Lanzkowsky's Manual of Pediatric Hematology and Oncology

Jonathan D. Fish, Jeffrey M. Lipton, Philip Lanzkowsky

• Publisher: Academic Press
• Year: 2016
• eBook ISBN: 9780128016749
• Edition: 6
• Topic: Medicine
• Subtopic: Oncology

Lanzkowsky's Manual of Pediatric Hematology and Oncology, Sixth Edition, is a comprehensive book on patient management, replete with algorithms and flow diagrams on diagnosis and management. Reflecting the considerable advances in the treatment and management of hematologic and oncologic diseases in children, the sixth edition of this successful clinical manual has been entirely updated to incorporate all current treatment protocols, new drugs, and management approaches. Its concise and easy-to-read format will enable readers to make accurate diagnoses and permit them to treat patients without having to reference larger medical textbooks.Based on the new standards of genetic classification and prognostic information that have arisen in the past five years, the sixth edition includes two new chapters (Diagnostic, Molecular, and Genomic Methodologies for the Hematologist, Transfusion Medicine) and several new expanded chapters that were previously sections in consolidated chapters (Myelodysplasia, Myeloid Leukemias, Lymphoid Leukemias, Hemolytic Anemia, and Disorders of Coagulation).- Presents a concise, systematic approach to all pediatric hematologic and oncologic disorders in one manual- Offers an alternative to bigger references which only cover either oncologic or hematologic disorders in twice as many pages- Presents an easy-to-read format: multiple tables, charts, and flow-diagrams for diagnosis and management of pediatric hematologic and oncologic disorders- Includes 2 new chapters and several expanded chapters: Diagnostic, Molecular and Genomic Methodologies for the Hematologist, Transfusion Medicine, Myelodysplasia, Myeloid Leukemias, and Lymphoid Leukemias

Chapter 1

Diagnostic Molecular and Genomic Methodologies for the Hematologist/Oncologist

Vijay G. Sankaran

Abstract

Over the past several years, molecular diagnostic testing in patients with hematologic and oncologic disorders has become increasingly sophisticated and prevalent. While in the past focused genetic tests were performed, in recent years the widespread use of genomic and molecular approaches in both research and clinical settings has shown potential to refine our understanding of pediatric blood disorders and cancer. This chapter provides an overview of the currently used molecular and genomic methods. In that way, the format for this chapter differs from those that describe specific disease entities. Thus the chapter can be read in its entirety as essential background for the modern practice of pediatric hematology/oncology. We will primarily focus on those genetic methods that are currently in use in clinical settings. Undoubtedly, in the coming years, the use of certain methods will evolve and new methods will become available. With this in mind, there are two goals in this chapter. We first aim to provide an overview of the types of currently used clinical genetic testing methods and specifically attempt to examine their utility in detecting specific changes at the molecular level that underlie both congenital and acquired conditions that are commonly seen by pediatric hematologists and oncologists. This overview will be important for clinicians to better understand how newly developed methods could supplant the currently used approaches in the coming years. The second goal of this chapter is to provide a basic understanding of the limitations that exist for the most common molecular and genomic methods in use, so that clinicians who receive these results can be sufficiently versed in these methods and avoid misinterpreting the results obtained from these tests.

Keywords

Pediatric hematology/oncology; WGS; WES; FISH; molecular lesions; array CGH; MLPA

Over the past several years, molecular diagnostic testing in patients with hematologic and oncologic disorders has become increasingly sophisticated and prevalent. While in the past focused genetic tests were performed, in recent years the widespread use of genomic and molecular approaches in both research and clinical settings has shown potential to refine our understanding of pediatric blood disorders and cancer. This chapter provides an overview of the currently used molecular and genomic methods. In that way, the format for this chapter differs from those that describe specific disease entities. Thus the chapter can be read in its entirety as essential background for the modern practice of pediatric hematology/oncology. We will primarily focus on those genetic methods that are currently in use in clinical settings. Undoubtedly, in the coming years, the use of certain methods will evolve and new methods will become available. With this in mind, there are two goals in this chapter. We first aim to provide an overview of the types of currently used clinical genetic testing methods and specifically attempt to examine their utility in detecting specific changes at the molecular level that underlie both congenital and acquired conditions that are commonly seen by pediatric hematologists and oncologists. This overview will be important for clinicians to better understand how newly developed methods could supplant the currently used approaches in the coming years. The second goal of this chapter is to provide a basic understanding of the limitations that exist for the most common molecular and genomic methods in use, so that clinicians who receive these results can be sufficiently versed in these methods and avoid misinterpreting the results obtained from these tests.

Clinical Molecular and Genomic Methodologies

Despite the large range of approaches that have been developed, all of the methods remain focused on the goal of identifying patients’ molecular lesions that underlie their disease. The methods can be broadly classified into two categories:

1. Direct testing: These approaches look for the presence of genetic mutations that directly contribute to disease.

2. Indirect testing: This category includes approaches that compare genomic or molecular markers in multiple affected individuals to unaffected individuals. These approaches often identify markers that may segregate with a disease, but the markers themselves may not cause the disease itself.

There may be overlap between these categories and certain methods may identify causal genetic mutations in some instances (direct testing), while only identifying segregating markers (indirect testing) in other cases.

Table 1.1 lists the commonly used genetic testing methodologies, along with the types of molecular lesions that they are able to identify.

Table 1.1

Overview of Molecular and Genomic Diagnostic Methodologies

Method	Common point mutations	Rare point mutations	Copy number variants	Uniparental disomy	Balanced inversions or translocations	Repeat expansions	Examples of use in pediatric hematology/oncology
Linkage analysis (using markers such as short tandem repeats)	X		X				Family pedigree with history of hereditary spherocytosis and interest in identifying causal gene
Fluorescent in situ hybridization			X		X		Acquired monosomy in myelodysplastic syndrome
Array comparative genomic hybridization			X	X			Testing for microdeletion in patient with hematologic and syndromic phenotype
Genome-wide single nucleotide polymorphism microarrays	X		X				Testing for small copy number variants in pediatric leukemia
Targeted polymerase chain reaction analysis	X	X				X	Testing for JAK2 V617F mutation in patient with a myeloproliferative disorder
Sanger sequencing	X	X					Molecular diagnosis of a patient with pyruvate kinase deficiency
Multiplex ligation-dependent probe amplification			X			X	Deletions in α- or δβ- thalassemia cases
Gene panel sequencing	X	X					Severe congenital neutropenia
Whole-genome or -exome sequencing	X	X	X				Unknown bone marrow failure syndrome

Linkage Analysis

While most methods are now focused on identifying the precise molecular cause of disease, indirect tests can be quite useful, particularly for mapping causes of a disease in a family. For example, testing for markers such as single nucleotide polymorphisms (SNPs) or short tandem repeats (STRs, which are 2–5 base long repetitive elements with varying numbers of repeats) that are found throughout the genome can be extremely useful as a way to identify likely causal genes, particularly in diseases where multiple possible causal genes have been implicated. For example, in hereditary spherocytosis a number of genes including ANK1, SPTB, SPTA, SLC4A1, and EBP42 are implicated in the disease. Many of these genes are quite large and while sequencing a panel of genes is certainly possible, in a large family, the use of either SNP-based methods or other markers such as STRs can identify the likely causal locus to help focus targeted sequencing efforts. These methods are commonly used for diagnostic mapping in resource-poor settings where whole-genome sequencing (WGS) methods may not be available and these methods can also be extremely useful in other settings. For example, if a family is being followed with a known disease, but no coding mutations are identified on targeted sequencing, these approaches can help validate that there is linkage to a specific gene and they may assist in the efforts to identify mutations in regulatory regions of the implicated gene. Specifically, segregation of markers that are in linkage with the causal mutation should only be found in affected family members and would suggest that the causal mutation is located nearby. These methods are also commonly used as an initial screen in families where possible cancer predisposition syndromes may exist and can help focus in-depth analysis on certain regions of the genome. Even in cases where whole-exome or -genome sequencing is performed, linkage can provide an excellent indirect approach to focus on marker genes that segregate appropriately in individuals who have a particular disease.

Fluorescent In Situ Hybridization

Fluorescent in situ hybridization (FISH) was developed in the 1980s and uses fluorescently labeled DNA probes to query whether entire chromosomes or parts of a chromosome may be duplicated or deleted in cells. The fluorescently labeled DNA probes are complementary to the region of interest on a chromosome and therefore specifically hybridize only to this region and not to others. FISH is commonly used to assess for gain or loss of chromosomes or large parts of chromosomes in patients with hematologic malignancies. Typically, a numbe...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• Dedication
• List of Contributors
• About the Editors
• Preface to the Sixth Edition
• Preface to the Fifth Edition
• Preface to the Fourth Edition
• Preface to the Third Edition
• Preface to the Second Edition
• Preface to the First Edition
• Introduction: Historic Perspective
• Chapter 1. Diagnostic Molecular and Genomic Methodologies for the Hematologist/Oncologist
• Chapter 2. Hematologic Manifestations of Systemic Illness
• Chapter 3. Classification and Diagnosis of Anemia in Children
• Chapter 4. Lymphadenopathy and Diseases of the Spleen
• Chapter 5. Anemia During the Neonatal Period
• Chapter 6. Iron-Deficiency Anemia
• Chapter 7. Megaloblastic Anemia
• Chapter 8. Bone Marrow Failure
• Chapter 9. General Considerations of Hemolytic Diseases, Red Cell Membrane, and Enzyme Defects
• Chapter 10. Extracorpuscular Hemolytic Anemia
• Chapter 11. Hemoglobinopathies
• Chapter 12. Polycythemia
• Chapter 13. Disorders of White Blood Cells
• Chapter 14. Disorders of Platelets
• Chapter 15. Disorders of Coagulation
• Chapter 16. Lymphoproliferative Disorders
• Chapter 17. Myelodysplastic Syndromes and Myeloproliferative Disorders
• Chapter 18. Acute Lymphoblastic Leukemia
• Chapter 19. Acute Myeloid Leukemia
• Chapter 20. Histiocytosis Syndromes
• Chapter 21. Hodgkin Lymphoma
• Chapter 22. Non-Hodgkin Lymphoma
• Chapter 23. Central Nervous System Malignancies
• Chapter 24. Neuroblastoma
• Chapter 25. Renal Tumors
• Chapter 26. Rhabdomyosarcoma and Other Soft-Tissue Sarcomas
• Chapter 27. Malignant Bone Tumors
• Chapter 28. Retinoblastoma
• Chapter 29. Germ Cell Tumors
• Chapter 30. Hepatic Tumors
• Chapter 31. Hematopoietic Stem Cell Transplantation
• Chapter 32. Management of Oncologic Emergencies
• Chapter 33. Supportive Care of Patients with Cancer
• Chapter 34. Evaluation, Investigations, and Management of Late Effects of Childhood Cancer
• Chapter 35. Psychosocial Aspects of Cancer for Children and Their Families
• Chapter 36. Blood Banking Principles and Practices
• Appendix 1. Hematological Reference Values
• Appendix 2. Biological Tumor Markers
• Index