Breast Cancer Research Protocols (Methods in Molecular Medicine)

533 Pages·2005·8.221 MB·English

by  Susan A. Brooks (Editor), Adrian Harris (Editor)

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Breast Cancer Research Protocols
M E T H O D S I N M O L E C U L A R M E D I C I N E™
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M E T H O D S I N M O L E C U L A R M E D I C I N E™
Breast Cancer
Research Protocols
Edited by
Susan A. Brooks
School of Biological and Molecular Sciences
Oxford Brookes University
Headington, Oxford, UK
Adrian Harris
Medical Oncology Unit
The Churchill Hospital
Headington, Oxford, UK
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Breast cancer research protocols / edited by Susan A. Brooks and Adrian
Harris.
p. ; cm. -- (Methods in molecular medicine, ISSN 1543-1894 ; 120)
Includes bibliographical references and index.
ISBN 1-58829-191-X (alk. paper)
1. Breast--Cancer--Laboratory manuals.
[DNLM: 1. Breast Neoplasms--genetics. 2. Breast Neoplasms--pathology. 3.
Biopsy--methods. 4. Clinical Protocols--methods. 5. Genetic Techniques.
WP 870 B82398 2005] I. Brooks, Susan A. II. Harris, Adrian, 1950- III.
Series.
RC280.B8B68788 2005
616.99'449'0072--dc22
2005010939
Preface
Throughout the world, breast cancer has increased in incidence in the last
decade. Conversely, there has been a decrease in mortality as a result of an
emerging combined therapy approach that includes systemic application
of treatment modalities, such as chemotherapy and hormone therapy, combined
with early detection. Based on the molecular understanding of the disease,
many new treatments have been introduced into the clinic in the last five years,
including antibody therapy to HER2, pure antiestrogens, and aromatase
inhibitors. It is vital for breast cancer research to be able to link studies on cell
lines and in preclinical models to studies of human tumor material. There are
also many new technologies involving large-scale analysis for the expression
of genes, such as proteomics and gene array analysis. Unprecedented opportuni-
ties have arisen for clinical application. Ultimately, these each need to be tested
in randomized prospective studies modulating the target of interest.
Breast Cancer Research Protocols aims to cover the majority of the tech-
niques that would be necessary for a research scientist or clinician intending
to enter into this field and initiate research, as well as to communicate with
others on methodologies and approaches. Because of the dramatic increase in
the diagnosis and therapy of breast cancer, it is particularly important that these
approaches be integrated for patient benefit in the long run.
Thus, this book of protocols initially covers how to collect and handle
human breast tumor samples appropriately, and how to extract them for their
constituent DNA, RNA, or proteins. Because samples are often quite small, the
technology of handling biopsies appropriately is important. The new methods
of analyzing gene expression are also described, as breast cancer has actually
been one of the areas where this has been successfully applied.
One of the most important areas in breast cancer treatment is understanding
prognosis and being able to select patients that are at highest risk. This is cov-
ered for all the major surgical and pathological criteria currently in use.
Validating targets and understanding the biology of newly discovered genes
is critical and methods of analyzing cells in vivo and in vitro are also described.
Based on these combined approaches, we hope that Breast Cancer Research
Protocols will contribute to further advances in effective management and
therapy of breast cancer in the future.
Susan A. Brooks
Adrian Harris
v

Contents
Preface ..............................................................................................................v
Contributors .....................................................................................................xi
PART I PREPARATION OF CELL AND TISSUE SAMPLES
1. Breast Tissue Banking: Collection, Handling, Storage,
and Release of Tissue for Breast Cancer Research............................3
Linda Snell and Peter H. Watson
2. Frozen Biopsy Collection and Storage: Frozen Biopsy Samples..........25
Russell Leek
3. Serial Biopsies/Fine-Needle Aspirates and Their Assessment.............. 29
Irene Boeddinghaus and Stephen R. D. Johnson
4. Breast Tissue Microarrays.................................................................... 43
Robert J. Springall and Cheryl E. Gillett
5. Preparation of Tumor Homogenates for Subsequent Preparation
of Cytosols, Membrane Fractions, RNA, and DNA......................... 51
Naomi Robertson and Russell Leek
6. Isolation of RNA From Tumor Samples: Single-Step Guanidinium
Acid–Phenol Method......................................................................55
Naomi Robertson and Russell Leek
7. Isolation of DNA From Tumor Samples .............................................. 61
Naomi Robertson and Russell Leek
8. Laser-Assisted Microdissection and Isolation of DNA and RNA......... 65
Ulrich Lehmann and Hans Kreipe
PART II MARKERS OF CLINICAL OUTCOME AND PROGNOSIS
9. Traditional and Established Indicators of Prognosis
and Treatment Success.................................................................... 79
Derek E. Roskell and Ian D. Buley
10. Sentinel Lymph Node Biopsy in Breast Cancer................................... 91
Celia Chao and Kelly M. McMasters
11. Pathological Evaluation of Axillary Sentinel Lymph Nodes
in Breast Cancer........................................................................... 113
Michael Z. Gilcrease and Aysegul Sahin
vii
viii Contents
12. Measurement of Estrogen Receptor Status by Immunocytochemistry
in Paraffin Wax Sections ................................................................ 127
Bharat Jasani, Anthony Douglas-Jones, Anthony Rhodes,
Susan Wozniak, Peter J. Barrett-Lee, Julia Gee,
and Robert Nicholson
13. Markers of Apoptosis......................................................................... 147
Fazlul H. Sarkar and Yiwei Li
14. Quantitative Angiogenesis in Breast Cancer ..................................... 161
Stephen B. Fox
PART III ANALYSIS OF TUMOR-DERIVED PROTEINS AND ANTIGENS
15. Immunohistochemistry...................................................................... 191
Cheryl E. Gillett
16. Detection of Aberrant Glycosylation in Breast Cancer
Using Lectin Histochemistry......................................................... 201
Tracey M. Carter and Susan A. Brooks
17. SDS-PAGE and Western Blotting to Detect Proteins
and Glycoproteins of Interest in Breast Cancer Research............. 217
Chloe Osborne and Susan A. Brooks
18. Breast Cancer Proteomics Using Two-Dimensional Electrophoresis:
Studying the Breast Cancer Proteome.............................................231
Miriam V. Dwek and Sarah L. Rawlings
19. Procedures for the Quantitative Protein Determination
of Urokinase and Its Inhibitor, PAI-1, in Human Breast Cancer
Tissue Extracts by ELISA................................................................ 245
Manfred Schmitt, Alexandra S. Sturmheit, Anita Welk,
Christel Schnelldorfer, and Nadia Harbeck
PART IV ANALYSIS OF GENES AND GENE EXPRESSION IN TUMOR SPECIMENS
20. Fluorescence In Situ Hybridization and Comparative Genomic
Hybridization................................................................................ 269
Patricia Gorman and Rebecca Roylance
21. Fluorescence In Situ Hybridization Assessment of c-myc Gene
Amplification in Breast Tumor Tissues.......................................... 297
Jan K. Blancato, Mary Steele Williams, and Robert B. Dickson
22. Detection of HER2 Gene Amplification by Fluorescence In Situ
Hybridization in Breast Cancer ...................................................... 309
John M. S. Bartlett and Amanda Forsyth
23. In Situ Hybridization Combined With Immunohistochemistry
to Localize Gene Expression......................................................... 323
Rosemary Jeffery, Toby Hunt, and Richard Poulsom
Contents ix
24. Quantitation of RNA by Ribonuclease Protection Assay................... 347
John W. Moore
25. Identification of Steroid Hormone-Regulated Genes
in Breast Cancer........................................................................... 363
Bruce R. Westley and Felicity E. B. May
26. Sequencing of the Tumor Suppressor Gene TP 53............................389
Barbro Linderholm, Torbjörn Norberg, and Jonas Bergh
27. Expression Profiling Using cDNA Microarrays.................................. 403
Chris Jones, Peter Simpson, Alan Mackay, and Sunil R. Lakhani
28. Gene Expression Analysis Using Filter cDNA Microarrays............... 415
Peter Simpson, Chris Jones, Alan Mackay, and Sunil R. Lakhani
PART V STUDYING CANCER CELL BEHAVIOR IN VITRO AND IN VIVO
29. Methods to Analyze the Effects of the Urokinase System
on Cancer Cell Adhesion, Proliferation, Migration,
and Signal Transduction Events .................................................... 427
Ute Reuning, Manfred Schmitt, Birgit Luber, Veronika Beck,
and Viktor Magdolen
30. Phospho-Specific Antibodies as a Tool to Study In Vivo Regulation
of BRCA1 After DNA Damage........................................................ 441
Kum Kum Khanna, Magtouf Gatei, and Gordon Tribbick
31. Models of Hormone Resistance In Vitro and In Vivo........................ 453
Jennifer MacGregor Schafer and V. Craig Jordan
32. Generation of Genetically Modified Embryonic Stem Cells
for the Development of Knockout Mouse Animal Model Systems .....465
Stephen D. Robinson, Stephen Wilson,
and Kairbaan M. Hodivala-Dilke
33. In Vivo Xenograft Models of Breast Cancer Metastasis..................... 479
Ursula Valentiner, Susan A. Brooks, and Udo Schumacher
34. Neoadjuvant Endocrine Therapy Models.......................................... 489
Juliette Murray, William R. Miller, and J. Michael Dixon
35. Primary Mouse Endothelial Cell Culture for Assays of Angiogenesis ... 503
Louise E. Reynolds and Kairbaan M. Hodivala-Dilke
Index ............................................................................................................ 511