Microarray Technology and Cancer Gene Profiling

by Mocellin, Simone, M.D., Ph.D.

• ISBN13: 9780387399775
• ISBN10: 0387399771
• Edition: 1st
• Format: Hardcover
• Copyright: 2006-12-30
• Publisher: Landes Bioscience

Summary

Cancer is a heterogeneous disease in most respects, including its cellularity, different genetic alterations and diverse clinical behaviors. Traditional molecular analyses are reductionist, assessing only one or a few genes at a time, thus working with a biologic model too specific and limited to confront a process whose clinical outcome is likely to be governed by the combined influence of many genes. The potential of functional genomics is enormous, because for each experiment, thousands of relevant observations can be made simultaneously. Accordingly, DNA array, as other high throughput technologies, might catalyze and ultimately accelerate the development of knowledge in tumor cell biology. Although in its infancy, the implementation of DNA array technology in cancer research has already provided investigators with novel data and intriguing new hypotheses on the molecular cascade leading to cancerogenesis, tumor aggressiveness and sensitivity to antineoplastic agents. Given the revolutionary implications that the use of this technology might have in the clinical management of cancer patients, principles of DNA array-based tumor gene profiling need to be clearly understood for the data to be correctly interpreted and appreciated. In the present book, written by leading experts in each field, the technical features characterizing this powerful laboratory tool are discussed, and the applications so far described in the field of oncology are reviewed.

Table of Contents

Microarray Technology
Manufacturing of Microarrays	p. 1
Abstract	p. 1
Introduction	p. 1
Nomenclature	p. 1
The DNA Microarray Library	p. 2
Robotic Printers	p. 3
Printing Pins	p. 5
Microarray Slide Substrates	p. 8
Personnel	p. 10
Conclusions	p. 10
Technological Platforms for Microarray Gene Expression Profiling	p. 12
Abstract	p. 12
Principles of Gene Microarray Technology	p. 12
Target Preparation and Hybridization	p. 14
Image Acquisition and Quantification	p. 16
Principles of Gene Microarray Data Analysis	p. 19
Abstract	p. 19
Introduction	p. 19
Study Design and Data Analysis	p. 20
Data Collection and Normalization	p. 23
Comparing Expression Data	p. 24
Gaining Weights...and Feeling Good About It!	p. 31
Abstract	p. 31
A Light Introduction	p. 31
P-Value Weighting	p. 32
Within-Spot Pixel Variance Weighting	p. 35
A Weighty Discussion	p. 37
Complementary Techniques: RNA Amplification for Gene Profiling Analysis	p. 39
Abstract	p. 39
Introduction	p. 39
Source Material Collection and RNA Isolation	p. 40
Single-Strand cDNA Synthesis	p. 41
Double-Stranded cDNA (ds-cDNA) Synthesis	p. 41
RNA Amplification	p. 42
Target Labeling for cDNA Microarray Using Amplified RNA	p. 45
RNA Amplification Protocol	p. 46
Complementary Techniques: Laser Capture Microdissection-Increasing Specificity of Gene Expression Profiling of Cancer Specimens	p. 54
Abstract	p. 54
Microdissection Technologies: The Past and the Present	p. 54
Why Microdissection?	p. 59
Slide Preparation for Microdissection	p. 62
In Our Laboratory	p. 63
Conclusions	p. 64
Complementary Techniques: Validation of Gene Expression Data by Quantitative Real Time PCR	p. 66
Abstract	p. 66
PCR-Based Analysis of Gene Quantitation	p. 66
Principles	p. 67
Main Issues	p. 68
Messenger RNA Cell Source	p. 71
Applications in the Oncology Field
Microarrays for Cancer Diagnosis and Classification	p. 74
Abstract	p. 74
Introduction	p. 74
Revealing Expression Profiles for Cancer Diagnosis and Classification	p. 75
Using Expression Profiles in the Clinic	p. 79
Perspective	p. 83
Gene Profiling for the Prediction of Tumor Response to Treatment: The Case of Immunotherapy	p. 86
Experimental Models, Human Polymorphisms and Cancer Heterogeneity	p. 86
Gene Profiling and T-Cell Phenotypes	p. 87
Interactions between Tumor Cells and TA-Specific T-Cells	p. 88
The Tumor Microenvironment	p. 89
Influence of Genetic Background and Cancer Heterogeneity on Immune Responsiveness	p. 89
Gene Profiling of Anticancer Immune Responses	p. 90
Conclusions	p. 92
Identification of Molecular Determinants of Tumor Sensitivity and Resistance to Anticancer Drugs	p. 95
Abstract	p. 95
Introduction	p. 95
Correlation of Gene Expression Data from DNA Arrays with Response to Anticancer Agents	p. 97
Conclusions	p. 102
SNP and Mutation Analysis	p. 105
Abstract	p. 105
Introduction	p. 105
Enzymatic Approach	p. 106
Polymerase Chain Reaction (PCR)	p. 106
Microarray Approaches	p. 107
Invader Assay	p. 108
Mass Spectrometry	p. 110
Sequencing	p. 111
Summary	p. 115
Cancer Development and Progression	p. 117
Abstract	p. 117
Cancer Development	p. 117
Science/Technology	p. 120
Complementary Approach	p. 123
In Vitro/Preclinical Research Using Microarray	p. 125
Clinical Studies	p. 127
Gene Expression Profiling in Malignant Lymphomas	p. 134
Abstract	p. 134
Introduction	p. 134
Gene Expression Profiling	p. 135
Gene Expression Analysis	p. 135
The Applications of Gene Expression Profiling to Leukemia and Lymphoma	p. 136
Tumor Immunology	p. 147
Abstract	p. 147
Tumor Immunology and the Post-Genomic Era	p. 147
Tumor Immune Escape	p. 148
New Targets for Tumor Immunotherapy	p. 149
Dendritic Cell Biology	p. 150
T Cell Biology	p. 150
Tumor Microenvironment	p. 151
Concluding Remarks	p. 154
Index	p. 157
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