Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas - 1st Edition - ISBN: 9780120884049, 9780080456256

Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas

1st Edition

Molecular Genetics: Liver and Pancreatic Carcinomas V3

Editors: M. Hayat
eBook ISBN: 9780080456256
Hardcover ISBN: 9780120884049
Imprint: Academic Press
Published Date: 17th August 2005
Page Count: 432
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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


Details

No. of pages:
432
Language:
English
Copyright:
© Academic Press 2005
Published:
Imprint:
Academic Press
eBook ISBN:
9780080456256
Hardcover ISBN:
9780120884049

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

Reviews

"...this book contains a wealth of information of value to the oncologist and biomedical scientist." --MICRON (2005) “Professor Hayat’s Handbook has the great potential of becoming the handbook of choice for clinician and basic scientists.” -–Akhouri A. Sinha, Ph.D., Professor of Genetics, University of Minnesota and Research Scientist at the VA Medical Center