Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas

Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas

Molecular Genetics: Liver and Pancreatic Carcinomas V3

1st Edition - August 17, 2005
This is the Latest Edition
  • Editor: M. Hayat
  • eBook ISBN: 9780080456256

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Description

Immunohistochemistry is the use of specific antibodies to stain particular molecular species in situ. This technique has allowed the identification of many more cell types than could be visualized by classical histology, particularly in the immune system and among the scattered hormone-secreting cells of the endocrine system, and has the potential to improve diagnosis, prognosis and therapeutic options of cancer. This book discusses all aspects of immunohistochemistry and in situ hybridization technologies and the important role they play in reaching a cancer diagnosis. It provides step-by-step instructions on the methods of additional molecular technologies such as DNA microarrays, and microdissection, along with the benefits and limitations of each method. The topics of region-specific gene expression, its role in cancer development and the techniques that assist in the understanding of the molecular basis of disease are relevant and necessary in science today.

Key Features

* The only book available that translates molecular genetics into cancer diagnosis
* The results of each Immunohistochemical and in situ hybridization method are presented in the form of color illustrations
* Methods discussed were either developed or refined by expert contributors in their own laboratories

Readership

Graduate and medical students in cancer research, oncology, pathology, biology, immunology, bioinformatics and endocrinology.

Table of Contents


  • Contents

    Authors and Coauthors of Volume 3

    Foreword

    Preface to Volume 3

    Prologue

    Selected Definitions

    Classification Scheme of Human Cancers

    I Molecular Genetics

    1 Quality Assurance in Immunohistochemistry

    Introduction

    Definitions

    Principles of Quality Control in Immunohistochemistry

    Technical Aspects of Individual Steps of Immunohistochemistry

    References

    2 Methodology of Microarray Data Analysis

    Introduction

    Microarray Data Normalization and Transformation

    Classification and Exploratory Data Analysis

    Postanalysis Follow-Up and Validation of Microarray Data

    Looking Ahead

    References

    3 Microarray-Based Gene Expression Analysis of Defined Tumor Areas

    Introduction

    Materials and MethodS

    Conclusion

    References

    4 Tissue Microarray and Quantitative Fluorescence Image Analysis in Tumor Biomarker Analysis

    Introduction

    Materials

    Methods

    Results and Discussion

    References

    5 Identification of Differentially Expressed Genes Using Rapid Subtraction Hybridization: Detailed Methodology for Performing Rapid Subtraction Hybridization

    Introduction

    Materials

    Methods

    Results and discussion

    References

    6 Noncontact Laser Microdissection and Pressure Catapulting: A Basic Tool in Genomics, Transcriptomics, and Proteomics

    The Force of Focused Light: An Introduction

    History and Principle of Laser Cutting and Laser Pressure Catapulting

    Sample Features and Specimen Preparation

    Technical Setup of the PALM MicroBeam System

    PALM RoboSoftware for Automated Microdissection and Catapulting

    Automated Cell Recognition

    RoboMover and RoboStage II

    Applications of PALM MicroBeam: Overview and Outlook

    References

    7 DNA Methylation Analysis of Human Cancer

    Introduction

    The Significance of DNA Methylation in Cancer

    Clinical Significance of DNA Methylation in Tumors

    Methods

    Quantification of Global Methylation

    Nonbisulfite Methods

    Bisulfite Methods

    Materials

    Procedure

    References

    8 Antiepidermal Growth Factor Receptor Antibody: Immunohistochemistry

    Introduction

    Materials and methods

    Results and discussion

    Conclusions

    References

    9 PTEN and Cancer

    Introduction

    Materials and Methods

    Materials

    Methods

    Materials

    Methods

    Results and Discussion

    Conclusion

    References

    10 The Role of p53 and p73 Genes in Tumor Formation

    Gene Architecture of the p53 Family

    TP63 and TP73 Play Important Roles in Development and Differentiation

    Transcriptional and Apoptotic Activity of p63 and p73

    Regulation of p73 and p63 Protein Stability and Transcriptional Activity

    Role of p73 and p63 in Tumors

    p73 Is Not a Classic Knudson-Type Tumor Suppressor

    Alteration of p73 Expression in Human Cancer

    Alteration of p63 Expression in Human Cancer

    Upstream Components That Signal to p73 and p63

    Mechanisms of Transdominance: Heterocomplex Formation and Promoter Competition

    p73 and Chemosensitivity

    Potential Application of p63/p73 in Gene Therapy of p53 Inactivated Tumors

    Conclusions

    References

    11 Analysis of Centrosome Amplification in Cancer

    Introduction

    Centrosome Structure

    The Centrosome Cycle

    Coordination of the Centrosome, DNA, and Cell Cycles

    The Centrosome and DNA Cycles Can Be Uncoupled

    Evidence for a Centrosome-Based Cell Cycle Checkpoint

    Centrosome Amplification in Cancer

    Centrosome Amplification, Aneuploidy, and Chromosomal Instability

    Methods for Analysis of Centrosome Amplification in Cancer

    Sample Procurement

    Morphological and Structural Methods

    Functional Assay for Microtubule Nucleation and Growth

    Conclusion

    References

    II Liver Carcinoma

    1 Liver Carcinoma

    Hepatocellular Carcinoma

    Genetics of Hepatocellular Carcinoma

    Biomarkers for Hepatocellular Carcinoma

    Aldose Reductase-like Protein

    Beta-Catenin

    Carcinoembryonic Antigen, CA19-9 and DU-PAN-2 Glycoproteins

    CCND1

    Cyclooxygenase-2

    Glypican-3

    Hepatitis B XAntigen

    HER-2/neu

    Inhibitor of Differentiation and DNA Binding Protein

    LKB1 Gene

    Metastatic Tumor Antigen1

    Mitogen-Activated Protein Kinase

    17p

    p53

    PTEN

    Smad4

    Squamous Cell Carcinoma Antigen

    Use of cDNA Microarray

    Use of Proteomics

    Role of Virus in Hepatocellular Carcinoma

    Hepatitis B Virus

    Hepatitis C Virus

    Risk Factors

    Treatment of Hepatocellular Carcinoma

    Hepatoblastoma

    References

    2 Mechanisms of Human Hepatocarcinogenesis: An Overview

    Introduction

    Epidemiology of Hepatocellular Carcinoma

    Natural History of Hepatocellular Carcinoma

    Chronic Hepatitis

    Cirrhosis

    Phenotypic Alterations and Dysplasia

    Emergence and Progression of Hepatocellular Carcinoma

    Etiology of Hepatocellular Carcinoma in Humans

    Hepatitis Viruses

    Aflatoxins

    Ethanol Consumption and Smoking

    Hepatotoxic Drugs and Chemicals

    Genetic Diseases of the Liver

    Cellular Pathogenesis of Hepatocellular Carcinoma

    Development of Monoclonal Hepatocyte Populations in Preneoplastic and Neoplastic Liver

    Increased Hepatocyte Proliferation in Preneoplastic and Neoplastic Liver

    Mechanisms Governing Increased Hepatocyte Proliferation in Preneoplastic and Neoplastic Liver

    Molecular Pathogenesis of Hepatocellular Carcinoma

    Genomic Alterations in Hepatocellular Carcinoma

    Cellular Protooncogenes and Hepatocarcinogenesis

    Tumor-Suppressor Genes and Hepatocarcinogenesis

    Conclusions and Perspective

    References

    3 Genes Involved in Hepatocellular Carcinoma

    Introduction

    Allelic Imbalance and Microsatellite Instability

    Cell Cycle Regulation

    p53 and Homologues

    Wnt Pathway: APC, β-Catenin, Axin 1, and E-Cadherin

    Alterations of the Transforming Growth Factor-β/Insulin-like Growth Factor-Axis

    Distinct Hepatocarcinogenic Pathways in Hepatocellular Carcinoma with or without Cirrhosis?

    Molecular Markers in the Differential Diagnosis of Hepatocellular Carcinomas

    References

    4 Role of p53 and ZBP-in Hepatocellular Carcinoma

    Introduction

    Materials

    Methods

    Handling of Liver Tissues

    Immunohistochemical Staining of p53 or ZBP-89

    Dual-Immunohistochemical Staining to Detect ZBP-89 and p53 in the Same Cell

    Co-Immunoprecipitation of p53 and ZBP-89

    Results and Discussion

    References

    5 Role of Immunohistochemical

    Expression of PTEN in Hepatocellular Carcinoma

    Introduction

    Immunohistochemical Protocols for PTEN Staining of Paraffin Sections

    PTEN Antibodies for Immunohistochemical Analysis

    Materials

    Methods

    Results and Discussion

    References

    6 The Potential Role of Bcl-2 and Protein Expression in Hepatocellular Carcinomas: A Clinicopathologic Study

    Introduction

    Materials and Methods

    Patients

    In situ Hybridization for Bcl-2 mRNA Detection

    Materials

    Preparation of the Reagents According to Manufacturer’s Instructions(Hybridization/Detection Complete System [IH-60002 (IHD-0052)]: Ultra Sensitivity, MBI)

    Protein Block Buffer

    Detergent Wash Buffer

    Methods

    Immunohistochemistry for Bcl-2 and Hepatocyte Protein

    Materials

    Preparation of Solutions

    Protocol (Methods)

    Results

    Conventional Pathology

    In situ Hybridization for Bcl-2 mRNA

    Immunohistochemistry for Bcl-2 Oncoprotein

    Statistical Analysis

    Discussion

    References

    7 The Detection and Clinical Aspect of Immunohistochemical Analysis of Bcl-xL Protein in Hepatocellular Carcinoma

    Introduction

    Materials and Methods

    Patients

    Western Blot Analysis

    Immunohistochemical Staining

    Immuno-Electron Microscopy

    Results

    Western Blot of Bcl-xL

    Immunohistochemical Staining

    Immuno-Electron Microscopy

    Discussion

    References

    8 Growth Arrest DNA Damage-Inducible Gene 45 β in Human Hepatocellular Carcinoma

    Introduction

    Epidemiology of Liver Cancer

    Hepatitis, Liver Cirrhosis, and Liver Cancer

    Hepatocarcinogenesis

    The Down-Regulation of GADD45β Gene Is Associated with Liver Cell Malignant Transformation

    GADD45 Gene Family Involves Program Cell Death When the Cell Is Damaged by Genotoxins

    Materials and Methods

    RNA Isolation, cRNA Preparation, and Microarray Hybridization

    Microarray Analyses

    Immunohistochemical Study of GADD45β, GADD45α, and Mutant p53 Protein

    Genomic GADD45β Promoter Sequence and Binding Elements Involved in Transcription Regulation

    p53 Tumor-Suppressor Gene Involved in Liver Disease and HCC Development

    References

    9 Immunohistochemical Detection of DNA Topoisomerase IIα in Hepatocellular Carcinoma

    Introduction

    Materials

    Antibodies

    Methods

    Results

    Immunohistochemical Detection and Labeling Index of Topo IIα

    Relationship between Topo IIα and Other Prognostic Factors (Ki-67 and p53)

    Topo IIα Labeling Index and Survival

    Discussion

    References

    10 Role of Immunohistochemical

    Expression of DNA Methyltransferases in Hepatocellular Carcinoma

    Introduction

    Materials

    For Immunohistochemical Staining

    For Cell Culture and Immunofluorescent Staining

    Methods

    Immunohistochemical Staining

    Cell Culture and Immunofluorescent Staining

    Results and Discussion

    References

    11 Role of Hepatitis B Surface Antigen in Hepatocarcinogenesis

    Introduction

    Hepatitis B Virus and Hepatocellular Carcinoma

    Hepatitis B Virus Integration and Hepatocarcinogenesis

    Role of Hepatitis B Surface Antigen and Hepatocellular Carcinoma

    Detection of Hepatitis B Surface Antigen in Hepatocellular Carcinoma

    Materials

    Immunohistochemistry

    Southern Blot Analysis

    Methods

    Immunohistochemistry

    Southern Blot Analysis

    Results and Discussion

    Detection of Hepatitis B Surface

    Antigen Expression by Immunohistochemistry

    Detection of Hepatitis B Virus Integration by Southern Blot Analysis

    References

    12 Immunohistochemical Detection of Host Gene Products Up-Regulated by Hepatitis B Virus X Antigen and Their Putative Roles in Hepatocellular Carcinoma

    Introduction

    Materials and Methods

    Cell Lines and Culture

    Plasmids

    Preparation of Recombinant Retroviruses and Infection of HepG2 Cells

    Detection of CAT Activity and HB XAg Polypeptide in HepG2 Cells

    Polymerase Chain Reaction, Select Complementary DNA Subtraction, Cloning, Sequencing, and Identification of a Complementary DNA from Putative HB XAg Effectors

    Patient Samples

    Cloning of Full-Length cDNA from Up- and Down-Regulated Genes and Identification of the Encoded Products

    Preparation and Use of Antisera

    Western Blot

    Detailed Protocols

    Preparation of Cell Lysates

    Electrophoresis

    Immunoblotting

    Tissue Staining

    Embedding

    Immunostaining Protocol for Tissue

    Results

    Discussion

    References

    13 Distinction between Well-Differentiated Hepatocellular Carcinoma and Benign Hepatocellular Nodular Lesions, Using Fine-Needle Aspiration

    Biopsy

    Introduction

    What Are the Diagnostic Problems and Pitfalls?

    Fine-Needle Aspiration Biopsy

    Normal Liver, Cirrhosis, and Steatosis

    Benign Hepatocellular Nodular Lesions

    Well-Differentiated Hepatocellular Carcinoma

    Ancillary Studies

    Special Stains

    Immunohistochemistry

    Other Immunostains

    Conclusions

    References

    14 Immunohistochemical Detection of EBAG9/RCAS1 Expression in Hepatocellular Carcinoma

    Introduction

    Identification of EBAG9/RCAS1

    EBAG9/RCAS1 Expression in Normal Tissue and Cancer Tissue

    Stepwise Evolution of Hepatocellular Carcinoma and EBAG9/RCAS1 Expression in Hepatocellular Carcinoma

    Materials

    Liver Samples

    Materials for Immunohistochemistry

    Method

    Liver Tissue Preparation

    Protocol for EBAG9/RCAS1 Immunostaining

    Results and Discussion

    EBAG9 Expression in Normal and Chronically Diseased Liver

    EBAG9 Expression in Hepatocellular Carcinoma

    Semiquantitative Classification of EBAG9 Expression in Hepatocellular Carcinoma Sections

    Correlation between EBAG9 Expression and Pathologic Variables

    EBAG9/RCAS1 Expression in the Process of Stepwise Hepatocellular Carcinoma Progession

    References

    15 Hepatocellular Carcinoma of Differential Viral Origin: Analysis by Oligonucleotide Microarray

    Introduction

    Materials and Methods

    Hepatocellular Carcinoma Samples

    Control Liver Samples

    Sample Preparation

    Complementary DNA Synthesis and in vitro Translation for Labeled Complementary RNA Probe

    Gene Expression Analysis by High-Density Oligonucleotide Array

    Procedure for Gene Selection

    Statistical Analysis

    Results and Discussion

    Clinicopathologic Characteristics of the Three Types of Hepatocellular Carcinomas

    Genes Selected in Each Comparison

    Molecular Features of B-type Hepatocellular Carcinoma

    Molecular Features of C-type Hepatocellular Carcinoma

    Molecular Features of Non-B, Non-C Hepatocellular Carcinoma

    References

    III Pancreatic Carcinoma

    1 Pancreatic Carcinoma: An Introduction

    Introduction

    Pancreatic Adenocarcinomas

    Mucinous Tumors of the Pancreas

    Intraductal Papillary Mucinous Tumors

    Pancreatic Neuroendocrine Tumors

    Acinar Cell Carcinomas

    Biomarkers for Pancreatic Cancer

    Importance of Biomarkers

    Biomarkers

    ADAM9

    BRAF

    BRCA2

    Cartilage Oligomeric Matrix Protein

    CDKN2A

    Cyclooxygenase-2

    Cysteine-Rich Secretory Protein-

    Cystic Fibrosis Transmembrane Conductance Regulator

    DPC4

    Epidermal Growth Factor Receptor

    Hypoxia-Inducible Factor-1 Alpha

    Id Protein

    Integrin

    Interleukin-8

    KOC

    K-ras Gene

    Maspin

    Metastasis-Associated Gene 1

    Microvascular Density

    Mucins

    Neurokinin-1 Receptor

    Nuclear Factor Kappa B

    Osteopontin

    p16

    p21WAF1/C1P1

    p27

    p53

    Rad51

    S100P

    S100A6

    Serine Proteinase Inhibitor

    Smad 4

    Stk11 Gene

    Telomerase

    Transforming Growth Factor Beta 1

    Vascular Endothelial Growth Factor

    Validity of Biomarkers in Clinical Practice

    References

    2 Detection of Genomic Imbalances in Endocrine Pancreatic Tumors Using Comparative Genomic Hybridization

    Introduction

    Materials

    Methods

    DNA Extraction from Frozen Tumor Sample

    DNA Extraction from Paraffin-Embedded Tumor Material

    DNA Labeling by Nick Translation

    Probe Mix Preparation

    Hybridization

    Post-Hydridization Wash and Counterstain

    Visualization and Analysis

    Statistics

    Results and Discussion

    Common Regions of Chromosomal Gains and Losses

    Genetic Changes in Association with Tumor Size and Malignant Outgrowth

    Comparative Genomic Hybridization Findings in Primary Tumors and Metastases

    Comparative Genomic Hybridization Differences in Endocrine Pancreatic Tumor Subtypes

    References

    3 Molecular Detection of Micrometastases in Pancreatic Cancer

    Introduction

    Materials

    Immunohistochemistry for Pan-Cytokeratin

    Molecular Detection by Mutated K-Ras

    Methods

    Immunohistochemistry for Pan-Cytokeratin

    Molecular Detection by Mutated K-Ras

    Results

    Immunohistochemical Examination

    Molecular Detection of Micrometastases

    Discussion

    References

    4 K-ras Protooncogene in Human Pancreatic Cancer

    Introduction

    Pancreatic Cancer

    K-ras Protooncogene

    Methods

    Immunohistochemistry

    Dot Blot Hybridization

    Single-Strand Confirmation Polymorphism (PCR-SSCP)

    DNA Auto Sequencing by ABI

    Systems

    Results and Discussion

    References

    5 Altered Expression of p27 Protein in Pancreatic Carcinoma

    Introduction

    Materials

    Methods

    Immunohistochemical Staining Using Streptavidin-Biotin Complex Method

    Results and Discussion

    References

    6 The Role of DUSP6/MKP-3 in Pancreatic Carcinoma

    Introduction

    Primary Function of DUSP6/MKP-3

    Materials

    Methods

    Immunohistochemistry Using the Indirect Peroxidase Method

    Results and Discussion

    Inactivation of DUSP6/MKP-3 in the Pancreatic Cancer

    Growth Suppressive Effect of DUSP6/MKP-3

    Conclusion and Future

    PerspectiveS

    References

    7 Expression of Carbohydrate Antigens in Pancreatic Cancer

    Introduction

    Materials

    Immunohistochemistry

    Polymerase Chain Reaction

    Methods

    Hematoxylin and Eosin Staining

    Immunohistochemistry

    Genetic Analysis for Detecting Point Mutations of K-ras at Codon 12

    Results

    Discussion

    CA 19-9

    DU PAN-2

    Span-1

    CA 15-3

    CA 50

    CA 72-4

    CA 125

    CA 242

    CA 494

    PAM4

    CAM 17-1

    TKH2

    A10

    References

    8 Role of Mucins in Pancreatic Carcinoma

    Introduction

    Materials

    Methods

    Discussion

    MUC1 in Pancreatic Adenocarcinomas

    MUC1 in Pancreatic Adenocarcinomas and Precursor Lesions

    MUC1 and MUC2 in Intraductal Papillary Mucinous Neoplasms

    MUC4 in Pancreatic Adenocarcinomas

    References

    9 Immunohistochemical Detection of Cyclooxygenase-2 in Pancreatic Ductal Adenocarcinomas and Neuroendocrine Tumors

    Introduction

    Materials

    Methods

    Results and Discussion

    References

    10 Immunohistochemical Detection of Activated Stat3 Protein in Pancreatic Cancer

    Introduction

    Materials

    Methods

    Tissue Slide Preparation

    Deparaffinization

    Rehydration

    Antigen Retrieval

    Tissue Digestion

    Immunostaining Procedure

    Specimen Analysis

    Results and Discussion

    References

    11 The Role of Transcription Factor E2F-1 in Pancreatic Ductal Carcinoma

    Introduction

    Materials

    Methods

    Results and Discussion

    References

    12 Role of Immunohistochemical Expression of Chromogranins in Pancreatic Endocrine Tumors

    Introduction

    Materials and Methods

    Results and Discussion

    References

    13 Role of Immunohistochemical Expression of Maspin in Pancreatic Carcinoma

    Introduction

    Materials

    Methods

    Results and Discussion

    References

    14 Role of Mast Cells in Pancreatic Carcinoma

    Introduction

    Role of Mast Cells in Nonpancreatic Tumors

    Role of Mast Cells in Pancreatic Cancer

    Materials

    Methods

    Immunostaining Procedure

    Evaluation of Immunohistochemical Findings

    Results and Discussion

    Demographic and Pathologic Characteristics of the Patient Population

    Mast Cell Count and Characterization in the Normal Pancreas and Pancreatic Cancer

    SCF and c-kit Expression in the Normal Pancreas and Pancreatic Cancer

    References

    15 Endocrine Cells in Invasive Ductal Adenocarcinoma of the Pancreas: An Immunohistochemical Study

    Introduction

    Materials

    Methods

    Results

    Incidence and Location of CgA-IR Cells

    Hormonal Reactivity of CgA-IR Cells

    Ki-67 Positivity of CgA-IR Cells in Adenocarcinoma

    Discussion

    References

    16 Fine-Needle Aspiration Biopsy of Pancreatic Adenocarcinoma

    Introduction

    Materials

    Methods

    Preparation of Slides

    Staining Methods

    Cytologic Criteria for Adenocarcinoma

    Evaluation of Fine-Needle Aspiration Biopsy Smears

    Results and Discussion

    Chronic Pancreatitis

    Pancreatic Endocrine Neoplasms

    Solid-Pseudopapillary Tumor of the Pancreas

    Acinar Cell Carcinoma

    Serous Microcystic Adenoma

    Mucinous Cystic Neoplasms

    Metastasis

    Conclusion

    References

    17 Immunohistochemical Expression

    of Mitogen-Activated Protein Kinase Kinase 2 in Pancreatic Cancer

    Introduction

    Materials

    Cell Lines

    Tissue Samples

    Antibodies

    Methods

    Immunofluorescent Staining of MEK2 in Pancreatic Cancer Cell Lines

    Immunohistochemical Staining of MEK2 Protein in Human Pancreatic Cancer Specimens

    Results and Discussion

    References

    Index


Product details

  • No. of pages: 432
  • Language: English
  • Copyright: © Academic Press 2005
  • Published: August 17, 2005
  • Imprint: Academic Press
  • eBook ISBN: 9780080456256

About the Editor

M. Hayat

M. Hayat
Dr. Hayat has published extensively in the fields of microscopy, cytology, immunohistochemistry, immunocytochemistry, and antigen retrieval methods. He is Distinguished Professor, Department of Biological Sciences, Kean University, Union, New Jersey, USA.

Affiliations and Expertise

Distinguished Professor, Department of Biological Sciences, Kean University, Union, NJ, USA