement for T2 mapping

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Figure 6.32 shows T2 maps obtained as adjacent 3-mm-thick slices. The maps have been cropped from larger originals, and sub-mm resolution can be inferred from the dimensions of the catheter, whose location is indicated by white circles. The useful mapping volume is limited by SNR to an approximate cylinder coaxial with the catheter. Black regions represent the limit of this volume (or of the specimen). Blue color (mean T2 value 32.2 ms; standard deviation 4.6 ms) represents liver parenchyma, green is fibrosis, yellow/brown (mean T2 value 63.2 ms; standard deviation 5.2 ms) is duct wall and tumor, and red is bile trapped at the distal end of the duct. The duct wall has thickened considerably, and wall irregularities follow polypoid internal structures. These results represent the first intraductal imaging of CCA. Figure 6.33 shows how maps may stacked to visualize extended disease in 3D. Here the catheter track is marked in red, and the tumor is encroaching from the left. Such data may ultimately allow improved surgical planning. T2 mapping of fixed specimens showed a general shorting of time constants, in agreement with the literature [82]. Fixed specimens also allowed a highly detailed correlation between T2 values and the results of histopathology, often involving optical microscope examination of the entire specimen cross section [110]. Signs of early stage disease Fig. 6.32 Adjacent T2 maps obtained from a resection specimen using a catheter receiver. The circles indicate the catheter location, and the color-bar shows T2 values

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Fig. 6.33 3D presentation of polypoid intraductal tumor obtained as stack of T2 maps. The red line indicates the catheter track, and the color-bar shows T2 values

characteristic of OV-induced CCA have been identified, including granulomatous inflammation around fluke eggs and neoplasia. Further work is required to establish method and patient benefit before any in vivo trials. Production-standard MRI compatible duodenoscopes and catheters must be developed, and their safety thoroughly proven. The effect of respiratory motion on relaxometry acquisitions must be investigated, for example, using a motion simulator. Procedures allowing the use of contrast agents must be investigated, possibly through artificial perfusion of resection specimens. A database of T2 values and their consistency must be established, and careful assessment made of the advantage of increased resolution. Protocols for in vivo use must then be developed, for example involving preliminary imaging to identify blocked ducts, and an ERCP suite co-located with MRI to place the duodenoscope and cannulate the ductal system with the patient under sedation. Although the effort involved is significant, the results above appear sufficiently promising to warrant further work.

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88. Joo I, Lee JM (2013) Imaging bile duct tumors: pathologic concepts, classification, and early tumor detection. Abdom Imag 38(6):1334–1350 89. Kim JY, Lee JM, Han JK, Kim SH, Lee JY, Choi JY et al (2007) Contrast-enhanced MRI combined with MR cholangiopancreatography for the evaluation of patients with biliary strictures: differentiation of malignant from benign bile duct strictures. J Magn Reson Imag 26(2):304–312 90. Hakansson K, Christoffersson JO, Leander P, Ekberg O, Hakansson HO (2002) On the appearance of bile in clinical MR cholangiopancreatography. Acta Radiol 43(4):401–410 91. Libove JM, Singer JR (1980) Resolution and signal-to-noise relationships in NMR imaging in the human body. J Phys E 13(1):38–44 92. Hoult DI (1979) The sensitivity of the zeugmatographic experiment involving human samples. J Magn Reson 34(2):425–433 93. Eryaman Y, Oner Y, Atalar E (2009) Design of internal MRI coils using ultimate intrinsic SNR. MAGMA 22(4):221–228 94. Gudbjartsson H, Patz S (1995) The Rician distribution of noisy MRI data. Magn Reson Med 34(6):910–914 95. Atalar E, Bottomley PA, Ocali O, Correia LC, Kelemen MD, Lima JA et al (1996) High resolution intravascular MRI and MRS by using a catheter receiver coil. Magn Reson Med 36(4):596–605 96. Martin AJ, Plewes DB, Henkelman RM (1992) MR imaging of blood vessels with an intravascular coil. J Magn Reson Imag 2(4):421–429 97. Weiss CR, Georgiades C, Hofmann LV, Schulick R, Choti M, Thuluvath P et al (2006) Intrabiliary MR imaging: assessment of biliary obstruction with use of an intraluminal MR receiver coil. J Vasc Interv Radiol 17(5):845–853 98. Gilderdale DJ, Williams AD, Dave U, deSouza NM (2003) An inductively-coupled, detachable receiver coil system for use with magnetic resonance compatible endoscopes. J Magn Reson Imag 18(1):131–135 99. Inui K, Nakazawa S, Yoshino J, Yamao K, Yamachika H, Wakabayashi T et al (1995) Endoscopic MRI: preliminary results of a new technique for visualization and staging of gastrointestinal tumors. Endoscopy 27(7):480–485 100. Kozarek R (2007) Biliary ERCP. Endoscopy 39(1):11–16 101. Kanal E, Shellock FG, Talagala L (1990) Safety considerations in MR imaging. Radiology 176(3):593–606 102. Shellock FG (2000) Radiofrequency energy-induced heating during MR procedures: a review. J Magn Reson Imag 12:30–36 103. Shamonina E, Kalinin VA, Ringhofer KH, Solymar L (2002) Magneto-inductive waveguide. Electron Lett 38:371–372 104. Syms RR, Young IR, Ahmad MM, Taylor-Robinson SD, Rea M (2013) Magneto-inductive catheter receiver for magnetic resonance imaging. IEEE Trans Biomed Eng 60(9):2421–2431 105. Syms RRA, Kardoulaki E, Rea M, Choonee K, Taylor-Robinson S, Wadsworth C, Young IR (2017) Magneto-inductive magnetic resonance imaging duodenoscope. Prog Electromagnet Res 159:125–138 106. Segkhoonthod K, Syms RRA, Young IR (2014) Design of magneto-inductive magnetic resonance imaging catheters. IEEE Sens J 14(5):1505–1513 107. Syms RRA, Young IR, Ahmad MM, Rea M (2012) Magnetic resonance imaging using linear magneto-inductive waveguides. J Appl Phys 112(11):114911 108. Kardoulaki EM, Syms RRA, Young IR, Rea M (2016) SNR in MI catheter receivers for MRI. IEEE Sens J 16(6):1700–1707 109. Khuntikeo N, Titapun A, Chamadol N, Boonphongsathien W, Sa-Ngiamwibool P, TaylorRobinson SD et al (2020) In vitro intraductal MRI and T2 mapping of cholangiocarcinoma using catheter coils. Hepat Med 12:107–114 110. Khuntikeo N, Titapun A, Chamadol N, Boonphongsathien W, Sa-Ngiamwibool P, TaylorRobinson SD et al (2020) Improving the detection of cholangiocarcinoma: in vitro MRIbased study using local coils and T2 mapping. Hepat Med 12:29–39

7

Surgery Narong Khuntikeo, Ake Pugkhem, Tharatip Srisuk, Vor Luvira, Attapol Titapun, Theerawee Tipwaratorn, Vasin Thanasukarn, Vivian Klungboonkrong, and Jitraporn Wongwiwatchai

Contents 7.1

7.2

Part I: Surgical Anatomy of the Liver and Biliary Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.1 Liver Anatomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.1.1 Surface and Relation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.1.2 Peritoneum Covering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.1.3 Plate System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.1.4 Ligament . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.1.5 Blood supply . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.1.6 Venous drainage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.1.7 Bile Duct . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.1.8 Porta Hepatis: Relation and Variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.1.9 Segmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.2 Pancreas Anatomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.2.1 Relation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.2.2 Pancreatic Duct . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.2.3 Ampulla of Vater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Part II Pre-operative Treatment for CCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.1 Clinical Presentation for Each Type in Different Symptoms . . . . . . . . . . . . . . . . 7.2.2 Liver Function Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.2.1 Basic Serum Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.2.2 Direct Liver Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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N. Khuntikeo (B) · A. Pugkhem · T. Srisuk · V. Luvira · A. Titapun · T. Tipwaratorn · V. Thanasukarn Department of Surgery, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand e-mail: [email protected] A. Titapun e-mail: [email protected] Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen 40002, Thailand V. Klungboonkrong · J. Wongwiwatchai Department of Radiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. Khuntikeo et al. (eds.), Liver Fluke, Opisthorchis viverrini Related Cholangiocarcinoma, Recent Results in Cancer Research 219, https://doi.org/10.1007/978-3-031-35166-2_7

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7.2.2.3 Child–Pugh Score . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.2.4 The Model for End-Stage Liver Disease (MELD) Score . . . . . . . . . . 7.2.2.5 Indocyanine Green (ICG) Clearance Test . . . . . . . . . . . . . . . . . . . . . . . . 7.2.3 Volume Assessment Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.3.1 Volumetric Threshold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.4 Pre-operative Biliary Drainage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.4.1 Pre-operative PTBD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.4.2 Pre-operative ENBD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.5 Portal Vein Embolization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3 Part III: Surgery for Intrahepatic Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.1 General Consideration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2 Patient Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.3 Margin of Resection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.4 Role of Lymph Node Dissection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.5 Vascular Resection for iCCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.6 Resection After Neoadjuvant Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.7 Resection of Tumor Recurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.8 Special Consideration for Intraductal Growth Subtype . . . . . . . . . . . . . . . . . . . . . 7.4 Part IV: Surgery for Perihilar Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4.1 Definition of Perihilar Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4.2 Assessment of Curative Resectability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4.3 Surgical Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4.3.1 Lymphadenectomy and Extrahepatic Bile Duct Resection . . . . . . . . . 7.4.3.2 Liver Parenchymal Transection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4.3.3 Liver Hanging Maneuver (LHM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4.3.4 Low CVP Hepatectomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4.3.5 Vascular Inflow and Outflow Occlusion . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4.3.6 Biliary-Enteric Reconstruction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4.3.7 Vascular Resection and Reconstruction . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4.4 Left and Right Trisectionectomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4.5 Hepatopancreatoduodenectomy (HPD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5 Part V: Surgical Treatment in Distal Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5.1 Assessment of Resectability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5.2 Arterial Approach Pancreaticoduodenectomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5.3 Extended Lymphadenectomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5.4 Resection Part . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5.5 Reconstruction Part . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6 Part VI: Minimally Invasive Surgery in Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . 7.6.1 Intrahepatic Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6.1.1 Position and Port Insertions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6.1.2 Liver Mobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6.1.3 Inflow Control and Outflow Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6.1.4 Lymphadenectomy and Hilar Dissection . . . . . . . . . . . . . . . . . . . . . . . . . 7.6.1.5 Parenchymal Transection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6.2 Distal Bile Duct Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6.2.1 Position and Port Insertions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6.2.2 Resection Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6.2.3 Reconstruction Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7 Part VII: Role of Liver Transplantation in Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . 7.7.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7.2 Hilar Cholangiocarcinoma and Liver Transplantation . . . . . . . . . . . . . . . . . . . . . . 7.7.3 Liver Transplantation Versus Liver Resection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7.4 Intrahepatic Cholangiocarcinoma and Liver Transplantation . . . . 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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Part I: Surgical Anatomy of the Liver and Biliary Tract

7.1.1

Liver Anatomy

The liver is the largest organ in the abdominal cavity weighing about 2% of total body weight and having a volume equal to 19.59 × body weight in the Thai population [1]. In other places globally difference equations are used to calculate liver volume. For instance, for the Japanese population liver volume is calculated by the equation, LV (ml) = 2.223 × BW (kg)0.426 × body height (BH) (cm) 0.682 [2] and LV (ml) = 1072.8 × BSA(m2) – 345.7 for the Caucasian population [3]. The overall shape of the liver is wedge shape with two ends, one points to right hypochondrium and another points to epigastrium.

7.1.1.1 Surface and Relation The liver is located at the right upper quadrant of the abdomen. The superior, anterior, posterior, and right surfaces are convex and continuous and may be grouped at the diaphragm surface. The rest of surface contains the inferior surface, or visceral surface. The entire superior surface is diaphragm associated. Inferior surfaces are associated with many organs, from the lateral to medial liver which are related to the hepatic flexor colon, right kidney, right adrenal gland, first part of the duodenum, inferior vena cava (IVC), aorta, lesser curvature of stomach, and esophagus, respectively. 7.1.1.2 Peritoneum Covering The liver is partially covered by the peritoneum and may have some bare areas mainly at the posterior surface and a little in the superior surface of the liver in triangular ligament, coronary ligament, and falciform ligament. 7.1.1.3 Plate System The plate system in the hepatic hilum area is described as the space that is composed of connective tissue between the extrahepatic structure and liver capsule, which includes the cystic plate, hilar plate, and umbilical plate. The cystic plate is the space between the gallbladder and the gallbladder fossa. This is similar to the hilar plate and umbilical plate to liver capsule. Knowing the plate system structure assists in determining the plane of dissection for cholecystectomy during the approach to encircle and control the glissonean pedicle by an extra fascial technique.

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7.1.1.4 Ligament There are two types of defined ligaments. 1. Ligament from the peritoneum covering the liver These are ligaments that are formed by peritoneum which include the triangular ligament, coronary ligament, and falciform ligament. The triangular ligament is the peritoneum at the edge of each liver wedge shape end, which then continues with the coronary ligament covering bare areas at the posterior surface of the liver. The falciform ligament is the peritoneum that covers the ligamentum teres that lies connected from the abdominal wall and the umbilical portion of left portal vein. 2. Ligament from obliteration of fetal vessels Ligaments from obliteration of the fetal vessel include the ligamentum teres and the ligamentum venosum (Arantius ligament). The ligamentum teres is an obliteration of the umbilical vessel that provides access of blood supply and drainage from the placenta. This ligament connects the umbilical portion of the left portal pedicle and abdominal wall in the periumbilical area. The ligamentum venosum is an obliteration of Ductus venosus that provides blood drainage from the umbilical vein into the inferior vena cava via the left hepatic vein, or junction of the left hepatic vein and the middle hepatic vein. This ligament is located between the left lobe of the liver and the caudate lobe, which is attached to the lessor omentum.

7.1.1.5 Blood supply Hepatic inflow is a dual blood supply from the hepatic artery (branch from celiac artery) and portal vein (draining blood from the whole intestine via the mesenteric veins). The ratio of inflow by portal vein and hepatic artery has been found to be a 70:30 ratio, respectively. Hepatic Artery

The hepatic artery arises from the celiac axis branching as the left gastric artery, splenic artery, and common hepatic artery that subsequently branches as the right hepatic artery and left hepatic artery for blood supply to the liver. Further branching of the right hepatic artery gives rise to the cystic artery and the middle hepatic artery which branches from the left hepatic artery. Only 60–80% of population, however, have this classical blood supply. In some people, the right hepatic artery may arise partially (i.e., the accessory right hepatic artery) or completely (i.e., replacing the right hepatic artery) from the superior mesenteric artery. These various arteries may lie in difference positions in the hepatoduodenal ligament and lie posterolateral to the common bile duct (Fig. 7.1A). Similarly, the left hepatic artery may arise partially (i.e., the accessory left hepatic artery) or completely (i.e., replacing left hepatic artery) from the left gastric artery entering the liver on lessor omentum (Fig. 7.1B).

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Fig. 7.1 Position of variated hepatic artery. A: Replaced or accessory right hepatic artery. B: Replaced or accessory Left hepatic artery

The classification of the variation of the hepatic artery was proposed by Michels in 1966 [4]. Recent reviews of the incidence of this variation show that 5.3% of cases have right hepatic artery variations. Replaced right hepatic arteries from superior mesenteric arteries were recorded in 3.7% of cases. Accessory right hepatic arteries were found in 1.6% of cases, whereas variation of the left hepatic arteries was found in 6.2% of cases. Replaced left hepatic artery branching from left gastric arteries was found in 3% of cases, and accessory left hepatic arteries were found in 3.2% of cases. Replaced right and left hepatic arteries have been recorded in 0.8% of cases, and in 1.2% of cases, the common hepatic arteries were found to arise from superior mesenteric arteries [5]. Portal Vein

The portal vein drains blood form the inferior mesenteric vein, splenic vein, and superior mesenteric vein from the origin of the portal vein (i.e., the junction of splenic vein and superior mesenteric vein). First branching from supra-pancreatic part is the superior pancreaticoduodenal vein followed by the left gastric vein (Coronary vein) which subsequently branches as the left and right portal veins. In some cases, caudate branches at bifurcation are observed. The right portal vein then branches as the right anterior portal vein and the right posterior portal vein. The left portal vein has two portions consisting of a transverse portion, the segment of left portal vein before entering the umbilical fissure, and an umbilical portion giving rise to tertiary branches which supply segment 2, 3 and 4 of the liver. The portal vein has been at times observed to have variations in branching and has been classified into the following three types: bifurcation type (type I), trifurcation type (type II), and independent right posterior type (type III). The three variation types have been found in 86%, 4.5%, and 19.5% of cases in the population, respectively (Fig. 7.2) [6].

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Fig. 7.2 Portal vein variations

7.1.1.6 Venous drainage Liver outflow is the draining of blood from inflow to the IVC by hepatic veins including the right hepatic vein, middle hepatic vein, and the left hepatic vein for blood draining to the supra-hepatic IVC and short hepatic vein and finally for blood draining to the retro hepatic IVC. Hepatic Vein Branch

Branching of the hepatic veins is complex, and hence, it is an important consideration to identify the specific veins which drain from each specific segment that are planned to be resected or not resected. A study has identified each hepatic vein branch and found that tributaries of the left hepatic vein were the left superficial vein (LSV) and the umbilical fissure vein (UFV). They also found that tributaries of the middle hepatic vein were two veins for segment 4 (inferior and superior) and two veins for segment 8 (the ventral vein and intermediate vein). Additionally, veins for segment 5 tributaries of the right hepatic vein were right superficial vein (RSV) and dorsal vein for segment 8. There were also two accessory veins, namely, the inferior right hepatic vein and the middle right hepatic vein [7].

7.1.1.7 Bile Duct Right Hepatic Duct

The right hepatic duct drains bile from the right liver lobe via the right anterior sectoral bile duct (segments 5 and 8) and right posterior sectoral bile duct (segments 6 and 7). The right hepatic duct can be classified as A to J types and has a grouping of presence or absence of the right hepatic duct and supra-portal and infra-portal right posterior sectoral bile ducts (Fig. 7.3).

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Fig. 7.3 Right hepatic duct variation: Modified from Kitami M [8]. RHD = right hepatic duct, RPSD = right posterior sectoral duct

Type A has been defined as the Classic type and is found in 73% of the population, having right posterior sectoral bile duct joining right anterior sectoral bile duct supraportally forming the right hepatic duct before joining the bile duct confluence. Type B was found in 12% of the population, having the right posterior sectoral bile duct joining the left hepatic duct supraportally. This results in the absence of right hepatic duct. Type C was found in 5% of population, having right anterior and posterior sectoral bile ducts and left hepatic duct as a trifurcation of bile duct confluence.

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Type D was found in 4% of population, having the right posterior sectoral bile ducts joining the right anterior sectoral bile duct infraportally, and forming the right hepatic duct as type A. Type E was found in 3% of population, having the right posterior sectoral bile duct joining the common hepatic duct infraportally. Types F–J depict other types of variation which are found in 2% of the population. The study found that if subjects had variations of the portal vein such as trifurcation or independent right posterior postal vein [6], more of the variation in the right hepatic duct also occurred. It has been documented that for type A, the incidence reduced from 63 to 32% and increased in variation type from 12 to 34%, 5 to 7%, 4 to 14%, and 3 to 4% from B–E types, respectively [8]. Left Hepatic Duct

Unlike right hepatic drainage, there is one large common bile duct for draining the left lobe of the liver. The left hepatic bile duct runs alongside the left portal vein as a transverse portion and umbilical portion branching to segments 2, 3, and 4 of the liver as a tertiary branch in the glissonean pedicle concept [9]. Another concept of branching of the left hepatic duct is that there are four main bile ducts of segments 4a, 4b, 2, and 3 of the liver. For segments 2 and 3, duct 2 is joined first (defined as the left lateral sectoral bile duct) before the bile duct joined segment 4 (defined as the left hepatic duct). With regards the portal vein, the infra-portal bile duct is very rare (3% of population), and variation has been recorded as types A–C as described below. Type A has been defined as the classic type found in 73% of the population, having a common trunk of segments 2 and 3 of the liver which is known as the left lateral sectoral duct as mentioned before and joined to branch at segment 4. Type B has been found in 5% of the population and has segments 2, 3, and 4 bile ducts joining as a trifurcation at the start of the umbilical portion. Type C has been found in 18% of population and has a bile duct segment 2 branch from the bile duct of segment 4 [8]. As for the right hepatic duct, the study found that if people have variations of the portal vein, such as trifurcation portal vein or independent right posterior postal vein [6], greater variation in the left hepatic duct also occurred [8]. For type A the incidence was reduced from 73 to 55% and increased in variation type from 5 to 9% and 18 to 27% for B and C types, respectively, which has been reported [8].

7.1.1.8 Porta Hepatis: Relation and Variation It is important to examine the relationship of bile ducts and vessels in the porta hepatis in individual patients who have been planned for surgical treatment in perihilar CCA. The porta hepatis contains hepatic arteries, portal veins, and bile

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ducts covering with the glisson sheath. There may also exist variations in portal veins such as bifurcation type (type I), trifurcation type (type II), and independent right posterior type (type III) [6]. However, within the Glisson sheath, numerous further types of variation may exist. Classical relationships between the right hepatic artery, bile ducts, and the portal vein have previously described that the portal vein is mostly found posteriorly to arteries and bile ducts. The right hepatic artery runs across the common hepatic artery posteriorly before entering the right hepatic hilum area. The middle hepatic artery enters the intrahepatic part at right side of the umbilical fissure. The left hepatic artery enters the intrahepatic part at left side of the umbilical fissure (Fig. 7.4). Consideration regarding these relationships for perihilar CCA is described below. • The left hepatic artery is located at a distance from the hilar area and is rarely involved with tumor. • The right hepatic artery is often involved with tumor because it lies across the common hepatic duct posteriorly even in tumors that in mostly involve the left hepatic duct (Bismuth-Corlette type IIIb). • If the replaced right hepatic artery is present and lies to the right side of bile duct away from the bile duct confluence, the tumor may not involve the right hepatic artery (12). The relation of the right posterior hepatic artery and the portal vein has been classified as infra-portal type (82.6%) and supra-portal type or mixed as variants Fig. 7.4 Classical relationships in porta hepatis

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Fig. 7.5 Right hepatic pedicle variation related to the portal vein (modified from Watanabe N) [6]

(17.4%). The relation of the right posterior sectoral bile duct and portal vein has been classified as classically supra-portal type (83.1%) and infra-portal or mixed as variants (16.9%) (Fig. 7.5) [6].

7.1.1.9 Segmentation Since 1954, Couinaud’s Segmentation of the liver has been described [10]. Many segmentations have been proposed and used in numerous studies and publications, until The Scientific Committee of the International Hepato-Pancreato-Biliary Association (IHPBA) meeting in December 1998 created a Terminology Committee to deal with the confusion in nomenclature of hepatic anatomy and liver resections [11]. This terminology was named The Brisbane 2000 terminology and described segmentation based on Couinaud’s Segmentation. Liver was divided into four sections by three hepatic veins including the right posterior section, right anterior section, left medial section, and the left lateral section. These were further subdivided into eight segments. The left lateral section contained segment 2 superiorly and segment 3 inferiorly, whereas the left medial section contained segment 4. The right anterior section contained segment 8 superiorly, segment 5 inferiorly, and the right posterior section contained segment 7 superiorly, segment 6 inferiorly.

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Segment 1 (Caudate Lobe)

The caudate lobe is area of the liver between the inferior vena cava (IVC) posteriorly and left pedicle, middle and left hepatic veins superiorly. The draining bile duct of caudate joins the biliary tree at or near the bile duct confluence on both the left and right hepatic ducts. Venous drainage of the caudate lobe is via multiple short hepatic veins to the inferior vena and drains directly to left and middle hepatic veins. There are three parts of the caudate lobe which is divided into Spiegel’s lobe, paracaval portion, and caudate process [12]. Spigel’s lobe is the part of caudate lobe that protrudes medially from inferior vena cava (IVC) and major blood supply is from the left portal pedicle. Paracaval portion is the caudate lobe located between both edges of IVC. The caudate process is the part fused with the right lobe of the liver located under the border of the middle and right hepatic veins.

7.1.2

Pancreas Anatomy

The pancreas located in retroperitoneum area lies across the “transpyloric plane [13]” or at the first Lumbar vertebra spine (L1) from C-loop of the second part of the duodenum to the splenic hilum. The pancreas can be divided into four parts, head, neck, body, and tail. The pancreatic head encompasses the SMA and SMV which is defined as the uncinate process.

7.1.2.1 Relation The right lateral of the pancreas attaches to the duodenum, whereas the left lateral is related to the spleen. The posterior relation is the splenic vein joined with SMV to form the portal vein and SMA, whereas the anterior is related with mesocolon. 7.1.2.2 Pancreatic Duct There are two pancreatic ducts, the major pancreatic duct and accessory pancreatic duct. Major Pancreatic Duct (Duct of Wirsung)

The main pancreatic duct lies posteriorly and inferiorly along the pancreatic tail to pancreatic head and joins with CBD in area of the major papilla (ampulla of Vater). Abnormalities of the junction of pancreatic duct and CBD are known as “pancreaticobiliary maljunction (PBM)”. PBM is a congenital malformation in which the pancreatic and bile ducts join outside the duodenal wall. Because of this, reciprocal reflux can occur between pancreatic juice and bile, resulting in retention of bile in biliary system causing choledochal cyst and biliary tract cancer, especially gallbladder cancer [14]. In an imaging study, the long common channel greater in length of more than 9 mm was consider as having this condition [15]. Patients with PBM have higher risk for biliary tract or gallbladder cancer. The mean age for the development of cancer was 50–60 years [16].

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Accessory Pancreatic Duct (Duct of Santorini)

The accessory pancreatic duct occurs branched from main pancreatic duct and drains pancreatic juice from part of the head of the pancreas to minor papilla. In about half of population, it has been found that the accessory pancreatic duct was obliterated and may be patent in about 41–52% of the population [17–19]. In some populations, major and accessory ducts have been found in 4–14% of cases to separately drain into the duodenum, which is known as “Pancreatic divisum” [20]. Additionally, that major pancreatic duct has been found to drain via the minor papilla causing recurrent pancreatitis because of the retention of pancreatic juice. For patients having both PBM and pancreatic divisum, the incidence of biliary tract cancer may be low since pancreatic juice reflux into the bile duct might be reduced by the flow of pancreatic juice into the duodenum through Santorini’s duct [21]. Another rare variant of the pancreatic duct system was “Ansa pancreatica” which is found in 0.8% of the population. This variant occurs when the accessory duct forms a reversed-S shape and connects with a side branch of the duct of main pancreatic duct [22]. This condition is also hypothesized to be a predisposing factor for recurrent pancreatitis, but the mechanism is currently unexplained.

7.1.2.3 Ampulla of Vater Ampulla of Vater is an orifice of the common channel of biliopancreatic duct opening to duodenum and covered with duodenal mucosa. It also has a sphincter around the ampulla to control the flow of bile and pancreatic juice. When observations in an endoscopic view are made over the duodenal mucosa covering, part of ampulla may be observed as frenulum, orifice, recessus, and infundibulum [23]. Infundibulum of Ampulla of Vater

Part of infundibulum may protrude into the duodenum in a different manner and can be classified into four types according to Haraldsson classification [24]. Type Type Type Type

1 2 3 4

regular papilla no infundibulum projecting infundibulum creased or ridged papilla

seen seen seen seen

in in in in

41.26% of the population. 8.74% of the population. 22.03% of the population. 27.97% of the population [25].

7.2

Part II Pre-operative Treatment for CCA

7.2.1

Clinical Presentation for Each Type in Different Symptoms

Jaundice: This symptom occurs in pCCA and dCCA because only obstruction that involves the common hepatic duct at the bile duct confluence downward to distal common bile duct will cause jaundice. Thus, iCCA is unlikely to cause jaundice

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except that it may cause decline of hepatic function, which has been found to occur in cases of widespread intrahepatic metastases. Hepatomegaly: As in jaundice, the complete obstruction of biliary tract will cause hepatomegaly in pCCA but less in dCCA because early distal common bile duct obstruction may have a dilated common bile duct acting as a reservoir. Hence, pCCA may present with hepatomegaly more than in for dCCA. Distended Gallbladder: Gallbladder distention caused by obstruction of the bile duct distally from cystic duct junction, for instance as in cases of dCCA, can occur and may be palpated. For patients with pCCA, they can also present with gallbladder distension if the tumor occurs from the hilar down to the cystic duct junction. Radical surgery is the only chance for long-term survival among treatment options for CCA. Resectability depends on the patient’s physiological fitness, liver function reserved, volume of liver remnant, and tumor stage. Cholangiocarcinoma patients who are assessed for potential curative resection may have obstructive jaundice, cholangitis, cirrhosis, malnutrition, and other problems that can lead to perioperative morbidity. The assessment of the patient’s status, liver function reserved, and pre-operative treatment are most important issues in the planning and preparation for surgery.

7.2.2

Liver Function Assessment

Major liver resection should be performed only in physically fit patients. It is also important that liver function is carefully assessed, before any operation, to prevent insufficient post-hepatic resection of the liver. Several methods can be used to assess the function of the liver.

7.2.2.1 Basic Serum Test This involves a basic liver function test including markers of three aspects of hepatic function, namely, synthetic function (albumin and INR), excretory function (bilirubin), and hepatocyte injury (AST, ALT). Abnormal serum tests may indicate impairment of each aspect of liver function. 7.2.2.2 Direct Liver Function Bile content can be observed and assessed for infection or function of drainage of the liver segment in perihilar CCA in patients by performing external pre-operative biliary drainage. For poor excretory function, hypersecretory bile may be observed as large amounts and the pale color of bile. Infected bile has a dark color, is turbid, or contains sludge. Low levels of total bilirubin excreted from future liver remnants have been found to be significantly associated with post-hepatectomy liver failure (Fig. 7.6) [26].

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Fig. 7.6 Bile color in each condition. A: Healthy bile, B: Infected bile, C: Stasis bile

7.2.2.3 Child–Pugh Score The Child–Pugh score was developed to assess the severity of cirrhosis and to predict the mortality risk of patients with cirrhosis and portal hypertension undergoing operative procedures [27]. This score is calculated from the laboratory test (albumin, INR, and bilirubin) and clinical findings (ascites and encephalopathy). The Child–Pugh score is classified into three categories: Child–Pugh A [score less than 7] is associated with good liver reserve, Child–Pugh B [score 7–10] is associated with impaired liver function, and Child–Pugh C reflects end-stage, decompensated liver cirrhosis. Cholangiocarcinoma patients with obstructive jaundice, coagulopathy, and malnutrition may have an impaired Child–Pugh score. Therefore, the Child–Pugh score in jaundiced CCA patients should be re-evaluated after adequate biliary drainage has been performed. Liver resection can be performed only in Child–Pugh A patients, while Child–Pugh’s B and C are a contraindication for performing liver resection due to the high risk of post-operative liver failure. 7.2.2.4 The Model for End-Stage Liver Disease (MELD) Score The MELD score has been used to determine a liver transplantation waiting list for allocation of organs. The score is calculated using the basic laboratory serum test including bilirubin, creatinine, and INR [28]. This score is also used for predicting

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post-operative complications, for instance, in patients with a MELD score of more than 8 which are likely to have perioperative mortality and decreased long-term survival [29].

7.2.2.5 Indocyanine Green (ICG) Clearance Test Indocyanine green (ICG) is an inert, water-soluble, fluorescent tricarbocyanine which when given intravenously to patients distributes in serum, binds with serum protein in close to 95% of cases, and requires clearance with hepatic excretory function. The study procedure was to injected subjects with a 0.5 mg/kg dose intravenously to generate a plasmatic concentration of 100 mg/mL [30, 31]. Subsequently, the concentration of serum ICG level was measured at 15 min (ICGR15) by an optical sensor measuring device on held on the fingertip [32]. For normal hepatic function in subjects, clearance if ICG was almost complete (> 97%) at 15 min after injection and was consider normal if was still less than 10% at 15 min after injection (normal value of ICGR15 is < 10%) [30, 33]. In patients with obstructive jaundice with serum bilirubin level > 3 mg/mL, the ICGR15 value maybe falsely high because serum bilirubin also binds to serum protein as carrier competitive. Therefore, measuring ICGR15 in this group of patients may be performed after adequate biliary drainage has been achieved. Use of an ICGR15 value followed the Makuuchi’s criteria [34, 35]. Makuuchi’s criteria divided ICGR15 value into five ranges to guide a safe operation in patients without ascites and serum bilirubin ≤ 1 mg/dl [30, 31, 34, 35] (Table 7.1). A study using these criteria guaranteed that no post-operative mortality occurred in a Japanese cohort of 1056 patients. Furthermore, only one patient developed post-operative hepatic insufficiency [36]. In 2019 Makuuchi’s criteria were expanded using both the ICG clearance tests, and measuring of future liver volume in ml was proposed [31]. The safety for liver resection was assured for patients when the ICG clearance rate (ICGK) of remnant liver (ICGK-rem) was ≥ 0.05. The ICGK was measured by sampling ICG concentration at three time points at 5, 10, and 15 min after injection. Three points were plotted on a semilogarithmic graph using a non-logarithmic scale for time (x-axis) and a logarithmic scale for ICG concentration (y-axis). The three points were connected, and a line was created by the least-squares method and the initial concentration of ICG was determined from y-axis intersection. The half-valued period (λ) of ICG concentration was also determined from this graph [37]. Table 7.1 Makuuchi’s criteria ICGR15 values

Safe operation

< 10%

Trisectionectomy

10%–19%

Left hepatectomy or single sectionectomy on right lobe

20%–29%

Single Couinaud’s segmentectomy

30%–39%

Limited resection (wedge resection)

≥ 40%

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ICGK was calculated by the following formula: ICGK = 0.693 / ICGλ (ICGλ = half-life of ICG) ICGK-rem was calculated by following formula: ICGK-rem = ICGK × FLR/TLV (FLR = future liver volume, TLV = total liver volume)

7.2.3

Volume Assessment Method

Liver volume measurement is currently determined by using three-dimensional simulation software (SYNAPSE VINCENT®, FUJIFILM, Tokyo, Japan) [38] to calculate the volume of liver according to tributaries of each portal vein on CT scan imaging. A transection line was simulated to include the tumor and tumorbearing portal vein territory along the hepatic vein in anatomical segmentectomy. Total liver volume (TLV) was defined as the volume of the non-cancerous liver parenchyma excluding tumors. In some cases, volume assessment using standard liver volume may be more appropriate, such as in patients that have large volume of tumor. The resected non-cancerous liver parenchyma may be observed in small amounts, and despite the future liver volume which has been left being very small, the percent of FLR may be observed as a high percentage of FLR. Thus, in such cases, using standard liver volume instead of total liver volume may more effective and appropriate. Standard liver volume (SLV) is determined by whole liver volume calculated by bodyweight and height difference in various formulas. For the Thai population, liver volume has been shown to be 19.59 times of body weight [1]. In other different ethnic populations, a different equation is employed to calculate of liver volume. For the Japanese population, liver volume is calculated by the equation, LV (ml) = 2.223 × BW (kg)0.426 × body height (BH) (cm)0.682 [2], whereas, LV (ml) = 1072.8 × BSA(m2) – 345.7 for the Caucasian population [3].

7.2.3.1 Volumetric Threshold The ICG clearance test may not be available in all treatment centers and may not be suitable for some patients, such as in patients with hyperbilirubinemia. In such cases, a volume assessment without ICGR15 value may be useful. Volumetric threshold has been defined as the lowest FLR allowed in different background liver statuses. For patients with normal liver accepted FLR volume is ≥ 20% of TLV, ≥ 30% of TLV in patients who have received extensive chemotherapy, and ≥ 40% of TLV in patients with hepatic fibrosis or cirrhosis [39]. In real life, our institute practice is that the FLR should be more than 30% in normal liver to prevent post-hepatectomy liver failure.

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Pre-operative Biliary Drainage

Most perihilar CCA patients present with jaundice problems due to biliary tract obstructions. The aims of pre-operative biliary drainage are to improve liver function and to reduce post-surgical complications. To date, there are three methods available for pre-operative biliary drainage in perihilar CCA patients, namely, percutaneous transhepatic biliary drainage (PTBD), endoscopic retrograde biliary drainage (ERBD), and endoscopic nasobiliary drainage (ENBD) [40–42]. Perihilar CCA patients who have obstructive jaundice and malfunction of the liver have a significant risk of morbidity and mortality as well as complications after radical surgery. Pre-operative biliary drainage has been shown to increase the resectability rate and post-operative survival in perihilar CCA patients. Another benefit of pre-operative biliary drainage for selective cholangiography is diagnosis of the longitudinal extension of tumor and usefulness in clarifying the complicated segmental anatomy of the intrahepatic bile ducts. This provides a sound basis and information for operative planning (Fig. 7.7). A previous study has revealed that internal biliary drainage is superior to external drainage in liver regeneration and function after hepatectomy in obstructive jaundice. However, PTBD is superior to internal drainage in complications such as cholangitis and pancreatitis [41–45]. There are experimental studies which have shown that PTBD is superior to ERBD in resectable hilar CCA or segmental cholangitis [40]. There are some studies, however, that suggested that ENBD should be the first line of management for biliary drainage due to decreased inflammation, less invasiveness, and the decreased risk of tract seeding compared to PTBD. If jaundice

Fig. 7.7 A. Cholangiogram obtained from the left PTBD catheter (white arrow) shows total obstruction at the left main hepatic duct from cholangiocarcinoma. Bile duct segments 2, 3, and 4 were free from tumor; therefore, this case was suitable for right hepatectomy with hilar and caudate resection. B. Cholangiogram obtained from the right posterior PTBD catheter (white arrow) shows total obstruction at the right hepatic duct, no contrast filled in segment 8 bile duct due to tumor invasion. This case was suitable for left trisectionectomy

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and cholangitis are still present, then PTBD is the choice as adjunctive treatment [40, 46]. There are, however, different suggestions and opinions that have been advanced in some previous studies. For instance, a study comparing preoperative endoscopy to percutaneous transhepatic biliary drainage in potentially resectable perihilar CCA has shown that there is no significance in terms of seeding metastases and survival. Decisions to undertake PTBD should not be based on development of seeding metastases as PTBD has a very low association with seeding metastases of 2–5% of patients after resection [47]. In our institute, pre-operative biliary drainage has been performed in patients with potential resectable perihilar CCA with jaundice or significant bile duct dilatation in the remnant liver. ENBD was considered as a first-line approach unless the patient had contraindication for ERCP or high-level obstruction, which is a technically difficult placement of an ENBD catheter. PTBD is considered for patients with unsuitable for ENBD, backup procedure of failed ENBD, treatment of segmental cholangitis, and adjunct treatment for slow recovery of jaundice. Supportive treatments are important to improve a patient’s nutritional status and recovering from jaundice. Oral nutritional support, ursodeoxycholic acid, and anxiolytic treatment have been routinely provided for pre-operative patients.

7.2.4.1 Pre-operative PTBD The roles in PTBD in CCA patients are for pre-operative biliary drainage in patients with resectable perihilar CCA as well as for palliative treatment. The Faculty of Medicine, Khon Kaen University, Thailand, has performed palliative PTBD since 1984 and pre-operative PTBD since 2002. The Indication for PTBD is as Follows

1. Pre-operative drainage. 2. Cholangitis treatment. 3. Palliative treatment. In general, the percutaneous transhepatic biliary drainage (PTBD) procedure is employed for pre-operative biliary draining, cholangitis treatment, and palliative drainage. The contraindication for PTBD is uncorrected coagulopathy and massive ascites. Pre-procedural patient’s Preparation

Patients should be admitted in hospital for pre-procedural preparation. 1. Pre-procedural laboratory test (liver function test, CBC, and coagulogram). 2. Adequate antibiotic coverage, as the manipulations in the obstructed bile duct system can increase the risk of cholangitis and sepsis. 3. Fasting 6 hours before the procedure.

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Steps of PTBD

After cleaning the patient’s abdomen with antiseptic techniques, ultrasound is conducted to select the appropriate bile duct. An anesthetic injection at the puncture site is administered followed by puncture with a needle into the selected bile duct under ultrasound guidance. After the needle is placed in the appropriate bile duct, bile is collected for laboratory testing, and subsequently contrast media is slowly injected for cholangiogram imaging. After the cholangiogram is obtained, a biliary drainage catheter is inserted via the Seldinger technique. Complications

A previous study has shown that complication rates following PTBD ranged from 3 to 10% and procedural mortality rates ranged from 0.1% to 0.8% including access-related complications, non-vascular complications, vascular complications, and catheter-related complications [48]. Hemobilia is the most common complication in many studies and can happen from both the arteries and veins. When the PTBD procedure is performed using appropriate techniques and materials, serious bleeding is significantly reduced. Our case with post-PTBD hemobilia is demonstrated in Fig. 7.8. Bile leaks are one of the common complications of the PTBD procedure. Leaks can occur around the catheter insertion site or into the peritoneum which may cause peritonitis or biloma. Leaks can also occur into the pleural cavity and cause pleuritis. Although PTBD is a sterile procedure, antibiotics before and after a procedure are recommended. If sepsis occurs, blood culture and appropriate drainage should be undertaken. Cholecystitis and pancreatitis could happen due to distal biliary manipulation and because of the placement of internal and/or external biliary drainage catheters. Results

From our experiences, we have found that the result depends on performance status, disease pathology, operator’s technique, experience of an interventionist, material, and a multidisciplinary team. The complications discussed above have been steadily decreasing as more experience is gained by multidisciplinary staff. Despite these factors, we have found that pre-operative PTBD can lower both post-operative morbidity and mortality.

7.2.4.2 Pre-operative ENBD Significant complications are associated with PTBD, such as, vascular injury and tumor seeding. Although vascular injury is not a common complication, it precludes planed curative surgery and causes fatality in severe cases. Tumor seeding via PTBD has been found in 2–5% of patients [41, 49, 50] and affects long-term survival outcome (Fig. 7.9). Endoscopic nasobiliary drainage has been frequently used for pre-operative biliary drainage for more than 10 years and became firstline treatment instead of PTBD because it is minimally invasive, and there are no chances of tumor seeding and no risk of vascular injury.

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Fig. 7.8 A: 63-year-old man with a history of perihilar IIIA S/P left PTBD 2 weeks previously developed hemobilia with Hct drop. Coronal view CTA image shows abnormal outpouching lesion from left hepatic artery (white arrow). B: 63-year-old man with a history of perihilar cholangiocarcinoma IIIA S/P left PTBD 2 weeks previously, developed hemobilia with Hct drop. Selective left hepatic angiogram shows abnormal outpouching lesion (black arrow). After coil embolization is done (arrow head). A post-embolization left hepatic angiogram shows total occlude of abnormal outpouching lesion

Fig. 7.9 A: Cholangiocarcinoma recurrent at a PTBD site after left PTBD and placement of internal metallic biliary stent (white arrow). B: tumor seeding at PTBD tract found during explore laparotomy (white arrow)

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Indications for ENBD more than pre-operative biliary drainage are treatment for other causes, because ENBD is minimally invasive external drainage, for instance for bile drainage color, and volume can be monitored, samples have taken, and samples have repeatedly sent for microbial study. The contraindication for ENBD was uncorrected coagulopathy, history of gastric bypass or duodenal obstruction, massive ascites, and multi-segment biliary obstruction. Steps of ENBD Procedure

1. Patients are prepared in the prone position; then, endoscopic retrograde cholangiopancreatography under sedation by anesthesiologist is performed. 2. Cannulate ampulla of Vater and insert hydrophilic tip guidewire into the common bile duct. 3. Perform retrograde cholangiography to evaluate obstruction site, followed by a guidewire passed through the stricture part into the selected bile duct. Care must be taken not to inject contrast into the bile duct that is not planned to be drained to avoid segmental cholangitis. 4. Insertion of the nasobiliary catheter followed by the guidewire into the obstructed bile duct and aspiration of bile for bacterial culture then check cholangiography again. 5. Re-locate the drainage catheter from mouth to nostril and connect to a vacuumed drainage bottle (Fig. 7.10). All patients were trained for catheter self-care before being discharged from hospital and scheduled follow-up was done every 1–2 weeks until the day of surgery. Additional drainage should be considered if serum bilirubin reduction was slow or segmental cholangitis occurred. Complications

Post-ERCP pancreatitis Patient’s clinical symptoms can be persistent abdominal pain and increasing levels of serum lipase and amylase. The incidence of post-ERCP pancreatitis following ENBD was 3%–15% and most cases had mild pancreatitis. Routine sphincterotomy should be avoided thus preserving the function of Oddi’s sphincter and preventing pancreatitis. The use of a NSAIDs’ rectal suppository can reduce post-ERCP pancreatitis in selected cases, namely, difficult cannulation, precut sphincterotomy, pancreatic cannulation, and pancreatic contrast injection [51]. Most of pancreatitis following ENBD was mild and can be managed by conservative treatment. Cholangitis Cholangitis following ENBD was due to two major causes, ENBD catheter malfunction and undrained segmental cholangitis. Catheter malfunction can occur by

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Fig. 7.10 Steps of ENBD procedure. A Cholangiogram of Bismuth IIIa perihilar CCA. B–C Cannulation ENBD catheter follow the guide wire into left hepatic duct. D Re-location catheter to nostril and connected to vacuumed drainage bottle

sludge obstruction, tube kinking, or dislodgement. Regular aspiration and flushing the ENBD catheter with sterile water can prevent tube obstruction. Patients should be well trained before discharge from hospital in the appropriate care and maintenance of the self-catheter. Persistent jaundice after ENBD indicates poor liver function or segmental cholangitis. Adjunct biliary drainage should be considered if a patient is clinically not improved by antibiotic and other supportive treatments. Adjunct ENBD in the contralateral lobe of liver or PTBD in the undrained segment can be performed depending on technical accessibility (Fig. 7.11).

7.2.5

Portal Vein Embolization

Cholangiocarcinoma needs advanced hepatobiliary surgical techniques with major hepatectomy for curative treatment. The incidence of post-hepatectomy liver failure (PHLF) may be as high as 10–20% after major hepatic resection in high-risk patients, and this is associated with a very high mortality rate [52]. Portal vein embolization (PVE) has a role in pre-operative preparation in CCA patients for radical surgery that will reduce post-operative mortality and morbidity.

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Fig. 7.11 Adjunct bilateral ENBD for perihilar cholangiocarcinoma patient with segmental cholangitis after pre-operative left ENBD

PVE is an interventional radiology procedure devised first by Makuuchi and Kinoshita in 1984 [53]. Makuuchi et al. introduced pre-operative PVE before major hepatectomy for hilar bile duct carcinoma in 1990 [54]. This procedure redirects portal blood to non-embolized segments or hepatic segments of the future liver remnant (FLR), which induced hepatocyte regeneration with hypertrophy and atrophic change of embolized segments (Fig. 7.12). The degree of hypertrophy after PVE differs depends on the degree of underlying liver disease. At 2 weeks post-PVE, patients with normal livers regenerate at a rate of 12–21 cm3 /day, compared with 9 cm3 /day in cirrhotic patients. In normal livers, sufficient hypertrophy occurs within 2–4 weeks, whereas cirrhotic patients can take ≥ 4 weeks [55, 56]. Induction of hypertrophy of estimated FRL may result in fewer complications and shorter hospital stays following major hepatectomy. At present, PVE is being used as a safe and effective standard for pre-operative management in patients with inadequate FLR for the prevention of PHLF. Many studies show that pre-operative PVE significantly induces hypertrophy of FLR and clinical impact to decrease the incidence of PHLF [57–63]. PVE was performed in case of inadequate FLR volume as follows [34, 64]: 1. Normal liver or indocyanine green retention test (ICG R15) < 10% with FLR/ TLV ≤ 30%. 2. Cirrhotic liver. 2.1. ICG R15 = 10–20% with FLR/ TLV ≤ 40%. 2.2. ICG R15 > 20% with FLR/ TLV ≤ 50%. Cross-sectional imaging (CT scan or MRI of upper abdomen) for procedural planning should be performed immediately prior to PVE to evaluate the extent of disease (e.g., extrahepatic disease or involvement of the planned FLR), FLR volume, and portal venous anatomy. Liver volumetry using CT scan is used to calculate liver volume and FLR (Fig. 7.13).

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Fig. 7.12 Portal vein embolization. (A) CT scan of upper abdomen before PVE shows relatively small size of left hepatic lobe (*). (B) CT scan of upper abdomen after right PVE shows hypertrophy of left hepatic lobe (*) and atrophy of right hepatic lobe (arrow). (C) Picture from surgery after PVE shows hypertrophy of left hepatic lobe (big arrow) and atrophy of right hepatic lobe (small arrow)

The relative contraindications for PVE are patients with distant metastases or periportal lymphadenopathy who cannot undergo hepatectomy, widespread intrahepatic disease, tumor invasion of the portal vein, tumor precluding safe transhepatic access, and uncorrectable coagulopathy. PVE is performed at least 2–4 weeks before scheduled hepatectomy. In jaundice patients, PVE is performed after the total bilirubin concentration has decreased to less than 5 mg/dl following biliary drainage [61]. The techniques to access the portal vein are via the percutaneous transhepatic ipsilateral approach (access through the portion of the liver to be resected)

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Fig. 7.13 A 65-year-old female patient with perihilar-type cholangiocarcinoma (CCA), BismuthCorlette type IIIA, planned for right trisegmentectomy. (A) CT scan of upper abdomen shows extension of perihilar CCA (arrow). (B) Portogram after right PVE shows occlusion of right portal vein with patent left portal vein (arrow). (C) CT volumetric program contouring lateral segment of left hepatic lobe as FLR (*) for calculated volume. (D) CT volumetric program after right PVE 30 days, contouring lateral segment of left hepatic lobe (*) which shows hypertrophy of FLR (FLR increase 77.1%)

(Fig. 7.14), percutaneous transhepatic contralateral approach (access through the portion of the FLR) (Fig. 7.15), and trans-ileocolic venous approach (access through the ileocolic vein). The percutaneous transhepatic approach is the most widely used because it is technically easier and does not require surgery as is the case for the trans-ileocolic venous approach. The advantage of the transhepatic ipsilateral approach is to reduce the risk of damage to the FLR and associated complications of vascular access. On the day of the procedure, prophylactic broad-spectrum antibiotics are administered intravenously for prevention of biliary sepsis. The peripheral branch of portal vein is accessed by ultrasound guidance with transhepatic approach under local anesthesia. After a vascular sheath is installed, flush portogram is performed to evaluate the anatomy of the portal system. The segmental branches of portal veins that have been identified to resect are catheterized and embolized. Many

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Fig. 7.14 PVE, percutaneous transhepatic ipsilateral approach (A) Portogram obtained from catheter (arrow) access through right hepatic lobe. (B) Portogram after right PVE shows occlusion of right portal vein with patent left portal vein (arrow)

Fig. 7.15 PVE, percutaneous transhepatic contralateral approach (A) Portogram obtained from catheter (arrow) access through right hepatic lobe. (B) Portogram after left PVE and anterior branches of right PVE show occlusion of left portal vein and anterior branches of right portal vein with patent posterior branches of right portal vein (arrow)

embolic materials have been used, including particles, N-butyl-cyanoacrylate (glue), gelatin sponges, coils, and vascular plugs. Embolization is performed until stasis or near-complete stasis is achieved. At the end of the procedure, the access tract or parenchymal tract is closed with gelatin sponges or coil embolization to prevent intra-abdominal bleeding. The overall complication rate after PVE is 2%–9% and includes non-targeted portal vein thrombosis, subcapsular hematoma, hemoperitoneum, hemobilia, pseudoaneurysm, arteriovenous fistula, and cholangitis [65, 66].

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At our interventional radiology unit, PVE was performed in CCA patients first in 2002 using the transileocecal venous approach. Currently, the percutaneous transhepatic approach for PVE is performed in all patients. Most patients have perihilar CCA, and as such, PVE is performed using the ipsilateral portal vein approach. Our studies have shown that pre-operative PVE before major hepatectomy is an effective procedure to increase FLR with a subsequent decreased risk of PHLF [63]. Furthermore, no procedure-related deaths have occurred.

7.3

Part III: Surgery for Intrahepatic Cholangiocarcinoma

7.3.1

General Consideration

Intrahepatic CCA is defined as the tumor arising in the bile ducts peripheral to secondary order bile ducts. According to gross morphology iCCA, it can be further divided into mass-forming (MF), periductal infiltration (PI), intraductal growth (IG), and mixed types (Fig. 7.16), which have different biological characteristics [67]. The symptoms of iCCA are usually non-specific, resulting in patients with iCCA usually presenting with late stage of the disease [68]. Current knowledge suggests that negative-margin resection is the ultimate goal of treatment of CCA [68–70].

7.3.2

Patient Selection

Selection of the patient for surgery is a crucial matter. The decision as to whether to perform an operation or not is mostly dependent on pre-operative cross-sectional imaging [71–73]. Pre-operative tissue biopsy is not necessary and should be avoided. The risks and benefits of the operations should be mandatory evaluated all cases on an individual basis. Given the relatively poor prognosis of the patients with iCCA and the relatively high risk of operative procedure required, a safe negative-margin (R0) resection with adequate future liver remnant should be ensured by careful review of pre-operative imaging and liver evaluation [68, 69, 72, 73]. The R0 resection rate varies across centers with a range of between 33.0 and 87.6% [74, 75] and is dependent on surgical experience, pre-operative planning, patient selection, gross morphology of the tumor, and pathological examination process. Multiple gross lymph node involvement and significant distant metastases (Fig. 7.17) are associated with poor prognosis after hepatic resection, which is generally accepted as an absolute contraindication for hepatic resection. The tumors that are involved in either inflow or outflow bilaterally are technically unresectable; consequently, they are considered as contraindication for resection. The outcome after surgery for multiple intrahepatic metastatic and vascular invasion iCCA is poor (Fig. 7.18) and, hence, should be considered as a relative contraindication for hepatic resection, except for only highly selected patients with limited burden of

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Fig. 7.16 Surgical specimen (a–c) and comparison diagram (d) of gross morphology of iCCA. a) Mass-forming types. b) Periductal infiltrating type; the tumor spreading along the bile duct wall. c) Intraductal growth type; the papillary tumors grow inside the lumen of dilated bile duct. d) Comparison of types of iCCA according to the growth pattern

the disease. Multi-focal tumor itself does not preclude hepatic resection since there has been a report of long-term survival in selected cases with limited multi-focal tumor. An aspect that should be considered is multiple intrahepatic lesions which could be either the multiplicity of the tumor itself, as we usually find in intraductal tumor [76], or intrahepatic metastases. The latter are associated with poor outcome after surgery and should be considered as a contraindication for hepatic resection. Diagnostic laparoscopy is advised to rule out peritoneal or disseminated disease undetected preoperatively (Fig. 7.19). Upon abdominal exploration, the surgeon should evaluate resectability by identifying peritoneal metastases, lymph node size and consistency, intrahepatic liver metastases, and the status of the future liver remnant. Multiple gross lymph node involvement and significant distant metastases are generally accepted as contraindications for hepatic resection.

7.3.3

Margin of Resection

R0 resection has been proven to be associated with better survival of the patients after resection. Previous studies reported no 5-year survival of patients with margin-positive disease. Even an R1 resection could worsen survival of the

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Fig. 7.17 Computed tomographic findings of clearly unresectable tumors. a) Primary massforming tumor (asterisk) with numbers of gross lymph node metastases (arrow head). b) Primary mass-forming tumor (asterisk) with numbers of subcutaneous metastases (arrow)

Fig. 7.18 Surgical specimen of resectable iCCA with poor prognostic indicators. a) Multiple ipsilateral intrahepatic tumors. b) iCCA grossly involving intrahepatic vessel (asterisk)

Fig. 7.19 Computed tomographic findings (a) and laparoscopic findings (b) of the patient with peritoneal carcinomatosis. a) CT scan depicted supra-diaphragmatic lymph node enlargement (arrow), raising the suspicion of peritoneal carcinomatosis. b) Diagnostic laparoscopy revealed iCCA (asterisk) and multiple nodules at parietal peritoneum of right diaphragm (arrowhead)

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Fig. 7.20 Intraoperative findings’ comparison of before (a) and after lymph node dissection (b) with no lymph node dissection (c). a) Hepatoduodenal ligament consists of common bile duct, hepatic artery, portal vein, and lymphatic tissues, covered by the peritoneum. b) After hepatoduodenal ligament was skeletonized. (removing all connective tissues leaving the vital structures including bile duct, hepatic artery, and portal vein) c) In the case of no lymph node dissection, all connective tissues with the vessel to liver remnant were left intact

patients, however, survival is still better than R2 or no resection [68, 77]. There is a trend toward increasing survival of patients with an increased distance of surgical margin. This effect has been observed at up to 1 cm of the margin. The benefit of a wider surgical margin is affected by other factors, such as lymph node metastases, the presence of lymphovascular invasion, vascular invasion, and multiplicity of the tumor. Currently, at least 5 mm of surgical margin is generally acceptable [77].

7.3.4

Role of Lymph Node Dissection

It appears that lymph node involvement is the strongest predictor of survival of patients. However, the role of routine lymph node dissection is still controversial because of the lack of clarity of the benefit as well as the difficulty of performing lymph node dissection (Fig. 7.20). A lymph node biopsy of at least 1–6 nodes is generally recommended [78], for prognostication of the patient and for making decisions of whether or not to give the patient adjuvant treatment. Even though most studies have shown that there is no survival benefit of routine lymph node dissection for iCCA, there are also several studies that have reported that routine lymph node dissection might provide some benefit, particularly in patients with a limited number of lymph node involvement [79]. Currently, only a minority of the patients receive adequate lymph node dissection for accurate staging. This could be due to (i) inaccurate pre-operative diagnosis of iCCA, (ii) trending toward minimally invasive hepatic resection, and (iii) advanced disease where it is unlikely to gain benefits of lymph node dissection.

7.3.5

Vascular Resection for iCCA

Vascular resection is considered as a predictor of poor outcome after surgery. The proportion of iCCA patients who require vascular resection is very low compared

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to patients with perihilar CCA (pCCA), which may indicate the large size of the tumor or lymph node involvement. However, it is not a contraindication for hepatic resection, as long as R0 resection is expected. The short- and long-term outcome of the iCCA patients with R0 vascular resection has been found to be comparable to patients without vascular resection.

7.3.6

Resection After Neoadjuvant Treatment

For patients who are not suitable for upfront surgery due to the tumor burden, neoadjuvant treatment is the most promising treatment approach (Fig. 7.21). A conversion rate of 8–100% [80, 81] has been recorded with some selected patients subsequently converted to resectable cases. The prognosis of the iCCA patients who respond to neoadjuvant treatment has been reported to be comparable with or even better than patients who received upfront surgery for initially resectable iCCA. The benefit of neoadjuvant treatment is to test the tumor biology and exclude the patients who are not suitable for hepatic resection. However, this approach is still not supported as a standard treatment by current available results. As surgery is currently the only treatment providing long-term survival, all patients who respond to neoadjuvant treatment should be reconsidered for hepatic resection.

7.3.7

Resection of Tumor Recurrence

Most of the patients with iCCA experience tumor recurrence after hepatic resection, at a recurrent rate of between 38 and 82%, [82, 83] with a median time of recurrence of 2–56 months [83–86]. The most common site of recurrence is within the liver remnant [84, 86]. However, there is no specific pattern of the recurrence of the tumor [84], indicating an initial role for adjuvant chemotherapy for prevention of recurrence in all sites, but the efficacy is still not promising [87]. The mainstay of treatment of recurrence is systemic therapy. However, in highly selected cases, there is a role for repeated resection for tumor recurrence where the reported rate of resection of recurrence was 7.4–28% [77, 88, 89]. The outcome of the patients receiving repeated resection is better than for patients who received other treatment modalities [85, 86, 88, 89]. The previous studies have shown that the survival of patients with successful resection of the recurrences had similar survival rates compared to non-recurrence patients after the first operation.

7.3.8

Special Consideration for Intraductal Growth Subtype

Intraductal growth type of CCA has now been considered as a new specific entity [69, 90]. Intraductal tumor of the bile duct can be divided, according to pathology, into more common intraductal papillary neoplasm of the bile duct (IPNB), and

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Fig. 7.21 Computed tomographic findings of a patient with advanced iCCA before (a, c), and after (b, d) receiving systemic chemotherapy. a, b) Primary tumor (asterisk) was significantly decreased in size. c, d) Bulky aortocaval lymph nodes (arrow) had significant shrinkage and calcification (arrowhead) after treatment. This patient later underwent right hepatectomy with extended lymph node dissection and still received a 4-year recurrence-free survival

less common intraductal tubular neoplasm of the bile duct (ITNB), based on the proportion of papillary and tubular components of the tumors. This section focuses on IPNB, which is more common as well as being associated with O. viverrini infection [69, 91]. IPNB is strongly associated with intraductal growth type of CCA and the papillary–polypoid type of perihilar and distal CCA (Fig. 7.22). These tumors might be the same pathological entities with different locations [69, 92]. Intraductal papillary neoplasm of the bile duct (IPNB) has some specific features which should be considered in the planning for hepatic resection. The unique features of IPNB include multiplicity [76], presence of skip lesions, slow progression, various degrees of mucin-producing a low proportion of lymph node involvement [69, 92, 93], presence of synchronous papillary lesions of gallbladder or pancreas [76, 94], and frequent intrabiliary recurrence after resection. These indicate that IPNB requires some specific pre-operative planning and operative procedure which differ from conventional cholangiocarcinoma operative procedures. Since IPNB arises from peribiliary glands which distribute along the biliary tree, gallbladder, and pancreas, it usually is found as multiple lesions [76] (Fig. 7.23), making achievement of R0 resection very difficult. Most of the

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Fig. 7.22 Surgical specimen of perihilar (a, b) and distal (c, d) cholangiocarcinoma comparing papillary–polypoid (a, c) and flat-nodular infiltrating (b, d) types. a) Papillary–polypoid tumor at perihilar region (asterisk), the tumor grows inward the bile duct lumen which corresponds to intraductal growth type of iCCA. b) Flat-nodular infiltrating tumor at perihilar region, the tumor infiltrating around bile duct wall (arrowhead), corresponds to periductal infiltrating type of iCCA. c) Papillary–polypoid tumor at distal common bile duct (triangle). d) Area of infiltrating tumor at distal common bile duct (framed)

IPNB has micro-papillary lesions extending from the main lesion across the resection margin. It is crucial to identify all lesions at the pre-operative period. If the surgeons suspect IPNB with multiple lesions, good-quality cholangiography is required, to determine the optimal resection point. The most effective way to determine the extension of the IPNB, from our experience, is cholangioscopy (choledochoscopy), either intraoperatively or per-orally. Most of the micro-papillary lesions missed by pre-operative imaging are usually detected by

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cholangioscopy (Fig. 7.24). Hence, decisions of the transection point of the bile duct should be finalized after cholangioscopy. Similar to other types of iCCA, the presence of lymph node metastases results in poor prognosis of the patients with IPNB [69]. As mentioned earlier, lymph node dissection might provide some survival benefit in patients with a limited number of lymph node involvement. Since IPNB is a slowly progressing tumor, we have found a limited number of lymph node involvement cases at the time of operation. However, routine lymph node dissection is not recommended in all IPNB patients, because the overall rate of lymph node involvement is quite low,

Fig. 7.23 Computed tomographic findings of the patient with diffuse papillary lesions of the biliary tree and the gallbladder in axial (a) and coronal section (b). The papillary lesions arising along the distribution of peribiliary glands, including intrahepatic, extrahepatic bile duct, and the gallbladder

Fig. 7.24 Pre-operative magnetic resonance imaging (a) and choledochoscopic findings (b) of the patient with simultaneous cystic IPNB and IPMN-P. a) Branch duct IPMN-P of the pancreas (asterisk) and cystic-biliary dilatation at right posterior section of the liver (arrowhead) were depicted. The common bile duct wall was smooth and appears normal (arrow). b) Diffuse micro-papillary lesions of the common bile duct found on choledochoscopy

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making unnecessary dissection in node-negative patients. Therefore, to predict the nature of the tumor and the rate of lymph node involvement, we have established a morphological classification of IPNB according to its morphology on pre-operative imaging divided into five common subtypes [92, 93], namely, (i) intrahepatic intraductal lesion (i.e., presence of an intraductal tumor with unilateral intrahepatic duct dilatation), (ii) extrahepatic intraductal lesion (i.e., presence of an intraductal tumor with bilateral intrahepatic duct dilatation), (iii) cystic variant (i.e., cystic tumor with a papillary tumor inside and the presence of bile duct communication), which has a radiological picture similar to a hepatic mucinous neoplasm, (iv) micro-papillary lesion (i.e., disproportional bile duct dilatation in the absence of any discernible tumor), and (v) macro-invasive IPNB (i.e., presence of a massforming tumor incorporated with intraductal tumor). For the later classification type, presence with the highest chance of lymph node involvement would provide some survival benefit following routine lymph node dissection [95]. One of the most difficult situations of IPNB is biliary papillomatosis involving both lobes of the liver, making resection of all tumor impossible. The ideal treatment for biliary papillomatosis is liver transplantation [94], but it is contraindicated when the tumor invades and metastases to the lymph nodes. To ensure the absence of invasive focus or lymph node metastasis at the time of liver transplant, parts of the tumor should be obtained and examined pathologically. There has been a study that has recommended conducting partial hepatectomy and lymph node sampling first and examination of the tumor to ensure non-invasive nature followed by liver transplant. This approach, however, has limited success because of some limitations, namely, (i) most IPNB is malignant at the time of presentation of patients, (ii) shortage of liver donors, and (iii) the result of this approach has not currently been replicated or validated. Nevertheless, we still recommend performing partial resection of the liver with more tumor burden/invasive first to remove the most threatening parts affecting patients. Owing to the slow progressive nature of IPNB, the remaining foci which at times consist of non-invasive types, the slow growing part in the liver remnant might take a period of time to cause problems to the patients.

7.4

Part IV: Surgery for Perihilar Cholangiocarcinoma

7.4.1

Definition of Perihilar Cholangiocarcinoma

Cholangiocarcinoma has several classification systems according to gross pathology, histology, or location of the tumor. The classification of CCA according to tumor site and invasion are the most relevant to surgical treatment consideration. Perihilar CCA was first described in 1965. Dr. Gerald Klatskin described clinical and pathologic features of 13 patients with adenocarcinoma at the porta hepatis that are often overlooked during laparotomy. The cause of death in these patients was liver failure or infection and proposed palliative internal bypass surgery for treatment [96].

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Fig. 7.25 A: perihilar cholangiocarcinoma, the mass was originated at hilar bile duct B: intrahepatic cholangiocarcinoma with hilar invasion, the mass was centered in the liver and had periductal extension to hilar bile duct

Cholangiocarcinoma was classified into intrahepatic and extrahepatic types until Nakeeb et al. [97] reported survival outcomes of CCA after resection and classified CCA into three types, intrahepatic, perihilar, and distal. Intrahepatic tumor was defined as the tumor that was confined in liver parenchyma without involving the extrahepatic bile duct, perihilar tumor was defined as the tumor that was involved or required resection of the hepatic duct bifurcation, and distal tumor was defined as the tumor that involved the distal bile duct, intrapancreatic bile, and amendable to pancreaticoduodenectomy. Nakeeb’s definition of perihilar CCA included two main categories of tumor, namely intrahepatic tumor involving hepatic duct bifurcation and extrahepatic tumor which originated at the bile duct bifurcation. Most intrahepatic tumors with hilar involvement have been found in a more advance stage with poor prognosis compared to extrahepatic tumors. In 2014, Ebata and colleagues restricted the definition of perihilar CCA to tumors that were located at the hilar bile duct, namely, the duct located topologically between the right side of the umbilical portion of the left portal vein and the left side of the origin of the right posterior portal vein. If a tumor was of a significant mass, the center of the mass had to be located between these landmarks (Fig. 7.25) [98].

7.4.2

Assessment of Curative Resectability

Perihilar CCA was classified according to longitudinal extension of tumor into four types by Bismuth 1975 (Table 7.2) [99]. Bismuth classification has been used to select the most appropriate procedure for patients to achieve margin negative resection. For Bismuth type IIIa, the procedure should be performed which is right hepatectomy with resection of the caudate lobe and bile duct, while left hepatectomy with caudate lobe and bile duct resection performed for Bismuth type IIIb. Bismuth type I and II right hepatectomy

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Table 7.2 Bismuth classification of perihilar cholangiocarcinoma Bismuth classification

Bile duct extension

Type I

Tumor involved the common hepatic duct, but did not involve the hepatic duct confluent

Type II

Tumor involved the hepatic duct confluent, proximal left and right hepatic ducts

Type IIIa

Tumor extended to the proximal right posterior duct and proximal right anterior duct and did not involve the left side

Type IIIb

Tumor extended to the proximal segment 4 duct and did not involve the right side

Type IV

Tumor extended to both sides, right posterior duct, right anterior duct, and segment 4 duct

with caudate lobe and bile duct resection should be undertaken, if possible, to assure that bile duct margins are free from tumor, while limited bile duct and caudate lobe resection without major hepatectomy can be performed in selected cases. Right or left trisectionectomy should be performed for Bismuth type IV depending on which side that tumor dominates. Since the information of bile duct extension is important, careful evaluation of bile duct extension is crucial in pre-operative planning for perihilar cholangiocarcinoma. Assessment of bile duct extension by CT or MRI should be done before performing biliary drainage to identify duct thickening and contrast-enhancing walls. In the case of collapsed bile ducts, accurately identifying the boundary of tumor is extremely difficult. Care must be taken for intrahepatic CCA as most of tumors have periductal invasions or intraductal spreading and therefore should be carefully checked for hilar invasion. If tumor invasion is beyond the umbilical fissure or right posterior duct, then operations should be planned following procedures for perihilar CCA. Cholangiograms provide important information about the anatomy of bile duct variations and longitudinal extension of tumors which enable accurate setting of the resection line of the bile duct margin. Cholangiograms can be obtained during pre-operative biliary drainage by either ERCP or PTBD. Stricture and irregular border seen in cholangiograms indicate the tumor-infiltrated area. MRCP is a less invasive procedure that also enables reconstruction of 3D images that form the basis of planning the operation, which have gradually replaced the role of conventional cholangiograms. Peroral cholangioscopy is another useful procedure to identify mucosal spreading that can be missed from cholangiograms, CT, or MRI. Peroral cholangioscopy performed via the endoscopic route and retrograde introduction of a small scope through the ampulla of Vater provide direct visualization of the biliary tract epithelium. Tissue biopsies can be taken from peroral cholangioscopy from suspicious lesion, but a major current limitation is that the scope cannot pass obstructions in bile ducts to assess the proximal margin of tumor (Fig. 7.26).

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Fig. 7.26 Images from spyglass peroral cholangioscopy of perihilar cholangiocarcinoma type IIIa, A–B Papillary mucosal spreading tumor of right intrahepatic bile duct to hilar bile duct, C Normal left bile duct mucosa

Bismuth classification and longitudinal tumor extension alone cannot evaluate resectability for perihilar CCA. Radial tumor extension, vascular invasion, and other factors should be included in any consideration. Jarnagin et al. proposed the criteria of unresectability for perihilar CCA which consisted of three major categories [100]. The first category was a patients’ factor, for instance, medically unfit for major surgery and liver cirrhosis were contraindicated. The second category was evidence of any distant organ metastasis including para-aortic lymph node, celiac lymph node, liver and other distant organs were contraindicated. The third category was local tumor-related factors that prohibited curative resection in perihilar CCA, including tumor extension to secondary bile duct bilaterally or Bismuth type IV, tumor encasement of the main portal vein, lobar atrophy with contralateral portal vein involvement, lobar atrophy with contralateral secondary bile duct extension, and Bismuth type IIIa or IIIb with contralateral portal vein involvement. With recent major improvements of surgical techniques in liver transplantation and development of perioperative treatment, more radical curative surgery can be performed with low mortality and achieving a good survival outcome. Vascular resection and reconstruction have been applied for tumors with vascular invasion and trisectionectomy for Bismuth type IV tumors. Jarnagin’s criteria of unresectability in local tumor-related factors have become less applicable in current surgical procedures. Kuriyama et al. [101] recently published a proposed resectability classification of localized perihilar CCA focused on biliary factors and vascular factors. Resectable tumors were defined as curative resection obtained from the free bile duct margin without the need for vascular resection, and borderline resectable tumor was defined as curative resection obtained from the free bile duct margin requiring vascular resection with safe reconstruction. Unresectable tumor was defined as curative resection which cannot be obtained from the free bile duct margin and/or safe vascular reconstruction which cannot be performed [101]. Local tumor-related factors are an issue for consideration in the pre-operative planning by the multidisciplinary team, such as patient and tumor factors as well as the team’s previous and ongoing experience and practical expertise.

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Surgical Technique

Because of complex anatomy, variations of bile ducts, vascular anatomy, local tumor invasion, curative resection for perihilar CCA have become the one of most complex surgeries in the hepatobiliary field. Curative surgery for perihilar CCA involves major liver resection with total caudate lobectomy, regional lymphadenectomy, extrahepatic bile duct resection, biliary-enteric reconstruction, combined vascular resection, and reconstruction. The primary goal of an operation is to achieve negative surgical margin, adequate removal of lymph nodes with lowest post-operative morbidity. The following sections of this chapter will describe steps and points of consideration for perihilar CCA curative surgery.

7.4.3.1 Lymphadenectomy and Extrahepatic Bile Duct Resection Complete examination for distant organ metastases should be carried out by diagnostic laparoscopy or laparotomy. Celiac lymph nodes and para-aortic lymph nodes should be palpated, and any suspicious metastatic lymph node should be sent for frozen specimen sectioning and pathological examination. Peritoneal seedings should be carefully checked at the cul-de-sac, mesentery, omentum and the dome of the diaphragm. If distant metastases have been confirmed, major liver resection must be aborted. A full Kocher’s maneuver and hepatoduodenal ligament dissection should be performed to identify vascular structure and tumor extension. At this step, the distal common bile duct should be divided at the intrapancreatic part and the bile duct stump has sent for pathological examination to assure a free margin. All regional lymph nodes, including common hepatic nodes (No. 8a, 8p), pericholedochal nodes (No. 12h, 12c, 12b), periportal nodes (No. 12a, 12p), and posterior pancreaticoduodenal nodes (No.13a), should be removed with all lymphatic tissue and sent for pathological examination. The portal vein and hepatic artery should be divided after all named vessels have been identified to prevent accidental injury by misidentification. All portal vein branches of the caudate lobe should be divided in this step. After finished lymphadenectomy and divided inflow vessels, liver should be mobilized; then, all short hepatic vein branches from caudate lobe to IVC should be divided to free the caudate lobe from IVC and proceeded to parenchymal transection. The proximal bile duct or intrahepatic bile duct margin should be divided after parenchymal transection has been completed to obtain a maximum margin. 7.4.3.2 Liver Parenchymal Transection Parenchymal transection is the most important step in hepatectomy as most of blood loss occurs at this step of surgery. Difficulties encountered at this step can prolong operation time, surgeon’s stress, lead to massive blood loss, and post-operative complications. Key principles to perform safe and easy parenchymal transection are (1) good planning and setting the landmark of the plane, (2) lowering central venous pressure (CVP), and (3) experienced techniques and instrumentation.

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Planning

Planning the resection plane and setting the landmark of the plane by using pre-operative CT/MRI or 3D simulations can provide a guide for the surgeon to perform transections with confidence and prevent accidental vascular injury. The surgeon should plan the resection plane to have at least 1 cm margin from the tumor with adequate hepatic venous drainage of remnant liver. Intraoperative ultrasound should be performed to identify the margin and vascular structures accordingly as dictated by the pre-operative plan. As the parenchymal resection plane is not in a straight line, we use three anatomical landmarks as reference points to prevent the surgeon losing direction during transection. The three anatomical landmarks used are the demarcation line, middle hepatic vein, and the finish line. 1. Demarcation line The first reference point is the demarcation line which occurs after the occluded or divided portal vein of the future resected liver. The parenchymal transection can be started from this line until small branches of the hepatic vein are found. The dissection plane proceeds along the branches of hepatic vein until the major branch has been exposed. 2. Middle Hepatic vein For left hepatectomy and right hepatectomy, the middle hepatic vein (MHV) is used as the second reference point. The right hepatic vein (RHV) is utilized as the second reference point for left trisectionectomy. The transection plane proceeds along the left side of MHV during left hepatectomy and the right side of MHV during right hepatectomy. Following division of the major hepatic vein branches of the resected liver, the transection plane proceeds in the direction from MHV to the finish line. 3. Finish line The last reference point or finish line for left hepatectomy and right hepatectomy with caudate resection is the ligamentum venosum. The finish line for right hepatectomy and left hepatectomy with caudate resection is the right border of the inferior vena cava. Parenchymal transection from MHV to the finish line results in deep poor exposure and bleeding. We have applied a liver hanging maneuver to facilitate this step.

7.4.3.3 Liver Hanging Maneuver (LHM) Jacques Belghiti described liver a hanging maneuver for anterior approach right hepatectomy in 2001 [102]. Anterior approach hepatectomy is performed during liver parenchymal transection without liver mobilization use in cases of large tumor or where tumors are involved in the retroperitoneal tissue. Utilizing the anterior

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approach has benefit in preventing tumor rupture, preventing systemic dissemination from tumor squeezing, and preventing hemodynamic instability from IVC compression or remnant liver pedicle torsion during mobilization of the liver. Disadvantages of the anterior approach technique are poor exposure and bleeding during transection in the deep part of liver parenchyma. LHM can facilitate this step by passing tape into the space anterior to retrohepatic IVC to hang the liver. The hanging tape provides better exposure, and it is easier to control bleeding and to guide transection direction. At 10–11 o’clock of IVC, the avascular space along the anterior surface of IVC allows surgical dissection to pass the hanging tape through this space. However, attempting LHM has a risk of subsequent bleeding 2–7% [103, 104], with the most common cause being the breaking of the liver capsule which is easily controlled by compression. Major bleeding may occur from the hepatic vein or IVC injury if the surgeon dissects in the wrong direction. The anatomy of IVC is that it is slightly tilted to left and upward in cranial side. Cases that are contraindicated for LHM involve a tumor in the retrohepatic IVC or dense adhesion obliteration of the avascular space. LHM has been adapted and applied for living donor transplantations and various types of anatomical liver resection by changing the entry and exit positions of the hanging tape to different spaces between the Glisson’s pedicle and the hepatic vein (Figs. 7.27, 7.28 and 7.29) [103]. Fig. 7.27 Hanging tape placement between MHV and right hepatic vein and along ligamentum venosum

188 Fig. 7.28 Hanging tape was relocated to above left hepatic duct

Fig. 7.29 Complete parenchymal transection, left hepatic duct was divided in last step to avoid contamination and achieve greatest length margin

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7.4.3.4 Low CVP Hepatectomy Parenchymal transection without bleeding is the key to perform good hepatectomy outcomes. Limited or no blood loss in this step allows for good visualization of the operative field, reducing a surgeon’s stress, faster operation time and reduces the risk of post-operative complications. The source of bleeding has been found to be mostly from back bleeding from the hepatic vein; therefore, the pressure in central vein directly controls the flow of bleeding. CVP should be as low as 4–5 mmHg during parenchymal transection. Low CVP can be achieved by anesthesiologic management, vascular inflow, and outflow occlusion techniques. Anesthesiologic Management

1. Reverse Trendelenburg position A reverse Trendelenburg position of the body during parenchymal transection can lower CVP to 3–4 mmHg. A study from Japan has reported CVP during hepatectomy compared to the supine position of a 10° reverse Trendelenburg position and infra-hepatic IVC clamp were 8 cmH2O, 5.6 cmH2O, and 5 cmH2O, respectively. There were no significant changes recorded in hemodynamics of the reverse Trendelenburg group; however, systolic blood pressure was found to be lower in IVC clamp group [105]. 2. Perioperative and intraoperative fluid restriction. 3. Reduced intrathoracic pressure achieved by setting the respirator to decrease airway pressure and turning off PEEP. 4. Diuretics may be considered if the CVP before parenchymal transection is higher than 8–10 mmHg.

7.4.3.5 Vascular Inflow and Outflow Occlusion A total hepatic pedicle clamp or Pringle’s maneuver is the most popular vascular inflow occlusion technique. Total hepatic pedicle clamping to occlude the portal vein and hepatic artery inflow results in good bleeding control but may result in ischemic injury to the remnant liver. A randomized control trial in 1999 examined the time and interval of Pringle’s maneuver during liver resection by comparing between the use of an intermittent clamp (15 minutes clamp, 5 minutes reperfusion) and a continuous clamp until complete resection [106]. The total ischemic time was found to be equal in both groups at 40 minutes, and there was no significant difference in overall blood loss. Cirrhotic patients in the continuous clamp group had liver failure after surgery with deaths of 5–10%. Therefore, during surgery, we perform the intermittent Pringle’s maneuver to control inflow in both cirrhotic and normal livers. Outflow occlusion during parenchymal transection reduces bleeding by block backflow from hepatic vein. Hepatic vascular exclusion is performed by clamping the supra-hepatic IVC and the infra-hepatic IVC and is an effective outflow

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occlusion method providing complete blockage of back flow; however, there is a higher risk of hemodynamic instability and post-operative complications [107]. The isolated infra-hepatic IVC clamp has been found to be the better method in terms of safety compared to hepatic vascular exclusion. Infra-hepatic IVC clamp effectively reduces CVP and blood loss compared to Pringle’s maneuver, but there is a higher risk of hemodynamic instability and pulmonary embolism [105, 108, 109] We therefore employ anesthesiologic management to lower CVP which is routinely performed with an intermittent Pringle’s maneuver. We apply an infrahepatic IVC clamp in very selected cases, such as, when tumors are involved or enclose the IVC.

7.4.3.6 Biliary-Enteric Reconstruction Biliary-enteric anastomosis after resection of perihilar cholangiocarcinoma is performed by hepaticojejunostomy (HJ) is the last step of operation that required meticulous surgical technique to prevent bile leakage and anastomosis stricture which may lead to serious complication and mortality. HJ anastomosis of remnant bile duct is often multiple and small segmental duct which is high risk for stricture. Biliary ductoplasty was performed to make multiple small orifices to single large bile duct orifices by separate bile duct wall or septum and reapproximate with interrupted PDS 5–0 (Fig. 7.30). We have always performed HJ anastomosis by the interrupted parachute technique with fine absorbable suture. Posterior wall anastomosis was performed firstly at the corner and serially placed suture without tying knot until completed suture posterior wall. Anterior wall anastomosis was performed after completed posterior wall. This technique provides good exposure for ensuring duct to mucosa in small bile duct anastomosis and easy to practice (Fig. 7.31). Trans-anastomosis external stent tubes can be placed in difficult anastomosis cases, such as small fragile or torn bile duct wall while suturing to reduce bile leakage risk should be performed.

Fig. 7.30 Biliary ductoplasty technique

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Fig. 7.31 Parachute technique for biliary-enteric hepaticojejunostomy anastomosis

7.4.3.7 Vascular Resection and Reconstruction Vascular invasion has previously indicated unresectable locally advanced disease. With advancement of surgical techniques and increased experience obtained from liver transplantation, CCA with vascular invasion has become resectable in selected cases. The classification of vascular invasion is based on considering the longitudinal extension of disease. Hence, portal vein invasion has been classified into PV1 to PV4 and arterial invasion into HA1 to HA4 [110]. Portal Vein Resection

Portal vein resection and reconstruction are now a routine procedure for perihilar tumors with portal vein invasion. The extension of portal vein invasion should be assessed by pre-operative CT/MRI and completely assessed again in the operative field. Portal vein resection should be performed only in cases where tumor cannot be freed from the portal vein during hilar resection. En bloc resection of tumors with portal vein without dissection (No-touch technique) can lead to unnecessary portal vein resection, and results of this operation are a high post-operative mortality of 16% [111]. Portal vein resection should be performed as a last step of the operation after complete parenchymal transection and division of hepatic ducts. After resection of the portal vein, the liver specimen can be removed, and portal

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vein reconstruction can be performed easier with good exposure. Most portal vein reconstructions can be performed by primary anastomosis without tension. Vein grafts are rarely needed for reconstruction. Care must be taken during right hepatectomy with portal vein resection as the portal vein anastomosis can be twisted after reconstruction. Marking of the reference point of the main portal vein and the left portal vein can prevent anastomosis torsion. Results of perihilar CCA resection with combined portal vein resection have been reported in several large cohorts. Ebata et al. have reported results of surgery in 574 perihilar cholangiocarcinoma patients and found that combined portal vein resection performed in 35.9% of cases had 6% mortality. There was no significant difference in the absent vascular resection groups [112]. Higuchi et al. reported results of combined vascular resection with curative resection in perihilar cholangiocarcinoma and found mortality rates in combined hepatic artery and portal vein resection of 16% and 5.4%, while absent vascular resections had only 1.7% mortality. The 5-year survival rate was poorer in portal vein resection 35.6% compared to 53.4% in the non-vascular invasion group; however, portal vein invasion with residual tumor (R1) had no chance of a 5-year survival [113]. We have published results of surgery in 153 perihilar CCA patients in 2015, where we showed that combined vascular resection which was performed in 7.8% of cases had no significant difference in survival outcome [70]. Hepatic Artery Resection

Previously combined hepatic artery resection is contraindicated due to a high postoperative mortality. Recent advances in the past decade in surgical techniques which have been applied in liver transplantation have led to increased usage in the combination of hepatic artery resection and reconstruction in many medical centers. Post-operative mortality as reported by Miyazaki et al. 2007, Nagino et al. 2010, and Higuchi et al. 2019 was found to be 33%, 2%, and 16%, respectively [113–115]. The 5-year survival rate for perihilar CCA with combined vascular resection was 30.3% which was a significantly poorer rate compared to cases without vascular resection of 47.6%, but better than unresectable cases where no patients survived for more than 3 years [114]. From current evidence, combined hepatic artery resection is still controversial and should be performed only in selected cases by highly experienced surgical teams with meticulous perioperative care. In our medical institute, combined hepatic artery and portal vein resection have been performed in several cases with acceptable outcome (Figs. 7.32 and 7.33).

7.4.4

Left and Right Trisectionectomy

Perihilar CCA with extensive bile duct extension up to the secondary branch of hepatic duct has been classified as Bismuth type IV and previously defined as unresectable locally advanced disease. Curative resection of Bismuth IV tumors can be performed by extended surgery to the right trisectionectomy (right hepatectomy

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Fig. 7.32 Intraoperative photography of left hepatectomy, caudate lobectomy with vascular resection. Transhepatic hilar approach was performed, and portal vein was reconstructed by end-to-end anastomosis (PV). Tumor involved right hepatic artery was shown (*). (HA; hepatic artery proper, LHA; left hepatic artery)

including segment 4) for right side predominate tumors and left trisectionectomy (left hepatectomy including segment 5 and segment 8) for left-side predominate tumors. Meticulous pre-operative plans should be made to achieve a negative bile duct margin, adequate liver remnant volume, safe vascular reconstruction, and safe biliary reconstruction (Figs. 7.34 and 7.35). Outcomes of right trisectionectomy were reported to achieve a negative margin of more than 80% and a 5-year survival rate was 27%–64% [116, 117]. Furthermore, outcomes of left trisectionectomy achieved a negative margin more than 72%–85% and a 5-year survival rate ranging between 39 and 46% [118, 119]. Left and right trisectionectomy, although complex surgery, can be performed to increase a negative margin and provide a longer survival outcome.

7.4.5

Hepatopancreatoduodenectomy (HPD)

This surgery combines major hepatectomy with pancreaticoduodenectomy, and it is performed as a treatment of perihilar tumor with extensive longitudinal spreading to distal bile ducts. HPD is a complex procedure which was first performed in Japan. Nimura et al. (1991) reported the findings of HPD for 24 patients with advanced bile duct cancer which resulted in a high operative mortality of

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Fig. 7.33 Completion photography of left hepatectomy with hepatic artery and portal vein resection. After complete arterial anastomosis and biliary-enteric anastomosis. (HA; hepatic artery, PV; portal vein, IVC; inferior vena cava)

12.5% and poor survival with a 2-year survival rate of only 17.9% [120]. Currently, surgical techniques and perioperative care have greatly improved, resulting in a decreased operative mortality and achievable long-term survival. For instance, Ebata et al. (2012) conducted HPD in 85 patients with CCA resulting in mortality of 2.4% with a 5-year survival rate of 37.4%. Shimizu et al. have recently reported a mortality after HPD for CCA patients of 5.4% and a 5-year survival of 37%. HPD still resulted in morbidity even in a highly experienced medical center with post-operative liver failure of 45%–75% and pancreatic fistula of 30%–70% [121, 122]. Results of HPD from Western countries are also not impressive, with operative mortality of 26%, morbidity of 87%, and liver failure 56% [123]. Hence, for long-term outcomes in locally advanced CCA, HPD has been shown to have high morbidity, especially liver failure. HPD should be considered for treatment in selected patients with adequate liver volume and function. The specimen and operative field after completed HPD for extensive perihilar CCA in Srinagarind Hospital, Khon Kaen University, Thailand, are shown in Figs. 7.36 and 7.37.

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Fig. 7.34 Pre-operative imaging of Bismuth IV perihilar cholangiocarcinoma with right side predominate. CT scan showing thickening of bile duct with contrast enhancement from segment 4 branches extended to right anterior duct (arrows), segment 2, 3 ducts were free

7.5

Part V: Surgical Treatment in Distal Cholangiocarcinoma

The principle of surgical treatment in patients with this type of cancer is the same as for cancer surrounding the ampulla of Vater, where all nearby tissues are removed (R0 resection). The first successful two-stage pancreaticoduodenectomy was performed by a German surgeon, Kausch (1912). However, this operation was previously well known by Allen Oldfather Whipple who reported a case series of pancreaticoduodenectomy in 1935 [124]. Since 2000, pancreaticoduodenectomy has been reported to improve morbidity and mortality, where recently many centers have reported operative mortalities of less than 5% [125].

7.5.1

Assessment of Resectability

According to the international consensus on the definition of resectable pancreatic ductal adenocarcinoma in 2017, we can assume the same principle of the criteria for resectability [126]. Our resectable cancers are defined as no distant metastasis nor major arterial involvement, with portal vein and superior mesenteric vein contact of less than 180 degree.

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Fig. 7.35 Photography of operative field after complete right trisectionectomy for perihilar cholangiocarcinoma. (HA; common hepatic artery, LHA; left hepatic artery, LPV; left portal vein, B2; segment 2 bile duct, B3; segment 3 bile duct)

7.5.2

Arterial Approach Pancreaticoduodenectomy

The arterial approach PD was first proposed by Nakao in 1993 which is also known as the mesenteric approach [127]. The arterial approach has been demonstrated in six directions by various surgeons [128]. Although currently no randomized comparative studies have been conducted, the non-randomized evidence shows longer survival using the arterial approach PD.

7.5.3

Extended Lymphadenectomy

In general, lymphadenectomy in periampullary cancer is the removal of lymph node stations 8, 12, 13, and 17. Lymphatic spreading can involve lymph nodes

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Fig. 7.36 This patient had pre-operative biliary drainage by metallic stent and performed right HPD. The specimen showed longitudinal spreading tumor from hilar to distal common bile duct (arrows) Fig. 7.37 Photograph of operative field after complete hepatopancreatoduodenectomy operation (MHV: middle hepatic vein, IVC: inferior vena cava, LPV: left portal vein, P: pancreatic stump)

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stations 9, 14, 15, and 16. In last decade three randomized studies have shown that there is no significant benefit in survival rate among extended and standard lymphadenectomy [129].

7.5.4

Resection Part

The patient is placed in supine position with the surgeon standing on the right side of patient. We suggest an inverted L incision for proper exposure; however, midline or bilateral subcostal incision can also be performed. The upper midline incision is started from the Xiphoid to 1 cm above the umbilicus, and a horizontal incision is made to the right anterior axillary line. The abdominal muscle is meticulously opened layer by layer. There are several self-retractors that have been used, namely, a bar-type retractor such as the Rochard retractor or Kent retractor, and a Frametype retractor, such as the Thompson retractor or Omni retractor. Intraoperative assessment is begun from top to bottom. All the peritoneal cavities are visualized and palpated for any small metastases. The entire liver is palpated and combined with intraoperative ultrasonography; the liver is assessed for the presence of metastases not seen by pre-operative imaging studies. The surrounding pancreatic head tissue is assessed for local resectability. The hepatic flexure colon is taken down and an extensive Kocherization is performed by mobilizing the duodenum, pancreas, the surrounding soft tissue, and lymph nodes (station 13) out of the retroperitoneum. This allows visualization of the inferior vena cava, left renal vein, and superior mesenteric artery at its origin with the aorta (Fig. 7.38). An arterial approach is commenced by using cauterization dissected from the root of superior mesenteric artery craniocaudal direction. The dissection of all soft tissues along to the right lateral aspect of the SMA is performed to maximize the negative oncological margin of the surgical resection. This surgical plane should be dissected in its periadventitial plane from the cranial border of the origin of the

Fig. 7.38 Left: right posterior arterial approach proximal superior mesenteric artery from origin of the aorta just above of left renal vein. Right: inferior arterial approach distal superior mesenteric artery from mesentery just below pancreatic neck

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SMA to the caudal border of the uncinate process. During this step, care must be taken to avoid injury of replacing the right hepatic artery which may be present at the proximal part of superior mesenteric artery. If assessment reveals no evidence of local tumor extension to the SMA margin implying potential resectability, the greater omentum is opened and the right gastroepiploic vessels are ligated followed by ligation of right gastric vessels. Transection of gastric antrum or first part of the duodenum can be done by GIA stapler or hand-sewn. Hepatic artery lymph nodes (stations 8a and 8p) are dissected just above the pancreatic body and the common hepatic artery is encircled by a vessel loop. The hepatoduodenal ligament is skeletonized, allowing visualization of vasculature and collection of lymph nodes (stations 8, 12). The common bile duct is transected just above cystic junction and the gallbladder is removed. The gastroduodenal artery (GDA) is temporally clamped to determine that there are no variations in hepatic arterial flow, and the GDA is divided and ligated. The proximal jejunum is mobilized from ligament of Treitz into the upper abdomen, and small mesenteric vessels are sealed and ligated. The proximal jejunum is transected by a GIA stapler or hand-sewn. The pancreatic neck is dissected from the lower border to identify the superior mesenteric vein and its branches. The middle colic vein can either be dissected gently out of the way or ligated and transected. The superior mesenteric vein is encircled by the vessels’ loop. The retropancreatic neck dissection should be gently dissected anterior to superior mesenteric vein in the caudocranial direction until the plane is connected with the previous upper border pancreatic dissection. Transection of the pancreatic neck can be done by a sharp knife or energy devices; however, the pancreatic duct should be individually transected with sharp cut to avoid thermal injury. The portal vein, superior mesenteric vein, splenic vein, and its branches should be identified and controlled with vessel loops. The superior mesenteric vein is retracted to the left side of patient. The surgeon should carefully dissect all branches around SMV perform individual ligations and divide the inferior and superior pancreaticoduodenal vein. Care must be taken not to injure the J1 branch and inferior mesenteric vein. The specimen is retracted to right side of patient, and the soft tissue between the uncinate process and SMA is opened with electrocautery or sealing devices to expose the periadventitia of the anterior surface of the SMA until it connects to the previous dissection. There are always one or two inferior pancreaticoduodenal arteries which should be ligated. The specimen is now free from the major vascular system. If a tumor is involved in the SMV, the vein should be retracted to the right side of patient. The surgeon should gently dissect all branches around the SMV, and sometimes, the first jejunal vein and inferior mesenteric vein need to controlled and ligated. The soft tissue anterior surface of the SMA should be opened with electrical cautery on the left side of SMV. The lateral border of SMA should be meticulously dissected, and the inferior pancreaticoduodenal arteries have identified and ligated. Retroperitoneal tissues can be dissected cranially using a sealing device, until connected to the previous arterial dissection behind the splenic vein and caudally to the dissection developed behind the SMV at the level of the first

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jejunal vein. At this point, the specimen is completely separated from SMA and is only adherent to the vein. If the resected portion of the vein is less than 4 cm, then primary anastomosis is feasible. Complete liver and mesenteric mobilization can be facilitated by primary anastomosis. Venous resection is performed after temporally controling the portal vein, superior mesenteric vein, and splenic vein by atraumatic clamps. For complex venous anastomosis, which may take a longer reconstructive time, additional control of superior mesenteric artery can reduce small bowel congestion. An end-to-end portal anastomosis can then be performed, with a running 4–0 or 5–0 polypropylene suture (Prolene). At the end of the anastomosis, a knot is tied by leave a growth factor of usually about half of the portal diameter.

7.5.5

Reconstruction Part

There are several techniques for reconstruction after pancreaticoduodenectomy. Each technique has their specific technical and physiological benefits. One of the most common sequences of reconstruction is started with the pancreas followed by the bile duct and then the stomach. There are two main types of reconstructions that can be performed as single loop or Roux-en-Y. The issues and controversies concerning evidence of pancreatic and biliary reconstruction are still debated. We prefer to use the jejunum for a single-loop reconstruction, by bringing the jejunal end through the original hole behind the mesenteric vessels. The pancreatic stump is subsequently freed 2–3 cm from retroperitoneal tissue. A two-layer end-to-side duct-to-mucosa pancreaticojejunostomy is performed and constructed using 3–4 interrupted transpancreatic U-sutures with 3–0 monofilament synthetic absorbable sutures (PDS) to the seromuscular layer of the posterior wall of the jejunal stump. The return transpancreatic stitch is done as a horizontal mattress suture. The sutures conducted with needles are not tied and held separately with clamps. After completing the posterior outer layer, a small jejunal opening is created at the opening of the pancreatic duct. The inner layer of the duct-to-mucosa pancreaticojejunostomy usually consists of between 6 and 8 simple interrupted sutures with 4–0 absorbable monofilament (PDS). An internal pancreatic duct stent is routinely used, except for very large pancreatic duct. Once all ducts to mucosal sutures are tied, the outer anterior horizontal mattress sutures by using the previous transpancreatic U-sutures are placed and tied. A pancreatic anastomosis is now completed. The biliary anastomosis is simply performed with an end-to-side hepaticojejunostomy approximately 15 cm away from the previous pancreatic anastomosis. The anastomosis can be performed with both a series of single layer of interrupted absorbable sutures or continuous running absorbable sutures. There is usually no need for a biliary stent; however, if the patient has a previous pre-operative external biliary drain, this can be left in place. The enteric anastomosis is typically performed by bringing up the distal jejunum approximately 30–45 cm away from the previous biliary anastomosis

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Fig. 7.39 Classical single-loop Child’s reconstruction for pyloric preserved pancreaticoduodenectomy (left) and pyloric resected pancreaticoduodenectomy (right)

through the retrocolic incision to create a duodenojejunostomy in patients who have undergone pylorus preservation. In the case of pyloric resected pancreaticoduodenectomy, Braun’s gastrojejunal anastomosis can be performed by both hand-sewn or GIA stapler-assisted anastomosis with defunctioning jejunojejunostomy approximately 15 cm below the gastric anastomosis preventing bile reflux gastritis. Alternatively, Roux-en-Y reconstruction can be carried out using the same principles for Braun’s anastomosis (Fig. 7.39).

7.6

Part VI: Minimally Invasive Surgery in Cholangiocarcinoma

Minimally invasive surgery in hepatopancreatobiliary surgery has been performed first by laparoscopic cholecystectomy on September 12, 1985, by Prof Dr Med Erich of Boblingen [130]. In 1991, H Reich reported the first laparoscopic liver resection in benign liver disease [131]. Over three decades, laparoscopic surgery has progressed regarding new surgical devices and surgical techniques. Since 2014, the recommendations for laparoscopic liver resection have been reported from the second international consensus conference which is known as the Morioka consensus [132]. Laparoscopic liver surgery has been increasing rapidly and has become widely accepted to treat malignant diseases. It is important to have high-resolution camera systems and specific laparoscopic instruments to facilitate complex surgeries. Many surgeons prefer 3D camera systems which provide a better visual depth of field and allow easier complex reconstruction [133]. However, 2D systems with 4 K resolution provide a great

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magnification and facilitate precise dissections [134]. There are several energy devices which support specific tissue sealing and dissecting. The ultrasonic dissector, which is commonly known as a harmonic scalpel, has an ability to coagulate and cut in the same time [135, 136]. However, it should be used with caution because of the heat of the active blade which can create serious thermal injury. The advanced bipolar sealing system has a major advantage regarding coagulation and can seal vessels up to 7 mm in diameter [136, 137]. The ultrasonic aspirator is an instrument that combines ultrasonic energy with water irrigation and aspiration which create vessel sparing cavitation of the parenchyma of the liver [138].

7.6.1

Intrahepatic Cholangiocarcinoma

As mentioned previously, liver surgery is the only potentially curative treatment for treatment for intrahepatic CCA. The systematic reviews of small case series have reported that laparoscopic liver surgery has reduced blood loss and hospital stay compared to open surgery [139]. However, the advantage of open surgery is being able to perform lymphadenectomy [140]. Recently, Ratti reported oncological outcomes of 150 laparoscopic matched cases versus 150 open surgeries. The short-term results of laparoscopic surgery were shown to reduced blood loss, post-operative complications, functional recovery, and hospital stay, and there were differences between the length of surgery, surgical margin, number of retrieved nodes, and mortality. Long-term outcomes demonstrated that there were no significant differences of disease-free survival and nor any significant differences in overall survival between the two groups. Furthermore, the disease recurrence rate and modality of recurrences were similar [141]. The indication for hepatic resection in laparoscopic surgery is not different from open surgery; however, our recommendation is to select the candidate base on Iwate criteria [142].

7.6.1.1 Position and Port Insertions There are several positions that can be applied for laparoscopic liver surgery depending on tumor location and type of operations. We recommend two operative setups, first the French position where the patient lies in modified lithotomy and the surgeon stands between the legs of the patient, and second the supine position where most surgeons stand on the right side of the patient. Type of operation will be guided by the positioning of port sites. We prefer four to six ports and at least two twelve-millimeter ports. The positioning of trocar is demonstrated in Fig. 7.40. 7.6.1.2 Liver Mobilization We recommend standard liver mobilization for open surgery. However, some surgeons prefer mobilization after a complete anterior approach. For right lobe hepatectomy, we start separation of ligamentum teres and falciform ligament. The dissection is created along the right coronary ligament over the upper part of the right lobe of liver. Subsequently, an assistant elevates the right lobe of liver so

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Fig. 7.40 Liver surgery. Position and port insertion

that the surgeon can divide the right triangular ligament, dissect the posterior part of the liver, and visualize the inferior vena cava. Left lobe hepatectomy is commenced by separation of the ligamentum teres and falciform ligament followed by dissection along the left coronary ligament until complete division of the left triangular ligament.

7.6.1.3 Inflow Control and Outflow Control There are several techniques of inflow control that have been described previously. Here, we describe two types, namely, total control and selective control. Total control, which is known as the Pringle maneuver, can be prepared with both intracorporeal control or extracorporeal control [143]. In our experience, extracorporeal control can shorten the time to apply occlusion and remove occlusion. Preparation for extracorporeal control is achieved by opening the lesser omentum and encircling the hepatoduodenal ligament by surgical cord tape. The tape is brought out through a 5-mm abdominal incision opposite to the site to the liver resection and covered with a small rubber tube (Fig. 7.41). During parenchymal transaction, intermittent occlusion (15-min occlusion and 5-min release periods) can be performed [144]. Selective control can be performed by various techniques. For intrahepatic CCA, we suggest that individual ligation can be performed, although the Glisson pedicle approach is also acceptable for lesions far from hepatic hilum [145]. 7.6.1.4 Lymphadenectomy and Hilar Dissection Lymphadenectomy for intrahepatic CCA is a controversial issue, and we prefer systematic lymphatic dissection, commencing with dissection of the common hepatic artery nodes (lymph node station 8) in line above the pancreatic neck. The dissection proceeds along the anterior to the common hepatic artery and its’ branches. Complete lymphatic dissection of hepatoduodenal nodes (lymph node station 12) is meticulously undertaken and proceeds along the hepatic artery, portal vein, and bile duct. At this point, combined kocherization facilitated extended

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Fig. 7.41 Extracorporeal Pringle tape

lymphatic dissection of the retropancreatic node (lymph node station 13) is performed. In the case of left lobe CCA with enlarged left gastric lymph nodes, sampling node dissection can be added. During lymphadenopathy, individual ligation of the hepatic artery and portal vein are done simultaneously. Combined pre-operative and intraoperative assessments are very important to understand the variation of anatomy before ligation and transection of hepatic inflow. The hepatic artery can be done safely with double clips. Although the portal vein and bile duct can be controlled with double clips, some surgeons prefer an endovascular stapler for transection. However, our preference is to transect only the portal vein first, and followed by the hepatic bile duct after parenchymal transection.

7.6.1.5 Parenchymal Transection The demarcation line is visualized over the surface of liver after complete hemihepatic inflow control. Intraoperative ultrasonography is also used as a guide for deeper parenchymal transection to identify the margin of the tumor and the middle hepatic vein, which is known as the intrahepatic landmark-guided transection [146]. Recently, an ICG-camera system has proven useful to guide anatomical parenchymal transection from superficial to a deep margin. Intraoperative indocyanine green should be injected before transection with a dose of 0.1–0.5 mg/kg [147].

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In general, parenchymal transection is performed anteroposterior and in a caudocranial direction. Within a 2-cm superficial transection can be performed by any kind of device. Our center prefers to use a two hand instrument technique. One hand is used to hold the ultrasonic shearing or advance bipolar which facilitates transection and the sealing small vascular blood vessels and another hand using a 10-mm ultrasonic aspirator which allows precise dissection and preserves small vascular structures (Fig. 7.42). During deeper parenchymal dissection, lowering central venous pressure and intermittent occlusion of hepatoduodenal ligament is performed to reduced blood loss in the operative field, and allows better visualization of the middle hepatic vein and its’ branches. Selective hepatic vein branches are clipped and divided. Bleeding in this step always occurs from the hepatic vein branch temporally which compresses and increases intra-abdominal pressure which can minimize blood loss. Small holes that appear on the hepatic vein can stop bleeding without interference; however, larger holes need to be clipped or sutured. After complete anterior midline separation of the liver, the middle hepatic vein is clearly observed along the cut surface and drains into IVC. In laparoscopic right hepatectomy, the caudate lobe is dissected between the inferior vena cava and the right hepatic bile duct which allows better exposure for transection of the right hepatic bile duct. Short hepatic vein branches are clipped and divided along the anterior to inferior vena cava. The dissection continues upward until the right hepatic vein and IVC ligament are completely encircled. These structures should be secured with an endovascular stapler; however, double large hem-o-lok clips or sutures are also acceptable. In laparoscopic left hepatectomy, an Arantius ligament is divided, and the parenchymal dissection is performed along these grooves to encircle the left hepatic bile duct and expose the left hepatic vein trunk. We use an endovascular stapler to transect the left hepatic bile duct and left hepatic vein, respectively. In general, a large specimen can be removed under a covered bag through a 10-cm transverse suprapubic incision. A drain is placed in subphrenic space.

Fig. 7.42 Parenchymal transection

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Distal Bile Duct Cholangiocarcinoma

Several studies have demonstrated the feasible of minimal invasive surgery in this procedure; however, only a few randomized controlled laparoscopic pancreaticoduodenectomy trials (LPD) versus open pancreaticoduodenectomy (OPD) have been established. C Palanivelu reported short-term outcomes of a randomized controlled trial in a specialized high-volume center, with laparoscopic surgery shown to reduce blood loss and hospital length of stay. However, there were no significant differences of overall complications between both groups [148]. In 2019, a multicenter randomized controlled trial known as LEOPARD-2 trial was terminated early because of a significant higher 90-day post-operative mortality with laparoscopic surgery compared to open surgery [149]. Recently, a 14 multicenter trials in China randomly assigned (1:1) 762 patients and showed that LPD was associated with a shorter length of stay and similar short-term morbidity and mortality rates to OPD [150]. Ki Byoung Song reported that 87.8% of patients had complete oncological resection and over 60% had a 5-years overall survival in distal bile duct cancer. But, these results need to be confirmed [151]. Indication for laparoscopic pancreaticoduodenectomy is defined as the same as resectable pancreatic cancer in open surgery. However, we recommend that only the tumor without vascular involvement and distant metastasis can be considered.

7.6.2.1 Position and Port Insertions Currently, French and supine positions are commonly used, with the surgeon usually standing between the legs of the patient. Five to six ports are inserted as in Fig. 7.43. Two or three of these are 12-mm ports which provide the basis to switch the camera to another different views during the procedure. 7.6.2.2 Resection Phase The camera is placed through the sub-umbilical port. Complete intra-abdominal evaluation is performed. Transabdominal sutures are placed on ligamentum teres and the gallbladder. These expose a clearer view of the subhepatic area. The gastrocolic ligament and gastrohepatic ligament are divided and the transverse colon is taken down. To complete extended kocherization, the duodenum and pancreas are mobilized to the midline to expose the inferior vena cava and aorta. During kocherization, the third part of duodenum and duodenojejunal junction is dissected from the direction of the right side and the proximal jejunum is brought to the right side behind the mesenteric vessels. For pre-operative clearly resectable cases, early transection of first part of the duodenum or gastric antrum with an endoGIA stapler facilitated better exposure of further dissection. The hepatic artery node (lymph node station 8) is dissected just above pancreatic neck. The right gastric vessels are transected and the hepatic arterial branches are dissected freely. The gastroduodenal artery is controlled and transected. The upper and lower borders of the pancreatic neck are dissected and the retropancreatic neck dissection should be gently dissected anterior to mesenteric vein in the caudocranial direction until connected to the upper border. The

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Fig. 7.43 Pancreatic surgery port position

pancreatic neck is encircled with vessel loop. The proximal jejunum is transected with endoGIA stapler. An assistant lifts the duodenum anteriorly which allows the surgeon to dissect the jejunal mesentery and uncinate process from the superior mesenteric vein. The branches of gastrocolic trunk of Henle are individual clipped and divided. Precise dissection should allow preservation of the first venous branch of jejunum and the right colic vein. The pancreatic neck is transected with an energy device, but care must be taken when a scissor is used to cut the pancreatic duct. Assistants pull the specimen to the right and retracted SMV to the left, so that the surgeon can precisely dissect the uncinate process and perineural plexus from the superior mesenteric artery (SMA). The inferior pancreaticoduodenal artery and superior pancreaticoduodenal vein are then clipped and transected. The dissection should be cautious about raising of replacing the right hepatic artery. The common bile duct is dissected free from other vasculature and all hepatoduodenal lymph nodes (lymph node station 12) are collected. Cholecystectomy is taken down and the common hepatic duct is temporally clamped and transected. The specimen is collected in a retrieval bag. Any bleeding should be checked and stopped.

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7.6.2.3 Reconstruction Phase We prefer to use the jejunum for a single-loop reconstruction by bringing up the jejunal limb through the resected canal behind mesenteric vessels. The length of the jejunal limb is measured by the end of the stump and must be placed a little longer over the pancreatic cut surface without any tension. Our recommendation is to perform a bilioenteric anastomosis the first instance; however, many surgeons may perform pancreatoenteric anastomosis. The enteric anastomoses are marked at both locations to prevent anastomotic tension. A small enterotomy is made, 4–0 absorbable monofilament suture (PDS) is sutured from the right side of the bile duct, and the knot is tied from the outside. A continuous suture is made for posterior anastomosis from right to left. The anterior anastomosis is performed in the same fashion as the posterior and the knot is tied at the end on left side of the anastomosis. However, closure is performed posteriorly for small hepatic duct less than 8-mm interrupted. The anastomosis is checked on both sides by flipping the jejunal limb. The pancreatic stump is freed 2–3 cm from retroperitoneal tissue. A two-layer end-to-side duct-to-mucosa pancreaticojejunostomy is performed by using 1 or 2 interrupted transpancreatic U-suture with 3–0 monofilament synthetic absorbable sutures (PDS) to the seromuscular layer of the posterosuperior wall of the jejunal stump and the return transpancreatic stitched as a horizontal mattress suture. Subsequently, these sutures are tied. After completing the upper half of the posterior outer layer, a small jejunal opening is created at the opening of the pancreatic duct. The inner layer of duct-to-mucosa pancreaticojejunostomy usually consisting of 6–8 simple interrupted sutures with 4–0 absorbable monofilament (PDS) is performed. An internal pancreatic duct stent is routinely used, except for the very large pancreatic duct. Once the duct-to-mucosal sutures are tied, the lower half of posterior outer layer is constructed with two interrupted transpancreatic U-sutures and tied. The interrupted sutures are made anteriorly and tied, and the pancreatic anastomosis is complete. The gastroenteric anastomosis is typically performed by bringing up the distal jejunum approximately 30–45 cm away from the previous biliary anastomosis through a retrocolic incision to create a side-to-side gastrojejunostomy by endoGIA stapler-assisted anastomosis. Closure is by continuous suture with 3– 0 monofilament synthetic absorbable sutures (PDS). An alternative defunctioning jejunojejunostomy can be made approximately 15 cm below the gastric anastomosis to prevented bile reflux gastritis. The specimen is removed under the bag through a 6–8 cm transverse suprapubic incision. Alternatively, the incision can be made 8–10 cm over the upper midline of abdomen to remove the specimen and any unexpected events can also be restored.

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7.7

Part VII: Role of Liver Transplantation in Cholangiocarcinoma

7.7.1

Background

Surgical resection of CCA typically results in 5-year overall survival rate of about 25 to 45% [152–154]. However, for the vast majority of CCA, once diagnosed, results in the diseases are being beyond the curative stage. From Khon Kaen University hospital’s data, only 6% of patients in the outpatient clinic were operable and only in 3% can an R0 margin be achieved. Post-operative morbidity rates have been reported as high as 70% [68]. There are many patients who are inoperable due to the involvement of bilateral structures or underlying parenchymal liver disease (e.g., primary sclerosing cholangitis). Among these patients, liver transplantation is an alternative treatment which can also act as a cure. One major cause of CCA is primary sclerosing cholangitis (PSC). Approximately one-third of patients with PSC develop after PSC is diagnosed, especially in the first year. However, most patients with CCA may have other causes as well, such as choledochal cyst and parasitic infections. From our data, most cases of CCA are related with O. viverrini liver fluke infection due to eating behavior of raw, partially cooked or fermented freshwater fish [see previous Chapters] [155]. The outcome of non-surgical treatment for Hilar CCA is disappointing, and most patients survive for less than one year [68]. A key factor for favorable prognosis is complete resection, which occurs between 25 and 40% of cases. Even with well-selected cases, the chance of getting a 5-year survival rate is not more than 40%. Furthermore, there are no pre-operative or post-operative treatments which would clearly change the outcome of surgery. In addition, half of the patients have disease recurrence after curative-intent resection [49, 70].

7.7.2

Hilar Cholangiocarcinoma and Liver Transplantation

Orthotopic liver transplantation (OLT) was introduced in the 1980s as therapeutic option and a promising outcome of R0 margins in patients were not candidates for liver resection due to locally advanced disease, unresectable disease, and underlying liver diseases. Prior to 2005, liver transplantation outcomes in hilar CCA were very disappointing. In 1998 in Pittsburgh USA was the first reported a 5-year survival of 25% in the absence of any neoadjuvant therapy. As a result of the long waiting time for transplantation, Nebraska University proposed a 100% neoadjuvant concurrent chemoradiation therapy (CCRT) in unresectable hilar CCA by biliary brachytherapy via percutaneous transhepatic drainage and intravenous infusion of 5-Fluorouracil (5-FU) until transplantation showed a better 5-year survival of 33%. However, pre-2005 the neoadjuvant therapy views were still not clearly debated and strict selection criteria for liver transplantation were not applied. Later, both neoadjuvant CCRT and strict selection criteria were reported by the Mayo

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group with the modified neoadjuvant CCRT regimen, and they proposed strict selection criteria called “Mayo’s protocol” [156, 157], namely, 1. 2. 3. 4.

Patient selection. Neoadjuvant therapy. Operative staging. Liver transplantation.

1. Patient Selection a) Diagnosis: Pathologically confirmed hilar CCA. b) Or malignant appearing stricture + one of the following: c) Mass at the site of stricture on imaging. d) Serum CA19-9 > 100 ng/mL (no cholangitis events). e) Polysomy on fluorescent in situ hybridization (FISH). f) Radial tumor < 3 cm and vertical extension above cystic duct. g) Absence of intrahepatic or extrahepatic metastases on imaging by crosssectional imaging and exclude lymph node metastasis by endoscopic ultrasound guide biopsy or fine-needle aspiration. h) Resectable cancer in setting of PSC. i) Absent contraindication for chemotherapy or radiation therapy. j) Candidate for liver transplantation and no contraindication for solid organ transplantation. k) Age > 18 years and < 70 years. 2. Neoadjuvant Therapy: a. External beam radiotherapy (EBRT): Target dose 4500 cGy, 150 cGy twice daily and radiosensitizer chemotherapy: intravenous 5-FU (500 mg/m2 /day for 3 days). b. Transcatheter irradiation with iridium (2000–3000 cGy cover 1 cm radius margin) after EBRT concurrent with intravenous 5-FU (225 mg/m2 /day). c. Oral capecitabine (2,000 mg/m2 /day) divided in two doses and continued for two weeks and omit 1 week until liver transplantation. d. Tumor biology was tested by timing and neoadjuvant CCRT. e. Neoadjuvant CCRT complication included biliary tract infection, progression of disease and dead. 3. Operative Staging: a. Explore laparotomy for staging was performed after completion of neoadjuvant therapy for 2–3 weeks. Lymph node sampling and peritoneal washing for cytology were done for exclude extrahepatic metastases. b. About 30% had extrahepatic metastases from operative staging and were excluded from protocol. 4. Liver Transplantation a. Staging laparotomy for contraindication of LT. b. Total hepatectomy without hepatic hilum dissection, prevent tumor spreading. Frozen section for distal margin.

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c. Consider vein graft, e.g., iliac vein graft for LDLT, harvest longer portal vein graft for DDLT. d. Irradiated recipient hepatic artery with higher rate of hepatic artery thrombosis (HAT) and hepatic artery stenosis (HAS) in LDLT and consider iliac artery jumped graft for arterial reconstruction in DDLT. e. Roux-en-Y hepaticojejunostomy for all cases. Tissue diagnosis is the most difficult part of diagnosis due to low yields of brushing or biopsy for Hilar Cholangiocarcinoma (hCCA). To diagnose these patients, it is necessary to determine the presence of malignant appearing stricture by cholangiography and only one of the following criteria: (1) mass at the site of stricture, (2) CA19-9 > 100, (3) polysomy on fluorescent in situ hybridization replaced, pathological reports to include them in protocols, and some have no pretreatment tissue diagnosis. However, the absence of tissue diagnosis before treatment does not affect 5-year survival, except in PSC which has a higher incidence of benign strictures [158]. For strict selection criteria in Mayo’s protocol, this process selected patients with early-stage hCCA who are inoperable due to unresectable disease or arise in the PSC patients. Prior or attempted resection and transperitoneal biopsy were excluded from the protocol. This protocol denied iCCA, hCCA with more than 3 cm tumor in size and vertical extension below the joining of the cystic duct. Pancreatoduodenectomy combined with OLT is justified to reach R0 resection as there are no longitudinal limitations [157]. Hence, patients who have tumor extended below the cystic duct but still in early stage of disease by other criteria should not be excluded from the protocol. Vascular encasement is not a contraindication for liver transplantation. For neoadjuvant therapy, several European transplantation centers cautiously reported the results of patients who did not undergo neoadjuvant treatment and subsequent LT based on only the strict selection criteria for LT. They reported an acceptable 5-year overall survival for negative lymph node status between 50 and 59% [156] and that positive lymph node status was 0%. These results showed a poorer prognosis of lymph node status to patient survival and raised the question of successful outcomes of neoadjuvant treatment in the Mayo protocol. Nevertheless, a combination of neoadjuvant CCRT and strict selection criteria is recommended before LT.

7.7.3

Liver Transplantation Versus Liver Resection

Most of unresectable hCCA cases where an R0 margin cannot be achieved by LR have significant limitations and have the poorest outcomes. However, historical data of LT for unresectable hCCA in the Mayo protocol have shown that the Mayo protocol offers the best results of LT compared to several centers of the LR groups. Comparisons and discussions of LT and LR should also include resectable hCCA.

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Even in the most experienced centers, an R0 margin rate was only 70% for resection compared to 90% with LT [159]. Moreover, LR showed higher 90-day mortality due to the complexity of hepatectomy, lower future liver remnants, and vascular reconstruction. “Waitlist dropout” is the one main problem of the LT group which may differ in different countries or regions. Even though reducing the rate of the waitlist dropout by MELD exception point like HCC was given for hCCA, significant numbers of dropout have been reported to be still as high as 23.9% at 12 months [160]. Pre-operative managements of hCCA for LR are simpler with biliary drainage and/or portal vein embolization compared to the complex process of neoadjuvant CCRT (EBRT and brachytherapy) and laparotomy staging in LT. Ethun et.al from a retrospective review of LT versus LR in 304 patient from a US multicenter cohort comparing the same group of early-stage hCC have reported that with tumor < 3 cm and negative lymph node status, the LT group showed superior outcome of 5-year survival and overall survival of 64% vs. 31% p-value < 0.001, respectively. They also showed that a superior outcome was achieved after the exclusion of PSC patients with 5-year survival of 41% vs. 27%, p-value = 0.049. Nevertheless, there is discussion of why none of the vascular resections and reconstructions in LR group showed higher mortality in LR group with a 5-year survival rate in this study which was less than the previously reported 50–60%. This has raised the question of whether LT should be considered as the preferred treatment even for resectable hCCA [161].

7.7.4

Intrahepatic Cholangiocarcinoma and Liver Transplantation

From historical data nearly all retrospective studies of early 2000, intrahepatic cholangiocarcinoma (iCCA) with LT had poor results due to a high recurrence rate of 60–90% and poor long-term survival 5-yr OS 0–42%. Most cases of iCCA from these studies were diagnosed by “explant liver pathology” with previously diagnosed HCC in cirrhotic liver. Moreover, these poor outcomes have also been described in patients with mixed-hepatocellular-cholangiocarcinoma (HCC-CC) which have higher rate of microvascular and macrovascular invasion than HCC. Sapisochin et al. conducted a multicenter retrospective study and reported excellent long-term results of single and a size less than 2 cm iCCA (very early stage), namely, a 5-yr OS of 65% and disease recurrence of only 18%, which are comparable with HCC and LT. However, there has been some conjecture that iCCA size < 2 cm in cirrhotic liver may be difficult to differentiate from HCC by imaging and that an iCCA size < 2 cm in non-cirrhotic liver is resectable. A study has shown that for a iCCA size > 2 cm group, a subset of non-incidental iCCA size > 2 cm may have better prognosis than incidental iCCA of size > 2 cm. Interestingly, the non-incidental iCCA group received neoadjuvant chemotherapy 69% which supported this better result and raised the question about the role of chemotherapy in iCCA and LT [162]. In order to answer this question, a UCLA study was undertaken by Hong et.al of 37 iCCA cases who received neoadjuvant and/or adjuvant chemotherapy. Results showed that of neoadjuvant and adjuvant CMT, adjuvant

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alone and no neoadjuvant, and adjuvant CMT have recurrence rates of 28%, 40%, and 50%, respectively [163]. Recently, two challenging studies by De Martin et.al and Lunsford et.al 2017 have reported recurrence outcomes of iCCA size > 2 cm. Firstly, De Martin et.al, France, multicenter examined 49 cases which included iCCA size > 2 cm and < 5 cm who underwent LT and reported a 5-yr OS 65% and recurrence rate of 21% [164]. Secondly, Lunsford et.al conducted only a prospective study for iCCA and where LT was performed for large tumor size > 5 cm and/or multi-focal iCCA and absence of vascular invasion, extrahepatic disease, or lymph node involvement, and allowing responsive chemotherapy by Gemcitabine and Cisplatin at least for 6 months. From this strategy, patients (n = 6 from 12) achieved a 5-yr OS of 83% after LT [165]; however, future studies need to be undertaken on a greater number of patients to confirm this result. Liver transplantation in hCCA has good long-term results based on an active pre-operative preparation process which includes “neoadjuvant CCRT and strict selection criteria”, especially for unresectable hCCA related to PSC. Studies of liver transplantation in non-PSC resectable hCCA are ongoing. Liver transplantation in iCCA has good results for very early-stage tumors (less than 2 cm), and recent reports of larger sized iCCA provide increased challenges to achieve outcomes compared to early-stage iCCA and HCC.

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8

Systemic Treatment for Cholangiocarcinoma Aumkhae Sookprasert, Kosin Wirasorn, Jarin Chindaprasirt, Piyakarn Watcharenwong, Thanachai Sanlung, and Siraphong Putraveephong

Contents 8.1 8.2

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neoadjuvant Treatment in Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.1 Resectability Criteria and Rationale of Neoadjuvant Therapy . . . . . . . . . . . . . . . 8.2.2 Clinical Data and Ongoing Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.3 Ongoing Clinical Trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.4 Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3 Adjuvant Systemic Treatment in Cholangiocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4 Systemic Treatment for Advanced or Unresectable Cholangiocarcinoma . . . . . . . . . . . . 8.4.1 Chemotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4.1.1 First-Line Chemotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4.2 Second-Line Chemotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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A. Sookprasert (B) · K. Wirasorn · J. Chindaprasirt · P. Watcharenwong · T. Sanlung · S. Putraveephong Medical Oncology Unit, Department of Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand e-mail: [email protected] K. Wirasorn e-mail: [email protected] J. Chindaprasirt e-mail: [email protected] P. Watcharenwong e-mail: [email protected] T. Sanlung e-mail: [email protected] S. Putraveephong e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. Khuntikeo et al. (eds.), Liver Fluke, Opisthorchis viverrini Related Cholangiocarcinoma, Recent Results in Cancer Research 219, https://doi.org/10.1007/978-3-031-35166-2_8

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Introduction

Cholangiocarcinoma (CCA) is a heterogenous group of epithelial cancers involving the biliary tree. Anatomically, CCA is classified into three subtypes; intrahepatic (iCCA), perihilar (pCCA), and distal CCA (dCCA). Intraheaptic CCA arises from the periphery of the second-order bile ducts, pCCA arises in the right, left, or the junction of the hepatic duct, while dCCA involves the common bile duct [1, 2]. Perihilar and distal CCA share similar characteristics including their common presentation; thus, they are referred as extrahepatic CCA (eCCA) [1, 3]. CCA is a rare cancer, particularly in Western countries. The incidence is low in Western countries (from 0.35 to 2 cases per 100,000 population per year), while the incidence is much higher (> 6 cases per 100,000 population per year) in endemic regions, such as China and Thailand [2, 4–6]. There are currently several well-established risk factors for CCA. In the USA and Europe, the main risk factors are primary sclerosing cholangitis and choledochal cysts. Bile duct stones also increase the risk of CCA, particularly in eCCA [7]. In Southeast Asia and East Asia, infection with liver flukes Clonorchis sinensis and Opisthorchis viverrini, both of which have been classified as group 1 carcinogens, remains a dominant risk factor. Chronic inflammation caused by parasitic infection leads to periductal fibrosis and subsequently contributes to cholangiocarcinogenesis [8, 9]. Certain viral infections, including HBV and HCV, are also related to the development of CCA [7, 9]. A number of recent studies have shown a spectrum of heterogeneous molecular alterations in biliary tract cancers. The genomic profiling of CCAs improves our knowledge in cholangiocarcinogenesis and treatment paradigm in the era of targeted therapy. The most common genetic alterations in iCCA are fibroblast growth factor receptor 2 (FGFR2) fusions and isocitrate dehydrogenase 1/2 (IDH1/ 2) mutations, whereas the most common genetic alterations in eCCA are TP53 mutations, KRAS mutations, and HER2 amplification [2, 10, 11]. IDH1/2 mutation is found approximately 10–20% in iCCAs. The gain of function of mutation of IDH1/2 leads to accumulation of the oncometabolite 2-hydroxyglutarate (2HG), which blocks normal cell differentiation and promotes carcinogenesis [10, 11]. Approximately, 10–20% of iCCAs are expected to harbor FGFR2 fusion. The fibroblastic growth factor (FGF) pathway is involved in multiple processes including angiogenesis, cell proliferation, migration, differentiation, and wound healing. FGFR2 fusion contributes to the increase of cellular proliferation and angiogenesis [10, 11]. The dimerization of HER receptor leads to activation of the intracellular tyrosine kinase domain, in turn causing the activation of downstream pathways, including the MAPK and PI3K/ATK pathways [12]. HER2 overexpression is commonly found in eCCAs (approximately 10–20%) and could be found around 5% in iCCAs [10, 11, 13]. Understanding the underlying pathogenesis and molecular drivers in CCAs broadens our therapeutic interventions and guides us to more precise treatments to improve clinical outcomes in CCA patients.

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Cholangiocarcinomas are usually asymptomatic in the early stages. Jaundice is the most common symptom in eCCA, while it is less frequent in iCCA, and it is associated with advanced stages. Approximately, 25% of iCCA are asymptomatic at the time of diagnosis or incidentally found during radiological surveillance [2, 14–16]. Most CCAs present in advanced stages due to late diagnosis, resulting in a poor prognosis. Despite recent advances in diagnosis and treatment for CCA, the five-year survival rate is approximately 10%. In iCCA, localized diseases provide a five-year survival rate of 25%, while metastatic diseases provide a five-year survival rate of 2%. Localized eCCAs provide five-year overall survival of 15%, and metastatic eCCAs provide five-year overall survival of 2% [17]. Currently, surgical resection is the only potentially curative treatment for localized CCA. However, generally, 30–50% of patients with resected CCA will later develop recurrent or metastatic disease [2, 15, 17]. Thus, adjuvant systemic treatment could provide benefit in terms of relapse-free and overall survival in resected CCA. Due to a dismal prognosis in advanced CCA, chemotherapy, targeted therapy, and immunotherapy play a major role as a palliative systemic treatment to improve survival and quality of life in the patients.

8.2

Neoadjuvant Treatment in Cholangiocarcinoma

As mentioned in previous chapters, surgery is the only potentially curative treatment for patients with either intrahepatic, perihilar, or distal cholangiocarcinoma [18]. Even with the screening program, many CCA patients present with more advanced disease and therefore are not candidates for complete resection [19]. Bile duct resection alone for hilar CCA leads to a high local recurrence rate, and the specific techniques employed have been discussed in the previous surgery Chapter [8]. For clearly unresectable diseases, systemic therapy is the major modality of treatment. However, some patients present with a potentially resectable disease that requires systemic treatment and/or radiation before definite surgery. The role of neoadjuvant treatment has grown increasingly controversial. The rationale of preoperative treatment of CCA is based on the hypothesis that it could increase the rate of complete resection (R0) and therefore the long-term overall survival. Since there is no specifically dedicated RCT in locally advanced BTC, the available evidence comes from retrospective and non-randomized prospective studies. There are concerns about the delay of the surgery and the negative events of the chemotherapy. Currently, no guidelines endorse the role of neoadjuvant treatment. In the following sections, we present the current evidence and the future implications of the preoperative treatment of CCA.

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Table 8.1 Examples of borderline resectability tumors from a surgical point of view Intrahepatic CCA

Perihilar CCA

Single mass with perihilar lymph node enlargement

Perihilar tumor with liver parenchyma invasion

Multiple masses occupying the ipsilateral side of Combined with ipsilateral intrahepatic CCA the liver Intrahepatic mass with contact to perihilar structure

Contact of the main portal vein or hepatic artery or IVC

Mass with contact to extrahepatic structure

Contact to extrahepatic structure

Large single tumor > 10 cm in diameter CCA: cholangiocarcinoma, IVC: Inferior vena cava

8.2.1

Resectability Criteria and Rationale of Neoadjuvant Therapy

Currently, there is no standard definition for resectable or borderline resectable for CCA cases. The criteria differ among the primary site and whether it is intrahepatic, hilar, or extrahepatic CCA. We recommended that all locally advanced or borderline resectable CCA cases are discussed by a multidisciplinary team. Generally, our institutional anatomical borderline resectable definition is curative resection that can be obtained with or without reconstruction of the portal vein and/or hepatic artery and no distant metastasis. The examples of borderline resectable according to surgical aspects are shown in Table 8.1. However, several other aspects include, but are not limited to, patient performance status, underlying disease, future liver remnants, nutritional status, and patient preference which are important factors that should be considered in the MDT team. The rationales of neoadjuvant treatment are shown in Table 8.2. The ultimate goal of the treatment is to downstage the tumor and the ability to convert the tumor to a resectable stage which would result in a long-term survival benefit. However, preoperative treatment means the delay of curative surgery and the toxicity from chemotherapy. Liver toxicity from systemic therapy must be considered since the liver of cholangiocarcinoma patients is usually compromised. Selecting the patient who would truly benefit is very important as mentioned earlier. Without good evidence of benefit and lack of randomized trials, currently, preoperative therapy is not recommended apart from a clinical study.

8.2.2

Clinical Data and Ongoing Data

Neoadjuvant chemotherapy in intrahepatic CCA Data of neoadjuvant treatment are mostly from retrospective studies with limited sample sizes. The chemotherapy backbone has been gemcitabine either as a single agent or combined with platinum with a response rate of 13–24%. This is

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Table 8.2 Rationales for neoadjuvant therapy Rationale

Things to consider

Downstaging of the tumor

Chemotherapy-induced liver toxicity

Convert from borderline resectable to resectable tumor

Delay surgery

Early treatment of micrometastatic disease

No evidence of survival benefit

In vivo sensitization of tumor Test of time

comparable to the response rate in advanced disease with the same chemotherapy regimen [20–22]. The study by Le Roy et al. included initially unresectable patients and evaluated the response and conversion to surgery rate. They found that patients who underwent surgery had a longer median overall survival of 36 versus 11 months [22]. A recent meta-analysis showed that preoperative chemotherapy resulted in an improved clinical benefit rate but there are limited data concerning resection rate [23]. The pooled analysis of studies published between 2000–2018 demonstrated a significantly longer survival in patients who underwent resection following down-staging compared to those who did not (29 versus 12 months, p < 0.001) [24]. Neoadjuvant treatment in extrahepatic CCA Surgery for extrahepatic CCA is technically challenging especially when patients present in a locally advanced stage with portal vein or hepatic artery invasion or infiltration of the biliary tract [25]. These neoadjuvant data have been from retrospective single-institution studies with a limited number of patients [26–29]. Chemotherapy combined with radiation is the major modality of the preoperative approach. The resectability rates were as high as 80–100%, but one must keep in mind that the patients underwent a rigorous selection process, and the studies were retrospective. The chemotherapy regimen used concurrently with RT is fluorouracil, gemcitabine, or S-1.

8.2.3

Ongoing Clinical Trials

Several studies are ongoing to evaluate the role of systemic chemotherapy as a neoadjuvant approach. Since the combination of a gemcitabine-cisplatin doublet in advanced disease resulted in only a 25% response rate (complete and partial response), several other combinations are being tested in the neoadjuvant approach as shown in Table 8.3. Two phase II studies are evaluating the response and resectability rate of a three-drug combination of mFOLFOXIRI (our institution) or Gem/Cis/nabpaclitaxel. Two randomized studies are evaluating the benefit of neoadjuvant chemotherapy versus upfront surgery; Gem-Ox then surgery versus surgery alone in intrahepatic CCA and gemcitabine-cisplatin then surgery versus surgery alone in all BTCs.

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Table 8.3 Selected clinical studies for neoadjuvant treatment for cholangiocarcinoma Phase Country

Systemic chemotherapy Patient population

N

Primary outcome

Selected secondary outcomes

2

Thailand mFOLFOXIRI

Borderline resectable CCA

25 Overall response rate

Resectability rate, treatment-related toxicity

2

Korea

Gemcitabine, cisplatin, and nabpaclitaxel

Resectable, high-risk iCCA

34 R0 resection rate

Radiological response rate

2

China

Gem-Ox → Surgery versus surgery

iCCA

100 EFS

OS, ORR, adverse events

3

Germany Gem-Cis → Surgery versus surgery (GAIN)

BTC

300 OS

QoL, PFS, OS, R0 resection rate

2

Korea

2

Germany Bintrafusp Alfa (NEOBIL)

BTC (resectable)

2

China

Resectable iCC

Gemcitabine + Localized Cisplatin ± Durvalumab BTC (DEBATE) (potentially resectable)

Toripalimab, GEM-Ox, Lenvatinib

45 R0 resection rate

OS, EFS, ORR, adverse events

24 Major Tumor response, pathological resectability rate, response adverse events 128 EFS

OS, ORR

Apart from chemotherapy, targeted agents and checkpoint inhibitors are also being tested as potential neoadjuvant approaches. The DEBATE trial is a phase 2 study comparing the R0 resection rate between Durvalumab + Gem-Cis vs Gem-Cis alone. Both arms will receive postoperative durvalumab as an adjuvant treatment. The study started in 2020 and is expected to be completed at the end of 2023. The NEOBIL study is currently evaluating the effect of bintrafusp alfa, a bifunctional fusion protein targeting TGF-β and PD-L1, in resectable biliary tract cancer. The primary endpoint is the major pathological response rate according to Becker score of at least < 10% of viable tumors. The study aims to enroll 24 patients, and it is expected to be completed in September 2023.

8.2.4

Future Perspectives

Targeted therapy The molecular profiling of advanced BTC has resulted in the discovery of multiple targets, such as FGFR, IDH1, and BRAF, which led to the approval of the drugs for BTC [2]. The response rates look promising; however, the data are largely from the phase 2 study [30–32]. Whether the targets of the locally advanced stage are

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the same as the metastatic stage or the targeted treatments would be of benefit is still questionable since, as yet, no clinical trials have been conducted. Immunotherapy The data of immunotherapy in BTC have not yet shown strong clinical benefit in the advanced setting in an unselected population. Apart from MSI, no predictive markers for use in immunotherapy in this setting have been identified yet [33, 34]. Currently, there are 2 ongoing phase II trials incorporating checkpoint inhibitors and chemotherapy as mentioned earlier, and the results will be available shortly.

8.3

Adjuvant Systemic Treatment in Cholangiocarcinoma

Currently, curative resection is the only option as a cure for CCA, specifically in early and localized CCA. Unfortunately, the result after attempted curative resection has not been good based on retrospective data from northeast Thailand which has a very high prevalence of this disease. Results showed that the median overall survival was only 17 months, one-, two-, and three-year overall survival was 65.6%, 45.2%%, and 35.4%, respectively [35].. Adverse prognostic features which can affect overall survival are high serum CEA at base line, positive margin, lymph node metastasis. In this retrospective study, patients who received adjuvant chemotherapy survived significantly longer than patients who did not, with a median survival of 21.6 months compared to 13.4 months with a HR = 0.71, p = 0.01. The adjuvant chemotherapy regimen in this trial was 5FU plus mitomycin C and 5FU plus leucovorin. Active chemotherapy in cholangiocarcinoma includes 5FU, gemcitabine, cisplatin, and capecitabine. These agents have been tested in adjuvant settings, but because of non-uniformity between studies and as there have been no large randomized control trials conducted, the results obtained have proven problematical. An accepted well-documented role of adjuvant chemotherapy for treatment of CCA was not available until the era of the BILCAP trial. The BILCAP trial is a large randomized trial that was done in the biliary tract of cancer (CCA) patients after at least R1 resection [36]. Patients with histologically confirmed CCA (both intrahepatic and extrahepatic) and gall bladder cancer who had at least R1 resection with good Eastern Cooperative Oncology Group (ECOG) 0, 1, 2 with adequate organ function were randomized for treatment by capecitabine 2500 mg/ m2 D1–D14 every 21 days for 8 cycles with observation. This trial met the primary endpoint, and patients who received adjuvant capecitabine had significantly improved OS, with a median OS 51.1 versus 36.4 with the HR an OS of 0.75, p = 0.028. Furthermore, progression-free survival was also significantly improved with a median PFS of 25.9 versus 17.6 months, HR = 0.71, p = 0.011. This trial led to the single agent, capecitabine, being classified as the standard adjuvant treatment after curative resection in CCA and gall bladder cancer. However, some conjecture still exists because another two randomized controlled trials, which had been done in the adjuvant setting in biliary tract cancers, had negative outcomes

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following capecitabine treatment. For instance, the first study was a BCAT trial that compared gemcitabine 1000 mg/m2 D1, 8, 15 every 28 days for 6 cycles with observation [37]. The median OS was not different between the 2 arms, 62.3 months for the gemcitabine arm versus 63.8 months in the observation arm, HR = 1.01, p = 0.964. Additionally, the PFS was not improved, and median PFS for gemcitabine versus observation was 36 and 39.9 months, respectively. The second study was another large trial involving treatment with PRODIGE-12/ ACCORD-18 for randomized patients compared to gemcitabine plus oxaliplatin versus observation alone [38]. Negative outcomes of treatments were also found in this trial. Even though there was a numerical improvement in OS, 75.8 versus 50.8, the HR was found to be 1.08, p-value 0.74. PFS; hence, a no significant improvement 30.4 versus 18.5, HR = 0.81, p = 0.74. All three major trials in adjuvant CCA are summarized in Table 8.4. Adjuvant chemotherapy after R0 or R1 resection is a major area of study in Thailand, specifically in the highest endemic area of the world, northeast Thailand. Comprehensive research is being conducted to compare gemcitabine alone versus gemcitabine plus cisplatin and other chemotherapeutic agents. We envisage that interim results should be available in the next few years. Table 8.4 Summary of large randomized trials in the adjuvant setting in cholangiocarcinoma Study

BCAT (37)

PRODIGE-12/ ACCORD-18 (38)

BILCAP (36)

Study design

RCT, phase III

RCT, phase III

RCT, phase III

Experimental versus control arm

Gem single agent versus surgery alone

Gemcitabine plus oxaliplatin versus surgery alone

Capecitabine versus surgery alone

Country

Japan

France

UK

N

226

196

447

Primary endpoint

OS

RFS

OS

Result

Negative

Negative

Positive

RFS (months)

36 versus 39.9 HR = 0.93 P = 0.693

30.4 versus 18.5 HR = 0.81 P = 0.48

25.9 versus 17.6 HR = 0.71 P = 0.011

OS (months)

62.3 versus 62.8 HR = 1.01 P = 0.964

75.8 versus 50.8 HR 1.08 P = 0.964

52.7 versus 36.1 HR = 0.75 P = 0.028

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8.4

Systemic Treatment for Advanced or Unresectable Cholangiocarcinoma

8.4.1

Chemotherapy

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Systemic chemotherapy has usually been applied in patients who are unresectable or advanced cholangiocarcinoma (CCA). In 1996, it was established that chemotherapy improved the overall survival and quality of life of patients. The benefit of first-line chemotherapy, which is better than best supportive care alone, was published in a trial that randomly assigned 90 patients with advanced biliary or pancreatic cancer (37 with biliary tract cancer) to fluorouracil-based chemotherapy or best supportive care alone (median overall survival (OS) 6 versus 2.5 months, respectively) [39].

8.4.1.1 First-Line Chemotherapy The standard treatment for first-line therapy in advanced CCA has been the combination of gemcitabine plus cisplatin which was established in a randomized phase III trial, namely the ABC-02 study. It included 410 patients with locally advanced or metastatic CCA, gallbladder cancer, or ampullary cancer who were randomly assigned to receive cisplatin (25 mg/m2 ) followed by gemcitabine (1000 mg/m2 on days 1 and 8, every three weeks for 8 cycles) or gemcitabine alone (1000 mg/ m2 on days 1, 8, and 15, every four weeks for 6 cycles) for a total of 24 weeks. The results revealed the patients who received the combination treatment had a median OS of 11.7 mo, which was significantly longer than 8.1 mo in the gemcitabine alone arm (HR = 0.64, 95%CI: 0.52–0.80, P < 0.001). The PFS also was significantly longer (median PFS: 8 versus 5 mo; P < 0.001) in patients who were treated with the doublet chemotherapy, and the tumor was controlled in significantly more patients (81.4% versus 71.8%; P = 0.049) [40]. In addition, there were many combination chemotherapy regimens for first-line therapy in advanced CCA but the clinical benefit obtained was found to be modest (Table 8.5). As stated in previous chapters, in Thailand, CCA is predominately associated with O. viverrini infection and has poor survival outcomes when compared with other groups in an integrated whole-genome and epigenomic analysis on an international scale [41, 42]. In a retrospective study of first-line chemotherapy in Thailand, 224 patients were included out of which, 143 patients (63.8%) received 5FU-based chemotherapy and 81 (36.2%) received a gemcitabine-based regimen. The overall survival was not significantly different between the 5FU arm vs the gemcitabine arm (9.6 versus 9.0 months, p = 0.20). In patients with measurable disease at baseline, the overall response rate was 24.4% versus 26.2% (5FU versus gemcitabine; p = 0.32). Additionally, the disease control rate was 24.4% versus 30.9% respectively (p = 0.50) [43]. Results from this study also correlated with a previous retrospective study in Thailand, where results showed that the median OS was 7.2 versus 10 months in 5FU-based and gemcitabine-based regimen, respectively (p = 0.36) [44]. The median OS for the first-line chemotherapy in advanced

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Table 8.5 Summary of first-line chemotherapy studies in advanced or unresectable CCA Trial (N)

Population Drugs (CCA/GB/ Ampulla(%)

ORR (%)

Median PFS (months)

Median OS (months)

ABC-02 [40] (N = 410) Ph.III

57.8/36.9/ 5.3

Gem-Cis versus Gem

26.1 versus 15.5 DCR 81.4 versus 71.8 (P = 0.49)

8.0 versus 5.0 (HR 0.63, P < 0.001)

11.7 versus 8.1 (HR 0.64, P < 0.001)

Okusaka et al. [45] (N = 83) Ph.II

56.0/39.0/ 5.0

Gem-Cis versus Gem

19.5 5.8 versus versus 3.1 11.9 HR 0.69, P = 0.07

11.2 versus 7.7 HR 0.69, P = 0.14

FUGA-BT [46] (N = 354) Ph.III (Non-inf HR 1.155, P 0.05)

57.0/39.0/ 4.0

Gem-Cis versus Gem-S1

29.8 5.8 versus versus 6.8 32.4 HR 0.86, 95%CI 0.70–1.07

13.4 versus 15.1 HR 0.95, P = 0.046

Kim et al. [47] (N = 222) Ph.III (Non-inf 6 mo PFS 15%)

72.5/27.5/0

Gem-Ox versus Cape-Ox

24.6 5.3 versus versus 5.8 15.7 6 mo PFS 44.6 versus 46.7

10.4 versus 10.6 (P = 0.131)

PrE0204 [48] (N = 74) Ph.II

100/0/0

Gem-nabpaclitaxel

30

12.7 (95% CI, 9.2–15.9)

KHBO1401-MITSUBA NA [49] (N = 246) Ph.III

Gem-Cis-S1 versus Gem-Cis

41.5 7.4 versus versus 5.5 15.0 (HR 0.75, P < 0.0001)

Shroff et al. [50] (N = 60) Ph.II

78/22/0

Gem-Cis-nabpaclitaxel 45

Mungwatthana et al. [43] (N = 224) Retrospective

100/0/0

Gem-based versus 5FU-base

7.7 (95% CI, 5.4–13.1)

11.8 (95% CI, 6.0–15.6)

24.4 NA versus 26.2

13.5 versus 12.6 (HR 0.79, P= 0.046) 19.2 (95% CI, 13.2-NR) 9.6 versus 9.0 (P = 0.20)

ORR: overall response rate, DCR: disease control rate, PFS: Progression-free survival, OS: Overall survival

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CCA in Thailand was slightly shorter than in other global studies, which may be due to differences in molecular alterations. Currently, in the other solid tumors, the rationale employed is a combination of treatments, namely chemotherapy plus immunotherapy, chemotherapy plus targeted therapy, or combination immunotherapy. Studies have shown that a combination of treatments has resulted in increasing the benefit of treatment for patients. Accordingly, there are many ongoing clinical studies concerning combination treatment for the first-line of treatment of unresectable or advanced CCA.

8.4.2

Second-Line Chemotherapy

Few prospective clinical trials have been conducted comparing specific chemotherapy regimens in second-line therapy for advanced CCA. The largest phase III study for the chemotherapy regimens in a second-line setting was the ABC-06 study. The study included the 162 patients who had disease progression while receiving or after receiving gemcitabine plus cisplatin, and who had a good performance status (ECOG 0–1), in the absence of potentially actionable molecular targets randomly assigned to active symptom control (ASC) with or without modified FOLFOX (mFOLFOX) regimen. It was found that mFOLFOX was associated with significantly better OS at 6 (51% versus 36%) and 12 months (26% versus 11%) and significantly improved median OS (6.2 versus 5.3 mo, HR 0.69, 95% CI 0.50–0.97) [51]. The alternative treatments for second-line treatment have been examined in phase II single-arm or retrospective studies (Table 8.6). In Thailand, a retrospective study has been conducted that included 19 patients with locally advanced or metastatic biliary tract cancer, but they did not respond favorably after being treated with gemcitabine plus cisplatin compared to patients treated with FOLFOX4. The median PFS was 2.6 mo, and the median OS was 6.2 mo. The response rate and the disease control rate were 15% and 77%, respectively [52]. Prognostic factors The prognostic factors in patients with advanced biliary tract cancer receiving firstline palliative chemotherapy were established by a Korean study of 213 patients. In a multivariate analysis, an intrahepatic primary site, metastatic disease, liver metastases, poor ECOG performance status, and elevated level of serum alkaline phosphatase were significant predictors of overall survival. This study used these five variables to develop a prognostic index to stratify patients into low, intermediate, and high-risk groups. There was a statistically significant difference between median OS values; 11.5, 7.3, and 3.6 mo, respectively. From the whole-genome and epigenomic landscapes of etiologically distinct subtypes of CCA studies, CCA in Thailand which is mostly associated with O. viverrini infection was classified in subgroups 1 and 2. The survival was poor when compared to the other groups [41]. The study investigating the prognostic factors of advanced CCA in Thailand showed male gender, metastatic disease,

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Table 8.6 Summary of second-line chemotherapy studies in advanced or unresectable CCA Trial (N)

Population (CCA/GB/ Ampulla (%)

Drugs

ORR (%)

Median PFS (months)

Median OS (months)

ABC-06 [51] (N = 162) Ph.III

72.2/21.0/6.8

mFOLFOX-ASC versus ASC

5 versus NA (DCR; 33 versus NA)

4.0 versus NA

6.2 versus 5.3 (HR 0.69, P = 0.031)

Zheng et al. [53] (N = 60) Ph.II

78/22/0

XELIRI versus Irinotecan

13.4 versus 6.7 (DCR; 63.3 vs 50)

3.7 versus 2.4 (HR 0.54, P = 0.036)

10.1 versus 7.3 (HR 0.63, P = 0.107)

Kim et al. [54] (N = 321) Retrospective

76.3/23/7/0

5FU-based

Single; 1.2 Doublet; 7.6

Single; 1.8 Doublet; 2.6 (P = 0.43)

Single; 6.5 Doublet; 6.2 (P = 0.87)

Sookprasert et al. [52] (N = 19) Retrospective

89.4/0/10.6

FOLFOX4

15

2.6

6.2

ORR: overall response rate, ASC: Active symptom control, DCR: disease control rate, PFS: Progression-free survival, OS: Overall survival

treatment options, liver cirrhosis, chronic HBV or HCV infection, and diabetes mellitus significantly affected survival outcomes [4, 55]. Furthermore, sarcopenia was diagnosed in 61.3% of the patients who had advanced CCA. Multivariable analysis adjusted for chemotherapy regimen and age revealed that a high appendicular muscle mass independently predicted significantly better survival outcomes (HR 0.40; 95% CI, 0.18–0.88; p = 0.023) [56]. 1. Targeted therapy

Molecular alterations in cholangiocarcinoma Lowery et al. analyzed tumor samples from Caucasian cholangiocarcinoma patients with next-generation sequencing. Of 195 patients, 78% were intrahepatic (iCCA) and 22% extrahepatic cholangiocarcinoma(eCCA). The most common genetic alterations in iCCA were IDH1 (30%), ARID1A (23%), BAP1 (20%), TP53 (20%), and FGFR2 gene fusions (14%). Moreover, alterations in CDKN2A/ B and ERBB2 were associated with negative prognostic implications [57]. The molecular subtypes of the CCA were proposed by another research group in 2017 including Thai, other Asian, and Caucasian patients. Jusakul et al. integrated whole-genome and epigenomic analyses of 489 CCA tissue samples globally and classified them into four clusters, Table 8.7. Clusters 1 and 2 or so-called ‘flukepositive CCA’ were enriched in ERBB2 amplification. Conversely, the clusters 3

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and 4 or ‘fluke-negative CCA’ exhibited high PD-1/PD-L2 expression, or epigenetic mutations (IDH1/2, BAP1) and FGFR/PRKA-related gene rearrangements [41]. This classification and knowledge led to the development of novel targeted therapy and immunotherapy. Since mid-2021, the US Food and Drug Administration (US FDA) approved pemigatinib and infigratinib for previously treated advanced CCA with FGFR2 fusion or rearrangement, and ivosidenib for an IDH1-mutation. From the results of the several solid tumor-agnostic studies, many target therapies and immunotherapies including pembrolizumab were approved for patients whose tumor carried high microsatellite instability (MSI-H), mismatch repair deficiency (dMMR), or high tumor mutational burden (TMB-H). The details of the current recommendation for targeted therapy drugs are as follows: Regorafenib Regorafenib is a multikinase inhibitor including EGFR, RAS, RAF, VEGFR, FGFR, and PDGFR. A phase II single-arm study enrolled CCA patients who were previously treated with at least one systemic chemotherapy to receive regorafenib at dose of 160 mg once daily, 21 days on, followed by 7 days off in a 28-day cycle. The median progression-free survival was 15.6 weeks (90% CI 12.9–24.7), and the median overall survival was 31.8 weeks (90% CI 13.3–74.3). A partial response was achieved in 5 patients (11%) [58]. The toxicity profiles were as expected. Despite regorafenib being considered as a recommended option in some guidelines, it should be kept in mind that it is not currently approved by the US FDA. Pemigatinib Pemigatinib is the selective, potent, oral inhibitor of FGFR1, 2, and 3. The FIGHT202 study was a phase II multiple cohorts study of the patients who received at least one previous treatment for locally advanced or metastasis CCA. A dose of 13.5 mg once daily of pemigatinib was given in 2 weeks on and 1 week off of 21-day cycle to the 3 cohorts of the patients including FGFR2 fusions or rearrangements, other FGF/FGFR alterations, and no FGF/FGFR alterations. Of 146 patients, 102 who carried FGFR2 fusions or alterations had an objective response rate of 35.5% (95% CI 26.5–45.4) including a 2.8% complete response. The median duration of response was 7.5 months (95% CI 5.7–14.5), median progression-free survival was 6.9 months (95% CI 6.2–9.6), and the median overall survival was 21.1 months (95% CI 14.8 to not estimable) [31]. There was no response in cohort 2 and 3, the overall survival was 6.7 and 4.0, respectively. Hyperphosphatemia at any grade was found in 60% of patients. The most frequent serious adverse events were abdominal pain (5%), pyrexia (5%), cholangitis (3%), and pleural effusion (3%). Infigratinib Infigratinib is the first-in-class, oral, selective pan-FGFR kinase inhibitor. BGJ398 was a multicenter, open-label, phase II study of patients with advanced or

1

Mostly fluke positive

Mixed

Mostly Thailand

– Highest single nucleotide variants burden – Enriched in TP53, ARID1A, BRCA1/2 mutations – Enriched in H3K27me3-associated promoter mutation – ERBB2 amplification – TET1 downregulation – EZH2 upregulation – CpG island hypermethylated

Poorer prognosis

Cluster

Fluke-association

Location

Country of origin

Genetic alteration

Prognosis

Table 8.7 Molecular subtypes in cholangiocarcinoma

Singapore, France

iCCA

Mostly fluke negative

3

Poorer prognosis

Poorer prognosis

– Enriched in TP53 – Highest CNA burden mutations – Immune-related pathways – PD-1, PD-L2, and BTLA – ERBB2 amplification upregulation – CTNNB1, WNT5B, AKT1 upregulation

Thailand, Singapore, Romania

Mixed

Mixed

2

Better prognosis

– Enriched in BAP1 and IDH1/2 mutations – Enriched in FGFR alterations – CpG shore hypermethylated

Singapore, France, Romania, Brazil

Mostly iCCA

Mostly fluke negative

4

236 A. Sookprasert et al.

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metastatic CCA containing FGFR2 fusions or other FGFR alterations whose disease had progressed while receiving prior therapy. The patients received infigratinib 125 mg once daily for 21 days followed by 7 days off in a 28-day cycle. After first reported in 2018 [32], the 2021-updated study included 108 patients, of which 83 had FGFR2 fusions. The central-reviewed objective response rate was 23.1% (95% CI 15.6–32.2) including 1 complete response and 24 partial responses. The median duration of response was 5.0 months (95% CI 0.9–19.), median progression-free survival was 7.3 months (95% CI 5.6–7.6) [59]. The most common treatment-emergent adverse events at any grade were hyperphosphatemia (76.9%), eye disorders (67.6%, excluding central serous retinopathy/ retinal pigment epithelium detachment, CSR/RPED), CSR/RPED (16.7%), stomatitis (54.6%), and fatigue (39.8%). Ivosidernib Ivosidenib is an oral, potent, targeted inhibitor of mutated IDH1. ClarIDHy was a phase III multicenter, randomized controlled trial of ivosidenib compared with a placebo in previously treated IDH1-mutant CCA. 185 patients received 500 mg daily of ivosidenib or placebo. The primary endpoint was progression survival which was significantly longer in the ivosidenib group compared with the placebo group, with a median 2·7 months (95% CI 1.6–4.2) vs 1.4 months (1.4–1.60), hazard ratio 0.37 (95% CI 0.25–0.5, one-sided p < 0.0001). The median overall survival was 10.8 months and 9.7 months for ivosidenib and placebo, respectively, HR 0.69 (95% CI 0.44–1.10, p = 0.060). There was a 2% partial response in the ivosidenib group (30). The most common adverse events included nausea (35%), fatigue (31%), cough (21%), and abdominal pain (22%). Tumor-agnostic therapy Additional to the aforementioned specific CCA studies, there are several tumoragnostic studies in previously treated solid tumors which harbor no satisfactory systemic therapy options. In summary, the study enrolled cancer patients of any primary sites who have the specific potentially targetable driver mutations to the study. BRAFV600E mutant CCA patients were treated as a part of a phase II singlearm, ROAR study [60]. In this study, 43 patients with BRAFV600E mutated biliary tract cancers received dabrafenib and trametinib. There were 51% (95% CI 36– 67) overall responses, 9 months (95% CI 5–10) median progression-free survival, and 14 months (95% CI 10–33) overall survival in this study. Another result from the phase II study of BRAFV600 mutant solid tumor excluding melanoma, thyroid, colorectal cancer, and non-small cell lung cancers showed an overall response of 38% (90% CI 22.9–54.9). There were four intrahepatic CCA of 38 patients who participated in this study [61]. Entrectinib and larotrectinib are TRK fusion inhibitors. Pooled analysis of 3 phase I/II studies of entrectinib (ALKA-372-001, STARTRK-1, and STARTRK-2) showed a 57% objective response rate of 54 adults with advanced or metastatic NTRK fusion-positive solid tumors. Even though there was only 1 CCA patient in

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this study, the study met the primary endpoint, and the drug was approved for treatment in curtained solid tumors, including CCA [62]. Moreover, there were 2 CCAs of 55 patients who participated in another pooled analysis of larotrectinib (phase I, SCOUT, NAVIGATE). This study showed an increased objective response of 75% (95% CI 61–85) [63]. A summary of recommended targeted therapy is shown in Table 8.8. 2. Immunotherapy Pembrolizumab, an anti-PD-1 monoclonal antibody, has been approved for MSI-H/ dMMR or TMB-H solid tumors that have progressed following prior treatment and that have no satisfactory alternative treatment options. KEYNOTE-158 was a phase II multiple cohorts study of pembrolizumab. This study was prospective and also had pre-planned retrospective analyses. Cohort K enrolled 233 patients with 27 tumor types who had MSI-H. Overall, the objective response rate was 34.3% (95% CI 28.3–40.8), median progression-free survival was 4.1 months (95% CI, 2.4– 4.9), and median overall survival was 23.5 months (95% CI, 13.5 to not reached). It was interesting that of 22 patients (9.4%) who had CCAs, seven had a partial response and two had a complete response [64]. A prospective biomarker analysis of KEYNOTE-158 through all cohorts was also performed. Of 790 patients who were evaluable for TMB by FoundationOne CDx assay, 102 (13%) had TMB-H which prespecified cut-off of at least ten mutations per megabase. The objective response rate was 29% (95% CI 21–39), 6% (5–8) for TMB-H, and non-TMB-H, respectively. There was no CCA patient involved in this analysis but the US FDA approved this drug as tumor-agnostic therapy [65]. Preliminary data of the LEAP005 study biliary tract patient cohort also showed the efficacy of pembrolizumab in combination with Lenvatinib. However, the study is ongoing and not yet approved [66]. Another anti-PD-1 immunotherapy is nivolumab. The efficacy of nivolumab in previously treated CCA was investigated in a multicenter phase II single-arm study. Of 54 patients, the investigator-assessed objective response rate was 22%, and the central-reviewed objective response rate was 11%. It is important to note that all patients who had responded to treatment had proficient mismatch repair protein. The median progression-free survival was 3.68 months (95% CI 2.30–5.69), and the median overall survival was 14.24 months (95% CI 5.98 to not reached) [67]. This treatment is also not yet approved. Several ongoing trials are investigating chemotherapy with or without immunotherapy and antiangiogenesis in a first-line setting of locally advanced or metastatic cholangiocarcinoma.

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Table 8.8 Targeted therapy and immunotherapy recommendation by the National Comprehensive Cancer Network guidelines in the locally advanced or metastatic cholangiocarcinoma Study drugs

Target

Regorafenib

Study

Phase Population

n

Results

Multikinase Sun et al. [58] inhibitors

II

Previously treated CCAs

43

ORR 11% mPFS 15.6 weeks mOS 31.8 weeks

Pemigatinib

FGFR1-3

FIGHT-202 [31]

II

Previous treated CCAs with FGFR2 fusions or rearrangement (cohort 1)

146 ORR 35.5% mPFS 6.9 months mOS 21.1 months

Infigratinib

FGFR1-3

BGJ398 [59]

II

Previous treated CCAs with FGFR2 fusions or other FGFR alterations

108 ORR 23.1% mPFS 7.3 months

Ivosidernib

IDH1

ClarIDHy [30]

III

Previous treated CCAs with IDH1-mutation

185 ORR 2% mPFS 2.7 months mOS 10.8 months

Dabrafenib and trametinib

BRAF MEK

ROAR [60]

II

Previously treated 43 BRAFV600E mutated biliary tract cancers

ORR 51% mPFS 9 months mOS 14 months

NCI-MATCH (EAY131-H) [61]

II

Previously treated BRAFV600 solid tumors

38

ORR 38% (4 CCAs)

Entrectinib

TRK

Pooled analysis [62]

I/II

Previously treated NTRK fusion tumors

54

ORR 57% (1 CCA)

Larotrectinib

TRK

Pooled analysis [63]

I/II

Previously treated NTRK fusion tumors

55

ORR 75% (2 CCAs)

KEYNOTE-158 II [64]

Previously treated MSI-H non-colorectal solid tumors

233 ORR 40.9% mPFS 4.2 months mOS 24.3 months (22 CCAs)

KEYNOTE-158 II [65]

Previously treated TMB-H non-colorectal solid tumors

102 ORR 29% (0 CCAs)

Previously treated biliary tract cancer

31

Pembrolizumab PD-1

Pembrolizumab PD-1 LEAP-005 [66] and lenvatinib Multikinase inhibitors

II

ORR 10%

(continued)

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Table 8.8 (continued) Study drugs

Target

Study

Phase Population

n

Results

Nivolumab

PD-1

Kim, 2020 [67]

II

54

ORR 11–22% mPFS 3.68 months mOS 14.24 months

8.5

Previously treated biliary tract cancer

Conclusion

Biliary tract cancers are associated with poor prognosis and many patients are diagnosed at an advanced stage. Several advances have been made in systemic treatments in recent years including adjuvant capecitabine and second-line chemotherapy. Molecular profiling of the tumors has led to the discovery of several druggable targets with promising results including IDH1, FGFR, BRAF, and NTRK mutation. Checkpoint inhibitors are being tested either as a single agent or combined with chemotherapy in cholangiocarcinoma, and the results are awaiting.

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9

Palliative Care in Cholangiocarcinoma Attakorn Raksasataya, Anucha Ahooja, Vivian Krangbunkrong, Apiwat Jareanrat, Attapol Titapun, and Narong Khuntikeo

Contents 9.1

9.2

General Principles in Palliative Care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.1 Definition of Palliative Care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.2 Model of Care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.3 Palliative Care Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.4 Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.5 Symptom Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.6 Pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.7 Dyspnea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.8 Nausea and Vomiting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.9 Communication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.10 Bereavement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1.11 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Palliative Biliary Drainage for Advance Stage Cholangiocarcinoma . . . . . . . . . . . . . . . . 9.2.1 Percutaneous Palliative Biliary Drainage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.2 Palliative PTBD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.3 Complication of Percutaneous Transhepatic Biliary Drainage . . . . . . . . . . . . . . . 9.2.3.1 Acute Cholangitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.4 Hemorrhage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.5 Pericatheter Leakage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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A. Raksasataya (B) Palliative Care Unit, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand A. Ahooja · V. Krangbunkrong Department of Radiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand A. Jareanrat · A. Titapun · N. Khuntikeo Department of Surgery, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand e-mail: [email protected] A. Titapun e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. Khuntikeo et al. (eds.), Liver Fluke, Opisthorchis viverrini Related Cholangiocarcinoma, Recent Results in Cancer Research 219, https://doi.org/10.1007/978-3-031-35166-2_9

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9.2.6 9.2.7 9.2.8 9.2.9 9.2.10 9.2.11 9.2.12 9.2.13 9.2.14 9.2.15 9.2.16 9.2.17 9.2.18 9.2.19 9.2.20 References

9.1

Palliative PTBS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Planning and Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Indications for PTBS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contraindication for PTBS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Instruments and Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Complications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endoscopic Biliary Stenting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Type of Metallic Biliary Stent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fully Covered Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Partly Covered Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uncovered Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Outcome of SEMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Surgical Biloenteric Bypass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Operative Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Right Sided Hepaticojejunostomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...........................................................................

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General Principles in Palliative Care

9.1.1 Definition of Palliative Care Palliative care is an approach that improves the quality of life of patients and their families who are facing problems associated with life-threatening illness. It prevents and relieves suffering through the early identification, correct assessment and treatment of pain and other problems, whether physical, psychosocial, or spiritual [1]. Hospice care is a program that gives special care to people who are near the end of life and have stopped treatment to cure or control their disease. Hospice offers physical, emotional, social, and spiritual support for patients and their families [2]. The goal of both cares is improving quality of life, symptoms control, and wellbeing of patients and their family. The place of care may be a hospital, nursing homes or the patient’s home (Table 9.1).

9.1.2 Model of Care For individual patients, palliative care can be initiated at the time of diagnosis of incurable diseases. At this point palliative specialists may not need to be consulted as the palliative approach could be introduced by an attending team. The palliative care approach includes the recognition that the illness is not curable, optimizes quality of life, involves basic symptoms control with good communication, and supports the patient and their family to cope with complex issues. When the illness progresses, the role of palliative care increases, however, the role of curative treatment subsequently decreases. The American Society of Clinical Oncology (ASCO) has published an Integration of Palliative Care into Standard Oncology Care: ASCO Clinical Practice Guideline Update. It has recommended that palliative care

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Table 9.1 Compare palliative care and hospice care Palliative care

Hospice care

Diagnosis

Serious illness in any stage of disease

Terminal stage of disease

Life expectancy

Any

Less than 6 months

Focus on

Quality of life and symptoms control

Quality of life and symptoms control

Support

Patient and family

Patient and family

Multidisciplinary team

Palliative doctor, nurse, social worker, chaplain, volunteer, and other medical specialists such as oncologist, surgeon, nephrologist

Palliative doctor, nurse, social worker, chaplain, and volunteer

Curative treatment

Curative treatment may or may not be available

No curative treatment

for patients with advanced cancer should be delivered through interdisciplinary palliative care teams, with consultation available in both outpatient and inpatient settings. Oncologists may note that they are providing palliative care themselves. Palliative care studies have all employed standardized symptoms, spiritual and psychosocial assessments, with an emphasis on early discussion of prognosis and treatment options to discern prognostic awareness and early discussion of hospice care [3]. For the service system, data from multicenter studies has revealed that 18.7% of in hospital patients met criteria for palliative care and only 17.3% of these were referred to a palliative specialist [4]. Palliative care service delivery is divided into the following categories. 1. Palliative care approach is provided by all health care professional to most patients. 2. Generalist palliative care is provided by health care professionals who have had some training in palliative care. Some patients will need this care level. 3. Specialist palliative care is provided by interdisciplinary fulltime health care professionals who have had advance training. The basic team is composed predominately of doctors and nurses, whereas the extended team includes doctors, nurses, pharmacists, psychologists, social workers, physiotherapists, and others. A small number of patients and family who have complex issues need this level of care [5–11]. Some patients may have a complex issue, and therefore, require the palliative care specialist level. After the issue is solved, the patient may step back to generalist palliative care or the palliative care approach. Palliative care need is different from person to person, from family to family, and can fluctuate along the course of illness.

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9.1.3 Palliative Care Criteria Palliative care is focused on the quality in life limited illness without a rigid time frame. Incidentally, a “surprise question” is widely used to identify patients who need palliative care. “Would I be surprised if this patient died in next year (12 months)” is a simple question that health care providers ask themselves. If the answer is “no,” the palliative care approach or palliative consultation should be delivered to patients [12, 13]. Systematic reviews in 2017 found this tool has an accuracy level of 74.8% [14], with sensitivity of 67.0%, specificity of 80.2%, and provides better discrimination in cancer patients when compared with non-cancer patients [15]. The supportive and palliative care indicator tool (SPICT) is an international tool, which has been translated into more than 10 languages and is used in palliative care services in 30 countries [16]. SPICT is divided to part 1 general indicators of poor or deteriorating health, and part 2 clinical indicators of one or multiple life-limiting conditions including cancer and organ failure.

9.1.4 Assessment Palliative care is a holistic care program, administered via impeccable assessment. The American Medical Association’s Education for Physicians on End-of-Life (EPEC) proposed 9 dimensions of whole patient assessment for palliative care, that include followings [17]. 1) 2) 3) 4) 5) 6) 7) 8) 9)

Illness/treatment summary Physical issues Psychological issues Decision making Communication Social issues Spiritual issues Practical issues Anticipatory planning for death

The Edmonton Symptom Assessment System (ESAS) was developed some 30 years ago, and it is a simple self-report symptom assessment tool, widely used and translated into 20 languages [18, 19]. The revised version (ESAS-r) contains pain, tiredness, drowsiness, nausea, lack of appetize, shortness of breath, depress, anxiety, well-being, and blank box for other symptoms that patients may have [20, 21]. Each symptom intensity can be rated from 0 (no)–10 (the worst) and can be interpreted into 4 groups: 0 = no, 1–3 = mild, 3–6 = moderate, and 7–10 = severe symptom intensity. Constipation is a common symptom that needs to be evaluated but may not need to be rated 0–10.

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9.1.5 Symptom Management In advanced cancer patients, more than half experienced pain, breathlessness, constipation, fatigue, anorexia, and dry mouth [22]. For CCA patients they experience multiple symptoms, for instance, anxiety 87.3%, fatigue 87.3%, abdominal pain 76.9%, etc. Patients reported that the most symptom distress symptom is abdominal pain 48.8% [23].

9.1.6 Pain Pain management after clinical evaluation of pain severity, can be classified into 2 types of pain: nociceptive pain or neuropathic pain. It is important to note that cancer pain can be either of the two types or both types. Recommendations according to the WHO guidelines for the pharmacological treatment of cancer pain include prescribing paracetamol, non-steroidal anti-inflammatory drugs (NSAID) and opioid combinations at the stage of initial of pain management, either alone or combination, depending on pain types/levels, and the WHO has also recommend these medications for maintenance pain control [24]. Weak opioids (codeine and tramadol) have a place in step 2 of the ladder. However, the European Association of Palliative Care (EAPC) has recommended strong opioids may be used instead of codeine or tramadol in step 2 of the ladder [25]. From the typical starting dose (Table 9.2), dose reduction is considered for an elderly/frail, pediatric, liver impaired or renal impaired patients. The typical starting dose and approximate opioid conversion rations may differ from country to country due to local guidelines, practicing experience, ethnicity, pharmacokinetics, pharmacodynamics, and genetic polymorphisms (Table 9.3). A dose reduction of around 50% of the calculated equivalent dose from the table of the new opioids is prudent when switching at high doses [24]. The anti-neuropathic pain medication starting dose are describe in Table 9.4.

9.1.7 Dyspnea Dyspnea is as known as shortness of breath and breathlessness. The American thoracic society consensus statement defines dyspnea as a subjective experience of breathing discomfort that consists of qualitatively distinct sensations that vary in intensity [26]. It is an individual perception and may not be related to respiratory rate, hypoxemia, and respiratory muscle usage. Medication for palliate dyspnea symptom are opioids and benzodiazepine (Table 9.5). In the last days of life, most patients experience a rattle, and gurgling respiration. It is usually called death rattle. It is caused by respiratory secretions that patients cannot expel. Anticholinergic medication is effective to dry secretions at the end of life (Table 9.6).

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Table 9.2 Typical starting dose of analgesic medication Medication

Typical starting dose

Note

Paracetamol

500–1000 mg oral q 4–6 h

Max dose 4000 mg/day

Ibuprofen

400 mg oral after meal 3 times/day

Consider adding proton pump inhibitor. Avoid in bleeding risk Maximum dose 3200 mg/day

Etoricoxib

60 mg oral once a day

Max. dose 120 mg/day

Celecoxib

100–200 mg oral twice a day

Max. dose 400 mg/day

Codeine

30 mg oral q 4–6 h

Max. dose 360 mg/day

Tramadol

50 mg oral q 8–12 h

Max. dose 400 mg/day

Morphine

5 mg orally q 4 h 2 mg IV/SC q 4 h

No max. dose

Fentanyl

0.5–1mcg/kg/hr drip IV/SC 25–100 mcg IV/SC prn q 1 h 12–25 mcg/hr TD patch q 72 h

Do not use TD patch in patients with severe cachexia, fevers or frequent sweating No max. dose

Oxycodone

10 mg oral q 12 h (extended release)

No max. dose

*

IV = intravenous, SC = subcutaneous, TD = transdermal, q = every

Table 9.3 Approximate opioids conversion ratio

First opioid

Second opioid

Ratio

Tramadol IV

Tramadol oral

1:1.5

Tramadol oral

Morphine oral

5–10:1

Codeine oral

Morphine oral

10:1

Morphine oral

Morphine IV, SC

2–3:1

Morphine IV

Fentanyl IV, SC

100:1

Morphine oral

Fentanyl TD

60 mg/day: 25mcg/ hour

Morphine oral

Oxycodone oral

1.5:1

Morphine oral

Hydromorphone oral

4–5:1

Hydromorphone oral

Hydromorphone IV, SC

2–5:1

*

IV = intravenous, SC = subcutaneous, TD = transdermal

In many centers these drugs are prescribed regularly around the clock. When patients are in the dying stage, administration of an anticholinergic prophylactic to prevent death rattle is more effective than late administration [28].

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Table 9.4 Anti-neuropathic pain medication Medication

Typical starting dose

Note

Amitriptyline

10–25 mg oral at bedtime

Anticholinergic side-effects including orthostatic hypotension, sedation, confusion, tachycardia, constipation, dry mouth

Nortriptyline

10–25 mg oral at bedtime

Max dose 150 mg/day

Duloxetine

30 mg oral at bedtime

Max dose 120 mg/day

Venlafaxine

37.5 mg oral at bedtime

Max dose 225 mg/day

Gabapentin

300 mg oral at bedtime

Max dose 3600 mg/day

Pregabalin

75 mg oral at bedtime

Max dose 3600 mg/day

Table 9.5 Opioids and benzodiazepine for dyspnea control

Table 9.6 Medication for death rattle control: modified from Pereira JL [27]

Medication

Typical starting dose

Morphine

2.5–5 mg oral every 4–6 h 1–3 mg IV/SC every 4–6 h

Hydromorphone

1 mg oral every 4–6 h 0.5 mg IV/SC every 4–6 h

Diazepam

2–5 mg oral/IV prn q 8 h as needed

Lorazepam

0.5–1 mg IV/SC/sublingual prn q 2 h as needed

Midazolam

2.5–5 mg IV/SC prn q 2 h as needed 0.5 mg/hour IV/SC drip

Medication

Typical starting dose

Hyoscine hydrobromide

0.2 mg–0.4 mg SC every 2–4 h prn as needed

Hyoscine butyl bromide

10–20 mg SC every 4–6 h prn as needed

Glycopyrrolate

0.2 mg–0.4 mg SC every 2–4 h prn as needed

Atropine

0.4 mg–0.8 mg SC every 2–4 h prn as needed

9.1.8 Nausea and Vomiting Nausea and vomiting are controlled by the vomiting center that get signals from 4 areas. 1. Chemoreceptor trigger zone, which is mediated by D2, 5HT3, NK1, mu (opioid) 2. Gastrointestinal track, which is mediated by D2, 5HT3, 5HT4, muscarinic 3. Vestibular system, which is mediated by muscarinic, H1

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Table 9.7 Nausea and vomiting medications: modified from a Raksasataya [30, 31] Medication

Typical starting dose

Max dose

Target receptors

Domperidone

10 mg oral 3 times before meal

60 mg/day

D2

Haloperidol

0.5–1 mg oral, IV, IM, SC at 20 mg/day bedtime

D2

Metoclopramide

10 mg q 6–8 h oral, IV/SC

D2, 5HT4

Cyclizine

25–50 mg PO, SC, PR q 8 h 200 mg/day

H2, muscarinic

Hyoscine hydrobromide

0.2 mg–0.4 mg IV, IM, SC q 2.4 mg/day 2–4 h prn as needed

Muscarinic

Hyoscine butyl bromide

10–20 mg SC q 4–6 h

120 mg/day

Muscarinic

Promethazine

12–25 PO, IV, SC, rectal q 4–8 h

150 mg/day

D2, H2, muscarinic

Chlorpromazine

25–50 mg oral, IM q 4–6 h 25–50 mg rectal q 6–8 h

2,000 mg/day

D2, H2, 5HT2, muscarinic

Prochlorperazine

5–10 mg PO, IM Q 6–8 h 25 mg rectal q 12 h

40 mg/day

D2, H2, 5HT2

Levomepromazine

5–25 mg PO, SC, IV, IM at bedtime

200 mg/day

D2, H2, 5HT2, muscarinic

Olanzapine

5–10 mg oral OD

20 mg/day

D2, 5HT2, muscarinic

Mirtazapine

15 mg oral at bedtime

45 mg/day

5HT2, 5HT3

Ondansetron

4–8 mg oral. IV q 12 h

24 mg/day

5HT3

120 mg/day

4. Cerebral cortex, which is mediated by GABA, H1 [29] Many medications could be prescribed for relief of symptoms. The principle is to select the correct medication for the specific receptor (Table 9.7). Dexamethasone 4–8 mg OD oral, IV, SC can relieve nausea/vomiting. However, the mechanism by which this is achieved is unclear. Delta-9-tetrahydrocannabinol (THC) from cannabis may relieve nausea/vomiting [32]. Clinicians should commence with slow THC and titrate slowly.

9.1.9 Communication Communication is a vital part of effective palliative care. Continuous practice is a primary key to achieve high levels of communication skills. Good communication skills enhance the shared decision-making model, empathy, patient/family satisfaction, good relationships/trust in a difficult time, and maintain the patient’s wishes, goals, and values in any circumstance. The protocols to deliver bad news and guide for empathic response are describe in Tables 9.8 and 9.9.

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Table 9.8 SPIKES protocol to deliver bad news: modified from Baile WF [33] Steps

Details

Setting

Arrange for some privacy Involve others significantly Sit down, make patient feel comfortable Make connection and relation with the patient Manage time constraints and interruptions

Perception

Determine what the patient knows about medical conditions Uses open-ended questions “What have you been told about your medical situation so far?”

Invitation

Ask patient if she/he wishes to know the details of the medical condition and/or treatment “Would you like me to give you all the information or just a short brief outline?”

Knowledges

Give information about disease, stage, previous treatment, current situation, and future plans Give information in non-medical language, small chunks, and at the same pace as the patient can understand

Emotion and empathy

Recognize and respond to patient emotions Show empathy to patients, e.g., using NURSE technique (Table 9)

Strategy and summary

Summary Give a chance for the last question from patient/family Set the date for next meeting Close discussion

Table 9.9 NURSE, the mnemonic of emphatic response: modified from Tulsky J [34] Techniques

Details

Naming

State the patient’s emotion “I think I can perceive how you feel, you feel…. Is it right?”

Understand

Empathize with and legitimize the emotion “This is a difficult situation, I think if I were you, I will feel like you”

Respect

Praise the patient for strength “I am very impressing, how good you can deal with this situation”

Support

Show your support “I will be here to help you get through this situation”

Explore

Ask the patient to elaborate on the emotion “Tell me more about how you feel”

Advance care planning is defined as a process that supports adults at any age or stage of health in understanding and sharing their personal values, life goals, and preferences regarding future medical care [35]. Evidence has shown that ACP has potential beneficial outcomes for patients and healthcare systems. Some barriers to advanced care planning is related to heath care professionals that include; hesitance to discuss end of life with patients, fear to take away patients’ hope, and the lack of training and skills in considering end of life communication [36]. RED-MAP

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Table 9.10 RED-MAP, advance care plan conversation guide: modify from Boyd K [37] Steps

Details

Example

Ready

Initiate conversation Can we talk about your health and care?

When would be a good time to talk? Who should join us? This about making good plans for your treatment and care

Expect

Evaluate function, perception, expectation of patient What do you know? What do you want to ask? What are you expecting…?

How have you been doing recently? What has changed? How do you see things going in the next days/ weeks/ months….? Some people think about what might happen if…? Can we talk about what might happen if you get less well?

Diagnosis Give information about diagnosis, stage, previous treatment, treatment option, and future plan We know… We do not know… Questions or worries?

What is happening with your (health problem) is…? We hope that…, but I am worried about… It is possible that you might not get better because… We do not know exactly when…, can we talk about that? Do you have questions or worries you would like us to talk about?

Matters

Evaluate wish, value, preference, acceptable quality of life, and goal of care What matters to you?

What is important to you that we should know? What would you like to be able to do? How would you like to be cared for? Is there anything you want/do not want?

Actions

Offer treatments to meet goals What can help… This does not work…

Things we can do are…. Options we have are… This does not work because… It will not help when/if…

Plan

Let us plan ahead for when/ if…

Can we make some plans so everyone knows what to do? Talking and planning “just in case” helps people get better care

is an advanced care planning conversation guide, that is widely used in Scotland [37] (Table 9.10).

9.1.10 Bereavement Bereavement is the state of loss resulting from death. Grief is the emotional response associated with loss. Mourning is the process of adaptation, including the cultural and social rituals prescribed as accompaniments [38]. These 3 phrases/ words are frequently used interchangeably.

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Grief and bereavement are a normal process of loss, and there is no definite time frame to indicate special intervention. Some elderly people spend a few years with normal grief. Whereas individuals who experience intense sorrow, significant impairment in social, occupational, or other important areas of functioning follow up is needed for early detection of pathological/complicated grief. It is a clinician’s task to identify and recognize such emotional stages, and provide support, empathy and ensure that the family can get back to usual life. If some cases are difficult to handle, the clinician can refer to a psychologist or other expert that can provide help.

9.1.11 Summary Palliative care is a vital part to deliver to incurable or advance stage cancer patients. It is a holistic approach which includes biological, psychological, social, and spiritual factors along the course of treatment until the end of life and bereavement. Maintaining good quality of life is essential by relieving pain, dyspnea, other suffering, and coping with difficult situation, family support, and psychosocial–spiritual support.

9.2

Palliative Biliary Drainage for Advance Stage Cholangiocarcinoma

Cholangiocarcinoma is mostly present with advance stage and only 10–20% are potentially appropriate for curative resection. Palliative chemotherapy and symptomatic care are treatment options for most patients to improve quality of life and prolong survival time. Obstructive jaundice is the most common symptom in advanced stage CCA that cause patients pain, pruritus, malnutrition, infection, and organ failure. Palliative biliary drainage is a principal procedure for the symptomatic care for the treatment of biliary obstruction. Palliative biliary drainage is a procedure used in advanced stage CCA in patients with significant symptoms including pain, severe pruritus, and cholangitis. Jaundice patients who do not have symptoms and expected life does not exceed 3 months are not considered for biliary drainage procedure. Other indications for palliative biliary drainage are to improve function status and liver function for further systemic chemotherapy. Palliative biliary drainage procedure has been divided into 3 approaches, namely, percutaneous approach, endoscopic approach, and surgical bypass.

9.2.1 Percutaneous Palliative Biliary Drainage Percutaneous palliative biliary drainage is performed by ultrasonographic guided puncture directly into the dilated intrahepatic bile duct through the skin. This

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Fig. 9.1 A DIY bathing kits

approach includes percutaneous transhepatic biliary drainage (PTBD) and percutaneous transhepatic biliary stenting (PTBS).

9.2.2 Palliative PTBD PTBD plays a major role in the treatment of proximal malignant biliary obstruction, especially in bismuth type III and IV perihilar CCA. PTBD has been shown to have better therapeutic success rate with fewer cholangitis complications compared to the endoscopic approach [39]. Our institute has been performing PTBD since 1984. We found that PTBD was an effective treatment to improve quality of life as it decreased disability and prolonged the patient’s survival in advance stage CCA [40]. However, caring for the PTBD drainage system may cause some difficulty in some patients, for instance there may be some problems of catheterrelated complications, such as wound and hygiene care, catheter dislodgement, and PTBD obstruction. To alleviate these complications, patients were trained for catheter care before being discharged from hospital. Furthermore, our team developed a bathing kit (Figs. 9.1 and 9.2) so that the catheter insertion site is kept dry and gives the patient more comfort during bathing. We have distributed this bathing kit to other hospitals throughout Thailand.

9.2.3 Complication of Percutaneous Transhepatic Biliary Drainage 9.2.3.1 Acute Cholangitis The exact etiology of postprocedural cholangitis is unknown. Multifactorial etiological includes ex-vitro infection from the drainage catheter, and reflux of intestinal flora during the procedure. Prevention during the procedure includes aseptic techniques, gentle manipulation of the guidewire, limited use of contrast media during cholangiograms, and the most important is prophylaxis antibiotics covering gram-negative bacilli. Bile aspiration for culture sensitivity must be performed.

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Fig. 9.2 Photograph shows our CCA bathing kit which keeps the catheter insertion site dry and give the patient more comfort during bathing

9.2.4 Hemorrhage Bleeding, usually transient after procedures can be caused by an accidental puncture to the vessel adjacent to the dilated bile duct. Conservative treatment can be performed in a patient with stable hemodynamic status. Temporally clamping the catheter for 24 h usually resolves bleeding. Severe bleeding causes hemodynamic instability and life-threatening conditions, and in such cases, laparotomy should be conducted to stop bleeding.

9.2.5 Pericatheter Leakage Pericatheter leakage is caused by a side hole of the catheter lying outside the bile duct. Intraabdominal leakage causes bile peritonitis and is usually found after a puncture in the patient with ascites. Diagnosis by cholangiogram shows leakage of contrast media into the peritoneal cavity. In such cases, the catheter should be repositioned with the guidewire and cholangiogram until contrast leakage is not evident.

9.2.6 Palliative PTBS PTBS is a procedure aimed at the palliative management of jaundice and the optional treatment in selected patients with malignant biliary obstruction. It has the benefit of bypassing the region of malignant obstruction and restores biliary drainage into the gastrointestinal tract to minimize loss of bile salts and electrolytes using plastic or metallic stents. The efficacy of PTBS with self-expandable metallic stent implantation is better than those of catheter drainage [41]. PTBS can decrease catheter-related complications and improve quality of life, as it does not require external drainage. Compared with plastic stents, metal stents are of larger diameter,

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have better long-term patency, and are more cost-effective [42, 43]. For the palliation of advanced hilar CCA (Bismuth III and IV), the outcomes of percutaneous stenting are superior to endoscopic stenting [44].

9.2.7 Planning and Preparation Imaging evaluation is the key to achieve procedural outcomes for patients. For instance, CT or MRI images are the basis for procedural planning. The purpose of reviewing these images is to determine the level of obstruction and assess functional liver parenchyma. Drainage of at least 50% or more of functional liver volume may be adequate, and typically the bile duct that drains the maximum amount of functional hepatic parenchyma should be targeted. Intravenous broad-spectrum antibiotics prophylaxis within one hour prior to the procedure is recommended to prevent transient bacteremia during or after procedure [44, 45]. At our institute, PTBD is the first management stage for palliative biliary drainage in perihilar cholangiocarcinoma Bismuth III-IV. After percutaneous catheter drainage, a cholangiogram is performed to evaluate the degree of obstruction. If the region of obstruction shows partial obstruction or a prior CT scan showed short segment of obstruction, then we will discuss with the patient about the PTBS option and arrange a schedule for PTBS installation.

9.2.8 Indications for PTBS 1. Obstructive jaundice due to unresectable malignant biliary obstruction, including the tumor which has invaded the hilar confluence. 2. Metastatic tumor at hilum, in which bile duct is compressed by tumor or enlarged lymph nodes. 3. Patients with high surgical risk resulting from a variety of factors, such as age, general asthenia, poor pulmonary or cardiac function, or technically unresectable perihilar CCA. 4. Patients who cannot tolerate or fail ERCP.

9.2.9 Contraindication for PTBS 1. 2. 3. 4.

Patients with poor systemic status. Massive ascites. Patients with a wide range of intrahepatic bile duct stricture. Patients with severe stenosis of the portal vein adjacent to the narrow site.

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9.2.10 Instruments and Procedures PTBS is performed under local anesthesia and moderate sedation by insertion of a 0.035 guidewire into the previously installed PTBD and subsequently, the PTBD catheter is exchanged with 5 French diagnostic catheters (Cobra catheter). Cannulation of the tract passing through the region of obstruction into the duodenum is done using a 0.035 guidewire whereupon a metallic stent is inserted. Usually, pre-stent dilatation of malignant biliary stricture is not required as this may cause tumoral bleeding leading to an early blockage of the stent [46] (Fig. 9.3 A– D). Following stent placement, the internal–external drainage catheter is inserted (temporary close catheter hub) as a fail-safe device in case of stent malfunction. Follow-up after biliary stenting involves clinical evaluation, laboratory investigation at 2 weeks. Cholangiogram through the internal–external drainage catheter to check patency of the stent at 2 weeks is performed followed by the removal of the internal–external drainage.

9.2.11 Complications Early complications of biliary stents are cholangitis, pancreatitis, and bleeding. Most common late complications are stent dysfunction, bleeding, cholecystitis and, less frequently, duodenal perforation. Recurrent jaundice or cholangitis due to obstruction of stents is the major complication of biliary stenting. The main cause of obstruction is biliary sludge and tumor ingrowth through the stent [47].

9.2.12 Endoscopic Biliary Stenting Endoscopic biliary drainage is the minimally invasive technique for the treatment of biliary obstruction by insertion of either plastic or self-expandable metallic stents (SEMS). CT and MRI scans of patients with suspected malignant biliary obstruction must be performed to assess resectability and these scans must be discussed in multidisciplinary team for thorough treatment planning. Incurable patients should be considered for palliative biliary drainage by ERCP with biliary stenting. The type of biliary stent is an important factor and should be carefully chosen for each patient according to the site of obstruction and the patient’s expected survival. The internal plastic biliary stent is made of polyethylene tube, with an approximate occlusion time of 3–4 months. Plastic stent occlusion is mostly caused by sludge deposition and bacterial biofilm adherence to stent surfaces. Replacement of plastic stents is required every 3–4 months; therefore, plastic stents were not recommended for patients with expected survival exceeding 3 months [44]. Plastic stents have low success rate of 54–84% in perihilar malignant obstruction compared to endoscopic SEMS which have a functional success rate up to 82–97% [42, 48]. Moreover, SEMS provide longer median survival time of 154 days compared to plastic stents of 43 days [42]. SEMS is the first line of

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Fig. 9.3 A: Cholangiogram via right PTBD shows malignant stricture at perihilar bile duct with contrast partially pass into the duodenum, B: 0.035 hydrophilic tip guidewire was passed through the stricture site into the duodenum, C: percutaneous type, self-expandable metallic stent was deployed followed by balloon dilation, D: Stent fully deployed in proper position then catheter and guidewire were removed

treatment in perihilar CCA, and the endoscopic approach is preferred in Bismuth type I and II. A percutaneous approach is preferred for Bismuth III and IV when there are technical difficulties in the endoscopic approach. Metallic biliary stents have stent patency longer than plastic biliary stents and external drainage catheter; therefore, it is the best option for palliative biliary drainage. SEMS is a wire mesh made of shape-memory metal alloy, nickeltitanium (Nitinol), or cobalt-chromium (stainless steel), which is mounted on a delivery system. SEMS was delivered in obstructed bile duct by ERCP and placed across the stricture site into the proximal bile duct. SEMS is deployed by retracting the cover sheath from a preloaded through the scope delivery system over the 0.035 guidewire with the ability to recapture a SEMS into the delivery catheter

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after partial deployment in case of inappropriate SEMS position deployment. After deployment, SEMS are expanded to a full diameter of 9–10 mm depending on angulation and stricture tightness. SEMS with high radial expansile force and flexibility is preferred for perihilar CCA. To achieve functional success in palliative biliary drainage in perihilar CCA, stent placement in atrophic liver or portal vein invaded liver segment should be avoided and 50% functional liver volume should be drained. Multiple SEMS may be needed in high grade perihilar obstruction, which can be performed by various techniques and different types of stents depending on stricture anatomy and endoscopist preference (Fig. 9.4).

Fig. 9.4 ERCP with bilateral SEMS by stent-in-stent technique with Niti-S large cell D-type biliary stent (Taewoong Corp., Seoul, Korea) A: Place the first stent into right hepatic duct B: Passing guidewire inside lumen of first stent through open mesh to left hepatic duct C: Deploy second stent into left hepatic duct D: Completion cholangiogram

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9.2.13 Type of Metallic Biliary Stent There are many types of biliary SEMS manufactured for various purposes and applications.

9.2.14 Fully Covered Type Fully covered stents are wire mesh SEMS with covering materials extending over the entire length of the stent. Fully covered SEMS can be removing after deployment and the covering film can prevent tumor growth into the stent lumen and occluded stent. Fully covered SEMS can only be placed in the distal bile duct. The proximal migration of fully covered stent may cause hilar bile duct blockage, segmental cholangitis, and cholecystitis.

9.2.15 Partly Covered Type SEMS with covering material with small areas at the ends left uncovered to decrease the chance of migration.

9.2.16 Uncovered Type Uncovered SEMS has no covering material, it can be placed in the intrahepatic bile duct without blockage of bile duct branches. Uncovered SEMS are completely buried inside the tumor to prevent stent migration. Therefore, the disadvantages are that uncovered SEMS are impossible to change or remove after fully deployed and early occlusion can occur by tumor ingrowth.

9.2.17 Outcome of SEMS The most common long-term complication of SEMS placement for malignant biliary obstruction is stent occlusion. Stent dysfunction has been diagnosed as the presence of two of the three of the following criteria including ultrasound showing new dilatation of intrahepatic or extrahepatic bile ducts; serum bilirubin ≥ 2 mg/dL with an increase ≥ 1 mg/dL compared to the value after initial successful drainage or elevation of alkaline phosphatases/gamma-glutamyl transferase to more than twice the upper limit of normal values with an increase of at least 30 U/L; and signs of cholangitis [49]. The most common mechanism of stent occlusion is tumor ingrowth, which is tumor growth through the open mesh of SEMS, that is more common in uncovered SEMS [50]. Tumor overgrowth, tumor growth and invasion blockage of the proximal or distal end of the stent is another cause in long-term stent occlusion. Sludge, mucus or debris obstruction

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can occur, but they are mostly concomitant with tumor ingrowth or overgrowth. The median patency of SEMS has been found to range between 5–6 months [48, 51] before the stents become obstructed. Management of occluded stents include balloon mechanical cleaning, plastic stent placement, metallic stent placement, and endobiliary radiofrequency ablation. Mechanical cleaning can be performed only in debris occlusion but occurs where mostly tumor ingrowth was concomitant, mechanical cleaning should be combined with other procedures. Second plastic stent placements have shorter patency of 60–90 days compared to second SEMS placements which have a patency of 100 days [50]. Endobiliary radiofrequency ablation is a novel intervention to ablate ingrowth tumor inside the stent lumen with safety and resulting in long-term patency equal to SEMS placement [52]. Endobiliary radiofrequency ablation can be repeatedly performed without compromising stent lumen, while second SEMS causes narrowing lumen diameter with more stent placement.

9.2.18 Surgical Biloenteric Bypass Surgical bypass has resulted in significant high perioperative mortality and morbidity of 0–17% and 17–55%, respectively [53]. Therefore, surgical bypass should be the last choice of palliative biliary drainage after percutaneous or endoscopic approach have failed. Surgical bypass can be performed in cases where distant metastasis are found during laparotomy. Surgical bypass can be performed by choledochojejunostomy for distal CCA or intrahepatic bile duct bypass for perihilar CCA. Extrahepatic bile duct bypass is not highly technically demanding, results in low morbidity, and is feasible with distal obstruction or perihilar Bismuth type I. Palliative surgical bypass for perihilar CCA is a complex surgery for each type of tumor, including Bismuth type IIIa, and is suitable for left hepaticojejunostomy or segment 3 bypass. Bismuth type IIIb requires right hepaticojejunostomy and Bismuth type IV requires right or left sectoral duct bypass. Surgical bypass for Bismuth type IV is mostly not effective because one anastomosis cannot drain enough functioning liver volume, therefore a bilateral hepaticojejunostomy bypass in Bismuth IV should be performed in very selected cases.

9.2.19 Operative Procedure Left sided hepaticojejunostomy Left hepaticojejunostomy can be performed at the left hepatic duct or segment 3 of the sectoral duct by the following steps: 1) Segment IV is retracted cranially to expose the area of the confluence at the base of segment IV 2) Bridge of liver tissue lies across the ligamentum teres joining segment III and IV, this can be divided

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3) Deep dissection along the glissoneon sheet allows left portal structures to be lowered from the base of segment IV. Portal vein to segment IV and middle hepatic artery may require exposing sufficient length of left hepatic duct. 4) Longitudinal incision made on the exposed duct with optimal length more than 2 cm 5) Roux-en Y jejunal limb prepared 6) Side-to-side single layer interrupted mucosal to mucosal anastomosis is performed using a 4–0 monofilament suture start with posterior layer 7) Placing the anterior row of sutures into the bile duct (leaving the needles attached) 8) Parachute technique performed to approximate posterior layer anastomosis and tied sutures 9) Anterior layer can be completed under direct vision

9.2.20 Right Sided Hepaticojejunostomy Right sided anastomosis requires identification of the right anterior sectoral duct by dividing the overlying liver parenchyma to approximately half-way of a parenchymal transection in usual hepatectomy. The right posterior sectoral duct is the most difficult to expose and perform anastomosis, therefore a right sided bypass should be performed on the right anterior sectoral duct. The steps of the anastomosis technique are the same as in left hepaticojejunostomy. Palliative biliary drainage provides good symptom control and prolongs the patient’s survival in advanced stage CCA with symptomatic obstructive jaundice. A non-invasive approach either percutaneous or endoscopic should be considered first. A multidisciplinary team care is critical to provide the most appropriate multimodality treatment for good quality of life and survival of the patients.

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Digital Innovations (Isan Cohort) Bandit Thinkhamrop, Kavin Thinkhamrop, Chaiwat Tawarungrueng, and Panuwat Prathumkham

Contents 10.1 10.2 10.3 10.4 10.5 10.6 10.7 10.8

Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . OV-CCA Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tele-Radiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathology Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Surgery Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Palliative Care Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Randomized Controlled Trial (RCT) Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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The roles that information technology can play are vital in cancer prevention and care. The Surveillance, Epidemiology, and End Results (SEER) Program, at https:/ /seer.cancer.gov, is an example of a comprehensive system that provides information on cancer statistics for the Ultra Sound (U.S.) diagnosed population [1]. Similarly, the Isan Cohort is a comprehensive system to combat Opisthorchis viverrini (OV)-associated cholangiocarcinoma (CCA) in the Thai population where the incidence of CCA is the highest in the world. Details of the design and early findings are available elsewhere [2]. In this chapter, we discuss the background, B. Thinkhamrop (B) Department of Epidemiology and Biostatistics, Faculty of Public Health, Khon Kaen University, Khon Kaen 40002, Thailand e-mail: [email protected] B. Thinkhamrop · K. Thinkhamrop · P. Prathumkham Health and Epidemiology Geoinformatics Research (HEGER), Faculty of Public Health, Khon Kaen University, Khon Kaen 40002, Thailand C. Tawarungrueng Epidemiology and Biostatistics Program, Faculty of Public Health, Khon Kaen University, Khon Kaen 40002, Thailand © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. Khuntikeo et al. (eds.), Liver Fluke, Opisthorchis viverrini Related Cholangiocarcinoma, Recent Results in Cancer Research 219, https://doi.org/10.1007/978-3-031-35166-2_10

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features of the system, and lessons learned in the implementation of the Isan Cohort.

10.1

Background

In 2013, a database system was designed for hard copy paper-based research. It was called CASCAP Tools which is located at www.cascap.in.th (Fig. 10.1). This website has been used since 2013 despite in recent times having being defined as the Isan Cohort. The former name was an abbreviation which stands for the Cholangiocarcinoma Screening and Care Program (CASCAP). The latter name was used to reflect the main population at risk of CCA, namely, the population in the northeastern region of Thailand which is also called “Isan”. However, the Isan Cohort becomes a brand name as the cohort members expanded beyond the Isan region. Indeed, it now covers all of the Thai population irrespective of the region. Once people meet the criteria for enrollment to the Cohort, they become members of the Isan Cohort. In essence, CASCAP Tools drive the Isan Cohort. The paper-based data collection system was used only in the first year since the inception of CASCAP as information technology was not well equipped within the region. For instance, the paper-based system was used in a mobile ultrasound screening campaign for CCA at an area without an established internet network. We found that using a paper-based design did not provide the basis to ensure a high quality of data as it was difficult to achieve since there was no real-time validation checking at the entry point of the data. Furthermore, delays occurred in the routine availability of reports for the healthcare providers and the policymakers. These difficulties resulted in increased costs in running the program and obtaining hard data. Fortunately, in 2014, the second year of implementation, the availability of information technology allowed us to improve the system so that it was completely online where there was no paper being used in the CASCAP activities, except for the informed consent processes where the participant’s signature must be an original on an appropriate paper document. This enabled the implementation of a validation check at the point of data entry. Following these introduced procedures, the data were enhanced in quality and were promptly available for reporting. Many add-on web applications were developed in the CASCAP Tools as user modules (Fig. 10.2). Like an application, a module is a collection of tools for specific tasks. Details of some important modules are described in the next section.

10.2

Overview

To reiterate, the CASCAP Tools drive the Isan Cohort. Figure 10.3 summarizes the modules that are the core system of CASCAP Tools. The OV-CCA module is the main system covering more than 80% of the activities as well as the size of the database. The Tele-Radiology module comprises both the web and the

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Fig. 10.1 Website of the Isan Cohort

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Fig. 10.2 Add-on modules within the CASCAP tools

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Fig. 10.3 Core system of CASCAP tools

mobile applications for hepatobiliary ultrasonography screening and for confirmation of diagnosis using a computerized tomography scan (CT-scan) or magnetic resonance imaging (MRI). The Pathology database is for documenting the pathological findings for each patient. The Surgery database is for clinical data collection as well as the treatments being provided to patients. The Palliative Care database is for following up the patients regarding their disease progressions and home care consultations. Importantly, these modules cover a spectrum of patient care from primary prevention to the end-of-life care. The last module, the Randomized Controlled Trial (RCT) database, is a research management system designed for an RCT under the CASCAP. All modules comprise tools for data management, monitoring of the services to comply with the CASCAP protocols, and evaluation of interventions. Each participating hospital can see all their data as well as real-time reports with sufficient details for guiding specific actions, if needed. Reports that combine the results from every hospital are also available for district, province, health region, and national levels. The important aspect here is that the participating hospitals need only do things to serve people under their own responsibilities. All other tasks that they were formerly required to perform, such as data analysis, report writing to the higher level of commands, and project evaluations, are undertaken and managed by the CASCAP Tools. Convincing phrases, such as, “Give less—Get more” are widely known among healthcare institutes by the time of scaling up the CASCAP. These real-time reports play a very important role as they reduce the effort required in data analysis for the hospitals as well as reflecting performances of the hospitals to effectively deliver their services which, in turn, maximizes the coverage of the services. The National Committee for CCA Eradication uses these reports for

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monitoring, evaluation, and guiding specific actions to solve the problems at the policy level. Figures 10.4 and 10.5 are examples of the reports. The following details how the Isan Cohort can be easily scaled up. First, any healthcare centers or hospitals can apply for an account at www.cascap.in.th. Each hospital will then be designated a unique URL. This is indeed a cloud health center or hospital for CCA. The whole and blank CASCAP database equipped with all modules is duplicated for a new account. All applications for CASCAP tasks as well as real-time reports are available and ready to use from the time of initiation of the site. The only requirement is that a website administrator needs to be officially authorized by the Director of the hospital. The site administrator will then be responsible for managing the members of the site as to who can access what modules in the system. This can be done without any necessary specialist training. Secondly, if a hospital desired to link their existing hospital information system (HIS) to the CASCAP database without the need of manually uploading the data, the administrator will need to install a database connector with some assistance from the CASCAP Tools developers. However, this is optional, and we found that few hospitals prevented any software to be installed to their HIS. In this case, the healthcare personnel of those hospitals must manually upload the data required by CASCAP that exists in their HIS to the CASCAP database. At this point, everything is set and ready to use. With this system in place, healthcare providers can do their assigned tasks according to the CASCAP protocols in a systematic manner. All data being entered are fully and efficiently utilized. With this system in place, healthcare providers can do their assigned tasks according to the CASCAP protocols in a systematic manner. All data being entered are fully and efficiently utilized. The routine data became the information for action—not for storage as it usually is. A number of reports that are available in real time can assist the providers to monitor their progress and guide them to take a specific action(s) if required. These have never existed in their current situations. In addition, when Cohort members may require patients transferring to a higherlevel hospital for further investigation(s) or receiving advanced treatments, the CASCAP Tools facilitate these processes seamlessly without borders between hospitals. This can be done in Thailand as every Thai citizen has their own 13-digit identification number. The CASCAP Tools become a transferring center across the country for CASCAP purposes. Significantly, it is a healthcare system based on a patient-centered approach. The adoption rate of CASCAP Tools among healthcare centers in Thailand was greatly accelerated after the endorsement of the Government of Thailand that CCA was a national agenda and priority. The Isan Cohort (and the tools therein) was used for implementing the Programs and is the core database for monitoring and evaluating interventions under the Programs. Five years after implementation, the CASCAP Tools were adopted by all provinces in the northeast region, half of the provinces in the north, and two provinces of the east region of Thailand. There were approximately 4000 healthcare centers that used the system with data for

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Fig. 10.4 Example of a real-time report for hepatobiliary ultrasonography screening

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Fig. 10.5 Example of real-time reports for geographical distribution of participants according to their status—hepatobiliary ultrasonography screening results or being diagnosed as having cholangiocarcinoma

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more than 20 million of the Thai population currently stored in the database. The database continues to expand in real time geographically as well as the expansion of the scope of services provided.

10.3

OV-CCA Module

This module facilitates healthcare providers at every level of care in performing activities according to the CASCAP protocols. However, it is mainly used by the primary and secondary care levels or at the healthcare centers and community hospitals. This is because the Thai National Health Security Office has assigned almost all Thai citizens to be under each of these healthcare facilities. Only a few have been assigned to a higher level of care. The module enables health providers to have access to all CASCAP data no matter at what level or where they provide service for their population. For instance, for a hospital without facilities for CTMRI, pathological testing, or CCA treatment, the data of their patients who have been treated at any referral hospital will be transferred to their database. Hence, they are fully informed of the health status of each of their patients within the population they serve. Important components of this module are as follows: • Database connector for automatically transferring the eligible population in the HIS database to the CASCAP database. This can also be done manually by uploading (or typing) directly the target population as the eligible population for OV-CCA screening. • Registration Tools for the process of: – Informed consent – Verbal screening by collecting baseline information and selected risk factors for OV and CCA • OV Screening Tools • Hepatobiliary Ultrasonography Screening – Mobile application – Web-based real-time reports of ultrasonography screening • Referral System • Data Quality Assurance System These tools facilitate healthcare providers to register their target population and conduct verbal screening for candidates for further screening. By utilizing this approach, informed consent forms and baseline information regarding risk factors of CCA can be obtained and stored in the database. Hence, they provide the basis for the inception of the Cohort. Subsequently, there are two main tools used to screen these Cohort members, namely, OV infection screening, and ultrasonography screening for any hepatobiliary abnormalities. For OV screening, there are tools for collecting and managing the specimens, both stool and urine, and tools for reporting the results. For the ultrasonography screening, details are provided in the next section.

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Tele-Radiology

The mobile application allows the ultrasound screening processes to be undertaken anywhere in remote geographical areas while the results being documented are available in a real-time manner. A large number of mobile devices can be used without the need of connecting to the internet as the data can be stored in the device at time of data entry, which can then be synchronized to the CASCAP database once connecting to the internet after arriving at home institutions. This was found to be very useful for a campaign where there were thousands of participants in lines of different services. The working processes can then be distributed, for instance, healthcare providers use their own mobile devices to install the application(s), thereby being able to do the tasks simultaneously. Consequently, the range of services can flow smoothly without any bottle necks. Ultrasound images are stored in the database allowing radiologists to examine images and provide diagnosis by general physicians, in real time, in case patients need further consultations or some important findings need to be reviewed according to the protocols. Additionally, patients whose data suggest that they had suspected CCA can be closely monitored and processed for further investigations via alert/warning algorithms for responsible healthcare providers. The Tele-Radiology module includes tools for the physicians to provide ultrasound screening for the Cohort members where work lists exist for them prior to an operation, an electronic form for documenting the findings, instructions for storing the ultrasound images to the CASCAP database, and consultation or discussion regarding the findings with the radiologists who work for the CASCAP. Such consultations comprise tools to retrieved and examine the images and comments or discussions for specific issues being addressed. Additionally, there are tools for transferring participants with suspected CCA for confirmatory diagnosis using CT or MRI. For patients with positive results, there are tools to transfer them to the hospital for treatment. Healthcare providers also have tools to monitor the processes with an alert or warning if there are patients that were not identified according to the protocols. In such cases, the system facilitates corrective actions by just a “click and make a phone call” to the responsible healthcare personnel so that no person is left undiagnosed.

10.5

Pathology Database

This module serves as a tool for pathologists under CASCAP to document findings regarding the results of pathological tests of all CCA patients that required this confirmatory diagnosis. Use of this tool has found that the proportion of CCA cases with pathologically confirmed CCA is increasing at an alarming rate which has never been recorded to date. These findings are not only helping the treatment process but are also an important basis for subsequently publishing important comprehensive data now and in future.

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It is the world largest database for pathologically confirmed OV related CCA cases.

10.6

Surgery Database

Regarding CCA treatment, there are tools for both patients from the screening cohort and walk-in patients. The tools comprise an in-transfer for the screening cohort and the enrolment process for walk-in patients. Subsequently, there is a form for documenting clinical findings, treatment, or the operation being provided for every visit. However, patients with late-stage CCA receive palliative treatment and then immediately transferred to end-of-life care.

10.7

Palliative Care Database

For the CCA end-of-life care, there are tools for the healthcare providers to know who their patients are at this stage and who and when to follow them. Patients will be followed according to the protocols until they pass away.

10.8

Randomized Controlled Trial (RCT) Database

This system utilizes 10 years of experience of the developers based on their development and operation of a system for a Randomized Control Trial (RCT) for pharmaceutical companies in Thailand. The system comprises essential international standards including the ICH-GCP and the 21 CFR Part 11. The system was subsequently used for the first RCT under the CASCAP entitled “Gemcitabine alone versus Gemcitabine plus Cisplatin as an adjuvant chemotherapy after curative intent resection of cholangiocarcinoma (GeCiCCA)”. Conclusion In conclusion, the Isan Cohort is a comprehensive system for combating CCA for the Thai population where the incidence of CCA is the highest in the world. It is a unique liver fluke related CCA caused by the group 1 carcinogen, OV. The Cohort members ranged from the OV-risk cohort, OV-positive cohort, cohort with various types of hepatobiliary abnormalities based on ultrasonography results, suspected CCA cohort, and the CCA patient cohort. For example, the current size of over a million members of a cohort with regular ultrasonography screening at least annually is unique. It is the largest database in the world that provides the foundation to provide answers in the future for currently unanswerable questions regarding OV related CCA. Furthermore, it will enable healthcare professional to understand with a high degree of accuracy how changes over time, for example, how long it takes liver fluke-infected people to develop CCA. The longer the period of follow-up, the more accuracy of the prediction we can obtain which might take many years.

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The instigation, development, and application of the Isan Cohort is indeed a Thai national asset, in particular, for future generations. Introduction of the Isan Cohort throughout liver fluke related CCA regions in Southeast Asia and elsewhere globally will assist in eliminating/controlling this insidious disease.

References 1. Gallicchio L, Elena JW, Fagan S, Carter M, Hamilton AS, Hastert TA, et al. (2020) Utilizing SEER cancer registries for population-based cancer survivor epidemiologic studies: a feasibility study. Cancer Epidemiol Biomarkers Prev 29(9):1699–709. Epub 2020/07/12. https://doi. org/10.1158/1055-9965.EPI-20-0153. PubMed PMID: 32651214; PubMed Central PMCID: PMCPMC7484198 2. Khuntikeo N, Chamadol N, Yongvanit P, Loilome W, Namwat N, Sithithaworn P, et al. (2015) Cohort profile: cholangiocarcinoma screening and care program (CASCAP). BMC Cancer 15:459. Epub 2015/06/10. https://doi.org/10.1186/s12885-015-1475-7. PubMed PMID: 26054405; PubMed Central PMCID: PMCPMC4459438

RAW ATTITUDES: Socio-Cultures, Altered Landscapes, and Changing Perceptions of an Underestimated Disease

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Carl Grundy-Warr, Ross H. Andrews, Narong Khuntikeo and Trevor N. Petney

Contents 11.1 An Ecologically Embedded and Socially Entwined Life Cycle . . . . . . . . . . . . . . . . . 282 11.2 An Underestimated and Neglected Parasite in World Public Health . . . . . . . . . . . . . . 283 11.3 Socio-Economic Dimensions of Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284 11.4 Developmental Landscapes and Anthropogenic Ecologies . . . . . . . . . . . . . . . . . . . . . 286 11.4.1 Aquaculture and Irrigation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 11.4.2 Dams and Reservoirs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 11.4.3 Roads and Rivers: Migration and Mobility . . . . . . . . . . . . . . . . . . . . . . . . . 294

Dedicated, long-term, inter-disciplinary research programs incorporating both scientific and social methodologies may help facilitate plans for grounded disease prevention strategies and health education measures in many parts of the region [34].

C. Grundy-Warr (*)  Department of Geography, National University of Singapore, Singapore, Singapore e-mail: [email protected] R. H. Andrews  Department of Surgery and Cancer, Faculty of Medicine, Imperial College, London, UK R. H. Andrews · T. N. Petney  Cholangiocarcinoma Research Institute, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand N. Khuntikeo  Cholangiocarcinoma Research Institute, Department of Surgery, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand T. N. Petney  Departments of Zoology and Paleontology and Evolution, State Museum of Natural History Karlsruhe, Erbprinzenstrasse 13, 76133 Karlsruhe, Germany © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 N. Khuntikeo et al. (eds.), Liver Fluke, Opisthorchis viverrini Related Cholangiocarcinoma, Recent Results in Cancer Research 219, https://doi.org/10.1007/978-3-031-35166-2_11

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11.5 Uneven Regional Knowledge About the Extent of Opisthorchis viverrini and Opisthorchiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296 11.6 Public Health Programs Tackling OV and CCA in Thailand . . . . . . . . . . . . . . . . . . . . 300 11.7 Raw Attitudes in Isan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301 11.8 Raw Attitudes in Lao PDR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310 11.9 Raw Attitudes in  Vietnam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311 11.10 Raw Attitudes in Cambodia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316 11.11 Altering Attitudes and Public Health Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338

The first paper that deployed the provocative term ‘raw attitudes’ revealed how culturally embedded raw- and partially cooked fish eating was in the Mekong region. This chapter seeks to explore these ‘raw attitudes’ further and to show how there are multiple moving parts to the story that entail appreciation of biophysical and social environments, as well as how these settings are being continually transformed, which further requires research mindsets and methodologies that gain deeper understanding of fish-borne zoonotic trematodes (FZTs) dynamics within complex anthropogenic ecologies. Such approaches require thinking through the multiple environmental, land-use change, developmental, socio-economic, and cultural factors that impinge upon FZT life cycles.

11.1 An Ecologically Embedded and Socially Entwined Life Cycle Ecological studies have found ‘high levels of population genetic variability in Opisthorchis viverrini in different wetlands in Thailand and Lao PDR’ indicating that ‘we have underestimated the complexity of this epidemiological situation’ [2]. The O. viverrini life cycle thrives in parts of the Lower Mekong Basin, with its many water bodies, rivers, streams, wetlands, irrigated paddy fields, and monsoonal flood-pulsed ecosystems, but there is scope for detailed research on differing aspects of liver-fluke ecology, its life cycle, and sundry anthropogenic influences relating to prevalence and human infection. This leads to a reflection about life cycles as being deeply rooted within ecosystems and impacted by anthropogenic influences upon those ecosystems [91]. Studying issues of prevalence and of human infection takes us into economic, social, and cultural arenas. This chapter considers physical and anthropogenic ecologies of the life cycle by building upon earlier work focusing on ‘raw attitudes’ relating to the cultural landscapes of wetlands and farm-fishing zones of the Mekong region [34]. The chapter also explores ‘interfaces between the environmental impact of humans’ and the ‘epidemiological cycle’ [91].

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11.2 An Underestimated and Neglected Parasite in World Public Health There is a tendency to consider O. viverrini ‘to represent a limited, local problem in Southeast Asia’ [2], which is misleading on three counts. First, the life cycle of O. viverrini is embedded in the physical landscapes of the Mekong region [113, 142]. Within the countries bordering the Mekong, there are an estimated 90 million people at risk of infection with an estimated 10 million being infected [2, 53]. Thus, it is apt to consider it widespread (in terms of prevalence, infection, and disease) rather than ‘limited’, particularly as there are potentially suitable O. viverrini life cycles that transcend multiple localities and boundaries. However, the perception of infection and disease as being ‘limited’ is primarily based upon the presence of ‘accurate spatial and temporal morbidity data’ [2], and available data sets relating to cholangiocarcinoma (CCA), the bile duct cancer that is caused by O. viverrini and invades human liver tissues. Thus, it is misleading to consider a problem as ‘limited’ and ‘local’ as there is growing evidence to suggest O. viverrini endemicity in multiple localities across international borders, and as reliable spatial–temporal data sets are underdeveloped in the region as a whole. Second, the parasite’s life cycle is not just physical but socially embedded as it is interdependent with human livelihoods, wetland socio-ecologies, farming-fishing cultures, and food choices (eating raw, semi-cooked, fermented fish dishes), particularly among people engaged in farming-fishing and in a region where freshwater fish are the major source of animal protein [34]. Third, accurate qualitative evidence about parasite and disease prevalence has received detailed coverage in northeast Thailand, central and southern Laos, and parts of Vietnam, but the evidence of O. viverrini endemicity is geographically far from complete. Studies that find evidence of an active life cycle, with snail, fish, and human infection elsewhere in the region, tend to provide snapshots of specific contexts at particular times. Some studies aim to apply quantitative modelling and ‘evaluate the epidemiological parameters’ in order to generate ‘evidence-based strategies for parasite control’ at different spatial scales [16]. Others apply highresolution risk maps to reveal uneven spatial concentrations of infection and risk [151]. Mass screening by abdominal ultrasonography has found that in endemic areas of northeast Thailand, 17.0% of individuals had periductal fibrosis and 1.2% had suspected CCA [15]. The Cholangiocarcinoma Screening and Care Program (CASCAP) in Thailand has amassed significant geo-spatial data for the analysis of CCA prevalence and hepatobiliary abnormalities down to sub-district levels [125, 127] with healthcare workers in many villages enrolling at-risk patients under a ‘CASCAP Tools’ web application [53]. Notwithstanding advancements in digital survey data and geographically specific scientific investigations, there remain zones of the Mekong region where detailed data on helminth infection is rare and O. viverrini infection is ‘underreported’ [49], particularly where ‘epidemiological studies are scarce and the spatial distribution of infection remains to be determined’ [30]. Trans-border prevalence

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of O. viverrini is established beyond the ‘core areas’ of infection [122], but there is not enough epidemiological data to know how many people in the region are actually infected with the parasite beyond extrapolations of what is known. Furthermore, the non-core areas of infection are in zones where there has been little to no research, which includes zones beyond the so-called core areas but sharing similar environments, biophysical characteristics, and agrarian practices. All of which strongly indicates that O. viverrini is indeed an underestimated parasite in the region and in world public health. Another critical issue relates to the fact that the countries of Southeast Asia harbour a mostly hidden burden of neglected tropical diseases (NTDs) [39]. The epidemiology of such diseases is often linked to parasite life cycles and various pathogens that are well suited to certain tropical environments and landscapes. Globally, NTDs ‘disproportionately affect the “bottom billion,” which refers to the approximately 1.4 billion people who live below the World Bank poverty figure of US$1.25 per day’ [40]. There are highly uneven socio-economic burdens relating to NTDs, and a neglected dimension of these diseases is the fact that they tend to impact upon poorer groups in society the most. Lower socio-economic status is associated with greater likelihood of disease [135]. Thus, epidemiological studies and public health interventions require sensitivity to socio-economic inequalities, which may include uneven access or abilities to access health services, and issues of poverty exacerbating disease and of disease-induced poverty [100].

11.3 Socio-Economic Dimensions of Disease Cholangiocarcinoma is an extremely aggressive cancer that is usually fatal [53], and opisthorchiasis [2] is a neglected and underestimated disease which, as the major risk factor for CCA, wreaks havoc for hundreds of thousands of individuals, households, and communities [121]. Aside of public health impacts, disease incurs direct and indirect socio-economic costs at multiple scales [50]. Cholangiocarcinoma is a primary cancer originating in the biliary epithelium and is extremely invasive, often metastasizes, and has a very poor prognosis [8, 111]. As [121] observe: ‘among the helminth parasitic infections none can rival the high mortality levels of opisthorchiasis’. Apart from obvious signs of morbidity, opisthorchiasis is associated with inflammatory changes to the liver during disease progression, which may have debilitating effects on human health. Ultrasound screening in O. viverrini endemic areas of northeast Thailand has found that some patients have hepatobiliary abnormalities, including enlargement of the left hepatic lobe and the gallbladder, and cases of periductal fibrosis are common [68–70]. Hepatobiliary problems would likely remain clinically silent without ultrasound and other tests, which undetected can lead to the development of CCA many years after initial infection with O. viverrini. As [53] report, most CCA patients are seen during late-stage disease with 5 year survival being less than 10%.

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Rural societies have been greatly affected by big transformations over the last 50 years, including demographic ones as younger people of working age have sought work in towns and cities, and agrarian transformations through changes in land tenure, commodification via commercial crops and plantations, and in some areas, loss of farmland to industries and urban expansion. In addition, people’s lifestyles are changing and there are generational implications as younger people are exposed through education and migration to different diets, habits, ways of living, and so on. These economic transformations have occurred differentially but over time affect all countries of the Mekong region. Thus, in northeast Thailand, screening of at-risk populations found that liver fluke infection has been particularly high for men and women over 50 years of age, most of whom had only primary school education, and the majority of whom are farmers who reported eating uncooked fish [53]. Younger people are influenced by the economic transformations of rural life and their schooling, especially high school takes them out of village contexts. Studies reveal that higher education is crucial in providing a ‘higher protective effect’ against contracting tropical diseases [124]. Other studies reveal significant gendered differences in attitudes to raw fish consumption with women playing greater roles in public health awareness campaigns [144]. Disproportionate socioeconomic burdens of disease fall upon individuals, families, and communities with CCA diagnosis, which are ‘unplanned and unexpected’ events [100], can turn relatively affluent families into paupers due to the loss of income-earning, the high costs of daily care, travel from home to hospitals, medical treatment, restructured livelihoods, disruptions to family life, and funeral expenses. As Khuntikeo et al. [50] put it: ‘We argue that the socioeconomic burden is high on many levels for affected households and families, for communities that are particularly adversely affected, and at the regional scale where we are only beginning to understand the cumulative impacts over time’. The COVID19 (SARS-CoV-2) global pandemic has adversely affected vast populations, although research reveals socioeconomic burdens to be highly uneven, with the worst affected groups often being the most vulnerable and poorest groups in society [132]. COVID-19 generated massive havoc on the world stage in a short span of time. In contrast, neglected tropical diseases create cumulative burdens over very long time periods. COVID-19 presented societies with sudden and sweeping threats to life which generated extraordinary public health responses involving massive funding reallocations and reorganisation of health priorities, whereas efforts of create coordinated responses to endemic neglected tropical diseases may take decades of incremental change to develop into fully national programs with long-term support [42, 43]. The very embeddedness of pathogens and parasitic life cycles within biophysical and socioeconomic landscapes creates vital challenges for public health and evidence-based control strategies. The following subsection examines large-scale developmental impacts occurring at sub-regional scales that can alter broader ecologies of which FTZ life cycles are a part.

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11.4 Developmental Landscapes and Anthropogenic Ecologies Anthropogenic alterations to physical landscapes through political economic projects do have impacts upon parasitic life cycles [91]. In the Mekong Basin, consideration of cross-border movements of water, fish, birds, stock animals, and humans should be an aspect of a larger-scale understanding of interconnections relating to human health and disease. In the following subsection, three material arenas relating to developmental changes are examined: aquaculture ponds, water channels, irrigation works, hydropower dams, and roads. Finally, the role of human movement and cross-border migration are explored. Since borders were opened up after the Cold War, developmental projects are transforming the socio-economic landscape of the entire Mekong region, and they create anthropogenic ecologies that impact upon parasite life cycles, human health, and disease. Land use changes, resource-induced landscape transformations, and human mobility have implications for the spread of O. viverrini infection.

11.4.1 Aquaculture and Irrigation Aquaculture is of increasing significance in the Mekong region. For example, in Lao PDR the contribution of aquaculture to fish supply has bypassed wild-capture fishery production since 1997 [29]. There exist five kinds of aquaculture, including the production of rice and fish grown together, fish ponds, cages, hatcheries, and integrated livestock and fish operations. Aquaculture is a form of farming fish, as opposed to wild-capture fisheries, and cultured fish are sent increasingly to translocal and transnational markets, and they rely on imports of juvenile fish for stocking farms. Within mainland Southeast Asia and Southwest China, there is growing demand for fingerlings to stock aquaculture farms, which increases the potential for importing FZT infected specimens from hatcheries to farms, and across borders. As observed by [91], ‘Clonorchis sinensis (a related human fish-borne liver fluke parasite) and O. viverrini can infect common freshwater aquaculture fish, such as the common carp (Cyprinis carpio), the grass carp (C. idellus), the silver carp (Hypophthalmichthys molitrix), the topmouth gudgeon (Pseudorasbora parva), the goldfish (Carassius auratus), and the Amur bream (Parabramis pekingensis)’, as well as one of the most common commercial cultured fish, tilapia (Oreochromis mossambicus) are susceptible to C. sinensis infection. Different studies from mainland Southeast Asia reveal that aquaculture contributes to the spread of fish-borne zoonotic trematodes (FZTs), including O. viverrini. Examination of cultured fishes from the Mekong Delta from carp poly-culture and intensive small-scale integrated vegetable–aquaculture–animal husbandry farming (VAC) systems reveal that FZT metacercariae were common and ‘belonged to the Heterophyidae family of trematodes, Haplorchis pumilio, H.

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taichui, Centrocestus formosanus, and Stellantchasmus falcatus’ [126]. They found the FZT infection was the highest in the flooding season in carp poly-culture. ‘Landscape determinants’ are highly relevant arenas for studying FTZs and water-borne trematodes [143]. Small aquaculture ponds and rice fieldponds may provide potential microhabitats for the full life cycle of O. viverrini (Figs. 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7 and 11.8). Studies have found such ponds to be ideal sites for perpetuating the life cycle of FZTs [10]. The Bithynia snail populations tend to thrive in certain conditions, and they may be distributed widely by irrigation water channels in rice fields. Seasonal flooding will affect not only the distribution of water but of snails as water can transport snails from one area to another, and may flow into ponds, particularly those nearby paddy fields. Studies of land use impacts of rice paddy cultivation and seasonality on life cycle dynamics are significant, for as Kopolrat et al. [59] reveal, ‘the prevalence of O. viverrini infection in Bithynia siamensis goniomphalos varies with the amount of rainfall, with peaks of infection occurring after the cool dry season, that is, after each rainy season’ [59]. Furthermore, anthropogenic changes to the agrarian landscape impact on the life cycle. As Kopolrat et al. [59] observe: ‘Bithynia siamensis goniomphalos and B. funiculata, first intermediate hosts of O. viverrini, most frequently inhabit shallow freshwater environments such as rice fields, roadside ponds, and irrigation canals with an underlying red-yellow podzolic soil’. This study revealed the potential for changes in agricultural practices such as dry season irrigation and double cropping to introduce snail primary hosts into areas

Fig. 11.1  Paddy fields in lowland areas of the Mekong Basin are ideal habitats for Bithynia snails

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Fig. 11.2  Irrigation channels provide highways for snail transport

Fig. 11.3  Snail in irrigation channel

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Fig. 11.4  Collecting Bithynia snails

Fig. 11.5  Local farmer fishing in ponds near his fields

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Fig. 11.6  Fish caught by farmer

Fig. 11.7  Aquaculture pond in Xepon District, Savannakhet Province, Lao PDR

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Fig. 11.8  Farmland in Isan at the beginning of the rainy season

where they were absent and may help boost their population density. Thus, rice paddy cultivation, irrigation channels, the presence of multiple natural ponds and man-made aquaculture ponds, combined with seasonal water-level fluctuations and anthropogenically modified hydrological conditions, potentially create ideal conditions for these Bithynia spp. and a variety of cyprinid fishes, the second hosts of O. viverrini. As farmers regularly fish in retention ponds, lakes, water channels, and paddy fields, the potential for humans to become infected then depends on how fish are prepared and consumed. Farmland is mostly at some distance from homes, and so open defecation in fields is a further factor that helps to continue the O. viverrini life cycle [106]. In Vietnam, studies show considerable incidence of FZT infection of juvenile fish in nurseries before being sold in local markets [24, 93]. Vietnamese researchers recognise the role of aquaculture in FZT transmission and why cultured fish should be part of public health strategies [92]. In northeast Thailand, Pitaksakulrat et al. [97] ‘detected O. viverrini and FZT metacercariae (Centrocestus formosanus and Haplorchis taichui) in two popular fish species, Barbonymus gonionotus (silver barb) and Cirrhinus mrigala (mrigal), from aquaculture farms’ in Khon Kaen province. Cultured fish species are not free of FTZs, and cross-infection to native freshwater species is possible. As observed by Sithithaworn et al. [114], ‘the prevalence of O viverrini in Cyclocheilichthys armatus—a native fish that coexists with aquaculture pond species— is ten times higher in Lao PDR than in Thailand (40 vs 3%), suggesting active and ongoing transmission of the disease after stocking with infected fish’. If conditions surrounding ponds are suitable for Bithynia snails and the ponds are stocked with fish species susceptible to infection by free-swimming cercariae, then fish culture further exposes humans to infection, especially if small ponds serve the host farm, family, and district markets, and the fish are used for making

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raw, semi-cooked, or fermented fish dishes. Conditions in many parts of central and southern Laos, central and southern Vietnam, lowland Cambodia, and north and northeast Thailand are suitable for the life cycle of O. viverrini to flourish on small holdings with livestock, cats and dogs, and fish culture ponds. Surrounding rural landscapes may contribute to the potential for water exchanges between natural ponds and those used for aquaculture. Water diversions and exchanges facilitate snail and cercarial transport. Madsen et al. [67] observe: ‘There is a considerable production of cercariae in snail hosts in various habitats surrounding fish ponds such as rice fields and small water canals and since ponds may exchange water with these habitats, there is a risk that metacercariae found in fish may be of allochthonous origin’.

11.4.2 Dams and Reservoirs Lao PDR has one of the most ambitious hydropower dam building projects in the world with 78 dams in operation and signed memorandums of understanding for 246 other hydropower projects that will export electricity through the Mekong Region via the regional energy grid (Radio Free Asia 09.08.2021). Transforming once free-flowing rivers into water that drives turbines for energy has multiple upand downstream ecological impacts, particularly in relation to migratory fish species, fish behaviour, flowing larvae, sediments, predictability of water levels, and flood-pulse dynamics [4, 6, 45, 57, 98, 104, 146]. Most of the wild-capture fisheries of the Mekong basin have migratory fishes, and migratory fish patterns are at a transboundary scale [23] (Fig. 11.9: Transboundary fish migrations in the Mekong Basin). As the majority of some 64 million people in the Lower Mekong still rely heavily upon wild-capture fisheries for sources of protein, the impacts of ongoing and future hydropower projects on migratory fish, fish habitats, spawning, and other aspects of ecology are critical to food security [89, 155].

Fig. 11.9  Transboundary fish migrations in the Mekong Basin

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Some of the short-term and cumulative impacts upon hydrology and fisheries are: – Reductions in biodiversity and abundance of fish species [23, 89]. – Dams may trap sediments and starve downstream rivers of sediment-associated nutrients, detritus, and organic debris has impacts for terrestrialaquatic zone productivity [57]. – Hydropeaking is caused by the daily operations of hydropower projects as they operate their turbines to generate electricity responding to fluctuations in energy demand in distant load centres. It creates short-term alterations in volume and flow which can damage fish habitats, destabilise riverbanks, and disturb fish behaviour. – Adverse impacts upon downstream seasonally inundated floodplains adjacent to the river channels that are major productive feeding, spawning, and nursing habitats [5, 61]. – Terrestrial-aquatic zones are very sensitive to unseasonal and sudden changes in water-level, and the impacts of multiple dams over time may weaken the annual flood-pulse, which is an ecological driver in the Tonle Sap and Lower Mekong [4, 61]. Developmental natural resource landscape changes produce complex consequences for migratory fishes, wild-capture fisheries, livelihoods, rice-fishing cultures, and food security [35]. These mean that dams do have human health and disease implications. For instance, the reservoirs created by dams may increase the risk of malaria and stretch periods of potential malaria transmission [148]. Ziegler et al. [153] observe: ‘Schistosomiasis naturally occurs in the mainstream Mekong in southern Laos and north-eastern Cambodia, and dam building could increase its prevalence’, as it did in China following the construction of the Three Gorges Dam [72]. Dams are altering fish habitats, migrations, productivity, and leading to expanded reliance upon aquaculture. For instance, in Lao PDR the government and development institutions have promoted small-scale rural aquaculture projects as an aspect of providing rural incomes and tackling food security needs, particularly as wild-capture fisheries productivity is impacted by hydropower [31]. The consequences of hydropower on hydrological cycles and FZT life cycles are complex, requiring understanding of long-term cumulative social and environmental impacts [120].

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11.4.3 Roads and Rivers: Migration and Mobility The migration of humans and their domestic animals has been the pathway for disseminating parasitic zoonoses throughout recorded history and will continue to have an impact on the emergence, frequency, and spread of infections [66].

Historical movements and connections Southwest China and mainland Southeast Asia have historically been forged through the movement and mobility of peoples, animals, fish, and traded goods, particularly across and along rivers, and long before the region was divided by international boundaries [33, 141]. Prior to strict boundary delimitations, people regularly criss-crossed spaces for trade and as migratory groups, and as such pathogens could move with people. International boundaries marking territorial forms of sovereignty became a means to control flows of people and goods. Regarding the Mekong River and the creation of a boundary between what became Thailand and Lao PDR, Thongchai [147] writes: ‘once the 1893 treaty was concluded, the Siamese authorities wanted the boundary just settled to mark the distribution of population as well. Those who belonged to the lords of the left bank of the Mekhong (French Indochina) but inhabited the other side (Siam) were permitted to return to their homeland. If they did not, they would become Siamese by residency’. Whilst the boundaries did eventually create national territories, crossborder movements of traders, migrants, refugees and tourists, particularly within borderlands where people share kith and kin, ethnic relations, and maintain strong ties that often circumvent political space [79, 131]. Trans-political ties and mobilities mean that national geo-bodies are often imperfect epidemiological containers and pathological landscapes may transcend political space [62]. Border Waterscapes: Cross-Border Interactivity The Mekong region encompasses a large zone which may be called border waterscapes and are frequently traversed, up and downstream, and across rivers [74]. Crossing points between key towns and nodes are major transit points for goods, migrant workers, and travellers. Away from official crossing points, there are permeable zones where undocumented and illegal crossings are possible, but also there exist many banal, locally licit, temporary crossings, particularly along the Mekong River in parts of the Thai-Lao border and the Southern Lao-Cambodia border. Traditionally, the riparian lowlands and island communities have shared the border river spaces as valuable sources for transboundary common property resources, particularly wild-capture freshwater fisheries [5, 35]. Research has found high O. viverrini prevalence rates in specific districts of border provinces, such as Nakhon Phanom [123] and Sakon Nakhon [21] on the Thai side of the Mekong border, the Mekong corridor areas of Southern Lao PDR [30], and the Khong Island district at the southern border of Champasack province, Lao PDR with Cambodia [140]. Various studies have focused on the cross-seasonal infection dynamics of O. viverrini metacercariae in cyprinid fish species in the river border zone of Mukdahan Province, Thailand, and

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Khammouane Province, Lao PDR [78], and O. viverrini infection in Bithynia siamensis goniomphalos [63] and cyprinid fish species commonly consumed in Nakhon Phanom [64]. People residing close to the river rely on small catches to supplement their incomes or for subsistence and consumption of raw and fermented fish remains a common part of riparian lives. Whilst the focus has been on different aspects of the life cycle of O. viverrini and its prevalence and infection, there is scope to explore how frequent but short interactions for fishing, trading, business meetings, services (health clinics), and other activities in border waterscapes may influence disease dynamics. Internal migration Internal migrations of people from Isan to other parts of Thailand may relate to the spread of O. viverrini. Rangsin et al. [99] studied Baan Nayao village in a rural area of Chachoengsao Province, South-Central Thailand, 228 km east of Bangkok. ‘Our study showed a relatively high incidence rate of O. viverrini infection of 21.6/100 person-years in the study community. The infection rate was similar to that reported in Khon Kaen Province (24%), an area in Northeastern Thailand in which opisthorchiasis is highly endemic’ [99]. Many people migrated from Isan into more central areas of Thailand during the 1950–1980s. This was a period when O. viverrini prevalence was high in many parts of the northeast [112, 133]. Many of the older immigrant generation enjoy eating koi pla and pla-som made from cyprinid fishes, which put them at higher risk of O. viverrini infection. Isan people have moved for work and residence to parts of central and south Thailand, with many working in Bangkok, and even in the city they can find Isan food outlets catering to migrant communities. However, if raw and semi-cooked fish dishes are less common in the cities where they work, during festivals (such as Songkran), and public holidays, a great many migrants return to their home provinces and villages where they partake of the foods they miss most. Transnational cross-border migration The Greater Mekong Subregion (GMS) with transborder, cross-regional connections have been very much a part of the changing regional landscape initially sponsored by the Asian Development Bank (ADB) [145]. This has multi-dimensions in the region and for host and sender nations for migrant workers. Regarding food cultures, Cambodian, Vietnamese, Chinese, Laotian, Thai, and Burmese workers who have worked on development projects across borders are very likely to have been exposed to the raw food dishes of the country and so possible infection with FZTs. Transnational migration and mobility represent another critical mode of possible spread of O. viverrini over time. Petney et al. [91] speculate that cross-border movements may be ‘responsible for the genetic similarities found in the Songkhram’ in northeast Thailand and Lao PDR populations of O. viverrini. Prior to SARSCoV-2 (COVID-19) pandemic, open borders associated with the ASEAN Economic Community (AEC) enabled easier transnational movement for migrant workers within the region. In borderlands with a long history of cross-border cultural and economic ties, epidemiological studies should consider potential transnational

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influences on life cycles and disease. Cross-border economic migration means that migrant workers are mobile agents in efforts to track infections and disease spread.  Kaewpitoon et al. [46] conducted a cross-sectional study among 249 participants including Thai, Cambodian, Laotian, and Burmese, who worked or lived in Nakhon Ratchasima province in northeast Thailand. The cohort included mostly young labourers, male and female, between the ages 21 and 40 years old, with almost 50% of the workers coming from Cambodia. After stool analysis, it was found that O. viverrini infections were detected in 30.77% of Laotians, 25.62% of Cambodians, and 5.56% of the Thai workers [46]. In total, ‘23.69% were infected by one or more helminthic infection. The majority of infections were O. viverrini, followed by hook-worm, Endolimax nana, Strongyloides stercoralis, Taenia spp., Blastocystis hominis, and Entamoeba histolytica’ [46]. The authors argued for a verbal screening test to assess risk as a way to prevent the spread of O. viverrini by foreign migrant workers. In addition, domestic migration for work is high throughout Thailand. Overseas migration Overseas migration does not alter the fact that many migrant workers prefer familiar food. Thus, in Singapore’s Golden Mile Complex, where migrants from Isan are one of the main groups of Thai foreign workers who utilise the complex on their days off, there is preference for eating sticky rice or khao niao and familiar Isan food, including fermented fish or pla-ra. Jars of pla-ra are imported and purchased by migrant workers and visitors to the complex. There are approximately 47,000 Thai migrant workers in the city-state, many of them from Isan, and Golden Mile is often called a ‘mini-Thailand’ as it caters to these workers. Most eateries in the complex offer Isan food and fermented fish dishes (see photo set, Golden Mile. Figs. 11.10, 11.11, 11.12, 11.13 and 11.14). So, even though the O. viverrini life cycle is not endemic, migration for work overseas modifies but does not completely change people’s yearning for food from their respective home places. A study of Thai male workers in Singapore used the term ‘village transnationalism’ to describe the life of many of these workers, who remain villagers at heart in spite of working on urban engineering and construction projects in a faraway city [55]. Food culture is a strong part of that rural identity. Absence from their rural homelands may thus serve to reinforce traditional food cultures, so that when migrants do return home, they crave to eat foods they have missed most, including, in some cases, raw fish dishes.

11.5 Uneven Regional Knowledge About the Extent of Opisthorchis viverrini and Opisthorchiasis There remains uneven geographical knowledge about the extent of O. viverrini prevalence and infection, with epicentres of relatively high-quality survey data and quantitative information relating to spatial and temporal morbidity data, prevalence, and cholangiocarcinoma (CCA) provided through surveys in

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Fig. 11.10  Typical small Thai food restaurant in Golden Mile Complex, Singapore

Fig. 11.11  Jars of fermented fish and fish paste on sale

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Fig. 11.12  Jars of fermented fish from Isan to Had Yai to Singapore

Fig. 11.13  Pla ra from one of the food stores in Golden Mile

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Fig. 11.14  Shope selling chilli paste and pla raa mostly to migrant workers from Isan

North and northeast Thailand by the Cholangiocarcinoma Screening and Care Program (CASCAP). Survey and screening of populations elsewhere in the Mekong region have a patchy temporal–spatial coverage. Numerous studies suggest that liver fluke related disease is being underreported, and over the past two decades, Opisthorchiasis is an ‘emerging’ health problem in Lao PDR [104, 111], Cambodia [73, 116, 131, 150], Myanmar [119], and Vietnam [18, 19, 80]. These studies reveal O. viverrini as endemic across borders, but due to partial survey coverage coupled with the low sensitivity of diagnostics the burden of disease is underestimated [16]. Raw attitudes across the region are not confined to fish [34], but also include beef, pork, and raw vegetables collected from potentially contaminated environs nearby homes. People catch frogs, crabs, tadpoles, clams, small shrimps, and snails, all aquatic animals found in the rivers, streams, ditches, ponds, lakes, and other water bodies near to their farms [77, 95]. Thus, in the farming-fishing zones of the Lower Mekong, there are socio-economic (livelihood) and biophysical environmental influences on food culture, and dietary habits are transnational, although it is possible to discern certain cultural nuances relating to eating raw, semi-cooked, and fermented fish. With the extension of public health education campaigns to remote rural areas, there has been growing awareness of risks from eating raw fish, but it takes many years for messaging to modify behaviour and practices. To illustrate different sociocultural and socio-economic dimensions relating to fish-borne zoonotic trematodes (FZTs), including O. viverrini, raw attitudes in Thailand, Lao PDR, Vietnam, and Cambodia are explored further.

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11.6 Public Health Programs Tackling OV and CCA in Thailand The public health issues relating to food-borne and fish-borne trematodes have been recognised in Thailand for decades since the USAID-supported Helminthiasis Control Units were started in the 1950s in five provinces (Nakhon Ratchasima, Udon Thani, Sakhon Nakhon, Ubon Ratchathani, and Songkhla), including an opisthorchiasis control program in the Sixth Five-year National Public Health Development Plan (1987–1991) under the auspices of the Department of Communicable Disease Control and a decade-long program (1984– 1994) focusing on reducing raw fish-eating habits in the Isan region [42, 43]. The eighth National Health Plan (1997–2001) led to the opisthorchiasis control program that was integrated into the Nationwide Disease Control Aims, with the objective of reducing the prevalence of O. viverrini infection to 20%) were ‘found in provinces from Preah Vihear to Takeo’. ‘O. viverrini infection remains underreported in Cambodia’ [49]. Bless et al. [9] found a high prevalence of large trematode eggs in schoolchildren. ‘In 2006, a stool survey was conducted by the National Center of Malaria, Parasitology and Entomology (CNM) of Cambodia in Ang Svay Chek Village in Takeo Province and Ampil Village in Kandal Province, southern Cambodia. The results showed 13.4% of the residents were positive for small trematode eggs’ [129]. Sohn et al. [116] report: ‘In May 2010, we analysed fecal samples from 1,993 persons in 3 villages (Ang Svay Chek, Kaw Poang, and Trartang Ang) in the Prey Kabas District, Takeo Province, Cambodia, ≈ 45 km south of Phnom Penh, to confirm the presence of O. viverrini flukes among humans. We found an egg-positive rate of 32.4% for small trematode eggs’. A high prevalence of O. viverrini (47.5%) was found among 1,799 residents of Takeo Province examined by the Kato-Katz technique [150]. O.viverrini research facilitated by the Korea-Cambodia International Collaboration on Intestinal Parasite Control in Cambodia (2006–2011) has led to growing evidence of liver-fluke prevalence in human populations. Sohn et al. [117] detail findings from a survey of seven riparian villages along the Mekong River (Roka Kandal A, Talous, Roka Kandal B, Sambok, Tmor Kre, Kratie

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Krong, and Acnouvat), including faecal examination and anthelmintic treatment sanctioned by the Ministry of Health, Cambodia. In total, they tested 2101 villagers. They observed: ‘The highest egg prevalence was found in Roka Kandal A village (10.4%) followed by Talous village (5.9%) and Roka Kandal village B (3.0%). In Roka Kandal A and Talous villages, adults (≥15 years) showed higher (p