Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas
Molecular Genetics: Liver and Pancreatic Carcinomas V3
- 1st Edition - April 4, 2013
- Editor: M. A. Hayat
- Language: English
- Paperback ISBN:9 7 8 - 0 - 1 2 - 3 8 8 5 4 9 - 4
- eBook ISBN:9 7 8 - 0 - 0 8 - 0 4 5 6 2 5 - 6
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… Read more
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.
* 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
* 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
Graduate and medical students in cancer research, oncology, pathology, biology, immunology, bioinformatics and endocrinology.
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
- Edition: 1
- Published: April 4, 2013
- Language: English
MH
M. A. 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, USARead Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas on ScienceDirect