In Situ Molecular Pathology and Co-expression Analyses

Gerard J. Nuovo

• Publisher: Academic Press
• Year: 2020
• ISBN:
  9780128206546
• Edition: 2
• Topic: Biological Sciences
• Subtopic: Human Anatomy & Physiology
• Index: Biological Sciences

Major advancements in the field of in situ molecular pathology have occurred since publication of the first edition. In Situ Molecular Pathology and Co-expression Analyses, Second Edition, continues to teach both the molecular basis for the improvements and the actual protocols. This is the unique feature that separates it from the pack of other "cook-book" type approaches. The fields of in situ hybridization and immunohistochemistry have expanded rapidly where computer-based analyses systems have greatly expanded the power of these methods. Further, knowledge of the marked improvements in the reagents themselves since the first edition can make the difference of excellent versus misleading data. The automated platforms require that researchers and diagnostic biomedical investigators have a good understanding of the basics of in situ based tests, protocols, and biochemistry for troubleshooting in order to maximize the use of these platforms. This second edition focuses attention on straightforward protocols used to simultaneously detect two or more proteins/nucleic acids within intact tissue by doing co-expression analyses. Practicing molecular pathologists, diagnostic pathologists, laboratory directors, and toxicologists, as well as clinicians and researchers in training, will benefit from this clear presentation of protocols and theoretical framework. Data derived from in situ hybridization and immunohistochemistry.

• Explains the theory and foundation of immunohistochemistry and in situ hybridization and presents easy-to-follow experimental protocols with tricks of the trade
• Includes two new chapters: Recent improvements in immunohistochemistry and in situ hybridization, Quality control for immunohistochemistry and in situ hybridization: How to know if the color change is signal or background
• The second edition also includes a detailed test to help one learn the basics of histologic interpretation of tissues and a separate detailed test in how to differentiate signal from background
• Includes chapter-ending summaries of Key Points to Remember, bringing beginners up to speed with any seasoned veteran in the field
• Thoughtfully tackles the molecular basis if IHC and ISH, along with application of that knowledge to improving the techniques is significant

Chapter 1

Introduction

Abstract

The major advancements in biomedical research over the past 30 years have been based in the broad field of molecular biology. DNA, RNA, and proteins are now routinely sequenced, altered, and their functions analyzed in great detail. The in situ-based methodologies (immunohistochemistry and in situ hybridization) were in their infancy 30 years ago. Today, they are mainstream, as evidenced by the tens of thousands of peer review articles published each year that have used either methodology. Still, much remains to be learned about how to maximize the power of immunohistochemistry and in situ hybridization. This book starts by building a foundation, assuming little or no prior knowledge in the area of molecular biology. I hope that, at the end of the book, you will have a fount of knowledge that allows you to be very knowledgeable and capable in the in situ-based molecular pathology methods.

Keywords

In situ hybridization; immunohistochemistry; signal; specificity; sensitivity

One of the most dramatic changes in my 30+ year career has been the explosion of the field of molecular pathology. Technological changes have resulted in the evolution of a field that basically did not exist 20 years ago, to the point that it is now a dominant player in both research and clinical medicine. There are two in situ-based tests: in situ hybridization and immunohistochemistry. Indeed, in clinical pathology, many diagnoses are dependent on performing an in situ-based test. Further, clinicians often depend on specific in situ-based results to make the definitive decision on how to treat a patient’s disease. For example, the her-2-neu immunohistochemical test is routinely done to decide whether a woman’s breast cancer will show reduced growth if treated with a specific drug called Herceptin that has minimal side effects, at least relative to standard chemotherapy.

The critical need for immunohistochemistry and in situ hybridization tests in the diagnostic and research arenas has led to the involvement of major biotechnical companies in this area. Large companies such as Enzo Biochem, Roche, Ventana Medical Systems, Dako, Leica, and others offer many products for in situ-based molecular pathology including automated platforms, and they, and many other large companies, market reagents for such tests. This has led to basically all diagnostic and most research laboratories using these automated in situ hybridization- and immunohistochemical-based systems. I can attest that 25 years ago the idea that automated machines could do in situ hybridization and immunohistochemistry was almost in the realm of science fiction!

As a result of these advances in the field of in situ-based molecular pathology, many more laboratories are either using these tests for their diagnostics or research, or wanting to incorporate them into their work. Hence, the purpose of this textbook. I hope that this book can make in situ-based molecular pathology more accessible and understandable to both the research and diagnostic laboratory. I hope to do this by focusing on two key goals: (1) to explain the theory and foundation of immunohistochemistry and in situ hybridization and (2) to present simplified protocols that are easy to follow for the different in situ-based protocols. I also include protocols for the identification of two or more DNA/RNA/protein targets in a given tissue.

This textbook has been written assuming a minimal prior knowledge of the topics of molecular pathology in general and in situ-based molecular pathology in particular. The first chapters focus more on the biochemistry of the processes inherent in any molecular pathology-based method, including the polymerase chain reaction (PCR) and hybrid capture solution phase detection of DNA or RNA, as well as Western blot detection of proteins. The biochemistry part, though, strongly emphasizes just the key parts you must understand to be able to “visualize” what is actually happening inside the intact cell when doing either immunohistochemistry or in situ hybridization. As all such methods, of course, use intact tissue, I also include a chapter to assist you in being better able to determine the cell type(s) that contain the target sequence of interest. Specifically, I include a chapter that is meant to teach the basics of histopathology to the nonpathologist. This second edition includes a thorough quiz on the interpretation of basic histopathology in the Appendix, which I hope will help the reader with little experience in this area become more adroit at examining tissue under the microscope. The second edition also includes two new chapters. One deals with differentiating signal from background. In this chapter, one will see that by combining their knowledge of histopathology with the color-based changes of the in situ molecular tests, they will rarely (hopefully, if ever) misinterpret background as signal. The other new chapter focuses on several major developments in the fields of in situ hybridization and immunohistochemistry that have been described since the first edition was published. After this basic introduction to these key topics, we move on to the practical applications of in situ hybridization, immunohistochemistry, and coexpression analyses.

Thus, it is certain that all readers will be able to either just breeze through or skip certain sections, depending on your training. It is my strong hope that all readers, after finishing this book, will not only want to try their hand at in situ-based molecular pathology but also have the confidence that they will be able to reason out the best way to solve the problems that arise when using any such methodologies. The end result, I hope, will be well worth the effort. For one, the power of the in situ-based molecular pathology tests is extraordinary. By knowing the cell type or types that contain the target of interest, you typically get tremendous insight into the role of the target that simply cannot be achieved by PCR, Western blots, or any of the other solution-based methods, as each of the latter tests requires the pulverization of the tissue as a prerequisite to doing the test. Also, with these methods, you can get the true pleasure of looking under a microscope and often seeing for the first time data that no one before has seen, especially when working with novel DNA/RNA or protein sequences. Thus, we can appreciate the wonder and excitement of Van Leeuwenhoek when he first examined microbes under the microscope. The fun and enjoyment of doing this is why I enjoy in situ hybridization and immunohistochemistry today every bit as much as, if not more so, when I started 30 years ago!

When I started writing this book, I realized that I had certain preconceived notions about in situ hybridization and immunohistochemistry. It seemed that the format of writing a book in this field was the perfect time to test such preconceived notions. For example, I had assumed for my entire career that if I was unable to get a good signal for either immunohistochemistry or in situ hybridization with an older block (usually defined as at least 10 years old), the target DNA/RNA/protein had simply degraded and that was that. I was trained (and it made perfect sense) to simply avoid such blocks of tissue, as they probably were not fixed properly at the time of biopsy and, more importantly, nothing could be done to “rejuvenate” the signal. Similarly, I was trained to assume that any RNA would quickly degrade in the tissue sections, either from just time-related degradation and/or RNase activity in the tissue/in situ solutions and, thus, to only use recently done formalin-fixed biopsies for RNA in situ hybridization analysis, and to also use strict “RNase-free” protocols. Although I could give you many such other examples, let me end with just one more. I was trained to rely primarily on one method to “expose the target” when doing in situ hybridization or immunohistochemistry. This method has many names, including antigen retrieval, cell conditioning, and liquid-based denaturation. Again, this made perfect sense because it was well documented that formalin fixation cross-linked cellular proteins to each other and to RNA/DNA. The logic went that this extensive cross-linking created many small pores that needed to be opened for the DNA/RNA probe or primary antibody and ancillary reagents to enter the cell and access the target. Of course, this theory became very popular when antigen retrieval first came on the scene about 25 years ago, and many proteins that were otherwise undetectable with immunohistochemistry became evident. Certainly, I clearly remember the importance of antigen retrieval to the anatomic pathologist in breast cancer, as the ER/PR and her-2-neu testing required this pretreatment to get an accurate idea of the signals that, in turn, had important implications for the treatment of the woman.

An important focus of this book is that all the preconceived notions noted in the preceding paragraph, despite making sense, are simply wrong! Look at Fig. 1.1. This is the result of in situ hybridization for HPV DNA in a tissue sample over 20 years old. When I first tested the block in 1992 (just when HPV could be successfully detected in situ), it produced an intense s...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• Contributors
• Chapter 1. Introduction
• Chapter 2. The basics of molecular pathology
• Chapter 3. The biochemical basis of in situ hybridization and immunohistochemistry
• Chapter 4. The basics of in situ hybridization
• Chapter 5. The basics of immunohistochemistry
• Chapter 6. Quality control for immunohistochemistry and in situ hybridization: how to know if the color change is signal or background
• Chapter 7. Recent improvements in immunohistochemistry and in situ hybridization
• Chapter 8. The basics of histologic interpretations of tissues
• Chapter 9. The recommended protocol for in situ hybridization
• Chapter 10. The recommended protocol for immunohistochemistry
• Chapter 11. Coexpression analyses
• Chapter 12. PCR in situ hybridization and RT in situ PCR
• Appendix (Discovery Life Sciences)
• Appendix 2. Locked nucleic acids—properties and applications
• Appendix 3. Spectral analyses
• Appendix 4. Enzo life sciences
• Appendix 5. Quiz on distinguishing signal from background
• Appendix 6. Test on basic histopathology
• Appendix 7. A broad-based approach to differentiate CIN from its mimics: the utility of in situ hybridization and immunohistochemistry
• Appendix 8. False-positive results in diagnostic immunohistochemistry are related to horseradish peroxidase conjugates in commercially available assays
• Index