Pediatric Hematopoietic Stem Cell Transplantation for Pharmacists: The Gold Standard to Practice [1st ed.] 9783030434908, 9783030434915

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Pediatric Hematopoietic Stem Cell Transplantation for Pharmacists: The Gold Standard to Practice [1st ed.]
 9783030434908, 9783030434915

Table of contents :
Front Matter ....Pages i-xiii
The Pharmacist in Pediatric Hematopoietic Stem Cell Transplantation (Carolina Witchmichen Penteado Schmidt)....Pages 1-2
Stem Cells (Carolina Witchmichen Penteado Schmidt)....Pages 3-13
Stem Cell Transplantation in Pediatrics (Carolina Witchmichen Penteado Schmidt)....Pages 15-51
Homing of Stem Cells to the Bone Marrow: Finding the Way Home (Carolina Witchmichen Penteado Schmidt)....Pages 53-66
Pediatric Graft-Versus-Host Disease (GvHD) and the Pharmacist (Carolina Witchmichen Penteado Schmidt)....Pages 67-71
Antibacterial, Antifungal, and Antiviral Prophylaxis for Children Undergoing HSCT (Carolina Witchmichen Penteado Schmidt)....Pages 73-83
Peripheral Blood Stem Cell Mobilization in Pediatric Stem Cell Transplantation (Carolina Witchmichen Penteado Schmidt)....Pages 85-87
Reprogrammed Cells in Pediatric HSCT (Carolina Witchmichen Penteado Schmidt)....Pages 89-90
Stem Cell Transplantation in Children up to 1 Year Old (Carolina Witchmichen Penteado Schmidt)....Pages 91-93
Clinical Pharmacy in Pediatric Stem Cell Transplantation (Carolina Witchmichen Penteado Schmidt)....Pages 95-100
Back Matter ....Pages 101-123

Citation preview

Pediatric Hematopoietic Stem Cell Transplantation for Pharmacists The Gold Standard to Practice Carolina Witchmichen Penteado Schmidt

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Pediatric Hematopoietic Stem Cell Transplantation for Pharmacists

Carolina Witchmichen Penteado Schmidt

Pediatric Hematopoietic Stem Cell Transplantation for Pharmacists The Gold Standard to Practice

Carolina Witchmichen Penteado Schmidt Curitiba Paraná Brazil

ISBN 978-3-030-43490-8    ISBN 978-3-030-43491-5 (eBook) https://doi.org/10.1007/978-3-030-43491-5 © Springer Nature Switzerland AG 2020 This work is subject to copyright. All rights are reserved 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

Foreword

The outcome for children with cancer has shown enormous improvement over the last decades. In economically privileged countries, overall survival rates have now reached 80% at 5 years from diagnosis, and most of these young people will become long-term survivors. Therefore, an optimal management of children with cancer is needed, based on knowledge of current treatment strategies. Treatment options in pediatric oncology are chemotherapy, radiotherapy, and surgery. However, some patients also require a stem cell transplantation to have a chance for cure. Treatment of childhood cancer patients affects not only medical doctors or nurses but also pharmacists. Thus more and more knowledge about pediatric oncology pharmacy is required in daily practice. But up to now there is no book available for pharmacists who are involved in the field of pediatric stem cell transplantation to find the information they are looking for. With this book Carolina Schmidt has closed that previous gap. After publishing several books about pediatric oncologic pharmacy like Drug Therapy and Interactions in Pediatric Oncology: A Pocket Guide, Chemotherapy in Neonates and Infants: Pharmacological Oncology for Children Under 1 Year Old, and Pediatric Oncologic Pharmacy: A Complete Guide to Practice, Carolina Schmidt addresses in this book a wide range of topics, for example, the background of hematopoietic stem cell transplantation, its meaning in the various kinds of childhood cancer, graft-versus-host disease, and antibacterial, antifungal, and antiviral prophylaxis. All in all Carolina Schmidt has written an important book in a clear and professional style again. For the first time there is a book intended for all pharmacists who deal with the topic of stem cell transplantation for children and who want to find all needed information in one place. So I hope that the book will be widely distributed. I thank Carolina Schmidt for her innovative book idea and for the result of her work. Christian Müller Gert and Susanna Mayer Foundation, Communication & Individual Grant Program Wuppertal, Germany v

Acknowledgments

This book was possible due to the support of a lot of people. I would like to thank Vanessa Shimabukuro, my dedicated Springer editor, Anila Vijayan and her team, and the Springer team, for dedicating their time to transform a manuscript into a beautiful and easy-to-read book; the professionals and institutions who bought this book, which was written with a lot of work and dedication, with them in mind, to  help in this journey to cure children; Frederico Schmidt, my husband, for his constant support; and my family, for supporting my work and Christian Müller, for the foreword.

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Contents

1 The Pharmacist in Pediatric Hematopoietic Stem Cell Transplantation����������������������������������������������������������������������������������������    1 2 Stem Cells ������������������������������������������������������������������������������������������������    3 2.1 General Aspects of Stem Cells����������������������������������������������������������    3 2.2 Types of Stem Cells��������������������������������������������������������������������������    6 2.3 Differentiation and Self-Renew��������������������������������������������������������    8 2.3.1 Differentiation����������������������������������������������������������������������    8 2.3.2 Progenitor Cells��������������������������������������������������������������������    8 2.4 Embryonic and Non-embryonic Stem Cells ������������������������������������    9 2.4.1 Embryonic Stem Cells and the Possibility to Use Them in Pediatric Oncology/Hematology����������������    9 2.5 Basic Concepts on Researching and Handling with Stem Cells������   11 2.5.1 NOD/SCID Mice������������������������������������������������������������������   11 2.5.2 Intraosseous and Intravenous Transplantation in Research����������������������������������������������������������������������������   12 2.5.3 Understanding β2mnull NOD/SCID Mice in Stem Cell Research��������������������������������������������������������������������������������   12 2.5.4 Understanding Pre-immune Sheep in Stem Cell Research������   13 3 Stem Cell Transplantation in Pediatrics������������������������������������������������   15 3.1 General Aspects of Stem Cell Transplantation in Pediatrics������������   15 3.1.1 Hematopoietic Stem Cells����������������������������������������������������   15 3.1.2 Stem Cell Sources ����������������������������������������������������������������   16 3.2 Regimens for HSCT in Pediatrics����������������������������������������������������   18 3.2.1 The “Big Picture” of Regimens for Allogeneic Versus for Autologous Pediatric Stem Cell Transplantation������������   18 3.2.2 Myeloablative, Non-myeloablative, and Reduced-­Intensity Pediatric Regimens for Hematopoietic Stem Cell Transplantation���������������������������������������������������������������������   19 3.3 Autologous Stem Cell Transplantation in Pediatrics������������������������   20 3.3.1 Acute Lymphoid Leukemia (ALL) ��������������������������������������   21 ix

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Contents

3.3.2 Acute Myeloid Leukemia (AML)����������������������������������������   21 3.3.3 Chronic Myeloid Leukemia��������������������������������������������������   22 3.3.4 Non-Hodgkin Lymphoma ����������������������������������������������������   22 3.3.5 Hodgkin Lymphoma ������������������������������������������������������������   23 3.3.6 Solid Tumors������������������������������������������������������������������������   24 3.3.7 Wilms Tumor������������������������������������������������������������������������   27 3.3.8 Osteosarcoma������������������������������������������������������������������������   27 3.3.9 Germ Cell Tumors����������������������������������������������������������������   27 3.3.10 Rare Tumors��������������������������������������������������������������������������   27 3.4 Allogeneic Stem Cell Transplantation in Pediatrics ������������������������   28 3.4.1 Donors����������������������������������������������������������������������������������   28 3.4.2 Risks for the Recipient of Allogeneic Hematopoietic Stem Cell Transplantation Regarding the Sources of Stem Cells������������������������������������������������������������������������   33 3.4.3 Acute Lymphoid Leukemia (ALL) ��������������������������������������   34 3.4.4 Biphenotypic Leukemia��������������������������������������������������������   38 3.4.5 Acute Myeloid Leukemia (AML)����������������������������������������   39 3.4.6 Chronic Myeloid Leukemia��������������������������������������������������   44 3.4.7 Non-Hodgkin Lymphoma ����������������������������������������������������   46 3.4.8 Hodgkin Lymphoma ������������������������������������������������������������   48 3.4.9 Juvenile Myelomonocytic Leukemia, Anemias, Myelodysplasia, Primary Immunodeficiency, and Inborn Error of Metabolism ������������������������������������������   50 4 Homing of Stem Cells to the Bone Marrow: Finding the Way Home����������������������������������������������������������������������������   53 4.1 Homing ��������������������������������������������������������������������������������������������   53 4.1.1 A Big Picture of Homing������������������������������������������������������   53 4.1.2 Homing First Step����������������������������������������������������������������   54 4.1.3 Homing Second Step: Activation of Lymphocyte Function-­Associated Antigen 1 (LFA-1), Very Late Antigen 4/5 (VLA-4/5), and CD44��������������������������������������   58 4.1.4 Homing Third Step: Cytoskeleton Rearrangement, Membrane Type 1 (MT1)-Matrix Metalloproteinase (MMP) Activation and Secretion of MMP2/9����������������������   62 4.1.5 Homing Fourth Step: Rolling and Firm Adhesion of Progenitors to Endothelial Cells in Small Marrow Sinusoids Under Blood Flow������������������������������������������������   63 4.1.6 Homing Fifth Step: Trans-Endothelial Migration Across the Physical Endothelium/Extracellular Matrix (ECM) Barrier ����������������������������������������������������������   63 4.1.7 Homing Sixth Step: Stem Cells Finalize Their Homing by Selective Access and Anchorage to Their Specialized Niches in the Extravascular Space of the Endosteum Region and Periarterial Sites������������������������������������������������   64

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4.2 Bone Marrow and Homing ��������������������������������������������������������������   64 4.3 Preconditioning for Stem Cell Transplantation and Homing������������   65 4.4 Radiation and Homing����������������������������������������������������������������������   66 5 Pediatric Graft-Versus-Host Disease (GvHD) and the Pharmacist����   67 5.1 Graft-Versus-Host Disease (GvHD) ������������������������������������������������   67 5.2 Thymus Biochemistry����������������������������������������������������������������������   67 5.3 Pediatric GvHD Prophylaxis������������������������������������������������������������   68 6 Antibacterial, Antifungal, and Antiviral Prophylaxis for Children Undergoing HSCT ����������������������������������������������������������������������������������   73 6.1 Antibacterial Prophylaxis������������������������������������������������������������������   73 6.2 Antifungal Prophylaxis ��������������������������������������������������������������������   75 6.3 Antiviral Prophylaxis������������������������������������������������������������������������   78 6.3.1 Cytomegalovirus ������������������������������������������������������������������   79 6.3.2 Varicella Zoster ��������������������������������������������������������������������   81 6.3.3 Herpesviridae Family ����������������������������������������������������������   82 7 Peripheral Blood Stem Cell Mobilization in Pediatric Stem Cell Transplantation����������������������������������������������������������������������������������������   85 8 Reprogrammed Cells in Pediatric HSCT����������������������������������������������   89 9 Stem Cell Transplantation in Children up to 1 Year Old ��������������������   91 9.1 Stem Cell Transplantation as a Treatment for Children up to 1 Year Old��������������������������������������������������������������������������������   91 9.2 What Changes for the Pharmacist When the HSCT Will Be Performed in a Neonate of Infant����������������������������������������   92 10 Clinical Pharmacy in Pediatric Stem Cell Transplantation����������������   95 10.1 Overview����������������������������������������������������������������������������������������   95 10.2 Standards����������������������������������������������������������������������������������������   96 References ��������������������������������������������������������������������������������������������������������  101 Index������������������������������������������������������������������������������������������������������������������  107

About the Author

Carolina Witchmichen Penteado Schmidt,  Pediatric Oncologic Pharmacist and Writer. MBA in Planning and Business Management and Oncological Hospital Pharmacy Specialist. She is author of Chubby’s Tale: The True Story of a Teddy Bear Who Beat Cancer (in the BookAuthority list of “81 Best Leukemia Books of All Time”) and of the Springer books Pediatric Oncologic Pharmacy: A Complete Guide to Practice (in the BookAuthority lists: Best Pharmacy Books of All Time, Best Pharmacy Books to Read in 2020, and Best Pharmacy ebooks of All Time), Chemotherapy in Neonates and Infants: Pharmacological Oncology for Children Under 1 Year Old, and Drug Therapy and Interactions in Pediatric Oncology: A Pocket Guide. She has extensive experience with chemotherapy in children, as well as in hospitals and clinics with pharmacy, drug interactions, pediatrics, oncology, hematology, neonatology, intensive healthcare, and infectology. She has academic experience in pharmacy, pediatrics, oncology, and hematology, teaching specialists such as pharmacists, physicians, and nurses. She always had a fascination for hematopoietic stem cell transplantation in pediatrics, having worked with several pediatric stem cell transplantations and writing about this theme for children in her book Chubby’s Tale: The True Story of a Teddy Bear Who Beat Cancer. She published several children’s books—under the pen name Carola Schmidt—about cancer, such as Bald Is Beautiful: A Letter for a Fabulous Girl.

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Chapter 1

The Pharmacist in Pediatric Hematopoietic Stem Cell Transplantation

Hematopoietic stem cell transplantation (HSCT) has been used for a variety of malignant and nonmalignant diseases of the blood and immune-system in adults, children, and even infants. The HSCT involves the ablation of the bone marrow of the recipient with high doses of chemotherapy, and in some cases also radiation, to allow engraftment of the donor cells. It is autologous when the cells are from the own patient and allogeneic when the cells comes from a donor. The ability to successfully transplant hematopoietic stem cells to reconstitute the hematopoietic system is a relatively new area and in constant development [34, 91]. Multidrug therapy is required in HSCT area. High-doses of chemotherapy are used to kill tumorous cells at the cost of profound myelosuppression. Multidrug therapy is used to overcome resistance and heterogeneity of malignant cells. Moreover, there are many supportive drugs used to prevent and treat a variety of infections and rejection. Therefore, in this area, not only pediatric oncologic pharmacists but also clinical pharmacists are necessary [91]. The area of hematopoietic stem cell transplantation has been changing. Because of with the improvement of therapeutic strategies, better chemotherapy trials, progress in genomic research—which has revealed numerous genetic changes that led to a better risk-stratification of the patients, and the improvement in prevention of infections and GvHD (graft-versus-host disease), the guidelines for transplantation changed. For some diseases, with the development of new drugs and better trials, transplantation became less used. For others, it increased the overall survival. The transplantation will be an even more individualized decision over the years. We pharmacists should have a wide range of knowledge to understand and work well with pediatric stem cell transplantation, such as cellular biology, which includes types of stem cells, their potency, sources of stem cells, and other concepts. Although hematopoietic stem cell transplantation differs of regenerative medicine, the basic knowledge in regenerative medicine helps us to understand better the stem cells, how they work, and the studies and new approaches in this area. Sometimes regenerative medicine almost interconnects with our area, bringing important concepts and findings to improve hematopoietic stem cell transplantation. Because of all this, © Springer Nature Switzerland AG 2020 C. W. P. Schmidt, Pediatric Hematopoietic Stem Cell Transplantation for Pharmacists, https://doi.org/10.1007/978-3-030-43491-5_1

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regenerative medicine is also discussed sometimes in this book. The cellular biology concepts help to understand not only the reprogramming of stem cells, why each type off cell can be used or why cannot, and when and how they should be used but also other aspects that influence all our work, including why and when each stem cell transplantation is done and what drugs will be used for. The broad knowledge of drugs used in stem cell transplantation is essential. All the processes that occur in a patient’s body after stem cells infusion are important to understand the necessity of preventive therapies, as well as monitoring and treatment of infections and rejection. It is not easy to make the best choices and follow all the changes in pediatric HSCT area. Commonly, there are no “right or wrong” decisions. The new regimens of conditioning with chemotherapy followed by HSCT, and the chemotherapy or immunosuppressive drugs chosen for prophylaxis, arise based on clinical trials and the data that shows what works, what does not work, and what can be improved for each patient (because depending on the risk, donor available, if the patient is in first or second complete remission, everything can change). Due to the complexity of this relatively new area, absence of data and constant changes, there are many spaces for pharmacists who are interested in pediatric HSCT. The follow-up with blood serum dosages can be valuable for these patients when they change from IV to oral doses or to define the best dose of each drug in a combination of drugs for a regimen. There is a great needing of oncology/hematology pharmacists and clinical pharmacists in this area, and, since everything is relatively new and in needing of researchers and new, modern guidelines, it is the role of the pharmacists to find their places and how they can help and be part of all that. Conditioning regimens for HSCT patients have very high doses of chemotherapy, which can lead to many late effects. And that is not over when the patients are cured, they need to be checked through their lifetimes. Follow up the patients through their lifetimes is another great area in needing of pharmacists. There are countries in which the late effects are constantly underrated because of the lack of investments and professionals. But it is a common complaint of adults who had pediatric malignancies even in the most developed countries that they would like to have a better understanding of late effects, more follow-­ups, and even support groups with other patients and multidisciplinary healthcare professionals.

Chapter 2

Stem Cells

2.1  General Aspects of Stem Cells There are different types of stem cells; but all of them have two abilities in common: self-renewal and differentiation. Those abilities allow their use for stem cell transplantation—to regenerate the host’s blood-forming system. The term “stem cell” seems to have first appeared in the scientific literature in 1868, written by the German biologist Ernst Haeckel. Studies and the search for stem cells probably came up in 1945, during the aftermath of the bombings in Hiroshima and Nagasaki. People who had a prolonged period exposed to lower doses of radiation could not regenerate they hematopoiesis with white blood cells and platelets, and they died. Higher doses of radiation also killed the stem cells of the intestinal tract, resulting in more rapid death. Based on that, researchers demonstrated that doses of whole-body X-irradiation caused the same radiation syndromes and death due to hematopoietic failure approximately 2 weeks after minimum radiation exposures. Shielding a single bone or the spleen from radiation prevented those irradiation syndromes. Thereafter, researchers showed that whole-body irradiated mice could be rescued from hematopoietic failure by injecting cells from blood-forming organs such as the bone marrow. In 1956, it was demonstrated that the injected bone marrow cells directly regenerates the blood-forming system, rather than releasing factors to cause the repairing of irradiation damage. It all led to the only known treatment nowadays for hematopoietic failure following whole-body irradiation, which is the stem cell transplantation to regenerate blood-forming system in the hosts [4, 36, 46, 48]. As it was mentioned in the previous paragraph, there are many types of stem cells; and the most important types for us who work in pediatric hematology/ oncology area are inside the family of hematopoietic stem cells. Hematopoietic stem cells of a human generate approximately 100 billion new hematopoietic cells each day. Experiments with genetic markers showed that multipotent/pluripotent stem cells (they can generate most of the cell types in the blood) must exist in the bone marrow. Besides the great applicability and importance of hematopoietic © Springer Nature Switzerland AG 2020 C. W. P. Schmidt, Pediatric Hematopoietic Stem Cell Transplantation for Pharmacists, https://doi.org/10.1007/978-3-030-43491-5_2

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stem cells for us, non-hematopoietic stem cells also have been used in studies on oncology and hematology, and some of these studies showed positive features to be applied in hematology and oncology treatments—such as the mesenchymal stem cells supportive role in hematopoiesis and their anti-inflammatory and immunomodulatory properties that improve the engraftment and treatment of graft-versus-host disease in patients receiving hematopoietic stem cell transplantation—and others showed negative features of these cells, such as the involvement of mesenchymal stem cells in leukemogenesis, with several genetic and functional abnormalities that were detected in the mesenchymal stem cells of leukemia patients, their leukemia-­enhancing effects, and also an induced chemotherapy-resistance in leukemia cells. The therapeutic value for hematology and oncology versus the harmful effects of non-hematopoietic stem cells—like mesenchymal stem cells—is still being evaluated, and new studies on this topic are necessary. The umbilical cord blood generally also has non-hematopoietic stem cells, such as endothelial progenitor cells, mesenchymal stromal cells, unrestricted somatic stem cells, very small embryonic-­like stem cells, multilineage progenitor cells, and neuronal progenitor cells. Hematopoietic and non-hematopoietic stem cells are largely researched also for tissue repair and regenerative medicine [4, 36, 46, 48–50]. Hematopoietic stem cells (HSCs) have the potential to migrate—through a process called homing, which is discussed in detail in Chap. 4—and repopulate, by self-renewal and multilineage differentiation capacities. The essential feature of self-renew allows stem cells to make copies with the same or very similar potential. That characteristic is unique, and more differentiated cells, such as hematopoietic progenitors, cannot do the same, even though most of the progenitors can expand significantly during a limited time. It is not well clear which key signals are involved in their self-renewal; however, the expression of telomerase, which is the enzyme necessary for maintaining telomeres (which are the DNA regions at the end of chromosomes to protect them from accumulating damage during the DNA replication), was shown as being associated with self-renewal of stem cells. The engraftment of stem cells in bone marrow, which is the rate of stem cells received during transplantation that were able to start growing and making new cells, is essential to the transplantation success. The morphology of stem cells usually is circular with a low cytoplasm to nucleus ratio. There are several specific markers of the general lineages; however, alkaline phosphatase is common to most stem cell types. Stem cells undergo mitosis, originating two different daughter cells [4, 36, 46, 48, 49]. During the embryonic development, blood-forming stem cells do homing from the fetal liver—via blood circulation—to the bone marrow, and then they repopulate the bone marrow with high levels of immature and maturing blood cells of all lineages. Most of these cells are released into the circulation, and a small pool of undifferentiated stem cells keep within the bone marrow. Stem cell motility is considered as the migration, homing, retention, and release of cells, and it is crucial for hematopoietic development and bone marrow repopulation. Definitive stem cells have the ability to migrate in the circulation and home to populate the host bone marrow to rescue lethally irradiated recipients; however, non-definitive

2.1  General Aspects of Stem Cells

5

stem cells require an additional maturation step in the form of in  vitro incubation—with stromal cells and cytokines—before they develop from pre-definitive into definitive stem cells to have the abilities of functional migrating and repopulating. These observations were made starting from studies with murine fetal livers. Hematopoietic stem cells were isolated from the yolk sac and aorta-gonad-mesonephros region before completion of the circulatory system. The vast majority of these stem cells were not definitive, but despite their inability to home to the bone marrow, stem cells isolated from the murine yolk sac could give rise to hematopoietic progeny if they were directly injected into the liver of newborn mice that were preconditioned as fetuses with low-dose chemotherapy. After birth, 0.5% of the human umbilical cord blood cells are immature CD34+ progenitor cells—which is a relatively high amount—including a minority of more primitive, undifferentiated CD34+/CD38−/low cells, suggesting high levels of hematopoietic stem cell migration via circulation during the late-stage embryonic development. A small pool of hematopoietic stem cells within the bone marrow continuously produces high levels of immature and maturing myeloid and lymphoid blood cells. They have limited lifetimes and are released into the circulation, and undifferentiated stem cells are maintained within the bone marrow throughout life. However, low levels of noncycling quiescent progenitor cells, including primitive stem cells, are also released into the peripheral blood [11]. The stem cell mobilization, homing, engraftment, and repopulation are sequential events with physiological roles that are essential to the success of hematopoietic stem cell transplantation (HSCT). Hematopoietic stem cells require a bone marrow microenvironment to regulate their migration, proliferation, and differentiation to maintain active the hematopoiesis throughout life. • Homing Stem cell homing is a coordinated process in which circulating hematopoietic cells actively cross the blood/bone marrow endothelium barrier, and they lodge at least transiently in the bone marrow compartment by adhesion before their proliferation. This is a complex biochemistry process that is discussed in detail in Chap. 4. Although many cell types can home to the bone marrow (BM)—which includes long-term repopulating human CD34+/CD38− stem cells, short-term repopulating CD34+/CD38+ progenitors, and mature specialized T-cells and neutrophils—only stem cells, which can home to their endosteal niches, initiate long-term repopulation. Both stem cell homing and engraftment/repopulation are regulated by mechanisms of migration and adhesion, such as stromal-derived factor 1/CXC chemokine receptor 4+signaling (SDF-1/CXCR4+signaling), very late antigen 4/vascular cell adhesion molecule 1 (VLA4/VCAM-1), and CD44/hyaluronic acid [4]. • Engraftment and Repopulation Stem cell engraftment requires cell division, and instead of homing—which various types of cells can do—only stem cells, which can home to their endosteal niches, can initiate a long-term repopulation. While homing does not require host

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preconditioning with DNA damage, durable engraftment with high levels of stem cells repopulation is dependent on this procedure [4]. The engraftment in bone marrow is essential to transplantation success. Progenitors isolated from the murine embryo were shown to lack directional migration and homing in adult mice that received transplants. These progenitors isolated are non-definitive, which means they cannot engraft the bone marrow, and fail to rescue lethally irradiated recipients. These progenitors require in vitro stimulation with stromal cells, which convert them into functional, bone marrow-repopulating stem cells—then having the potential to migrate and homing potential. The migration of mobilized human CD34+ cells toward a gradient of the chemokine stromal-­ derived factor-1 (SDF-1) in vitro correlates with their in vivo repopulation potential in patients who received autologous transplants and in immune-deficient non-obese diabetic or severe combined immunodeficient mice [4]. Evidence of donor replacement of the recipient’s bone marrow begins with the increased white blood cell counts; therefore, the patient starts to lose the transfusion dependency. Engraftment occurs when the recipient achieves three consecutive days of absolute neutrophil counts >0.5 × 109/L. When the volume of CD34+ stem cells is higher, the engraftment occurs earlier, such as after HSCT with peripheral blood cells. Engraftment syndrome occurs with the fast rise in white blood cells, and the symptoms are fever, rash, pulmonary symptoms, and weight gain secondary to capillary leak. A short course of intravenous steroids can alleviate the symptoms since there is no infection. Primary engraftment failure is diagnosed when there is a lack of engraftment within 6 weeks after transplantation. To determine the marrow engraftment and primary engraftment failure, chimerism studies (of peripheral blood or bone marrow), measure of donor, and recipient cell percentages (to compare them in the recipient) can be used. Secondary graft failure is the graft rejection and occurs after the initial wave of engraftment secondary to the continued presence of recipient cytotoxic T-cells. Chimerism studies can be used to determine that the majority of cells are of the host origin [91]. Causes of graft failure include nonmyeloablative regimen, low volume of stem cells, high immunogenicity (mismatched HSCT), infection, recurrence of an underlying hematological malignancy, and the use of myelosuppressive drugs during engraftment—such as for GvHD.

2.2  Types of Stem Cells Stem cells have self-renew capacity, and they also can differentiate into the various types of cells with specialized functions that a human body has. Stem cell groups can be first divided, according to their origin, into embryonic (from the blastocyst stage embryo), fetal or perinatal (from cord blood, placenta, amniotic fluid, amniotic membranes, or fetal tissues), or adult stem cells (from adult tissues). Stem cells are also subdivided according to their differentiation potential, which means the number of cell types that can be differentiated from them [46–48, 51]:

2.2  Types of Stem Cells

7

• Totipotent: Stem cells can divide and can differentiate into any cell of human tissues. Because totipotency is the highest differentiation potential, it allows cells to form both embryo and extra-embryonic structures. An example of a totipotent cell is a zygote. These cells can later develop either into any of the three germ layers or form a placenta. After approximately 4  days, the blastocyst’s cells become pluripotent. The totipotent stem cells are those embryo cells at 1–3 days. • Pluripotent: They can differentiate into any cell of all germ layers but not into extraembryonic structures, such as the placenta. However, some multipotent cells are capable of conversion into unrelated cell types, and some authors suggest naming them pluripotent cells. Two examples of pluripotent stem cells are embryonic stem cells and induced pluripotent stem cells derived from the epiblast layer of implanted embryos. Their pluripotency is a continuum, starting from completely pluripotent cells such as embryonic stem cells and induced pluripotent stem cells, ending on representatives with less potency (multipotent, oligopotent, or unipotent cells.) The pluripotent stem cells are blastocyst cells (5–14 days). • Multipotent: They have the ability to differentiate into multiple closely related cell types only. An example of multipotent stem cell is the hematopoietic stem cell, which can differentiate into several types of blood cells. After differentiation, the hematopoietic stem cell becomes an oligopotent cell. This differentiation ability is restricted to cells of its lineage. However, some multipotent cells are capable of conversion into unrelated cell types, and authors suggest naming them pluripotent cells. Multipotent stem cells are present in fetal tissues, cord blood, and also adult cells. • Oligopotent: They can differentiate into specific cell types, and an example is myeloid stem cell, which can divide into white blood cells but not red blood cells. • Unipotent: These stem cells have the self-renewal capabilities that characterize stem cells but have the narrowest differentiation capabilities and a special property of dividing repeatedly. These cells are only able to form one cell type, for example, dermatocytes. Correlating the categories of origin and potency, we have [46–48]: • Embryonic stem cells, which can be subdivided into totipotent or pluripotent • Fetal stem cells, which can be divided into pluripotent or multipotent • Adult stem cells, which can be pluripotent, oligopotent, or unipotent There is no clinical use of embryonic-derived stem cells for now, because of technical barriers that include not only the ethical issues. Although this type of stem cells has a higher ability for development and differentiation than those from adult tissues, recognizing the actual hematopoietic stem cells in cultures is problematic, reflecting the variability in the markers and altered reconstitution behavior of these cells, which are expected to mimic fetal hematopoietic stem cells. There is also a potential risk of including undifferentiated cells in a graft of stem cells when using these cell sources. Adult stem cells are the cells of choice for regenerative medicine; but embryonic stem cells have the ability to differentiate into different types

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of cells and show great applicability. Published studies reported that contributions of adult stem cells to cell types outside their tissue of origin are very rare, and the concept of plasticity—which is the presumed ability of tissue specific stem cells to differentiate into cell types different from those of their tissue of origin—maybe could explain that. That concept came when studies led to claim that hematopoietic stem cells have the capacity to differentiate into a wider range of tissues than previously researchers thought, and it was suggested that, following reconstitution, bone marrow stem cells can differentiate not only into blood cells but also muscle cells (skeletal myocytes and cardiomyocytes), brain cells, liver cells, skin cells, lung cells, kidney cells, intestinal cells, and pancreatic cells. However, results suggested that the normal tissue regeneration relies predominantly on the function of cell type-­specific stem or progenitor cells. Evidence on stem cell plasticity has been published, and this subject has been discussed. There are currently many studies on adult stem cell plasticity, positive and negative evidence. Adult stem cells maintain the tissue homeostasis and usually differentiate into a restricted range of progenitors and terminal cells that replace the local parenchyma; they are also involved in injury repair and sustaining cellular turnover [46, 48].

2.3  Differentiation and Self-Renew 2.3.1  Differentiation The stem cells can differentiate through signals to influence their specialization process, and they can be divided into external, such as physical contact between cells or chemical secretion by surrounding tissue, and internal, which are controlled by genes in DNA. Stem cell activity depends on the organ in which they are in. Their division is constant in bone marrow, but in some other organs, such as pancreas, stem cells can divide only under special physiological conditions [51].

2.3.2  Progenitor Cells One of the most important properties of hematopoietic stem cells is the capacity of differentiation into progenitors—to have a reduced differentiation capacity and an extensive proliferative potential, as well as their most important characteristic: the inability to self-renew. Therefore, progenitor cells have to be constantly regenerated from the hematopoietic stem cells (HSCs). The common lymphoid progenitor (CLP) has the potential to generate all of the lymphoid but not the myeloerythroid cells; and the common myeloid progenitor (CMP) has the potential to generate all of the mature myeloerythroid but not the lymphoid cells. Progenitors have clinical potential and are objects of studies. Growth factors and cytokines conduct a complex system to HSC differentiation. Studies showed that once HSCs had proceeded

2.4  Embryonic and Non-embryonic Stem Cells

9

with differentiation, they were not able to revert to a self-renewing state. Moreover, specific signals, in a strict regulation, seem to be needed to maintain HSCs, pointing to the proliferative potential present in HSCs—deregulation could result in malignant diseases, such as leukemia or lymphoma [48].

2.4  Embryonic and Non-embryonic Stem Cells 2.4.1  E  mbryonic Stem Cells and the Possibility to Use Them in Pediatric Oncology/Hematology Embryonic Stem Cells Embryonic stem cells are originated during embryonic development, in the blastocyst stage embryo, which has a self-replication potential and cell pluripotency in that stage, causing a following differentiation into cells of the three germ layers that form all membranes in the body. A blastocyst is formed after the fertilization, on the fusion of sperm and ovum. Its inner wall is lined with short-lived embryonic stem cells. Blastocysts are composed of two cell types: the inner cell mass—which develops into epiblasts and induces the development of a fetus—and the trophectoderm, which forms the extraembryonic support structures needed for the successful origin of the embryo, such as the placenta. As the trophectoderm begins to form a specialized support structure, the inner cells remain undifferentiated, pluripotent, and proliferative. Human embryonic stem cells are derived from the inner cell mass. During the process of embryogenesis, cells form the germ layers endoderm, mesoderm, and ectoderm. After human embryonic stem cells differentiate into one of the germ layers, they start to have a limited potency to only the cells of the germ layer, becoming multipotent stem cells. Then, pluripotent stem cells occur all over the organism as undifferentiated cells and can proliferate by the formation of the next generation of stem cells, as well as differentiate into specialized cells under certain physiological conditions [46, 51]. Research with Embryos and the Reprogramming (of Embryonic and Non-­ embryonic) Stem Cells Research with embryo stem cells started in the mid-1800s and is ongoing despite the ethical controversies. The tipping point in research with stem cells came up in 2006, when scientists Shinya Yamanaka and Kazutoshi Takahashi discovered that it is possible to reprogram multipotent adult stem cells to the pluripotent state using retrovirus-mediated transduction of mouse fibroblasts with four transcription factors (Oct-3/4, Sox2, KLF4, and c-Myc), which are mainly expressed in embryonic stem cells, and they induced the fibroblasts to become pluripotent. This new type of stem cells was named induced pluripotent stem cell. This process avoided using fetus. Embryonic stem cells are used by researchers to study the possibility to develop new sources to try to decrease the ethical issues about them, as well as to understand the usability of embryonic stem cells for pharmaceutical tests about

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drugs in pregnancy, heart diseases treatment, miscarriage understanding, and other possibilities. Embryonic stem cells are considered to treat diseases caused by tissue dysfunction or loss, and this type of research with embryonic stem cells seems to be more frequent than studies with embryonic stem cells to treat oncological and hematological diseases. There are studies with embryonic stem cells in hematology to try to develop immunotherapy with an “off-the-shelf” human embryonic stem cell (hESC)-derived natural killer cell for the treatment of acute myeloid leukemia (AML), which is in progress at the University of California and conducted by the researcher Dan Kaufman. This study is being developed with in vitro and mouse models and is based on that hESC-derived NK cells provide an approach to treat relapsed or refractory AML that is resistant to current chemotherapy options. hESCderived NK cells provide a standardized, homogeneous, “off-the-shelf” cellular immunotherapy source that should be used as an allogeneic adoptive transfer treatment for AML patients who have never achieved remission with standard induction therapy or relapsed after previous chemotherapy. hESC-derived NK cells acts on tumor killing its cells by direct cytotoxicity, antibody-dependent cell-mediated cytotoxicity, induction of apoptosis, and production of cytokines [45, 51]. Regarding the studies with embryonic stem cells for uses out of the oncology and hematology, studies are being developed intending to have structures for research with less controversial sources than embryos left over from in  vitro fertilization procedures; and researchers have achieved new ways to have stem cells, such as the development of structures that mimic early embryos. The embryo-like structures were developed and are the first to produce rudimentary reproductive cells and also go through stages that resemble several landmarks in early human development. While some researchers are studying reprogramming of adult stem cells, other research groups are seeking to make sophisticated artificial embryo-like structures that can be used to study early-stage embryonic development. Researchers intend that these embryo-like stem cells someday will be used by used by pharmaceutical companies to test whether drugs are safe for pregnant women, and by physicians to investigate why some patients have multiple miscarriages. These studies have their own ethical issues, because although these structures are developed in vitro, they develop an embryo, and many people consider it as an individual. Embryo-­ like structures were developed using colonies of human pluripotent stem cells from embryos plus others that were made from skin cells reprogrammed to an embryo-­ like state. Using a mix of biochemical signals at the right time, Jianping Fu—who is a bioengineer at the University of Michigan who led the research—and his team were able to coax the colonies of human pluripotent stem cells, which can differentiate into other cell types to mimic the first step by which homogenous cells of an early embryo become various tissue types. The method worked only about 5% of the time. To increase that to 95%, he and his team replaced the conventional culture plates used to grow colonies of pluripotent stem cells with a small device containing different materials channels. The middle one was filled with a gel and lined with support posts that anchor the colonies of pluripotent cells. The colonies were loaded through another channel, and the third channel was used to deliver a couple of dozen of biochemical signals at precise times. The result was that the

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embryo-like structures produced using the last process were more similar to natural embryos. The primitive streak was better defined, and precursors of the cells that go on to form eggs and sperm emerged. After the modern process of reprogramming adult stem cells was discovered, according to the research of scientists Shinya Yamanaka and Kazutoshi Takahashi as cited before, it is possible to induce terminally differentiated cells to become pluripotent again even though pluripotency can occur naturally only in embryonic stem cells. This process of direct reprogramming converts differentiated somatic cells into induced pluripotent stem cells (iPSC) lines that can form all cell types of an organism. This reprogramming is focused on the expression of oncogenes, such as Myc and Klf4 (Kruppel-like factor 4). There is a histone alteration; and downregulation of genes, including that by p53, promoting genome stability. There is a mutagenic risk due to all this process, which probably can be avoided with reprogramming of stem cells, because although there were demonstrated single mutations in the non-genetic region, there were observed non-­ retrotransposon insertions, which led to the conclusion that current reprogramming methods can produce fully pluripotent iPSCs with no severe genomic alterations. For the reprogramming of cells to iPSCs, fibroblasts were used as a source at first. Since biopsy was needed to achieve these types of cells, the technique underwent further research to investigate whether more accessible cells could be used, such as peripheral blood cells, keratinocytes, and renal epithelial cells found in urine. The best stem cell source still appears to be the fibroblasts, because its stimulation can be fast and well controlled A suggested alternative strategy to stem cell transplantation is to stimulate patient’s endogenous stem cells to divide, or differentiate, occurring naturally when skin wounds are healing. Pancreatic exocrine cells already were reprogrammed to functional, insulin-producing beta cells [44, 51].

2.5  B  asic Concepts on Researching and Handling with Stem Cells 2.5.1  NOD/SCID Mice Studies with stem cells are commonly done in mice. The most used mice for this type of research are the non-obese diabetic/severe combined immunodeficiency (NOD/ SCID). The SCID mutation was discovered in a colony of mice in 1980 by the Fox Chase Cancer Center team (Philadelphia, PA). This recessive autosomal mutation impairs a lymphoid differentiation. These mice are deficient in T- and B-cells but their myelopoiesis is not affected. On NOD mice, the functions of antigen-­ presenting, myeloid, and natural killer cell are impaired. Most homozygotes do not have any detectable IgM, IgG1, IgG2a, IgG2b, IgG3, or IgA.  Some NOD mice with prkdcscid mutation can become leaky from the spontaneous development of functional T and B lymphocytes. The presence of the prkdcscid mutation does not allow for the phenotypical expression of the type I diabetes that characterizes the

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NOD background. NOD/SCID mice tend to survive under specific and opportunistic pathogen-free (SOPF) conditions for 8–9 months, because they develop thymic lymphoma [5].

2.5.2  I ntraosseous and Intravenous Transplantation in Research Intraosseous infusion for transplantation in human patients has been studied compared with intravenous infusion, showing that intraosseous infusion of stem cells, although it is a safe procedure, it generally is similar to the intravenous infusion regarding the results, so it is not necessary due to the simplicity of an intravenous procedure. In mice, the results are different, and it should be considered in studies. Intraosseous transplantation of human CD34+- and CD34−-enriched cord blood progenitors in immune-deficient mice resulted in much higher seeding efficiencies of both short- and long-term repopulating cells than intravenous transplantation, instead of in human, in whom? was not observed significant differences between intraosseous or intravenous transplantation. Structural and size-related differences between murine and human bones may take a role; murine bones are smaller and have compact bone marrows, while human bones have less compact bone marrows containing fat cells, bigger blood vessels, and higher blood volume [4, 6].

2.5.3  U  nderstanding β2mnull NOD/SCID Mice in Stem Cell Research The biology of human progenitor cell homing has been studied in NOD/SCID (nonobese diabetic/severe combined immune-deficient) mice that were preconditioned with total body irradiation. The first studies were realized with SCID mice that were irradiated. These mice received transplantation of normal and leukemic human progenitor cells. The researchers facilitated high levels of multilineage myeloid and lymphoid human hematopoiesis in the murine bone marrow and identification of leukemic CD34+/CD38− stem cells—which are called SCID leukemia-initiating cell (SLIC)—obtained from patients with acute myeloid leukemia. Later, researchers started to study in NOD/SCID mice, with additional reduced immunity, in which was studied the identification of primitive, normal human CD34+/CD38− SCID repopulating cells (SRCs), CD34−/CD38− SRCs, and SLIC from different subtypes of acute myeloid leukemia that could initiate the disease in mice that received transplantation. β2 microglobulin-deficient (β2mnull) NOD/SCID mice have further reduced innate immunity, due to the lack of natural killer cells. CD34+/CD38+ short-­ term repopulating cells (STRC) enriched in mobilized peripheral blood were identified in studies by researchers with B2mnull NOD/SCID mice or by inactivation of NK-cell activity in NOD/SCID mice [4].

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Although mechanisms of human stem cell homing were characterized in immune-deficient NOD/SCID and β2mnull NOD/SCID mice, there is a xenogeneic barrier-dependent limitation in these mice, which cause a low frequency of homing progenitors. There is a positive correlation between engraftment in patients and NOD/SCID or β2mnull NOD/SCID mice [4].

2.5.4  U  nderstanding Pre-immune Sheep in Stem Cell Research The biology of human progenitor cell homing has been also studied with pre-­ immune sheep, which uses pre-immune sheep fetuses. These fetuses are implanted with immature human CD34+ and CD34− cells with no preconditioning. It allows the short- and long-term stem cell engraftment in sheep recipients of both prime and secondary transplants. In this model, immature human bone marrow CD34+enriched cells transplanted to sheep fetuses can be detected 24–48  h later in the patient’s bone marrow [4].

Chapter 3

Stem Cell Transplantation in Pediatrics

3.1  G  eneral Aspects of Stem Cell Transplantation in Pediatrics 3.1.1  Hematopoietic Stem Cells The concepts of cellular biology and the classification of stem cells were described in the previous chapter. In this chapter, the focus will be only on hematopoietic stem cells, which we can use today for stem cell transplantation in pediatric oncological/ hematological patients. Hematopoietic stem cells are the most thoroughly characterized tissue-specific stem cells and have been experimentally studied for more than 50 years. The transplantation we work with in pediatric oncology/hematology area uses multipotent hematopoietic stem cells; they can generate all functional hematopoietic lineages in blood, including erythrocytes, leukocytes, and platelets; therefore, its transplantation solves the inappropriate functioning of the hematopoietic system in diseases such as leukemia and anemia. Hematopoietic stem cells for transplantation are collected from the bone marrow, peripheral blood, or umbilical cord blood. The transplantation can be autologous, with stem cells from the patient, or allogenic, with stem cells from a donor. When the donor is an identical twin, the transplantation is named syngeneic [51]. The induced pluripotent stem cells (iPSC), which are adult stem cells reprogrammed into a pluripotent status through a technic that is described in detail in the subchapter “Research with Embryos and the Reprogramming (of Embryonic and Non-embryonic) Stem Cells,” in the previous chapter of this book, are a new promise in pediatric oncology/hematology, since using the patient’s cells can solve problems like the high rate of rejection, and it was suggested that iPSC can solve the issue of the development of malignancies by transplanted stem cells [51].

© Springer Nature Switzerland AG 2020 C. W. P. Schmidt, Pediatric Hematopoietic Stem Cell Transplantation for Pharmacists, https://doi.org/10.1007/978-3-030-43491-5_3

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3.1.2  Stem Cell Sources Stem cells for HSCT are collected from the bone marrow, peripheral blood, or umbilical cord blood; and the most common source is peripheral blood [91]. Umbilical Cord Blood Stem Cells Umbilical cord blood (UCB) is a reliable source of hematopoietic stem cells for transplantation. Stem cells from this source are used either for autologous or allogeneic hematopoietic stem cell transplantation (HSCT). Nowadays, stem cells are mostly collected from peripheral blood. Although umbilical cord blood is a rich source of hematopoietic stem cells, it is usually discarded at birth, and the volume of cells is the lowest if UCB is compared with the other sources, because of the volume of blood. Stem cells from cord blood require less stringent HLA-matching criteria (six loci, rather than ten as when bone marrow cells are used). Cord blood stem cells can be cryopreserved, which provides a solution to patients in urgent need of transplantation. These factors are particularly advantageous for non-Caucasian patients, because of the difficulty to find a match. The safety and efficacy of stem cells from umbilical cord blood have been widely studied and established for both children and adults for a variety of indications. Cells from this source have a lower risk of graft-­versus-­host-disease (GvHD), being the lowest of all sources, as well as greater protection against disease relapse in various cases. The most important disadvantage of using umbilical cord blood as a source is the low yield of stem cells when compared to bone marrow or peripheral blood. Using fewer stem cells than the ideal amount can delay the hematological recovery, as well as increase the engraftment failure and risk of infection rates. This also results in increased hospitalization times treatment costs. Double umbilical cord blood transplantation is often employed to decrease these risks [34, 91]. There are three types of umbilical cord blood banks: public, private, and hybrid. Public banks store sources received from donors, which are then listed on international registries and made available for any potential HLA-match recipient pending or in the future. Private banks, also called family banks, store sources for exclusive future use by the donor or their matched relative. This limited use translates to low recall rates on UCB units. The overall potential of stem cells from autologous cord blood is limited; and the indication of the stored cord blood to autologous stem cell transplantation, like other sources, is not possible for every disease and case. However, many families are not well advised and can be driven by subjective and emotional factors when they decide to store the umbilical cord blood. Hybrid banks offer a combined public and private storage. Either the private bank offers a public donation or the public bank offers a private storage option. There are different models of hybrid banks: 25% of privately stored umbilical cord blood samples are donated to the public system in accordance with national legislation (Turkish model); umbilical cord blood samples are stored privately, but if an unrelated match is found, the unit can then be donated to the public (Spanish model); harvested umbilical cord blood units can be divided into

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17

two—one portion for exclusive use and the other for public use (Virgin model)— and umbilical cord blood samples are stored for private use and at a later stage are released to the public following consent from the donor. The public side of hybrid banks is generally cross-subsidized by income generated from its private activities for all models [34]. Peripheral Blood Stem Cells Nowadays, stem cells are mostly collected from peripheral blood, following a 4–5-­ day regimen with a mobilizing agent such as filgrastim, which is a granulocyte colony-stimulating factor (G-CSF). This drug mobilizes stem cells from the bone marrow to peripheral blood. Peripheral blood stem cells are collected through apheresis. These cells are identified by the presence of the cell surface marker CD34. CD means a cluster of differentiation. The number required to ensure engraftment in general is 5 × 106 CD34+ cells/kg. The peripheral blood, after the G-CSF mobilization, has the highest yield of CD34+ cells compared with the other sources, and the engraftment when peripheral blood cells are used occurs earlier than when using cells from another sources [34, 36, 91]. The use of bone marrow stem cells versus peripheral blood stem cells was largely compared. The engraftment is faster with peripheral blood stem cells than bone marrow stem cells. Some studies have reported that stem cells from peripheral blood are associated with a higher risk of developing GvHD than from bone marrow or umbilical cord blood. However, the GvHD is associated with a lower risk of relapse, reflecting the ability of the immune response to simultaneously attack the malignant cells, which is called a graft-versus-malignancy effect. Disease-free and overall survival seem not to differ between those two sources. Research comparing bone marrow and peripheral blood sources from HLA-matched related donors for transplantation after myeloablative conditioning and conventional post-grafting immunoprophylaxis for GvHD with a calcineurin inhibitor plus methotrexate showed comparable outcomes except for an increase of 6–15% in the incidence of chronic GvHD after transplantation with peripheral blood sources. However, a study intending to investigate the same but with a non-­ablative conditioning showed those rates of acute and chronic GvHD, as well as overall survival, were similar after transplantation of bone marrow and peripheral blood sources. Another study compared outcomes with bone marrow and peripheral blood as stem cell sources for transplantation from haploidentical donors after myeloablative conditioning and concluded that, since regulatory T-cells have a role in modulating GvHD, the low incidence of severe chronic GvHD found with a regimen of GvHD prophylaxis (which includes post-transplant cyclophosphamide) can be explained by the resistance of these cells to cyclophosphamide [35, 36, 91]. Bone Marrow The stem cells are collected from the bone marrow through the pelvic bone of the donor under general anesthesia. There is a donor inconvenience and a difficult logistic in bone marrow stem cells compared with peripheral blood stem cell donation [36].

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The use of bone marrow stem cells and peripheral blood stem cells was largely compared. The engraftment is slower with bone marrow stem cells than peripheral stem cells. Some studies have reported that stem cells from the bone marrow are associated with a decreased incidence in GvHD than stem cells from peripheral blood. However, the GvHD is associated with a lower risk of relapse, reflecting the ability of the immune response to simultaneously attack the malignant cells, which is called graft-versus-malignancy effect. Disease-free and overall survival seem not to differ between those two sources [36].

3.2  Regimens for HSCT in Pediatrics The differences between autologous HSCT (cells from the patients are collected) and allogeneic HSCT (there is a donor), as well as the characteristics of the matching donors, are described in the next subchapters. Here, there is an introduction about the regimens that will be discussed in the next subchapters about auto- and allo-HSCT. Pre-regimens for HSCT pediatric patients include high-dose chemotherapy, with or without irradiation, to maximize malignant cells killing for all patients and also to cause sufficient immunosuppression for those who receive allo-HSCT, to overcome rejection. Post-regimens with chemotherapy are used for GvHD prophylaxis [91].

3.2.1  T  he “Big Picture” of Regimens for Allogeneic Versus for Autologous Pediatric Stem Cell Transplantation Since the majority of allogeneic transplantations are performed for the treatment of malignant diseases, protocols should provide tumor cytoreduction and disease eradication. The regimen chosen for allo-HSCT must be immunosuppressive to overcome host rejections of the stem cells received. Most of the high-dose chemotherapy regimens have been used in patients with hematological malignancies in the setting of allogeneic transplantations. The drugs used in the conditioning regimen and post-transplant immunosuppression may reduce graft rejection, relapse rates, and toxicity in pediatric hematopoietic stem cell recipients. Radiation has an important role too, which can be observed in the increased homing of stem cells to the bone marrow. The difference found in non-irradiated and irradiated mice, although there are similar levels of stem cell retention between both, was a significantly decreased donor-derived repopulation levels found in non-irradiated mice, which require transplantation of higher doses of cells. The role of irradiation for homing is described in Chap. 4 [4, 52–54, 59].

3.2  Regimens for HSCT in Pediatrics

19

3.2.2  Myeloablative, Non-myeloablative, and Reduced-­Intensity Pediatric Regimens for Hematopoietic Stem Cell Transplantation A simple definition of myeloablative, non-myeloablative, and reduced-intensity conditioning is described below and then discussed in detail [59]. • Myeloablative: These protocols cause irreversible cytopenia. There is a requirement for stem cell support. • Non-myeloablative: These protocols cause minimal cytopenia. This type of regimen can be given without stem cell support. • Reduced-intensity conditioning: These regimens do not fit the criteria of myeloablative or non-myeloablative, because cytopenia caused by this type of regimen is reversible. Stem cell support is necessary. Myeloablative regimens cause irreversible cytopenia; therefore, there is a requirement for stem cell support. The myeloablative regimen of choice depends on the disease as well as the stem cell source. Two goals need to be attained by the conditioning regimen prior to hematopoietic stem cell transplantation: a reduction of the malignant cell mass in case of patients with malignant disease (which is the majority of allogeneic transplants), and the other goal is the disease eradication. In the allogeneic setting, the preparative regimen also needs to be immunosuppressive enough to secure engraftment and prevent graft rejection [68]. Conventional ablative allo-HSCT depends on tolerated chemotherapy and radiotherapy. Reduced-intensity preparative regimen followed by stem cell infusion was associated with mixed chimerism followed by full chimerism with a documented graft-versus-leukemia (GVL) effect—which is the ability of donor T-cells to eliminate host leukemic cells after allo-HSCT—in patients with hematologic malignancies, and graft-versus-tumor (GVT) effect, which is the ability of donor T-cells to eliminate residual malignant cells of the tumor after allo-HSCT, in the setting of solid tumors, both increasing the relapse rates in patients who do not develop GvHD and who receive T-cell-depleted grafts, which are grafts that passed through a treatment to eliminate T-cells and decrease the risk of GvHD, or those receiving grafts from identical twins and having documentation of high remission rates after donor lymphocyte infusions [59–61]. Non-myeloablative and reduced-intensity regimens for allo-HSCT opened a new era for elderly patients and those with comorbidities. The reduced-intensity conditioning had been used for about 40% of all allo-HSCT and that number is increasing. Some of the most known reduced-intensity conditioning regimens include fludarabine and intermediate doses of alkylating agents—such as thiotepa, melphalan, and busulfan. The reduced-intensity regimens decrease complications of allo-­HSCT such as pancytopenia, mucositis, and organ damage. Non-myeloablative regimens usually contain low-dose (2 Gy) total body irradiation (TBI) with or without fludarabine. Such conditioning regimens may cause mild myelosuppression, low treatment-related toxicity, and antitumor responses. It has been suggested that

20

3  Stem Cell Transplantation in Pediatrics

reduced-intensity regimens have improved survival and decreased incidence of relapse more than non-myeloablative conditioning regimens. However, some data has shows similar outcomes between these two regimens [59]. For ALL, which is the most common disease in pediatric hematology/oncology, fractionated total body irradiation (fTBI) is the gold standard of conditioning in the most common myeloablative regimens, due to its considerable antileukemic potential. It is indicated for ALL in its first, second, or subsequent remission—despite the well-established long-term sequelae of fTBI. Regimens with only chemotherapy are also indicated in pediatrics, and they are widely used for children under 2 years old with ALL, AML, myelodysplasias, and severe aplastic anemia. Conditioning regimens are modified according to new studies [68]. Myeloablative conditioning for ALL in the matched related, matched unrelated, and mismatched settings commonly include TBI, which in most cases is fTBI, combined with cyclophosphamide, which has been extensively used because of the antileukemic and immunosuppressive potential of this regimen, as well as the lack of cross-reactivity with other modalities. It has been used cumulative doses of radiation up to 10–14 Gy, reducing the pulmonary cumulative dose down to 8–9 Gy, and fTBI to patients above the age of 2 years. In the pre-transplant conditioning for pediatric ALL, fTBI is commonly used in combination with 60 mg/kg etoposide for patients above 2–3 years old. For patients who are above 2–3 years old and are not eligible for fTBI, such as those with a history of high-dose irradiation, chemotherapy-­only regimens are used, such as busulfan in combination with cyclophosphamide and melphalan. Although radiation is not recommended for neonates and infants (children under 1-year-old) and most groups currently employ chemotherapy-only regimens for patients under the age of 1 year—as recommended by the Interfant group—or 2–3 years old, regimens containing fTBI have been employed also for patients under 18 months old, particularly in the unrelated setting. Most patients who use chemotherapy-only regimens receive busulfan in combination with cyclophosphamide plus another drug, such as melphalan, thiotepa, etoposide, or alemtuzumab [68]. In pediatric haplo-SCT for ALL, as well as for AML, both standard fTBI-based regimens and busulfan with cyclophosphamide and thiotepa have been successfully employed in conjunction with the CD34+ cells [68].

3.3  Autologous Stem Cell Transplantation in Pediatrics The patient’s own stem cells are used for hematopoietic autologous stem cell transplantation (auto-HSCT). The stem cell source matters and the options are evaluated regarding the disease and the donor (autologous or allogeneic). For auto-HSCT, the most used source is peripheral blood. Stem cells are collected several times in the weeks before a pre-transplantation trial that can include a high dose of chemotherapy and/or radiation. This type of transplantation has limitations not only regarding the types of cancer and the right time to perform it but also the patient’s bone marrow condition, which should be disease-free. Since the bone marrow will be rescued with stem cell transplantation, it is allowed to perform very aggressive

3.3  Autologous Stem Cell Transplantation in Pediatrics

21

chemotherapy trials. Autologous transplantation in pediatrics is often performed for high-risk neuroblastoma, high-risk brain tumors—such as medulloblastoma and PNET (primitive neuroectodermal tumor)—metastatic retinoblastoma, recurrent high-risk germ cell tumors, relapsed Hodgkin and non-Hodgkin lymphoma, and relapsed Wilms tumor [24, 33, 91].

3.3.1  Acute Lymphoid Leukemia (ALL) Based on the evidence, autologous HSCT is not recommended for ALL in first complete remission (instead of allo-HSCT). At the second or posterior complete remission of patients at high risk, it can be evaluated by a specialist [27, 28]. PH+ ALL Although allogeneic hematopoietic stem cell transplantation was considered a standard treatment for Ph+ (positive Philadelphia chromosome) ALL achieving complete remission after induction containing tyrosine kinase inhibitors, the outcomes of myeloablative autologous hematopoietic stem cell transplantation for PH+ ALL patients are comparable to the allogeneic, both in first molecular remission. The autologous transplantation seems to be an attractive treatment option, potentially allowing for circumvention of allogeneic transplantation sequelae. Irrespective of the type of transplantation, trials including total body irradiation should be considered the first choice of conditioning for Ph+ ALL [26, 27].

3.3.2  Acute Myeloid Leukemia (AML) Although hematopoietic stem cell transplantation has been playing an important role for treatment of AML, as well as improving the outcomes in children, the patients who receive autologous transplantation have worse survival than those who received allogeneic transplantation. With the modern trials, autologous hematopoietic stem cell transplantation has been less performed for pediatric AML than chemotherapy alone. Allogeneic transplantation remains an important treatment for many pediatric AML patients. However, there are protocols in which autologous hematopoietic stem cell transplantation in pediatrics is generally not recommended for low-risk patients, while it is a standard treatment for high-risk patients in first complete remission; and it depends on the decision of the specialist in first complete remission for very high-risk children. In second complete remission of AML, in the same protocols, autologous transplantation is a standard treatment; however, it is generally not recommended after this remission. It all means that each case should be individually evaluated, regarding risk stratification, patient response to chemotherapy, remission, and the availability of a donor [28, 29]. Allo-HSCT (HCT) with myeloablative conditioning is the gold standard of treatment, but it is associated with a high incidence of non-relapse mortality. A study

22

3  Stem Cell Transplantation in Pediatrics

compared outcomes between myeloablative and reduced-intensity regimens in one of the largest reported cohorts of pediatric patients receiving allo-HSCT for AML (and therapy-related myelodysplastic syndrome), to evaluate if reduced-intensity regimens may reduce that type of mortality, and it concluded that outcomes were poor due to a high rate of non-relapse mortality with myeloablative conditioning and a high rate of disease-related death after reduced-intensity conditioning. The conclusion was that novel strategies that lead to a reduction in transplant-related mortality are needed, while providing sufficient disease control, to improve survival in these patients [62].

3.3.3  Chronic Myeloid Leukemia Autologous hematopoietic stem cell transplantation is generally not recommended for pediatric chronic myeloid leukemia patients [29].

3.3.4  Non-Hodgkin Lymphoma High dose chemotherapy plus HSCT is a treatment used for more aggressive types of or recurrent non-Hodgkin lymphomas in children. Although there is a limitation to perform autologous transplantation—because using the child’s own stem cells is often not possible if the lymphoma is in the bone marrow—autologous transplantation still is nowadays more used than allogeneic to treat non-Hodgkin lymphoma [24]. For patients in first complete remission, autologous hematopoietic stem cell transplantation is generally not recommended for patients classified as low risk and depends on the decision of the specialist and institutional guideline for those classified as high risk. In second complete remission of non-Hodgkin lymphoma, autologous transplantation can also be evaluated [28]. A retrospective analysis of transplantation activity between 2008 and 2014 in the United States showed that 16% (from the total 506) of all autologous hematopoietic stem cell transplantations performed from 2008 to 2014 were to treat lymphoma. Within this group, the number of autologous transplants for non-Hodgkin lymphoma showed a decline from 2008 to 2014 (26% in 2008 and 15% in 2014) [31]. An institutional retrospective analysis, which evaluated the role of transplantation in children with non-Hodgkin lymphoma between 1982 and 2004 (including patients with lymphoblastic lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma, anaplastic large-cell lymphoma, peripheral T-cell lymphoma, and undifferentiated non-Hodgkin lymphoma), with the different sources from donors when the transplantation was allogeneic, showed that disease-free survival was 55% and 53% with a median follow-up of 9.75 years, and the outcomes were similar in patients receiving autologous and allogeneic transplantation. Both allogeneic and autologous transplantation offered the prospect of durable, disease-free survival for a significant proportion

3.3  Autologous Stem Cell Transplantation in Pediatrics

23

of those patients with relapsed or refractory non-Hodgkin lymphoma, and the survival rate was superior among patients with chemotherapy-­sensitive disease [32].

3.3.5  Hodgkin Lymphoma A retrospective analysis of transplantation activity between 2008 and 2014 in the United States showed that 16% (from the total 506) of all autologous hematopoietic stem cell transplantations performed from 2008 to 2014 were to treat lymphoma. Within this group, Hodgkin lymphoma was the most common (79%). Autologous transplantation for Hodgkin lymphoma showed increased activity over 2008–2014 (74% in 2008 and 84% in 2014) [31]. Autologous transplantation, together with high-dose chemotherapy, is the standard treatment for relapsed Hodgkin lymphoma in pediatric patients with non-­ localized disease after frontline therapy [31]. Patients who received transplantation for Hodgkin lymphoma (either autologous or allogeneic) who had failed in the previous HSCT can benefit from brentuximab vedotin—which is a CD30-targeting antibody-drug conjugate that has demonstrated effectiveness as a monotherapy for patients with relapsed or refractory Hodgkin lymphoma and systemic anaplastic large-cell lymphoma in trials. Although this drug has been used as a consolidative treatment and is an approved option for adult patients with relapsed or refractory classical Hodgkin lymphoma after failure of autologous stem cell transplantation or at least two prior chemotherapy regimens when HSCT is not an option, and for relapsed or refractory systemic anaplastic large-cell lymphoma following failure of at least one previous chemotherapy regimen, patients in pediatrics who had no other alternative can be considered young adults had been receiving this drug followed by HSCT as an off-label alternative. Then, more studies in pediatrics with this drug have been performed and published. A study with 7–18-year-old patients with relapsed or refractory classical Hodgkin lymphoma or systemic anaplastic large-cell lymphoma, for whom standard treatment was unavailable or no longer effective, evaluated brentuximab vedotin and concluded that it has manageable toxicity and is associated with clinically meaningful responses in pediatric patients, and this drug allows either subsequent autologous or allogeneic HSCT in some patients who were initially ineligible for HSCT. One of the primary goals of using brentuximab vedotin in pediatrics is to reduce the long-term, treatment-related toxicities, which are commonly associated with other combined modalities or chemotherapy-only regimens, such as cardiovascular mortality, secondary cancer, and infertility. It is interesting to have an option to avoid the cumulative effects of radiation and anthracycline doses [93–95] (Table 3.1). Table 3.1  Brentuximab vedotin doses in pediatric patients with relapsed or refractory classical Hodgkin lymphoma or systemic anaplastic large-cell lymphoma followed by auto- or allo-HSCT Dose 1–8 mg/kg

Administration IV

Days Once every 3 weeks for up to 16 cycles [95]

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3  Stem Cell Transplantation in Pediatrics

3.3.6  Solid Tumors High-dose chemotherapy with autologous stem cell rescue has been applied in very-­poor-­risk pediatric solid tumors, such as neuroblastoma and Ewing sarcoma. Pediatric neuroblastoma of high risk has a poor prognosis, and more than half of patients experience disease recurrence. High-dose chemotherapy—in a myeloablative therapy—and the following auto-HSCT (mostly with sources from peripheral blood) as a rescue can improve the survival with low relapse rates in patients with these solid tumors without a history of prior auto-HSCT; and the recurrence of the disease is the most common cause of treatment failure. Allo-­HSCT after a prior auto-HCT appears to offer minimal benefit to patients with neuroblastoma. Ewing sarcoma is the second most common type of primary bone malignancy in children and young adults, and its survival rates for localized disease have improved to more than 70% with aggressive chemotherapy. However, there is still little improvement in survival rates for patients with metastatic or recurrent Ewing sarcoma. The treatment of tumors of this family is multimodal. Axial location and metastasis are prognostic factors. Induction chemotherapy with the VIDE (vincristine, ifosfamide, doxorubicin, and etoposide) regimen is feasible in most patients, with a low risk for early progression, with substantial but manageable toxicity [83]. Promising data is available about the use of high-dose busulfan for neuroblastoma and high-dose busulfan or treosulfan for Ewing sarcoma. High-dose thiotepa is less commonly used. This treatment for results in an encouraging outcome without toxic mortality for these patients [83]. HD BU–MEL (high-dose busulfan and melphalan) regimen, which is also known as BU–Mel HDT, to perform as conditioning therapy for pediatric autoHSCT in patients with Ewing sarcoma (high risk) is described in Table 3.2. The conventional consolidation with these drugs (CC BU–MEL) had worse results, so it is not described in this book. Probability of relapse-free survival in median observation time was significantly worse in high-risk pediatric patients with Ewing sarcoma who were given CC BU–MEL than those who received HD BU–MEL [52, 84].

Table 3.2  HD BU–MEL (high-dose busulfan and melphalan) pediatric HSCT conditioning regimen for Ewing sarcoma Drug Busulfan

Administration Dose Oral 1 mg/kg/dose every 6 h (4 mg/kg/day)

Melphalan IV

140 mg/m2

Days Total of 16 doses, on days 3–6 prior to transplantation. This dose and frequency are used both for patients 4 years old

Dose 0.8 mg/kg/dose 1 mg/kg/dose 0.8 mg/kg/dose

Route IV IV IV

Administration Every 6 h Every 6 h Every 6 h

Total number of doses 16 doses 16 doses 16 doses

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3  Stem Cell Transplantation in Pediatrics

Table 3.10  Reduced-intensity conditioning for allogeneic HSCT in pediatric patients (i.e., infants, children, or adolescents), who are ineligible for myeloablative conditioning, with busulfan, fludarabine, and antithymocyte globulin

Drug Busulfan

Busulfan

Fludarabine

Days (counting negative days as the days prior to transplant, which is performed on Total number of day 0) Dose Administration doses 0.8 mg/kg/dose IV Only the first −7 if related dose. Then, the donor; −10 if plasmatic unrelated donor dosage test or cord should be recipient performed to evaluate the next dosages IV, every 6 h 7 doses (for a With −3 and −2 if total of 8 doses adjustment related donor; with the starting based on the −6 and −5 if dose) unrelated donor serum dosage performed after the first dose 30 mg/m2 −7 to −2 (if IV, once daily Total dose is 180 unrelated donor mg/m2 or cord blood source is used, days −10 to −5) −1 2.5 mg/kg IV, as a single Total dose is 2.5 dose mg/kg

Thymoglobulin—rabbit ATG for recipients of bone marrow or peripheral blood cells from related donors −4 through −1 Thymoglobulin—rabbit ATG for recipients of unrelated donors (either bone marrow, cord blood, or peripheral blood cells [54])

2.5 mg/kg

IV, daily

Total dose is 10 mg/kg

This conditioning regimen was part of the trial ONC0313 of 2009 and is used for patients who are ineligible for myeloablative therapy. It is done in combination with fludarabine and antithymocyte globulin (rabbit). The youngest patient treated in studies with this trial was 2 years old. It is used for acute and chronic leukemias, myelodysplasia, or lymphomas. AML patients do not need to be in remission; they can have M1 or M2 marrows to perform this protocol [54].

3.4  Allogeneic Stem Cell Transplantation in Pediatrics

43

or treat relapse in AML in pediatrics, but it was documented that it has lower nonrelapse mortality and lower relapse in patients with AML in first complete remission (CR1) treated with BuCy than those treated with TBI and cyclophosphamide (TBICy). After these studies, the use of TBI-based conditioning regimen in the AML pediatric population was reduced, as long-term TBI-related toxicity represents a significant burden for surviving children. Regimens containing busulfan together with melphalan and cyclophosphamide as conditioning for AML pediatric patients followed by allo-HSCT were proposed many years ago, being the cyclophosphamide dose 120 mg/kg (because of the high mortality rate with doses of 200 mg/kg in previous studies of other groups) and 140 mg/m2 melphalan used previously for pediatric myelodysplastic syndrome. BuCyMel has, therefore, been incorporated into national prospective/retrospective AML studies, and the conclusion for now is that BuCyMel, although increasing the intensity of conditioning regimen for children with AML in CR1 might be a key factor in preventing relapse, has been reported as severely toxic. Retrospective studies are important because of the high rate of patients that have data collected through the years, and the evaluation of the study groups for new approaches can be guided by past results. The post-HSCT relapse risk still represents the most frequent and less treatable complication for AML patients [52–55, 100]. Busulfan doses in pediatrics are described in Tables 3.7, 3.8, and 3.9, and the most common potential risks for pediatric HSCT patients are fever and chills, pruritus, anaphylaxis, and serum sickness [91]. Formula 3.1  Adjustment of busulfan dose for pediatric hematopoietic stem cell transplantation conditioning: é target AUC ( micromolar × minute ) ù Adjusted dose ( mg ) = Actual dose ( mg ) ´ ê ú êë actual AUC ( micromolar × minute ) úû In pediatric haplo-HSCT for AML, as well as for ALL, both standard fTBI-based regimens and busulfan with cyclophosphamide and thiotepa have been successfully employed in conjunction with the CD34+ cells. And PARP1 inhibitors also have been researched for AML. Patients with AML often achieve remission but subsequently die of relapse due to chemotherapy-resistant leukemic stem cells. Driven by the hypothesis that leukemic stem cells must also escape immune-surveillance to initiate and maintain cancer, and investigating the interplay with NKG2D—which is a danger detector expressed by cytotoxic lymphocytes, such as natural killer cells—that recognizes stress-induced ligands (NKG2DL) of the MIC and ULBP protein families on AML cells, researchers suggested overcoming the leukemic stem cell mechanisms using PARP1 inhibitors. In conjunction with functional natural killer cells, PARP1 inhibitors promise to eradicate leukemic stem cells and promote immune-mediated cure of AML, because it was shown that leukemic cells can have a natural killer cell recognition, by selectively suppressing the surface expression of NKG2DL and other immune-stimulatory molecules. Absence of NKG2DL can identify leukemic stem cells across genetic AML subtypes, including CD34-­ negative AML. PARP1 inhibitors act by overcoming this immune evasion [68, 70].

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3  Stem Cell Transplantation in Pediatrics

3.4.6  Chronic Myeloid Leukemia Chronic myeloid leukemia (CML) is not common in children. However, young patients with this disease have low morbidity with hematopoietic stem cell transplantation, in part because it can reduce the prolonged use of tyrosine kinase inhibitors, which can cause significant morbidity when prolonged. Transplantation is the only curative treatment that was proved for pediatric CML. Indications for hematopoietic stem cell transplantation in children include advanced disease, presence of resistant mutation, or failure to achieve complete remission 1 year after initiation of tyrosine kinase inhibitor therapy. The most common guidelines indicate the transplantation with cells from a sibling or compatible non-related donor in chronic or advanced phase as the standard therapy. Allogeneic hematopoietic stem cell transplantation, especially in the early phase of the disease, is recommended for patients who have intra-familial or non-relative donors with full human leukocyte antigen (HLA) compatibility. The success of tyrosine kinase inhibitors in children with CML, as well as the achieved long lifespan expectancy, has been causing discussions related with the necessity of transplantation and its timing. Children with a prognosis of longer life expectancies are likely to receive prolonged tyrosine kinase inhibitors during periods of active growth and sexual development, increasing the cumulative risk for morbidities. Because of that, transplantation is more common in children and younger adults than adults. The indication for transplantation in patients in the first chronic phase is not well established [29, 30]. A CIBMTR cohort analysis did a retrospectively research evaluating outcomes in 449 CML patients (0–29 years) with early disease and clinical remission who received myeloablative allogeneic transplantation. Special attention was given to the analysis of age impact and the use of tyrosine kinase inhibitors pre-­ transplantation to identify prognostic factors. In the tyrosine kinase inhibitor era, the transplantation is recommended in patients in first complete remission who are resistant or intolerant to those drugs. The 5-year overall survival rate was 75% (for all patients 1 antigen incompatible. For patients in second complete remission, allogeneic transplantation is a standard treatment with cells from a sibling or compatible non-relative donor; and if these donors are not available, the possibility of using cells from a non-relative donor who is >1 antigen incompatible can be evaluated by a specialist [28]. Approximately 5% of all allogeneic transplantations (from a total of 217) were performed to treat lymphoma. Non-Hodgkin lymphoma was the most common of all these lymphomas (85%). According to a retrospective analysis of transplantation activity between 2008 and 2014  in the United States, the number of allogeneic transplantation performed for non-Hodgkin lymphoma declined in 2011, and it increased every year thereafter. The number of allogeneic transplantations for non-Hodgkin lymphoma performed with sources from unrelated bone marrow (46) and unrelated umbilical cord blood (36) was comparable to that of sibling marrow transplants (39) [31]. An institutional retrospective analysis, which evaluated the role of transplantation in children with non-Hodgkin lymphoma between 1982 and 2004 (including patients with lymphoblastic lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma, anaplastic large-cell lymphoma, peripheral T-cell lymphoma, and undifferentiated non-Hodgkin lymphoma), with the different sources of donor when the transplantation was allogeneic, showed that disease-free survival rates were 55% and 53% with a median follow-up of 9.75 years, and the outcomes were similar in patients receiving autologous and allogeneic transplantation. Both allogeneic and autologous transplantation offered the prospect of durable, disease-free survival for a significant proportion of those patients with relapsed or refractory non-Hodgkin lymphoma, and the survival rate was superior among patients with chemotherapy-­ sensitive disease [32]. Non-Hodgkin Patients Who Are Ineligible for Myeloablative Conditioning Pediatric patients who are ineligible for myeloablative conditioning can receive a regimen with busulfan, fludarabine, and antithymocyte from rabbit (trial ONC0313 of 2009) that is described in Table 3.12. The youngest patient treated in studies with this trial was 2 years old. It is used for acute and chronic leukemias, myelodysplasia, or lymphomas. Patients with non-Hodgkin lymphoma need responsive disease with no persistent masses more than 5 cm to perform this protocol [54].

3.4  Allogeneic Stem Cell Transplantation in Pediatrics

47

Table 3.12  Reduced-intensity conditioning for allogeneic HSCT in non-Hodgkin pediatric patients (i.e., infants, children, or adolescents), who are ineligible for myeloablative conditioning, with busulfan, fludarabine, and antithymocyte globulin

Drug Busulfan

Busulfan

Fludarabine

Days (counting negative days as the days prior to transplant, which is performed on Total number of day 0) Dose Administration doses 0.8 mg/kg/dose IV Only the first −7 if related dose. Then, the donor; −10 if plasmatic unrelated donor dosage test or cord should be recipient performed to evaluate the next dosages IV, every 6 h 7 doses (for a With −3 and −2 if total of 8 doses adjustment related donor; with the starting based on the −6 and −5 if dose) unrelated donor serum dosage performed after the first dose 30 mg/m2 −7 to −2 (if IV, once daily Total dose is 180 unrelated donor mg/m2 or cord blood source is used, days −10 to −5) −1 2.5 mg/kg IV, as a single Total dose is 2.5 dose mg/kg

Thymoglobulin—rabbit ATG for recipients of bone marrow or peripheral blood cells from related donors −4 to −1 Thymoglobulin—rabbit ATG for recipients of unrelated donors (either bone marrow, cord blood, or peripheral blood cells [54])

2.5 mg/kg

IV, daily

Total dose is 10 mg/kg

48

3  Stem Cell Transplantation in Pediatrics

3.4.8  Hodgkin Lymphoma Hematopoietic stem cell transplantation is generally not recommended for Hodgkin lymphoma patients in first complete remission. After first relapse and second complete remission, it depends on the evaluation of the specialist with sources from a sibling donor. If they are not available, the possibility of using sources from a compatible non-relative donor is experimental and can be evaluated by a specialist; however, it is not recommended to use sources from a relative or non-relative donor who is >1 antigen incompatible [28]. Approximately 5% of all allogeneic transplantations (from a total of 217) were performed to treat lymphoma. According to a retrospective analysis of transplantation activity between 2008 and 2014 in the United States, the allogeneic transplantation for Hodgkin lymphoma demonstrated a decline in 2010, followed by an increase in 2011, and then it showed a slow decline again [31]. Patients with cases of Hodgkin lymphoma who had failed in previous HSCT can benefit from brentuximab vedotin—which is a CD30-targeting antibody–drug conjugate that has demonstrated effectiveness as a monotherapy for patients with relapsed or refractory Hodgkin lymphoma and systemic anaplastic large-cell lymphoma in trials (Table 3.13). Although this drug is used as a consolidative treatment and is an approved option for adult patients with relapsed or refractory classical Hodgkin lymphoma after failure of autologous stem cell transplantation or at least two prior chemotherapy regimens when HSCT is not an option, and for relapsed or refractory systemic anaplastic large-cell lymphoma following failure of at least one previous chemotherapy regimen, patients in pediatrics who had no other alternative and were young adults had been receiving this drug followed by HSCT as an off-label alternative. Then, more studies in pediatrics with this drug have been performed and published. A study with 7–18-year-old patients with relapsed or refractory classical Hodgkin lymphoma or systemic anaplastic large-cell lymphoma, for whom standard treatment was unavailable or no longer effective, evaluated brentuximab vedotin and concluded that it has manageable toxicity and is associated with clinically meaningful responses in pediatric patients, and this drug allows either subsequent autologous or allogeneic HSCT in some patients who were initially ineligible for HSCT. One of the primary goals of using brentuximab vedotin in pediatrics is to reduce the long-term, treatmentrelated toxicities, which are commonly associated with other combined modalities or chemotherapy-only regimens, such as cardiovascular mortality, secondary cancers, and infertility. It is interesting to have an option to avoid the cumulative effects of radiation and anthracycline doses [93–95].

Table 3.13  Brentuximab vedotin doses in pediatric patients with relapsed or refractory classical Hodgkin lymphoma or systemic anaplastic large-cell lymphoma followed by auto- or allo-HSCT Dose 1–8 mg/kg

Administration IV

Days Once every 3 weeks for up to 16 cycles [95]

3.4  Allogeneic Stem Cell Transplantation in Pediatrics

49

Hodgkin Patients Who Are Ineligible for Myeloablative Conditioning Pediatric patients who are ineligible for myeloablative conditioning can receive a regimen with busulfan, fludarabine, and antithymocyte from rabbit (trial ONC0313 of 2009) that is described in Table 3.14. The youngest patient treated in studies with this trial was 2 years old. It is used for acute and chronic leukemias, myelodysplasia, or lymphomas. Patients with Hodgkin lymphoma have to have responsive disease with no persistent masses more than 5 cm to perform this protocol [54].

Table 3.14  Reduced-intensity conditioning for allogeneic HSCT in pediatric patients (i.e., infants, children, or adolescents), who are ineligible for myeloablative conditioning, with busulfan, fludarabine, and antithymocyte globulin

Drug Busulfan

Busulfan

Fludarabine

Days (counting negative days as the days prior to transplant, which is performed on Total number of day 0) Dose Administration doses 0.8 mg/kg/dose IV Only the first −7 if related dose. Then, the donor; −10 if plasmatic unrelated donor dosage test or cord should be recipient performed to evaluate the next dosages IV, every 6 h 7 doses (for a With −3 and −2 if total of 8 doses adjustment related donor; with the starting based on the −6 and −5 if dose) unrelated donor serum dosage performed after the first dose −7 through −2 30 mg/m2 IV, once daily Total dose is 180 (If unrelated mg/m2 donor or cord blood source is used, days −10 through −5) −1 2.5 mg/kg IV, as a single Total dose is 2.5 dose mg/kg

Thymoglobulin—rabbit ATG for recipients of bone marrow or peripheral blood cells from related donors −4 through −1 Thymoglobulin—rabbit ATG for recipients of unrelated donors (either bone marrow, cord blood, or peripheral blood cells [54])

2.5 mg/kg

IV, daily

Total dose is 10 mg/kg

50

3  Stem Cell Transplantation in Pediatrics

3.4.9  J uvenile Myelomonocytic Leukemia, Anemias, Myelodysplasia, Primary Immunodeficiency, and Inborn Error of Metabolism Patients with Juvenile Myelomonocytic Leukemia or Myelodysplasia Who Are Ineligible for Myeloablative Conditioning Pediatric patients who are ineligible for myeloablative conditioning can receive a regimen with busulfan, fludarabine, and antithymocyte from rabbit (trial ONC0313 of 2009) that is described in Table 3.15. The youngest patient treated in studies with this trial was 2 years old. It is used for acute and chronic leukemias, myelodysplasia, or lymphomas. Patients with juvenile myelomonocytic leukemia (JMML) or myelodysplasia (MDS) should have less than 5% blasts to receive this conditioning regimen followed by allo-HSCT [54]. Besides chemotherapy-only regimens with busulfan, cyclophosphamide, and melphalan, patients with juvenile myelomonocytic leukemia and myelodysplastic syndrome (MDS) have the option to receive fTBI-containing regimens, with the former showing a seemingly lower incidence of non-relapse mortality [68]. Anemias Pediatric patients with Fanconi anemia commonly receive (low-dose) cyclophosphamide 20 mg/kg and 5 Gy thoraco-abdominal irradiation as conditioning for HSCT with sources from sibling donors. A limited number of patients receive regimens with only cyclophosphamide. For patients with sources from unrelated donors, the dose of cyclophosphamide is higher, being a medium dose (40–45 mg/kg), and these patients also receive—in combination—either thoraco-abdominal or fTBI and antiT serotherapy. A reduction in treatment-related toxicity has been achieved when fludarabine-containing regimens are used, and many of these regimens also contain cyclophosphamide and fTBI (4.5 Gy) [68]. Pediatric patients with severe aplastic anemia commonly receive non-fTBI-­ containing regimens as conditioning. Those with sources from unrelated donors receive fludarabine, cyclophosphamide, and ATG; and those with sources from a sibling receive cyclophosphamide with or without ATG. Some fludarabine-containing regimens are used in the sibling setting as an essentially non-myeloablative approach. 2–6 Gy TBI with cyclophosphamide, fludarabine, and ATG is also used in the unrelated donor setting. Although there is an increased risk of graft rejection, the use of partial T-cell depletion has been reported in pediatric hematopoietic stem cell transplantation for pediatric severe aplastic anemia [68]. Primary Immunodeficiency The recommendation of the European Society for Blood and Marrow Transplantation and the European Society for Primary Immunodeficiencies supports the use of busulfan and cyclophosphamide with effective T-cell depletion particularly in the mismatch setting [68].

3.4  Allogeneic Stem Cell Transplantation in Pediatrics

51

Table 3.15  Reduced-intensity conditioning for allogeneic HSCT in pediatric patients (i.e., infants, children, or adolescents), who are ineligible for myeloablative conditioning, with busulfan, fludarabine, and antithymocyte globulin

Drug Busulfan

Busulfan

Fludarabine

Days (counting negative days as the days prior to transplant, which is performed on Total number of day 0) Dose Administration doses 0.8 mg/kg/dose IV Only the first −7 if related dose. Then, the donor; −10 if plasmatic unrelated donor dosage test or cord should be recipient performed to evaluate the next dosages IV, every 6 h 7 doses (for a With −3 and −2 if total of 8 doses adjustment related donor; with the starting based on the −6 and −5 if dose) unrelated donor serum dosage performed after the first dose 30 mg/m2 −7 to −2 (If IV, once daily Total dose is 180 unrelated donor mg/m2 or cord blood source is used, days −10 to −5) −1 2.5 mg/kg IV, as a single Total dose is 2.5 dose mg/kg

Thymoglobulin—rabbit ATG for recipients of bone marrow or peripheral blood cells from related donors −4 to −1 Thymoglobulin—rabbit ATG for recipients of unrelated donors (either bone marrow, cord blood, or peripheral blood cells [54])

2.5 mg/kg

IV, daily

Total dose is 10 mg/kg

Inborn Errors of Metabolism Regimens for inborn errors of metabolism may vary. The feasibility of standard busulfan with cyclophosphamide has been widely used, for example, for mucopolysaccharidosis type 1H (Hurler) [68].

Chapter 4

Homing of Stem Cells to the Bone Marrow: Finding the Way Home

4.1  Homing 4.1.1  A Big Picture of Homing Once stem cells are introduced in the patient’s blood, they proceed with a systematic migration to their bone marrow, through a process called homing. Since “homing instinct,” according to the Merriam-Webster’s dictionary, is an ability to return home from a great distance, which is related to an animal with a homing instinct, homing is a very didactic word to also call the process through stem cells to find their way home starting from the blood. Although stem cells need to achieve the patient’s bone marrow, they are infused through the patient’s vein, like transfusions, because of the homing process—in which stem cells will find the bone marrow. The stem cell mobilization, homing, and repopulation are sequential events with physiological roles; and homing is essential for successful engraftment and repopulation. The stem cell homing process is the first and a rapid process; it can be measured in hours and no longer than 1–2 days. Homing occurs as migration of stem cells across the endothelial vasculature, through the blood, to the bone marrow niches, and to different organs as well. In homing, the circulating hematopoietic cells actively cross the blood/bone marrow endothelium barrier and lodge at least transiently in the bone marrow compartment by activation of adhesion interactions prior to their proliferation. The active navigation required in this process is the homing. The term homing is not only used for hematopoietic stem cell transplantation (HSCT). Similarly, homing is required for fetal bone marrow seeding by hematopoietic progenitors during development. Homing also has roles in adult bone marrow homeostasis, and it is amplified during stress-­induced recruitment of leukocytes from bone marrow reservoir and stem cell mobilization—which is a part of the host defense and reparation [4].

© Springer Nature Switzerland AG 2020 C. W. P. Schmidt, Pediatric Hematopoietic Stem Cell Transplantation for Pharmacists, https://doi.org/10.1007/978-3-030-43491-5_4

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The multistep process of homing is coordinated, and a “big picture” of the whole process can be seen as these steps below, and they are more detailed described on the next paragraphs after the overview of steps [4]: 1. Signaling by chemokines and cytokines, in which stromal-derived factor 1 (SDF-1) and stem cell factor (SCF) play a role. 2. Activation of adhesion molecules, in which lymphocyte function-associated antigen 1 (LFA-1), very late antigen 4/5 (VLA-4/5), and CD44. 3. Cytoskeleton rearrangement, membrane type 1 (MT1)–matrix metalloproteinase (MMP) activation and secretion of MMP2/9. 4. Rolling and firm adhesion of progenitors to endothelial cells in small marrow sinusoids under blood flow. 5. Trans-endothelial migration across the physical endothelium/extracellular matrix (ECM) barrier. 6. Stem cells finalize their homing by selective access and anchorage to their specialized niches in the extravascular space of the endosteum region and periarterial sites.

4.1.2  Homing First Step The signaling by chemokines and cytokines, in which stromal-derived factor 1 (SDF-1) and stem cell factor (SCF) play a role. Chemokines and Cytokines Cytokines and chemokines promote the cell signaling of inflammation. They are extracellular molecular regulators that mediate both immune cell recruitment and complex intracellular signaling control mechanisms of inflammation. There are many cytokines, such as interleukine (IL) families, tumor necrosis factor (TNF), and others. They can be classified based on many characteristics [10]. Chemokines are a family of small cytokines. The expression of chemokines, its receptors, and the adhesion of molecules contribute to the selective migration and tissue specificity of leukocytes. Chemokines are a group of small (8–12 kDa) proteins with a presence of 3–4 conserved cysteine residues. They are subdivided into four families, based on the positioning of the N-terminal cysteine residues. The C–X–C subfamily is classified by the separation of the first two cysteines by a variable amino acid; the C–C subfamily has cysteine residues adjacent to each other. The majority of the known chemokines are in the CXC and C–C subfamilies. The third group of chemokines, which is subfamily C, lacks the first and third cysteines and possesses only a single cysteine residue in the conserved position—subfamily C includes the lymphocyte-specific chemotactic peptide XCL1 (lymphotactin). The fourth subfamily of chemokines, which is CX3C, has the two N-terminal cysteine residues separated by three variable amino acids—this family has only one member is humans, which is CX3CL1 (fractalkine), and that is the unique in possessing a mucin-like glycosylated stalk, allowing it to exist as a soluble or membrane-bound chemokine [10].

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SDF-1 SDF-1 is a chemokine that is also known as CXCL-12 and plays a role in cell trafficking, promoting the CD34+ stem cell homing. These cells are attracted toward a gradient of SDF-1. When an organ is injured, its cells highly express SDF-1, and this increasing is localized. This localized increasing in SDF-1 levels in the injured organ leads to recruitment and retention of circulating CD34+progenitor cells at the injury site via chemotactic attraction toward the gradient of SDF-1. Approaches in research of tissue engineering are used to improve the regeneration of the damaged tissue/ organ in tissue engineering, introducing SDF-1 in tissue, as direct protein incorporation into scaffolds, and transplanting SDF-1-overexpressing cells. There are other different techniques of SDF-1 introduction in tissues to research the regeneration, and they have proved to be effective in recruitment of various stem/progenitor cells. Thereby, the role of SDF-1 in the regeneration of tissues has been shown [7, 10]. SCF and c-Kit Receptor The SCF is a cytokine also known as kit ligand, mast cell growth factor and steel factor. It binds to the SCF receptor, which is c-kit. During embryonic life, SCF and c-kit receptor RNA are expressed along the migratory pathways of primordial germ cells and melanocytes in sites of the hematopoiesis—such as yolk sac, fetal liver, and bone marrow—gut, and central nervous system. SCF is important in diverse early developmental processes and also in adult life. SCF production by marrow endothelial cells and fibroblasts is important to the maintenance of normal basal hematopoiesis. The c-kit receptor is broadly distributed within the hematopoietic cells and can be found also in tissues. This receptor is present on hematopoietic progenitors, including those of B and T lymphocytes. The c-kit receptor can physically associate with the cytoplasmic domain of the EPO receptor in cells that are responsive to cytokines, and that may provide a reason for the potent synergistic effects of both SCF and erythropoietin on erythropoiesis. The c-Kit receptor has an extracellular domain with five immunoglobulin-like motifs, a single short membrane-­spanning domain, and a cytoplasmic domain with tyrosine kinase activity. The SCF trigs its biological effect as a hematopoietic cytokine after binding to c-kit. SCF structure can be soluble or transmembrane forms—and both are active. After SCF binding to c-kit, these receptors pass by a homodimerization and intermolecular tyrosine phosphorylation, creating docking sites for SH2-containing signal transduction molecules (SH2 is the Src homology 2, a protein domain of sequence-specific phosphotyrosine-binding module present in many signaling molecules that mediate cellular localization, substrate recruitment, and regulation of kinase activity). Soluble c-kit receptor naturally occurs in molecules, which counteracts the effects of SCF. It is released by human hematopoietic cells, mast cells, as well as endothelial cells, and it circulates in normal human plasma concentrations near to 325  ng/ mL.  Soluble c-kit receptor in plasma exceeds SCF by a 30-fold molar ratio [7–10]. Neoplastic human hematopoietic cells in AML, anaplastic large-cell lymphoma, and Hodgkin lymphoma can display the c-kit receptor. The density of this receptor is highest in erythroid leukemia cells, which may express 50,000–100,000 c-kit receptors per cell [8].

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There is a role of mutations in the c-kit in gastrointestinal stromal tumors. These mutations activate the kinase enzymatic activity of c-kit. A kinase inhibitor targeted against c-kit, such as imatinib, can be very effective in these notoriously chemotherapy-resistant tumors [8]. SDF-1 and SCF Signaling and Homing The chemokineSDF-1 and its receptor, CXCR4, play a role in definitive fetal liver stem cell homing to the bone marrow, as well as in hematopoietic retention and repopulation of this organ during embryonic development. Various studies in mice demonstrated essential roles for SDF-1 and CXCR4 interactions in murine embryonic development and also in steady-state adult hematopoiesis (regulation of leukocyte trafficking and stem cell self-renewal). One of those studies shown that murine T-cells that overexpress human CXCR4 and CD4 are mostly localized within the bone marrow of transgenic mice, demonstrating a central role for this chemokine in homing and retention of hematopoietic cells within the bone marrow microenvironment, probably by chemotaxis, through the activation of adhesion molecules and influencing their cell cycle status. Studies showed that a lack of SDF-1 or its receptor, CXCR4, in murine embryos causes multiple defects that are lethal, including impaired bone marrow lymphoid and myeloid hematopoiesis. Although this chemokine is a pre-B-cell growth factor and B-lymphopoiesis in the fetal liver was also impaired because of that, T-cell development in the embryonic thymus and myeloid development in the fetal liver are not affected. Fetal liver cells isolated from CXCR4null embryos can engraft the marrow of transplanted adult wild-type mice that are preconditioned with total body irradiation, but the stem cell self-renewal, retention of maturing hematopoietic cells within the bone marrow, and migration patterns of myeloid and lymphoid progenitor cells, including thymic T-cell development, are defective. While the levels of maturing myeloid and lymphoid cells in the bone marrow remained low even when a tenfold increase of fetal liver cells was transplanted, the levels of immature CXCR4null cells were normal. Transplantation of CXCR4null cells recovered from the bone marrow of these mice into serially transplanted secondary recipients resulted in reductions in the levels of engraftment compared to the results from serially transplanted wild-type cells, demonstrating an essential role of SDF-1 and CXCR4 in definitive stem cell repopulation and development in transplanted adult mice. CXCR4null progenitor cells recovered from the murine fetal liver do not qualify as hematopoietic stem cells. In addition to their inability to home and repopulate the bone marrow of the developing embryo, these cells also fail to give rise to high levels of multi-lineage myeloid and lymphoid cells in the bone marrow and peripheral blood of primary and serially transplanted secondary murine recipients, which are essential for a repopulating cell to qualify as a pluripotent stem cell with self-renewal potential. While CXCR4null cells do not migrate to a gradient of SDF-1 in vitro in transwell assays, SDF-1 mediates adhesion and migration in threedimensional ECM-like gels of CXCR4null cells, suggesting these cells can partially compensate an absence of CXCR4. Moreover, the same research also demonstrated that micro particles derived from activated platelets expressing functional CXCR4

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adhere to the bone marrow endothelium-bound SDF-1 and also have the capacity to bind to murine stem cells and immature human CD34 cells via their plateletbinding sialo-mucin P-selectin (CD162) and integrin Mac-1 (CD11b–CD18) antigen. These interactions increase the adhesion of human and murine progenitors to the endothelium-bound SDF-1 and also increase in vivo murine stem cell homing and repopulation. These results suggest that CXCR4 platelets in the circulation of adult wild-type mice transplanted with CXCR4null fetal liver cells could also increase homing and engraftment of CXCR4null progenitor cells in response to SDF-1 signaling. Murine progenitor cells that overexpressed SDF-1 when transplanted resulted in increased myeloid and B-lymphoid hematopoiesis, while T-cell development was impaired, most probably due to the anti-­apoptotic survival properties of this chemokine [11]. Purified adult murine hematopoietic stem cells migrate to SDF-1 and not to any other known chemokines, which reveals a central role for SDF-1 and CXCR4 interactions in adult murine hematopoiesis. All these results suggest that SDF-1 is a key regulator of murine stem cell migration, homing, and anchorage of repopulating cells to the bone marrow, and it is also important for the releasing of maturing cells into the blood circulation [11]. SCF is required to make stem cells responsive to other colony-stimulating factors (CSFs). The STC synergizes with other cytokines such as erythropoietin, interleucin­3, granulocyte-colony-stimulating factor (G-CSF), and granulocyte-­macrophage-­ stimulating factor (GM-CSF) to promote a direct colony growth of burst-forming unit-erythroid (BFU-E), colony-forming unit-granulocyte/macrophage (CFU-GM), and colony-forming unit (CFU)-granulocyte/ erythroid/ macrophage/ megakaryocyte (GEMM). SCF can act on a more primitive cell, like pre-colony-forming unit cell (pre-CFU-C), capable of generating the direct colony-­forming cells. SCF can also promote progenitor cell survival, accelerate stem cell entry into the cell cycle, and be chemotactic and chemokinetic factors for these cells [8, 12]. Studies with primates treated with SCF showed a 10- to 100-fold increase in the number of circulating progenitor cells. The effects of SCF when combined with G-CSF were even more pronounced. The treatment with SCF increases the progenitor cells of many types, including BFU-E, CFU-GM, CFU-Meg, and CFU-GEMM in the bone marrow [8]. SCF is a component of the niche that supports and controls hematopoietic stem cells and progenitor cells in the bone marrow. Transplanted hematopoietic stem cells and progenitor cells have to re-establish the physiologic interactions between c-kit receptors and SCF present in their niches. Research showed that the blocking of c-Kit receptors—with the anti-c-Kit antibody ACK2—significantly reduced the transplantation success. The significance of c-kit for the initial lodgment of transplanted hematopoietic stem cells within the endosteal bone marrow region and to the functional positioning of hematopoietic stem cells to the niche was already demonstrated. The inherent small differences in the c-kit expression level in hematopoietic stem cells influence their engraftment after transplantation. However, a study was done to find out if diminished numbers of c-kit receptors on hematopoietic stem cells and progenitor cells, after their internalization induced by the

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binding of SCF, would jeopardize the transplantability of hematopoietic stem cells and progenitor cells. They used a battery of functional assays to evaluate the capacity of hematopoietic stem cells and progenitor cells with markedly different c-kit expression levels to be transplanted. This study was conducted using the ability of SCF to downregulate c-kit receptors by inducing their internalization and degradation, and, in striking contrast to the researchers’ expectations, grossly diminished c-kit expression level in hematopoietic stem cells and progenitor cells did not affect the homing, lodgment, and engraftment of transplanted hematopoietic stem cells and progenitor cells. They demonstrated the ability of hematopoietic stem cells and progenitor cells to rapidly replace c-kit receptors consumed after their activation by SCF and that even markedly reduced numbers of c-kit receptors did not hamper the ability of hematopoietic stem cells and progenitor cells to be transplanted. However, the tested hypothesis, assuming that depressed c-kit expression in hematopoietic stem cells and progenitor cells in regenerating bone marrow significantly contributes to their inferiority in transplantation assays, could not be ultimately rejected on the basis of their negative results regarding the impact of c-kit downregulation on hematopoietic stem cells and progenitor cells transplantation, because of the SCFinduced downregulation of c-kit was a transient phenomenon lasting less than 12 to 14 hours, whereas the whole process of engraftment of transplanted HSPCs may require more time. It is known that the c-kit expression level is decreased in regenerating bone marrow, causing worse performance than that of a normal bone marrow. Surprisingly, as it was concluded before, the results of the experiments testing the homing of transplanted cells to bone marrow of recipient mice and their short-term and long-term engraftment did not reveal any defects in hematopoietic stem cells and progenitor cells with severely reduced numbers of c-kit receptor molecules— which could be ascribed to the quick replacement of the consumed c-kit receptors by SCF. This research could conclude that the exposure of hematopoietic stem cells and progenitor cells to SCF and diminished numbers of c-kit receptors in their cell membranes do not compromise the capacity of hematopoietic stem cells and progenitor cells to reconstitute damaged hematopoietic tissue [13].

4.1.3  H  oming Second Step: Activation of Lymphocyte Function-Associated Antigen 1 (LFA-1), Very Late Antigen 4/5 (VLA-4/5), and CD44 Lymphocyte Function-Associated Antigen 1 (LFA-1) The LFA-1 is an integrin receptor that is expressed only by leukocytes. This receptor is involved in every step of the leukocyte-endothelial cell adhesion cascade— which are rolling, firm adhesion, transmigration, and sub-endothelial migration. It also cognates the interactions between T-cells and antigen-presenting cells. Its main ligand is intercellular adhesion molecule-1 (ICAM-1) that is expressed on

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endothelium, sub-endothelial cells, and leukocytes. The Adhesiveness of LFA-1 is controlled by receptor-transduced signals, as well as by adhesion-strengthening signals emanating from ligand binding. The LFA-1 conformation is then modified, precluding adhesion events in the bloodstream or at sites distant from the inflammatory target. In the inactive form, the LFA-1 α/β heterodimer (CD11a/CD18) has a bent conformation, which causes the ligand-binding headpiece less accessible. The active conformation of LFA-1 has an extended structure that exposes the ligandbinding site on the I domain of the α subunit headpiece. The extended conformation is recognized by the reporter antibodies KIM-127 and 24, and they bind to epitopes from β subunit and α/β headpiece. The switch between active and inactive conformers is controlled by a membrane-proximal motif on the cytoplasmic domain of the β subunit, which competes with the intracytoplasmic region on the α subunit promoting a low-affinity state and also competes with the cytoskeletal protein talin promoting a high-affinity state [14]. Very Late Antigen 4/5 (VLA-4/5) The very late antigens (VLA) are integrins, which are proteins that act as matrix receptors to tie the matrix to the cell’s cytoskeleton. VLAs are heterodimers of at least six unique alpha chains sharing a common beta chain. Although some transmembrane proteoglycans function as co-receptors for matrix components, the principal receptors on animal cells for binding most extracellular matrix proteins are the integrins, including collagens, fibronectin, and laminins integrins. In blood cells, integrins also serve as cell-cell adhesion molecules, helping the cells bind to other cells and to the extracellular matrix. Integrins, like other cell adhesion molecules, differ from cell-surface receptors for hormones and other extracellular soluble signal molecules in that they usually bind their ligand—with lower affinity. If the binding were too tight, cells would presumably become irreversibly glued to the matrix and would be unable to move, which is a problem that does not arise if attachment depends on large numbers of weak adhesions (Velcro principle). Like other transmembrane cell adhesion proteins, integrins do more than just attach a cell to its surroundings. They also activate intracellular signaling pathways to communicate to the cell the character of the extracellular matrix that is bound. The very late antigen 4 (VLA-4) is expressed by numerous cells of hematopoietic origin and possesses a key function in the cellular immune response, such as by mediating leukocyte tethering, rolling, binding, and transmigration of the vascular wall at inflammatory sites.VLA-4 also is a signaling of target to interfere with pathological inflammations. Leukemic cells and solid tumors that express VLA-4 make use of these adhesive functions and confer VLA-4 a progressive role in the metastatic spread. The VLA-4 has multiple functions for the tumor, such as in leukocyte recruitment to micrometastases, the protection of tumors from immune surveillance, and contribution to a chemoresistance. However, therapeutic interferences with VLA-4 in cancer sciences are difficult to be developed, because of the marked impact on the physiological immune response. The very late antigen 5 (VLA-5) is a fibronectin receptor and a member of the integrin supergene family. As the most VLAs, VLA-5

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is involved in cell attachment to the extracellular matrix. A hypothesis of hematopoietic development is that maturing cells leave the bone marrow because of the loss of VLA-5 during differentiation [16–18]. CD44 and CD44s CD44 is a transmembrane glycoprotein, a cell adhesion molecule encoded by one gene, which includes more than 20 isoforms and is produced by alternative mRNA splicing and/or post-translational modifications. CD44 standard (CD44s), a signaling receptor, is the most abundant isoform and is expressed by most mammalian cells. CD44s is involved in hematopoietic regulation. It is also involved in hyaluronan—an important component of the hematopoietic stem cell niche—uptake and degradation, angiogenesis, wound healing, tissue formation, and patterning. In contrast, CD44 variants (CD44v) are upregulated in neoplasia, involved in tumor metastasis and aggression. CD44s binds the extracellular matrix components hyaluronan—which is the most common ligand, collagen, fibronectin, and transmembrane receptors such as E-selectin. Various cells express high levels of CD44s; however, they do not constitutively bind hyaluronan, with N-glycosylation, and carbohydrate-sulfation modulating binding capacity. The hyaluronan synthases HAS1, HAS2, and HAS3 synthetizes hyaluronan at the inner face of the plasma membrane and extruded to the outer surface, where it is secreted. CD44 is the most common receptor for hyaluronan. The hyaluronan uptake and degradation are CD44-­ dependent. Hyaluronan participates in hematopoietic stem cell lodgment in the endosteal region and also acts in stem cell proliferation and differentiation. CD44, hyaluronan, and SDF-1 interaction affects hematopoietic stem cells and progenitor cell trafficking. CD44s is known to be extensively expressed in fetal hematopoietic organs, and important roles for CD44s in adult hematopoiesis had been identified. In mice, blocking CD44s inhibited the lymphopoiesis in long-term bone marrow cultures, as well as the T-precursor trafficking to the thymus and lymph nodes, and memory cell activation. CD44s expression is downregulated during myeloid and erythroid development and involved in the retention of hematopoietic progenitors in bone marrow and spleen [21]. LFA-1, CD44 and CD44s, VLA-4, VLA-5, and Homing Studies showed that the engraftment of non-obese/severe with combined immunodeficiency (NOD/SCID) mice by human stem cells is dependent on the VLA-4, VLA-5, and—to a lesser degree—LFA-1. It was proved that the treatment of human CD34+ cells with antibodies to either VLA-4 or VLA-5 prevented that the engraftment would occur, and treatment with anti-LFA-1 antibodies significantly reduced the levels of engraftment. SDF-1 activates the CD34+ cells, which bear the chemokine receptor CXCR4, and it leads to a firm adhesion and trans-endothelial migration dependent on LFA-1/ICAM-1 (intracellular adhesion molecule-1) and VLA-4/ VCAM-1 (vascular adhesion molecule-1). There is a SDF-1-induced polarization and also an extravasation of CD34+/CXCR4+ cells through the extracellular matrix underlining the endothelium, and these two processes are dependent on both VLA-4 and VLA-5. The repopulating of human stem cells functionally expresses LFA-1, VLA-4, and VLA-5 [15].

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After considering that adhesion molecules implicate in the interactions of hematopoietic stem cells, and also progenitor cells, with the bone marrow extracellular matrix and stromal cells, a study examined the role of VLA-5 in stem cell mobilization and homing after stem cell transplantation. CBA/H mice were used in studies with VLA-5. CBA/H mice were developed by Strong in 1920 from a cross of a Bagg albino female and a DBA male. Strain CBA was selected for a low mammary tumor incidence and C3H for a high incidence. Now they are widely distributed and used as a general purpose strain. Differences between sub-strains are probably too large to be accounted for by mutation, and genetic contamination in the past is probable. The major recognized sub-strains are CBA/Ca or CBA/H. This sub-strain is used in most British research. Research with normal bone marrow from CBA/H mice concluded that 79  ±  3% of cells from the mice’s bone marrow in the lineage negative fraction expresses VLA-5. After mobilization with a trial including cyclophosphamide and G-CSF, the number of VLA-5 expressing cells in mobilized peripheral blood cells decreased to 36% (±4%). The lineage negative fraction of mobilized peripheral blood cells migrating in vitro toward SDF-1α demonstrated a further decrease to 3% (±1%) of VLA-5 expressing cells. Thereby, a downregulation of VLA-5 on hematopoietic cells during mobilization is suggested. This study then labeled the mobilized peripheral blood cells (with PKH67-GL) and transplanted in lethally irradiated recipients. Three hours after transplantation, an increase in VLA-5 expressing cells was observed, which remained stable until 24 hours post-­transplantation. Although the increase might implicate an upregulation of VLA-5, they did not exclude selective homing of VLA-5+ cells as a possible explanation. They determined the percentage of VLA-5 expressing cells immediately after transplantation in the peripheral blood of the recipients and were not able to observe any increase in VLA-5+ cells in the first 3 hours posttransplantation. Then the researchers separated the mobilized peripheral blood cells in VLA-5+ and VLA-5− cells and plated the cells out in clonogenic assays for progenitor (CFU-GM) and stem cells (CAFC-day35). It could be shown that 98.8% of the progenitor cells and 99.4% of the stem cells were present in the VLA-5+ fraction. They concluded that VLA-5 is not downregulated during the process of mobilization and that the observed increase in VLA-5 expressing cells after transplantation is caused by selective homing of VLA-5+ cells. To discover more about the role of VLA-5 in the process of homing, they treated the bone marrows and mobilized peripheral blood cells with an antibody to VLA-5. After VLA-5 blocking of mobilized peripheral blood cells, an inhibition of 59% in the homing of progenitor cells in bone marrow was found, and homing in the spleen of the recipients was only inhibited by 11%. For bone marrow cells, an inhibition of about 60% in the bone marrow was observed. Homing of bone marrow cells in the spleen was not affected after VLA-5 blocking. Based on these data, they concluded in this study that the mobilization of hematopoietic progenitor or stem cells does not coincide with downregulation of VLA-5. The observed increase in VLA-5 expressing cells after transplantation is caused by preferential homing of VLA-5+ cells. Homing of progenitor or stem cells to

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the bone marrow after transplantation might require adhesion interactions that can be inhibited by blocking VLA-5 expression. Homing to the spleen, for the researchers of this study, seemed to be independent of VLA-5 expression, and these data were indicative for different adhesive pathways in the process of homing to bone marrow or spleen [19, 20]. CD44s is involved in hematopoietic regulation, including progenitor cell proliferation and homing to bone marrow. CD44 has been shown to regulate hematopoietic stem cells and the bone marrow microenvironment by influencing matrix assembly; cytokine and chemokine capture and/or release; cytoskeletal linker protein binding (e.g., ankyrin, ezrin, radixin, and moesin) and signal transduction; and matrix degradation via protease production to influence hematopoietic stem cell adhesion, homing, migration, quiescence, mobilization, and resistance to oxidative stress [21].

4.1.4  H  oming Third Step: Cytoskeleton Rearrangement, Membrane Type 1 (MT1)-Matrix Metalloproteinase (MMP) Activation and Secretion of MMP2/9 The cell-surface proteolytic enzyme membrane type 1 (MT1)-matrix metalloproteinase (MMP) is expressed by human CD34  +  progenitors. It is activated by SDF-1 and G-CSF and is needed for SDF-1-induced migration and to induce homing and mobilization. Proteolytic enzymes, which also regulate cell migration and can both turn on and off the locomotion of human CD34+ cells (also mediating stem cell recruitment and organ localization), are highly expressed by various cell types during steady-state and injury conditions. It regulates hematopoietic cell migration and tissue localization. MMP-2/9enzymes are secreted by circulating CD34+ cells. Bone marrow CD34+ cells do not secrete MMP-2/9. The circulating CD34+ cells secreting MMP-2/9 facilitate their migration toward SDF-1 in vitro, and—according to that and experiences with human cells—MMP-2/9 inhibition reduces homing of CB CD34+ cells. Although isolated CB CD34+ cells do not express MMP-2/9, upon incubation with MMP-2/9-rich conditioned media, they gain an increased CXCR4 expression, as well as SDF-1-directed migration. If incubated with SCF, they have increased expression of MMP-2/9 and studies showed that incubating with SCF increased homing and repopulation in NOD/ SCID mice [4]. CD26, a cell-surface peptidase, is expressed on the surface of bone marrow CD34+ cells, a sub-fraction of cord blood CD34+ cells that are mainly CXCR4-­ expressing cells and other cells. CD26 cleaves and inactivates many ligands, such as SDF-1. CD26 seems to be mostly responsible for inactivation of SDF-1 in the circulation [4].

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4.1.5  H  oming Fourth Step: Rolling and Firm Adhesion of Progenitors to Endothelial Cells in Small Marrow Sinusoids Under Blood Flow Hematopoietic stem cells maintain a unique relationship with endothelial cells. Endothelial cells are specialized cells that make up the inner cellular lining of blood vessels and lymphatics; and they exhibit structural, molecular, and functional heterogeneity between and within organs. They act as an active barrier to deliver oxygen and nutrients to tissues, as a conduit for blood cell trafficking and playing a role in innate and adaptive immunity. Endothelial cells act in hematopoietic stem cells development, homeostasis, and regeneration. Endothelial cells act as a highway for hematopoietic stem cells migration and are important niches. Endothelial cells express angiocrine factors, such as growth factors, chemokines, and extracellular matrix components that regulate stem cells maintenance, self-renewal, differentiation, and regeneration after injury. Their normal function in bone marrow is required for maintaining hematopoiesis in vivo [23]. Hematopoietic stem cells migrate over long distances between niches. After entering circulation, stem cells home to the next site of blood development. Endothelial cells direct stem cells to the bone marrow and function as a molecular brake to slow cell migration under hemodynamic shear stress. Homing receptors facilitate stem cells tethering to the endothelium. Chemokine receptors activate adhesion molecules and their ligands on stem cells and endothelium, which facilitate the firm adhesion, as well as allow the endothelial transmigration, which is the fifth step of homing. Hematopoietic stem cells actively enter the bone marrow, migrate through the extravascular space, and lodge in the marrow niche [23].

4.1.6  H  oming Fifth Step: Trans-Endothelial Migration Across the Physical Endothelium/Extracellular Matrix (ECM) Barrier Rolling and firm adhesion of progenitors to endothelial cells in small marrow sinusoids is followed by trans-endothelial migration across the endothelium/extracellular matrix barrier. Selectins play an important role in bone marrow homing of hematopoietic stem cells. They regulate initial tethering and rolling of progenitors cells along the endothelial wall of blood vessels. The inhibition of VE-cadherin disturbs the integrity of bone marrow derived endothelial cell monolayers, which is important for trans-endothelial migration. SDF-1-mediated integrin activation induces firm adhesion of the hematopoietic stem cells to the endothelial wall and firmly attached stem cells can transmigrate through the endothelial layer, and basal

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lamina, consisting of the integrin substrates fibronectin, collagen, and laminin. Engrafted cells reside within the bone marrow niche and are able to self-renew and differentiate into all mature blood lineages [22].

4.1.7  H  oming Sixth Step: Stem Cells Finalize Their Homing by Selective Access and Anchorage to Their Specialized Niches in the Extravascular Space of the Endosteum Region and Periarterial Sites The last stage of homing is when hematopoietic stem cells anchor to the specialized niches within the bone marrow compartment near osteoblasts and initiate a long-­ term repopulation [22].

4.2  Bone Marrow and Homing The endothelium of the bone marrow is the first anchoring site for homing cells. It has molecules of adhesion and stimulates chemokines. Specialized cell structures regulate cell trafficking into small blood vessels; and chemotherapy and/or irradiation can disrupt the physical bone marrow endothelium barrier, allowing erythrocyte passage from the circulation to the extravascular hematopoietic space, as shown in mice. G-CSF is used not only to induce the mobilization but also to increase the bone marrow permeability, and creating larger gaps, as shown with murine bone marrow endothelial cells. According to studies with mice, in the endosteum region of bone marrow, the chemokine SDF-1 and its receptor CXCR4 are expressed by immature human osteoblasts, which are essential for stem cell seeding during fetal development and definitive repopulation in adults that receive transplants. SDF-1 and its receptor CXCR4—which are better described in Homing chapter—are expressed by many cell types, such as immature and maturing hematopoietic cells, stromal cells lining the stem cell-rich endosteum region, and human Stro1+ mesenchymal progenitors; and that expression occurs because of SDF-1 and CXCR4 expression are regulated by the hypoxia-inducible factor-1 (HIF-1) and the bone marrow is partially hypoxic. Irradiation was shown to increase SDF-1 and stem cell factor (SCF) secretion within 24–48 hours in murine bone marrow and spleen. The murine injured liver was shown to secret SDF-1 to the circulation, and the SDF-1 functionally passed the endothelium barrier—in a CXCR4-dependent manner into the bone marrow—and recruited hematopoietic progenitors to the circulation. It was tested to intravenously inject human SDF-1 in preconditioned but non-irradiated mice, which increased the homing of human mobilized and cord blood CD34+-enriched cells to the bone marrow and spleen of the mice. After 2 hours, it was observed murine stem cells adherent to microdomains in bone marrow that expresses SDF-1 [4].

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The release of stem cells into the circulation and their homing back to the bone marrow are important sequential steps in steady-state homeostatic processes. The stem cell niches occupied by quiescent, undifferentiated cells in the bone marrow, which are stationary, may be an oversimplification of a more dynamic situation in which there is a higher migratory turnover of stem cells involving their release, migration in the circulation, homing, and re-engraftment of bone marrow as part of proliferation and differentiation. The hematopoietic osteoclasts play a role continuously destructing the bone surface by forming absorption pits, and stromal osteoblasts remodel the new bone formation. There are also different types of hematopoietic and stromal cells within the bone marrow, which secrete a wide array of proteolytic enzymes in response to stimulation with cytokines or chemokines. The extracellular matrix of bone marrow, as well as adhesion molecules, cytokines, and chemokines, is degraded and cleaved by these proteolytic enzymes, and this also facilitates trans-endothelium cell migration [11].

4.3  P  reconditioning for Stem Cell Transplantation and Homing The chemotherapy with or without irradiation used before the HSCT induces not only cell death in the bone marrow, spleen, and blood, but it also damages the physiological bone marrow endothelium barrier. The tissue damage strongly increases the levels of secreted chemokines, cytokines, and proteolytic enzymes in many organs, as part of the regeneration and repair process, which impacts the stem cell homing—although homing does not depend on DNA damage—and its repopulation; the engraftment depends on DNA damage. Chemotherapy and irradiation commonly acts damaging DNA; this DNA damage, as well as the stress caused by chemotherapy and irradiation, also leads to proliferation and mobilization of quiescent stem cells from the bone marrow to the circulation. Thereby, the protocols used for stem cell transplantation are chosen with these characteristics in mind. Cyclophosphamide—which is an alkylating agent of the nitrogen mustard type, and its active metabolite, phosphoramide mustard, alkylates or binds to DNA— proceeds cross-linking in DNA and RNA strands and also inhibits synthesis of protein. Cyclophosphamide appears to be cell-cycle nonspecific. It is largely used prior to transplantation, because it induces stem cell mobilization and destroys malignant cells prior to transplantation. The DNA damage and elimination of malignant cells also increase secretion of survival, migration, and angiogenic factors, such as SDF-1—which can increase CXC chemokine receptor 4+ (CXCR4+) malignant stem cell survival, as well as proliferation, invasion, and metastasis. Due to the pre-transplantation treatment, the bone marrow microenvironment is broken during cell mobilization, and it should be re-established to enable stem cells to home to the bone marrow and also the retention. Granulocyte-colony-­stimulating factors (G-CSFs), like filgrastim, induce mobilization and increase the levels of stem cell engraftment in the bone marrow [1, 4].

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4.4  Radiation and Homing Radiation has an important role in homing. Because other organs, such as the heart and brain, also induce a shift in SDF-1 expression by endothelial cells in the liver and HIF-1 activation during stress, both that and the secretion of other chemokines, cytokines, and proteolytic enzymes induce stem cell homing and repopulation in opposite directions. Although the stem cell homing to other organs and mobilization induced by DNA damaged from radiation can reduce the direct homing to bone marrow and the early retention of progenitors within bone marrow, irradiated mice were shown to require much lower cell doses for donor cell repopulation. Durable repopulation by transplanted stem cells is enhanced in recipients preconditioned with radiation, eliminating host stem cells and hematopoiesis. Radiation is important also to send stress signals, following conditioning with DNA damage, as part of the repair process, which can also influence definitive stem cells in a time-­ dependent manner by enhancing their migration, proliferation, and differentiation; the time interval of transplantation after radiation has major impacts on repopulation and survival [4].

Chapter 5

Pediatric Graft-Versus-Host Disease (GvHD) and the Pharmacist

5.1  Graft-Versus-Host Disease (GvHD) Graft-versus-host disease (GvHD) is a reaction of donor immune cells against host tissues. It does not occur in autologous transplantation. The activated donor T-cells damage host cells after an inflammatory cascade that begins with the preparative regimen. Acute GvHD is a major obstacle for patients undergoing allo-HSCT. It principally involves the skin, gastrointestinal tract, liver, and thymus. The acute GvHD symptoms are dermatitis (skin rash), cutaneous blisters, crampy abdominal pain with or without diarrhea, persistent nausea and vomiting, and/or hepatitis. These symptoms commonly initiate on the first 100 days after allo-HSCT or later. Formerly, the acute GvHD was defined as occurring within the first 100  days following allo-HSCT and chronic GvHD as occurring after 100 days. Today, with the development of new strategies, such as reduced intensive conditioning, this definition is less clear. Current classification includes both late acute GvHD, which occurs after 100 days, and the overlap syndrome, which contains features of both. The acute GvHD diagnosis is clinically done, and the histologic confirmation is important, especially if the symptoms are atypical or involve just the liver or gut [1].

5.2  Thymus Biochemistry The thymus is the primary lymphoid organ responsible for T-lymphocyte production. The thymus has diverse T-cell receptors, capable of reacting with harmful foreign antigens but recognizing, as well as tolerating, self-antigens. The complete and undisrupted thymic microenvironment is essential for normal T-lymphocyte development, and it is dependent on input from the developing thymocytes (the thymic crosstalk). Bone marrow progenitor cells enter the thymus on a homing © Springer Nature Switzerland AG 2020 C. W. P. Schmidt, Pediatric Hematopoietic Stem Cell Transplantation for Pharmacists, https://doi.org/10.1007/978-3-030-43491-5_5

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process, with P-selectin and platelet P-selectin glycoprotein ligand on the progenitor cells appearing to play an important role in that. Interaction between notch-1 receptor and delta-like 4 ligand expressed by the cortical and medullary thymic epithelial cells (cTECs) occurs. At this stage, thymocytes express a triple-negative phenotype (without CD3, CD4, and CD8 surface markers). Following the expansion of the triple-­negative cells, controlled by signals such as interleukin (IL)-7 and FMS-like tyrosine kinase 3 ligand (FLT3L), they gain both CD4 and CD8 and acquire a double-positive phenotype with a heterodimeric T-cell antigen receptor αβ (TCRαβ) complex. The T-cell antigen receptor (TCR) diversity is achieved by genetic rearrangements of the TCR loci, and it is estimated to be in the region of 1020 α-β chain combinations. These genetic combinations have the potential to generate self-­reactive TCRs, which carry the risk of autoimmunity. Thymocytes have a rigorous two-stage selection process to identify and remove potentially damaging self-­reactive T-lymphocytes. The first stage, which is a positive selection, occurs in the cortex. Double-positive thymocytes are exposed to a self-peptide/major histocompatibility (MHC) complex presented by cTECs. Thymocytes recognize this complex and proceed to the next stage of development, ensuring recognition of antigen in association with self-MHC molecules. If the TCR does not recognize the complex, or recognizes—but not with high affinity—the T-lymphocyte will undergo apoptosis or death by neglect. Following positive selection, the surviving thymocytes migrate to the medulla. This process is regulated by chemokine receptor 7 and the corresponding ligands CCL19 and CCL21 expressed by medullary thymic epithelial cells (mTECs), and they subsequently lose either CD4 or CD8; that depends on which MHC class they are associated during positive selection to become single-positive cells. The second stage of TCR selection, which is the negative selection, occurs in the medulla where simple positive thymocytes are exposed to a self-peptide/MHC complex presented by mTECs and dendritic cells. Medullary TECs possess the unique ability of a wide range of peripheral tissue-restricted self-­ antigens (TRAs)’ ectopic expression—also called promiscuous gene expression. It is partly controlled by the autoimmune regulator (AIRE) transcription factor and Fezf2 transcription factor, and it forms a “molecular mirror of peripheral self.” TCRs that react with high affinity to the TRA/MHC complexes are deleted, because they have the potential to elicit autoimmunity. Re-encounter of peptides present on both cTECs and mTECs, also called shared peptides, leads to thymocyte deletion, which is a mechanism thought to increase peptide/MHC diversity. Negative selection is an indispensable part of central tolerance, and T-lymphocytes become tolerant of themselves [1].

5.3  Pediatric GvHD Prophylaxis Pediatric GvHD prophylaxis regimens include immunosuppressive drugs and chemotherapy. These regimens commonly include a calcineurin inhibitor, such as cyclosporine. When sources from related donors are used, common protocols

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suggest cyclosporine continued through day 100 after HSCT and then weaned over the course of 10–12 weeks. When the sources are from unrelated donors, cyclosporine is continued through day 180 after HSCT, and then it is weaned over the same time period. Patients who receive cells from unrelated donors also received corticosteroid prophylaxis (1 mg/kg prednisolone), which should start shortly after HSCT, and it can be weaned at day 21 after transplantation over 10–12 weeks if no GvHD occurred. Methotrexate (15 mg/m2 on D1 after transplantation, followed by 10 mg/ m2 on D3, 6, and 11 after transplantation) is commonly given. Patients who receive sources from umbilical cord blood commonly receive calcineurin inhibitor together with mycophenolate mofetil or corticosteroids, and that protocol is commonly used without methotrexate [73]. The treatment for GvHD can include methylprednisolone IV or orally (1–2 mg/ kg/day). In corticosteroid-refractory patients, it is usual to perform additional immunomodulatory modalities, such as infliximab or extracorporeal photopheresis. In corticosteroid-responsive patients, corticosteroids are slowly tapered when stable response is achieved [73]. Cyclophosphamide can be used as part of GvHD prophylaxis regimen. It is used in combination with cyclosporine in acute and chronic GvHD prophylaxis, mostly in AML pediatric patients undergoing allo-HSCT, as described in Table 5.1. A study to evaluate outcomes for children with high-risk leukemia undergoing HSCT using cyclosporine (with no methotrexate or mycophenolate mofetil) concluded that dose adjustments were highly variable between patients and also for each individual. The mean daily doses that allowed to reach a target trough blood concentration (TBC)—although it is not proved that the pharmacokinetic parameter that best correlates with GvHD outcomes is TBC—around 120 ng/mL were 3.53 mg/kg (range 1.50–5.50 mg/kg) in small children (0–8 years old) and 2.38 mg/kg (range 0.83–5.70 mg/kg) in older children (9–19 years old). The study used an initial dose of 2 h IV infusion of 1.5 mg/kg cyclosporine two times daily. Then, subsequent doses were determined individually, using a Bayesian approach. When oral administration could be tolerated, cyclosporine was given orally every 12 h. In the absence of GvHD, cyclosporine dosage was tapered, starting 2 months following the transplantation. In the same study, corticosteroids added only in patients with at least grade II acute GvHD, which was prednisolone 1–2 mg/kg/day, Table 5.1  Prophylaxis for acute and chronic GvHD with cyclophosphamide and cyclosporine Drug Dose Cyclophosphamide 50 mg/ kg/once daily Cyclosporine 3 mg/ kg/day

Administration IV

Period On third and fourth days (2 doses)

IV (concentration should not exceed 2.5 mg/mL and the administration of this drug is done over 2–6 h)

Starting from the 5th post-­ transplant day. Cyclosporine is tapered beginning from the 45th post-operative day and then completely discontinued on the 90th post-transplant day [96]

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depending on the stage, either immediately for severe GvHD or after 48 h of increased cyclosporine doses with adequate TBC of cyclosporine in patients with poorly responding to the moderate GvHD. A changing from IV to oral doses of cyclosporine should be carefully evaluated, because the dosages of oral and IV cyclosporine are commonly different. A study (which was not performed in pediatrics) concluded that patients who receive voriconazole should be converted from cyclosporine IV to oral at a ratio of 1:1. In patients who are receiving fluconazole but without azole co-­medication, the ratio is 1:1.3 to prevent subtherapeutic cyclosporine concentrations. All in all, the ratio of 1:1 is commonly used. Since there remains little consensus on the administration schedule, dosage, and monitoring of cyclosporine, the role of a clinical pharmacist to evaluate the concentration of the drug in blood and perform institutional protocols of dosage and blood monitoring together with the multidisciplinary team is important. It has been reported that a dosing schedule of cyclosporine administered three times daily in children helped to avoid deleterious peak levels with less fluctuation in blood concentration; and these results were obtained without any significant change in the average concentration of the drug. Data suggest that a three-time daily dose is useful for pediatric HSCT recipients. Studies showed that cyclosporine administration as a continuous IV infusion may be more effective and is associated with less serious side effects [96–99]. Since the introduction of haploidentical stem cell transplant (haplo-SCT) into a pediatric BMT program in 2015 by the Banner University Medical Center, Tucson, USA, 69.2% of recipients had been ethnic or racial minorities, and researchers from there described their experience with the first 13 pediatric and young adult patients with hematologic malignancies who have undergone T-cell-replete haploSCT after myeloablative conditioning. They had previously documented that in experimental haplo-SCT, post-transplant bendamustine was at least as effective as post-transplant cyclophosphamide to prevent graft-versus-host disease (GvHD) and led to superior graft-versus-leukemia effects. Preliminary findings from this study demonstrated that partial substitution of post-transplantation bendamustine for post-transplantation cyclophosphamide is feasible and safe after haplo-SCT as an immune-modulatory strategy to alleviate GvHD, and this substitution potentially preserves graft-versus-leukemia effects [58]. Protocols to treat ALL and AML are very different. But regarding conditioning regimens for HSCT of patients with ALL and AML, unlike the protocols to treat these diseases in pediatric patients who will not undergo transplantation, conditioning regimens before HSCT and regimens to GvHD prophylaxis often are the same for patients with ALL and AML. These regimens commonly are indicated for “acute leukemias.” AML is the most common indication for haplo-SCT, which was 76% according to a retrospective study of the Acute Leukaemia Working Party EBMT, and approximately 45% of the patients were transplanted in CR1. Post-­ transplant cyclophosphamide (PT-Cy) has been used successfully in the setting of unmanipulated haplo-SCT as GvHD prophylaxis. Other not standardized immunosuppressive drugs are used in combination, and the post-transplant initiation

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day varies. However, the time of starting of immunosuppression drugs after haploSCT in combination with PT-Cy was shown to influence the results of unmanipulated haplo-SCT. The early starting of cyclosporine A and mycophenolate mofetil (CSA + MMF) at day+1 was shown to decrease the risk of chronic GvHD, as well as improved GVHD-free/ relapse-free survival (GRFS) [69].

Chapter 6

Antibacterial, Antifungal, and Antiviral Prophylaxis for Children Undergoing HSCT

Infections in pediatric patients who undergo HSCT can be caused by virus, bacteria, fungus, or protozoa. The most common infections in these patients include: • On the first 30 days: Gram-negative aerobes and anaerobes, Staphylococcus epidermidis, Aspergillus sp., Candida, and herpes simplex type 1 reactivation. • On days 30–120: C. albicans, C. tropicalis, other Candida spp., Aspergillus, Trichosporon sp., Fusarium sp., Pneumocystis jiroveci, cytomegalovirus, adenovirus, Epstein–Barr virus, human herpesvirus 6, and Toxoplasma sp. [91].

6.1  Antibacterial Prophylaxis The most frequent causes of febrile neutropenia are bacteria and fungi. These pathogens are confirmed by culture in some cases, while in others the infections are suspected on clinical findings. Common antibacterial protocols for pediatric HSCT include fluoroquinolone, trimethoprim-sulfamethoxazole, or cephalosporin. Fluoroquinolone seems to not be significantly associated with increased Clostridium difficile infection or invasive fungal disease, but it was proved that its use in HSCT prophylaxis increases the resistance to fluoroquinolone among bacteremia isolates. There is heterogeneity in fluoroquinolone effect on bacteremia. Prophylaxis with trimethoprim-sulfamethoxazole should be done administering a dose every day. The prophylaxis against bacterial infection can be daily and for Pneumocystis jiroveci pneumonia is intermittent; therefore systemic antibiotic prophylaxis with trimethoprim-­sulfamethoxazole can require administration at least once daily. In countries where there is a high rate of Toxoplasma gondii infection, daily doses may be preferred. Although trimethoprim-sulfamethoxazole is efficient for prophylaxis, its use in HSCT prophylaxis is proved to increase the trimethoprim-­ sulfamethoxazole resistance in bacteremia isolates. Cephalosporin prophylaxis reduces bacteremia but does not significantly reduce infection-related mortality. © Springer Nature Switzerland AG 2020 C. W. P. Schmidt, Pediatric Hematopoietic Stem Cell Transplantation for Pharmacists, https://doi.org/10.1007/978-3-030-43491-5_6

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Fluoroquinolone vs cephalosporin and levofloxacin vs ciprofloxacin were proved to have similar effects in terms of bacteremia prevention [74, 81]. Trimethoprim-sulfamethoxazole is the drug of choice for first prophylaxis of Pneumocystis jiroveci pneumonia (PCP). PCP-related mortality rate is significantly reduced with this prophylaxis. There is also a well-documented efficacy of trimethoprim-­sulfamethoxazole to prevent PCP in HIV-infected patients. However, trimethoprim-sulfamethoxazole prophylaxis was shown to do not markedly reduce all-cause mortality in hematology patients. Prophylaxis with trimethoprim-­ sulfamethoxazole commonly is done with doses 2–3 times weekly during the entire period at risk, from engraftment to ≥6 months and as long as immunosuppression is ongoing. It can keep longer in patients who continue to receive immunosuppressive drugs or have chronic GvHD. Data indicate that children may benefit equally from a once-weekly regimen. Primary prophylaxis is also usually performed during the period of treatment-induced immunosuppression or until the CD4+ cell count achieves >200 cells/mm3. Given the potential for marrow toxicity, prophylaxis with this drug should not be started during the pre-engraftment period in HSCT recipients—with the possible exception of the conditioning period. Table 6.1 describes the doses for prophylaxis and patients undergoing HSCT [82]. Table 6.1  Prophylaxis for bacterial infections in pediatric patients undergoing HSCT Drug Sulfamethoxazole-­ trimethoprim (first choice for allogeneic HSCT) [52, 82]

Dapsone [52, 82]

Atovaquone [82] Pentamidine [82]

Prophylaxis The most common is 2–3 times weekly during the entire period at risk, from engraftment to ≥6 months and as long as immunosuppression is ongoing. Data indicate that children may benefit equally from a once-weekly regimen 150/750 mg/m2/day or 5/25 mg/kg/day orally 1–7 days/week. This daily dose can be given as a single dose or divided into 2 doses (every 12 hours). If the drug is given every day, the protocol is to divide the dose in 2 doses/ day. When the patient receives the drug 3 days/week, these 3 days can be consecutive or alternating There is an indication for Pneumocystis prophylaxis for HIV+ or HIV-exposed patients who are 4 weeks to 24 months old: 30 mg/kg/day 4–24 months old: 45 mg/kg/day Aerosol (age >5 years): 300 mg once/month Intravenously: 4 mg/kg every 4 weeks

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6.2  Antifungal Prophylaxis Fungal infections in immunosuppressed patients can be difficult to treat; therefore, the emphasis has been placed on prevention. Approaches include preventing exposure through environmental strategies, such as high efficiency particulate air filters, and preventing disease with the use of antifungal prophylaxis. Patients commonly receive prophylaxis against Pneumocystis jiroveci and also fluconazole as prophylaxis against fungal infection. Routine fungal surveillance cultures are commonly obtained, and the prophylaxis is adjusted based on sensitivities of isolates [73, 79]. For children undergoing allogeneic HSCT, the prophylaxis for fungus with fluconazole and other drugs that have been used is described in Table 6.1. Those drugs that did not show real benefits to be used on pediatric HSCT are not described on the table. Children who cannot receive fluconazole can receive an echinocandin drug. There is a concern about fluconazole in prophylaxis for patients undergoing allogeneic HSCT because of its lack of anti-mold coverage. Allogeneic HSCT patients are at risk for both invasive yeast infections, such as Candida sp., and mold infections. Since fluconazole is active only against some yeasts, several trials have investigated broader-spectrum agents. However, these trials showed that overall agents with broader spectrum had no benefit over fluconazole. Mold-active agents were shown by studies to do not influence overall mortality, and they also have important limitations including drug interactions, toxicity, and costs, and some have been limited data in children; therefore fluconazole remains the recommended agent. Recommendations may change as data becomes available in the future. Another lack of covering of fluconazole is regarding a decreased effectiveness against certain Candida species, including C. glabrataand and C. krusei. Since the introduction of routine fluconazole prophylaxis in HSCT, the incidence of non-albicans Candida appears to be increasing. The gaps in coverage of fluconazole should be taken in mind when treating patients with persistent fever. Patients known to be colonized with non-albicans fluconazole-resistant species should have the potential for disease from these species considered early [79]. The standard therapy, based on studies and available data, generally is to use fluconazole for children undergoing either allogeneic or autologous HSCT, even if acute GvHD grade II–IV or chronic extensive GvHD in children