Table Of ContentM E T H O D S I N M O L E C U L A R M E D I C I N ETM
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Melanoma Techniques and 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 ETM
John M. Walker, Series Editor
69. Gene Therapy Protocols, 2nd ed., edited 53. Renal Cancer: Methods and Protocols,
byJeffrey R. Morgan, 2002 edited by Jack H. Mydlo, 2001
68. Molecular Analysis of Cancer, edited by 52. Atherosclerosis: Experimental Methods and
Jacqueline Boultwood and Carrie Fidler, 2002 Protocols,edited by Angela F. Drew, 2001
67. Meningococcal Disease: Methods and 51. Angiotensin Protocols, edited by Donna
Protocols,edited by Andrew J. Pollard and H. Wang, 2001
Martin C. J. Maiden, 2001
50. Colorectal Cancer: Methods and
66. Meningococcal Vaccines: Methods and Protocols, edited by Steven M. Powell,
Protocols,edited by Andrew J. Pollard and 2001
Martin C. J. Maiden, 2001
49. Molecular Pathology Protocols, edited by
65. Nonviral Vectors for Gene Therapy: Anthony A. Killeen, 2001
Methods and Protocols, edited by Mark A,
48. Antibiotic Resistance Methods and
Findeis,2001
Protocols,edited by Stephen H. Gillespie,
64. Dendritic Cell Protocols, edited by Stephen 2001
P. Robinson and Andrew J. Stagg, 2001
47. Vision Research Protocols,edited by P.
63. Hematopoietic Stem Cell Protocols, Elizabeth Rakoczy, 2001
edited by Christopher A. Klug and Craig T.
46. AngiogenesisProtocols,edited byJ.
Jordan,2001
Clifford Murray, 2001
62. Parkinson’s Disease: Methods and Protocols,
45. Hepatocellular Carcinoma: Methods and
edited by M. Maral Mouradian, 2001
Protocols,edited byNagy A. Habib, 2000
61. Melanoma Techniques and Protocols:
44. Asthma: Mechanisms and Protocols, edited by
Molecular Diagnosis, Treatment, and
K. Fan Chung and Ian Adcock, 2001
Monitoring,edited by Brian J. Nickoloff, 2001
43. Muscular Dystrophy: Methods and
60. Interleukin Protocols, edited by Luke A. J. Protocols,edited byKatherine B. Bushby
O’Neill and Andrew Bowie, 2001 and Louise Anderson, 2001
59. Molecular Pathology of the Prions, edited 42. Vaccine Adjuvants:Preparation Methods
byHarry F. Baker, 2001 and Research Protocols, edited by Derek T.
O’Hagan, 2000
58. Metastasis Research Protocols: Volume 2,
Cell Behavior In Vitro and In Vivo,edited 41. Celiac Disease: Methods and Protocols,
bySusan A. Brooks and Udo Schumacher, edited by Michael N. Marsh, 2000
2001
40. Diagnostic and Therapeutic Antibodies,
57. Metastasis Research Protocols: Volume 1, edited by Andrew J. T. George and
Analysis of Cells and Tissues, edited by Susan Catherine E. Urch, 2000
A. Brooks and Udo Schumacher, 2001
39. Ovarian Cancer: Methods and Protocols, edited
56. Human Airway Inflammation: Sampling byJohn M. S. Bartlett, 2000
Techniques and Analytical Protocols, edited
38. Aging Methods and Protocols, edited by
byDuncan F. Rogers and Louise E. Donnelly,
Yvonne A. Barnett and Christopher R.
2001
Barnett, 2000
55. Hematologic Malignancies: Methods and
37. Electrochemotherapy, Electrogenetherapy,
Protocols,edited by Guy B. Faguet, 2001
and Transdermal Drug Delivery:
54. Mycobacterium tuberculosis Protocols, edited Electrically Mediated Delivery of Molecules to
byTanya Parish and Neil G. Stoker, 2001 Cells, edited by Mark J. Jaroszeski, Richard
Heller, and Richard Gilbert, 2000
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 ETM
Melanoma
Techniques
and Protocols
Molecular Diagnosis, Treatment,
and Monitoring
Edited by
Brian J. Nickoloff, ,
MD PhD
Loyola University Medical Center, Maywood, IL
Foreword by
Leroy Hood, MD, PhD
President and Director, Institute for Systems Biology
Seattle, WA
Humana Press Totowa, New Jersey
© 2001 Humana Press Inc.
999 Riverview Drive, Suite 208
Totowa, New Jersey 07512
All rights reserved.
No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any
means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permis-
sion from the Publisher. Methods in Molecular Medicine™ is a trademark of The Humana Press Inc.
All authored papers, comments, opinions, conclusions, or recommendations are those of the author(s), and
do not necessarily reflect the views of the publisher.
This publication is printed on acid-free paper. (cid:39)
ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials.
Cover illustrations: Clinical appearance of advanced cutaneous melanoma (upper left corner). Microarray
demonstrating genes that are overexpressed (red boxes) and underexpressed (green boxes) in melanoma
tumor cells relative to normal skin (lower left box). Vaccine approach to treating metastatic melanoma using
antigen-pulsed dendritic cells injected either intradermally or intranodally (center box). Histological appear-
ance of cutaneous melanoma with malignant cells arising in epidermis and invading dermis (upper right
panel). Quantitative reverse transcriptase/polymerase chain reaction detecting cytokines present in meta-
static melanoma lesion (lower right panel). Illustrations courtesy of Dr. Brian J. Nickoloff.
Cover artwork and layout: Heide Bauer and Brian Bonish
Series design: Patricia F. Cleary
For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact
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Library of Congress Cataloging in Publication Data
Main entry under title:
Methods in molecular medicine™.
Melanoma: methods and protocols / edited by Brian J. Nickoloff
p. cm. —(Methods in molecular medicine; 61)
Includes bibliographical references and index.
ISBN 0-89603-684-7 (alk. paper)
1. Melanoma—Laboratory manuals. I. Nickoloff, Brian J., 1953– II. Series.
RC280.M37 M447 2001 00-063353
616.99'477–dc21
Foreword
This book is about melanoma—its biology, immunology, and pathology, as
well as the initial use of powerful genomic tools to study its fundamental molecu-
lar and genetic characteristics. The study of cancer will be profoundly impacted
by the Human Genome Project. I would like to discuss some of these changes.
The first draft of the human genome sequence was announced in June 2000, and
we have just scratched the surface of the changes it will engender in medicine. A
relevant question is what are the long-term effects of the Human Genome Project
for medicine? I would argue that there are three, and each of these three point
toward the view that systems biology will dominate biology and medicine of the
21st century. First, the Human Genome Project introduced a new type of sci-
ence—discovery science. Discovery science takes a biological system (e.g., the
genome) and defines all of its elements (e.g., the sequences of the 24 human chro-
mosomes). Thus, it creates a rich infrastructure from which the classical hypoth-
esis-driven science can be done more effectively. The effective integration of
discovery- and hypothesis-driven science is a key for systems approaches to biol-
ogy and medicine.
Second, the Human Genome Project has provided a “periodic table of life.”
This genetics parts list includes the identification of the 40,000 to 80,000 or so
human genes and access to the genetic polymorphisms that differentiate most
humans from one another (on average, one change every 500 nucleotides). Most
of these polymorphisms have no effect on the human phenotype, but a few pre-
dispose to late onset diseases such as cancer, cardiovascular disease, and many
immunological diseases. Identifying the relevant polymorphisms and under-
standing their consequences will transform medicine of the 21st century, mov-
ing us initially from reactive to predictive medicine, and ultimately to preventive
medicine.
Finally, the Human Genome Project has catalyzed a series of paradigm
changes in how we view biology and medicine that lead us to conclude that sys-
tems approaches will revolutionize biology and medicine of the 21st century.
• Biology is an informational science. This leads to the view biological
information is complex and hierarchal (DNA(cid:65)RNA(cid:65)protein(cid:65)protein
interactions(cid:65)informational pathways(cid:65)informational networks(cid:65)etc.).
The challenge is to capture and integrate information from these differ-
ent levels so that systems approaches (see below) can be applied.
v
vi Foreword
• High-throughput biological analyses are transforming biology. DNA
sequencing, DNA arrays, genotyping, and now proteomics, are examples
of high-throughput biology that can gather enormous amounts of infor-
mation. High-throughput biology is key to systems biology because sys-
tems biology requires that all the elements in a biological system be
defined before they can be understood.
• Computer science and applied mathematics are critical to capturing, stor-
ing, analyzing, graphically displaying, modeling, and transmitting biologi-
cal information. Powerful new computational tools have been developed
over the past 10 years. Being able to handle and analyze most amounts of
biologic information is central to systems biology.
• Model organisms are the Rosetta Stones for deciphering biological sys-
tems. The Human Genome Project had underscored the striking unity of
life; that is, organisms as diverse as fruit flies or humans share many of
the same informational pathways and networks. Because systems must
be perturbed in order to understand how they function, and because it is
generally impossible to experiment on humans, it becomes important to
study biological systems in model organisms.
What is systems biology? Let us consider the following metaphor. Sup-
pose you wished to understand how Manhattan functions as a city. You would
have to gather information about its infrastructure (roads, buildings, docks, com-
munication channels, etc.), power requirements, the movements of people and
animals, the nature of communications, etc. All of these data would have to, in
some manner, be integrated and modeled before ultimately predicting the
behavior of the city. So it is with systems approaches to biological systems and
networks—information must be gathered from the various hierarchal biological
levels, integrated, and ultimately modeled mathematically so that both the struc-
ture and systems (emergent) properties of the biological system can be predicted.
A cancer such as melanoma arises as a consequence of mutations in par-
ticular informational pathways (e.g., cell cycle, DNA repair, developmental,
etc.). These mutations either may be inherited (germline) or may arise during
the lifetime of the individual (somatic). Two conclusions can be drawn from
a systems view of cancer. First, mutations in different informational path-
ways may give rise to the same general category of tumor (e.g., melanoma).
However, the mutations in each distinct pathway will initiate distinct sub-
types of tumors. Thus, for each general category of cancer, one must define
the distinct subtypes, for they may give rise to tumors with different prog-
noses (e.g., relatively benign or rapidly metastatic), perhaps ultimately requir-
Foreword vii
ing distinct approaches to therapy. This process of identifying the subtypes of
a particular tumor is termed stratification.
Second, many tumors progress through additional mutations and become
more invasive and malignant as time passes. So, for a given tumor subtype, it
is important to know how far progression has advanced.
A systems approach to cancer allows one to identify diagnostic markers
for stratification and progression, potential therapeutic targets (generally on
the cell surface), and molecules (genes) that may play a direct role in the neo-
plastic process itself. The idea is to use the high-throughput tools of genomics
(DNA sequencing, DNA arrays) or proteomics to examine the changes in gene
expression patterns in normal cells and cancer cells and in many different types
of cancer cells of the same type (e.g., melanoma). These comparative analyses
will reveal the differences (quantitative and qualitative) in gene expression pat-
terns between normal and cancer cells, will facilitate the stratification of can-
cers of the same general type, and, with the analysis of multiple tumor samples,
permit the analysis of changes in gene expression patterns as a particular sub-
type of tumor progresses. From these subtractive or comparative analyses,
diagnostic markers for stratification and progression can be identified. These
markers may be secreted (detectable with antibodies), on the cell surface
(detectable with antibodies or PCR), or in the cell (detectable by PCR). In a
similar view, unique cell surface molecules may be identified on cancer cells
that could serve as targets for therapeutic intervention (e.g., immunologic).
Finally, subtractive analyses of the type described above may reveal
interesting candidate genes for the neoplastic process. Ultimately, the sys-
tems approach will allow these genes (proteins) to be placed in the context of
their informational pathways and through an understanding of these pathways,
superior therapeutic strategies may be designed.
Systems approaches to cancer are already impacting the field (1,2). Through-
out Melanoma Techniques and Protocols: Molecular Diagnosis, Treatment, and
Monitoring, we can see the initial forays of genomics and systems approaches into
the study of melanoma. More will be coming very quickly. It appears very likely
that systems approaches to diseases will transform the practice of medicine in the
early part of the 21st century, moving us irrevocably toward preventive medicine.
Leroy Hood, MD, PhD
1. Golub TR, Slonim DK, Tamayo P, Huard C, Gaasenbeek M, Mesirov JP, Coller H, Loh ML, Downing
JR, Caligiuri MA, Bloomfield CD, and Lander ES. (1999) Molecular Classification of Cancer: Class
Discovery and Class Prediction by Gene Expression Monitoring. Science286,531–536.
2. Bittner M, Melzer P, Chen Y, Jiang Y, Seftor E, Hendrix M, Trent J. (2000) Molecular Classification
of Cutaneous Malignant Melanoma by Gene Expression Profiling. Nature 406, 536–540.
Preface
Finding a cure for melanoma will be dependent on a greater understanding
of the complex molecular interactions that begin in the skin, prior to melanoma’s
ruthless spread and distribution at distant metastatic sites. Melanoma is one of the
most virulent forms of cancer that is now at epidemic proportions worldwide. It is
increasingly responsible for deaths and loss of livelihood for countless individuals
in their second, third, or fourth decades. With the completion of the sequencing of
the 3.1 billion nucleotides that comprise the Human Genome Project, it is now
possible to intensify efforts at deciphering the biochemical cascade of events in
melanoma, beginning with the relevant genes and the proteins they encode. One
of the main challenges is not only to come to grips with the rapid pace of techno-
logical advances, but to integrate the new tools of modern genomics with a sound
biological perspective that has clinical relevance.
The aim of Melanoma Techniques and Protocols: Molecular Diagnosis,
Treatment, and Monitoring a volume in the Methods in Molecular Medicine™
series, is to provide a comprehensive and up-to-date summary of the most impor-
tant advances in the field pertaining to melanoma. Each author was instructed to
provide clear-cut experimental protocols (including detailed “Notes” on points
that often are not spelled out in regular publications), to ensure that investigators
outside the field could successfully use these techniques in their own laboratories.
As can be appreciated by surveying the table of contents, a highly diverse group of
authors were enlisted to provide expert reviews in their respective areas including
perspectives in clinical medicine, molecular biology, tumor immunology, and
pathology. Authors who could write from first-hand experience were selected to
ensure that relevant expertise and direct knowledge of the technique and literature
were presented for the reader.
This book is divided into four major categories including: Biology, Diagno-
sis, Treatment, and Monitoring of Patients with Melanoma. For each category,
“cutting-edge” techniques are presented, and discussed within a biological con-
text so that the complexities of melanoma may be better understood by each reader
who completes the entire book. Though it is true that only modest clinical
improvements have been made for melanoma patients, despite the exhilarating
progress in the dissection of many molecular mysteries, it is hoped that this book
will be seen as providing forward-thinking perspectives and experimental pro-
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