Current Cancer Research

Richard J. CoteRam H. Datar Editors

Circulating Tumor CellsAdvances in Basic Science and Clinical Applications

More information about this series at http://www.springer.com/series/7892

Current Cancer Research

Series Editor: Wafi k El-Deiry

Richard J. Cote • Ram H. Datar Editors

Circulating Tumor Cells Advances in Basic Science and Clinical Applications

ISSN 2199-2584 ISSN 2199-2592 (electronic) Current Cancer Research ISBN 978-1-4939-3361-7 ISBN 978-1-4939-3363-1 (eBook) DOI 10.1007/978-1-4939-3363-1

Library of Congress Control Number: 2015959021

Springer New York Heidelberg Dordrecht London © Springer Science+Business Media New York 2016 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfi lms 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 specifi c 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, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made.

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Editors Richard J. Cote University of Miami Jackson Memorial Hospital Miami , FL , USA

Ram H. Datar University of Miami Miami , FL , USA

Metastasis is the process that defi nes cancer. Studies in understanding circulating tumor cells have been at the forefront of efforts to favorably impact the morbidity and mortality due to metastatic spread of the cancer. In the making of this book, an effort made possible by the world’s leading investigators, it is important to remember and recognize those who we aim to cure. We dedicate this volume to those countless cancer sufferers across the world, and hope that this sharing of knowledge will move us closer to alleviating this emperor of maladies.

This book is dedicated to mentors and colleagues. Dr. Lloyd J. Old, Dr. Edward J. Beattie, and Dr. A. Munro Neville are entirely responsible for any contribution I may have made to this fi eld. Dr. Clive R. Taylor allowed me to pursue these studies when most of the scientifi c community considered micrometastases and CTC to be of no consequence. And Dr. Ram H. Datar has been my longtime collaborator and friend. I am also deeply grateful to my family, Annie, Nick, Juliet, and Gracie, who have supported me throughout this endeavor and more.

Richard J. Cote, MD, FRCPath, FCAP

I would like to express my gratitude to Dr. Richard Cote for introducing me to the fascinating fi eld of cancer metastasis! Dr. Cote has mentored me throughout as we collaboratively tackled the diffi cult clinical problem over many years and developed lasting associations with various thought leaders in the fi eld, many of whom have actively contributed to this volume. I also take this opportunity to thank my parents and parents-in-law, wife Bharati, our son and daughter-in-law Nakul and Aditi, respectively, and our granddaughter Mallika, who all were persistent and unwavering in their support throughout.

Ram H. Datar, MPhil, PhD

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Foreword

Metastatic spread of cancer is among the most leading causes of cancer-associated morbidity and mortality, and circulating tumor cells form the very basis of this pro-cess [1]. There was an emergent need for a volume that can serve as an authoritative compilation of information about circulating tumor cells (CTC) from thought lead-ers, which this volume has directly addressed.

Over the past decade, a variety of ultrasensitive assays have been developed to detect CTC in the peripheral blood and disseminated tumor cells (DTC) found in the bone marrow of cancer patients at the single-cell level. CTC can be distinguished and enriched from the surrounding leukocytes by either physical properties (e.g., density and size) or biological properties (e.g., expression of epithelial proteins such as EpCAM or cytokeratins). CTC/DTC are usually detected by immunostaining or RT-PCR assays, and more recently by the EPISPOT assay which measures the num-ber of cells releasing/secreting tumor-associated marker proteins. Chapters 1 – 4 in this volume serve as a comprehensive survey of these detection technologies. The epithelial-mesenchymal transition (EMT) is an emerging important issue in CTC research. For example, at present, most assays rely on epithelial markers and may miss CTC undergoing EMT. New markers such as the actin bundling protein plas-tin- 3 that are not downregulated during EMT and not expressed in normal blood cells might overcome this important limitation and, therefore, increase the sensitiv-ity of CTC assays. Chapter 8 provides an overview of this important emerging area.

Interestingly, the bone marrow seems to be a common homing organ for cells derived from various epithelial tumors including breast and prostate cancer. However, a signifi cant fraction of DTC remain over years in a “dormant” stage, and little is known about the conditions required for the persistence of dormancy or the escape from the dormant phase into the active phase of metastasis formation, con-cepts which are reviewed in Chaps. 5 – 7 of the present volume. Recent fi ndings indicated that a subset of EpCAM low , CD44 high , CD47 + , c-Met + CTC obtained

viii

from the peripheral blood of breast cancer patients might have an increased ability to colonize bone marrow in immunodefi cient mice. However, it is unclear whether these CTC are metastasis-inducer cells because they were obtained from patients with advanced metastatic disease and extraordinarily high CTC counts. While further in-depth molecular analyses of CTC are expected to answer such questions, the current state of phenotypic and genotypic knowledge about CTC is nicely summarized in Chaps. 9 and 10 of this volume, while Chap. 10 addresses the need for functional characterization of CTC.

While bone marrow is an informative site for study, sampling of peripheral blood is much more convenient and less intrusive than collection of bone marrow samples, and many research groups are currently assessing the clinical utility of CTC for determining prognosis and monitoring of systemic therapy. In particular, monitor-ing of CTC during and after systemic adjuvant therapy (e.g., chemotherapy, hor-monal therapy, antibody therapy) might provide unique information for the clinical management of the individual cancer patient and allow an early change in therapy years before the appearance of overt metastases signals incurability. There is an unmet need for biomarkers for real-time monitoring of the effi cacy of systemic adjuvant therapy in individual patients. In particular, early changes in CTC counts might indicate success or failure of a particular therapy given to an individual patient. Chapters 11 – 14 and 16 have each covered distinct but critical issues per-taining to the relevance of CTC in monitoring therapeutic response.

Besides CTC, the analysis of ctDNA and circulating microRNAs may provide complementary information as “liquid biopsy.” This information can be used as companion diagnostics to improve the stratifi cation of therapies and to obtain insights into therapy-induced selection of cancer cells. CANCER-ID is a newly formed European consortium funded by the Innovative Medicines Initiative (IMI) with cur-rently 33 partners from 13 countries aiming at the establishment of standard proto-cols for and clinical validation of blood-based biomarkers ( www.cancer-id.eu/ ). It brings together experts from academic and clinical research, innovative Small-to-Medium sized Enterprises (SMEs), diagnostics companies, and the pharmaceutical industry, thus providing a unique setting for showing clinical utility of “liquid biop-sies.” Although systemic therapies are aimed to eliminate metastatic cells, the cur-rent stratifi cation is usually performed on the primary tumors for practical reasons. However, there is increasing evidence that the phenotype and genotype of primary and metastatic cancer cells are discordant. Thus, the molecular analysis of CTC iso-lated from peripheral blood samples as “liquid biopsy” will reveal characteristics of metastatic cancer cells. This information can be used as companion diagnostics to improve the stratifi cation of therapies and to obtain insights into therapy-induced selection of cancer cells. Chapter 15 in the present volume reviews the various tech-nical and regulatory issues relevant to the use of CTC as companion diagnostics.

Foreword

ix

In conclusion, research on CTC opens a new avenue for detecting, understanding, and fi ghting early metastatic spread of tumor cells with important implications for future therapies. CTC have enormous potential as new biomarker and as the subject of basic research. Although CTC are already used in numerous clinical trials, their clinical utility is still under investigation. The present contributions by international experts in this book highlight the potential and current challenges of CTC research.

Hamburg, Germany Klaus Pantel

References

1. Alix-Panabieres C, Pantel K (2014) Challenges in circulating tumour cell research. Nat. Rev. Cancer 14: 623–631

Foreword

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Pref ace

Circulating tumor cells (CTC) were fi rst observed and described as epithelial tumor cells found in blood over a century ago [1]. But reliable methods have been devel-oped only recently to interrogate this rare population in blood. These methods are developed either utilizing unique antigen expression on CTC, such as the only FDA- cleared technology CellSearch™ [2], or utilizing their unique physical properties including size [3], density [4], electrical properties [5], etc. After an overview of the current status of our knowledge about CTC in the introductory chapter ( Chap. 1 ), Datar and coauthors present details of affi nity-based and non-affi nity-based CTC capture technologies in Chaps. 2 and 3 , respectively.

As more and more in-depth molecular and functional characterization of CTC studies has been carried out, the validity of CTC capture based on EpCAM expres-sion is being questioned. CTC population is discovered to be heterogeneous and the gene expression levels vary from cell to cell even within the same patient sample [6]. To address this heterogeneity, an increasing number of studies have begun to look beyond CTC enumeration to elucidate the subpopulations among CTC. Details on molecular characterization of CTC that can help resolve this heterogeneity are described by Lianidou and her colleagues in Chap. 4 . A potential subpopulation that is worth studying in CTC is the cancer stem cell population. Cancer stem cell population in CTC is further discussed by Wicha and his colleagues in Chap. 5 .

CTC in circulation may assume one or more of several optional states: they could undergo elimination (by anoikis, apoptosis, necrosis, or immune attacks), success-fully invade into secondary site, only to stay dormant or locked in mesenchymal states, or invade into a secondary site and metastasize by rapid proliferation. CTC dormancy studies are discussed in more detail in Chap. 6 ( Allan and Chambers ) and Chap. 7 ( Barkan and Chambers ). An important process involved in tumor metastasis that brings EpCAM-based CTC capture in question is the Epithelial- Mesenchymal Transition (EMT). In this process, tumor cells can potentially down-regulate their expression of epithelial markers including EpCAM and E-Cadherin and gain a mesenchymal and more invasive phenotype. In Chap. 8 , Thiery and colleagues discuss more about this EMT process as a mechanism through which CTC establish distant metastasis.

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An ability to perform phenotypic and genotypic studies of CTC is expected to lead to the development of assays with clinical applications to benefi t cancer manage-ment. Chapter 9 ( Dandachi and colleagues ) and Chap. 10 ( Magbanua and Park ) provide detailed discussions on phenotypic and genotypic analysis of CTC. In addi-tion to molecular characterization of CTC, another interesting direction is the func-tional characterization of CTC employing technologies that enable viable CTC capture and culture. Cote and colleagues present a detailed review of functional characterization of CTC in Chap. 11 .

With the emerging technologies to enumerate CTC from cancer patients, the clinical utilities of CTC have been investigated extensively in the past decade. One well-validated clinical application of CTC is their prognostic value at baseline. A detailed review on prognostic implications of CTC in breast cancer can be found in Chap. 12 ( Smerage ). Although CTC have been well validated as prognostic mark-ers for various cancer types, clinical applications for CTC as a surrogate endpoint, their use as a predictive marker to guide therapy, or use as an early detection marker are areas that are still largely unexplored and require large-scale clinical trials for validation. Although it is perhaps still a little early, it is not hard to envision CTC as powerful biomarkers as “liquid biopsy” which can provide valuable information via a minimally invasive blood draw. Chapter 13 ( Cristofanilli ) and Chap. 14 ( Polzer and Klein ) discuss in more detail the clinical applications of CTC. Chapter 15 ( Huang and Lackner ) tackles a crucial concept for pharmaceutical industry, that of developing CTC assay as a companion diagnostic for either a pre-approved or an under-development anticancer drug. This chapter thus emphasizes an early and close partnership between the drug development sponsors and the CTC diagnostic companies to successfully navigate the regulatory landscape through phase II and III clinical studies, which in turn would allow for synchronized regulatory review of the drug and CTC assay. Finally, Chap. 16 ( Kulkarni and Jeffrey ) summarizes the clinical applicability of CTC, while providing considerations for the future clinical trials.

As will be clearly evident, this volume is a result of a highly scholastic activity, with all of the contributors being thought leaders in the fi eld, with decades of exten-sive contributions to the study of molecular biology of metastasis and the clinical applications of these critical fi ndings.

References

1. Ashworth T (1869) A case of cancer in which cells similar to those in the tumours were seen in the blood after death. Aust Med J 14(3):146–149

2. Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Matera J, Miller MC, Reuben JM, Doyle GV, Allard WJ, Terstappen LW, Hayes DF (2004) Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med 351(8):781–791. doi: 10.1056/NEJMoa040766

3. Zheng S, Lin H, Liu JQ, Balic M, Datar R, Cote RJ, Tai YC (2007) Membrane microfi lter device for selective capture, electrolysis and genomic analysis of human circulating tumor cells. J Chromatogr A, 1162(2):154–161. doi: 10.1016/j.chroma.2007.05.064

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4. Gertler R, Rosenberg R, Fuehrer K, Dahm M, Nekarda H, Siewert JR (2003) Detection of cir-culating tumor cells in blood using an optimized density gradient centrifugation. In: Allgayer H, Heiss M (eds) Molecular staging of cancer. Springer, Berlin, pp 149–155

5. Becker FF, Wang X-B, Huang Y, Pethig R, Vykoukal J, Gascoyne P (1995) Separation of human breast cancer cells from blood by differential dielectric affi nity. Proc Natl Acad Sci 92(3):860–864

6. Powell AA, Talasaz AH, Zhang H, Coram MA, Reddy A, Deng G, Telli ML, Advani RH, Carlson RW, Mollick JA, Sheth S, Kurian AW, Ford JM, Stockdale FE, Quake SR, Pease RF, Mindrinos MN, Bhanot G, Dairkee SH, Davis RW, Jeffrey SS (2012) Single cell profi ling of circulating tumor cells: transcriptional heterogeneity and diversity from breast cancer cell lines. PLoS One 7(5):e33788. doi: 10.1371/journal.pone.0033788

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Acknowledgements

We thank the contributors to this ambitious undertaking; all are outstanding scientists and clinical investigators who are truly thought leaders in the fi eld of metastasis and CTC research. It has been a privilege to work with them, and it is to their credit that this authoritative and timely volume of outstanding contributions has come to pass.

This work would never have been initiated without the enduring support of Springer Science. We are highly appreciative of Ms. Fiona Sarne, Editor (Cancer Research); Ms. Joy Evangeline Bramble, Publishing Editor (Biomedical Sciences and Cancer Research); and Mr. Michael Koy, Project Coordinator for Book Production at Springer; it has been a pleasure to work closely with each of them. This diffi cult enterprise has seen its completion through the often insistent but always extremely helpful communications from Fiona, Joy, and Lesley. We sin-cerely thank them for their constant encouragement and assistance.

A volume with multiple chapters from busy and successful scientists and clini-cians poses special challenges, and this one would not have been possible without the assistance and guidance of Ms. Patricia Malinowski and Ms. Gladys Garcia- Greenberg. Patricia ungrudgingly and effi ciently handled the multiple communica-tions to each of the contributing lead authors, who were nearly always in different time zones and often different continents! It is to her great credit and patience that all of our contributors expressed their admiration for her skills and help. Gladys, joining us towards the end of this undertaking, employed her outstanding linguistic skills in manuscript editing. We owe our being punctual to these two wonderful colleagues with excellent organizational and interpersonal skills, and we are sincerely grateful.

Finally, we would like to express our sincere appreciation to our loving families—Annie, Juliet, Nicholas, and Gracie Cote, and Bharati, Nakul, Aditi, and Mallika Datar—who uncomplainingly and patiently supported us throughout this endeavor.

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Part I Technologies for CTC Identification

1 Significance of Studying Circulating Tumor Cells . . . . . . . . . . . . . . . . . . 3 Ram H. Datar , Zheng Ao , and Richard J. Cote

2 Affinity-Based Enrichment of Circulating Tumor Cells . . . . . . . . . . . . 17 Zheng Ao , Richard J. Cote , and Ram H. Datar

3 Size-Based and Non-Affinity Based Microfluidic Devices for Circulating Tumor Cell Enrichment and Characterization . . . . . . 29 Zheng Ao , Kamran Moradi , Richard J. Cote , and Ram H. Datar

4 Molecular Assays for the Detection and Molecular Characterization of CTCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 Evi S. Lianidou , Athina Markou , and Areti Strati

5 Cancer Stem Cells and Circulating Tumor Cells: Molecular Markers, Isolation Techniques, and Clinical Implications . . . . . . . . . . 75 Ebrahim Azizi , Sunitha Nagrath , Molly Kozminsky , and Max S. Wicha

Part II Fundamental Studies of CTC

6 Circulating Tumor Cells and Tumor Dormancy . . . . . . . . . . . . . . . . . 101 Alison L. Allan and Ann F. Chambers

7 Prevention of Conversion of Tumor Dormancy into Proliferative Metastases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 Dalit Barkan and Ann F. Chambers

8 Genesis of Circulating Tumor Cells Through Epithelial–Mesenchymal Transition as a Mechanism for Distant Dissemination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 Bee Luan Khoo , Prashant Kumar , Chwee Teck Lim , and Jean Paul Thiery

Contents

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Part III CTC Analysis

9 CTC Analysis: FISH, ISH, Array-CGH, and Other Molecular Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 Verena Tiran , Marija Balic , and Nadia Dandachi

10 Genome-Wide Gene Copy Number Analysis of Circulating Tumor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 Mark Jesus M. Magbanua and John W. Park

11 Perspectives on the Functional Characterization and In Vitro Maintenance of Circulating Tumor Cells . . . . . . . . . . . . 215 Anthony Williams , Ramdane Harouaka , Siyang Zheng , Chris Albanese , Richard Schlegel , Yu-Chong Tai , Ram H. Datar , and Richard J. Cote

12 Prognostic Implications of CTC in Breast Cancer . . . . . . . . . . . . . . . 233 Jeffrey B. Smerage

Part IV Potential Clinical Applications of CTC

13 CTC in Advanced Breast Cancer Prognosis, Monitoring, and Clinical Utility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 Massimo Cristofanilli

14 Evolution of Metastatic Disease: The Need for Monitoring and Emerging Therapeutic Opportunities . . . . . . . . . . . . . . . . . . . . . . 271 Bernhard Polzer and Christoph A. Klein

15 CTCs for Biomarker and Companion Diagnostic Development . . . . 293 Shih-Min A. Huang and Mark R. Lackner

16 Perspectives on Clinical Applications of CTCs . . . . . . . . . . . . . . . . . . 315 Rajan P. Kulkarni and Stefanie S. Jeffrey

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325

Contents

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Chris Albanese , Ph.D. Departments of Oncology , Pathology and Preclinical Imaging Research Laboratory, Lombardi Comprehensive Cancer Center, Georgetown University , Washington , DC , USA

Alison L. Allan , Ph.D. Departments of Oncology & Pathology , London Regional Cancer Program, University of Western Ontario , London , ON , Canada

Zheng Ao , Ph.D. Sheila and David Fuente Graduate Program in Cancer Biology , University of Miami Miller School of Medicine , Miami , FL , USA

Ebrahim Azizi , Pharm.D., Ph.D. Cancer Stem Cells Research , University of Michigan , Ann Arbor , MI , USA

Marija Balic , M.D., Ph.D. Division of Oncology, Department of Internal Medicine , Medical University of Graz , Graz , Austria

Dalit Barkan , Ph.D. Laboratory of Tumor Dormancy and Metastasis, Department of Biology and Human Biology, Faculty of Natural Sciences , University of Haifa, Mount Carmel , Haifa , Israel

Ann F. Chambers , Ph.D. Departments of Oncology, Medical Biophysics and Pathology & Laboratory Medicine, London Regional Cancer Program , London Health Sciences Centre/University of Western Ontario, London, Ontario , London , ON , Canada

Richard J. Cote , M.D., F.R.C.Path., F.C.A.P. Department of Pathology & Laboratory Medicine , University of Miami Miller School of Medicine , Miami , FL , USA

Department of Biochemistry and Molecular Biology , University of Miami Miller School of Medicine , Miami , FL , USA

Department of Pathology, Jackson Memorial Hospital, Miami, FL, USA

The Dr. John T. Macdonald Foundation Biomedical Nanotechnology Institute, University of Miami Miller School of Medicine , Miami , FL , USA

Contributors

xx

Massimo Cristofanilli , M.D., F.A.C.P. Department of Medicine and Translational Research & Precision Medicine , Robert Lurie Cancer Center-Northwestern University , Chicago , IL , USA

Nadia Dandachi , Ph.D. Division of Oncology, Department of Internal Medicine , Medical University of Graz , Graz , Austria

Ram H. Datar , M. Phil., Ph.D. Department of Pathology & Laboratory Medicine , University of Miami Miller School of Medicine , Miami , FL , USA

Department of Biochemistry and Molecular Biology , University of Miami Miller School of Medicine , Miami , FL , USA

The Dr. John T. Macdonald Foundation Biomedical Nanotechnology Institute , University of Miami Miller School of Medicine , Miami , FL , USA

Ramdane Harouaka , Ph.D. Department of Internal Medicine, Comprehensive Cancer Center , University of Michigan , Ann Arbor , MI , USA

Shih-Min A. Huang , Ph.D. Department of Oncology Biomarker Development , Genentech Inc. , San Francisco , CA , USA

Stefanie S. Jeffrey , M.D. Department of Surgery and Surgical Oncology Research , Stanford University School of Medicine , Stanford , CA , USA

Bee Luan Khoo , M.Sc. Mechanobiology Institute , National University of Singapore , Singapore

Christoph A. Klein , M.D. Experimental Medicine and Therapy Research , University of Regensburg , Regensburg , Germany

Fraunhofer Project Group “Personalized Tumor Therapy” ITEM Regensburg, Regensburg, Germany

Molly Kozminsky , M.S.E. Department of Chemical Engineering , Biointerfaces Institute, Translational Oncology Program, University of Michigan , Ann Arbor , MI , USA

Prashant Kumar , Ph.D. Institute of Molecular and Cell Biology , A*STAR (Agency for Science, Technology and Research) , Singapore

Rajan P. Kulkarni , M.D., Ph.D. Department of Medicine , David Geffen School of Medicine at UCLA , Los Angeles , CA , USA

Mark R. Lackner , Ph.D. Department of Oncology Biomarker Development , Genentech Inc. , San Francisco , CA , USA

Evi S. Lianidou , Ph.D. Analysis of Circulating Tumor Cells Lab, Laboratory of Analytical Chemistry, Department of Chemistry , University of Athens , Athens , Greece

Chwee Teck Lim , Ph.D. Mechanobiology Institute , National University of Singapore , Singapore

Contributors

xxi

BioSystems and Micromechanics (BioSyM) IRG, Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore

Department of Mechanical Engineering, National University of Singapore, Singapore

Department of Bioengineering, National University of Singapore, Singapore

Mark Jesus M. Magbanua , M.S., Ph.D. Division of Hematology/Oncology, Department of Medicine , University of California San Francisco , San Francisco , CA , USA

Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA, USA

Athina Markou , Ph.D. Analysis of Circulating Tumor Cells Lab, Laboratory of Analytical Chemistry, Department of Chemistry , University of Athens , Athens , Greece

Kamran Moradi , Ph.D. Department of Pathology & Laboratory Medicine , University of Miami Miller School of Medicine , Miami , FL , USA

Sunitha Nagrath , Ph.D. Department of Chemical Engineering & Biomedical Engineering, Biointerfaces Institute , Translational Oncology Program, University of Michigan , Ann Arbor , MI , USA

Klaus Pantel Department of Tumor Biology , University Medical Center Hamburg-Eppendorf , Hamburg , Germany

John W. Park , M.D. Division of Hematology/Oncology, Helen Diller Family Comprehensive Cancer Center , University of California San Francisco , San Francisco , CA , USA

Bernhard Polzer , M.D. Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany

Richard Schlegel , M.D., Ph.D. Departments of Oncology and Pathology, Lombardi Comprehensive Cancer Center , Georgetown University Medical Center , Washington , DC , USA

Jeffrey B. Smerage , M.D., Ph.D. Division of Hematology and Oncology, Breast Oncology Program of the Comprehensive Cancer Center , University of Michigan Health and Hospital System , Ann Arbor , MI , USA

Areti Strati , Ph.D. Analysis of Circulating Tumor Cells Lab, Laboratory of Analytical Chemistry, Department of Chemistry , University of Athens , Athens , Greece

Yu-Chong Tai , Ph.D. Department of Electrical & Mechanical Engineering , California Institute of Technology (CalTech) , Pasadena , CA , USA

Jean Paul Thiery , Ph.D. Department of Biochemistry, Yong Loo Lin School of Medicine , National University of Singapore , Singapore , Singapore

Contributors

xxii

Senior Principal Investigator, Cancer Science Institute (CSI), National University of Singapore, Singapore, Singapore

Research Director, Institute of Molecular and Cell Biology (IMCB), A*STAR, Proteos, Singapore

Verena Tiran , B.Sc., M.Sc. Division of Oncology, Department of Internal Medicine , Medical University of Graz , Graz , Austria

Max S. Wicha , M.D. Department of Internal Medicine, University of Michigan Comprehensive Cancer Center , Ann Arbor , MI , USA

Anthony Williams , Ph.D. Section of Urology, Department of Surgery , University of Chicago - Pritzker School of Medicine , Chicago , IL , USA

Department of Pathology & Laboratory Medicine, University of Miami Miller School of Medicine, MiaMI FL, USA

Siyang Zheng , Ph.D. Departments of Biomedical Engineering, Electrical Engineering, Materials Research Institute and Micro & Nano Integrated Biosystem (MINIBio) Laboratory , Pennsylvania State University, University Park , PA , USA

Contributors

xxiii

Richard J. Cote , M.D., F.R.C.Path., F.C.A.P. is Professor and Joseph R. Coulter Jr. Chair of the Department of Pathology & Laboratory Medicine. He is also Professor in the Department of Biochemistry and Molecular Biology and Chief of Pathology at Jackson Memorial Hospital. Additionally, he serves as the Director of the Dr. John T. Macdonald Foundation Biomedical Nanotechnology Institute at the University of Miami Miller School of Medicine. He is a board certifi ed Pathologist, who has served over 20 years in senior academic, consultative, and director/clinical roles with leading universities, hospitals, and healthcare enterprises, primarily in the area of cancer care.

Dr. Cote obtained degrees in chemistry and biology at the University of California at Irvine and received his medical degree from the University of Chicago – Pritzker School of Medicine. He completed his residency at New York Hospital – Cornell University Medical College. His training included a clinical fellowship in pathology at Memorial Sloan-Kettering Cancer Center, a research fellowship in Human Tumor Immunology at Memorial Sloan-Kettering Cancer Center, and a fellowship in Molecular Pathology at New York University School of Medicine. Prior to joining the University of Miami in 2009, he was Professor, Department of Pathology; Professor, Department of Urology; Director, Genitourinary Cancer Program; Director, Laboratory of Immunology and Molecular Pathology; and Director of the USC Biomedical Nanoscience Initiative at the University of Southern California Keck School of Medicine/Norris Cancer Center in Los Angeles.

Dr. Cote’s research is focused on the elucidation of cellular and molecular path-ways of tumor progression and response to therapy. He has special interests in micro-metastases detection and characterization and in the pathology of breast and genitourinary tumors. His laboratory is also focused on technology development, where he and his colleagues have developed immunohistochemical and molecular methods, such as antigen retrieval. Most recently, Dr. Cote and his colleagues at the University of Southern California, Caltech, and UC Berkeley have developed nanoscale technologies for cancer diagnostic applications, including bionanosensors for the detection of serum tumor markers, and technologies for the capture and

About the Authors

xxiv

characterization of circulating tumor cells. As a result of these efforts, he established the Biomedical Nanoscience Program at USC and the Dr. John T. Macdonald.

Biomedical Nanotechnology Institute at the University of Miami (which recently received a $7.5 million naming gift from the Dr. John T. Macdonald Foundation), for the development of novel diagnostic platforms and targeted therapeutics. He is the recipient of over $43 million in peer-reviewed grant support and holds numerous patents for cancer-related and nanoscale technologies. He has led three of the largest clinical trials in breast, lung, and bladder cancer, which were based on discoveries from his research. Dr. Cote is the author of over 300 publications and participates on numerous scientifi c advisory boards for both academic- and industry-related institutions. He is a frequent lecturer and is the co-author of the standard textbooks Immunomicroscopy: A Diagnostic Tool for the Surgical Pathologist (now in its third edition) and Modern Surgical Pathology (now in its second edition).

He also serves as a member and advisor to a large number of national and inter-national study groups, cancer programs and societies, including the National Cancer Institute. He is the founder of several technology-based companies; is listed in “US News and World report Top 1 % of Doctors”, “Best Doctors in America”, “America’s Top Doctors”, and “South Florida Super Doctors”; and is a Fellow of the Royal College of Pathology.

Ram H. Datar , M.Phil., Ph.D. is Associate Professor in the Departments of Pathology and Laboratory Medicine and Biochemistry & Molecular Biology. He also serves as the Co-Director of the Dr. John T. Macdonald Foundation Biomedical Nanotechnology Institute at the University of Miami Miller School of Medicine. He received his B.S., M.S., and M.Phil., degrees in molecular biology from Pune University, India, in 1979, 1981, and 1984, respectively. Datar received his Ph.D. in Applied Biology from the University of Bombay (1996) on gene regulation in oral cancers. Following his Ph.D. degree studies, Dr. Datar served as a scientist at the Department of Atomic Energy of the Government of India for 14 years. In 1998, he joined the faculty of the Department of Pathology at the University of Southern California. In 2007, Dr. Datar joined the Bioscience Division of the Oak Ridge National Laboratory as a Senior Scientist until his relocation to Miami in early 2009.

Dr. Datar’s areas of research focus include cancer molecular pathology and bio-medical nanotechnology. Specifi cally, he has been involved in various aspects of detection and molecular characterization of occult metastases in cancers. For many years, he has also been involved in the development and application of micro- and nanoscale devices for biomedical diagnostic applications. Dr. Datar has played a piv-otal role in the development of the microfi lter device for capture and characterization of the circulating tumor cells (CTC), various nanotechnology sensor platforms to detect clinical biomarkers, and is among the inventors of these technologies with numerous awarded patents and in-process patent applications.

He has authored more than 70 original papers, and his publications have received more than 2200 citations. He is a reviewer for a number of international biomedical journals and has served as editor for Current Issues in Molecular Biology, Caister Academic Press, UK. He also serves on numerous scientifi c advisory boards, NIH Study Sections, and NSF Special Panels.

About the Authors

Part I Technologies for CTC Identifi cation

3© Springer Science+Business Media New York 2016 R.J. Cote, R.H. Datar (eds.), Circulating Tumor Cells, Current Cancer Research, DOI 10.1007/978-1-4939-3363-1_1

Chapter 1 Signifi cance of Studying Circulating Tumor Cells

Ram H. Datar , Zheng Ao , and Richard J. Cote

Abstract Circulating Tumor Cells (CTC) are tumor cells released into blood. They are considered the pivotal component of the metastatic cascade and are being exten-sively studied only in the last decade or so. Understanding the biological and clini-cal impact of CTC is likely to reveal important information of the metastatic process and contribute to better management of cancer. We briefl y discuss here the current clinical utility of CTC and their emerging clinical applications.

Keywords Current and emerging clinical applications of CTC • Epithelial– mesenchymal transition (EMT) • Circulating cancer stem cells • CTC genomics • CTC culture

R. H. Datar , M.Phil., Ph.D. (*) Department of Pathology & Laboratory Medicine, University of Miami Miller School of Medicine , Biomedical Research Building , #714, 1501 NW 10th Avenue , Miami , FL 33136 , USA

Department of Biochemistry and Molecular Biology , University of Miami Miller School of Medicine , Miami , FL 33136 , USA

The Dr. John T. MacDonald Foundation Biomedical Nanotechnology Institute , University of Miami Miller School of Medicine , Miami , FL 33136 , USA e-mail: rdatar@med.miami.edu

Z. Ao , Ph.D. Sheila and David Fuente Graduate Program in Cancer Biology , University of Miami Miller School of Medicine , Miami , FL 33136 , USA

R. J. Cote , M.D., F.R.C.Path., F.C.A.P. Department of Pathology & Laboratory Medicine , University of Miami Miller School of Medicine , Miami , FL 33136 , USA

Department of Biochemistry and Molecular Biology , University of Miami Miller School of Medicine , Miami , FL 33136 , USA

Department of Pathology , Jackson Memorial Hospital , Miami , FL , USA

The Dr. John T. MacDonald Foundation Biomedical Nanotechnology Institute , University of Miami Miller School of Medicine , Miami , FL 33136 , USA

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1.1 Introduction

Circulating Tumor Cells (CTC) are tumor cells released into blood. They are con-sidered the pivotal component of the metastatic cascade and are being extensively studied only in the last decade or so. Understanding the biological and clinical impact of CTC is likely to reveal important information of the metastatic process and contribute to better management of cancer. We briefl y discuss here the current clinical utility of CTC and their emerging clinical applications.

1.2 Current Clinical Applications

Ashworth fi rst reported tumor cells in a patient’s peripheral blood over one and a half century ago [ 1 ]. However, the study of these tumor cells has always been hampered by the rare existence of this cell population amid the excess of hema-topoietic cells in blood. Various CTC isolation technologies have been developed only relatively recently, based on various principles such as affi nity-based cap-ture technologies including CellSearch™ [ 2 ], or non-affi nity based technologies such as size based microfi ltration [ 3 ], density-based gradient centrifugation [ 4 ], or electrical property-based dielectrophoresis (DEP) [ 5 ]. Most of the clinical data pertaining to clinical utility of CTC was collected utilizing CellSearch™, the only FDA-cleared technology for CTC enumeration for breast, colon, and pros-tate cancers.

1.2.1 CTC Enumeration at Baseline as Prognostic Marker

One well-validated clinical application of CTC is assessment of their prognostic value by CellSearch assay at pretreatment baseline. Patients with higher than 5 CTC per 7.5 mL blood were shown to have shorter progression-free survival and shorter overall survival in a study analyzing baseline CTC level in a cohort of 177 meta-static breast cancer patients in 2004 [ 2 ]. Subsequently, similar results were seen for metastatic prostate cancer in 2008 [ 6 ] and metastatic colorectal cancer in 2009 [ 7 ]. Several follow-up studies have confi rmed the prognostic value of CTC, and as a result, CTC has been proposed in the new 2010 edition of the tumor-node- metastasis (TNM) cancer staging system manual as cM 0 (i+) [ 8 ], which is yet to be included in the clinical guidelines. The following paragraphs briefl y discuss emerging clinical applications of CTC.

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1.3 Emerging Clinical Applications

1.3.1 CTC as Surrogate Endpoint for Clinical Trials

One important potential clinical utility of CTC is their use as a surrogate endpoint for clinical trials. If approved to be informative, interrogating CTC with minimally invasive blood draws at follow-up time points to monitor treatment will greatly benefi t the cancer management, and several clinical trials are designed to test the feasibility of this notion. For example, CTC enumeration analysis at follow-up vis-its for the same cohort of patients examined by Cristofanilli et al. above [ 2 ] showed CTC to be prognostic of progression-free survival and overall survival [ 9 ]. Similarly, another clinical trial in breast cancer also indicated that CTC as a surrogate endpoint is more reproducible and robust than radiographic response [ 10 ]. In prostate cancer, a clinical trial conducted in a cohort of 263 metastatic castration resistance prostate (mCRPC) cancer patients—the SWOG S0421 trial also indicated that, patients with rising CTCs at week 3 have signifi cantly worse overall survival as compared with those with less or equal number of CTC at the week 3 follow-up visit [ 11 ]. In another study in mCRPC setting, CTC in combination with serum lactate dehydro-genase (LDH) level was shown to be a better surrogate for survival than PSA level [ 12 ]. Thus, so far, clinical trials attempting to interrogate CTC as a surrogate end-point for clinical trials have shown some promising results, prompting further extensive follow-up studies.

1.3.2 CTC as Predictive Marker to Guide Treatment

As similarly encouraging evidence supports the value of CTC as prognostic markers in various cancers, an obvious question is: Can we use CTC to guide treatment selection? More specifi cally, can we use CTC measurement at baseline, or at fol-low- up time-points, to predict patient’s response to treatment and thus guide ther-apy? An example is the SWOG S0500 clinical trial [ 13 ].

Results from the SWOG S0500 Phase III clinical trial (see Fig. 12.2 , Jeffrey B. Smerage, for Trial Schema) were presented at the 2013 ASCO San Antonio Breast Cancer Conference. The trial was designed primarily to determine in a fi rst line chemotherapy setting whether women with metastatic breast cancer and ele-vated CTCs by CellSearch assay (≥5 per 7.5 mL of whole blood) after 3 weeks of fi rst-line chemotherapy derive increased benefi t (overall survival and progression- free survival) from changing to an alternative chemotherapy regimen at the next cycle, instead of waiting for clinical evidence of progressive disease before changing to an alternative chemotherapy regimen. The trial was not designed to compare chemotherapies. The underlying hypothesis was that treatment decisions can be made based on CTC levels, with the belief that a signifi cant number of patients resistant to their fi rst line of therapy would respond to a second-line therapy.

1 Signifi cance of Studying Circulating Tumor Cells

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Patients may benefi t by switching early to a new line of therapy through avoiding the cumulative toxicities of ineffective therapy while spending more time on active therapy, thus improving quality of life and potentially tolerating future therapies better. Contrary to expectation, the patient data from randomized arms did not differ with respect to progression-free or overall survival. Given the very poor survival outcomes for this population, it was concluded that this population likely has a dis-ease that is generally resistant to cytotoxic mechanisms. However, trial data did demonstrate a large, clinically signifi cant, and statistically signifi cant difference in prognosis for patients in whom the CTC remained elevated after one cycle of fi rst-line chemotherapy. This is a population that should be considered for clinical trials of novel agents or novel treatment strategies early in the course of their disease.

In summary, the SWOG-S0500 trial validated the hypothesis that the group of patients with elevated CTC at baseline and 21 days after starting the fi rst chemo-therapy has a worse prognosis with regard to progression-free and overall survival, while low baseline CTC levels indicate a very good prognosis. The trial also showed that switching to a different chemotherapy sooner does not improve outcomes. For these patients, a clinical trial to investigate new targeted therapies should be consid-ered, since chemotherapy is not effective in this population of patients.

To address the clinical utility of CTC in another direction, the ongoing METABREAST trial aims at identifying patients without the need for aggressive treatment if they have low CTC at baseline level. In this study, CTC were measured at baseline, and patients receive chemotherapy if they are detected with >5 CTC, otherwise they will receive endocrine therapy [ 14 ].

1.3.3 CTC as a Marker for Early Detection of Solid Tumors

In addition to investigating CTC as a surrogate endpoint and predictive marker, other studies focus on the possibility of using CTC for early detection for solid tumors. As reported in mouse model breast cancer research, tumor cells can “leave home early” [ 15 ] and establish metastasis without the necessity of experiencing the steps of transformation at primary sites [ 16 ]. Another study in pancreatic cancer transgenic mouse model revealed that CTC can enter blood stream even before tumor formation [ 17 ]. These observations encourage the notion that CTC could be used for early detection of cancer, as harbingers of impending malignancy. However, preliminary data from pilot clinical trials has stimulated some disputes. For exam-ple, a study probing for CTC in patients with benign colon diseases has detected CTC in 11.3 % of the 53 patients analyzed, which could be false-positive results [ 18 ]. Another potential problem of using CTC for early detection of cancer is the extremely low CTC count in early stage patients. The cut-off of CTC count in a nonmetastatic breast cancer setting by CellSearch is determined to be 1 per 7.5 mL blood draw, which, although it is prognostic [ 19 ], can be easily missed depending on the sampling of the blood and the analysis process. One solution to interrogate such a low level of CTC is to examine larger volume of blood. This can either be

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