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Ovarian Cancer
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 TM
M E T H O D S I N M O L E C U L A R B I O L O G Y™
Ovarian Cancer
Methods and Protocols
Edited by
John M. S. Bartlett
Department of Surgery, University of Glasgow, Glasgow, Scotland
Humana Press Totowa, New Jersey

iv
© 2000 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 permission from the Publisher. Methods in Molecular Biology™ 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.
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Cover design by Patricia F. Cleary
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Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1
Library of Congress Cataloging in Publication Data
Ovarian cancer : methods and protocols / edited by John M. S. Bartlett.

p.; cm.--(Methods in molecular medicine; 39)
Includes bibliographical references and index.
ISBN 0-89603-574-3 (alk. paper)
1. Ovaries--Cancer--Molecular aspects--Research--Methodology. 2. Tumor
markers--Research--Methodology. 3. Ovaries--Cancer--Genetic
aspects--Research--Methodology. I. Bartlett, John M.S. II. Series
[DNLM: 1. Ovarian Neoplasms--genetics. 2. Genetics, Biochemical. 3. Models,
Biological. 4. Tumor Markers, Biological. WP 322 O9647 2000]
RC280.O8 O926 2000
616.99' 465--dc21
00-040718
CIP
Preface
If there is one aspect of current cancer research that represents a major chal-
lenge in both novice and experienced researchers, it is the rapid advance in our
understanding of the disease. Researchers can be required to switch from analysis
of gene expression to kinetics of protein activation, from genetic studies to the
analysis of protein funtion. Cancers are highly complex disease systems and
researchers aiming to understand the functioning of cancer systems require access
to a wide range of laboratory techiques from a broad range of research disciplines.
Increasingly, however, published methods are incomplete or refer back to a series
of previous publications each containing only a small part of the complete proto-
col. The aim of Ovarian Cancer: Methods and Protocols is to provide for ovarian
cancer researchers in the first instance, a laboratory handbook that will facilitate
research into cancer systems by providing a series of expert protocols, with proven
efficacy, across a broad range of technical expertise. Thus, there are sections on
tumor genetics and cellular signal transduction, as well as sections on apoptosis
and RNA analysis.
The value of Ovarian Cancer: Methods and Protocols to the ovarian cancer
researcher will, I trust, be considerably enhanced by (1) the provision of a series
of overviews relating to the biology, diagnosis, and treatment of this important
neoplasm, and (2) the provision of a series of technical overviews introducing
each part that provides an expert review of the applications and pitfalls of the
various techniques included.
Ovarian Cancer: Methods and Protocols aims to provide a resource for both
the novice scientist/clinician coming to grips with laboratory-based research for
the first time, as well as for those more experienced investigators seeking to diver-
sify their technological base. Often, we are constrained less by our ideas than by
our abilities to carry forward those ideas using different technologies.
No volume can exhaustively cover every aspect of biological research, and
there will be gaps that one or another research group will identify. Each section
could readily be expanded (and in some cases has been) into a book in its own
right. However, I have sought to include a spectrum of techniques that will allow
the acquisition of key skills in each area covered. The aim is to give the researcher
an understanding of the technical issues covered in each section such that they can
then extrapolate their expertise into salient techniques in these areas.
As with all volumes in the Methods in Molecular Medicine series, clear
instructions in the perfomance of the various protocols is supplemented by addi-
tional technical notes that provide valuable insights into the working of the tech-
nique in question. Though often brief, these notes provide essential details that
allow a successful outcome.
I would like to express my gratitude to all those who have contributed to this
volume, who have been patient over the period required to collate their contribu-
v
vi Preface
tions. I am also grateful to Professor John Walker for his encouragement and guidance
as series editor. Finally, I would like to thank my wife, Dorothy for patiently proof
reading manuscripts and for being understanding on the many occasions when I
arrived home late during the preparation of this volume.
John M. S. Bartlett
vi
vii
Contents
Preface ............................................................................................................. v
Contributors ..................................................................................................... ix
PART I. INTRODUCTION TO OVARIAN CANCER
1 The Epidemiology of Ovarian Cancer
Emily Banks ......................................................................................................... 3
2 Familial Ovarian Cancer
Ronald P. Zweemer and Ian J. Jacobs ........................................................... 13
3 The Molecular Pathogenesis of Ovarian Cancer
S. E. Hillary Russell .......................................................................................... 25
4 Alterations in Oncogenes, Tumor Suppressor Genes,
and Growth Factors Associated with Epithelial Ovarian Cancers
Robert C. Bast, Jr. and Gordon B. Mills ........................................................ 37
5 Pathological Assessment of Ovarian Cancer
Alistair R. W. Williams ...................................................................................... 49
6 Tumor Markers in Screeening for Ovarian Cancer
Steven J. Skates, Ian J. Jacobs, and Robert C. Knapp ............................... 61
7 Primary Surgical Management of Ovarian Cancer
Dennis S. Chi and William J. Hoskins ............................................................ 75
8 Recent Insights into Drug Resistance in Ovarian Cancer
Thomas C. Hamilton and Steven W. Johnson .............................................. 89
PART II. TUMOR MARKERS
9 Markers of Tumor Burden: An Overview
Joseph E. Roulston ........................................................................................ 109
10 Bioactive Interleukin-6 Levels in Serum and Ascites as
a Prognostic Factor in Patients with Epithelial Ovarian Cancer
Günther Gastl and Marie Plante .................................................................... 121
11 ELISA-Based Quantification of p105 (c-erb-B2, HER2/neu) in Serum
of Ovarian Carcinoma
Harald Meden, Arjang Fattahi-Meibodi, and Dagmar Marx ....................... 125
12 Enzyme Immunoassay of Urinary β-core Fragment of Human Chorionic
Gonadotropin as a Tumor Marker for Ovarian Cancer
Ryuichiro Nishimura, Tamio Koizumi, Hiranmoy Das,
Masayuki Takemori, and Kazuo Hasegawa ............................................ 135
PART III. MODEL SYSTEMS
13 Ovarian Cancer Models: Technical Review
Simon P. Langdon, Joanne Edwards, and John M. S. Bartlett ................. 145
vii
viii Contents
14 Establishment of Ovarian Cancer Cell Lines

Simon P. Langdon and Sandra S. Lawrie .................................................... 155
15 Subcloning of Ovarian Cancer Cell Lines
Thomas W. Grunt ............................................................................................ 161
16 Culture and Characterization of Human Ovarian Surface Epithelium
Nelly Auersperg and Sarah L. Maines-Bandieria ....................................... 169
17 MTT Growth Assays in Ovarian Cancer
Daniel M. Spinner ............................................................................................ 175
18 In Vitro Invasion Assays
Setsuko K. Chambers .................................................................................... 179
19 p53 Transfectants in Ovarian Cancer
Faina Vikhanskaya and Massimo Broggini ................................................. 187
20 Estrogen-Responsive Ovarian Cancer Xenografts
Alison A. Ritchie and Simon P. Langdon .................................................... 193
21 The Use of Matrigel in the Establishment of Ovarian Carcinoma
Cell Lines as Xenografts
Peter Mullen and Simon P. Langdon ............................................................ 199
PART IV. CYTOGENETICS
22 Cytogenetics of Ovarian Cancer: Technical Overview
Thomas Liehr .................................................................................................. 207
23 Interphase Cytogenetics in Frozen Ovarian Tumor Tissue
Thomas Liehr .................................................................................................. 215
24 Interphase Cytogenetics in Paraffin-Embedded Ovarian Tissue
Susann Neubauer and Thomas Liehr .......................................................... 223
25 Rapid Identification of Chromosomes Using Primed
In Situ Labeling (PRINS)
GopalRao V. N. Velagaleti .............................................................................. 229
26 Gene Amplification of c-ermB-2 Detected by FISH
S. Robert Young, Wei-Hua Liu, and Zong-Ren Wang ................................ 237
27 Chromosome Microdissection for Detection of Subchromosomal
Alterations by FISH
Xin-Yuan Guan and Jeffrey M. Trent ............................................................ 247
28 Quantitation of FISH Signals in Archival Tumors
Amanda D. Watters and John M. S. Bartlett ................................................ 253
29 Comparative Genomic Hybridization for the Analysis of Unbalanced
Chromosomal Abnormalities in Ovarian Cancer
Lucy J. Curtis .................................................................................................. 261
PART V. MOLECULAR GENETIC IMBALANCES IN OVARIAN TUMORS
30 Molecular Genetics of Ovarian Cancer: A Technical Overview
William Foulkes and Andrew N. Shelling .................................................... 273
31 Extraction of DNA from Microdissected Archival Tissues
James J. Going ............................................................................................... 291
ix
Contents
32 RFLP Molecular Analysis of the Urokinase-Type Plasminogen

Activator Gene
Bernd Muhlenweg, Andreas Schnelzer, Beyhan Türkmen,
Ernst Lengyel, Ute Reuning, Henner Graeff,
Manfred Schmitt, and Viktor Magdolen ................................................... 299
33 PCR Microsatellite Analysis of LOH in Ovarian Tumors
Jayne Devlin and Margaret A. Knowles ....................................................... 307
34 Molecular Genetic Analysis of Flow-Sorted Ovarian
and Microdissected Ovarian Tumor Cells: Improved Detection
of Loss of Heterozygosity
Edwin C. A. Abeln and Willem E. Corver ..................................................... 315
35 SSCP and Sequence Analysis of p53 Mutation
in Ovarian Tumors
Anil K. Sood and Richard E. Buller .............................................................. 323
36 Multiplex PCR (MPCR) Screening Can Detect Small Intragenic
p53 Deletion and Insertion Mutations
Ingo B. Runnebaum and Shan Wang-Gohrke ............................................. 329
37 Transfer of Human Chromosome 3 to an Ovarian Carcinoma
Cell Line Identifies Regions Involved in Ovarian Cancer
Paola Rimessi and Francesca Gualandi ...................................................... 337
38 Double-and Competitive-Differential PCR
for Gene Dosage Quantitation
Burkhard Brandt, Alf Beckmann, Antje Roetger,
and Frank Gebhardt ................................................................................... 347
39 One Dimensional Genome Scanning—Detection of Genomic
Changes in Ovarian Carcinoma
Anthony Magliocco and Murray Brilliant ..................................................... 357
40 Mapping of Tumor Suppressor Genes in Ovarian Cancer
Ian G. Campbell and Emma J. Bryan ........................................................... 365
41 Detection of the Replication Error Phenotype in Ovarian
Cancer—PCR Analysis of Microsatellite Instability
Gillian L. Hirst and Robert Brown ................................................................ 375
42 Immunostaining Human Paraffin-Embedded Sections
for Mismatch Repair Proteins
Melanie Mackean and Robert Brown ........................................................... 383
43 Quantitative PCR Detection of c-erbB-2 Gene Amplification
Karl Dobianer, Michael Medl, and Jürgen Spona ....................................... 389
PART VI. MRNA ANALYSIS
44 mRNA Anaysis: A Technical Overview
John M. S. Bartlett .......................................................................................... 399
45 In Situ Hybridization Detection of TGR-β mRNA
Anders Gobl and Rudi Henriksen ................................................................. 411
46 mRNA Detection by In Situ RT-PCR
H. Anne Waller and A. Kay Savage .............................................................. 417
x Contents
47 RNase Protection Assay Analysis of mRNA for TGFβ1–3 in

Ovarian Tumors
John M. S. Bartlett .......................................................................................... 431
48 RT-PCR Quantitation of HSP60 mRNA Expression
Raymond P. Perez, Lakshmi Pendyala, Zeyad Elakawi,
and Mahmoud Abu-hadid .......................................................................... 439
49 The Effects of Butyrate and the Role of c-myc in N.1 Ovarian
Carcinoma Cells Determined by Northern Blotting
Georg Krupitza ................................................................................................ 449
50 Differential Display
Grant C. Sellar, Genevieve J. Rabiasz, Barbara Smith,
and Jennifer Southgate ............................................................................. 459
PART VII. PROTEIN EXPRESSION
51 Measurement of Protein Expression: A Technical Overview
Jonathan R. Reeves and John M. S. Bartlett .............................................. 471
52 Measurement of IGF-1 Receptor Content in Tissues
and Cell Lines by Radioimmunoassay (RIA) and ELISA Techniques
Eberhard P. Beck, Laura Sciacca, Guiseppe Pandini,
Wolfram Jaeger, and Vincenzo Pezzino .................................................. 485
53 Radioreceptor Measurement of ER/PR
Giovanni Scambia, Gabriella Ferrandina, G. D'Agostino,
A. De Dilectis, and Salvator Mancuso ..................................................... 493
54 nm23 Protein Expression by Western Blotting in Patients with Epithelial
Ovarian Carcinoma
Giovanni Scambia, Maria Marone, Gabriella Ferrandina,
Gabriella Macchia, and Salvatore Mancuso............................................ 499
55 Detection and Quantitation of Matrix Metalloproteases by Zymography
Thomas M. Leber and Rupert P. M. Negus .................................................. 509
56 Simultaneous Flow Cytometric Detection of DNA
and Cellular Protein Molecules
Willem E. Corver and Cees J. Cornelisse .................................................... 515
57 c-erbB-2 Immunohistochemistry in Paraffin Tumors
Gamal H. Eltabbakh ........................................................................................ 529
58 Quantitative Immunohistochemistry of Estrogen and Progesterone
Receptor Positivity in Ovarian Tumors: A Rapid and Reliable
Stereological Approach
Gerrit A. Meijer, Paul J. van Diest, Jane Brugghe,
and Jan P. A. Baak ...................................................................................... 535
59 Radioimmunohistochemistry: Quantitative Analysis of Cell Surface
Receptors in Frozen Sections
Jonathan R. Reeves........................................................................................ 545
PART VIII. SIGNAL TRANSDUCTION
60 Signal Transduction: A Technical Overview
M. Jane Arboleda and Dennis J. Slamon ..................................................... 555
xi
61 Immunoprecipitation to Determine JAK Kinase Activation in Response

to Interleukins in Ovarian Cancer
Takashi Murata ................................................................................................ 567
62 Detection of ErbB Receptor Family Tyrosyl Phosphorylation in Ovarian
Carcinoma Cells
Anne W. Hamburger ....................................................................................... 571
63 Phosphotyrosine Kinase Assays as a Prescreen for Inhibitors of EGFr
Joanne Edwards and John M. S. Bartlett .................................................... 577
64 Direct Kinase Assay Screening for Inhibitors of MAP Kinase
Joanne Edwards and John M. S. Bartlett .................................................... 583
65 Phophatidylinositol Kinase Activity in Ovarian Cancer Cells
Atsushsi Imai, Hiroshi Takagi, Atshushi Takagi,
and Teruhiko Tamaya ................................................................................ 589
66 Induction of c-fos Gene Expression by Urokinase-Type
Plasminogen Activator in Human Ovarian Carcinoma Cells
Inna Dumler ..................................................................................................... 597
67 Phosphotyrosine Phosphatase Activity in Ovarian Carcinoma Cells:
Stimulation by GnRH in Plasma Membrane
Atsushsi Imai, Shinji Horibe, Atsushi Takagi, and Teruhiko Tamaya ..... 601
68 Phosphatidylinositol 4-Kinase Assay in Ovarian Carcinoma Cells
Seiji Isonishi, Aiko Okamoto, Kazunori Ochiai, Yoshio Saito,
and Kazuo Umezawa .................................................................................. 607
69 Calcium Mobilization in Ovarian Cancer Cells in Response
to Lysophospholipids
Yan Xu and Derek S. Damron ........................................................................ 611
70 Compartmentalized Protein Kinase C Activation
in Ovarian Carcinoma Cells
Alakananda Basu and Giridhar R. Akkaraju ............................................... 621
71 A Novel and Simple Method to Assay the Activity of Individual
Protein Kinases in a Crude Tissue Extract
Basem S. Goueli, K. Hsiao, and Said A. Goueli .......................................... 633
72 Photoaffinity Detection of cAMP Binding Proteins in Ovarian Cancer
David J. Burns and Martin J. Hulme ............................................................. 645
PART IX. APOPTOSIS
73 Apoptosis: A Technical Overview
Susan M. Quirk ................................................................................................ 651
74 Detection of Low Levels of DNA Fragmentation in Ovarian Carcinoma Cells
Amanda J. McIlwrath ...................................................................................... 659
75 Measurement of Apoptotic Cells by Flow Cytometry (Tunnel Assay)
Michael G. Ormerod........................................................................................ 665
76 Detection of Apoptosis in Ovarian Cells In Vitro and In Vivo Using
the Annexin V-Affinity Assay
Manon van Engeland, Stefan M. van den Eijnde, Thijs van Aken,
Christl Vermeij-Keers, Frans C. S. Ramaekers, Bert Schutte,
and Christl P. M. Reutelingsperger .......................................................... 669
xii Contents
77 Use of Diphenylamine in the Detection of Apoptosis

Randall K. Gibb and Cicek Gercel-Taylor .................................................... 679
78 Activation of Caspase Protease During Apoptosis
in Ovarian Cancer Cells
Zhihong Chen, Mikihiko Naito, Tetsuo Mashima, Seimiya
Hiroyuki, and Takashi Tsuruo ................................................................... 681
79 Differential Expression of Apoptosis-Associated Genes bax
and bcl-2 in Ovarian Cancer
Dagmar Marx and Harald Meden .................................................................. 687
80 DNA-Dependent Protein Kinase in Apoptosis
John J. Turchi ................................................................................................. 693
81 Detection of Telomerase Activity by PCR-Based Assay
Satoru Kyo ....................................................................................................... 701
PART X. IMMUNO AND GENE THERAPY PROTOCOLS
82 Immunogical and Gene Therapy for Ovarian Cancer: A Technical Overview
Charles Gourley and Hani Gabra .................................................................. 713
83 Developing Anti-HER2/neu Single-Chain Fv Fragments
from Phage Display Libraries
Gregory P. Adams and Robert Schier .......................................................... 729
84 Direction of the Recognition Specificity of Cytotoxic T Cells Toward
Tumor Cells by Transduced, Chimeric T-Cell Receptor Genes
Martina Maurer-Gebhard, Marc Azémar, Uwe Altenschmidt,
Matjaz Humar, and Bernd Groner ............................................................. 749
85 Eradication of c-erbB-2 Positive Ovarian Cancer Cells Mediated
by Intracellular Expression of Anti c-erbB-2 Antibody
Jessy Deshane, Ronald D. Alvarez, and Gene P. Siegal ........................... 757
86 c-erbB-2 Antisense Oligonucleotides Inhibit Serum-Induced Cell
Spreading of Ovarian Cancer Cells
Kai Wiechen ..................................................................................................... 769
87 E1A-Mediated Gene Therapy
Mien-Chie Hung, Duen-Hwa Yan, and Su Zhang ........................................ 775
88 p53 Adenovirus as Gene Therapy for Ovarian Cancer
Jennifer L. Carroll, J. Michael Mathis, Maria C. Bell,
and Joseph T. Santoso .............................................................................. 783
89 Bispecific Antibody MDX-210 for Treatment of Advanced Ovarian
and Breast Cancer
Peter A. Kaufman, Paul K. Wallace, Frank H. Valone, Wendy A. Wells,
Vincent A. Meoli, and Mark S. Ernstoff .................................................... 793
Index ........................................................................................................... 807
xiii
Contributors
EDWIN C. A. ABELN • Centraal Bureau voor Schimmelcultures, RNAAS Institute,

Baarn, The Netherlands
MAHMOUD ABU-HADID • Division of Medicine, Roswell Park Cancer Institute, Buffalo, NY
GREGORY P. ADAMS • Department of Medical Oncology, Fox Chase Cancer Center,
Philadelphia, PA
GIRIDHAR R. AKKARAJU • Department of Molecular Biology and Immunology, Uni-
versity of North Texas Health Science Center, Fort Worth, TX
UWE ALTENSCHMIDT • Institute for Experimental Cancer Research, Tumor Biology
Center, Freiburg, Germany
RONALD D. ALVAREZ • Departments of Obstetrics and Gynecology, University
of Alabama, Birmingham, AL
M. JANE ARBOLEDA • Department of Medicine, Division of Haematological Oncology,
UCLA School of Medicine, University of Califorina, Los Angeles, CA
N ELLY AUERSPERG • Department of Obstetrics and Gynecology, University of British
Columbia, BC Women’s Hospital, Vancouver, Canada
GIRIDHAR R. AKKARAJU • Department of Pharmacology, University of Pittburgh
School of Medicine, Pittsburgh, PA
MARC AZÉMAR • Institute for Experimental Cancer Research, Tumor Biology Center,
Freiburg, Germany
JAN P. A. BAAK • Department of Quantitative Pathology, Academisch Ziekenhuis
VrijeUniversiteit De Boelelaan, Amsterdam, The Netherlands
EMILY BANKS • ICRG Cancer Epidemiology Unit, Radcliffe Infirmary, Oxford, UK
JOHN M. S. BARTLETT • Department of Surgery, Glasgow Royal Infirmary, Glasgow, UK
ROBERT C. BAST, JR. • Department of Obstetrics and Gynecology, Duke University
Medical Center, NC
ALAKANANDA BASU • Department of Molecular Biology and Immunology, University
of North Texas Health Science Center, Fort Worth, TX
EBERHARD P. BECK • Leitender Oberarzt, Städtische Frauenklinik Berk, Stuttgart, Germany
A LF B ECKMANN • Klinisher Chemiker, Institut für Klinische Chemie und
Laboratoriumsmedizin, Münster, Germany
MARIA C. BELL • Department of Cellular Biology and Anatomy, Louisiana State
University Medical Center-Shreveport, Shreveport, LA
B URKHARD B RANDT • Klinisher Chemiker, Institut für Klinische Chemie und
MURRAY BRILLIANT • Department of Pathology, Royal University Hospital Saskatoon,
Saskatchawan, Canada
xiii
xiv Contributors
MASSIMO B ROGGINI • Laboratory of Cancer Pharmacology, Mario Negri Institute

for Pharmacological Research, Milan, Italy
ROBERT BROWN • CRC Department of Medical Oncology, Beatson Laboratories,
Glasgow University, Glasgow, UK
JANE BRUGGHE • Department of Quantitative Pathology, Academisch Ziekenhuis
EMMA J. BRYAN • Department of Obstetrics and Gynecology, University of Southampton
Princess Anne Hospital, Southampton, UK
DAVID J. BURNS • Breast Cancer Research Unit , Western General Hospital,
Edinburgh, Scotland
RICHARD E. BULLER • Department of Pharmacology, University of Iowa Hospitals
and Clinics, Iowa City, IA
IAN G. C AMPBELL • Department of Obstetrics and Gynecology, University
of Southampton Princess Anne Hospital, Southampton, UK
JENNIFER L. CARROLL • Department of Cellular Biology and Anatomy,
and Obstetrics and Gynecology, Louisiana State University Medical Center-
Shreveport, Shreveport, LA
SETSUKO K. CHAMBERS • Department of Obstetrics and Gynecology, Yale School
of Medicine, New Haven, MA
ZHIHONG CHEN • Laboratory of Biomedical Research, Institute of Molecular
and Cellular Bioscience, University of Tokyo, Tokyo, Japan
DENNIS S. CHI • Gynecology Academic Office, Gynecology Service, Memorial
Sloan-Kettering Cancer Center, New York, NY
CEES J. CORNELISSE • Department of Pathology, Leiden University Medical Centre,
Leiden, The Netherlands
WILLEM E. CORVER • Centraal Bureau voor Schimmelcultures, RNAAS Institute,
Baarn, The Netherlands
LUCY J. CURTIS • Department of Pathology, Molecular Medicine Centre, Edinburgh
University, Edinburgh, Scotland
G. D’A GOSTINO • Department of Obstetrics and Gynecology, Laboratory of
Antienoplastic Pharmacology Zeneca, Catholic University of the Sacred Heart,
Rome, Italy
DEREK S. DAMRON • Department of Cancer Biology, Lerner Research Institute,
Cleveland Clinic Foundation, Cleveland, OH
HIRANMOY DAS • Department of Obstetrics and Gynecology, Hyogo Medical Center
for Adults 13–70, Akashi, Japan
A. D E D ILECTIS • Department of Obstetrics and Gynecology, Laboratory of
Antienoplastic Pharmacology Zeneca, Catholic University of the Sacred Heart,
Rome, Italy
JESSY DESHANE • Departments of Pathology, University of Alabama, Birmingham, AL
JAYNE DEVLIN • School of Biomedical Sciences, University of Ulster, Coleraine,
Northern Ireland
KARL DOBIANER • Department fo Cellular Endocrinology, Ludwig Bolzmann Institute
for Experimental Endocrinology, General Hospital of Vienna, Vienna, Austria
xv
Contributors
INNA DUMLER • Charité-Franz Volhard Clinic, and Max-Delbrück Center

for Molecular Medicine, Humboldt University at Berlin, Berlin, Germany
JOANNE EDWARDS • University Department of Surgery, Glasgow, Scotland
ZEYAD ELAKAWI • Division of Medicine, Roswell Park Cancer Institue, Buffalo, NY
GAMAL H. ELTABBAKH • Department of Gynecologic Oncology, Fletcher Allen
Health Care/University of Vermont, Burlington, VT
MARK S. ERNSTOFF • Section of Hematology/Oncology, Dartmoth Hitchcock Medical
Center, Lebanon, NH
ARJANG FATTAHI-MEIBODI • Department of Obstetrics and Gynecology, University
of Gottingen, Gottingen, Germany
GABRIELLA FERRANDINA • Department of Obstetrics and Gynecology, Laboratory
of Antienoplastic Pharmacology Zeneca, Catholic University of the Sacred Heart,
Rome, Italy
WILLIAM FOULKES • Department of Medicine, Human Genetics, and Oncology,
Montreal General Hospital and Sir M. B. Davis-Jewish General Hospital,
McGill University, Montreal, Quebec, Canada
H ANI G ABRA • ICRG Medical Oncology Unit, Western General Hospital,
Edinburgh, Scotland
G ÜNTHER G ASTL • Department of Hemtaology and Oncology, University
of Innsbruck, Innsbruck,
FRANK GEBHARDT • Klinisher Chemiker, Institut für Klinische Chemie und
CICEK GERCEL-TAYLOR • Division of Gynecologic Oncology, Department of Obstetrics
and Gynecology, University of Louisville, Louisville, KY
RANDALL K. GIBB • Division of Gynecologic Oncology, Department of Obstetrics
and Gynecology, University of Louisville, Louisville, KY
ANDERS GOBL • Department of Internal Medicine, University of Uppsala Hospital,
Uppsala, Sweden
JAMES J. GOING • Department of Pathology, University of Glasgow, Glasgow, Scotland
BASEM S. GOUELI • Department of Obstetrics/Gynecology, Jikei University School
of Medicine, Tokyo Japan
SAID A. GOUELI • Department of Obstetrics/Gynecology, Jikei University School
of Medicine, Tokyo Japan
CHARLES GOURLEY • Imperial Cancer Research Fund, Medical Oncology Unit,
Western General Hospital, Edinburgh, Scotland
HENNER GRAEFF • Klinischer Forschergruppe der Frauenklinik der Technischen
Universität München, Muenchen, Germany
BERND GRONER • Chemotherapeutisches Forschungsinstitut, Georg Speyer Haus,
Frankfurt, Germany
THOMAS W. GRUNT • Department of Oncology, University Clinic of Internal Medicine,
Vienna, Austria
FRANCESCA GUALANDI • Institute of Microbiology, School of Medicine, University
of Ferrara, Ferrara, Italy
XIN-YUAN GUAN • Laboratory of Cancer Genetics, National Human Genome
Research Unit, National Institutes of Health, Betheda, MD
xvi Contributors
ANNE W. HAMBURGER • Division of Cell and Molecular Biology, Greenebaum Center

for Cancer Research, University of Maryland, Baltimore, MD
THOMAS C. H AMILTON • Department of Medical Oncology, Fox Chase Cancer
Center, Philadelphia, PA
KAZUO HASEGAWA • Department of Obstetrics and Gynecology, Hyogo Medical
Center for Adults 13–70, Akashi, Japan
RUDI HENRIKSEN • Department of Women’s and Children’s Health, Obstetrics
and Gynecology, University of Uppsala Hospital, Uppsala, Sweden
SEIMIYA HIROYUKI • Cancer Chemotherapy Center, Japanese Foundation for Cancer
Research, Tokyo, Japan
GILLIAN L. HIRST • CRC Department of Medical Oncology, Beatson Laboratories,
Glasgow University, Glasgow, Scotland
SHINJI HORIBE • Department of Obstetrics and Gynecology, Gifu University School
of Medicine, Gifu, Japan
WILLIAM J. HOSKINS • Gynecology Academic Office, Gynecology Service, Memorial
Sloan-Kettering Cancer Center, New York, NY
K. HSAIO • Signal Transduction Group, Promega, and Department of Pathology
and Laboratory Medicine, University School of Medicine, Madison, WI
MARTIN J. HULME • Breast Cancer Research Unit , Western General Hospital,
Edinburgh, Scotland
MATJAZ HUMAR • Institute for Experimental Cancer Research, Tumor Biology
Center, Freiburg, Germany
MIEN-CHIE HUNG • Department of Cancer Biology, Section of Moecular Cell Biology
and Department of Surgical Oncology, The University of Texas, Houston, TX
ATSUSHSI IMAI • Department of Obstetrics and Gynecology, Gifu University School
SEIJI ISONISHI • Department of Obstetrics/Gynecology, Jikei University School
of Medicine, Minato-ku, Tokyo, Japan
IAN J. JACOBS • Gynecology Cancer Research Unit, St. Bartholomew’s Hospital,
West Smithfield, London, UK
WOLFRAM JAEGER • Department of Obstetrics and Gynecology, University
of Erlangen-Nurmberg, Universität Straβe, Erlangen, Germany
STEVEN W. JOHNSON • Department of Medical Oncology, Fox Chase Cancer Centre,
Philadelphia, PA
PETER A. KAUFMAN • Section of Hematology/Oncology, Dartmoth Hitchcock Medical
Center, Lebanon, NH
ROBERT C. KNAPP • Division of Gynecologic Oncology, Brigham and Women’s
Hospital, Boston, MA
MARGARET A. KNOWLES • ICRF Cancer Medicine Research Unit, St. James’s University
Hospital, Leeds, UK
TAMIO KOIZUMI • Department of Obstetrics and Gynecology, Hyogo Medical Center
for Adults 13–70, Akashi, Japan
GEORG KRUPITZA • Department of Haematopathology, Institute of Clinical Pathology,
Universtiy of Vienna, Vienna, Austria
xvii
Contributors
SATORU KYO • Department of Obstetrics and Gynecology, Kanazawa University,

Kanazawa, Japan
SIMON P. LANGDON • Imperial Cancer Research Fund, Medical Oncology Unit,
SANDRA S. LAWRIE • Imperial CancerResearch Fund, Medical Oncology Unit, Western
General Hospital, Edinburgh, Scotland
THOMAS M. LEBER • Imperial Cancer Research Fund, Biological Therapies Laboratory,
London, UK
ERNST LENGYEL • Klinischer Forschergruppe der Frauenklinik der Technischen
THOMAS LIEHR • Institut für Humangenetik, Jena, Germany
WEI-HUA LIU • Department of Obstetrics and Gynecology, University of South
Carolina School of Medicine, Columbia, SC
GABRIELLA MACCHIA •Department of Obstetrics and Gynecology, Laboratory
Rome, Italy
MELANIE MACKEAN • Department of Medical Oncology, Western General Hospital
NHS Trust, Edinburgh Scotland
VIKTOR MAGDOLEN • Klinischer Forschergruppe der Frauenklinik der Technischen
ANTHONY MAGLIOCCO • Department of Pathology, Royal University Hospital Saskatoon,
Saskatchawan, Canada
SARAH L. MAINES-BANDIERIA • Department of Obstetrics and Gynecology, University
of British Columbia, BC Women’s Hospital, Vancouver, Canada
SALVATORE MANCUSO • Department of Obstetrics and Gynecology, Laboratory
Rome, Italy
M ARIA M ARONE • Department of Obstetrics and Gynecology, Laboratory
Rome, Italy
DAGMAR MARX • Department of Obstetrics and Gynecology, University of Gottingen,
Gottingen, Germany
TETSUO MASHIMA • Laboratory of Biomedical Research, Institute of Molecular
J. MICHAEL MATHIS • Department of Cellular Biology and Anatomy, and Division
of Gynecologic Oncology, Department of Obstetrics and Gynecology, Louisiana
State University Medical Center-Shreveport, Shreveport, LA
MARTINA MAURER-GEBHARD • Institute for Experimental Cancer Research, Tumor
Biology Center, Freiburg, Germany
AMANDA J. MCILWRATH • CRC Department of Medical Oncology, Glasgow, Scotland
HARALD MEDEN • Department of Obstetrics and Gynecology, University of Gottingen,
Gottingen, Germany
MICHAEL MEDL • Department of Obstetrics and Gynecology, Lainz Medical Center,
Vienna, Austria
Contributors
xviii
VINCENT MEOLIO • Section of Hematology/Oncology, Dartmoth Hitchcock Medical

Center, Lebanon, NH
GERRIT A. MEIJER • Department of Quantitative Pathology, Academisch Ziekenhuis
GORDON B. MILLS • Department of Obstetrics and Gynecology, Duke University
Medical Center, NC
TAKASHI MURATA • First Department of Internal Medicine, School of Medicine,
Yokohama City University, Yokohama, Japan
BERND MÜHLENWEG • Klinischer Forschergruppe der Frauenklinik der Technischen
PETER MULLEN • Imperial CancerResearch Fund, Medical Oncology Unit, Western
MIKIHIKO NAITO • Laboratory of Biomedical Research, Institute of Molecular
RUPERT P. M. NEGUS • Imperial Cancer Research Fund, Biological Therapies
Laboratory, London, UK
SUSANN NEUBAUER • Klinik und Poliklinik für Strahlentherapie, Universitätsstr,
Erlangen, Germany
RYUICHIRO NISHIMURA • Department of Obstetrics and Gynecology, Hyogo Medical
KAZUNORI OCHIAI • Department of Obstetrics/Gynecology, Jikei University School
AIKO OKAMOTO • Department of Obstetrics/Gynecology, Jikei University School
MICHAEL G. ORMEROD • Reigate, United Kingdom
GUISEPPE PANDINI • Istituto di Medicina Interna, Malattie Endocrine e del Metabolismo,
Universita di Catania, Catania, ItalyLAKSHMI PENDYALA • Division of Medicine,
Roswell Park Cancer Institue, Buffalo, NY
LAKSHMI PENDYALA • Division of Medicine, Roswell Park Cancer Center, Buffalo, NY
RAYMOND P. PEREZ • Section of Hematology/Oncology, Department of Medicine,
Darthmouth-Hitchcock Medical Center, Lebanon, NH
VINCENZO PEZZINO • Istituto di Medicina Interna, Malattie Endocrine e del Metabolismo,
Universita di Catania, Catania, Italy
MARIE PLANTE • Gynecology Service, L’Hotel-Dieu de Quebec, Quebec City, Canada
SUSAN M. QUIRK • Department of Animal Science, Cornell University, Ithaca, NY
GENEVIEVE J. RABIASZ • Imperial Cancer Research Fund Medical Oncology Unit,
FRANS C. S. RAMAEKERS • Department of Molecular Cell Biology and Genetics,
University of Maastricht, Maastricht, Netherlands
JONATHAN R. REEVES • Delaware, Ontario, Canada
UTE REUNING • Klinischer Forschergruppe der Frauenklinik der Technischen
CHRISTL P. M. REUTELINGSPERGER • Department of Molecular Cell Biology and Genetics,
Contributors xix
PAOLA RIMESSI • Institute of Microbiology, School of Medicine, University of Ferrara,

Ferrara, Italy
ALISON A. RITCHIE • Imperial Cancer Research Fund, Medical Oncology Unit,
A NTJE R OETGER • Klinisher Chemiker, Institut für Klinische Chemie und
JOSEPH E. ROULSTON • Department of Reproductive and Developmental Sciences,
The University of Edinburgh, The Royal Infirmary, Edinburgh, Scotland
INGO B. RUNNEBAUM • Molecular Biology Laboratory, Department of Obstetrics
and Gynecology, University of Ulm, Ulm, Germany
S. E. HILLARY RUSSELL • Department of Oncology, The Queen’s University of
Belfast, Belfast City Hospital, Belfast, Northern Ireland
YOSHIO SAITO • Department of Applied Chemistry, Faculty of Science and Technology,
Keio University, Kohoku-ku, Yokohama, Japan
JOSEPH T. SANTOSO • Division of Gynecologic Oncology, University of Texas Medical
Branch, Galveston, TX
A. KAY SAVAGE • Department of Histopathology, Royal Free Hospital School
of Medicine, Hampstead, London
GIOVANNI SCAMBIA • Department of Obstetrics and Gynecology, Laboratory
of Antienoplastic Pharmacology Zeneca, Catholic University of the Sacred
Heart, Rome, Italy
ROBERT SCHIER • Xerion Pharmaceuticals GmbH, Martinsreid, Munich, Germany
ANDREAS SCHNELZER • Klinischer Forschergruppe der Frauenklinik der Technischen
MANFRED SCHMITT • Klinischer Forschergruppe der Frauenklinik der Technischen
BERT SCHUTTE • Department of Molecular Cell Biology and Genetics, University
of Maastricht, Maastricht, Netherlands
LAURA SCIACCA • Istituto di Medicina Interna, Malattie Endocrine e del Metabolismo,
Universita di Catania, Catania, Italy
GRANT C. SELLAR • Imperial Cancer Research Fund Medical Oncology Unit, Western
ANDREW N. SHELLING • Department of Obstetrics and Gynecology, Research Centre
in Reproductive Medicine, National Women’s Hospital, Auckland, New Zealand
GENE P. SIEGAL • Departments of Pathology and Biology, University of Alabama,
Birmingham, AL
STEVEN J. SKATES • General Medicine Division, Massachusetts General Hospital
and Harvard Medical School, Boston, MA
DENNIS J. SLAMON • Department of Medicine, Division of Haematological Oncology,
UCLA School of Medicine, University of Califorina, Los Angeles, CA
BARBARA SMITH • Department of Biology, Jack Birch Unit of Molecular Carcinogenesis,
University of York, York, UK
ANIL K. SOOD • Department of Obstetrics and Gynecology, Division of Gynecologic
Oncology, University of Iowa Hospitals and Clinics, Iowa City, IA
xx Contributors
JENNIFER SOUTHGATE • Department of Biology, Jack Birch Unit of Molecular

Carcinogenesis, University of York, York, UK
DANIEL M. SPINNER • Abteilung Frauenheilkunde und Geburtshilfe I, Universitats
Klinik Freiburg, Freiburg, Germany
JÜRGEN SPONA • Department fo Cellular Endocrinology, Ludwig Bolzmann Institute
for Experimental Endocrinology, General Hospital of Vienna, and Department
of Obstetrics and Gynecology, University of Vienna, Vienna, Austria
HIROSHI TAKAGI • Department of Obstetrics and Gynecology, Gifu University School
ATSUSHI TAKAGI • Department of Obstetrics and Gynecology, Gifu University School
MASAYUKI TAKEMORI• Department of Obstetrics and Gynecology, Hyogo Medical
TERUHIKO TAMAYA • Department of Obstetrics and Gynecology, Gifu University
School of Medicine, Gifu, Japan
JEFFREY M. TRENT • Laboratory of Cancer Genetics, National Human Genome
Research Unit, National Institutes of Health, Betheda, MD
TAKASHI TSURUO • Laboratory of Biomedical Research, Institute of Molecular and
Cellular Bioscience, University of Tokyo, and Cancer Chemotherapy Center,
Japanese Foundation for Cancer Research, Tokyo, Japan
JOHN J. TURCHI • Department of Biochemistry and Molecular Biology, School
of Medicine, Wright State University, Dayton, Ohio
BEYHAN TÜRKMEN • Klinischer Forschergruppe der Frauenklinik der Technischen
KAZUO UMEZAWA • Department of Applied Chemistry, Faculty of Science and
Technology, Keio University, Kohoku-ku, Yokohama, Japan
FRANK H. VALONE • Section of Hematology/oncology, Dartmoth Hitchcock Medical
Center, Lebanon, NH
THIJS VAN AKEN • Department of Molecular Cell Biology and Genetics, University
of Maastricht, Maastricht, Netherlands
CHRISTL VERMEIJ-KEERS • Department of Molecular Cell Biology and Genetics,
GOPALRAO V. N. VELAGALETI • Division of Medical Genetics, Department of Pediatrics,
Univeristy of Tennessee, Memphis, TN
PAUL J. VAN DIEST • Department of Quantitative Pathology, Academisch Ziekenhuis
Vrije Universiteit De Boelelaan, Amsterdam, The Netherlands
STEFAN M. VAN DEN EIJNDE • Department of Molecular Cell Biology and Genetics,
MANON VAN ENGELAND • Department of Molecular Cell Biology and Genetics,
GOPALRAO V. N. VELAGALETI • Division of Genetics, Department of Pediatrics,
University of Texas Medical Branch, Gavelson, TX
FAINA VIKHANSKAYA • Laboratory of CancerPharmacology, Mario Negri Institute
for Pharmacological Research, Milan, Italy
Contributors xxi
PAUL K. WALLACE • Section of Hematology/oncology, Dartmoth Hitchcock Medical

Center, Lebanon, NH
H. ANNE WALLER • Imperial College School of Medicine at St. Mary’s, Sussex
Gardens, London
ZONG-REN WANG • Department of Obstetrics and Gynecology, University of South
SHAN WANG-GOHRKE • Molecular Biology Laboratory, Department of Obstetrics
and Gynecology, University of Ulm, Ulm, Germany
AMANDA D. WATTERS • Department of Surgery, Glasgow Royal Infirmary, Glasgow, UK
WENDY A. WELLS • Section of Hematology/oncology, Dartmoth Hitchcock Medical
Center, Lebanon, NH
KAI WIECHEN • Institute of Pathology, University Clinic Charitie Faculty of Medicine,
Humboldt University of Berlin, Berlin, Germany
ALISTAIR R. W. WILLIAMS • Department of Pathology, Edinbrugh University,
Edinburgh, Scotland
YAN XU • Department of Cancer Biology, Lerner Research Institute, Cleveland
Clinic Foundation, Cleveland, OH
DUEN-HWA YAN • Department of Cancer Biology, Section of Moecular Cell Biology
S. ROBERT YOUNG • Department of Obstetrics and Gynecology, University of South
SU ZHANG • Department of Cancer Biology, Section of Moecular Cell Biology
R ONALD P. ZWEEMER • Department of Obstetrics and Gynecology, University
Hospital Vrije Universiteit, Amsterdam, Netherlands
Epidemiology of Ovarian Cancer 3
1
The Epidemiology of Ovarian Cancer
Emily Banks
1. Introduction
Ovarian cancer is the most common fatal cancer of the female reproductive tract in
industrialized countries. At the time of writing, it is the fourth most common cause of
cancer death in women in the U.K., after breast, lung, and colorectal cancer, with a
lifetime risk of approximately 2% (1). It tends to present at an advanced stage, with
limited prospects for treatment and generally poor survival.
The histological classification of ovarian cancer is complex, with a large number of
histological subtypes. Because of the rarity of each type, tumor studies have tended to
group the types into broader categories of “epithelial” and “nonepithelial” tumors.
“Borderline” tumors are distinguished by the absence of stromal invasion. They are
considered to be an earlier or less malignant form of ovarian cancer and have similar
epidemiological characteristics to epithelial tumors, with a better prognosis.
Generally speaking, ovarian cancer incidence increases with age and is more com-
mon in women with a family history of the disease. Reproductive and hormonal factors
appear to be the other main determinants of risk, with a decline in risk associated with
increasing parity, oral contraceptive use, hysterectomy, and sterilization by tubal liga-
tion. For other factors, such as the use of hormone replacement therapy, fertility drug
treatment, breast feeding, and infertility, the evidence remains equivocal. This chapter
will discuss the epidemiology of ovarian cancer, starting with a brief outline of patterns
of incidence and time trends, before reviewing the evidence to date regarding risk fac-
tors for nonepithelial and epithelial tumors. In view of the sparsity of data regarding
risk factors for nonepithelial tumors, the bulk of the chapter relates to epithelial ovarian
cancer. This chapter presents a general summary; those requiring a more detailed review
are directed to an earlier publication (2).
2. International and National Variations and Time Trends

National incidence and registry data usually combine all histological types of ova-
rian cancer, although epithelial types tend to dominate the findings as they represent
80 to 90% of tumors (3). Figure 1 presents the age-adjusted annual incidence rates of
ovarian cancer from a range of cancer registries (1). Ovarian cancer rates vary enor-
mously from country to country and appear to relate to their respective reproductive
patterns. Incidence rates are high in most of the industrialized countries of Europe,
From: Methods in Molecular Medicine, Vol. 39: Ovarian Cancer: Methods and Protocols
Edited by: J. M. S. Bartlett © Humana Press, Inc., Totowa, NJ
3
4 Banks
Fig. 1. Age-adjusted annual incidence of ovarian cancer at selected cancer registries.
North America, and Oceania, where women have relatively few children (with the
exception of rates in Italy, Japan, and Spain). Ovarian cancer is less common in Asian
and African countries with higher fertility rates. Rates of ovarian cancer also vary
among different ethnic groups within a particular country. Migration studies have
shown that ovarian cancer rates tend to approach those of the country of adoption rather
than the country of origin. This suggests that variations within countries are unlikely to
be fully explained by racial or genetic differences.
The changing reproductive patterns of Western women are thought to be behind the
increases in ovarian cancer witnessed in these countries for most of this century.
Changes in incidence are likely to reflect trends in family size (and other factors) from
some decades previously. For instance, women who were of reproductive age during
the 1930s Depression had a relatively small average family size and consequently
higher ovarian cancer risk in later life. Many Western countries have seen recent
decreases in ovarian cancer incidence, in the face of continuing declines in fertility.
Some authors have proposed that this phenomenon relates to increasing oral contracep-
tive pill use (4). In contrast, most of the poorer, lower-incidence countries have seen
recent increases in ovarian cancer rates.
3. Nonepithelial Ovarian Cancer
Nonepithelial tumors account for around 7–10% of all malignant ovarian tumors
and are divided into germ cell and sex-cord stromal tumors. They are rare, with an
incidence of approximately six per million women per year, and little is known about
their risk factor profiles (5).
Fig. 2. Annual incidence of ovarian cancer by age in England and Wales, 1983–1987.
Malignant germ cell tumors are most common in adolescents and young women,
with a peak in incidence at around 15–19 years of age. They may be associated with in
utero exposure to hormones, young maternal age, and high body mass in the woman’s
mother (6). There are suggestions that parity, recent birth, incomplete pregnancy (mis-
carriage and abortion), oral contraceptive use, alcohol consumption, and a family his-
tory of the disease may influence risk, but findings to date are generally nonsignificant
and based on very small numbers of cases (5,7).
Malignant sex-cord stromal tumors have more in common with epithelial ovarian
cancer in that they are more frequent in older women and the oral contraceptive pill
appears to have a protective effect. However, in contrast to epithelial tumors, findings
(once again, based on small numbers) suggest that increasing parity does not appear to
protect against these tumors (5,7).
4. Epithelial Ovarian Cancer
4.1. Personal Characteristics
4.1.1. Age
Figure 2 shows the log incidence of ovarian cancer by age. Epithelial ovarian can-
cer is rare among girls and young women and increases exponentially with age (8),
until reaching a plateau in incidence around age 50 to 55. Rates increase more slowly in
later life (9,10).
4.1.2. Socioeconomic Status
Some studies have found higher risks of epithelial ovarian cancer in women of higher
socioeconomic status (11), although this finding is believed to be the result of these
women having fewer children (12–14).
6 Banks
4.1.3. Weight/Body Mass Index
Results regarding the relationship between body mass index (BMI=weight(kg)/
height(m)2 ) or weight and ovarian cancer are conflicting and inconclusive, and may
depend on aspects of study design, such as choice of control group (15). Most studies
find no association between weight or BMI and epithelial ovarian cancer (16–18),
although some find increasing risk of disease with increasing obesity (14,19). Because
the disease process itself can affect body size, study design must address this issue.
4.1.4. Genetic/Familial Factors
For more than a century, researchers have reported on rare families with multiple
cases of ovarian cancer. In addition, a relationship between breast cancer and ovarian
cancer has been reported, both within families and within individuals (20). Clarifica-
tion of these findings has come with the discovery of the oncogenes BRCA1 and BRCA2,
which have been shown to be related to inherited breast and ovarian cancer, through
germline mutations in these genes (21–23). Although these rare mutations confer
extremely high risks of disease, women reporting a general family history of ovarian
cancer are only three to four times more likely to develop ovarian cancer than those
without such a family history (20). Whereas these findings are of scientific and
aetiological interest, inherited ovarian cancer accounts for only a small proportion of
those contracting the disease (less than 5%), and the vast majority of cases are spo-
radic, occurring among women with no family history of ovarian cancer (21).
4.2. Reproductive Factors
4.2.1. Menarche and Menopause
The majority of studies have not found any effect of age at first menstrual period
(menarche) on epithelial ovarian cancer risk, with one notable exception. Rodriguez et al.
(24) found a statistically significant decrease in fatal ovarian cancer (all histological
types combined) with menarche after age 12, compared with menarche at a younger age.
The age-specific incidence curve (Fig. 2) suggests a lessening of the rate of increase
in ovarian cancer around the age of menopause, but direct evidence of an effect of
menopause on risk has proved somewhat elusive. A study pooling a number of Euro-
pean studies (25) reports a doubling in the relative risk of ovarian cancer associated
with an age at menopause of 53 or greater compared with menopause under 45 years
old, and notes a significant trend of increasing risk of ovarian cancer with later age at
menopause. However, the pooled U.S. case-control studies found no trend in ovarian
cancer risk with increasing time since last menses (15) and Purdie et al. (14) found no
significant effect of age at menopause on ovarian cancer risk in Australia.
4.2.2. Parity and Gravidity
Early classic studies observed high rates of epithelial ovarian cancer among nuns
and low rates among groups with generally high parity, including Mormons and
Seventh-Day Adventists. The association of increasing parity with decreasing ovarian
cancer risk is now well established (12) and applies to populations in North America
(13,15), Europe (26,27), and Asia (28). Overall, published results show a 40% reduc-
tion in ovarian cancer risk associated with the first term pregnancy and trends consis-
tent with a 10–15% average reduction in risk with each term pregnancy (15). A Swed-
ish study found that the risk of ovarian cancer is reduced soon after childbirth and this
protective effect appears to diminish with time (26). The effects of incomplete preg-
nancy (induced abortion and miscarriage) and the effects of the timing of childbirth
(such as age at birth of first and/or last child, and birth spacing) require further investi-
gation.
4.2.3. Breast Feeding
The effect of breast feeding on ovarian cancer incidence is disputed, and further
research is needed on this subject. An analysis based on six U.S. case-control studies
(15) found a reduced risk of ovarian cancer in women who breast fed compared to
those who had not, after controling for parity and oral contraceptive use. Other studies
are inconsistent and generally do not support these findings (2).
4.2.4. Oral Contraceptive Use
One of the most interesting and striking findings in the epidemiology of epithelial
ovarian cancer over the last 20 years is that of the protective effect of the oral contra-
ceptive pill. Studies show consistent results of an approximately 40% reduction in the
risk of ovarian cancer with any use of the oral contraceptive pill, and a 5–10% decrease
in risk with every year of use (15,29). This protective effect appears to last for at least
15–20 yr after cessation of use and applies to parous as well as nulliparous women. The
use of the oral contraceptive pill has been widespread in many countries and the inci-
dence of ovarian cancer has been decreasing, in parallel with increases in oral contra-
ceptive pill use.
4.2.5. Hormone Replacement Therapy
Because the age-specific incidence of ovarian cancer suggests that the rate of inci-
dence slows around the time of the menopause, exposure to exogenous hormones in the
postmenopausal period could plausibly offset this apparent beneficial effect. Earlier
studies of hormone replacement therapy (HRT) tended to compare women who had
ever used HRT with never users, and findings are generally consistent with no effect
(2,15). However, as more is understood about the effect of oestrogenic and
progestagenic hormones on cancer, emphasis has shifted to looking at the effect of
current HRT use on ovarian cancer. A pooled analysis of case-control data from the
United States found a protective effect of current HRT use in one subgroup, although
findings were generally negative (15). In 1995, Rodriguez et al. (24) reported on the
findings of the only prospective study in this area, which found a 70% increase in risk
of ovarian cancer in long-term current HRT users, compared to never users.
Women who use HRT are known to differ from nonusers in a number of ways that
may affect their background risk of ovarian cancer. In particular, they are more likely
to have had a hysterectomy and to have used the oral contraceptive pill in the past,
compared to never users (30) and many previous studies have not accounted for these
preexisting differences. Further research is needed into the effects of current HRT use,
past use, and use of combined oestrogen and progestagen preparations. Other hormonal
preparations, such as diethylstilboestrol and depot-medroxyprogesterone acetate do not
appear to affect epithelial ovarian cancer risk.
8 Banks
4.2.6. Infertility
Women with fertility problems tend to have few children, and because low parity
confers an increased risk of epithelial ovarian cancer, investigating the effect of infer-
tility independent of parity has proved problematic. In addition, some researchers have
found an increased risk of ovarian cancer in women who have been treated with fertil-
ity drugs (see Subheading 4.2.7.) and that once this drug-treated subgroup is excluded,
infertility itself does not affect ovarian cancer risk (15).
Bearing this in mind, there appears to be a fairly consistent relationship between
various measures of infertility and an increased risk of ovarian cancer, although this
increased risk seems to be confined to women who have never succeeded in becoming
pregnant or having a child (2).
4.2.7. Fertility Treatment

All of the studies of ovarian cancer and fertility drug treatment are based on very
small numbers and findings must be interpreted with caution. In addition, disentan-
gling the effects of fertility drugs from the effects of infertility and low parity has been
extremely difficult, if not impossible, with the current data.
Case reports in the late 1980s raised concerns that use of drugs that stimulate ovula-
tion, such as clomiphene citrate, may increase a woman’s risk of ovarian cancer. Anxi-
ety was further heightened by the U.S. pooled case-control studies, which showed a
2.8-fold increase in ovarian cancer risk in infertile women who had been treated with
fertility drugs compared to women without a history of infertility. The risk was particu-
larly high (more than 20-fold) among women who had been treated with these drugs,
but had never become pregnant, compared with never-pregnant women without fertil-
ity problems (15). Other studies have shown more moderate increases in risk, and a
grouped meta-analysis of the published data in this area shows that at least part of the
purported effect of fertility drugs results from the relative infertility of the women
taking them (2).
4.2.8. Oophorectomy, Hysterectomy, and Sterilization

Previous unilateral oophorectomy has been associated with a decrease in risk of
ovarian cancer (9). The majority of studies also show a 30–40% reduction in the risk
of ovarian cancer with simple hysterectomy (without removal of the ovaries), which is
present after controling for parity and oral contraceptive use. There is evidence to sug-
gest that this protective effect is lasting, with no apparent trend in risk with time since
hysterectomy (15), although some authors dispute this. Tubal ligation has been noted
to protect against ovarian cancer in a number of studies (11) with reported reductions
in risk ranging from 40% to 80% (2,11), although some studies have not found this to
be the case.
It has been suggested that the apparent protective effect of simple hysterectomy may
be the result of misclassification bias, where women reporting hysterectomy only may
have had an accompanying oophorectomy that they were not aware of (11). Other
researchers have hypothesized that hysterectomy and tubal ligation allow visualization
and removal of ovaries noted to have a diseased appearance at surgery (15). This argu-
ment is to some extent countered by the finding of a sustained benefit of simple hyster-
ectomy for many years following the operation. Hysterectomy and tubal ligation may
also act by impairing ovarian blood supply and inducing anovulation or by preventing
passage of carcinogens from the vagina to the ovary, via the uterus (31).
4.2.9. Ovulation: Lifetime Frequency
Many of the reproductive findings with respect to epithelial ovarian cancer are con-
sistent with Fathalla’s “incessant ovulation” hypothesis (32). This hypothesis relates a
woman’s risk of ovarian cancer to her lifetime frequency of ovulation, and proposes
that ovulation causes trauma to the ovarian epithelium and stimulation of mitoses
through exposure to oestrogen-rich follicular fluid, which can result in neoplastic
transformation (or promotion of initiated cells).
Pregnancy, oral contraceptive use, breast feeding, late menarche, and early meno-
pause all cause a decrease in a woman’s frequency of ovulation, whereas ovarian stimu-
lation with fertility drugs causes increased ovulation. Some studies have used figures
relating to these to estimate and evaluate the effect of total duration of ovulation (or
“ovulatory age”) on ovarian cancer incidence. These studies have generally found an
increasing risk of ovarian cancer with increasing duration of ovulation, but find that the
degree of protection against ovarian cancer conferred by factors such as the oral con-
traceptive pill and pregnancy is greater than would be expected based on the duration
of anovulation caused (33).
4.3. Environmental Factors
4.3.1. Diet
Many dietary factors have been investigated in relation to epithelial ovarian cancer;
although, only a high intake of saturated fat and a low intake of vegetables have been
consistently associated with an increased risk. Earlier findings of associations between
milk and lactose intakes, galactose and ovarian cancer have been refuted and evidence
to date suggests that neither coffee nor alcohol intake is consistently related to risk (2).
The effect of meat and fish consumption is unclear.
4.3.2. Smoking
Smoking is known to affect a woman’s hormonal milieu and two studies have found
increases in ovarian cancer among cigarette smokers, compared to nonsmokers (14,34).
However, the majority of studies investigating this issue have shown no association
and the effect of smoking on ovarian cancer is likely to be small in comparison with its
important effects on lung cancer and cardiovascular disease.
4.3.3. Talc
A number of studies have found a significant association between the use of talcum
powder on the perineum and ovarian cancer (2,14). This coupled with the chemical
similarity of talc and asbestos (a known carcinogen) and the finding of talc particles in
normal and cancerous ovaries (35) has lead to concerns that this relationship is causal.
Other studies have not found an association between talc use and ovarian cancer.
4.3.4. Viruses
Earlier claims of a relationship between low rates of mumps virus (and other child-
hood diseases) and ovarian cancers have not generally been sustained (28,36,37), and
have been confused by conflicting serology findings.
10 Banks
4.3.5. Ionizing Radiation
Women receiving pelvic irradiation for treatment of metropathia hemorrhagica or
for inducing menopause are at an increased risk of pelvic cancer in general, but not of
ovarian cancer in particular (38,39). No elevation in risk of ovarian cancer has been
found in case-control studies looking at both diagnostic and therapeutic irradiation
(11,18,40).
4.4. Conclusions
The main established factors influencing epithelial ovarian cancer risk, such as age,
parity, oral contraceptive use, and hysterectomy have limited potential for modifica-
tion or public health intervention. For this reason, factors such as HRT, fertility drugs,
and breast feeding are of particular interest. Larger studies and further pooled analyses
are likely to clarify their effects.
References
1. Parkin, D. M., Muir, C. S., Whelan, S. F., et al., (eds.) (1992) Cancer Incidence in Five Continents.
IARC Scientif. Lyon, France.
2. Banks, E., Beral, V., and Reeves, G. (1997) The epidemiology of epithelial ovarian cancer: a review.
Int. J. Gynecol. Cancer 7, 425–438.
3. Mant, J. W. F. and Vessey, M. P. (1994) Ovarian and Endometrial Cancers in Trends in Cancer
Incidence and Mortality (Doll, R., Fraumeni, Jr. J. F., and Muir, C. S., eds.), Cold Spring Harbor
Laboratory, Plainview, NY, pp. 287–307.
4. Beral, V., Hannaford, P., and Kay, C. (1988) Oral contraceptive use and malignancies of the genital
tract. Results from the Royal College of General Practitioners’ oral contraceptive study. Lancet ii,
1331–1334.
5. Horn Ross, P. L., Whittemore, A. S., Harris, R., and Itnyre, J. (1992) Characteristics relating to
ovarian cancer risk: collaborative analysis of 12 U.S. case-control studies. VI. Non-epithelial can-
cers among adults. Collaborative Ovarian Cancer Group. Epidemiol. 3, 490–495.
6. Walker, A. H., Ross, R. K., Haile, R. W. C., and Henderson, B. E. (1988) Hormonal factors and risk
of ovarian germ cell cancer in young women. Brit. J. Cancer 57, 418–422.
7. Albrektsen, G., Heuch, I., and Kvale, G. (1997) Full-term pregnancies and incidence of ovarian
cancer of stromal and germ cell origin: a Norwegian prospective study. Brit. J. Cancer 75, 767–770.
8. Adami, H. O., Bergstrom, R., Persson, I., and Sparen, P. (1990) The incidence of ovarian cancer in
Sweden, 1960–1984. Amer. J. Epidemiol. 132, 446–452.
9. Booth, M. and Beral, V. (1985) The epidemiology of ovarian cancer in Ovarian Cancer (Hudson, C. N.,
ed). Oxford University Press, New York, pp. 22–44.
10. Ewertz, M. and Kjaer, S. K. (1988) Ovarian cancer incidence and mortality in Denmark 1943–1982.
Int. J. Cancer 42, 690–696.
11. Booth, M., Beral, V., and Smith, P. (1989) Risk factors for ovarian cancer: a case control study. Brit.
J. Cancer 60, 592–598.
12. Beral, V., Fraser, P., and Chilvers, C. (1978) Does pregnancy protect against ovarian cancer? Lancet
i, 1083–1087.
13. Risch, H. A., Marrett, L. D., and Howe, G. R. (1994) Parity, contraception, infertility, and the risk of
epithelial ovarian cancer. Amer. J. Epidemiol. 140, 585–597.
14. Purdie, D., Green, A., Bain, C., Siskind, V., Ward, B., Hacker, N., et al. (1995) Reproductive and
other factors and risk of epithelial ovarian cancer: an Australian case-control study. Int. J. Cancer
62, 678–684.
15. Whittemore, A. S., Harris, R., and Itnyre, J. (1992) Characteristics relating to ovarian cancer risk:
collaborative analysis of 12 US case-control studies. II. Invasive epithelial ovarian cancers in white
women. Collaborative Ovarian Cancer Group. Amer. J. Epidemiol. 136, 1184–1203.
16. Franceschi, S., La Vecchia, C., Helmrich, S. P., Mangioni, C., and Tognoni, G. (1982) Risk factors
for epithelial ovarian cancer in Italy. Amer. J. Epidemiol. 115, 714–719.
17. Hildreth, N. G., Kelsey, J. L., LiVolsi, V. A., Fischer, D. B., Holford, T. R., Mostow, E. D., et al.
(1981) An epidemiologic study of epithelial carcinoma of the ovary. Amer. J. Epidemiol. 114,
398–405.
18. Koch, M., Jenkins, H., and Gaedke, H. (1988) Risk factors of ovarian cancer of epithelial origin: a
case control study. Cancer Detect. Prev. 13, 131–136.
19. The Centers for Disease Control Cancer and Steroid Hormone Study. (1983) Oral contraceptive use
and the risk of ovarian cancer. JAMA 249, 1596–1599.
20. Amos, C. I. and Struewing, J. P. (1993) Genetic epidemiology of epithelial ovarian cancer. Cancer
71, 566–572.
21. Friedman, L. S., Ostermeyer, E. A., Lynch, E. D., Szabo, C. I., Anderson, L. A., Dowd, P., et al.
(1994) The search for BRCA1. Cancer Res. 54, 6374–6382.
22. Jacobs, I. and Lancaster, J. (1996) The molecular genetics of sporadic and familial epithelial ovarian
cancer. Int. J. Gynecol. Cancer 6, 337–355.
23. Gayther, S. A., Mangion, J., Russell, P., Seal, S., Barfoot, R., Ponder, B. A., et al. (1997) Variation
of risks of breast and ovarian cancer associated with different germline mutations of the BRCA2
gene. Nat. Genet. 15, 103–105.
24. Rodriguez, C., Calle, E. E., Coates, R. J., Miracle McMahill, H. L., Thun, M. J., and Heath, C. W., Jr.
(1995) Estrogen replacement therapy and fatal ovarian cancer. Amer. J. Epidemiol. 141, 828–835.
25. Franceschi, S., La Vecchia, C., Booth, M., Tzonou, A., Negri, E., Parazzini, F., et al. (1991) Pooled
analysis of 3 European case-control studies of ovarian cancer: II. Age at menarche and at meno-
pause. Int. J. Cancer 49, 57–60.
26. Adami, H. O., Hsieh, C. C., Lambe, M., Trichopoulos, D., Leon, D., Persson, I., Ekbom, A., and
Janson, P. O. (1994) Parity, age at first childbirth, and risk of ovarian cancer. Lancet 344,
1250–1254.
27. Negri, E., Franceschi, S., Tzonou, A., Booth, M., La Vecchia, C., Parazzini, F., et al. (1991) Pooled
analysis of 3 European case-control studies: I. Reproductive factors and risk of epithelial ovarian
cancer. Int. J. Cancer 49, 50–56.
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epithelial ovarian cancer in Beijing, China. Int. J. Epidemiol. 21, 23–29.
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analysis of 3 European case-control studies of epithelial ovarian cancer: III. Oral contraceptive use.
Int. J. Cancer 49, 61–65.
30. Lancaster, T., Surman, G., Lawrence, M., Mant, D., Vessey, M., Thorogood, M., et al. (1995) Hor-
mone replacement therapy: characteristics of users and non-users in a British general practice cohort
identified through computerised prescribing records. J. Epidemiol. Community Health 49, 389–394.
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talc: a case-control study. Cancer 50, 372–376.
32. Fathalla, M. F. (1971) Incessant ovulation—A factor in ovarian neoplasia? Lancet ii, 163.
33. Risch, H. A., Weiss, N. S., Lyon, J. L., Daling, J. R., and Liff, J. M. (1983) Events of reproductive
life and the incidence of epithelial ovarian cancer. Amer. J. Epidemiol. 117, 128–139.
34. Doll, R., Gray, R., Hafner, B., and Peto, R. (1980) Mortality in relation to smoking: 22 years’ obser-
vations on female British doctors. Brit. Med. J. 280, 967–971.
35. Henderson, W. J., Joslin, C. A. F., Turnbull, A. C., and Griffiths, K. (1971) Talc and carcinoma of
the ovary and cervix. J. Obstet. Gynaecol. Brit. Comm. 78, 266–272.
36. West, R. O. (1966) Epidemiologic study of malignancies of the ovaries. Cancer 19, 1001–1007.
37. McGowan, L., Parent, L., Lednar, W., and Norris, H. J. (1979) The woman at risk for developing
ovarian cancer. Gynecol. Oncol. 7, 325–344.
38. Brinkley, D. and Haybrittle, J. L. (1969) The late effects of artificial menopause by X-radiation.
Brit. J. Radiol. 42, 519–521.
39. Darby, S. C., Reeves, G., Key, T., Doll, R., and Stovall, M. (1994) Mortality in a cohort of women
given X-ray therapy for metropathia haemorrhagica. Int. J. Cancer 56, 793–801.
40. Newhouse, M. L., Pearson, R. M., Fullerton, J. M., Boesen, E. A. M., and Shannon, H. S. (1977) A
case control study of carcinoma of the ovary. Brit. J. Prev. Soc. Med. 31, 148–153.
Familial Ovarian Cancer 13
2
Familial Ovarian Cancer
Ronald P. Zweemer and Ian J. Jacobs
1. Introduction
Ovarian cancer represents the fifth most significant cause of cancer-related death for
women and is the most frequent cause of death from gynecological neoplasia in the
Western world. The incidence of ovarian cancer in the United Kingdom (U.K.) is over
5000 new cases every year, accounting for 4275 deaths per year (1). The lifetime risk
of ovarian cancer for women in the U.K. is approximately 1 in 80. Most (80–90%)
ovarian tumors are epithelial in origin and arise from the coelomic epithelium. The
remainder arise from germ-cell or sex cord/stromal cells. A hereditary component in
the latter group is rare, but includes granulosa-cell tumors in patients with Peutz–
Jeghers syndrome (2) and autosomal dominant inheritance of small-cell carcinoma of
the ovary (3,4). Because of their limited contribution to familial ovarian cancer, these
nonepithelial tumors will not be considered further in this chapter.
Epithelial ovarian cancer has the highest case fatality rate of all gynecological
malignancies, and an overall five-year survival rate of only 30%. This poor prognosis
is largely because of the fact that 75% of cases present with extra-ovarian disease,
which in turn, reflects the absence of symptoms in early-stage disease. Advanced stage
ovarian cancer (stage IV) has a five-year survival rate of approximately 10% whereas
early stage (stage I) ovarian cancer has a five-year survival rate of at least 85%. These
figures suggest that there may be a survival benefit from the detection of ovarian can-
cer at an early stage. To be able to develop appropriate screening strategies for ovarian
cancer, there is a need to understand the processes of carcinogenesis and tumor pro-
gression. For ovarian cancer, there are no recognizable precancerous lesions that could
be targeted for screening purposes; this contrasts with other types of cancer (e.g.,
colorectal or cervical cancer) where many of the critical histological alterations in the
development of cancer have been identified. In these cancer types, the precancerous
lesions have subsequently been linked to specific molecular genetic events (5). Because
very little is still known about the morphological and molecular genetic steps involved
in initiation and progression of epithelial ovarian cancer, detection and treatment of
premalignant lesions is not yet feasible.
Three large randomized controlled trials of screening for ovarian cancer in the gen-
eral population are currently underway. Because of the potential survival benefit from
From: Methods in Molecular Medicine, Vol. 39: Ovarian Cancer: Methods and Protocols
Edited by: J. M. S. Bartlett © Humana Press, Inc., Totowa, NJ
13
14 Zweemer and Jacobs
the detection and treatment of early-stage disease, these studies aim to detect early-
stage cancer, rather than premalignant disease. However, none of the current studies
have yet reached the stage at which information about the impact on mortality is avail-
able. To optimize the efficacy of screening, it may be desirable to target women at the
highest risk of developing the disease. Most of the established risk factors for ovarian
cancer are associated with the theory of “incessant ovulation” (6,7) and include
nulliparity, an increased number of ovulatory cycles, early menarche (age at first men-
struation), and late menopause (age of last menstruation). Oral contraceptive use and
multiparity as well as breast feeding reduce the risk of ovarian cancer. It has long been
recognized, however, that the most important risk factor for ovarian cancer besides
age, is a positive family history for the disease. In recent years, two genes associated
with a genetic predisposition for breast and ovarian cancer, the BRCA1 and BRCA2
genes, have been identified. This has led to a growing awareness among the public as
well as the medical profession that cancer may be hereditary and the demand for risk
counseling and molecular testing has increased dramatically. This chapter aims to pro-
vide an integrated overview of both the clinical and molecular genetic background of
familial and hereditary ovarian cancer.
2. Familial and Hereditary Contribution to the Ovarian Cancer Burden

As ovarian cancer affects approximately 1% of women some families will have a
history of ovarian cancer in two or more family members or in combination with a
common cancer diagnosed at a young age, just by chance. About 15% of all ovarian
cancer patients report a positive family history for the disease and can be included in a
working definition of “familial ovarian cancer.” Such examples of familial ovarian
cancer could be explained by chance, common lifestyle, or exposure to carcinogenic
factors or a shared genetic susceptibility. However, an estimated 5–10% of all ovarian
cancer cases are thought to be the result of an autosomal-dominant susceptibility factor
with high penetrance. These cases can be defined as “hereditary ovarian cancer.”
3. Clinical Diagnosis
The initial evidence for a hereditary component in ovarian cancer was derived from
three observations. First, a family history of ovarian cancer was found to confer the
greatest risk of all known factors for developing the disease (8,9). This effect is espe-
cially strong in families with more than one relative affected. Analysis of population-
based series of ovarian cancer cases has shown that the risk of ovarian cancer in a
woman who has a first-degree relative (mother or sister) with the disease is 1 in 30 by
the age of 70. This risk is around one in four when two first-degree relatives are affected
(10,11). Second, population-based epidemiological studies have shown that there is a
significant excess of specific types of cancer in the relatives of ovarian cancer patients.
These include additional ovarian cancer cases, breast cancer, colorectal, and stomach
cancer (12). Finally, many case reports have identified families with multiple cases of
ovarian cancer. The first of these describes ovarian cancer in twins (13). Others have
described families with multiple cases of ovarian cancer, often in combination with
other types of cancer (14). The occurrence of ovarian cancer in these families is best
explained by an autosomal-dominant inheritance factor.
3.1. Clinical Syndromes
In families where there is insufficient evidence to diagnose autosomal-dominant
disease, ovarian cancer can occur alone or in combination with other types of cancer.
These familial cancers are to be distinguished from families where autosomal-
dominant inheritance of ovarian cancer is likely. In the latter families, epidemiological
studies have provided evidence for three distinct clinical, autosomal-dominant cancer
syndromes.
1. Hereditary breast-ovarian cancer (HBOC). Families with a pattern of autosomal-
dominant inheritance of ovarian and (usually early-onset) breast cancer.
2. Hereditary ovarian cancer (HOC). Families with clear autosomal-dominant inheritance of
ovarian cancer, but without apparent excess of breast cancer.
3. Hereditary nonpolyposis colorectal cancer (HNPCC). Families with an autosomal-
dominant pattern of early-onset colorectal cancer often in combination with endometrial
cancer and sometimes ovarian cancer.
4. Molecular Genetic Diagnosis

The final proof that a genetic predisposition is responsible for familial clustering of
a disease was initiated by extensive genetic linkage analysis of several large families.
Hall et al. (15) identified a susceptibility locus on chromosome 17q21 in several fami-
lies with autosomal-dominant breast cancer. Narod et al. (16) confirmed linkage to the
same marker in breast–ovarian cancer families. The putative gene was named BRCA1
(BReast CAncer1). Subsequent analyses showed this gene to be responsible for over
80% of families with cases of breast and ovarian cancer or ovarian cancer alone (17).
The discovery of a candidate gene by Miki et al. (18) was confirmed by several studies
describing the segregation of inactivating germline mutations in this gene with the
breast and ovarian cancer cases in these families. In accordance with the notion that the
BRCA1 gene acts as a tumor suppressor gene, allelic deletions affecting the 17q21
locus have invariably been shown to involve the wild-type allele (19).
4.1. BRCA1
The BRCA1 gene consists of 22 coding exons distributed over 100 kb of genomic
DNA. It has 5592 bp of coding sequence and encodes a protein of 1863 amino acids.
To date, more than 300 distinct mutations have been described and scattered through-
out the gene. Although there are some well-defined founder mutations (20,21), there
are no specific hot-spots in the gene and only a minority of mutations are recurrent.
Approximately 80% of all mutations are nonsense or frameshift mutations causing a
truncation of the protein. Some have suggested a relation between the position of the
mutation and penetrance as well as tissue specificity. Gayther et al. (22) found a sig-
nificant correlation between the localization of the mutation in the gene and the ratio of
breast and ovarian cancer cases within a family. They found that mutations on the three
prime third of the gene conveyed a lower risk of ovarian cancer. Apart from this study,
genotype–phenotype correlations within BRCA1 have not been confirmed. Another
possibility is that environmental circumstances and/or modifier genes may influence
the penetrance of a specific type of cancer in germline mutation carriers. Phelan et al.
(23) suggested that the risk of ovarian cancer may be increased in women with a BRCA1
16 Zweemer and Jacobs
mutation who carried one of two rare variants of the HRAS variable number of tandem
repeats (VNTRs) compared to women with the common allele.
It has become clear that mutations in the BRCA1 gene are responsible for the major-
ity of HBOC and HOC families and, therefore, the clinical distinction between these
two syndromes may have become obsolete. Initially it was anticipated that somatic
mutations in BRCA1 would be as important in sporadic ovarian cancer as germline
mutations are in hereditary cases. This seemed likely as loss of heterozygosity analysis
of unselected ovarian cancers has constantly revealed a very high frequency of LOH on
chromosome 17q (24,25). However, thus far only a few somatic mutations have been
detected in sporadic ovarian cancer cases (26). The explanation for the high frequency
of LOH of the 17q locus in these cases remains unclear and may be because of another
tumor suppressor gene in the vicinity of BRCA1 as suggested by the LOH-results of
Jacobs et al. (27).
4.2. BRCA2
Localization and cloning of the BRCA2 gene followed soon after the identification
of BRCA1. In 1994, Wooster et al. (28) localized the gene at chromosome 13q12–13.
Only months later, the same group identified the gene by showing segregation of inac-
tivating mutations of mostly breast cancer in families linked with the 13q locus (29).
The BRCA2 gene consists of 26 coding exons distributed over approximately 70 kb of
genomic DNA. It has 10.254 bp of coding sequence and encodes a 3418 amino acid
protein which has little homology to previously identified proteins (30). To date, some
100 distinct mutations have been described and as is the case for BRCA1 these are
scattered throughout the coding sequence and apart from several distinct founder muta-
tions (31,32) there are no specific hot-spots. The most frequent type of BRCA2 muta-
tions are frameshifts, most commonly deletions. It appears that missense mutations are
rarer than in BRCA1. The contribution of BRCA2 to hereditary breast cancer (HBC)
appears to be similar to the contribution of BRCA1 whereas only a minority of cases of
HBOC and HOC are caused by BRCA2 germline mutations. Although the overall
penetrance for ovarian cancer in BRCA2 germline mutation carriers is estimated at
approximately 25% (17), Gayther et al. (33) found evidence for an “ovarian cancer
cluster-region” in exon 11. Mutations in this OCCR were suggested to confer a higher
risk of ovarian cancer. To a lesser extent than is the case for BRCA1, LOH at the BRCA2
locus is frequent in sporadic ovarian cancer (34) and somatic mutations of BRCA2 are
rare in ovarian cancer.
4.3. Function of BRCA1 and BRCA2
The 7.8 kb mRNA BRCA1-transcript is expressed most abundantly in the testis and
thymus and at lower levels in the breast and ovary. The mRNA BRCA2-transcript shows
a similar tissue-specific expression (30,35). Although BRCA1 and BRCA2 are unre-
lated at the sequence level, there are some intriguing similarities. Both have a large
exon 11, which contains more than half of the coding sequence. In both genes, transla-
tion site starts at codon 2 and both are relatively A-T rich. Defining the biochemical
and biological functions that are responsible for tumorigenesis in large genes such as
BRCA1 and BRCA2 has proven to be difficult. Both genes probably have several func-
tional domains. The presence of a “zinc-finger” motif suggests a role as a transcription
factor for the BRCA1 protein. BRCA2 has homology with known transcription factors
(36). Similar motifs have been found in genes directly controlling cellular proliferation
and in that respect it is important that BRCA1 has been found to inhibit cell growth
(37). The similarity between BRCA1 and BRCA2 also includes their ability to bind and
complex with Rad51, a protein involved in the repair of double-strand DNA breaks
(38,39). For both BRCA1 and BRCA2, a similar “granin” motif has been described,
suggesting that the proteins are secreted in secretory vesicles (40). The localization of
the BRCA1 protein, however, is unclear, conflicting reports have localized the protein
in the nucleus as well as the cytoplasm (41,42). Explaining the function of both BRCA1
and BRCA2 in tumorigenesis remains a major challenge and will be the subject of
research activity for some time.
4.4. BRCA1 and BRCA2 Mutation Testing

The risk of a mutation and the penetrance of this mutation determine an individuals
risk of (hereditary) cancer. The level of cancer-risk at which to offer a woman testing
for germline mutations in BRCA1 or BRCA2 is arbitrary and the decision of whether or
not a test should be considered is also depend on the purpose it serves for patients or
healthy family members.
The chance that cancer in a given family is because of a BRCA-germline mutation
can be estimated from data collected by the Breast Cancer Linkage Consortium (17). In
summary, the risk of detecting a mutation increases with the following: a) an increas-
ing number of affected relatives; b) a young age at diagnosis; and c) occurrence of
related cancers in successive generations.
Furthermore, the chance of detecting a BRCA1 mutation in a given family increases
when ovarian cancer is frequent, when patients with both breast and ovarian cancer are
present, and when bilateral breast cancer cases occur. The risk of a BRCA2 mutation
increases when male breast cancer occurs in a family. In specific populations, muta-
tions may also be detected in far less remarkable families especially in populations
with a high population frequency of founder mutations, such as the Ashkenazi Jewish
population. In this population, up to 39% of ovarian cancer patients with a minimal or
negative family history have been found to be caused by BRCA1 or BRCA2 germline
mutations (31).
DNA testing for cancer predisposition may serve several purposes. Especially for
breast cancer patients, the treatment modality and follow-up strategies may be modi-
fied if the disease is resulting from a genetic predisposition. For ovarian cancer, there is
currently no evidence that treatment should differ if the disease is hereditary in nature.
Healthy carriers of predisposing mutations may benefit from screening or preventative
surgery. The clearest advantage of testing is obtained in at-risk family members who
test negative after a mutation has been identified in the family. For this group preven-
tative measures are no longer indicated. Finally, patients and at-risk relatives may wish
to be tested on behalf of their children.
Nondirective counseling and education based on prior risk assessment is aimed at
reaching a decision whether or not an individual would like to pursue genetic testing.
For the initial mutation testing, the cooperation and consent of live affected relatives
will usually be required. It is important to test all available affected family members
because coincidental cases of either breast or ovarian cancer (phenocopies) may occur.
When a mutation is identified in a family, carrier status for individual unaffected fam-
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MR. FORTUNE’S
PRACTICE
BY
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CONTENTS
CASE PAGE
The
I Ascot . . . . . 1
Tragedy
The
President
II . . . . . 33
of San
Jacinto
The
III Young . . . . . 64
Doctor
The
IV Magic . . . . . 98
Stone
The
V Snowball . . . . . 126
Burglary
The
VI Leading . . . . . 153
Lady
The
VII Unknown . . . . 185
Murderer
CASE I
THE ASCOT TRAGEDY
T
HAT is what it would have been called in the evening papers if they
had known all about it. They did not. They made the most of the
mystery, you remember; it was not good for them or you to know
that the sequel was a sequel. But there is no reason why the flats should
not be joined now.
So let us begin at Ascot on the morning of that Cup Day. One of our fine
summers, the course rather yellow, the lawns rather brown, a haze of
heat over the distant woodland, and sunshine flaming about the flounces
and silk hats. There were already many of both in the Royal Enclosure (it
was a year of flounces), and among them, dapper, debonair, everybody’s
friend, the youngest middle-aged man in Europe. He, of course, is the
Hon. Sidney Lomas, the Chief of the Criminal Investigation Department,
though mistaken by some outsiders for a comic actor of fame. Tripping
back from a joke with the stewards, he discovered, sprawling solitary on
the end of one of the seats, Mr. Fortune, the adviser of him and all other
official and important people when surgery, medicine or kindred sciences
can elucidate what is or is not crime. No one looks more prosperous than
Reginald Fortune. He is plump and pinkly healthy, he and his tailor treat
each other with respect, his countenance has the amiability of a nice boy.
But on this occasion Lomas found fault with him. “Why, Fortune, you’re
very pensive. Have you lost the lady of your present affections? Or backed
a wrong ’un?”
“Go away. No fellow has a right to be as cool as you look. Go quite away.
I feel like the three fellows in the Bible who sang in the furnace. How can
you jest, Lomas? I have no affections. I cannot love, to bet I am
ashamed. I always win. Half-crowns. Why is the world thus, Lomas?”
“My dear fellow, you’re not yourself. You look quite professional.”
Reggie Fortune groaned. “I am. This place worries me. I am anatomical,
ethnological, anthropological.”
“Good Gad,” said Lomas.
“Yes. A distressing place, look at it”; he waved a stick.
The people in the Royal Enclosure were as pleasant to behold as usual.
Comely girls and women who had been comely passed in frocks of which
many were pretty and few garish; their men were of a blameless,
inconspicuous uniformity.
“What is he?” said Reggie Fortune. “I ask you. Look at his feet.”
What Lomas saw was a man dressed like all the rest of them and as well
set up, but of a darker complexion. He did not see anything remarkable.
“The big fellow?” he said. “He is a little weak at the knee. But what’s the
matter with him?”
“Who is he?” said Reggie Fortune.
Lomas shrugged. “Not English, of course. Rather a half-caste colour, isn’t
he? From one of the smaller legations, I suppose, Balkan or South
American.” He waved a hand to some elegant aliens who were at that
moment kissing ladies’ hands with florid grace. “They all come here, you
know.”
“I don’t know,” said Reggie Fortune peevishly. “Half-caste? Half what
caste? Look at his feet.” Now the man’s feet, well displayed beneath white
spats, were large and flat but distinguished by their heels, which stuck
out behind extravagantly. “That is the negro heel.”
“My dear Fortune! The fellow is no more a negro than I am,” Lomas
protested: and indeed the man’s hair was straight and sleek and he had a
good enough nose, and he was far from black.
“The negro or Hamitic heel,” Reggie Fortune drowsily persisted. “I suspect
the Hamitic or negro leg. And otherwise up above. And it’s all very
distressing, Lomas.”
“An Egyptian or perhaps an Arab: probably a Foreign Office pet,” Lomas
consoled him. “That would get him into the Royal Enclosure.”
Lomas was then removed by a duchess and Reggie Fortune tilted his hat
still farther over his eyes and pondered whether it would be wise to drink
before lunch and was dreamily aware of other people on his seat, an old
man darkly tanned and soldierly in the custody of a little woman brilliantly
dressed and terribly vivacious. She chattered without a pause, she made
eyes, she made affectionate movements and little caresses. The old man
though helpless seemed to be thinking of something else. And Reggie
Fortune sketched lower and still lower estimates of human nature.
They went away at last when everybody went away to gather in a crowd
at the gates and along the railings for the coming of the King. You will
please to observe that the time must have been about one o’clock.
Reggie Fortune, one of the few, remained on his seat. He heard the
cheering down the course and had sufficient presence of mind to stand
up and take off his hat as the distant band began to play. Over the heads
of the crowd he saw the red coats of the postilions and a gleam of the
grey of the team as the King’s carriage swept round into the enclosure.
The rest of the procession passed and the crowd melted away. But one
man remained by the railings alone. He was tall and thin and he leaned
limply against the railings, one arm hanging over them. After a little while
he turned on his heel and fell in a heap.
Two of the green-coated wardens of the gate ran up to him. “Oh, Lord,”
Reggie Fortune groaned, “why did I be a doctor?” But before he could get
through the flurry of people the man was being carried away.
The gift of Lomas for arriving where he wants to be displayed itself.
Lomas slid through the crowd and took his arm, “Stout fellow! Come
along. It’s Sir Arthur Dean. Touch of sun, what?”
“Arthur Dean? That’s the Persia man, pundit on the Middle East?”
“That’s the fellow. Getting old, you know. One of the best.”
Into the room where the old man lay came the shouting over the first
race. By the door Lomas and an inspector of police talked in low tones,
glancing now and then at Reggie, who was busy.
“Merry Man! Merry Man! Merry Man!” the crowd roared outside.
Reggie straightened his bent back and stood looking down at his patient.
Lomas came forward. “Anything we can get you, Fortune? Would you like
some assistance?”
“You can’t assist him,” said Reggie. “He’s dead.”
“Merry Man!” the crowd triumphed. “Merry Man!”
“Good Gad!” said Lomas. “Poor fellow. One of the best. Well, well, what is
it? Heart failure?”
“The heart generally fails when you die,” Reggie mumbled: he still stared
down at the body and the wonted benignity of his face was lost in
expressionless reserve. “Do you know if he has any people down here?”
“It’s possible. There is a married son. I’ll have him looked for.” Lomas sent
his inspector off.
“I saw the old man with a woman just before he died,” Reggie murmured,
and Lomas put up his eyeglass.
“Did you though? Very sudden, wasn’t it? And he was all alone when he
died.”
“When he fell,” Reggie mumbled the correction. “Yes, highly sudden.”
“What was the cause of death, Fortune?”
“I wonder,” Reggie muttered. He went down on his knees by the body, he
looked long and closely into the eyes, he opened the clothes . . . and to
the eyes he came back again. Then there was a tap at the door and
Lomas having conferred there came back and said, “The son and his wife.
I’ll tell them. I suppose they can see the body?”
“They’d better see the body,” said Reggie, and as Lomas went out he
began to cover and arrange it. He was laying the right arm by the side
when he checked and held it up to the light. On the back of the hand was
a tiny drop of blood and a red smear. He looked close and found such a
hole as a pin might make.
From the room outside came a woman’s cry, then a deep man’s voice in
some agitation, and Lomas opened the door. “This is Mr. Fortune, the
surgeon who was with your father at once. Major Dean and Mrs. Dean,
Fortune.”
Reggie bowed and studied them. The man was a soldierly fellow, with his
father’s keen, wary face. But it was the woman Reggie watched, the
woman who was saying, “I was with him only half an hour ago,” and
twisting her hands nervously.
“Most of that half-hour he has been dead. Where did you leave him,
madam?” Reggie said.
Husband and wife stared at him. “Why, in the Royal Enclosure, of course.
In the crowd when the King came. I—I lost him. Somebody spoke to me.
Yes, it was Sybil. And I never saw him again.”
Reggie stepped aside from the body. She shuddered and hid her face in
her hands. “His eyes—his eyes,” she murmured.
Major Dean blew his nose. “This rather knocks one over,” he said. “What’s
the cause of death, sir?”
“Can you help me?” said Reggie.
“I? What do you mean?”
“Nothing wrong with his heart, was there?”
“Never heard of it. He didn’t use doctors. Never was ill.”
Reggie stroked his chin. “I suppose he hadn’t been to an oculist lately?”
“Not he. His eyes were as good as mine. Wonderful good. He used to
brag of it. He was rising seventy and no glasses. Good Lord, what’s that
got to do with it? I want to know why he died.”
“So do I. And I can’t tell you,” said Reggie.
“What? I say—what? You mean a post-mortem. That’s horrible.”
“My dear Major, it is most distressing,” Lomas purred. “I assure you
anything in our power—sympathize with your feelings, quite, quite. But
the Coroner would insist, you know; we have no choice.”
“As you were saying,” Reggie chimed in, “we want to know why he died.”
Major Dean drew a long breath. “That’s all right, that’s all right,” he said.
“The old dad!” and he came to his father’s side and knelt down, and his
wife stood by him, her hand on his shoulder. He looked a moment into the
dead face, and closed the eyes and looked long.
From this scene Reggie and Lomas drew back. In the silence they heard
the man and woman breathing unsteadily. Lomas sighed his sympathy.
Mrs. Dean whispered, “His mouth! Oh, Claude, his mouth!” and with a
sudden darting movement wiped away some froth from the pale lips.
Then she too knelt and she kissed the brow. Her husband lifted the dead
right hand to hold it for a while. And then he reached across to the key
chain, took off the keys, slipped them into his pocket and helped his wife
to her feet.
Reggie turned a still expressionless face on Lomas. Lomas still exhibited
grave official sorrow.
“Well—er—thanks very much for all you’ve done,” Major Dean addressed
them both. “You’ve been very kind. We feel that. And if you will let me
know as soon as you know anything—rather a relief.”
“Quite, quite.” Lomas held out his hand; Major Dean took it. “Yes, I’m so
sorry, but you see we must take charge of everything for the present.” He
let the Major’s hand go and still held out his own.
Dean flushed. “What, his keys?”
“Thank you,” said Lomas, and at last received them.
“I was thinking about his papers, you know.”
“I can promise you they’ll be safe.”
“Oh, well, that settles it!” Dean laughed. “You know where to find me,”
and he took his wife, who was plainly eager to speak to him, away.
Lomas dandled the keys in his hand. “I wonder what’s in their minds? And
what’s in yours, Fortune?”
“Man was murdered,” said Reggie.
Lomas groaned, “I was afraid you had that for me. But surely it’s not
possible?”
“It ought not to be,” Reggie admitted. “At a quarter to one he was quite
alive, rather bored perhaps, but as fit as me. At a quarter past he was
dead. What happened in between?”
“Why, he was in sight the whole time——”
“All among the most respectable people in England. Yet he dies suddenly
of asphyxia and heart failure. Why?”
“Well, some obscure heart trouble——” Lomas protested.
“He was in the pink. He never used doctors. You heard them say so. He
hadn’t even been to an oculist.”
“A fellow doesn’t always know,” Lomas urged. “There are all sorts of heart
weakness.”
“Not this sort.” Reggie shook his head. “And the eyes. Did you see how
those two were afraid of his eyes? Your eyes won’t look like that when
you die of heart failure. They might if an oculist had put belladonna in ’em
to examine you. But there was no oculist. Dilated pupils, foam at the
mouth, cold flesh. He was poisoned. It might have been aconitine. But
aconitine don’t kill so quick or quite so quiet.”
“What is aconitine?”
“Oh, wolf-bane. Blue-rocket. You can get it from other plants. Only this is
too quick. It slew him like prussic acid and much more peacefully. Some
alkaloid poison of the aconite family, possibly unclassified. Probably it was
put into him by that fresh puncture in his hand while he was packed in
the crowd, just a scratch, just a jab with a hollow needle. An easy murder
if you could trust your stuff. And when we do the post-mortem we’ll find
that everything points to death by a poison we can’t trace.”
“Thanks, so much,” said Lomas. “It is for this we employ experts.”
“Well, the police also must earn their bread. Who is he?”
“He was the great authority on the Middle East. Old Indian civilian long
retired. Lately political adviser to the Government of Media. You know all
that.”
“Yes. Who wanted him dead?” said Reggie.
“Oh, my dear fellow!” Lomas spread out his hands. “The world is wide.”
“Yes. The world also is very evil. The time also is waxing late. Same like
the hymn says. What about those papers son and co. were so keen on?”
Lomas laughed. “If you could believe I have a little intelligence, it would
so soothe me. Our people have been warned to take charge of his flat.”
“Active fellow. Let’s go and see what they found.”
It was not much more than an hour before a policeman was letting them
into Sir Arthur Dean’s flat in Westminster. An inspector of police led the
way to the study. “Anything of interest, Morton?” Lomas said.
“Well, sir, nothing you could call out of the way. When we came, the
servants had heard of the death and they were upset. Sir Arthur’s man,
he opened the door to me fairly crying. Been with him thirty years, fine
old-fashioned fellow, would be talking about his master.”
Lomas and Reggie looked at each other, but the inspector swept on.
“Then in this room, sir, there was Sir Arthur’s executor, Colonel Osbert,
getting out papers. I had to tell him that wouldn’t do. Rather stiff he was.
He is a military man. Well, sir, I put it to him, orders are orders, and he
took it very well. But he let me see pretty plain he didn’t like it. He was
quite the gentleman, but he put it to me we had no business in Sir
Arthur’s affairs unless we thought there was foul play. Well, of course, I
couldn’t answer that. He talked a good deal, fishing, you might say. All he
got out of me was that I couldn’t allow anything to be touched. So he
said he would take it up with the Commissioner and went off. That’s all,
sir.”
“Who is he?” said Reggie.
“His card, sir. Colonel Osbert, late Indian Army.”
“Do you know if he was who he said he was?” Lomas asked.
The inspector was startled. “Well, sir, the servants knew him. Sir Arthur’s
man, he let him in, says he’s Sir Arthur’s oldest friend. I had no reason to
detain him.”
“That’s all right, Morton,” said Lomas. “Well, what time did you get here?”
“Your message came two o’clock, sir. I should say we were here by a
quarter past.”
Lomas nodded and dismissed him. “Quick work,” he said with a cock of
his eye at Reggie.
“We can time it all by the King. He drove up the course at ten past one.
Till the procession came Sir Arthur was alive. We didn’t pick him up till
five minutes after, at the least. No one knew he was dead till you had
examined him. No one knew then but me and my men. And yet Colonel
Osbert in London knows of the death in time to get round here and get to
work on the dead man’s papers before two-fifteen. He knew the man was
dead as soon as we did who were looking at the body. Damme, he has
very early information.”
“Yes. One to you, Lomas. And a nasty one for Colonel Osbert. Our active
and intelligent police force. If you hadn’t been up and doing and sent
your bright boys round, Colonel Osbert might have got away with what he
wanted. And he wouldn’t have had to explain how he knew too much.”
“When was the poison given? Say between five to one and ten past. At
that time the murderer was in the Royal Enclosure. If he had his car
waiting handy, could he get here before two-fifteen?”
“Well—if his car was a flier, and there were no flies on his chauffeur and
he had luck all the way, I suppose it’s possible. But I don’t believe in it. I
should say Osbert didn’t do the job.”
Lomas sprang up and called the inspector. He wanted to know what
Colonel Osbert was wearing. Colonel Osbert was in a lounge suit of grey
flannel. Lomas sat down again and lit a cigarette. “I’m afraid that will do
for an alibi, Fortune,” he sighed. “Your hypothetical murderer was in the
Royal Enclosure. Therefore——”
“He was in topper and tails, same like us. The uniform of respectability. Of
course, he could have done a change in his car. But I don’t think it. No.
Osbert won’t do. But what was he after?”
Lomas stood up and looked round the room. It had the ordinary furniture
of an old-fashioned study and in addition several modern steel chests of
drawers for filing documents. “Well, he set some value on his papers,”
Lomas said.
“Lots of honest toil before you, Lomas, old thing.” Reggie smiled, and
while Lomas fell to work with the keys he wandered about picking up a
bowl here, a brass tray there. “He kept to his own line,” he remarked.
“Everything is Asiatic.”
“You may well say so,” Lomas groaned, frowning over a mass of papers.
But Reggie’s attention was diverted. Somebody had rung the bell and
there was talk in the hall. He made out a woman’s voice. “I fancy this is
our young friend the daughter-in-law,” he murmured.
Lomas looked up at him. “I had a notion you didn’t take to her, Fortune.
Do you want to see her?”
“God forbid,” said Reggie. “She’s thin, Lomas, she’s too thin.”
In a moment or two a discreet tap introduced Inspector Morton. “Mrs.
Dean, deceased’s daughter-in-law, sir,” he reported. “Asked to see the
man-servant. I saw no objection, me being present. They were both
much distressed, sir. She asked him if Colonel Osbert had been here.
Seemed upset when she heard he was here before us. Asked if he had
taken anything away. The servant told her we weren’t letting anything be
touched. That didn’t seem to satisfy her. She said something nasty about
the police being always too late. Meant for me, I suppose.”
“I rather fancy it was meant for me,” said Reggie. “It’s a bad business.”
“I don’t think the Colonel got away with anything, sir. He was sitting down
to the diary on the table there when we came in.”
“All right.” Lomas waved him away. “Damme, it is a bad business. What
am I to do with this, Fortune?” He held up papers in a strange script,
papers of all sorts and sizes, some torn and discoloured, some fresh.
Reggie went to look. “Arabic,” he said. “And this is Persian.” He studied
them for a while. “A sort of dossier, a lot of evidence about some case or
person. Lomas old thing, you’ll have to call in the Foreign Office.”
“Lord, we can translate them ourselves. It’s the mass of it!”
“Yes, lot of light reading. I think I should have a talk to the Foreign Office.
Well, that’s your show. Me for the body.”
Lomas lay back in his chair. “What’s in your head?”
“I won’t let anything into my head. There is no evidence. But I’m
wondering if we’ll ever get any. It’s a beautiful crime—as a crime. A
wicked world, Lomas old thing.”
On the day after, Reggie Fortune came into Lomas’s room at Scotland
Yard and shook his head and lit one of Lomas’s largest cigars and fell into
a chair. “Unsatisfactory, highly unsatisfactory,” he announced. “I took
Harvey down with me. You couldn’t have a better opinion except mine,
and he agrees with me.”
“And what do you say?”
“I say, nothing doing. He had no medical history. There was nothing the
matter with the man, yet he died of heart failure and suffocation. That
means poisoning by aconitine or a similar alkaloid. But there is no poison
in the price list which would in a quarter of an hour kill quietly and
without fuss a man in perfect health. I have no doubt a poison was
injected into him by that puncture on the hand, but I don’t know what it
was. We’ll have some analysis done, of course, but I expect nothing of
that. There’ll be no trace.”
“Unique case.”
“I wouldn’t say that. You remember I thought General Blaker was
poisoned. He was mixed up with Asiatics too. There were queer
circumstances about the death of that Greek millionaire in Rome two
years ago. The world’s old and men have been poisoning each other for
five thousand years and science only began to look into it yesterday.
There’s a lot of drugs in the world that you can’t buy at the chemist’s.”
“Good Gad,” Lomas protested, “we’re in Scotland Yard, not the Arabian
Nights. What you mean is you can’t do anything?”
“Even so. Can you? Who wanted him dead?”
“Nobody but a lunatic. He had no money to leave. He was on the best
terms with his son. He was a popular old boy, never had an enemy. He
had no secrets—most respectable—lived all his life in public.”
“And yet his son snatched at his keys before he was cold. And his dear old
friend Osbert knew of his death before he was dead and made a bee-line
for his papers. By the way, what was in his papers?”
Lomas shrugged. “Our fellows are working at ’em.”
“And who is Osbert?”
“Well, you know, he’s coming to see me. He put in his protest to the
Commissioner, and they were going to turn him down, of course. But I
thought I’d like to listen to Colonel Osbert.”
“Me too,” said Reggie.
“By all means, my dear fellow. But he seems quite genuine. He is the
executor. He is an old friend, about the oldest living. Not a spot on his
record. Long Indian service.”
“Only son and daughter don’t seem to trust him. Only he also is a bit
Asiatic.”
“Oh, my dear Fortune——” Lomas was protesting when Colonel Osbert
came.
You will find a hundred men like him on any day in the service clubs. He
was small and brown and neat, even dapper, but a trifle stiff in the joints.
His manner of speech was a drawl concluding with a bark.
Reggie lay back in his chair and admired the bland fluency with which
Lomas said nothing in reply to the parade-ground demands of Colonel
Osbert. Colonel Osbert wanted to know (if we may reduce many
sentences to one) what Lomas meant by refusing him possession of Sir
Arthur Dean’s papers. And Lomas continued to reply that he meant
nothing in particular.
“Sudden death at Ascot—in the Royal Enclosure too,” he explained.
“That’s very startling and conspicuous. The poor fellow hadn’t been ill, as
far as we can learn. Naturally we have to seek for any explanation.”
So at last Osbert came out with: “What, sir, you don’t mean to say, sir—
suspect foul play?”
“Oh, my dear Colonel, you wouldn’t suggest that?”
“I, sir? Never entered my head. Poor dear Arthur! A shock, sir. A blow!
Getting old, of course, like the rest of us.”
“Ah, had he been failing?” said Reggie sympathetically.
“Well, well, well. We none of us grow younger, sir.” Colonel Osbert shook
his head. “But upon my soul, Mr. Lomas, I don’t understand the action of
your department.”
“I’m so sorry you should say that,” Lomas sighed. “Now I wonder if you
have particular reason for wanting Sir Arthur’s papers at once?”
“My good sir, I am his executor. It’s my duty to take charge of his papers.”
“Quite, quite. Well, they’re all safe, you know. His death must have been
a great shock to you, Colonel.”
“Shock, sir? A blow, a blow. Poor dear Arthur!”
“Yes, too bad,” Lomas mourned: and voice and face were all kindly
innocence as he babbled on: “I suppose you heard about it from his son?”
Colonel Osbert paused to clear his throat. Colonel Osbert stopped that
one. “Major Dean? No, sir. No. Point of fact, I don’t know who the fellow
was. Some fellow called me up on the ’phone and told me poor dear
Arthur had fallen down dead on the course. Upon my soul, I was knocked
over, absolutely knocked over. When I came to myself I rushed round to
secure his papers.”
“Why, did you think somebody would be after them?”
“My dear sir!” Colonel Osbert protested. “Really, now really. It was my
duty. Arthur was always very strict with his papers. I thought of his
wishes.”
“Quite, quite,” Lomas purred, and artless as ever he went on: “Mrs. Dean
was round at the flat too.”
“God bless my soul!” said Colonel Osbert.
“I wonder if you could tell me: is there anyone who would have an
interest in getting hold of his papers?”
Colonel Osbert again cleared his throat. “I can tell you this, sir. I don’t
understand the position of Mrs. Dean and her husband. And I shall be
glad, I don’t mind owning, I shall be very glad to have poor dear Arthur’s
papers in my hands.”
“Ah, thank you so much,” said Lomas, and with bland adroitness got
Colonel Osbert outside the door.
“He’s not such a fool as he looks,” Reggie murmured. “But there’s better
brains in it than his, Lomas old thing. A bad business, quite a bad
business.”
And then a clerk came in. Lomas read the letter he brought and said:
“Good Gad! You’re an offensive person, Fortune. Why did you tell me to
go to the Foreign Office? Here is the Foreign Office. Now we shall be in
the affair for life. The Foreign Office wants me to see His Excellency
Mustapha Firouz.”
“Accompanied by Sindbad the Sailor and Chu Chin Chow?” said Reggie.
“Who is he?”
“Oh, he’s quite real. He’s the Median Minister. He—Why what is it now?”
The question was to the clerk, who had come back with a card.
“Says he’s anxious to see you immediately, sir. It’s very urgent, and he
won’t keep you long.”
“Major Dean,” Lomas read, and lifted an eyebrow.
“Oh rather. Let ’em all come,” said Reggie.
It was Major Dean, and Major Dean ill at ease. He had a difficulty in
beginning. He discovered Reggie. “Hallo! I say, can you tell me anything?”
he blurted out.
“I can’t,” said Reggie sharply. “I don’t know why your father died,” and
Major Dean winced.
“I thought you had something to tell us, Major,” Lomas said.
“Do you believe he was murdered? I’ve a right to ask that.”
“But it’s a very grave suggestion,” Lomas purred. “Do you know of anyone
who had a motive for killing your father?”
“It’s this filthy mystery,” the Major cried. “If he was murdered, I suppose
he was poisoned. But how?”
“Or why?” said Reggie.
The Major fidgeted. “I dare say he knew too much,” he said. “You know
he was the adviser to the Median Government. He had some pretty
serious stuff through his hands. I don’t know what. He was always great
on official secrecy. But I know he thought it was pretty damning for some
one.”
“Ah, thanks very much,” Lomas said.
But the Major seemed unable to go.
“I mean to say, make sure you have all his papers and stick to ’em.”
Lomas and Reggie studied him. “I wonder why you say that?” Lomas
asked. “The papers would naturally pass to Colonel Osbert.”
“I know. Osbert was the guv’nor’s best pal, worse luck. I wouldn’t trust
him round the corner. That’s what I mean. Now I’ve done it, I suppose”;
he gave a grim chuckle. “It is done, anyway”; and he was in a hurry to
go.
Reggie stood up and stretched himself. “This is pretty thick,” said he, “and
we’ve got His Excellency the Pasha of Nine Tales on the doorstep.”
Into the room was brought a man who made them feel short, a towering
man draped in folds of white. Above that flowing raiment rose a majestic
head, a head finely proportioned, framed in hair and beard of black
strewn with grey. The face was aquiline and bold, but of a singular calm,
and the dark eyes were veiled in thought. He bowed to each man twice,
sat down and composed his robe about him, and it was long before he
spoke. “I thank you for your great courtesy”: each word came alone as if
it was hard to him. “I have this to say. He who is gone he was the friend
of my people. To him we turned always and he did not fail. In him we had
our trust. Now, sir, I must tell you we have our enemies, who are also, as
it seems to us, your enemies. Those whom you call the Turks, they would
do evil to us which would be evil to you. Of this we had writings in their
hands and the hands of those they use. These I gave to him who is gone
that he should tell us what we should do. For your ways are not our ways
nor your law our law. Now he is gone, and I am troubled lest those
papers fall again into the hands of the Turks.”
“Who is it that Your Excellency fears? Can you tell me of any man?”
Lomas said.
“I know of none here. For the Turks are not here in the open and this is a
great land of many people. Yet in all lands all things can be bought at a
price. Even life and death. This only I say. If our papers go to your King
and the Ministers of your King it is well and very well. If they are
rendered to me that also may be well. But if they go I know not where, I
say this is not just.”
“I can promise Your Excellency they will go before the Foreign Office.”
The Median stood up and bowed. “In England I never seek justice in
vain,” he said.
And when he was gone, “Good Gad, how little he knows,” said Lomas.
“Well, Fortune?” but Reggie only lit a cigar and curled himself up on the
sofa. “What I like about you is that you never say I told you so. But you
did. It is a Foreign Office touch,” and still Reggie silently smoked. “Why,
the thing’s clear enough, isn’t it?”
“Clear?” said Reggie. “Oh Peter! Clear?”
“Well, Sir Arthur had in his hands papers damaging to these blood-and-
thunder Young Turks. It occurred to them that if he could die suddenly
they might arrange to get the papers into their hands. So Sir Arthur is
murdered, and either Osbert the executor or Major Dean the son is bribed
to hand over the papers.”
“In the words of the late Tennyson,” said Reggie,
“And if it is so, so it is, you know;
And if it be so, so be it.
But it’s not interesting, Lomas old thing.”

“It would be interesting to hear you find a flaw in it,” said Lomas.
Reggie shook his head. “Nary flaw.”
“For my part,” said Lomas with some heat, “I prefer to understand why a
crime was committed. I find it useful. But I am only a policeman.”
“And so say all of us.” Reggie sat up. “Then why talk like a politician? Who
did it and how are we going to do him in? That’s our little job.”
“Whoever it was, we’ve bilked him,” said Lomas. “He has got nothing for
his pains. The papers will go before the Foreign Office and then back to
the Median Legation. A futile crime. I find a good deal of satisfaction in
that.”
“You’re easy pleased then.” Reggie’s amiability was passing away. “A futile
crime: thanks to the active and intelligent police force. But damn it, the
man was murdered.”
“My dear Fortune, can I help it? It’s not the first and it won’t be the last
murder in which there is no evidence. You’re pleased to be bitter about it.
But you can’t even tell me how the man was murdered. A poison
unknown to the twentieth-century expert. No doubt that annoys you. But
you needn’t turn and rend me. There is also one more murderer unknown
to the twentieth-century policeman. But I can’t make evidence any more
than you. We suspect either Osbert or Major Dean had a hand in it. But
we don’t know which and we don’t know that either was the murderer. If
we could prove that they were mixed up with the Young Turks, if we knew
the man they dealt with we should have no case against them. Why, if we
could find some Young Turk hireling was in the Royal Enclosure we should
have no proof he was the murderer. We couldn’t have,” Lomas shrugged.
“Humanly speaking, it’s a case in which there can be no conviction.”
“My only aunt, don’t I know that?” Reggie cried. “And do you remember
what the old Caliph said, ‘In England I never seek justice in vain’? Well,
that stings, Lomas—humanly speaking.”
“Great heavens, what am I to do? What do you want to do?”
Reggie Fortune looked at him. The benign face of Reggie Fortune was set
in hard lines. “There’s something about the voice of a brother’s blood
crying from the ground,” he said slowly.
“My dear fellow! Oh, my dear fellow, if you are going to preach,” Lomas
protested.
“I’m not. I’m going to tea,” said Reggie Fortune. “Elise has got the trick of
some new cakes. They’re somewhat genial.”
They did not meet again till the inquest.
It was horribly hot in court. The newspaper reporters of themselves would
have filled, if given adequate space, a larger room. They sat in each
other’s pockets and thus yielded places to the general public, represented
by a motley collection of those whom the coroner’s officer permitted
himself to call Nosey Parkers: frocks which might have come out of a
revue chorus beside frocks which would well become a charwoman. And
the Hon. Sidney Lomas murmured in the ear of his henchman
Superintendent Bell, “I see several people who ought to be hanged, Bell,
but no one who will give us the chance.”
Mr. Reginald Fortune, that eminent surgeon, pathologist and what not,
called to the witness-box, was languid and visibly bored with the whole
affair. He surveyed the court in one weary, dreamy glance and gazed at
the coroner as if seeking, but without hope, some reason for his
unpleasant existence. Yes, he had seen Sir Arthur immediately after
death. He had formed the opinion that Sir Arthur died of asphyxia and
heart failure. Yes, heart failure and asphyxia. He was, however, surprised.
From the reporters’ table there was a general look of hungry interest. But
one young gentleman who had grown fat in the service of crime breathed
heavily in his neighbour’s ear: “Nothing doing: I know old Fortune. This is
a wash-out.”
Mr. Fortune had lost interest in his own evidence. He was looking sleepily
round the court. The coroner had to recall his wandering mind. “You were
surprised, Mr. Fortune?”
“Oh, ah. Well, I couldn’t explain the suddenness of the attack, the
symptoms and so forth. So with the assistance of Dr. Harvey I made a
further examination. We went into the matter with care and used every
known test. There is no evidence to be found that any other factor was
present than the natural causes of death.”
“But that does not explain the sudden failure of the heart.”
“I don’t explain it,” said Reggie. “I can’t.”
“Medicine,” said the coroner sagely, “still has its mysteries. We must
remember, gentlemen, that Sir Arthur had already completed our allotted
span, the Psalmist’s threescore years and ten. I am much obliged to you,
Mr. Fortune.”
And after that, as the fat young gentleman complained, there was nothing
in it. The jury found that Sir Arthur’s death was from natural causes and
that they sympathized with the family. So much for the Ascot mystery.
There remains the sequel.
When the court broke up and sought, panting, the open air, “He is neat,
sir, isn’t he?” said Lomas’s henchman, Superintendent Bell. “Very adroit, is
Mr. Fortune. That couldn’t have been much better done.” And Lomas
smiled. It was in each man’s simple heart that the Criminal Investigation
Department was well rid of a bad business. They sought Reggie to give
him lunch.
But Reggie was already outside; Reggie was strolling, as one for whom
time has no meaning, towards the station. He was caught up by the
plump young reporter, who would like you to call him a crime specialist.
“Well, Mr. Fortune,” he said in his ingratiating way, “good morning. How
are you, sir? I say, you have put it across us in the Dean case.”
The crime specialist then had opportunities for psychological study as Mr.
Fortune’s expression performed a series of quick changes. But it settled
down into bland and amiable surprise. “My dear fellow,” said Mr. Fortune,
“how are you? But what’s the trouble? There’s nothing in the Dean case,
never was.”
“No, that’s just it. And we were all out for a first-class crime story. After all
the talk there’s been, natural causes is pretty paltry.”
Reggie laughed. “Sorry, sorry. We can’t make crimes for you. But why did
you talk? There was nothing to talk about.”
“I say, you know, that’s a bit thick,” the crime specialist protested.
“My dear chap,” said Reggie modestly, “if the doctor on the spot hadn’t
happened to be me, you would never have thought of the case. Nothing
else in it.”
“Oh, well, come now, Mr. Fortune! I mean to say—what about the C.I.D.
holding up all the old man’s papers and turning down his executor?”
Reggie was not surprised, he was bewildered. “Say it again slowly and
distinctly,” he entreated, and when that was done he was as one who
tries not to laugh. “And very nice too. My dear fellow, what more do you
want? There’s a story for you.”
“Well, it’s never been officially denied,” said the young man.
“Fancy that!” Reggie chuckled.
“But between ourselves, Mr. Fortune——”
“It’s a great story,” Reggie chuckled. “But really—Well, I ask you!” and he
slid away.
In the hotel lounge he found Bell and Lomas and cocktails. “Pleasure
before business, as ever,” he reproached them, and ordered one for
himself.
“And what have you been doing, then?” Lomas asked.
“I have been consoling the Fourth Estate. That great institution the Press,
Mr. Lomas, sir. Through one of Gilligan’s young lions. Out of the mouths of
babes and sucklings——”
“I wish you wouldn’t talk to reporters,” Lomas complained.
“You’re so haughty. By the way, what was Ludlow Blenkinhorn doing
here?” He referred to a solicitor of more ability than standing. “Osbert
was here and his solicitor, the young Deans and their solicitor. Who was
old Blenkinhorn representing?”
Bell and Lomas looked at each other. “Didn’t see the fellow,” said Lomas.
“Mr. Fortune’s quite right, sir. Blenkinhorn was standing with the public.
And that’s odd, too.”
“Highly odd. Lomas, my dear old thing, I wish you’d watch Blenkinhorn’s
office and Osbert’s flat for any chaps who look a bit exotic, a bit foreign—
and follow him up if you find one.”
Lomas groaned. “Surely we’ve done with the case.”
“Ye-es. But there’s some fellow who hasn’t. And he has a pretty taste in
poisons. And he’s still wanting papers.”
“We’ve nothing to act on, you know,” Lomas protested.
“Oh, not a thing, not a thing. But he might have.” Lomas nodded and
Superintendent Bell went to the telephone.
When Mr. Fortune read “The Daily Post” in the morning he smiled upon
his devilled kidneys. Its report of the inquest was begun with a little
pompous descriptive work. “The mystery of the Ascot Tragedy was solved
yesterday. In the cold sanity of the coroner’s court the excitement of the
last few days received its quietus. Two minutes of scientific evidence from
Mr. Fortune—” and so on until young omniscience worked up to its private
little scoop. “The melodramatic rumours of sensational developments in
the case have thus only availed to expose the fatuity of their inventors.”
(This was meant for some rival papers.) “It may now be stated bluntly
that nothing in the case ever gave rise to speculation among well-
informed people, and that the stories of impounding documents and so
forth have no foundation in fact.”
But about lunch time Mr. Fortune received a curt summons from the Hon.
Sidney Lomas and instantly obeyed it. “Well, you know, I thought I should
be hearing from you,” he smiled. “I felt, as it were, you couldn’t live
without me long.”
“Did you, by Jove!” said Lomas bitterly. “I’ve been wishing all the morning
you had been dead some time. Look at that!” He tossed across the table a
marked copy of “The Daily Post.”
“Yes, I was enjoying that at breakfast. A noble institution, the British
Press, Lomas. A great power. If you know how to use it.”
“I wish to God you wouldn’t spoof reporters. It’s a low taste. And it’s a
damned nuisance. I can’t contradict the rag and——”
“No, you can’t contradict it. I banked on that,” Reggie chuckled.
“Did you indeed? And pray what the devil are you at? I have had Osbert
here raving mad——”
“Yes, I thought it would stir up Osbert. What’s his line?”
“Wants the papers, of course. And as you very well know, confound you,
they’re all at the Foreign Office, the cream of them, and likely to be. He
says we’ve no right to keep them after this. Nonsense, of course, but
devilish inconvenient to answer. And at last the old man was quite
pathetic, says it isn’t fair to him to give out we haven’t touched the
papers. No more it is. He was begging me to contradict it officially. I could
hardly get rid of him.”
“Busy times for Lomas.”
“Damme, I have been at it all the morning. Old Ludlow Blenkinhorn
turned up, too.”
“I have clicked, haven’t I?” Reggie chuckled.
“Confound you. He says he has a client with claims on the estate and is
informed by the executor that all papers have been taken by us. Now he
has read your damned article and he wants to know if the executor is
lying.”
“That is a conundrum, isn’t it? And who is Mr. Ludlow Blenkinhorn’s
client?”
“He didn’t say, of course.”
“What a surprise. And your fellows watching his office, do they say?”
Lomas took up a scrap of paper. “They have sent us something. A man of
foreign or mulatto appearance called on him first thing this morning. Was
followed to a Bayswater lodging-house. Is known there as Sherif. Mr. A.
Sherif. Thought to be an Egyptian.”
“The negro or Hamitic heel!” Reggie murmured. “Do you remember,
Lomas old thing?”
“Good Gad!” Lomas dropped his eyeglass. “But what the devil can we
do?”
“Watch and pray,” said Reggie. “Your fellows watch Sherif and Blenkinhorn
and Osbert and you pray. Do you pray much, Lomas?”
They went in fact to lunch. They were not long back when a detective
speaking over the telephone reported that a man of mulatto appearance
had called on Colonel Osbert. Reggie sprang up. “Come on, Lomas. We’ll
have them in the act and bluff the whole thing out of them.”
“What act?”
“Collusion. This Egyptian-Syrian-negroid-Young Turk and the respectable
executor. Come on, man.”

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But it’s not interesting, Lomas old thing.”

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