Table Of ContentBreast 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™
John M. Walker, SERIES EDITOR
112255.Myeloid Leukemia:Methods and Protocols, 110044. Stroke Genomics: Methods and Reviews,
edited by Harry Iland, Mark Hertzberg, edited by Simon J. Read and David Virley,
and Paula Marlton, 2006 2004
112244.Magnetic Resonance Imaging:Methods and 110033.Pancreatic Cancer: Methods and Protocols,
Biological Applications, edited by Pottumarthi edited by Gloria H. Su, 2004
V. Prasadi, 2006 110022.Autoimmunity: Methods and Protocols,
112233.Marijuana and Cannabinoid Research: edited by Andras Perl, 2004
Methods and Protocols, edited by Emmanuel 110011.Cartilage and Osteoarthritis: Volume 2,
S. Onaivi, 2006 Structure and In Vivo Analysis, edited by
112222.Placenta Research Methods and Protocols: Frédéric De Ceuninck, Massimo Sabatini,
Volume 2, edited by Michael J. Soares and and Philippe Pastoureau, 2004
Joan S. Hunt, 2006 110000.Cartilage and Osteoarthritis: Volume 1,
112211.Placenta Research Methods and Protocols: Cellular and Molecular Tools, edited by
Volume 1, edited by Michael J. Soares and Massimo Sabatini, Philippe Pastoureau, and
Joan S. Hunt, 2006 Frédéric De Ceuninck, 2004
112200.Breast Cancer Research Protocols,edited by 9999.Pain Research: Methods and Protocols,
Susan A. Brooks and Adrian Harris, 2006 edited by David Z. Luo, 2004
111199.Human Papillomaviruses: Methods and 9988.Tumor Necrosis Factor: Methods and
Protocols,edited by Clare Davy and John Protocols, edited by Angelo Corti and Pietro
Doorbar, 2005 Ghezzi, 2004
111188.Antifungal Agents: Methods and Protocols, 9977.Molecular Diagnosis of Cancer: Methods and
edited by Erika J. Ernst and P. David Rogers, Protocols, Second Edition, edited by Joseph E.
2005 Roulston and John M. S. Bartlett, 2004
111177.Fibrosis Research: Methods and Protocols, 9966.Hepatitis B and D Protocols: Volume 2,
edited by John Varga, David A. Brenner, Immunology, Model Systems, and Clinical
and Sem H. Phan, 2005 Studies, edited by Robert K. Hamatake and
111166.Inteferon Methods and Protocols,edited by Johnson Y. N. Lau, 2004
Daniel J. J. Carr, 2005 9955.Hepatitis B and D Protocols: Volume 1,
111155.Lymphoma: Methods and Protocols, edited by Detection, Genotypes, and Characterization,
Timothy Illidge and Peter W. M. Johnson, 2005 edited by Robert K. Hamatake and Johnson
111144.Microarrays in Clinical Diagnostics,edited by Y. N. Lau, 2004
Thomas O. Joos and Paolo Fortina, 2005 9944.Molecular Diagnosis of Infectious Diseases,
111133.Multiple Myeloma: Methods andProtocols, Second Edition, edited by Jochen Decker and
edited by Ross D. Brown and P. Joy Ho, 2005 Udo Reischl, 2004
111122.Molecular Cardiology: Methods andProtocols, 9933.Anticoagulants, Antiplatelets, and
edited by Zhongjie Sun, 2005 Thrombolytics,edited by Shaker A. Mousa,
111111.Chemosensitivity: Volume 2, In Vivo 2004
Models, Imaging, and Molecular Regulators, 9922.Molecular Diagnosis of Genetic Diseases,
edited by Rosalyn D. Blumethal, 2005 Second Edition, edited by Rob Elles and Roger
111100.Chemosensitivity: Volume 1, In Vitro Mountford, 2004
Assays, edited by Rosalyn D. Blumethal, 2005 9911.Pediatric Hematology: Methods and
110099.Adoptive Immunotherapy: Methods and Protocols,edited by Nicholas J. Goulden
Protocols, edited by Burkhard Ludewig and and Colin G. Steward, 2003
Matthias W. Hoffman, 2005 9900.Suicide Gene Therapy: Methods and
110088.Hypertension: Methods and Protocols, Reviews,edited by Caroline J. Springer, 2004
edited by Jérôme P. Fennell and Andrew 8899.The Blood–Brain Barrier: Biology and
H. Baker, 2005 Research Protocols, edited by Sukriti Nag, 2003
110077.Human Cell Culture Protocols, Second 8888.Cancer Cell Culture: Methods and Protocols,
Edition, edited by Joanna Picot, 2005 edited bySimon P. Langdon, 2003
110066.Antisense Therapeutics, Second Edition, 8877.Vaccine Protocols, Second Edition, edited by
edited by M. Ian Phillips, 2005 Andrew Robinson, Michael J. Hudson, and
110055.Developmental Hematopoiesis: Methods Martin P. Cranage, 2003
and Protocols, edited by Margaret H. 8866.Renal Disease: Techniques and Protocols,
Baron, 2005 edited by Michael S. Goligorsky, 2003
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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Gene Amplification in Breast Tumor Tissues," by Patricia Gorman and Rebecca Roylance.
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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
Contributors
PETER J. BARRETT-LEE • Oncology Unit, Velindre NHS Trust, Cardiff, UK
JOHN M. S. BARTLETT • Division of Cancer Sciences and Molecular
Pathology, University Department of Surgery, Endocrine Cancer Group
and HER2 Reference Laboratory, University of Glasgow, Glasgow Royal
Infirmary, Glasgow, UK
VERONIKA BECK • Clinical Research Unit, Department of Obstetrics
and Gynaecology, Technical University of Munich, Munich, Germany
JONAS BERGH • Radiumhemmet, Karolinska Institute and Hospital,
Stockholm, Sweden
JAN K. BLANCATO • Institute for Molecular and Human Genetics,
Georgetown University Medical Center, Washington, DC
IRENE BOEDDINGHAUS • Cape Town, South Africa
SUSAN A. BROOKS• School of Biological and Molecular Sciences, Oxford
Brookes University, Headington, Oxford, UK
IAN D. BULEY • Department of Cellular Pathology, The John Radcliffe
Hospital, Oxford, UK
TRACEY M. CARTER • School of Biological and Molecular Sciences, Oxford
Brookes University, Headington, Oxford, UK
CELIA CHAO • Division of Surgical Oncology, Department of Surgery,
University of Texas Medical Branch, Galveston, TX
ROBERT B. DICKSON • Institute for Molecular and Human Genetics,
Georgetown University Medical Center, Washington DC
J. MICHAEL DIXON • Edinburgh Breast Unit, Western General Hospital,
Edinburgh, UK
ANTHONY DOUGLAS-JONES • Department of Pathology, University of Wales
College of Medicine, Cardiff, UK
MIRIAM V. DWEK • Department of Applied and Molecular Biosciences,
School of Biosciences, University of Westminster, London, UK
AMANDA FORSYTH• Division of Cancer Sciences and Molecular Pathology,
University Department of Surgery, Endocrine Cancer Group and HER2
Reference Laboratory, University of Glasgow, Glasgow Royal Infirmary,
Glasgow, UK
STEPHEN B. FOX • Nuffield Department of Clinical Laboratory Sciences,
University of Oxford, John Radcliffe Hospital, Oxford, UK
MAGTOUF GATEI • Signal Transduction Laboratory, Cancer and Cell Biology
Division, The Queensland Institute of Medical Research, Queensland,
Australia
xi
