Breast Surgery A Companion to Specialist Surgical Practice

A COMPANION TO SPECIALIST SURGICAL PRACTICE
Series Editors
O. James Garden
Simon Paterson-Brown
Breast Surgery
FIFTH EDITION
Edited by
J. Michael Dixon
BSc(Hons) MBChB MD FRCS FRCSEd FRCPEd(Hon)
Professor of Surgery, University of Edinburgh;
Clinical Director, Edinburgh Breakthrough Unit;
Consultant Surgeon, NHS Lothian Edinburgh Breast Unit,
Western General Hospital, Edinburgh, UK
Edinburgh London New York Oxford Philadelphia St Louis Sydney Toronto 2014

Fifth edition © 2014 Elsevier Limited. All rights reserved.
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as may be noted herein).
First edition 1997
Second edition 2001
Third edition 2005
Fourth edition 2009
Fifth edition 2014
ISBN 978-0-7020-4959-0
e-ISBN 978-0-7020-4967-5
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Notice
Knowledge and best practice in this field are constantly changing. As new research and experience broaden our
understanding, changes in research methods, professional practices, or medical treatment may become necessary.
Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any
information, methods, compounds, or experiments described herein. In using such information or methods they should be
mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility.
With respect to any drug or pharmaceutical products identified, readers are advised to check the most current
information provided (i) on procedures featured or (ii) by the manufacturer of each product to be administered, to
verify the recommended dose or formula, the method and duration of administration, and contraindications. It is the
responsibility of practitioners, relying on their own experience and knowledge of their patients, to make diagnoses, to
determine dosages and the best treatment for each individual patient, and to take all appropriate safety precautions.
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vii
Douglas J.A. Adamson, MBChB, MD, PhD, FRCP
(Edin), FRCR
Consultant Clinical Oncologist and Honorary Senior
Lecturer, Tayside Cancer Centre, Ninewells Hospital,
Dundee, UK
Andrew D. Baildam, MD, FRCS
Professor of Breast and Oncoplastic Surgery,
Queen Mary, University of London, London, UK
Nicola L.P. Barnes, MD, FRCS
Specialist Registrar Breast Surgery, South
Manchester University Hospital, Manchester, UK
Tom Bates, FRCS
Honorary Professor of Surgery, Centre for
Professional Practice, University of Kent,
Canterbury, UK
Nigel J. Bundred, MD, FRCS
Professor in Surgical Oncology, University of
Manchester; Consultant Surgeon, University Hospital
of South Manchester NHS Foundation Trust,
Manchester, UK
Anees B. Chagpar, MD, MSc, MPH, MA
Associate Professor, Department of Surgery
Director, The Breast Center – Smilow Cancer
Hospital at Yale; Yale Comprehensive Cancer
Center – Diversity/Disparities Program Director, Yale
Interdisciplinary Breast Fellowship, Yale University
School of Medicine, New Haven, CT, USA
Daniel Xavier Choi, MD
United States Air Force, Air Mobility Command, 60th
Surgical Operations Squadron, Travis Air Force Base,
Fairfield, California, USA
Krishna B. Clough, MD
Chief of Surgery, Paris Breast Center, Paris, France
Tim Davidson, ChM, MRCP, FRCS
Consultant Breast Surgeon, University Department of
Surgery, Royal Free Hospital, London, UK
John A. Dewar, MA, FRCR, FRCP
Consultant Clinical Oncologist and Honorary
Professor of Clinical Oncology, Ninewells Hospital
and Medical School, Dundee, UK
J. Michael Dixon, BSc(Hons), MBChB, MD,
FRCS, FRCSEd, FRCPEd(Hon)
Professor of Surgery, University of Edinburgh; Clinical
Director, Edinburgh Breakthrough Unit; Consultant
Surgeon, NHS Lothian, Edinburgh Breast Unit,
Western General Hospital, Edinburgh, UK
Ian O. Ellis, BMBS, BMedSc, FRCPath
Professor of Cancer Pathology and Honorary
Consultant Pathologist, University of Nottingham,
School of Medicine, City Hospital, Nottingham, UK
D.Gareth R. Evans, MD, FRCP
Honorary Professor of Medical Genetics and
Cancer Epidemiology, The University of Manchester;
Consultant in Medical Genetics and Cancer
Epidemiology, Central Manchester Hospitals NHS
Foundation Trust and The Christie NHS Foundation
Trust, Saint Mary's Hospital, Manchester, UK
Lesley Fallowfield, BSc, DPhil, FMedSci
Professor in Psycho-oncology and Director,
Sussex Health Outcomes Research and Education
in Cancer (SHORE-C), Brighton and Sussex
Medical School, University of Sussex,
Brighton, UK
Rosalie Fisher, MBChB, FRACP
The Breast Unit, Royal Marsden Hospital,
London, UK
Gerald Gui, MS, FRCS, FRCS(Ed)
Academic Surgery (Breast Unit), Royal Marsden NHS
Trust, London, UK
Valerie Jenkins, BSc, DPhil
Sussex Health Outcomes Research and Education
in Cancer (SHORE-C), Brighton and Sussex Medical
School, University of Sussex, Brighton, UK
Contributors

Contributors
viii
Gabriel J. Kaufman, MD
Department of breast cancer and reconstructive
surgery, Paris Breast Center, Paris, France
Ian Kunkler, MA, MB, BCHIR, FRCR, FRCPE
Professor of Clinical Oncology, University of
Edinburgh; Consultant in Clinical Oncology, Western
General Hospital, Edinburgh, UK
Ava Kwong, MBBS(Lon), BSc(St Andrews),
FRCS, FRCS (Edin), FCSHK, FHKAM (surgery)
Clinical Associate Professor, Chief of Division of
Breast Surgery, The University of Hong Kong, Hong
Kong, China
Pamela Levack, MBChB, BMedBiol, MRCGP, FRCP
Consultant in Palliative Medicine, Ninewells Hospital;
Honorary Senior Lecturer in Surgery and Molecular
Oncology, University of Dundee, Dundee, UK
R. Douglas Macmillan, MBChB, MD, FRCS
Oncoplastic Breast Surgeon, Associate Clinical
Director, Nottingham Breast Institute, Nottingham City
Hospital, Nottingham, UK
Monica Morrow, MD, FACS
Chief, Breast Service, Department of Surgery;
Anne Burnett Windfohr Chair of Clinical Oncology,
Memorial Sloan-Kettering Cancer Center; Professor
of Surgery, Weill Medical College of Cornell
University, New York, NY, UK
Claude Nos, MD
Department of breast cancer and reconstructive
surgery, Paris Breast Center, Paris, France
Cameron Raine, MBChB, MD, FRCS(plastics)
Department of Plastic Surgery, St John's Hospital at
Howden, Livingston, UK
Richard M. Rainsbury, MBBS, BSc, MS, FRCS
Consultant Oncoplastic Breast Surgeon, Royal
Hampshire County Hospital, Winchester, UK
Emad A. Rakha, MB BCh, PhD, FRCPath
Clinical Associate Professor and Honorary
Consultant Pathologist University of Nottingham,
School of Medicine, City Hospital, Nottingham, UK
Rajendra S. Rampaul, MBChB, MD, FRCS
Consultant Oncoplastic Breast Surgeon, The Breast
Centre, Woodbrook, Trinidad and Tobago
John F.R. Robertson, MBChB, FRCS
Professor of Surgery, University of Nottingham;
Consultant Surgeon, City Hospital,
Nottingham, UK
Mark Schaverien, MBChB, MD, MSc, MEd,
FRCS(Plast)
Specialist Registrar, Department of Plastic Surgery,
Ninewells Hospital, Dundee, UK
Ian Smith, MD, FRCP, FRCPE
Professor of Cancer Medicine; Head, The Breast
Unit, Royal Marsden Hospital, London, UK
Sarah Tang, MBBS, MA, MRCS
Specialist Registrar in Breast Surgery, Breast Unit,
Royal Marsden Hospital, London, UK
Alastair M. Thompson, BSc(Hons), MBChB, MD,
FRCSEd
Professor of Surgical Oncology, Dundee Cancer
Centre, University of Dundee, Dundee, UK
Steven Thrush, MBBS, FRCS(Gen Surg)
Consultant Surgeon, Worcester Breast Unit,
Worcestershire Royal Hospital, Worcester, UK
A. Robin M. Wilson, MBChB, FRCR, FRCP(E)
Consultant Radiologist, Clinical Radiology, The Royal
Marsden, London, UK

ix
Series Editors' preface
It is now some 17  years since the first edition
of the Companion to Specialist Surgical Practice
series was published. We set ourselves the task of
meeting the educational needs of surgeons in the
later years of specialist surgical training, as well
as consultant surgeons in independent practice
who wished for contemporary, evidence-based
information on the subspecialist areas relevant
to their general surgical practice. The series was
never ­intended to replace the large reference sur-
gical textbooks which, although valuable in their
own way, struggle to keep pace with changing
surgical practice. This Fifth Edition has also had
to take due account of the increasing speciali-
sation in ‘general’ surgery. The rise of minimal
access surgery and therapy, and the desire of
some subspecialties such as breast and vascular
surgery to separate away from ‘general surgery’,
may have proved challenging in some countries,
but has also served to emphasise the importance
of all surgeons being aware of current devel-
opments in their surgical field. As in previous
editions, there has been increasing emphasis on
­evidence-based practice and contributors have
endeavoured to provide key recommendations
within each chapter. The eBook versions of the
textbook have also allowed the technophile
­improved access to key data and content within
each chapter.
We remain indebted to the volume editors and all
the contributors of this Fifth Edition. We have en-
deavoured where possible to bring in new blood to
freshen content. We are impressed by the enthusiasm,
commitment and hard work that our contributors
and editorial team have shown and this has ensured
a short turnover between editions while maintaining
as accurate and up-to-date content as is possible. We
remain grateful for the support and encouragement
of Laurence Hunter and Lynn Watt at Elsevier Ltd.
We trust that our original vision of delivering an up-
to-date affordable text has been met and that read-
ers, whether in training or independent practice, will
find this Fifth Edition an invaluable resource.
O. James Garden, BSc, MBChB, MD, FRCS(Glas),
FRCS(Ed), FRCP(Ed), FRACS(Hon), FRCSC(Hon),
FRSE
Regius Professor of Clinical Surgery, Clinical
Surgery School of Clinical Sciences, The University
of Edinburgh and Honorary Consultant Surgeon,
Royal Infirmary of Edinburgh
Simon
Paterson-Brown,
MBBS,
MPhil,
MS,
FRCS(Ed), FRCS(Engl), FCS(HK)
Honorary Senior Lecturer, Clinical Surgery School
of Clinical Sciences, The University of Edinburgh
and Consultant General and Upper Gastrointestinal
Surgeon, Royal Infirmary of Edinburgh

xi
I wrote in the last edition of how writing or ­editing
a book is like running a marathon. At the outset
when planning to run a marathon or to edit a
book, you set out a program and timetable and to
achieve a good outcome all you have to do is to
follow carefully the schedule you spent so much
time perfecting. But anyone who has run a mara-
thon or who has edited a multi-author text knows
that it is never that simple. There are competing
demands on time as well as unexpected problems to
deal with. Getting all the authors to deliver accord-
ing to the carefully designed schedule is never easy,
often through no fault of their own. The last few
chapters, like the last few miles of a marathon, are
the most difficult. The Fifth Edition has now run
its course and despite ups and downs and a variety
of problems, it is now here before you. Just as there
is pride in finishing a marathon so I have pride in
the efforts of the many who have contributed so
much to bring you the best edition yet of the Breast
section of the Companion Series. Although the
target audience is trainee and consultant surgeons
with a special interest in breast disease, I believe
this book will be valuable to many more groups.
Nurses working in breast clinics and operating de-
partments and any doctor involved in breast clin-
ics or the oncological management of breast cancer
should find it of great value.
Breast diseases and breast cancer are fortunate in
having a high public profile and being well sup-
ported financially. This irritates some colleagues
working in other areas but what is impressive in
breast cancer are the huge strides and advances in
our understanding and treatment that this invest-
ment has produced. This means that for many
women breast cancer is a treatable condition rather
than the death sentence it once was. Despite these
advances, there remains huge anxiety amongst
women about breast cancer and it remains women's
number one fear. Reducing the level of fear can only
be achieved through education about the advances
that have been achieved. These new advances are
all here in this Fifth Edition, which provides all the
­necessary ­information for doctors and nurses in-
volved in the care of patients with breast conditions.
The chapters on imaging, including breast screen-
ing and pathology, have been revised and updated.
More has been added on the controversies surround-
ing breast screening. Critics of screening express
concern at the numbers of potentially unnecessary
interventions and the potential over-treatment of
cancers that would not have caused problems in the
patient's lifetime. This is all addressed in the new
revamped chapter. Benign disease is more common
than breast cancer yet is often covered superficially
in many texts. The chapter on benign conditions has
been expanded to include a number of benign con-
ditions which are not common but will not be di-
agnosed unless there is knowledge of them. A more
detailed discussion of the management of nipple dis-
charge including ductoscopy is included in a com-
pletely new chapter by new authors.
The chapters dealing with breast-conserving sur-
gery and oncoplastic surgery have been revamped.
Written by recognised experts in their fields, these
chapters represent the current state of the art in
breast-conserving surgery. In the last edition, a
chapter on mastectomy was added. Far too many
mastectomies are performed through incisions that
leave a poor cosmetic outcome and this excellent
chapter has been reworked. It is essential reading
for all breast surgeons and now includes Goldilocks
mastectomy. The chapter on uncommon cancers
provides new and important information in an area
that is not well covered in many texts. One area
where practice continues to evolve and there have
been dramatic changes since the Fourth Edition has
been the management of the axilla. The Z0011 trial
has divided opinion on the two sides of the Atlantic.
A new transatlantic author presents a balanced
view of how the Z0011 trial should be interpreted
and how it has changed management of the axilla.
Not everyone has been keen to reduce the number
of axillary clearances and new trials in the UK and
the USA are planned, but what do we do in the
­meantime? Read the chapter to find out.
Editor's preface

Editor's preface
xii
There continue to be advances in our knowledge
of the genetics of breast cancer. A rewritten chapter
with the addition of a new author presents the lat-
est knowledge, not only on genetics but also on how
to manage such patients surgically. In the USA more
women than ever are choosing mastectomy in prefer-
ence to breast conservation, with these women often
choosing bilateral mastectomy in part because of the
increasing availability of good quality breast recon-
struction. A brand new chapter with new authors
covers the advances in breast reconstruction. There
have been extensive revisions to the chapter on ductal
carcinoma in situ. Building on the success of splitting
the chapters on systemic therapy into early, locally ad-
vanced and metastatic therapy, all three chapters have
been rewritten or revised. The chapter on the role of
systemic therapy in early breast cancer is authoritative
as well as comprehensive and would sit well in any
major text on oncology. Partial breast radiotherapy
after breast-conserving surgery has gained increas-
ing popularity in the USA. Whether all older patients
need radiotherapy after breast-conserving surgery
continues to be debated. Controversy as to which
patients benefit from postoperative chest wall radio-
therapy after mastectomy abounds. There are new
ongoing trials in these areas and a new chapter by an
author who is leading some of these trials shows that
the Fifth Edition is not merely a rewrite but a new
book. The chapter on psychosocial issues provides
excellent information and advice and reflects the ex-
pertise of the authors and is an important part of the
book. Medical negligence cases continue to increase
and so the book finishes with some sound advice and
vignettes of what to watch out for to avoid negligence
claims. Another chapter written by people at the top
of their game and a must-read.
The content of this book has evolved over
many  years. Various authors over this time have
been kind enough to share their knowledge, clinical
expertise and understanding because they all want
to do better for patients and this continues to be the
main motivation behind this book. It has been reas-
suring to see people carrying, reading and quoting
from the Fourth Edition. I hope and trust the Fifth
Edition will be as well received. Seeing the editions
evolve over time brings its own rewards. Ultimately
it will be you, the reader, who will judge whether the
efforts of the very many to get this book to the finish
line have been worthwhile. Your feedback is always
welcome. If there are topics we should have covered
which have been omitted, or this text fails to meet
your expectations, please let me know. If you enjoy
this book, please tell your friends and colleagues.
Acknowledgements
I would like to say a big thank you to all those who
have contributed to this Fifth Edition. The various
authors past and present have been enormously co-
operative and have produced consistently high-qual-
ity work. I would specifically like to thank Monica
McGill, who has coordinated each chapter with the
authors and made the numerous changes to the text
as well as coping with the late addition of numer-
ous new figures which, I believe, have added to the
book's utility. I would also like to thank Lynn Watt,
Development Editor, for her keen eye and endless
patience with me. There are a number of people who
have influenced me over my career who deserve spe-
cial mention. I first became aware of the complexi-
ties of the breast by working with Professor David
Page, a pathologist from Nashville. Professor Page
spent a sabbatical in Edinburgh during the year I did
pathology and his enthusiasm for his topic infected
me. There are a number of surgeons who have been
my heroes and who have taught me that success only
comes through hard work and dedication and these
include the late John Bostwick III, who worked in
Atlanta, and my friend and contemporary, Krishna
Clough from Paris. Professor Patrick Forrest in
Edinburgh taught me that the patient is the focus
and is paramount. I am fortunate to have been
taught and known other great individuals, too many
to mention. I hope they know who they are and I
thank them. I would like to pay a special tribute to
the many patients who have placed their faith and
trust in me. They continue to inspire me and are the
reason that we all need to try harder, learn more and
do better for them. Finally, I would like to thank my
family for their support. Writing and editing takes
time away from those who are there when everyone
has left. Pam, my wife, has been the rock on whom I
have been able to build many projects including this,
the Fifth Edition. Oliver and Jonathan, my sons,
have helped by understanding that medicine is not
just a profession.
Michael Dixon
Edinburgh

xiii
Critical appraisal for developing evidence-based
practice can be obtained from a number of sources,
the most reliable being randomised controlled clini-
cal trials, systematic literature reviews, meta-­analyses
and observational studies. For practical purposes
three grades of evidence can be used, analogous to
the levels of ‘proof’ required in a court of law:
1. Beyond all reasonable doubt. Such evidence
is likely to have arisen from high-quality
randomised controlled trials, systematic reviews
or high-quality synthesised evidence such as
decision analysis, cost-effectiveness analysis or
large observational datasets. The studies need
to be directly applicable to the population of
concern and have clear results. The grade is
analogous to burden of proof within a criminal
court and may be thought of as corresponding
to the usual standard of ‘proof’ within the
medical literature (i.e. P < 0.05).
2. On the balance of probabilities. In many
cases a high-quality review of literature
may fail to reach firm conclusions due to
conflicting or inconclusive results, trials of poor
methodological quality or the lack of evidence
in the population to which the guidelines apply.
In such cases it may still be possible to make
a statement as to the best treatment on the
‘balance of probabilities’. This is analogous
to the decision in a civil court where all the
available evidence will be weighed up and
the verdict will depend upon the balance of
probabilities.
3. Not proven. Insufficient evidence upon which to
base a decision, or contradictory evidence.
Depending on the information available, three
grades of recommendation can be used:
a. Strong recommendation, which should be
followed unless there are compelling reasons to
act otherwise.
b. A recommendation based on evidence of
effectiveness, but where there may be other
factors to take into account in decision-
making, for example the user of the guidelines
may be expected to take into account patient
preferences, local facilities, local audit results or
available resources.
c. A recommendation made where there is no
adequate evidence as to the most effective practice,
although there may be reasons for making a
recommendation in order to minimise cost or
reduce the chance of error through a locally
agreed protocol.
Evidence which is associated with either a strong
recommendation or expert opinion is highlighted in
the text in panels such as those shown above, and is
distinguished by either a double or single tick icon,
respectively. The references associated with double-
tick evidence are highlighted in the reference lists at
the end of each chapter along with a short summary
of the paper's conclusions where applicable.
The reader is referred to Chapter  1, ‘Evidence-
based practice in surgery’ in the volume, Core Topics
in General and Emergency Surgery of this series, for
a more detailed description of this topic.
Evidence-based practice in surgery
Evidence where a conclusion might be reached
‘on the balance of probabilities’ and where there
may be other factors involved which influence the
recommendation given. This will normally be based
on less conclusive evidence than that represented
by the double tick icons:
• III. Evidence from non-experimental descriptive
studies, such as comparative studies and
case–control studies
• IV. Evidence from expert committee reports or
opinions or clinical experience of respected
authorities, or both.
Evidence where a conclusion can be reached
‘beyond all reasonable doubt’ and therefore where
a strong recommendation can be given.
This will normally be based on evidence levels:
• Ia. Meta-analysis of randomised controlled trials
• Ib. Evidence from at least one randomised
controlled trial
• IIa. Evidence from at least one controlled study
without randomisation
• IIb. Evidence from at least one other type of
quasi-experimental study.

1
1
A. Robin M. Wilson
R. Douglas Macmillan
The role of imaging in breast diagnosis including
screening and excision of impalpable lesions
Introduction
Breast cancer is a major health problem. Worldwide
it has an increasing incidence, with over 1 million
newly diagnosed cases each year, and is the common-
est cancer to affect women and the commonest cause
of cancer death in women. Breast cancer mortality
in the UK is among the highest in the world, with
approximately 28 deaths per 100 000 women per
annum. This equates to around 48 000 new breast
cancers diagnosed and 11 500 deaths ­attributable
to breast cancer each  year. Approximately 1 in
9 women in the UK will develop breast cancer at
some time during their life.1
Strategies for diagnosing and managing breast
cancer are based on our current understanding of
breast disease epidemiology. Around 5% of breast
cancer is hereditary, mainly associated with the
BRCA1 and BRCA2 gene defects. This type of
breast cancer tends to occur in younger women.
The remaining 95% of breast cancer is sporadic
and its incidence increases with age. Breast cancer
is rare under the age of 35 years and over 80% of
breast cancer occurs in women over the age of 50.
The main causes of sporadic breast cancer are be-
lieved to be environmental factors. Recognised
risk factors include early menarche, late meno-
pause, nulliparity, and long-term use of the con-
traceptive pill and hormone replacement therapy
(HRT).
As there is a poor understanding of the causes of
breast cancer, primary prevention is currently not a
realistic or achievable option. It is known that ear-
lier diagnosis of breast cancer is more likely to result
in a favourable outcome. Tumour size at diagnosis,
grade and lymph node stage are the best predictors
of outcome. Regardless of tumour type or grade, the
smaller a breast cancer is at the time of diagnosis,
the more likely it is that it has not spread beyond the
breast. As a result the current strategy for reducing
breast cancer mortality is to seek diagnosis as early
as possible.
Early detection and improvements in treatment
have led to a 30% reduction in breast cancer mor-
tality in the UK in all age groups over the past
30 years.2
Early diagnosis is achieved by encouraging women
to present as soon as possible to breast clinics when
they develop breast symptoms and through regular
breast cancer screening. Breast imaging is funda-
mental to both.
Imaging in symptomatic
breast practice
Based on mortality statistics from the 1980s and
1990s, although the UK did not have the highest
incidence of breast cancer it did have the highest
death rate.

Chapter 1
2
Imaging is required at all three stages of this pro-
cess, and mammography and ultrasound have a
pivotal role to play. About 60% of breast cancer
is diagnosed in symptomatic breast referral clin-
ics. These clinics follow protocols that define the
triple test, the combination of clinical assessment,
imaging (mammography and ultrasound) and core
­biopsy or needle cytology, as the required standard.4
‘One-stop’ clinics are recommended at which all the
necessary tests required to make a diagnosis, includ-
ing needle biopsy, are performed at one clinic visit.
In order to achieve the earliest possible diagnosis of
symptomatic breast cancer, women are encouraged
through a variety of health promotion methods to
present to these clinics as soon as they develop any
change in their breasts. The clinical and imaging
­assessments in these clinics should be performed
by appropriately trained and experienced staff. All
such staff do not need to be doctors but they need
to be able to give an independent opinion and be
trained to an appropriate level.
Breast imaging techniques
Mammography
X-ray mammography has been the basis of breast
imaging for more than 30 years. The sensitivity of
mammography for breast cancer is age dependent.
The denser the breast, the less effective this method
is for detecting early signs of breast cancer. Breast
density tends to be higher in younger women and
increased density obscures early signs of breast can-
cer. The sensitivity of mammography for breast can-
cer in women over 60 years of age approaches 95%,
while mammography can be expected to ­detect
less than 50% of breast cancers in women under
40 years of age.5
Mammography uses ionising radiation to obtain an
image and therefore should only be used where there
is likely to be a clinical benefit. Consensus is that the
benefits of mammography in women over the age of
40 years are likely to far outweigh any oncogenic ef-
fects of repeated exposure. Screening of women over
the age of 40 by mammography is ­accepted practice.
However, in symptomatic practice, there is rarely an
indication for performing mammography in women
under the age of 35 unless there is a strong clinical
suspicion of malignancy. In many centres, all women
over the age of 35 presenting to breast clinics un-
dergo mammography as a routine. Practice is chang-
ing and ultrasound alone is being used increasingly
for the assessment of women with focal breast symp-
toms in women under 40 years of age and even in
some women aged 40–50. Mammography is routine
in all women in the screening age group attending
symptomatic clinics who have not had a screening
mammogram in the past year.
Most mammography in the UK is now carried
out using digital image acquisition.6–9 There are
major benefits from acquiring mammograms in di-
rect digital format.9 Compared with conventional
film/screen mammography, the benefits of full-field
digital mammography include better imaging of
the dense breast, the application of computer-aided
­detection and a number of logistical advantages
providing potential for more efficient mammogra-
phy services.10,11 The much wider dynamic range of
digital mammography means that visualisation of
the entire breast density range on a single image is
easily achievable. In the clinical setting, compara-
tive studies have shown that digital mammography
performs in general as well as film/screen mam-
mography but is better in younger women and in
women with dense breasts.6,7
Mammography is the basis of stereotactic breast
biopsy, which can be carried out using a dedi-
cated prone biopsy table or by using an add-on
device to a conventional upright mammography
unit. Stereotaxis is used to biopsy impalpable le-
sions that are not clearly visible on ultrasound (e.g.
microcalcifications).
Ultrasound
High-frequency (≥10 MHz) ultrasound is a very
­effective diagnostic tool for the investigation of ­focal
breast symptoms.12 Ultrasound does not ­involve
Recognising that breast cancer diagnosis
and treatment required significant improvement,
in 1995 the UK Department of Health published
guidelines for improving outcomes in breast cancer.
These guidelines were updated in 2002 and 2010 by
the National Institute for Clinical Excellence (NICE).3
The guidelines emphasise the following three key
issues in breast cancer care:
• accurate and timely diagnosis;
• appropriate treatment decided by accurate staging
of disease;
• appropriate follow-up of patients undergoing
treatment.

The role of imaging in breast diagnosis
3
­ionising radiation and is a very safe imaging tech-
nique. It has a high sensitivity for breast pathology
and also a very high negative predictive value.13
Ultrasound is the technique of choice for the fur-
ther investigation of focal symptomatic breast prob-
lems at all ages. Under 40 years of age, when the
risk of breast cancer is very low, it is usually the only
imaging technique required. Over 40, when the risk
of breast cancer begins to increase, it is often used in
conjunction with mammography. Ultrasound is less
sensitive than mammography for the early signs of
breast cancer and is therefore not used for popula-
tion screening. However, ultrasound does increase
the detection of small breast cancers in women who
have a dense background pattern on mammogra-
phy.5 In the screening setting there is clear evidence
that the addition of ultrasound improves small
cancer detection rates, particularly in women with
dense breasts, but there is currently insufficient evi-
dence of any mortality benefit and insufficient re-
sources to allow for routine ultrasound screening
of women with dense breasts on mammography.
Adding ultrasound to mammography or magnetic
resonance imaging (MRI) screening does increase
cancer detection but also significantly increases the
false-positive rate. Ultrasound is the technique of
first choice for biopsy of both palpable and impal-
pable breast lesions visible on scanning.
Ultrasound is now used routinely to assess the ax-
illa in women with breast cancer in most units.
Axillary nodes that show abnormal morphology
can be sampled accurately by fine-needle aspiration
(FNA) or needle core biopsy.16,17 Discussion of the
role of axillary ultrasound and FNA and core bi-
opsy can be found in Chapter 7.
Doppler ultrasound adds little to breast diag-
nosis and is not widely used. Three-dimensional
­ultrasound of the breast is said to increase the ac-
curacy of biopsy and the detection of multifocal
disease but is not widely available. Elastography
is a new application of ultrasound technology that
allows the accurate assessment of the stiffness of
breast tissue. It is being evaluated at present and
may prove to be a useful tool in excluding signifi-
cant abnormalities, for instance in assessment of
asymptomatic abnormalities detected by ultrasound
screening.
Magnetic resonance
mammography (MRM)
MRM is now widely available. In order to image the
breast the patient is scanned prone and injection of
intravenous contrast is required. MRM is the most
sensitive technique for detection of breast cancer,
­approaching 100% for invasive cancer and up to
92% for ductal carcinoma in situ (DCIS), but it has
a high false-positive rate.18,19,20 Rapid acquisition of
images facilitates assessment of signal enhancement
curves that can be helpful in distinguishing benign
from malignant disease. Significant overlap in the
enhancement patterns is seen, so needle sampling of
the lesions detected is often required. Magnetic
­resonance-guided breast biopsy is available in a few
centres but most breast lesions seen on MRM that
are larger than 5 mm can be seen on ­ultrasound if
they are clinically significant.
MRM is the best technique for imaging women
with breast implants. It is also of benefit in identi-
fying recurrent disease where conventional imag-
ing and biopsy have failed to exclude recurrence.
Provided it is carried out more than 18 months after
surgery, MRI will accurately distinguish between
scarring and tumour recurrence. MRI is recom-
mended to detect multifocality prior to conserva-
tion surgery for women with lobular cancers and
those with occult cancer on mammography or with
a significant discrepancy of size on conventional
imaging. However, it should not be used routinely
and a randomised trial (COMICE) showed no ben-
efit in reducing re-excision rates but did increase
High-resolution ultrasound easily distinguishes
between most solid and cystic lesions and can
differentiate benign from malignant lesions with
a high degree of accuracy. However, in most
circumstances, solid lesions seen on ultrasound
require needle sampling for accurate diagnosis.
Up to 50% of patients with axillary metastatic
disease can be diagnosed by ultrasound combined
with FNA or core biopsy, which may avoid the
need for sentinel node biopsy in some women with
pathologically proven nodal involvement.14,15
MRM is the best method for screening younger
women (under 40 years) at increased risk of breast
cancer but, because of cost, it is unlikely to be used
for general population screening.21–24

Chapter 1
4
­significantly the mastectomy rates for those who had
MRI. Breast MRI is of value in assessing response of
large or locally advanced breast cancers to neoadju-
vant chemotherapy. MRI of the axilla can demon-
strate axillary metastatic disease but its sensitivity is
not sufficient to replace surgical staging of the axilla.
For advanced breast cancer, MRI is the technique of
choice for assessing spinal metastatic disease.
Computed tomography
Computed tomography (CT) has no current proven
role in primary imaging of the breast. but dedicated
low-dose breast CT is being introduced; its clinical
efficacy has yet to be determined. CT is used to diag-
nose and stage systemic spread of breast cancer and
to assess response of metastatic disease to treatment,
particularly lung, pleural and liver metastases. Some
patients with breast cancer do have their cancers di-
agnosed incidentally during a routine CT scan.
Isotope imaging
Breast scintigraphy is not widely used because of its
lack of sensitivity compared with other techniques.
It is used in a few centres to stage the breast prior
to surgery and to assess response to treatment.
Scintigraphy is widely used to diagnose and assess
the presence of skeletal metastatic disease. It is now
often combined with a low-resolution non-contrast
CT scan.
Positron emission scintigraphy, particularly when
combined with CT, is a technology that may have a
role in future in staging breast cancer and monitor-
ing response to treatment. It is widely used in the
USA but elsewhere, at present, it continues to be
regarded as a research tool and its specific uses and
indications in assessing breast disease are still being
defined.
Breast cancer screening
Aim
The aim of breast screening is to reduce mortality
through early detection. Randomised controlled tri-
als and case–control studies carried out between the
1960s and 1980s have demonstrated that popula-
tion screening by mammography can be expected to
reduce overall breast cancer mortality by around
25% and by 35–40% in those who participate.
The mortality benefit of screening is greatest in
women aged 55–70 years.27,28 The mortality benefit
of screening women aged between 40 and 55 is ap-
proximately 20%. Screening women under the age
of 40 has not been shown to provide any mortality
benefit.27,28
Population screening
Breast screening has been introduced in many
countries over the past 25 years. In most countries,
screening is recommended in all women aged 40 and
over but in countries that provide population-based
screening, women of 50 and over are specifically
targeted. Breast cancer screening was introduced in
the UK in 1987 and provides screening by invita-
tion, free at the point of delivery, to all women be-
tween the ages of 50 and 70.1 Women over 70 can
attend but are not invited. Over 70% of the invited
population need to attend for a significant overall
mortality benefit to be achieved. Women under the
age of 50 are not offered screening in the UK un-
less they are at increased risk. A randomised trial of
extending the screening invitation to the age range
47–73 years was started in 2010. The results of this
trial will not be available until after 2020. From
2010 screening with annual MRI in addition to
mammography has been offered to women at very
high risk of breast cancer, including BRCA1 and
BRCA2 gene carrriers.
Method and frequency
The validity of these trials was questioned
in 2000–2002 but subsequent reviews by the
Swedish combined trials group and a World Health
Organisation International Agency for Research on
Cancer committee of experts have reaffirmed the
mortality benefit of mammographic screening and
determined that criticisms of the mammographic
screening trials were unjustified.25–30
The optimum screening method is two-view
mammography; clinical examination of the breast
and breast self-examination have not been shown
to contribute to mortality reduction through early
detection and so are not recommended.9,31,32

The role of imaging in breast diagnosis
5
Women aged 50–70 in the UK are invited for
mammography every 3  years. There has been
some concern that this screening interval is too
long. Mammography can be expected to detect
breast cancer approximately 2  years before it
becomes clinically apparent. The frequency of
mammographic screening is determined by the
lead-time of breast cancer. Based on the average
growth time of breast cancer in different ages,
this means that mammographic screening should
ideally be carried out  yearly in women aged
40–50, every 2 years in women aged 50–60 and
every 3 years thereafter. However, the UK breast
screening frequency trial completed in 1995 did
not show any predicted benefit for women aged
50–64 screened every year compared with those
screened every 3  years.33 Screening once every
3 years can be expected to detect approximately
two-thirds of all breast cancers that will arise
during the 3-year screening interval.
One-third of breast cancers present in the inter-
val between screens and are called interval can-
cers. Half of these present in the third year after
screening.
Factors affecting the effectiveness
of screening
HRT increases breast density and in a proportion of
women this reduces the sensitivity of mammography
for breast cancer.9,32–40 Up to 25% of women tak-
ing combined oestrogen/progestogen preparations
continuously show increased density on mammogra-
phy. This effect is significantly less with other HRT
preparations. As well as reducing sensitivity HRT
also reduces the specificity of mammographic screen-
ing. HRT also increases the risk of developing breast
cancer.34
Quality assurance
Breast screening programmes should have inbuilt
quality assurance; the UK NHS Breast Screening
Programme is subject to comprehensive quality
assessment and all 100 screening units across the
country have to comply with nationally defined
standard guidelines. There are national targets for
screening set by a central Department of Health
Advisory Committee (Table 1.1).
The screening process
Women invited for screening attend either a static
or mobile screening unit where two-view mammog-
raphy is performed. The images are then double
read within a few days. The vast majority of women
(95%) are informed by letter within 2  weeks of
­attendance that their mammograms show no evi-
dence of breast cancer. Those women in the appro-
priate age range will be invited for screening 3 years
later. They are advised to contact their general prac-
titioner as soon as possible if they become aware of
any change in their breasts in the meantime.
The screening process includes a fully integrated
multidisciplinary assessment process for all screen-
detected mammographic abnormalities; screening
programmes should ideally retain responsibility
through to definitive diagnosis. Approximately
5% of women screened are recalled for further
assessment of a problem identified at screening.
Some women are recalled for further assessment
of a clinical sign or symptom identified at the time
of screening but the vast majority of women are
recalled because of a mammographic abnormality.
The common types of mammographic abnormality
and their positive predictive value for cancer are shown
in Table 1.2. Well-defined masses are almost always
benign and do not require recall, whereas ­ill-defined
masses and spiculated lesions ­always require further
assessment (Figs 1.1 and 1.2). Clustered microcalcifi-
cations account for a high proportion of recalls that
result in needle biopsy. More than 20% of screen-de-
tected breast cancer is DCIS, mostly high or intermedi-
ate grade, and most of this type of cancer is detected by
the presence of clustered microcalcifications (Fig. 1.3).
Invasive cancer is usually represented on mammog-
raphy by ­either an ­ill-defined or spiculated mass. It
is ­essential to detect these lesions at small size as they
more commonly represent grade 2 or 3 invasive cancer.
Three-quarters of women recalled undergo fur-
ther imaging (mammography and/or ultrasound)
and clinical assessment before being reassured and
The most important cancers detected at
screening are high-grade DCIS, as most cases
of this type will progress to grade 2 or 3 invasive
breast cancer within the following 3 years, and
grade 2 and 3 invasive breast cancers under 10 mm
in diameter, as at this size these tumours are much
less likely to have metastasised.41,42

Chapter 1
6
Table 1.1  •  NHS Breast Screening Programme: screening targets, June 2005
Objective
Criteria
Minimum standard
Target
To maximise the number of
eligible women who attend
for screening
Percentage of eligible women
who attend for screening
• ≥70% of invited women to
attend for screening
• 80%
To maximise the number of
cancers detected
(a) Rate of invasive cancers
detected in eligible women
• Prevalent screen ≥2.7 per
1000
• Prevalent screen >3.6 per
1000


• Incident screen ≥3.1 per
1000
• Incident screen ≥4.2 per
1000

(b) Rate of cancers detected
that are in situ carcinoma
• Prevalent screen ≥0.4 per
1000



• Incident screen ≥0.5 per
1000


(c) Standardised detection
ratio
• ≥0.85
• ≥1.0
To maximise the number of
small invasive cancers
Rate of invasive cancers
less than 15 mm in diameter
detected in eligible women
invited and screened
• Prevalent screen ≥1.7 per
1000
• Prevalent screen ≥2.3 per
1000


• Incident screen ≥1.7 per
1000
• Incident screen ≥2.5 per
1000
To achieve optimum image
quality
(a) High-contrast spatial
resolution
• ≥12 lp/mm


(b) Minimal detectable
contrast:



5–6 mm detail
• ≤1.2%
• ≤0.8%

0.5 mm detail
• ≤5%
• ≤3%

0.25 mm detail
• ≤8%
• ≤5%

(c) Aim film density
• 1.5–1.9

To limit radiation dose
Mean glandular dose per film
for a standard breast at clinical
settings
• ≤2.5 mGy

To minimise the number of
women undergoing repeat
examinations
Number of repeat
examinations
• ≤3% of total examinations
• <2% of total examinations
To minimise the number of
women screened who are
referred for further tests*
(a) Percentage of women
who are referred for
assessment
• Prevalent screen <10%
• Incident screen <7%
• Prevalent screen <7%
• Incident screen <5%

(b) Percentage of women
screened who are placed
on early recall
• <0.5%
• ≤0.25%
To ensure that the majority
of cancers, both palpable
and impalpable, receive a
non-operative tissue diagnosis
of cancer
Percentage of women
who have a non-operative
diagnosis of cancer by
cytology or needle histology
after a maximum of two visits
• ≥80%
• >90%
To minimise the number
of unnecessary operative
procedures
Rate of benign biopsies
• Prevalent screen <3.6 per
1000
• Prevalent screen <1.8 per
1000

The role of imaging in breast diagnosis
7
discharged. The remaining 25% proceed to needle
biopsy and this results in a diagnosis of around six
cancers per 1000 women screened. Interval follow-
up of uncertain mammographic findings is dis-
couraged; the emphasis is on obtaining a definitive
diagnosis by the use of image-guided breast biopsy.
Over 90% of breast cancers should be diagnosed
prior to first surgery. Despite advances in breast
needle biopsy techniques, a small proportion of
women still require open surgical biopsy for diag-
nosis (up to 0.25% of women screened).
Sign
PPV (%)
Well-defined mass
<1
Ill-defined mass
35–50
Spiculated mass
50–90
Architectural distortion
20–40
Asymmetric density
<2
Clustered microcalcifications
15
Table 1.2  •  Positive predictive value (PPV) for
malignancy of mammographic signs
Objective
Criteria
Minimum standard
Target


• Incident screen <2.0 per
1000
• Incident screen <1.0 per
1000
To minimise the number
of cancers in the women
screened presenting between
screening episodes
Rate of cancers presenting in
screened women:
Expected standard


(a) In the 2 years following a
normal screening episode
• 1.2 per 1000 women screened in the first 2 years

(b) In the third year follow-
ing a normal screening
episode
• 1.4 per 1000 women screened in the third year
To ensure that women are
recalled for screening at
appropriate intervals
Percentage of eligible
women whose first offered
appointment is within
36 months of their previous
screen
• >90%
• 100%
To minimise anxiety for
women awaiting the results of
screening
Percentage of women who
are sent their result within
2 weeks
• >90%
• 100%
To minimise the interval from
the screening mammogram to
assessment
Percentage of women who
attend an assessment centre
within 3 weeks of attendance
for the screening mammogram
• >90%
• 100%
To minimise diagnostic delay
for women who are diagnosed
non-operatively
Proportion of women for whom
the time interval between
non-operative biopsy and
result is 1 week or less
• ≥90%
• 100%
To minimise the delay for
women who require surgical
assessment
Proportion of women for whom
the time interval between the
decision to refer to a surgeon
and surgical assessment is
1 week or less
• ≥90%
• 100%
To minimise any delay for
women who require treatment
for screen-detected breast
cancer
Percentage of women who
are admitted for treatment
within 2 months of their first
assessment visit
• >90%
• 100%
Table 1.1  •  (cont.) NHS Breast Screening Programme: screening targets, June 2005

Chapter 1
8
Figure 1.2  •  (a,b) Digital mammograms showing a spiculated mass in the right breast. The appearances are typical of
an invasive carcinoma. The mass contains microcalcifications and there is evidence of skin tether. (c) Doppler ultrasound
image showing the typical features of an invasive carcinoma with an irregular mass containing abnormal central vessels.
a
b
c
Figure 1.1  •  (a,b) Digital mammograms showing multiple
well-defined masses, the typical appearance of simple breast
cysts. (c) Ultrasound image showing typical features of a
simple cyst, i.e. a well-circumscribed anechoic mass with
distal acoustic enhancement.
a
b
c

The role of imaging in breast diagnosis
9
The performance of the NHS Breast Screening
Programme in 2010 is shown in Table  1.3. The
screening programme was predicted to produce a
25% reduction in mortality (1750 cancers per year)
directly attributable to early detection through
screening by the year 2010.
Adverse effects
Receiving an invitation for screening and attend-
ing for mammography are not associated with
any ­significant anxiety. However, recall for further
­assessment does cause measurable anxiety, although
this has largely subsided by 3 months.
The numbers of women who undergo open sur-
gical biopsy for what proves to be benign disease
should be kept to a minimum. Considerable train-
ing and investment in equipment has resulted in a
fourfold decline in benign surgical biopsies gener-
ated through the screening programme, although
rates of benign biopsy vary from unit to unit. False-
positive recall and benign surgical biopsy are both
more likely in younger women.
Overdiagnosis refers to the detection by screen-
ing of breast cancers that require treatment but that
would never have threatened the life of the woman.
This is inevitable in screening and there continues
to be considerable debate about what proportion
of screen-detected breast cancers fall into this cat-
egory. It is likely that most low-grade DCIS and
some special low-grade invasive cancers represent
overdiagnosis, and detection of these cancers results
in unnecessary treatment and unnecessary morbid-
ity associated with knowledge of the diagnosis of
cancer. The consensus view is that overdiagnosis ap-
plies to no more than 10% of screen-detected breast
cancer and that at this level this does not negate the
overall mortality benefit of breast screening. Women
attending for screening must be informed fully
about both the likely positive and negative effects
of screening. Efforts continue to reduce the morbid-
ity of screening and to understand better the natural
history of cancers so that potential over-treatment
is minimised. Even the most ardent critics of breast
Figure 1.3  •  Digital mammograms showing a cluster of
casting microcalcifications in the left breast. The appearances
are typical of high-grade ductal carcinoma in situ.

2008/2009
2009/2010
Total number of women invited (50–70)
2 642 511
2 662 298
Acceptance rate
73.8%
73.5%
Number of women screened (invited)
1947.424
1 954 815
Number of women screened (self-referral)
44,070
43 410
Total number of women screened
1 991 494
1 998 225
Number of women recalled for assessment
87,400
82 650
Percentage of women recalled for assessment
4.4%
4.1%
Number of benign surgical biopsies
1644
1519
Number of cancers detected
15 673
15 517
Number of in situ cancers detected
3253
3064
Number of cancers less than 15 mm
6460
6544
Standardised detection ratio (invited only)
1.45
1.44
Table 1.3  •  NHS Breast Screening Programme: results 2010
From NHS Breast Screening Programme Annual Review 2011. Available at

Chapter 1
10
screening believe screening should continue but they
argue it needs to be refined. This is accepted by those
who organise and run screening programmes.
Screening women at
increased risk
Women at increased risk of developing breast cancer
due to a proven inherited predisposing genetic muta-
tion, family history (with no proven genetic mutation),
previous radiotherapy (e.g. mantle radiotherapy for
Hodgkin's lymphoma) or benign risk lesions (atypical
hyperplasia, lobular intraepithelial neoplasia) may be
selected for screening at young age. Whether it is pos-
sible to identify other substantially increased risk
groups by summating various other epidemiological
factors (e.g. age at menarche, body mass index, age at
first pregnancy, alcohol intake) continues to be de-
bated. The cut-off point at which clinical management
of a woman is altered is often referred to as moderate
risk. Those individuals likely or proven to be carriers of
a predisposing genetic mutation are termed high risk.
Such cut-offs are useful as guidelines for specialist
referral, but most risk factors require clinical inter-
pretation before risk management is discussed with
the individual.
Unfortunately, the problem with screening young
women at increased risk of breast cancer is that
no screening test has as yet been shown to reduce
mortality in such women. Screening in this group is
therefore a management option for which an exact
benefit cannot be quoted for an individual woman.
Screening should not be offered to those who fall
below the moderate-risk cut-off.
Methods of screening young women at
increased risk: mammography
A national evaluation study of mammographic
screening for young women with a family history of
breast cancer has been conducted (FH01 study). The
study compared screening in women aged ­40–44
who were at moderate or high risk with the control
arm of the age trial (population-based trial of screen-
ing women in their forties in which the control arm
was not screened); this showed a non-­significant
mortality benefit for screening. It demonstrated that
screening was likely to reduce deaths from breast
cancer. The FH02 study is currently looking at the
effectiveness of screening women aged 35–39.
BRCA1-related breast cancer is usually high grade
and often has a ‘pushing’ margin. It rarely presents
with associated DCIS. The mammographic features
are therefore usually of a mass lesion with no as-
sociated microcalcification and no architectural
distortion (Fig.  1.4a). Such cancers often present
symptomatically as interval cases. BRCA2-related
cancers are more similar to sporadic cases and may
be more likely to be detected by mammography.
Ultrasound screening significantly improves sensi-
tivity when there is a dense mammographic back-
ground pattern but has a lower positive predictive
value and has not been shown to be a useful screen-
ing modality. Ultrasound features of BRCA1 can-
cers are often benign or indeterminate (Fig. 1.4b). If
mammographic screening is performed, it should be
repeated annually in women under age 50.
Methods of screening young women
at high risk: MRI
MRI is the most sensitive method of imaging young
women but has significant resource implications.44,48
The specificity of MRI has been a ­concern, although
with second-look recall (after which many poten-
tially abnormal findings may resolve), targeted
­ultrasound and the slowly increasing availability of
MRI-guided biopsy, this may be less of a problem
than initially thought. The MARIBS study evaluating
MRI in addition to mammography and ­several other
studies have shown that MRI is the most sensitive
screening test for young high-risk women, but it is
arguable whether the cancers ­detected are sufficient
to change the outcome of these young women.49 On
the basis of its better performance compared with
NICE guidelines have been produced (latest
version 2006) to classify risk groups and guide
care.43 They state that women at or near population
risk should be managed in primary care (defined
as <3% risk for women aged 40–49). Women at
moderate risk are those with a 10-year risk of 3–8%
between 40 and 49 years or a lifetime risk >17%,
and those at high risk are defined as those with a
10-year risk of >8% between 40 and 49 years or a
lifetime risk >30%.
Mammography has a greater positive
predictive value in young women at high risk when
compared with age-matched controls but lacks
sensitivity. This may be a particular problem in
women with BRCA1 mutations.24,44–47

The role of imaging in breast diagnosis
11
­mammography in increasing sensitivity, NICE has
recommended annual MRI surveillance for women
aged 30–39 years with a 10-year risk of >8% and
women aged 40–49 years with a 10-year risk >20%.43
MRI is also recommended for high-risk women with
a dense mammographic background pattern.
Age to start screening in young women
at increased risk
The age for starting screening should be based on
risk rather than the age of affected relatives. For
women at moderate risk, screening should start
at age 40. This can seem paradoxical if the reason
moderate risk has been established is because there
is one first-degree relative affected in their thirties.
However, if this is the only relative affected then the
individual is only at moderate risk and the emphasis
of management should be on reassurance rather than
screening. For women at high risk, screening may
be started at age 30. Such high-risk women should
ideally be managed in a specialist setting where
­experience with MRI screening is available. In the
UK such women may be best managed within the
NHS Breast Screening Programme. Women must be
advised about the limitations of screening at young
age. This is particularly relevant to known mutation
carriers for whom there is no evidence that screening
improves survival (cf. risk-reducing surgery).
Image-guided breast biopsy
Needle biopsy is highly accurate in determining the
nature of most breast lesions.50–53 Patients with ­benign
conditions avoid unnecessary surgery; ­carrying out
Figure 1.4  •  (a,b) Digital mammograms showing a small
circumscribed but ill-defined mass in the upper outer right
breast. (c) Ultrasound image of the same mass. Core biopsy
showed grade 3 invasive ductal carcinoma.
a
b
c

Chapter 1
12
open surgical biopsy for diagnosis should be regarded
as a failure of the diagnostic process. For patients who
are proven to have breast ­cancer, needle biopsy pro-
vides accurate understanding of the type and extent
of disease so ensuring that patients, and the doctors
treating them, are able to make informed treatment
choices. Needle biopsy not only provides accurate in-
formation on the ­nature of malignant disease, such as
histological type and grade, but also facilitates pre-
treatment ­assessment of tumour biology.
Which biopsy technique?
The current methods available for breast tissue
diagnosis are FNA cytology, needle core biopsy,
vacuum-assisted biopsy (VAB) and open surgical
biopsy.
FNA versus needle core biopsy
There has been much debate about the comparative
benefits of FNA and core biopsy,50–53 but 14 G 22-mm
automated core biopsy provides significantly greater
sensitivity, specificity and positive predictive value
than FNA. Results with core biopsy are particularly
superior to FNA in stereotactic biopsy of microcalci-
fications and architectural distortions.
The overall better performance achievable with
core biopsy compared with FNA is illustrated
in the performance of the NHS Breast Screening
Programme in the UK. In 1994, using FNA as the
primary diagnostic technique, fewer than 10% of
90 units were able to achieve the target of 70% pre-
operative diagnosis rate for cancer. By 2003, most
units had converted to automated core biopsy and
all units achieved the minimum standard and the
majority exceeded the expected standard of 90%
preoperative diagnosis rate.1
Vacuum-assisted biopsy (VAB)
The predominant reasons for not achieving an accu-
rate diagnosis by needle biopsy are sampling error
(missing the target) and failure to retrieve sufficient
representative material. These problems have been
largely addressed by the development of larger
­directional core techniques that yield significantly
greater volumes of tissue.54–56
VAB is a very successful method for improving the
diagnostic accuracy of borderline breast lesions and
lesions at sites in the breast difficult to biopsy using
other techniques. VAB has been shown to understage
both in situ and invasive cancer approximately half
as often as conventional core biopsy (typically 10%
vs. 20%).57,58 The VAB technique has a higher sen-
sitivity because it allows sampling of lesions at sites
that are difficult to biopsy using either FNA or core
biopsy and because the amount of tissue harvested
is at least five times greater per core specimen.
The indications for VAB include:
• very small mass lesions;
• architectural distortions;
• failed ‘conventional’ core biopsy;
• small clusters of microcalcifications;
• papillary and mucocele-like lesions;
• diffuse non-specific abnormality;
• excision of benign lesions;
• sentinel node sampling.
Core biopsy and VAB are now the recom-
mended techniques for sampling calcifications
and ­mammographic architectural distortions.59,60
For calcifications it is imperative that there is
proof of representative sampling with specimen
radiography. If calcification is not demonstrated
on the specimen radiograph and the histology is
­benign, then management cannot be based on this
­result as there is a high risk of sampling error; the
­procedure must ­either be repeated or open surgical
biopsy carried out.
Guidance techniques for breast
needle biopsy
Ultrasound provides real-time visualisation of
the ­biopsy procedure and visual confirmation of
­adequate sampling. Between 80% and 90% of breast
abnormalities will be clearly visible on ­ultrasound
and amenable to biopsy using this technique. For
impalpable abnormalities not visible on ultrasound,
stereotactic X-ray-guided biopsy is required. A few
lesions are only visible on MRI and require magnetic
resonance-guided biopsy.61 A number of different
approaches have been developed for this proce-
dure using both closed and open magnets. VAB is
Ultrasound guidance is the technique of
choice for biopsy of both palpable and impalpable
breast lesions; it is less costly, easy to perform and
more accurate than freehand or other image-guided
techniques.50

The role of imaging in breast diagnosis
13
the biopsy technique recommended for magnetic
­resonance-guided sampling.
The negative predictive value of combined nor-
mal mammography and ultrasound is extremely
high; where there is a clinically palpable abnormal-
ity and mammography and ultrasound are entirely
normal, the likelihood of malignancy is low (<1%).
However, in these circumstances it remains prudent
in the presence of a localised clinical abnormality
to carry out freehand needle biopsy to exclude the
occasional diffuse malignant process, such as classi-
cal invasive lobular carcinoma or low-grade DCIS,
that may be occult on both mammography and
ultrasound.
For stereotactic procedures it is prudent to mark
the biopsy site for future reference. Gel pellets or cel-
lulose combined with a metallic marker are preferred
and can be placed during the procedure to mark the
biopsy site. These markers have the advantage of be-
ing visible on ultrasound so that repeat biopsy or lo-
calisation for surgery can be subsequently performed
under ultrasound rather than X-ray guidance. These
markers dissolve and are reabsorbed in a few weeks,
leaving a small metal marker in case delayed X-ray
identification of the biopsy site is required.
Number of samples
A simple rule for satisfactory sampling using needle
techniques is to obtain sufficient material to achieve
a diagnosis.59,60,62 For ultrasound-guided core bi-
opsy, a diagnosis may be possible on a single core.
Showing on ultrasound that the needle has passed
through the centre of the abnormality and by ex-
amining the sample with the naked eye, it is usually,
but not always, possible to confirm whether a sat-
isfactory sample has been obtained. Because of this
and the knowledge that some lesions are heteroge-
neous, more extensive sampling of a lesion increases
sensitivity. The number of core specimens obtained
should reflect the nature of the abnormality being
sampled. For ultrasound-guided biopsy where there
is a suspicion of carcinoma, it is recommended that
multiple core specimens are obtained.
As stereotactic biopsy is used for abnormalities
that are difficult to define on ultrasound and are
therefore more difficult to sample, a minimum of
five core specimens should be obtained. Ensuring
that calcification is present in at least three separate
cores and/or five separate flecks of calcification are
retrieved from the area of suspicion is essential to en-
sure an accurate ­diagnosis. For calcifications many
prefer VAB, although some units perform 14-gauge
core biopsy as their initial biopsy technique.
When there is diagnostic uncertainty after core,
VAB can be used to obtain larger tissue volumes
(a 7-gauge VAB probe will retrieve approximately
300 mg per core). Ten- to seven-gauge mammotomy
probes can be used and are preferred for therapeutic
removal of breast lesions such as fibroadenomas.
Biopsy results
It is important that the results of FNA and/or core
needle breast biopsy are always correlated with
the clinical and imaging findings before clinical
management is discussed with the patient. This is
best achieved by reviewing each case prior to any
­surgical or other therapeutic procedure at multidis-
ciplinary meetings.50
Surgery for clinically occult
breast lesions
Wire-guided excision
The number of impalpable, clinically occult breast
lesions detected by screening is increasing. Accurate
localisation techniques are required to facilitate
their surgical excision. The hooked wire is the most
commonly employed technique and has proved very
reliable but does have inherent associated prob-
lems. There are various designs of localisation wire
in common use. All have some form of anchoring
device such as a hook with a splayed or barbed tip.
The wire is deployed under stereotactic or ultra-
sound guidance within a rigid over-sheath cannula,
which is then removed once positioning is satisfac-
tory (Fig. 1.5). The patient is then transferred to the
operating theatre with the wire in situ. Most wires
are very flexible and when the cannula is removed
the wire may assume a quite circuitous course, es-
pecially after stereotactic insertion when the breast
is released from compression. In a very fatty breast
in which there is no solid lesion or the wire has not
transfixed the lesion, care must be taken to avoid
displacing the wire. A cosmetically considered in-
cision is placed near to the tip of the wire and an
excision performed. Accurate wire placement is
­essential and ideally the shortest possible length of

Chapter 1
14
wire should be within the breast. In recent practice
this has been greatly facilitated by the use of radio-
opaque and ultrasound visible markers placed at the
time of initial stereotactic biopsy such that wire lo-
calisation can be performed under ultrasound guid-
ance (Fig. 1.6). In addition, for superficial lesions a
skin marker may be more appropriate.
Although lesions may be clinically occult prior to
surgery, most mass lesions will be palpable intra-
operatively. Procedures that can be surgically more
challenging are wide local excisions for DCIS with
no mass lesion. In such cases, where the distribution
of disease is often more eccentric, careful excision
planning is necessary. Inserting more than one wire
and bracketing the lesion with three or four wires
can be useful.
If the procedure is being performed to establish a
diagnosis, a representative portion of the lesion is
excised through a small incision, so leaving a sat-
isfactory cosmetic result if the lesion proves to be
benign (the European surgical quality assurance
guidelines require such diagnostic surgical excision
specimens to weigh less than 30 g). For diagnostic
excisions of very small lesions, a therapeutic wide
excision may (after discussion with the patient) be
considered appropriate, as the resulting cosmetic ef-
Figure 1.5  •  Digital mammogram showing a Reidy localisation wire placed under ultrasound guidance transfixing and
marking an impalpable mass to facilitate surgical wide local excision (as shown in Fig. 1.4).
a
b
Figure 1.6  •  (a) Ultrasound showing a cluster of gel pellets placed at the site of a previous stereotactic biopsy. The
clear visibility of the pellets facilitates ultrasound localisation for surgery of abnormalities that would normally require
X-ray localisation. (b) Mammogram after ultrasound localisation in the same case showing accurate placement of the
marker wire.
a
b

The role of imaging in breast diagnosis
15
fect of removing an extra rim of normal surrounding
tissue may be insignificant. Protocols vary for thera-
peutic excisions, but in general the lesion should be
excised with a 10-mm macroscopic margin of nor-
mal tissue. Intraoperative specimen radiography is
essential, both to check that the lesion has been re-
moved and, if cancer has been diagnosed, to ensure
that adequate wide local excision has been achieved.
Some surgeons experienced in this imaging tech-
nique have also used intraoperative ultrasound. Not
only can excision be guided but the margins of a
wide local excision specimen can also be assessed
intraoperatively using ultrasound.
Radioisotope occult lesion
localisation
Radioisotope occult lesion localisation (ROLL) has
been advocated as an alternative to the hooked-wire
technique.63 ROLL was first described by the Milan
group using 99mTc-labelled human ­macroaggregate
albumin, using scintigraphy and a hand-held gamma
probe to guide surgical excision. The Nottingham
method has modified the Milan technique and uses
radio-opaque contrast injected with the radiolabel
and immediate check mammography (Fig.  1.7).
Subsequently some centres have combined ROLL
with sentinel node biopsy. ROLL uses essentially the
same equipment as sentinel node biopsy. It has been
described using macroaggregate (which does not
migrate from the injection site) or low-molecular-
weight colloid (which does migrate and is normally
used for sentinel node biopsy). In both situations
it is radiolabelled with 99mTc and injected directly
into the lesion. The threshold of the signal proces-
sor on the gamma detector is then adjusted so that
an audible signal is heard only when the probe is
directly over the lesion. The probe then directs exci-
sion intraoperatively.
In a randomised trial of ROLL versus wire
­localisation, 2% of ROLL patients had a failed
Figure 1.7  •  (a) Mammogram of the right breast showing a small impalpable cancer. (b) Mammogram after injection of
radionuclide mixed with X-ray contrast confirming satisfactory localisation (ROLL).
a
b

Chapter 1
16
­technique due to intraductal injection of ­radiolabelled
colloid and dye that gave a ductogram ­appearance on
check mammography in both cases.64,65 As the radio-
opaque dye is absorbed rapidly, both cases were suc-
cessfully converted to wire localisation. The main
differences between ROLL and wire guidance were
that both surgeons and radiologists found ROLL eas-
ier to perform overall and patients found ROLL less
painful. There has been no significant difference in
accuracy of marking, operating time, mean specimen
weight, intraoperative re-excision or second thera-
peutic operation in the majority of reports, although
a recent European trial reported greater volumes of
tissue were excised using ROLL than standard wire lo-
calisation. Some studies have suggested that obtaining
clear margins may be significantly easier with ROLL.
In reality there is little to choose between ROLL and
wire ­localisation. ROLL may be a more suitable tech-
nique in the localisation of non-mass lesions (e.g.
DCIS), although even for these lesions when multiple
wires are placed any advantage is small.
There are various methods described for combining
ROLL with sentinel node biopsy.66–68 Low-molecular-
weight colloid can be injected at a different site, at
the same site with a different radiolabel, or into the
­tumour. With intratumoral injection of 99mTc nano-
colloid, only one injection is required and high success
rates have been reported. Combined with radio-
opaque contrast, this modification of the Nottingham
method has proved simple and successful.
Oncoplastic considerations
for screen-detected lesions
Oncoplastic surgery aims to provide optimum ef-
fectiveness of surgical treatment for breast cancer
with minimum effect on quality of life. As the 10-
year survival of screen-detected disease is estimated
at 87%, women do live a long time with the effects
of breast cancer surgery on body image, quality
of life and self-esteem. The degree to which these
outcomes are affected is strongly related to the cos-
metic outcome of surgery.
When assessing a woman for surgery for screen-
detected cancer, there should be a strong emphasis
towards the aesthetic outcome. Women should be
offered the full range of oncoplastic procedures
available to achieve a good outcome, including
where appropriate the quality-of-life benefits from
breast reduction. In addition, consideration should
be given to whether such procedures which reduce
re-operation rates may be desirable to achieve an
excellent cosmetic outcome. The latter consider-
ation may be particularly relevant for DCIS, for
which over 30% require further surgery follow-
ing an initial attempt at breast-conserving surgery
that fails because of involved excision margins. For
women with larger areas of DCIS, therapeutic mam-
moplasty may be the only option for achieving com-
plete excision and a satisfactory cosmetic outcome.
Such procedures should be available to all women.
Key points
• Breast imaging is an essential part of modern multidisciplinary breast diagnosis.
• Mammography is the technique of choice for population breast screening.
• Screening is targeted at women aged 50–70 years and can be expected to reduce mortality
through early detection by approximately 25%.
• The aim should be to achieve as near as possible 100% non-operative diagnosis of breast
problems.
• Both palpable and impalpable breast lesions are best sampled under image guidance.
• Automated core biopsy is the sampling technique of first choice.
• Ultrasound is the guidance technique of first choice.
• Digital stereotactic core biopsy should be reserved for sampling lesions not visible on ultrasound.
• A 14-gauge core biopsy can provide a definitive diagnosis in more than 90% of cases and should
be the preferred method.
• Mammotomy can provide the diagnosis in most of the remainder.
• Stereo-guided vacuum-assisted biopsy is particularly effective for small clusters of indeterminate
microcalcifications and calcifications in sites difficult to access with core biopsy.

The role of imaging in breast diagnosis
17
References
1. Office of National Statistics. Available from: http://

and-mortality/2008-2010/stb-cancer-incidence-and-
mortality-in-the-united-kindom--2008-2010.html;
2012.
2. Blanks RG, Moss SM, McGahan CE, et al. Effects
of NHS breast screening programme on mortality
from breast cancer in England and Wales, 1990–8:
comparison of observed with predicted mortality.
Br Med J 2000;321:1724–31.
3. National Institute for Health and Clinical Excellence.
NICE Clinical Guideline 80: Early and locally ad-
vanced breast cancer. 2009. Available from http://
guidance.nice.org.uk/CG80/NICEGuidance/pdf/
English;[accessed 23.07.12].
4. Willett AM, Michell MJ, Lee MJR. Available from
Best Practice Guideline for Patients Presenting
with Breast Symptoms. 2010. [accessed 23.07.12]

media/4585/best_practice_diagnostic_guidelines_
for_patients_presenting_with_breast_symptoms.pdf
5. Kolb TM, Lichy J, Newhouse JH. Comparison
of the performance of screening mammography,
­physical examination, and breast US and evaluation
of factors that influence them: an analysis of 27,825
patient evaluations. Radiology 2002;225:165–75.
6. Skaane P, Young K, Skjennald A. Comparison of film-
screen mammography and full-field mammography
with soft-copy reading in a population-based screening
program: the Oslo II study. Radiology 2002;225:267.
7. Pisano ED, Gatsonsis C, Hendrick E, et  al.
Diagnostic performance of digital versus film mam-
mography for breast cancer screening. N Engl J Med
2005;353:1773–83.
8. James JJ. The current status of digital mammogra-
phy. Clin Radiol 2004;59:1–10.
9. Committee on Technologies for the Early Detection
of Breast Cancer. Mammography and beyond:
­developing technologies for the early detection of
breast cancer. Washington, DC: National Academy
Press; 2001.
10. Legood R, Gray A. NHSBSP Equipment Report
0403. A cost comparison of full-field digital mam-
mography with film-screen mammography in breast
cancer screening. Sheffield: NHS Breast Screening
Programme Publications; 2004.
11. Gur D, Sumkin JH, Rockette HE, et al. Changes in
breast cancer detection and mammography recall
rates after the introduction of a computer-aided de-
tection system. J Natl Cancer Inst 2004;96:185–90.
12. Wilson ARM, Teh W. Mini symposium: Imaging
of the breast. Ultrasound of the breast. Imaging
1998;9:169–85.
13. Lister D, Evans AJ, Burrell HC, et  al. The ac-
curacy of breast ultrasound in the evaluation of
clinically benign discrete breast lumps. Clin Radiol
1998;53:490–2.
14. Berg WA, Blume JD, Cormack JD, et  al. ACRIN
6666 Investigators. Combined screening with ultra-
sound and mammography vs mammography alone
in women at elevated risk of breast cancer. JAMA
2008;299:2151–63.
15. Berg WA, Zhang Z, Lehrer D, et  al. Detection of
breast cancer with addition of annual screening
ultrasound or a single screening MRI to mammog-
raphy in women with elevated breast cancer risk.
JAMA 2012;307:1394–404.
16. Damera A, Evans AJ, Cornford EJ, et al. Diagnosis
of axillary nodal metastases by ultrasound guided
core biopsy in primary operable breast cancer.
Br J Cancer 2003;89:1310–3.
17. Houssami N, Ciatto S, Turner R, et al. Preoperative
ultrasound-needle biopsy of axillary nodes in invasive
breast cancer: meta-analysis of its accuracy and util-
ity in staging the axilla. Ann Surg 2011;254:243–51.
18. Kuhl C. The current status of breast MR imaging. Part 2.
Clinical applications. Radiology 2007;244:672–91.
19. Sardanelli F, Boetes C, Borisch B, et  al. Magnetic
resonance imaging of the breast: recommendations
of the EUSOMA working group. Eur J Cancer
2010;46:1296–316.
20. Kuhl CK, Schrading S, Bieling HB, et al. MRI for di-
agnosis of pure ductal carcinoma in situ: a prospec-
tive observational study. Lancet 2007;370:485–92.
• VAB is an effective and well-tolerated sampling technique for breast diagnosis and can also be
used to completely excise benign lesions.
• All breast needle biopsy results should be discussed at prospective multidisciplinary meetings
where the pathology results are correlated with the clinical and imaging findings.
• Accurate image-guided localisation and skills in wide local excision are required for the surgical
treatment of impalpable breast lesions.
• The aim of surgery is to excise the cancer completely and produce an excellent cosmetic
outcome.
• Oncoplastic surgery and therapeutic mammoplasty in appropriately selected women increases the
rate of excellent cosmetic outcomes.

Chapter 1
18
21. Kuhl CK, Schmutzler RK, Leutner CC, et  al.
Mammography, breast ultrasound and magnetic
resonance imaging for surveillance of women at
high familial risk for breast cancer. J Clin Oncol
2005;23:8469–76.
22. Warner E, Plewes DB, Shumak RS, et al. Comparison
of breast magnetic resonance imaging, mammogra-
phy, and ultrasound for surveillance of women at
high risk for hereditary breast cancer. J Clin Oncol
2001;19:3524–31.
23. Stoutjesdijk MJ, Boetes C, Jager GJ, et al. Magnetic
resonance imaging and mammography in women
with a hereditary risk of breast cancer. J Natl Cancer
Inst 2001;93:1095–102.
24. Brekelmans CTM, Seynaeve C, Bartels CCM,
et  al. Effectiveness of breast cancer surveil-
lance in BRCA1/2 gene mutation carriers and
women with high familial risk. J Clin Oncol
2001;19:924–30.
25. Olsen O, Gotzsche PC. Cochrane review on screen-
ing for breast cancer with mammography. Lancet
2001;358:1340–2.
26. Olsen O, Gotzsche PC. Systematic review of ­screening
for breast cancer with mammography. Available at

27. WHO handbook on cancer prevention. 7th ed
Lyons: IARC Press; 2002.
A comprehensive review of all the available data on the
effectiveness of breast cancer screening in reducing
breast cancer mortality.
28. Nystrom L, Andersson I, Bjurstam N, et al. Long-
term effects of mammographic screening: update
overview of the Swedish randomised trials. Lancet
2002;359:909–19.
Long-term follow-up of the combined Swedish trials
showing significant mortality benefit after more than
20 years.
29. Tabar L, Vitak B, Tony HH, et  al. Beyond ran-
domised controlled trials: organised mammographic
screening substantially reduces breast carcinoma
mortality. Cancer 2001;91:1724–31.
30. Duffy S, Tabar L, Chen HH, et al. The impact of
organised mammographic screening on breast car-
cinoma mortality in seven Swedish counties. Cancer
2002;95:458–69.
31. Hackshaw AK, Paul EA. Breast self-examination
and death from breast cancer: a meta-analysis.
Br J Cancer 2003;88:1047–53.
32. Smith RA, Saslow D, Sawyer KA, et al. American
Cancer
Society
guidelines
for
breast
can-
cer screening: update 2003. CA Cancer J Clin
2003;53:141–69.
33. Breast Screening Frequency Trial Group. The fre-
quency of breast cancer screening: results from
the UKCCCR randomized trial. Eur J Cancer
2002;38:1458–64.
34. Million Women Study Collaborators. Breast cancer
and hormone replacement therapy in the Million
Women Study. Lancet 2003;362:419–27.
Report of significantly increased risk of breast cancer in
women taking HRT in the UK.
35. Perrson I, Thurfjell E, Holmberg I. Effect of ­estrogen
and estrogen–progestin replacement regimes on mam-
mographic breast parenchymal density. J Clin Oncol
1997;15:3201–7.
36. Sendag F, Cosan Terek M, Ozsener S, et al. mammo-
graphic density changes during different postmeno-
pausal hormone replacement therapies. Fertil Steril
2001;76:445–50.
37. Evans A. Hormone replacement therapy and mam-
mographic screening. Clin Radiol 2002;57:563–4.
38. Litherland JC, Stallard S, Hole D, et  al. The ef-
fect of hormone replacement therapy on the sen-
sitivity of screening mammograms. Clin Radiol
1999;54:285–8.
39. Kavanagh AM, Mitchell H, Giles GG. Hormone re-
placement therapy and accuracy of mammographic
screening. Lancet 2000;355:270–4.
40. Litherland JC, Evans AJ, Wilson ARM. The effect of
hormone replacement therapy on recall rate in the
National Health Breast Screening Programme. Clin
Radiol 1997;52:276–9.
41. Evans AJ, Pinder SE, Ellis IO, et al. Screen detected
ductal carcinoma in situ (DCIS): over-diagnosis or
obligate precursor of invasive disease? J Med Screen
2001;8:149–51.
42. Evans AJ, Burrell HE, Pinder SE, et  al. Detecting
which invasive cancers at mammographic screening
saves lives? J Med Screen 2001;8:86–90.
43. National
Institute
for
Clinical
Excellence.
Guidance on cancer services. Familial breast
cancer. London: National Institute for Clinical
Excellence; 2006.Available from
org.uk;[accessed 30.10.12].
44. Robson M. Breast cancer surveillance in women
with hereditary risk due to BRCA1 or BRCA2 muta-
tions. Clin Breast Cancer 2004;5:260–8.
45. Tilanus-Linthorst M, Verhoog L, Obdeijn I-M,
et al. A BRCA1/2 mutation, high breast density and
prominent pushing margins of a tumour indepen-
dently contribute to a frequent false-negative mam-
mography. Int J Cancer 2002;102:91–5.
46. Warner E, Plewes DB, Hill KA, et al. Surveillance of
BRCA1 and BRCA2 mutation carriers with magnetic
resonance imaging, ultrasound, mammography, and
clinical breast examination. JAMA 2004;292:1317–25.
47. Hamilton LJ, Evans AJ, Wilson ARM, et al. Breast
imaging findings in women with BRCA1- and
BRCA2-associated breast cancer. Clin Radiol
2004;59:895–902.
48. Kriege M, Brekelmans CTM, Boetes C, et  al.
Efficacy of MRI and mammography for breast

The role of imaging in breast diagnosis
19
­cancer ­screening in women with a familial or genetic
predisposition. N Engl J Med 2004;351:427–37.
49. Leach MO, Boggis CR, Dixon AK, et al. Screening
with magnetic resonance imaging and mammog-
raphy of a UK population at high familial risk of
breast cancer: a prospective multicentre cohort study
(MARIBS). Lancet 2005;365:1769–78.
50. Teh W, Wilson ARM. Definitive non-surgical breast
diagnosis: the role of the radiologist. Clin Radiol
1998;53:81–4.
51. Britton PD. Fine needle aspiration or core biopsy.
Breast 1999;8:1–4.
52. Britton PD, McCann J. Needle biopsy in the NHS
Breast Screening Programme 1996/7: how much and
how accurate? Breast 1999;8:5–11.
53. Vargas HI, Agbunag RV, Khaikhali I. State of the art
of minimally invasive breast biopsy: principles and
practice. Breast Cancer 2000;7:370–9.
54. Heywang-Kobrunner SH, Schaumloffel U, Viehweg
P, et al. Minimally invasive stereotaxic vacuum core
breast biopsy. Eur Radiol 1998;8:377–85.
55. Brem RF, Schoonjans JM, Sanow L, et al. Reliability
of histologic diagnosis of breast cancer with
stereotactic vacuum-assisted biopsy. Am Surg
2001;67:388–92.
56. Parker
SH,
Klaus
AJ,
McWey
PJ,
et 
al.
Sonographically guided directional vacuum-assisted
breast biopsy using a handheld device. AJR Am J
Roentgenol 2001;177:405–8.
57. Kettritz U, Rotter K, Murauer M, et al. Stereotactic
vacuum biopsy in 2874 patients: a multicenter study.
Cancer 2004;100:245–51.
58. Brenner RJ, Bassett LW, Fajardo LL, et al. Stereotactic
core needle breast biopsy: a multi-institutional pro-
spective trial. Radiology 2001;218:866–72.
59. Wilson ARM. Therapeutic applications of vacuum
biopsy. Breast Cancer Res 2008;4:31.
60. Wilson R, Kavia S. Comparison of large-core vac-
uum-assisted breast biopsy and excision systems.
Recent Results Cancer Res 2009;173:23–41.
61. Kuhl CK, Morakkabati N, Leutner CC, et al. MR
imaging-guided large-core (14-gauge) needle biopsy
of small lesions visible at breast MR imaging alone.
Radiology 2001;220:31–9.
62. Fishman JE, Milikowski C, Ramsinghani R, et al. US-
guided core-needle biopsy of the breast: how many
specimens are necessary? Radiology 2003;226:779–82.
63. Luini A, Zurrida S, Paganelli G, et al. Comparison of
radioguided excision with wire localisation of occult
breast lesions. Br J Surg 1999;86:522–5.
64. Rampaul RS, Bagnall M, Burrell H, et  al.
Radioisotope for occult lesion localisation: results
from a prospective randomised trial of ROLL versus
wire guidance in occult lesions of the breast. Br J
Surg 2004;91:1575–7.
65. Rampaul RS, Burrell H, Macmillan RD, et  al.
Intraductal injection of the breast: a potential pitfall
of radioisotope occult lesion localisation. Br J Radiol
2002;76:425–6.
66. Patel A, Pain SJ, Britton P, et al. Radioguided occult
lesion localisation (ROLL) and sentinel node biopsy
for impalpable invasive breast cancer. Eur J Surg
Oncol 2004;30:918–23.
67. Tanis PJ, Deurloo EE, Valdes Olmos RA. Single in-
tralesional tracer dose for radio-guided excision of
clinically occult breast cancer and sentinel node.
Ann Surg Oncol 2001;8:850–5.
68. Gray RJ, Giuliano R, Dauway EL, et  al.
Radioguidance for nonpalpable primary lesions and
sentinel lymph node(s). Am J Surg 2001;182:404–6.

20
2
Rajendra S. Rampaul
Emad A. Rakha
John F.R. Robertson
Ian O. Ellis
Pathology and biology of breast cancer
Introduction
Management of women with breast carcinoma has
undergone significant changes over the past 20 years.
The pathology and biology of breast cancer influ-
ences diagnosis, selection of primary and adjuvant
treatment, formulation of follow-up protocols, prog-
nosis and provision of counselling and reassurance.
Screening and public education have accounted for a
major shift in the number and type of the breast car-
cinomas detected today. Cancers are now of smaller
size and more often lymph node negative. There is an
increasing need to discriminate accurately the risk of
recurrence and select appropriate adjuvant systemic
therapies. Modern clinical practice employs a signifi-
cant input from histopathological data to assist in
the decision-making process for selecting treatments.
This can be achieved by identifying accurate prog-
nostic and predictive factors. A prognostic factor is
defined as any patient or tumour characteristic that
is predictive of the patient's outcome. Outcome is
usually measured in terms of cancer-specific survival
or disease-free survival. A predictive factor is defined
as any patient or tumour characteristic that is predic-
tive of the patient's response (outcome) to a specified
treatment. The factors currently employed in breast
cancer prognostication and prediction each possess
independent prognostic information and predictive
power and may be combined into an index, making
it more ‘user friendly’, informative and ­reproducible.
A prognostic index is defined as quantitative set of
values based on results of a prognostic model. There
are several reasons for the use of such prognostic in-
dices in breast cancer, which include the ability:
1. to separate patients into groups with signifi-
cantly differing survival probabilities;
2. to separate patients into groups that include a
‘cured’ group and a group with poor survival;
3. to place a sufficient percentage of cases into
each group;
4. to be applicable for all operable breast cancers
– small, screen detected as well as symptomatic
and young age;
5. to be prospectively validated;
6. to be capable of use in all units and to be
inexpensive.
In this chapter common pathological features of
breast cancer are reviewed and their role in guiding
patients' management considered.
Traditional factors
Lymph node stage
Involvement of local and regional lymph nodes by
metastatic carcinoma is one of the most important
prognostic factors in breast cancer. The revised

Pathology and biology of breast cancer
21
TNM staging system for breast cancer has con-
firmed that the absolute number of axillary lymph
nodes involved in metastatic cancer (‘positive lymph
nodes’) is one of the most important prognostic fac-
tors in breast cancer.1 Lymph node stage has been
used consistently as a guide for therapy. However,
lymph node stage is considered as a time-dependent
factor; the longer the tumour has been growing the
more likely it is that lymph nodes are involved by
spread. It has also been reported that, when taken
alone, lymph node stage is incapable of defining ei-
ther a cured group or one with close to 100% mor-
tality from breast cancer.
The clinical assessment of nodal status (as in
TNM classification) is unreliable.2,3 Palpable nodes
may be enlarged because they show benign
­reactive changes whilst nodes bearing tumour de-
posits can be impalpable. Although axillary so-
nography is a moderately sensitive and fairly
specific technique in the diagnosis of axillary met-
astatic involvement,4 histological examination of
the lymph nodes from the axilla should be carried
out in all patients with primary operable invasive
breast cancer. It is well known that patients who
have histologically confirmed lymph node in-
volvement have a significantly poorer prognosis
than those without nodal metastases. The 10-year
survival is reduced from 85% for patients with no
nodal involvement to 40% for those with involve-
ment of multiple nodes. Prognosis worsens the
greater the number of nodes involved, and the
level of nodal involvement can provide useful in-
formation; metastasis to the higher level nodes
(level II or III) in the axilla and particularly those
at the apex (level III) carries a worse prognosis
(Fig. 2.1).
Optimal management of the axilla is discussed in
Chapter 9.
A refinement of lymph node sampling is pro-
vided by the technique of sentinel lymph node
(SLN) biopsy. There have been several studies
to examine the validity of this technique. The
ALMANAC trial provides level I evidence for UK-
based practice.3
From a pathological perspective, there are unre-
solved questions about how to optimally assess the
sentinel node. Studies have shown that the mean
number of sentinel nodes is close to 3. As the num-
ber of nodes removed during SLN biopsy is fewer,
pathologists are now challenged to process these
few nodes optimally. Several methods have been
studied: routine paraffin histology; intraoperative
frozen section; immunohistochemistry and more
intensive methods such as serial sectioning and
molecular methods including polymerase chain
reaction (PCR) and reverse transcription–PCR
(RT–PCR).
At present there is no consensus for the optimal
handling of the SLN in the laboratory. Most cen-
tres have developed their own ‘in-house’ protocols,
which are invariably tied to the institution's research
ambitions. This ad hoc approach can influence in-
terpretation of results, as the amount of material as-
sessed between centres is not uniform. In a number
of large studies, using different histopathological
techniques, the SLN false-negative rate (defined as
how often the SLN is negative for malignancy when
cancer is present in the rest of the axilla) varies be-
tween 0% and 11%.
In Nottingham the MRC ALMANAC protocol
is employed to assess all sentinel node biopsies.
Studies show that overall survival is
decreased as the number of nodes that are involved
increases.5–7
Lymph node stage
Node negative
1–3 nodes positive
>3 nodes positive
Probability
1.0
0.6
0.8
0.4
0.0
0.2
0
250
100
150
200
50
Survival in months
p<0.0001
Figure 2.1  •  Overall survival for patients in the NTPBCS
according to lymph node stage (P < 0.0001).
Sentinel node biopsy or blue dye directed
axillary sampling has been shown to provide
accurate prognostic information and has an
extremely low rate of lymphoedema.2 Of 1275
patients undergoing axillary sampling, only 0.04%
suffered symptomatic complaints of arm swelling
and lymphoedema.

Chapter 2
22
These nodes are cut into slices about 3 mm thick,
taken perpendicular to the long axis of the node
to maximise the assessment of the marginal si-
nus, with one node per cassette. The majority of
nodes can be completely embedded in one cas-
sette. Larger nodes have alternate slices embed-
ded and may require more than one cassette.
Large, obviously involved nodes have one section
taken.
Intraoperative frozen sections or imprint cytology
of axillary lymph nodes have been advocated by
some authors. Conventional frozen sections have a
high false-negative rate of between 10% and 30%.
More intensive intraoperative assessment with se-
rial sections and immunohistochemistry has been
described,8 but is time consuming and labour in-
tensive. Frozen section is best applied to selected
cases; for example, if the node is macroscopically
abnormal and this is confirmed histologically to
be metastatic carcinoma, further axillary surgery
can be performed immediately. Some studies have
found low false-negative rates of 2–3% with intra-
operative imprint cytology,9 but not all have been
able to achieve this level of accuracy. Lymph node
status can be assessed peroperatively using ultra-
sound combined with FNA or core biopsy, and
this reduces the need for preoperative frozen sec-
tion and imprint cytology to assess axillary nodes
intraoperatively.
RT–PCR is more sensitive than immunohisto-
chemistry at detecting metastatic tumour in axil-
lary nodes. Two types of methods have been used
to detect tumour cells with molecular techniques.
First, a genetic defect such as chromosomal rear-
rangement or mutation can be used. The problem
with this is that no single genetic defect is seen in
all breast carcinomas. The second method is to use
a molecular marker that is present in tumour cells
but not in the adjacent tissue. To identify a marker
that has this specificity and is expressed in the ma-
jority of tumours is difficult. It may be necessary
to use a panel of markers. A major problem with
PCR is the potentially high false-positive rate due
to the sensitivity of the method and potential for
contamination. In addition, it is not possible with
PCR to determine whether the DNA comes from
cells that are viable. An advantage of haematoxylin/
eosin sections and immunohistochemistry is that the
morphology of the cells can be examined and ma-
lignancy confirmed. A major unresolved ­question is
whether carcinoma detected only by PCR or RT–
PCR has the same prognostic significance as haema-
toxylin/eosin positivity.
The detection rate of micrometastases in axillary
lymph nodes has been reported to range from 9% to
46%.10 Studies have used serial sectioning with or
without immunohistochemical stains to detect mi-
crometastatic foci and these methods have increased
detection rates.
The European Working Group for Breast Screening
Pathology11 has formulated working guidelines for
the assessment and pathological work-up of SLNs
in breast cancer. From a literature review available
at that time, the committee concluded that it was
not possible to determine the significance of micro-
metastasis or isolated tumour cells. They noted that
approximately 18% of cases are associated with
other nodal (non-sentinel node) metastases. False-
negative rates are most often determined via im-
munohistochemistry (IHC). However, at present it
is recommended that an intensive work-up is not
justified on a population level. The committee did
suggest multilevel assessment and, where resources
permit, intraoperative assessment, although the re-
sults of Z0011 (see Chapter 7) suggest that some
node-positive patients may not require axillary dis-
section and this brings the whole issue of intraop-
erative assessment into question. The current view
is that IHC should not be used routinely to assess
axillary lymph nodes.
Tumour size
Tumour size is one of the most powerful predic-
tors of prognostic factor in breast cancer.12,13 The
frequency of nodal metastases in patients with
tumours <10 mm is 10–20%,13 and node-negative
patients with tumours <10 mm have a 10-year
disease-free survival rate of greater than 90%.14
Tumour size is a time-dependent prognostic factor
that depends on the period between tumour de-
velopment and detection, and on the balance be-
tween tumour cell proliferation and death (tumour
growth rate). It is well known that the association
between increasing tumour size and increasing
number of positive lymph nodes and worse out-
come is highly statistically significant.13 Therefore,
the main aim of population screening with mam-
mography is to detect smaller tumours that are

Pathology and biology of breast cancer
23
likely to have a better outcome than those that
present symptomatically (of a larger size). Patients
with smaller tumours have a better long-term sur-
vival than those with larger tumours (Fig.  2.2).
Estimation of tumour size has assumed particular
importance since the introduction of ­population
screening. In most studies the frequency of axillary
lymph node metastasis in small <15 mm (so-called
minimally invasive carcinoma (MIC)) invasive
carcinomas is 15–20%,15–17 compared with over
40% in tumours measuring 15 mm or more.18
During the prevalent round of breast screening
even more favourable results are obtained, with
the frequency of axillary lymph node metastasis
ranging from 0% to 15%.19–22 The Nottingham
Tenovus Primary Breast Cancer Study (NTPBCS)
has generated data that suggest the cut-off point
of 10 mm is not the best discriminator for MIC.
Life-table analysis of survival curves found no dif-
ference between tumours measuring up to 9 mm
and those measuring 10–14 mm. This indicates
that <15 mm may be a more realistic watershed
in defining small, invasive, carcinomas of good
prognosis. It is clear that pathological tumour size
is a valuable prognostic factor and it has become
an important quality assurance measure for breast
screening.21,23–26 It is also used in part to judge the
ability of radiologists to detect small impalpable
invasive carcinomas.
Differentiation
Modern pathologists have recognised that invasive
carcinomas can be divided according to their degree
of differentiation. There are two ways to achieve
this: (i) by allocating a histological type according
to the architectural pattern of the tumour; (ii) by
assigning a grade of differentiation based on semi-
quantitative evaluation of structural characteristics.
Certain histological types of invasive carcinoma
carry a favourable prognosis. Tubular, mucinous,
invasive cribriform, medullary and tubulolobu-
lar types, together with rare tumour types such as
adenoid cystic carcinoma, adenomyoepithelioma
and low-grade and squamous carcinoma, have all
been reported to have a more favourable outcome
than invasive carcinoma of no special type (ductal
NST). Undoubtedly, assessment of histological type
provides prognostic information in breast cancer.
However, this effect is relatively small in multivari-
ate analysis27 when compared with the prognostic
value of histological grade; histological type may
prove to be more useful in increasing our under-
standing of the biology of breast cancers.28
Invasive lobular carcinoma (ILC) is of particular
importance. It comprises approximately 5–15% of
breast cancers and appears to have a distinct biology.
It is less common than invasive carcinoma of no spe-
cial type, also called invasive ductal carcinoma (IDC).
0.8
0.7
0.5
0.4
0.3
0.2
0.1
0.6
1.0
0.0
0.9
Cum survival
12
24
36
48
60
72
84
96 108 120 132 144 156 168 180
0
Months
Survival by size
0–2 cm
2.01–5 cm
Figure 2.2  •  Overall survival according to size.

Chapter 2
24
Tumour grade
The Nottingham method (Elston and Ellis) is a
modification and improvement of previously exist-
ing morphological grading systems and is able to
provide greater objectivity of grade.30
In brief, assessment of grade considers three tumour
characteristics: tubule formation, nuclear pleomor-
phism and mitotic counts. A numerical scoring sys-
tem of 1–3 is used for each factor individually. The
three scores are added together to produce scores of
3–9 on which an overall tumour grade is assigned:
• Grade 1 – well differentiated = score 3–5 points.
• Grade 2 – moderately differentiated = score 6–7
points.
• Grade 3 – poorly differentiated = score 8–9
points.
It should be noted that grade is valuable irrespective
of histological type.
There is a highly significant correlation between tu-
mour grade with long-term prognosis (Fig. 2.3); pa-
tients with grade 1 tumours have a 93% chance of
surviving 10 years after diagnosis, whereas in patients
with grade 3 tumours this is reduced to 70%. It has
now been shown conclusively that the Nottingham
method, with its more objective criteria, has ex-
cellent reproducibility when used by experienced
pathologists.
Interestingly, grade is not included in the recent
revision of the TNM staging system of breast cancer
as its value is questioned in certain settings such as
lobular type.
Recently, Rakha et al.29 examined a large
group of 5680 breast tumours (415 patients (8%)
with pure ILC and 2901 (55.7%) with IDC (no
special type)) and demonstrated that, compared
to IDC, patients with ILC tended to be older and
have tumours that were more frequently of lower
grade (typically grade 2; 84%), hormone-receptor
positive (86% compared to 61% in IDC), of larger
size and with absence of vascular invasion.
More patients with ILC compared with IDC were
placed in the good Nottingham Prognostic Index
group (40% compared to 21% in IDC). ILC
showed indolent but progressive behavioural
characteristics with nearly linear survival curves
that crossed those of IDC after approximately
10 years of follow-up, thus eventually exhibiting
a worse long-term outcome. Interestingly, ILC
showed a better response to adjuvant hormonal
therapy (HT), with improvement in survival in
patients who received HT compared with matched
patients with IDC.
0.8
0.7
0.5
0.4
0.3
0.2
0.1
0.6
1.0
0.0
0.9
Cum survival
12
24
36
48
60
72
84
96 108 120 132 144 156 168 180
0
Months
Grade
1
2
3
Figure 2.3  •  Overall survival according to grade.

Pathology and biology of breast cancer
25
Vascular invasion
The presence of tumour emboli in vascular and lym-
phatic spaces has emerged as an important prognos-
tic factor. Several studies have now shown that the
presence of vascular invasion correlates closely with
local and regional lymph node involvement.33,34
It has been suggested that it can provide prognos-
tic information as powerful as lymph node stage.
Reproducibility is the limiting factor in its wide-
spread adoption and routine clinical assessment. In a
Nottingham study of assessing vascular invasion, the
issue of reproducibility was addressed specifically.
Of 1704 cases, a subset of 400 cases was ­examined
by two or more pathologists. There was a 77% over-
all inter-observer agreement on histological features
and an 85.8% overall agreement in the classification
of vascular invasion. Other studies have reported a
similarly high concurrence between pathologists.35,36
The assessment of vascular invasion is subjective but
there is good evidence that a high rate of ­concurrence
can be obtained as long as strict criteria are used.
Lymphatic and vascular invasion is considered to be
a valuable surrogate for lymph node stage in cases
where nodes have not been removed for examina-
tion. In patients whose axillary nodes are tumour
free on histological examination there is a correla-
tion between the presence of lymphovascular inva-
sion (LVI) and early recurrence.
Molecular/predictive factors
Despite the overall association between molecular
markers with prognosis and outcome, they are lim-
ited in their ability to capture the nuances of the
complex cascade of events that drive the clinical be-
haviour of breast cancer. Morphological factors are
unable to predict response to systemic treatments,
and tumours of apparently homogeneous morpho-
logical characteristics still vary in response to ther-
apy and have distinct outcomes. The use of strategies
­ranging from hormone therapy to chemotherapy
and recently to novel receptor-directed therapies
and vaccines relies on the expression of predictive
factors by the cancer, either individually (e.g. oestro-
gen receptors (ERs), progesterone receptors (PRs),
human epidermal growth factor receptor (HER-2)
Vascular invasion
Negative
Positive
Probability
1.0
0.6
0.8
0.4
0.2
0
250
100
150
200
50
Survival in months
p<0.0001
Figure 2.4  •  LVI and breast cancer-specific survival.
Recently, Lee et al.37 assessed the prognostic
value of LVI in a group of 2760 patients with node-
negative breast cancer with long-term follow-up
(median 13 years). This study demonstrated a
strong association between poor histological grade
and younger age with LVI-positive cancers. LVI
was prognostically significant and was independent
of grade, size and type for overall survival (see
Fig. 2.4).
The value of grade was addressed by Rakha
et al.31 in a large study of grade and overall survival
in a large and well-characterised consecutive series
of operable breast cancer (n = 2219 cases), with a
long-term follow-up (median 111 months) using the
Nottingham histological grading system. Histological
grade was strongly associated with both breast cancer-
specific survival (BCSS) and disease-free survival (DFS)
in the whole series as well as in different subgroups
based on tumour size (pT1a, pT1b, pT1c and pT2)
and lymph node (LN) stages (pN0 and pN1 and pN2).
The authors were able to demonstrate differences in
survival between different individual grades (1, 2 and
3). In multivariate analyses histological grade was an
independent predictor of both BCSS and DFS.
Additionally, the usefulness of routinely
assessing grade in invasive lobular cancer was
examined by this group in 4987 patients,32 of whom
517 were pure ILC cases. The majority of ILC was
of classical type or mixed lobular variants (89%).
Most ILC cases were moderately differentiated
(grade 2) tumours (76%), while a small proportion
of tumours were either grade 1 or 3 tumours (12%
each). There were significant associations between
histological grade and other clinicopathological
variables of poor prognosis such as larger tumour
size, positive lymph node status, vascular invasion,
oestrogen receptor and androgen receptor
negativity, and p53 positivity. Multivariate analyses
showed that histological grade was an independent
predictor of BCSS and disease-free interval.

Chapter 2
26
and epidermal growth factor receptor (EGFR))
or globally (e.g. high-throughput gene expression
­assays). These molecular markers are used not only
to guide treatments but are also being used to moni-
tor response and detect relapse. It is envisaged that
molecular predictive markers may form the basis of
tailoring an individual's adjuvant therapy based on
the genetic fingerprint of their cancer.
Oestrogen receptors (ERs)/
progesterone receptors (PRs)
Knowledge of the expression of hormone receptors
and their subcellular regulatory pathways is one of
the classic examples of the use of translational medi-
cine in breast cancer to further diagnosis and treat-
ment of this disease. The degree of ER expression is
used to predict an individual's response to hormone
therapy. Both ERs and PRs are steroid receptors that
are located in the cell nucleus. Oestrogen and proges-
terone are considered to diffuse into cells, or be trans-
ported to the nucleus. Genes, regulated by steroid
receptors, are involved in controlling cell growth,
and it is currently believed that these effects are the
most relevant to ERs and that ER expression influ-
ences the behaviour and treatment of breast cancer.38
Elucidation of downstream effect on genes that are
known to be influenced by hormones has led to the
inclusion of these ER-regulated genes in high-density
oligonucleotide array panels.39 This may better de-
fine those pathways that are endocrine responsive
and may lead to improved therapies with reduced
side-effects and a greater potential for cure.40
In unselected patients, approximately 30% will re-
spond to endocrine therapy. A tumour with both ER
and PR expression has a 78% chance of respond-
ing to hormone therapy while those cancers that are
ER/PR negative respond rarely, if ever.41–43 Due to
their close relationship with histological grade, ERs
are not of independent prognostic significance.43
Methods of measuring ERs and PRs
There are a multitude of methods available; how-
ever, in clinical practice most are based on two dis-
tinct strategies. The first is an older method based
on ligand-binding methods, i.e. radiolabelled steroid
ligand is used to detect the receptor. The second relies
on the recognition of the receptor protein by specific
antibodies. Several studies examined the correlation
between assay and outcome/response and studies
have suggested that IHC analyses have a number of
advantages.42 IHC is currently the most commonly
used method because it can be performed on paraffin-
embedded material and allows assessment of the ex-
pression specifically in either invasive or in situ cancer.
Interpretation of assays
The optimal way to score IHC for ERs and PRs re-
mains controversial. One method is to use an ‘H’
score (histo score), which takes into account the fre-
quency of positive cells as well as intensity of staining
and multiplies the two. The Allred score categorises
the percentage of cells (scored from 0 to 5) with the
intensity (scored from 0 to 3) and adds these two
scores to give a numerical score from 0 to 8. Others
simply estimate the percentage of positively stained
tumour nuclei. It is important to appreciate that all
methods are subjective and, at best, semiquantita-
tive. ER/PR status in the histopathological report
has a clearly defined role as a predictive factor for
the response of systemic endocrine therapy. ERs
and PRs should be assessed on all breast tumour
specimens. As these assays have become simpler, and
less costly, they are now available for all patients.
Progress still needs to be made in standardisation of
assay technique, objectivity and reproducibility.
Type 1 growth factor receptors
Activation of growth factor receptors was first
found to be important in human cancers by iden-
tifying the homology between the viral oncogene
v-erbB and EGFR. Sequence similarity between the
erbB-1 gene that encodes EGFR resulted in the iso-
lation of a second growth factor receptor, the hu-
man ortholog neu, HER-2 (erbB-2). Screening of
genomic DNA and messenger RNAs with probes
allowed isolation of two additional relatives of the
human ERB1 gene. They were subsequently named
erbB-3 (HER-3) and erbB-4 (HER-4).
To date there are four known receptors that belong
to this growth factor receptor family, HER-1–4, and
ligands for some of these have been identified. The
family works in a co-receptor dimerisation type ac-
tivation pathway.
The first two members of this family have been
studied extensively in breast cancer. There are lim-
ited data on the role of HER-3 and HER-4 as well
as their ligands. This family of signalling molecules
is currently of immense clinical interest as the first

Pathology and biology of breast cancer
27
two members are targets against which novel bio-
receptor agents have been developed, including
Iressa®, Tarveca® and Herceptin®.
EGFR
EGFR expression has been well studied in breast
cancer. Consequent on major differences in study
designs, the EGFR assay used and confounding
­factors such as adjuvant therapy, there is no consen-
sus on its prognostic value. With the development of
tyrosine kinase inhibitor (TKI)- and EGFR-directed
biotherapies, there is now a growing impetus to not
only standardise assays for detecting EGFR but also
to delineate accurately its prognostic and predictive
potential.
Methods of testing
Several techniques directed at DNA, RNA, protein
(and functionally active (activated) as well) or se-
rum can be employed to identify EGFR expression
in breast cancer.
Of all such methods, IHC is the most practical.
The advantages of IHC in EGFR as in HER-2 test-
ing include reproducibility, ease of interpretation
and the low cost compared to other techniques. It
can also be employed on archival tissue. Protein lev-
els can be quantified by Western analysis or enzyme
immunoassay; however, architecture of tissue is lost
in these procedures and there may be contamination
by normal tissue cells or ductal carcinoma in situ.
Serum EGFR levels have been examined in breast
carcinoma; however, few data are available.44,45
Due to the paucity of relevant data, it is difficult to
draw conclusions on the current value of measuring
EGFR in the serum of patients.
Prognostic and predictive value
The prognostic value of EGFR in breast cancer has
been examined in several large studies. There remains
no consensus on its prognostic value. The lack of a
clear picture has been attributed to several factors:
lack of a standard assay (monoclonal vs. polyclonal),
lack of a cut-off for positivity and great variation in
study designs (size, follow-up and influence of ad-
juvant therapy). Klijn et  al.46 reviewed data from
over 5000 patients where EGFR had been assessed.
Findings from this review demonstrated a great het-
erogeneity of study design, levels of cut-offs for posi-
tive and negative, and, not surprisingly, differences in
its prognostic value in these various studies.
The predictive value of this marker for hormone
resistance or responsiveness is better defined. EGFR
tumours are considered more resistant to endocrine
therapy, and there exists an inverse relationship
with EGFR-negative cancers (they are more likely
ER positive), being more often sensitive to endo-
crine manipulation. Though some studies have been
able to demonstrate that EGFR status is a predic-
tor of tamoxifen failure and even response rates,50
there are conflicting data from well-designed level
II studies51 that have shown no value of EGFR in
predicting the efficacy of tamoxifen in high-risk
postmenopausal women.
Perhaps the most exciting use of EGFR status and
indeed the driving impetus for clinicians and scien-
tist to measure it accurately is its putative role in
defining those who should respond to novel EGFR-
directed therapies.
HER-2
HER-2 is an important target in the development
of a variety of new cancer therapies, which include
monoclonal antibody (mAb)-based therapy, small-
molecule drugs directed at the internal tyrosine
kinase portion of the HER-2 oncoprotein, and vac-
cines. The most widely known HER-2-directed ther-
apy is trastuzumab (Herceptin; Genentech, South
San Francisco, CA, USA). Trastuzumab is a human-
ised recombinant mAb that specifically targets the
HER-2 extracellular domain. There are a variety of
techniques available to determine HER-2 status in
breast cancer, some of which are employed for re-
search purposes only. In diagnostic pathology labo-
ratories HER-2 status is assessed routinely either
by IHC, which assesses expression of the HER-2
oncoprotein, and fluorescence in situ hybridisation
(FISH), which measures the number of HER2 gene
copies per chromosome 17 or gene amplification.
Modifications of ISH using colorimetric detection
are being developed, including chromogenic in situ
hybridisation (CISH) and silver-enhanced in situ hy-
bridisation (SISH).
Recently, Tsutsui et al.47 reported on a large
series of 1029 patients (with adjuvant therapy)
and found EGFR to be of independent prognostic
value irrespective of nodal status. In contrast,
studies by Rampaul et al.48 and Ferrero et al.49
(without adjuvant therapy) showed EGFR to be
of no prognostic value (these latter series also
incorporated grade and had longer follow-up).

Chapter 2
28
Methods of HER-2 testing
Current guidelines for HER-2 testing52 specify the
methods that are suitable to detect either the HER-2
protein (by IHC) or gene amplification (using FISH
or other in situ methods). Guidelines stress the need
for stringent, reproducible and consistent criteria
for testing.
Immunohistochemistry for HER-2 testing
Among the methods in use for determining HER-2
status, IHC is the most widely used. In studies em-
ploying various commercially available antibod-
ies, a wide variety of sensitivity and specificity in
fixed paraffin-embedded tissues is seen.53,54 Antigen
retrieval techniques are currently not standardised
and they introduce the potential for false-positive
staining. Nonetheless, IHC possesses many advan-
tages to support its widespread adoption: (i) it al-
lows for the preservation of tissue architecture and
so can be used to identify local areas of overexpres-
sion within a heterogeneous sample, and can dis-
tinguish between HER-2 positivity in in-situ and
invasive cancer; (ii) it is applicable to routine patient
samples, facilitating use as a diagnostic test, and this
allows prospective and retrospective research stud-
ies of HER-2 status to be undertaken.
Two Food and Drug Administration (FDA)-
approved IHC tests for determining HER-2 status
are available: HercepTest (DAKO, Carpeteria, CA,
USA), based upon a polyclonal antibody; and CB11
(Pathway, Ventana Medical Systems, Tucson, AZ,
USA), based upon a monoclonal antibody. The
National Comprehensive Cancer Network guide-
lines55 classify an IHC score of 0 or 1+ as represent-
ing HER-2-negative status, 3+ as positive, while 2+ is
equivocal. Positive staining is defined as strong, con-
tinuous membranous expression of HER-2 in at least
10% of tumour cells. However, a joint report from
the American Society of Clinical Oncology (ASCO)
and the College of American Pathologists (CAP)52
specified a threshold of >30% strong circumferen-
tial membrane staining for a positive result. If both
uniformity and a homogeneous, dark circumferen-
tial pattern are seen, resultant cases are likely to be
amplified by FISH as well as positive for HER-2 pro-
tein expression. The equivocal range for IHC (score
2+), which may include up to 23% of samples, is
defined as complete membrane staining that is either
non-uniform or weak in intensity but with obvious
circumferential distribution in at least 10% of cells.
Equivocal or inconclusive results should be tested by
FISH. Consistent with previous guidelines, a nega-
tive HER-2 test is defined as either an IHC result
of 0 or 1+ for cellular membrane protein expression
(no staining or weak, incomplete membrane staining
in any proportion of tumour cells), though up to 5%
of 1+ on IHC may be FISH positive.
In situ hybridisation for HER-2 testing
FISH and CISH measure directly the number of
HER2 genes per chromosome 17, and when there
is a chromosome centromeric enumeration probe
(CEP) included, the copy number of chromosome
17 gene amplification is defined as an increase in
HER-2/CEP17 ratio above 2.0. ISH results are
semiquantitative, counting the number of signals
in non-overlapping interphase nuclei of the lesion
using either single-colour (HER-2 probe only, e.g.
Ventana Inform) or dual-colour hybridisation (us-
ing HER-2 and chromosome 17 centromere probes
simultaneously (e.g. Abbott, Chicago, USA, DAKO,
Copenhagen, Denmark, etc.), the latter making it
easier to distinguish true HER-2 amplification from
chromosomal aneuploidy. ISH allows simultane-
ous morphological assessment, where evaluation
of gene amplification can be restricted to invasive
carcinoma cells. Many studies have compared FISH
and IHC in the evaluation of HER-2 and have dem-
onstrated concordance between the two techniques
of up to 91%. Two studies have shown that FISH
predicts more accurately HER-2 positivity than IHC
when applied to molecularly characterised breast
cancers.56,57
Three FISH tests are FDA approved for selecting
patients for treatment with trastuzumab. The Path
Vysion (Vysis Inc., Downers Grove, IL, USA) and
PharmDx (Dako) tests require a ratio (HER-2 to
CEP17) of 2.2 or greater for the sample to be con-
sidered amplified and both include an HER2 gene
probe and a chromosome 17 probe. The INFORM
test (Ventana Medical Systems) requires that at least
5.0 gene copies of HER2 be present if a sample is
to be considered amplified as this kit uses a single
HER2 gene probe without a chromosome 17 probe.
Recommended guidelines for HER-2 assessment
in the UK have recently been updated and the reader
is referred to these guidelines for further details.58
In brief, a two-phase testing algorithm based on
IHC assay as the primary screen with FISH being re-
served for equivocal cases is currently ­recommended.

Pathology and biology of breast cancer
29
This is based on evidence showing very good
concordance between IHC and FISH results on
breast carcinomas from 37 laboratories when tested
in experienced reference centres.59
Chromogenic in situ hybridisation (CISH/SISH)
CISH and SISH are colorimetric methods to de-
tect gene amplification that can be viewed using a
­standard light microscope. Concordance between
FISH and SISH, for the validation of HER2 gene
status, is about 96% (kappa = 0.754).
Prognostic significance and association with
other prognostic factors
The seminal work by Slamon et  al.60 in 1987
showed that HER2 gene amplification indepen-
dently predicted overall survival (OS) and disease-
free survival (DFS) in a multivariate analysis in
node-positive patients. Since then most large stud-
ies have confirmed this relationship in multivariate
analysis. It is now well established that there is a
significant correlation between HER-2 overexpres-
sion/amplification and poor prognosis for patients
with nodal metastasis. There was until recently no
consensus on the prognostic value of HER-2 in
node-negative breast cancer patients, a group most
often diagnosed through screening and represent-
ing a subgroup that could potentially benefit from
appropriate adjuvant therapy. More recent stud-
ies have, however, shown a consistently poorer
prognosis when comparing HER-2-positive versus
-­negative small node-negative breast cancers.
Rilke et  al.61 reported on the prognostic signifi-
cance of HER-2 expression and its relationship with
other prognostic factors. Using specimens from 1210
consecutive patients treated between 1968 and 1971
at a single institution (National Cancer Institute
of Milan), with no systemic adjuvant therapy and
20-year follow-up, they found overexpression of
HER-2 in 23% and showed a negative impact on
survival of node-positive but not node-negative pa-
tients. Analysis of HER-2 in relation to the presence
of lymphoplasmacytic infiltrate (LPI; favourable
prognosis) and nodal status demonstrated that in
node-negative, LPI-negative patients, HER-2 over-
expression showed the same level of correlation with
poor prognosis as with those patients with nodal
metastasis. However, in the patients with node-
negative disease and LPI positivity, HER-2 over-
expression correlated with good prognosis. Some
studies have reported a prognostic value for HER-2 in
node-negative patients in selected subgroups,62–64
whereas others have shown no correlation.65,66
Mirza et al.66 have published a systematic review
of prognostic factors in node-negative disease. Data
for HER-2 showed a lack of standardisation of as-
says and no association with survival. However,
HER-2 status was shown to be of independent
prognostic significance in several large studies with
long-term follow-up, with worstened prognosis for
those node-negative patients with HER-2-positive
signalling.67,68,69
There is at present no agreement on the associa-
tion between HER-2 and other prognostic factors.
Several studies have shown a lack of association
between HER-2 status and tumour size,60,70,71 al-
though some do report a correlation.61,66,72–76
Prediction of response to therapy: hormonal
therapy
Transfection of normal breast cancer cells with the
HER2 gene has been shown to result in acquisition
of oestrogen-independent growth that is insensitive
to tamoxifen.77,78
A number of clinical studies, using various end-points
such as time to relapse, more rapid spread to other
sites, and DFS or OS, have reported an association
between HER-2 positivity and resistance to hormonal
therapy.79–83 Some reports have described specific re-
sistance to tamoxifen in HER-2-overexpressing tu-
mours.81,82 The 20-year update of the Naples GUN
Trial81 found that HER-2 overexpression not only
predicted resistance to tamoxifen, but that HER-2-
positive patients had a worse outcome on tamoxifen
therapy compared with those who were untreated.
Several studies have also shown a reduction in
response rates to hormonal therapy. Metastatic
breast cancer that overexpressed HER-2, measured
by high plasma levels of extracellular domain, dem-
onstrated a substantial reduction in response rate
to hormonal therapy.84 Other studies have failed to
find an association or even a trend between HER-2
status and response to hormonal therapy.85–87
Elledge et al.87 examined the response to tamoxifen
in 205 tumours with ER-positive disease. In HER-
2-positive compared to HER-2-negative patients,
they found no significant evidence for a poorer re-
sponse, time to treatment failure or survival. In a
more recent study, the relationship between HER-2
overexpression and response to tamoxifen was

Chapter 2
30
examined in the adjuvant setting in 741 (650 ER
positive, 91 ER negative/PR positive) of the total
of 1572 patients in the CALGB 8541 Trial who
had HER-2 measured.88 Tamoxifen significantly
improved response, DFS and OS, irrespective of
HER-2 status. However, tamoxifen was not ran-
domised within this trial and all patients received
one of three regimens of doxorubicin. Thus these
data on tamoxifen resistance have limitations to
their interpretation.
With regards to HER-2 and prognosis, not only
is there clinical value in its positive expression but
also in its absence – in so-called ‘triple-negative phe-
notype’ cancers (TNP), i.e. cancers that are HER-2,
ER and PR negative. It also denotes a biologically
different subgroup. The recognition of basal phe-
notype in these triple-negative cancers is of growing
importance, with several lines of evidence support-
ing the view that triple-negative tumours and basal
phenotype are not interchangeable but rather dis-
tinct entities. Ninety-one per cent of TNP tumours
display a significant association with the basal-like
centroid and about 20% of non-TNP tumours clus-
ter together with TNP tumours in the ‘basal-like’
cluster; these data support the view that the major-
ity of, but not all, TNP tumours have a basal-like
phenotype and that the majority of, but not all,
basal-like tumours have a TNP phenotype.89 Triple-
negative breast cancers have a relapse pattern that
is very different from hormone-positive breast can-
cers: the risk of relapse is much higher for the first
3–5 years but drops sharply and substantially be-
low that of hormone-positive breast cancers after
that. This relapse pattern has been recognised for
all types of triple-negative cancers for which suffi-
cient data exist, although the absolute relapse and
survival rates differ across subtypes.
Previous studies have shown that the expression
of ‘basal markers’ (i.e. CK5/6, CK14, CK17 and/
or EGFR) is associated with a poor prognosis, re-
gardless of hormone receptor expression. The ex-
pression of basal markers (basal cytokeratins and
EGFR) in TNP tumours (core basal phenotype)
also correlates with a worse prognosis and identi-
fies a clinically distinct subgroup within the TNP
group. Moreover, it should be noted that identifi-
cation of a subgroup of tumours solely based on
the lack of expression of immunohistochemistry
(for example, TNP) risks misassignment based on
technical artefacts.89
An in-depth discussion of these issues is beyond
the scope of this work and the reader is directed to
detailed reviews by Rakha et al.89
Histopathology of patients
with BRCA1 and BRCA2
mutations
The cancers that develop in patients with a genetic
predisposition to breast cancer as a result of mu-
tations in the breast cancer susceptibility genes
BRCA1 and BRCA2 are of great clinical interest.
Identification of histological features that could in-
dicate a genetic predisposition would be useful in
providing an insight into the function of these genes
and may aid in identifying those in whom screen-
ing for genetic mutations would be useful. There is
a general agreement that BRCA1-related cancers
are more frequently ‘medullary-like’ carcinomas and
high grade compared to those in patients without this
genetic alteration. Cancers associated with BRCA1
mutations have a significantly higher mitotic rate, a
larger proportion of tumour with a continuous push-
ing margin and more lymphocytic infiltration than
sporadic breast cancers.90,91 These are also likely to
be less positive for ERs and PRs and are more of-
ten aneuploid, have a high S-phase fraction, show
greater accumulation of p52 protein and expression
of basal cytokeratins (CK5/6, CK14 and CK17) than
do sporadic breast cancers.92–97 However, none of
these features alone or in combination can be used
to identify a cancer as being from a BRCA1 gene
mutation carrier. Pathological features reported in
BRCA2-related breast cancer have been less con-
sistent. Marcus et al.92 noted a significantly higher
proportion of tubulolobular cancers in BRCA2 mu-
tation carriers than in other patients. However, it has
been reported in another study that tubular carcino-
mas are less common in BRCA2 mutation carriers.94
Armes et  al.97 investigated the histological pheno-
types of breast carcinoma in women under 40 years
of age with and without BRCA1 and BRCA2 germ-
line mutations. It was found that the pleomorphic
variant of invasive lobular carcinoma was more
common in patients with BRCA2 mutations. Others
have reported that BRCA2-related cancers tend to be
of high histological grade,94,98 whereas others have
not noted any significant difference in grade between
BRCA2-related cancers and controls.99

Pathology and biology of breast cancer
31
High-throughput molecular
techniques as prognostic
and predictive tools in breast
cancer
Genomics
Mechanisms of genetic aberrations
The sequencing of the entire human genome has
permitted the introduction of high-throughput
­technologies that allow survey of thousands of genes
and their products in a single assay. Together with
powerful analytical tools this has opened new av-
enues for classifying breast cancer into biologically
and clinically distinct groups based on DNA copy
number alterations and gene expression patterns.
Cancer development is driven by the accumulation
of DNA changes within the genome. DNA repair
defects lead to a genome-wide genetic instability and
this can drive further cancer progression. Genomics
(the study of the human genome), transcriptomics
(the study of the transcriptome (mRNAs)) and pro-
teomics (the analysis of the protein complement
of the genome) are the three branches of molecu-
lar biology that are being explored in an effort to
improve understanding, diagnosis, prognostication
and provision of new therapeutic targets for the
treatment of breast cancer.
The genetic fingerprint provides information on
normal cellular processes and morphological/phe-
notypic expression. When this message is altered,
it forms the nidus for the development and progres-
sion of cancer. These alterations can be caused by
DNA mutations, chromosomal aberrations, epigen-
etic modification and protein interactions. DNA
mutations can lead to a change in gene function.
These mutations are often due to base substitutions
that may directly cause a stop codon. As a result the
gene is only partly transcribed and any functional
protein production terminated. Chromosomal in-
stability can be due to DNA modification, mutation
or viral genome integration. Changes in chromo-
some numbers are also seen and cancers can be an-
euploid (60–90 chromosomes) or near diploid (46
chromosomes). In solid tumours virtually all chro-
mosomal rearrangements are unbalanced, the net
result being loss or gain in certain parts of the chro-
mosome. By screening the genome, current losses
and gains can be identified. This information can
be used to investigate regions of overlap to reveal
the genes that are involved in malignant transfor-
mation and cancer progression. In some situations,
the DNA copy number and code remain intact but
the accessibility of this DNA for transcription is af-
fected by epigenetic modifications. This process can
occur through DNA methylation. It directly silences
genes and interferes with the binding of transcrip-
tion factors by changing the chromatin structure
around the gene. Global hypomethylation is a char-
acteristic feature of the genome of a cancer cell.
However, some sequences can be hypermethylated,
such as CpG islands, which tend to lie around the
transcription start sites of approximately half of
all human genes. DNA hypomethylation has been
shown to correlate with chromosome instability.
Chromatin architecture remodelling is essential for
gene transcription; thus the prognostic significance
of quantitative chromatin changes.100,101
Assessment of genomic status in
breast cancer
Several high-throughput molecular techniques have
been developed to assess the status of the genome
in a given cell population, in terms of copy number,
DNA sequencing and structure. Large-scale DNA
or gene copy number alterations can be assessed
using loss of heterozygosity (LOH) and compara-
tive genomic hybridisation (CGH) studies as well as
other techniques. LOH can be used to identify chro-
mosomal regions where allelic losses are more fre-
quent. In one study,102 a panel of 150 polymorphic
microsatellite markers from throughout the whole
genome was used to identify such regions. These
data were correlated with clinicopathological fea-
tures and showed four specific loci that correlated
with lymph node metastasis (11q23–24, 13q12,
17p13.3 and 22q13). Microsatellites can also be
used to check the ability of cells to repair DNA rep-
lication errors. Microsatellite instability (MSI) can
be employed to identify both genetic and epigenetic
modification. It has been shown that both genetic
and epigenetic alterations of hMSH2 and hMLH1
contribute to genomic instability and tumour pro-
gression in sporadic breast cancer.
Chromosome-based CGH can be used to identify
the loss of one or both copies of a given gene, as well
as regions of amplification. In addition, the technique
provides information on the number of copies of any
part of each chromosome throughout the whole ge-
nome. There have been several studies using CGH

Chapter 2
32
in breast carcinoma,103,104 and those with clinical
follow-up have led to the identification of genetic
changes related to prognosis. However, the resolu-
tion of every chromosome-based CGH technique
was limited to approximately 10 million base pairs
(Mbp). This limitation makes it difficult to link copy
number changes to the genes involved. Another limi-
tation to this technique is the need to perform karyo-
typing for target identification in every experiment.
Therefore, array CGH has been developed to over-
come the problems of chromosome-based CGH.
Array CGH does not require a normal metaphase
spread but rather an array of DNA fragments
(100 bp to 100 kb) and their precise chromosomal
locus. This approach can provide resolutions up
to 1 Mb. Albertson et al. utilised this technique to
map the recurrent breast cancer amplicon at chro-
mosome 20q12.3. They were able to demonstrate
that what was previously described as a single am-
plicon is in fact two distinct amplicons (ZNF217
and CYP24). A novel amplicon at 17q21.3 that is
implicated in the amplification and overexpression
of the HOXB7 gene in breast cancer has also been
recently characterised using array CGH.105
Assessment of gene expression in
breast cancer
In gene expression profiling, each gene is usually
represented by a single element (created with a
complementary DNA (cDNA) or oligonucleotide
for the gene studied) and with high-density oligo-
nucleotide cDNA microarray technology many
thousands of gene-specific mRNAs can be measured
in parallel in a single tissue sample. The principle
of gene expression cDNA array is comparable to
array CGH, other than that it uses cDNA gener-
ated from mRNA. Similar to the two main types
of expression microarrays (cDNA microarrays and
oligonucleotide microarrays), serial analysis of gene
expression (SAGE) can provide profiling of the tran-
scriptome by taking a raw count of sequence tags,
each representing a transcript in an RNA popula-
tion with each one representing one gene.
Broadly speaking, molecular profiling of breast
cancers by gene expression microarrays can be
performed in one of two ways: unsupervised or
supervised analysis. Unsupervised analysis is used
mainly for partitioning tumour samples into groups
or classes on the basis of gene expression profiles
regardless of other features. The main objective of
this approach is to determine whether discrete sub-
sets of cancers can be defined on the basis of gene
expression profiles, and whether new classes can be
identified (class discovery) that may have clinical
significance and to develop a new molecular tax-
onomy. Supervised analysis, on the other hand, is
used to allocate tumours to specific groups based
on clinical or pathological features (e.g. clinical out-
come or response to therapy). There are two main
types of supervised analysis: class comparison and
class prediction. The former aims to identify the
transcriptomic differences between two classes of
tumours, whereas the development of a ‘gene signa-
ture’ is the ultimate goal of the latter.
In breast cancer, the class discovery studies were
pioneered by the Stanford group,106 which proved
the principle that breast cancer could be classified
into molecularly distinct groups based upon gene ex-
pression profiles and their similarity to normal cell
counterparts. Multiple independent studies have con-
firmed and expanded the original results. According
to these studies, breast cancer was classified into
two main classes (ER-positive and ER-negative tu-
mours) and each one can be classified into multiple
molecularly distinct subclasses. For example, the
ER-negative tumours encompass three subgroups,
one overexpressing HER-2, one with tumours ex-
pressing genes characteristic of basal/myoepithelial
cells (basal-like cancer), and one with a gene expres-
sion profile similar to normal breast tissue. cDNAs
have also been used to distinguish cancers with
BRCA1/BRCA2 mutations107,108 and to determine
ER status,109 lymph nodes status110,111 and prog-
nostic subgroups in node-negative breast cancer.112
These recent studies demonstrate the ability of cDNA
to have a direct translational use in clinical practice.
Proteomics
The genetic code does not inform on which pro-
teins are expressed by a cancer, or whether they are
functional, if expressed. Post-translational modifi-
cations such as glycosylations or phosphorylation
of proteins affect their expression and function at
the protein level, which cannot be detected by as-
sessing the status of the genome or transcriptome in
a given cell population. Therefore, proteomics aims
to assess changes at the protein level in which the
proteome is displayed. Proteomics is quickly evolv-
ing to provide supportive and critical information

Pathology and biology of breast cancer
33
to data generated through genomic approaches in
high-throughput formats. This can be achieved by
using different techniques such as two-dimensional
polyacrylamide gel electrophoresis, isotope-coded
affinity tagging, surface-enhanced laser desorption
ionisation and matrix-associated laser desorption
ionisation–time of flight.
These global expression profiling approaches
yield candidate proteins and related genes that
require verification through application of other
techniques. Tissue microarrays (TMAs) provide
a method for high-throughput protein expression
analysis of large cohorts of archival samples that
can be readily linked to clinicopathological and
long-term follow-up databases. TMAs have expe-
dited the validation of the prognostic and predictive
significance of several candidate biomarkers. The
technique allows for a composite slide of up to 1000
cores of tissue to be constructed into one paraffin
block. The advantages are obvious in screening for
novel protein expression. There are concerns on the
validity of the cores as being representative samples
and indeed this aspect has not been well studied,
with only one study examining a large sample size
with correlation to corresponding larger histology
sections.103 This technology and its application have
been extensively reviewed.113
Clinical use of the high-
throughput molecular
techniques
It is envisaged that gene expression profiles may be
able to guide decisions on the choice of hormonal,
chemotherapeutic or targeted agents for each indi-
vidual in the future. Presently, one of the best ex-
amples of the use of genomics and proteomics in
clinical practice is the use of HER-2 expression/
amplification to select patients for trastuzumab.
Such profiles are useful to identify prognostically
significant genes, which studies have demonstrated
can perform better than traditional markers.104,114
In metastatic disease, expression patterns can be
used to establish the origin of the metastatic le-
sion in patients with more than one primary.
Transcription profiling has also been shown to dif-
ferentiate accurately breast cancers with germ-line
mutations in BRCA1/BRCA2 from those without
such mutations. Such findings, if validated in other
studies, open the way for molecular phenotyping
in high-risk families. In addition, gene expression
profiling has been used to develop expression pre-
dictors (class prediction studies) that can be used
for many types of clinical management decisions,
including risk assessment, diagnostic testing, prog-
nostic stratification and treatment selection. There
are two types of class prediction studies in breast
cancer: prognostic and predictive class prediction.
Prognostic class prediction includes (i) poor-prognosis
gene signatures that can discriminate between a good
and a poor outcome by comparison between highly
aggressive and less aggressive primary tumours,
and (ii) recurrence score gene signatures that define
tumours based on the risk of disease relapse. The
predictive class prediction includes predictors of re-
sponse to therapy.115,116
An example of a prognostic class prediction study
is that reported by van't Veer et al. Expression pro-
files of 117 primary breast cancers were compared
with known prognostic factors and matched with
5  years of follow-up data. Twenty-five thousand
genes were used to generate expression profiles,
which separated the tumours into two groups.
Group 1 developed distant metastasis in 34% and
in group 2 70% developed distant metastases. From
the 25000-gene set, 70 genes had great accuracy in
predicting recurrent disease. Multivariate analysis
with grade, size, LVI, age and ER showed the poor-
prognosis microarray profile to be an independent
predictor of recurrent disease. This approach was
further tested in 295 patients and again the use
of gene profiling was able to accurately identify a
poor-prognosis group.101,109 MammaPrint® assay,
which is based on the Amsterdam 70-gene gene
signature, is currently used as a molecular diagnos-
tic test for breast cancer prognostication and pre-
diction. Another predictor calculates a recurrence
score on the basis of the expression of 21 known
genes, with the use of RT–PCR in formalin-fixed,
paraffin-embedded tissue. The ‘Oncotype DX as-
say’ has been developed by researchers at Genomic
Health. Oncotype DX is currently used as a diag-
nostic test to quantify the likelihood of disease re-
currence in women with early-stage breast cancer
and assesses the likely benefit from tamoxifen and
certain types of chemotherapy. Oncotype DX em-
ploys a mathematical algorithm called the recur-
rence score (range 1–100) to calculate continuous
risk for relapse and death for patients receiving

Chapter 2
34
adjuvant tamoxifen, and has recently been shown
in a large population-based case–control study
to be an effective predictive test for ER-positive,
node-negative breast cancer patients treated or un-
treated with tamoxifen and no chemotherapy.117
In addition, a number of microarray studies have
been published which identified other prognostic
signatures with clinical significance. For example,
Change et al.118 have used a ‘wound-response gene
expression signature’ to stratify breast cancer pa-
tients based on the hypothesis that features of the
molecular programme of normal wound healing
might play an important role in cancer metastasis.
They found that this gene signature can improve
the risk stratification of early breast cancer over
that provided by standard clinicopathological fea-
tures. A gene expression signature of hypoxia re-
sponse, derived from studies of cultured mammary
epithelial cells' ‘hypoxia gene signature’, showed
a strong predictor of clinical outcomes in breast
cancer.119
Although proteomics has a long history that pre-
dates profiling at the RNA level, difficulties in pro-
tein identification and the lack of reproducibility of
several assay platforms have limited the utility of
such profiling systems. In breast cancer research,
however, proteomics has begun to take on a role
in the monitoring of response, prediction of resis-
tance and relapse in patients treated with novel bio-
directed therapies.120
Clinical use of prognostic
factors in patient
management
Prognosis in breast cancer depends on the presence
of spread of disease and on the inherent aggressive-
ness of the tumour. The latter depends on a number
of intrinsic biological characteristics, some of which
have already been evaluated, such as morphological
features, growth rate and hormone responsiveness.
Accurate prognostication is now required on an in-
dividual patient basis and this can only be achieved
by using a Prognostic Index that includes both time-
dependent and biological factors. The best way to
obtain such an index is to take potential factors
that have been shown to have some value in uni-
variate analysis and submit them to multivariate
analysis. This has been the approach in deriving the
Nottingham Prognostic Index (NPI), which is based
on three factors using the following formula:
(
)
(
)
(
)
=
×
+
+
NPI
Pathological tumour size cm
0.2
Lymph node stage 1,2 or 3
Histological grade 1,2 or 3
Arbitrary cut-off points of 3.4 and 5.4 are used to divide
patients into six prognostic groups: excellent (EPG),
good (GPG), moderate (MPG) I and II, poor (PPG) and
very poor (VPG) (Tables 2.1–2.3, Figs 2.5 and 2.6).
The NPI provides extremely powerful prognostic in-
formation within the NTPBCS and was able to dem-
onstrate its utility and reproducibility in studies from
other centres. In this respect Henson et al.,18 in a ret-
rospective analysis of prognostic data in over 22000
women from the SEER Programme of the National
Cancer Institute in the USA, confirmed that a

Percentage in prognostic group

1980–86
1990–99
EPG
12
14
GPG
19
21
MPG I
29
28
MPG II
24
22
PPG
11
11
VPPG
5
4
Table 2.1  •  NPI showing percentage in each prognostic
group
Prognostic groups: EPG, excellent prognosis; GPG, good
prognosis; MPG, moderate prognosis; PPG, poor prognosis;
VPG, very poor prognosis.

10-year
survival
(%)
±95% CL
P (log
rank)
EPG
96
2

GPG
93
4
0.14
MPG I
82
4
<0.0001
MPG II
75
4
0.0007
PPG
53
8
<0.0001
VPG
39
12
0.003
All
80
2

Table 2.2  •  Differences in 10-year survival for each
prognostic group
Prognostic groups: EPG, excellent prognosis; GPG, good
prognosis; MPG, moderate prognosis; PPG, poor prognosis;
VPG, very poor prognosis.
CL, confidence limit.

Pathology and biology of breast cancer
35
combination of lymph node stage and histological
grade improved prediction of prognosis. In a similar
way to the NTPBCS, Chevallier et al.121 have identified
‘young’ age, tumour size and histological grade as fac-
tors to be added to lymph node stage in the prediction
of recurrence; these factors were used to divide lymph
node-negative patients into three prognostic groups.
One of the strengths of the NPI is the fact that
it has been verified prospectively in the NTPBCS.
Further confirmation of its value has been provided
by its validation in two large multicentre studies in-
volving nearly 11000 patients in total.122,123 Such
studies demonstrate the inherent power of the path-
ological factors used in the NPI, which has become
the most widely used index for the management of
patients with breast cancer, certainly in the UK.
Other pathology-based prognostic indices used
in breast cancer include Adjuvant!Online (AO)124
and the St Gallen criteria,125 and more recently
Predict (a new UK prognostic model that predicts
survival following surgery for invasive breast can-
cer).126 This tool is a population-based validation
of the prognostic model PREDICT for early breast
cancer.

1980–86
± 95% CL
1990–99
±95% CL
RRR (death)
% ARR
(death)
EPG
88
6
96
2
0.67
8
GPG
72
8
93
4
0.75
21
MPG I
61
6
82
4
0.54
21
MPG II
42
6
75
4
0.57
35
PPG
14
8
53
8
0.45
39
VPG
12
10
39
12
0.31
27
All
55

80

0.56
25
Table 2.3  •  Relative risk reduction by NPI
Prognostic groups: EPG, excellent prognosis; GPG, good prognosis; MPG, moderate prognosis; PPG, poor prognosis; VPG, very poor
prognosis.
ARR, absolute risk reduction; CL, confidence limit; RRR, relative risk reduction.
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Proportion surviving
180
168
156
144
132
120
108
96
84
72
60
48
36
24
12
0
Months
MPGI
MPGII
PPG
VPG
GPG
EPG
Figure 2.5  •  Survival by NPI (1980–86).

Chapter 2
36
Adjuvant!Online ()
was developed in the USA and published in 2001;
users can input information on a patient's age, ER
status, tumour grade, tumour size and number of
positive nodes, and obtain predictions of 10-year
OS (the likelihood of being alive 10 years after the
diagnosis of breast cancer was first carried out),
BCSS (the likelihood of not dying of breast can-
cer within 10  years of diagnosis) and event-free
survival (EFS; the likelihood of surviving 10 years
without recurrence (local, regional or distant), a
second primary breast cancer, or death from breast
cancer), both with and without any proposed ad-
juvant therapy. The performance of AO has been
evaluated in cohorts of patients in Germany,
Canada and the UK. It has an advantage over the
NPI in that it integrates treatment, and for this rea-
son AO is the programme most commonly used by
oncologists.
The current St Gallen-derived algorithm for selec-
tion of adjuvant systemic therapy for early breast
cancer patients includes tumour size and grade,
nodal status, menopausal status, peritumoral ves-
sel invasion, endocrine status and HER-2 status.
Predict, which is similar in concept to AO but is
based on UK patients, has been developed at the
University of Cambridge. This tool is an alternative
updated method of assessing probability of survival
for UK patients.
All of the above discussion applies to primary op-
erable breast cancer. Prognostic factors have also
been explored in locally advanced and metastatic
breast cancer, albeit not to the same extent. In a
small study of locally advanced pancreatic cancer
patients (n = 60) treated with either tamoxifen or
radiation therapy and with crossover upon failure,
response to therapy correlated significantly with
histological grade (P = 0.02), ER (P = 0.02), PR sta-
tus (P = 0.02), mitotic index (P = 0.01) and tumour
ploidy (P = 0.04). Survival from initial therapy cor-
related significantly with ER (P  =  0.01) and PR
status (P = 0.04).127 In these patients, histological
grade, mitotic index, tumour ploidy, and ER and PR
status of the primary tumour may predict response
and prognosis.
These authors also explored the prognostic value
of certain factors as a composite index to guide
decision-making in metastatic disease.128 The ad-
vanced breast cancer (ABC) index comprises the
grade, ER status, site of initial metastasis and
disease-free interval. This index was tested both ret-
rospectively and prospectively and shown to sepa-
rate groups where the survival between each was
highly significant (P < 0.001).
1.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Proportion surviving
180
168
156
144
132
120
108
96
84
72
60
48
36
24
12
0
Months
MPGI
MPGII
PPG
VPG
GPG
EPG
Figure 2.6  •  Survival by NPI (1990–99).

Pathology and biology of breast cancer
37
Breast cancer histopathology minimum dataset.
Figure 2.7  •  Breast cancer histopathology minimum dataset. DCIS, ductal carcinoma in situ.

Chapter 2
38
Conclusion
Modern management of breast cancer requires a
multifaceted understanding of both the clinico-
pathological features and the molecular portraits
of the cancer. Emerging evidence is suggesting that
genomic approaches to prognosticating patients can
be complementary to standard prognostic tools.
Existing combinations of such factors are either
largely clinical (e.g. NPI) or purely molecular (e.g.
Oncotype DX), but a hybridised index is likely to
yield more information. Updating the traditional
NPI and incorporating well-established prognostic
factors such as grade, stage and tumour size with
biological factors have produced NPI+. This new
tool, which combines updated methods for assess-
ment of histological grade and tumour stage with
biological class determination, has recently been
shown to provide significant prediction of outcome
in all biological classes.129
The need to accurately prognosticate each patient
continues to drive research efforts to unravel the
‘perfect’ prognostic index. This information will al-
low us to use adjuvant therapies to influence and
improve such prognosis for a better outcome and
hopefully cure.
Contents of the final surgical
pathology report: the
minimum dataset
The Royal College of Pathologists' minimum da-
taset for breast cancer was originally developed in
recognition that certain histopathological features
of both in situ and invasive carcinoma are directly
related to clinical outcome and may therefore be im-
portant in deciding the most appropriate treatment,
including extent of surgery and use of and choice
of adjuvant therapy. In addition, histopathological
features can be used to monitor breast screening
programmes, the success of which is reflected by
more favourable prognostic features of the cancers
detected and also changing patterns of disease, par-
ticularly identified by cancer registries.
The minimum set of data should be used by pa-
thologists reporting all breast cancers, both screen
detected and those presenting symptomatically.
The Royal College of Pathologists minimum data-
set has been approved by the NHS Breast Screening
Programme
and
the
European
Commission
Working Group, the British Association of Surgical
Oncologists, the British Breast Group and the United
Kingdom Association of Cancer Registries (Fig. 2.7).
Key points
• Histopathological features such as size, grade and lymph node stage are key to prognosticating
and guiding adjuvant therapy in breast cancer patients.
• The type 1 growth factor family (e.g. EGFR and HER-2) are becoming key molecular features in
the design of novel biologically targeted therapies.
• The combination of standard pathological features and each cancer's genetic fingerprint will enable
us in the future to individually tailor each patient's therapy.
References
1. Singletary SE, Allred C, Ashley P, et al. Revision of the
American Joint Committee on Cancer staging system
for breast cancer. J Clin Oncol 2002;20:3628–36.
2. Rampaul RS, Mullinger K, Macmillan RD, et  al.
Incidence of clinically significant lymphoedema as a
complication following surgery for primary operable
breast cancer. Eur J Cancer 2003;39(15):2165–7.
3. Mansel RE, Fallowfield L, Kissin M, et  al.
Randomized multicenter trial of sentinel node bi-
opsy versus standard axillary treatment in operable
breast cancer: the ALMANAC Trial. J Natl Cancer
Inst 2006;98(9):599–609.
4. Alvarez S, Anorbe E, Alcorta P, et  al. Role of so-
nography in the diagnosis of axillary lymph node
metastases in breast cancer: a systematic review. Am
J Roentgenol 2006;186:1342–8.
5. Rampaul RS, Evans AJ, Ellis IO, et al. Long term
regional recurrence and survival after axillary
nodal sampling for breast cancer. Eur J Cancer
2003;1(4):23(abstr.).
6. Shukla HS, Melhuish J, Mansel RE, et al. Does lo-
cal therapy affect survival rats in breast cancer? Ann
Surg Oncol 1999;6(5):455–60.

Pathology and biology of breast cancer
39
7. Macmillan RD, Rampaul RS, Lewis S, et  al.
Preoperative ultrasound-guided node biopsy and
sentinel node augmented node sample is best prac-
tice. Eur J Cancer 2004;40(2):176–8.
8. Veronesi U, Galimberti V, Zurrida S, et al. Sentinel
lymph node biopsy as an indicator for axillary
dissection in early breast cancer. Eur J Cancer
2001;37(4):454–8.
9. Veronesi U, Paganelli G, Viale G, et  al. Sentinel
lymph node biopsy and axillary dissection in breast
cancer: results in a large series. J Natl Cancer Inst
1999;91(4):368–73.
10. Schwartz GF, Guiliano AE, Veronesi U; Consensus
Conference Committee. Proceeding of the consensus
conference of the role of sentinel lymph node bi-
opsy in carcinoma or the breast April 19–22, 2001,
Philadelphia, PA, USA. Breast J 2002;8(3):124–38.
11. Cserni G, Amendoeira I, Apostolikas N, et  al.
Discrepancies in current practice of pathological
evaluation of sentinel lymph nodes in breast can-
cer. Results of a questionnaire based survey by the
European Working Group for Breast Screening
Pathology. J Clin Pathol 2004;57(7):695–701.
12. Fisher ER, Fisher B, Sass R, et al. Pathologic findings
from the National Surgical Adjuvant Breast Project
(Protocol No. 4). XI. Bilateral breast cancer. Cancer
1984;54:3002–11.
13. Carter CL, Allen C, Henson DE. Relation of tu-
mor size, lymph node status, and survival in 24,740
breast cancer cases. Cancer 1989;63:181–7.
14. Bennett RL, Evans AJ, Kutt E, et  al. Pathological
and mammographic prognostic factors for screen
detected cancers in a multi-centre randomised, con-
trolled trial of mammographic screening in women
from age 40 to 48 years. Breast 2011;20(6):525–8.
15. Foulkes WD, Grainge MJ, Rakha EA, et al. Tumor size is
an unreliable predictor of prognosis in basal-like breast
cancers and does not correlate closely with lymph node
status. Breast Cancer Res Treat 2009;117(1):199–204.
16. O'Dwyer PJ. Editorial. Axillary dissection in pri-
mary breast cancer; the benefits of node clearance
warrant reappraisal. Br Med J 1992;302:360–1.
17. Blamey RW. Clinical aspects of malignant disease.
In: Elston CW, Ellis IO, editors. Systemic pathology.
The Breast. 3rd ed. London: Churchill Livingstone;
1998. p. 501–13.
18. Henson DE, Ries L, Freedman LS, et al. Relationship
among outcome stage of disease and histologic grade
for 22,616 cases of breast cancer. Br Cancer Res
Treat 1991;22:207–19.
19. Kutianawala MA, Sayed M, Stotter A, et al. Staging
the axilla in breast cancer: an audit of lymph-node
retrieval in one UK regional centre. Eur J Surg Oncol
1998;24:280–2.
20. Steele RJC, Forrest APM, Gibson T. The efficacy of
lower axillary sampling in obtaining lymph node
status in breast cancer: a controlled randomized
trial. Br J Surg 1985;72:368–9.
21. Dixon JM, Dillon P, Anderson TJ, et  al. Axillary
sampling in breast cancer: an assessment of its ef-
ficacy. Breast 1998;7:206–8.
22. Cabanas RM. An approach for the treatment of pe-
nile carcinoma. Cancer 1977;39:456–66.
23. Royal College of Radiologists. Quality assurance
guidelines for radiologists. 1990 NHS BSP Publications
no. 15.
24. Royal College of Radiologists. Quality assur-
ance guidelines for radiologists. 1997 NHS BSP
Publications no. 15. January.
25. European Commission. European guidelines for
quality assurance in mammography screening. 2nd
ed Luxembourg: Office for Official Publications of
the European Communities; 1996.
26. Mansel RE, Goyal A, Newcombe RG. ALMANAC
Trialists Group. Internal mammary node drain-
age and its role in sentinel lymph node biopsy: the
initial ALMANAC experience. Clin Breast Cancer
2004;5(4):279–84.
27. Rosen PP, Groshen S, Saigo S, et al. Pathological prog-
nostic factors in stage I (TIN0M0) and stage II (TINIM0)
breast carcinoma: a study of 644 patients with median
follow-up of 18 years. J Clin Oncol 1989;7:1239–51.
28. Parker C, Rampaul RS, Pinder SE, et al. E-Cadherin
as a prognostic indicator in primary breast cancer. Br
J Cancer 2001;14(85):1958–63.
29. Rakha EA, El-Sayed ME, Powe DG, et al. Invasive
lobular carcinoma of the breast: response to
hormonal therapy and outcomes. Eur J Cancer
2008;44(1):73–83.
30. Elston CW, Ellis IO. Pathological prognostic fac-
tors in breast cancer I. The value of histological
grade in breast cancer: experience from a large
study with long-term follow-up. Histopathology
2002;41(3A):154–61.
31. Rakha EA, El-Sayed ME, Lee AH, et al. Prognostic sig-
nificance of Nottingham histologic grade in invasive
breast carcinoma. J Clin Oncol 2008;26(19):3153–8.
32. Rakha EA, El-Sayed ME, Menon S, et al. Histologic
grading is an independent prognostic factor in inva-
sive lobular carcinoma of the breast. Breast Cancer
Res Treat 2008;111(1):121–7.
33. Pinder SE, Ellis IO, Galea M, et al. Pathological prog-
nostic factors in breast cancer III. Vascular invasion:
relationship with recurrence and survival in a large
study with long-term follow-up. Histopathology
1994;24(1):41–7.
34. Truong
PT,
Yong
CM,
Abnousi
F,
et 
al.
Lymphovascular invasion is associated with re-
duced locoregional control and survival in women
with node-negative breast cancer treated with
mastectomy and systemic therapy. J Am Coll Surg
2005;200(6):912–21.
35. Haybittle JL, Blamey RW, Elston CW, et al. A prog-
nostic index in primary breast cancer. Br J Cancer
1982;45:361–6.

Chapter 2
40
36. Ellis IO, Bell J, Todd J, et al. Evaluation of immu-
noreactivity with monoclonal antibody NCRC-II in
breast carcinoma. Br J Cancer 1987;56:295–9.
37. Lee AH, Pinder SE, Macmillan RD, et al. Prognostic
value of lymphovascular invasion in women with
lymph node negative invasive breast carcinoma. Eur
J Cancer 2006;42(3):357–62.
38. Gee JM, Eloranta JJ, Ibbitt JC, et al. Overexpression
of TFAP2C in invasive breast cancer correlates with
a poorer response to anti-hormone therapy and re-
duced patient survival. J Pathol 2009;217(1):32–41.
39. Sarwar N, Kim JS, Jiang J, et  al. Phosphorylation
of ERalpha at serine 118 in primary breast cancer
and in tamoxifen-resistant tumours is indicative
of a complex role for ERalpha phosphorylation
in breast cancer progression. Endocr Relat Cancer
2006;13(3):851–61.
40. Gee JM, Shaw VE, Hiscox SE, et  al. Deciphering
antihormone-induced compensatory mechanisms
in breast cancer and their therapeutic implications.
Endocr Relat Cancer 2006;13(Suppl. 1):S77–88.
41. Dowle CS, Owainati A, Robins A, et al. The prog-
nostic significance of the DNA content of human
breast cancer. Br J Surg 1987;74:133–6.
42. Putti TC, El-Rehim DM, Rakha EA, et al. Estrogen
receptor-negative breast carcinomas: a review of
morphology and immunophenotypical analysis.
Mod Pathol 2005;18(1):26–35.
43. Dowsett M, Allred C, Knox J, et  al. Relationship
between quantitative estrogen and progesterone
receptor expression and human epidermal growth
factor receptor 2 (HER-2) status with recurrence in
the Arimidex, Tamoxifen, Alone or in Combination
trial. J Clin Oncol 2008;26(7):1059–65.
44. Kumar RR, Meenakshi A, Sivakumar N. Enzyme
immunoassay of human epidermal growth factor
(hEGFR). Hum Antibodies 2001;10:143–7.
45. Eberhard DA, Huntzicker E, et  al. Epidermal
Growth Factor receptor immunohistochemistry:
assay selection and amplification to breast cancers.
ASCO 2002;no. 1791 (Abstr.).
46. Klijn JGM, Berns PMJ, Schmitz PI, et al. The clini-
cal significance of epidermal growth factor receptor
in human breast cancer: a review of 5232 patients.
Endocr Rev 1992;13:3–17.
47. Tsutsui S, Ohno S, Murakami S, et  al. Prognostic
value of epidermal growth factor and its relationship
to the ER status of 1029 patients with breast cancer.
Breast Cancer Res Treat 2002;71:67–75.
48. Rampaul RS, Pinder SE, Robertson JF, et al. EGFR
expression in operable breast cancer: is it of prognos-
tic significance? Clin Cancer Res 2004;10(7):2578.
49. Ferrero JM, Ramaioli A, Largillier R, et  al.
Epidermal growth factor receptor expression in 780
breast cancer patients: a reappraisal of the prognos-
tic value based on an eight-year median follow-up.
Ann Oncol 2001;12(6):835–41.
50. Nicholson RI, McCelland RA, Gee JMW, et  al.
Epidermal growth factor receptor expression in
breast cancer. Association with response to endocrine
therapy. Breast Cancer Res Treat 1994;29:117–25.
51. Knoop A, Bentzen SM, Nielsen MM, et  al. Value
of epidermal growth factor receptor, HER-2, p53
and steroid receptors in predicting the efficacy of
Tamoxifen in high risk postmenopausal breast can-
cer patients. J Clin Oncol 2001;19:3376–84.
52. Wolff AC, Hammond ME, Schwartz JN, et  al.
American Society of Clinical Oncology/College of
American Pathologists guideline recommendations
for human epidermal growth factor receptor 2 test-
ing in breast cancer. J Clin Oncol 2007;25:118–45.
53. Ratcliffe N, Wells W, Wheeler K, et al. The combina-
tion of in situ hybridization and immunohistochemi-
cal analysis: an evaluation of Her2/neu expression in
paraffin-embedded breast carcinomas and adjacent
normal-appearing breast epithelium. Mod Pathol
1997;10:1247–52.
54. Busmanis I, Feleppa F, Jones A, et  al. Analysis of
cerbB2 expression using a panel of 6 commercially
available antibodies. Pathology 1994;26:261–7.
55. Carlson RW, Moench SJ, Hammond ME, et  al.
HER2 testing in breast cancer: NCCN Task Force
report and recommendations. J Natl Compr Canc
Netw 2006;4(Suppl. 3):S1–24.
56. Bartlett JM, Going JJ, Mallon EA, et al. Evaluating
HER2 amplification and overexpression in breast
cancer. J Pathol 2001;195:422–8.
57. Press MF, Slamon DJ, Flom KJ, et al. Evaluation of
HER-2/neu gene amplification and overexpression:
comparison of frequently used assay methods in a
molecularly characterized cohort of breast cancer
specimens. J Clin Oncol 2002;20:3095–105.
58. Walker RA, Bartlett JM, Dowsett M, et al. HER2
testing in the UK: further update to recommenda-
tions. J Clin Pathol 2008;61(7):818–24.
59. Dowsett M, Bartlett J, Ellis IO, et  al. Correlation
between immunohistochemistry (HercepTest) and
fluorescence in situ hybridization (FISH) for HER-2
in 426 breast carcinomas from 37 centres. J Pathol
2003;199:418–23.
60. Slamon DJ, Clark GM, Wong SG, et  al. Human
breast cancer: correlation of relapse and survival
with amplification of the HER-2/neu oncogene.
Science 1987;235(4785):177–82.
61. Rilke F, Colnaghi MI, Cascinelli N, et al. Prognostic
significance of HER-2/neu expression in breast can-
cer and its relationship to other prognostic factors.
Int J Cancer 1991;49(1):44–9.
62. Viani GA, Afonso SL, Stefano EJ, et  al. Adjuvant
trastuzumab in the treatment of her-2-positive early
breast cancer: a meta-analysis of published random-
ized trials. BMC Cancer 2007;7:153.
63. Carter P, Presta L, Gorman CM, et al. Humanization
of an anti-p185HER2 antibody for human cancer
therapy. Proc Natl Acad Sci U S A 1992;89:4285–9.

Pathology and biology of breast cancer
41
64. Disis ML, Knutson KL, Schiffman K, et  al. Pre-
existent immunity to the HER-2/neu oncogenic
protein in patients with HER-2/neu overexpressing
breast and ovarian cancer. Breast Cancer Res Treat
2000;62:245–52.
65. Ward RL, Hawkins NJ, Coomber D, et al. Antibody
immunity to the HER-2/neu oncogenic protein
in patients with colorectal cancer. Hum Immunol
1999;60:510–5.
66. Mirza AN, Mirza NQ, Vlastos G, et al. Prognostic
factors in node-negative breast cancer: a review of
studies with sample size more than 200 and follow-
up more than 5 years. Ann Surg 2002;235(1):10–26.
67. Bernards R, Destree A, McKenzie S, et al. Effective
tumor immunotherapy directed against an onco-
gene-encoded product using a vaccinia virus vector.
Proc Natl Acad Sci U S A 1987;84:6854–8.
68. Amar S, McCullough AE, Tan W, et  al. Prognosis
and outcome of small (≤1 cm), node-negative breast
cancer on the basis of hormonal and HER-2 status.
Oncologist 2010;15(10):1043–9.
69. Chia S, Norris B, Speers C, et  al. Human epider-
mal growth factor receptor 2 overexpression as a
prognostic factor in a large tissue microarray se-
ries of node-negative breast cancers. J Clin Oncol
2008;26:5697–704.
70. Kraus MH, Popescu NC, Amsbaugh SC, et  al.
Overexpression of the EGF receptor-related proto-
oncogene erbB-2 in human mammary tumor cell
lines by different molecular mechanisms. EMBO J
1987;6:605–10.
71. Ring CJ, Blouin P, Martin LA, et al. Use of transcrip-
tional regulatory elements of the MUC1 and ERBB2
genes to drive tumour-selective expression of a pro-
drug activating enzyme. Gene Ther 1997;4:1045–52.
72. Yu DH, Hung MC. Expression of activated rat neu
oncogene is sufficient to induce experimental metas-
tasis in 3T3 cells. Oncogene 1991;6:1991–6.
73. Bria E, Cuppone F, Fornier M, et al. Cardiotoxicity
and incidence of brain metastases after adjuvant
trastuzumab for early breast cancer: the dark side of
the moon? A meta-analysis of the randomized trials.
Breast Cancer Res Treat 2008;109(2):231–9.
74. Chang H, Riese 2nd DJ, Gilbert W, et al. Ligands for
ErbB-family receptors encoded by a neuregulin-like
gene. Nature 1997;387:509–12.
75. Juhl H, Downing SG, Wellstein A, et  al. HER-2/
neu is rate-limiting for ovarian cancer growth.
Conditional depletion of HER-2/neu by ribozyme
targeting. J Biol Chem 1997;272:29482–6.
76. Deshane J, Siegal GP, Wang M, et al. Transductional
efficacy and safety of an intraperitoneally delivered
adenovirus encoding an anti-erbB-2 intracellular
single-chain antibody for ovarian cancer gene therapy.
Gynecol Oncol 1997;64:378–85.
77. Schmidt M, Hynes NE, Groner B, et al. A bivalent
single-chain antibody-toxin specific for ErbB-2 and
the EGF receptor. Int J Cancer 1996;65:538–46.
78. Park JW, Hong K, Carter P, et al. Development of
anti-p185HER2 immunoliposomes for cancer ther-
apy. Proc Natl Acad Sci U S A 1995;92:1327–31.
79. Chen SY, Yang AG, Chen JD, et al. Potent antitu-
mour activity of a new class of tumour-specific killer
cells. Nature 1997;385:78–80.
80. Newby JC, Johnston SRD, Smith I, et al. Expression
of epidermal growth factor and C-erb-2 during
the development of tamoxifen resistance in human
breast cancer. Clin Cancer Res 1997;3:1643–51.
81. Nicholson RI, McCelland RA, Finlay P, et  al.
Relationship between EGF-R, C-erb-2 protein
expression and Ki67 immunostaining in breast
cancer and hormone sensitivity. Eur J Cancer
1993;29A:1018–23.
82. Giai M, Roagna R, Ponzone R, et al. Prognostic and
predictive relevance of C-erb-2 and ras expression in
node positive and negative breast cancer. Anticancer
Res 1994;14:1441–50.
83. Archer SG, Eliopoulos SA, Spandidos D, et  al.
Expression of ras p21, p53 and C-erb-2 in advan-
vced breast cancer and response to first line hor-
monal therapy. Br J Cancer 1995;72:1259–66.
84. Pegram MD, Konecny GE, O'Callaghan C, et  al.
Rational combinations of trastuzumab with che-
motherapeutic drugs used in the treatment of breast
cancer. J Natl Cancer Inst 2004;96(10):739–49.
85. Muss H, Berry D, Thor A, et al. Lack of interaction
of tamoxifen (T) use and ErbB-2/HER-2/neu (H)
expression in CALGB 8541: a randomized adjuvant
trial of three different doses of cyclophosphamide,
doxorubicin and fluorouracil (CAF) in node-positive
primary breast cancer (BC). Proc Am Soc Clin Oncol
1999;18:68a.
86. Paik S, Bryant J, Park C, et al. ErbB-2 and response
to doxorubicin in patients with axillary lymph node
positive, hormone receptor-negative breast cancer.
J Natl Cancer Inst 1998;90:1361–70.
87. Elledge RM, Green S, Ciocca D, et al. HER-2 ex-
pression and response to tamoxifen in estrogen
receptor-positive breast cancer: a Southwest Oncology
Group Study. Clin Cancer Res 1998;4(1):7–12.
88. Ravin PM, Green S, Albain V, et al. Initial report of
the SWOG biological correlative study of CerbB-2
expression as a predictor of outcome in a trial com-
paring adjuvant CAF with tamoxifen (T) alone. Proc
Am Soc Clin Oncol 1998;17:97a.
89. Rakha EA, Reis-Filho JS, Ellis IO. Basal-like
breast cancer: a critical review. J Clin Oncol
2008;26(15):2568–81.
90. Johannsson OT, Idvall I, Anderson C, et al. Tumour
biological features of BRCA1-induced breast and
ovarian cancer. Eur J Cancer 1997;33:362–71.
91. Chappuis PO, Nethercot V, Foulkes WD. Clinico-
pathological
characteristics
of
BRCA1-
and
BRCA2-related breast cancer. Semin Surg Oncol
2000;18:287–95.