immunohistochemical profile of primary invasive carcinoma
TRANSCRIPT
1
IMMUNOHISTOCHEMICAL PROFILE OF PRIMARY INVASIVE CARCINOMA OF
THE FEMALE BREAST SEEN AT LAUTECH TEACHING HOSPITALS, OSOGBO
AND OGBOMOSO, NIGERIA(JANUARY 2005-DECEMBER 2014).
BEING A DISSERTATION SUBMITTED TO THE NATIONAL POSTGRADUATE
MEDICAL COLLEGE OF NIGERIA IN PARTIAL FULFILMENT OF THE
REQUIREMENTS FOR THE AWARD OF THE FELLOWSHIP OF THE NATIONAL
POSTGRADUATE MEDICAL COLLEGE OF NIGERIA (FMCPath)
BY
DR OLUOGUNWAHEED AKANNI
DEPARTMENT OF MORBID ANATOMY AND HISTOPATHOLOGY
LAUTECH TEACHING HOSPITAL,
PMB 4007, OGBOMOSO,
OYO STATE,
NIGERIA.
NOVEMBER, 2016
2
DECLARATION
This is to certify that this study titled IMMUNOHISTOCHEMICAL PROFILE OF
PRIMARY INVASIVE CARCINOMA OF THE FEMALE BREAST SEEN AT
LAUTECH TEACHING HOSPITALS, OSOGBO AND OGBOMOSO, NIGERIAwas
performed by me in the Department of Histopathology, LAUTECH Teaching Hospitals,
Ogbomoso and Osogbo. This project has not been submitted to any other College for
consideration.
……………………………………… ……………………
Date
DrOluogunWaheed A. (MBBS)
3
CERTIFICATION
This isto certify that we supervised Dr W.A. OLUOGUN in the conduct of thisdissertation
titled ‘’IMMUNOHISTOCHEMICAL PROFILE OF PRIMARY INVASIVE
CARCINOMA OF THE FEMALE BREAST SEEN AT LAUTECH TEACHING
HOSPITALS, OSOGBO AND OGBOMOSO, NIGERIA (JANUARY 2005-DECEMBER
2014)’’.
……………………….
Professor K. A. Adelusola
MBBS, FMCPath.
……………………………
Dr. D. Sabageh
MBBS, FMCPath.
4
ATTESTATION
The study titled ‘’IMMUNOHISTOCHEMICAL PROFILE OF PRIMARY INVASIVE
CARCINOMA OF THE FEMALE BREAST SEEN AT LAUTECH TEACHING
HOSPITALS, OSOGBO AND OGBOMOSO, NIGERIA (JANUARY 2005-DECEMBER
2014)’’ wascarried out by DrOluogunWaheedAkanni in the Department of Histopathology,
LAUTECH Teaching Hospital, Ogbomoso.
……………………………………… ………………..
Dr D. Sabageh (MBBS, FMCPath) Date
Senior Lecturer/Consultant Pathologist
HOD Department of Histopathology
LAUTECH Teaching Hospital,
Ogbomoso,
Oyo state.
5
DEDICATION
I dedicate this project to the Almighty God, to the memory of my late father
Mr.BadmusOluogun and to every member of the Oluogun family.
6
ACKNOWLEDGEMENT
I wish to acknowledge the immense efforts of my supervisors Prof K.A. Adelusola and Dr D.
Sabageh for their guidance, encouragement and dedication to this work. Prof. Adelusola’s
wealth of experience in tutoring and advising me in all aspects of this work is worth
mentioning. I need to write about this gentleman whose humble way of life has touched me in
many ways. My special gratitude to my head of Department Dr D. Sabageh who has shown an
exceptional characteristic of managing intellectuals. I appreciate the way you handle some
things in the Department.I thankDrs O.O. Atandaand T. O. Babatunde for their assistance in
proof-reading and contribution to this work. My gratitude also goes to my other teachers in the
Department of Histopathology: Prof. W.O. Odesanmi, Prof A.S Anjorin, Prof O.S. Ojo, Prof.
B.J. Olasode, Dr A.O. Komolafe, Dr G.O Omoniyi-Esan and Dr. O.O.Odujoko for all they
have imparted to my life.
I am grateful to all my colleagues in the Department for all the support and assistance given to
me. Dr Suleiman is my brother and friend and DrAdekunle is my jolly fellow. I wish to
appreciate DrsAderibigbe, Aladesanmi and Haruna for their assistance and support. I also wish
to thank MrOdetundeAbayomi and MrOkedereJide for the assistance rendered in processing
the immunohistochemistry at IMRAT (Institute for Advanced Medical Research and Training),
Ibadan.
I appreciate the management of LAUTECH Teaching Hospitals Ogbomoso and Osogbo for
their support.I wish to give gratitude to my wife Funke and my children for their
understanding.
Above all to the Almighty God who has given us the gift of life and the knowledge to possess
the land.
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TABLE OF CONTENTS
Page
TITLE PAGE i
DECLARATION ii
CERTIFICATION iii
ATTESTATION iv
DEDICATION v
ACKNOWLEDGEMENT vi
TABLE OF CONTENTS vii
LIST OF TABLES x
LIST OF FIGURES xi
APPENDICES xii
ABBREVIATIONS xiii
ABSTRACT xv
CHAPTER ONE
Introduction
1.1 Background Information 1
1.2 Rationale for the study 4
1.3 Aims and Objectives of the Study 5
CHAPTER TWO
Review of the Literature
2.1 Historical background and Epidemiology 6
2.1.1 Incidence of primary invasive breast carcinoma 8
2.1.2 Age at diagnosis of primary invasive breast carcinoma 9
2.1.3 Sex distribution, racial influence and socio-economic status
8
of primary invasive breast carcinoma 10
2.2Carcinogenesis of primary invasive breast carcinoma 11
2.3Traditional pathological features of primary invasive breast carcinoma 13
2.3.1 Site and size of the primary tumour 13
2.3.2 Axillary lymph node involvement 14
2.3.3 Histological classification and histological grading of primary
epithelial cancer 15
2.3.4 The role of inflammation in primary invasive breast
carcinoma 16
2.3.5 Oestrogen and progesterone receptors in primary invasive
breast carcinoma 17
2.3.6 HER-2/neu in primary invasive breast carcinoma 20
2.3.7 p53 protein in primary invasive breast carcinoma 22
2.3.8 Recent advances in the diagnosis of primary invasive
breast carcinoma 25
2.3.9 Invasive lobular carcinoma morphological diversity and
variants 30
2.3.10 Molecular pathology and Cytogenetics 31
2.3.11 Sentinel node biopsy 31
9
2.3.12 Lymphovascular invasion 32
2.3.13 Role of cytology in the evaluation of prognostic markers 32
2.3.14 Nottingham prognostic index 33
CHAPTER THREE
Materials and Methods 34
CHAPTER FOUR
Results 40
CHAPTER FIVE
Discussion 69
Conclusion 72
REFERENCES 74 APPENDICES .
88
10
LIST OF TABLES
Table1. Frequency Distribution of Histologic Types of Breast Cancers
Table2. Grading of breast cancers using Nottingham histologic grade
Table3. Nottingham histologic grade of Breast cancer
Table4. Distribution of cases of breast cancer according to lymph node positivity
Table5. Frequency Distribution according to ER Expression Status
Table6. Frequency Distribution according to PR Expression Status
Table7. Frequency Distribution according to HER-2 Expression Status
Table8. Frequency Distribution according to p53 Expression Status
Table9. Comparison of Nottingham grade and ER, PR, Her2/neu and p53 Positivity
Table10.1. Nottingham Prognostic Index (NPI) Components
Table10.2. Nottingham Prognostic Index (NPI)
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LIST OF FIGURES
Figure1. Age Distribution of Female Breast Cancer in Ogbomoso and Osogbo
Figure2. Molecular Classification of Primary Invasive Breast Carcinoma
Figure3. Photomicrograph of ER Positive Invasive Ductal Carcinoma X40
Figure4. Photomicrograph of PR Positive Invasive Ductal Carcinoma X40
Figure5. Photomicrograph of Her-2 Positive Invasive Ductal Carcinoma X40
Figure6. Photomicrograph of p53 Positive Invasive Ductal Carcinoma X40
Figure7. Photomicrograph of Invasive Ductal Carcinoma H & Ex40
Figure8. Photomicrograph of Invasive Lobular Carcinoma H & Ex40
Figure9. Photomicrograph of Mucinous Carcinoma H& E x 40
Figure10. Photomicrograph of Tubular Carcinoma H& E x40
Figure11. Photomicrograph of Apocrine Carcinoma H&E x40
Figure12. Photomicrograph of Medullary carcinoma H&E x40
Figure13. Photomicrograph of Metaplastic carcinoma H&E x40
Figure14. Photomicrograph of Malignant Phyllodes H&E x40
Figure15. Photomicrograph of Carcinosarcoma H&E x40
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LIST OF APPENDICES
Appendix1. WHO Histological Classification of Primary Epithelial Breast Cancer
2013
Appendix2. Nottingham Breast Cancer Grading Scheme Tabulated
Appendix3. Quick Score for ER and PR
Appendix4. Her-2/neu expression scoring method
Appendix5. p53 expression scoring method
Appendix6. Ethical approval
13
ABBREVIATIONS
MRI - Magnetic Resonance Imaging
BRCA 1- Breast Cancer Susceptibility Gene 1
BRCA 2- Breast Cancer Susceptibility Gene 2
DNA - Deoxyribonucleic acid
ER - Oestrogen Receptor
PR - Progesterone Receptor
HER-2 - Human Epidermal Growth Factor Receptor 2
Tp53 - 53 kDa protein
NHANES - National Health and Nutrition Examination Survey
CHEK 2 - Checkpoint Kinase 2
PTEN - Phosphatase and Tensin homolog
TNM - Tumour, Nodes, Metastasis
NSABP - National Surgical Adjuvant Breast Project
TMA - Tissue Microarray Analysis
MAPK - Mitogen-Activated Protein Kinase
STAT - Signal Transducer and Activator of Transcription
PKC - Protein Kinase C
CER - Cytoplasmic Estrogen Receptor
FISH - Flourescence in situ Hybridization
IHC - Immunohistochemistry
SERMS - Selective Estrogen Receptor Modulators
EGFR - Epidermal Growth Factor Receptor
SMA - Smooth Muscle Actin
CSC - Cancer Stem Cell
14
NHSBSP - National Health Services Breast Screening Programme
UICC - International Union against Cancer
AJCC - American Joint Committee on Cancer
LVI - Lymphovascular Invasion
NPI - Nottingham Prognostic Index
DD - Distilled Deoinized
IMRAT - Institute for Advanced Medical Research and Training
DAB - Diaminobenzidine
H-Score - Histochemical Score
LUMA - Luminal A
LUMB - Luminal B
TN - Triple Negative
NST - No Special Type
HCL - Hydrochloric Acid
WT - Wild Type
WHO - World Health Organization
G1 Phase - Growth Phase
S Phase - Synthetic Phase
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ABSTRACT
Introduction- Breast cancer is the commonest malignancy in women in both industrialized
and developing countries and the commonest malignancy in Nigerian women. The aim of
this study was to determine the immunohistochemical profile of primary invasive
carcinomas of the female breast seen at the LAUTECH Teaching Hospitals in Osogbo and
Ogbomoso over a ten year period.
Methods- All histologically diagnosed breast cancer cases in the Department of
Histopathology of the LAUTECH Teaching Hospitals, Ogbomoso and Osogbo, Oyo and
Osun state respectively between 2005 and 2014 were recruited into the study. Relevant
information was obtained from histology report cards. Cases with inadequate clinical or
pathology data were excluded from the study. Sections were recut for histological grading
and immunohistochemistry using the indirectimmunoperoxidase method was done on 205
cases. The panel of antibodies used was for oestrogen receptor, progesterone receptor, Her-
2/neu and p53.
Results- Three hundred and forty three cases of breast cancer were seen during the study
period. Out of these, 200 had immunohistochemistry done on them. The age range of the
patients was 20-89 years (mean -49 years). Majority (52.7%) of cases occurred after 50
years of age. Both breasts were equally affected. Large tumour size (> 2.0 cm) was seen in
91.3% of cases. Infiltrating ductal carcinoma (NOS) was the commonest histological
variant and was seen in 88.9% of the cases. Majority of the cases were intermediate grade
cancer (71.0%). High grade and low grade cancer were seen in 22.0% and 7.0% of the
cases respectively. Lymph node metastasis was seen in 23.9% of the cases.
Positivity for oestrogen and progesterone receptors was high and was seen in 62.0% and
70.7% respectively. Her-2/neu positivity was in only 34.2%. p53 positivity was
16
76.1%.Two hundred and five cases had complete set of immunohistochemistry stains. Of
these, 48 (23.9%) were triple negative.
Conclusion-The immunohistochemical profile of female breast cancer in Ogbomoso and
Osogbo shows high percentage of oestrogen and progesterone receptors; Her-2/neu and p53
positivity.
CHAPTER ONE
INTRODUCTION
1.1 BACKGROUND INFORMATION
Breast cancer is a global public health problem being the most common malignancy in women
worldwide.2 It is 9 times more common in women than in men.3 It is also the commonest
malignant tumour in Nigeria.4 One of every fifteen women in Europe and one of every eight
women in the United States of America will develop breast cancer in her lifetime.1,3 The
incidence in Nigeria is yet to be determined but ranges from 3.5% to 13.5% of breast cancer
cases in hospital based reports.5 The diagnosis of breast cancer at earlier stages is associated
with a more favourable overall prognosis.6
The following methods can be used in the detection of breast carcinoma: self-breast
examination, physical examination, mammography, genetic screening, ultrasound and magnetic
resonance imaging (MRI).
The majority of the breast lesions present as lumps for which differentiation between benign
and malignant lesions is often difficult using clinical examination alone.7
17
The factors that predispose to development of breast cancer include genetic and non-genetic
risk factors. Women with a positive family history of breast cancer have higher risk of
developing breast cancer. Certain genes that predispose to the development of breast cancer are
found in some families. Examples are BRCA I and BRCA 2 genes.8
The p53 gene is located on the short arm of chromosome 17 and encodes a 375 amino acid
nuclear phosphoprotein that prevents propagation of genetically altered cells.8 Wild type p53 is
a tumour suppressor protein that plays a vital role in regulating genomic stability by controlling
the cell cycle and inducing apoptosis when cell damage is beyond repair.9 Normally, cells with
wild type p53 are able to delay progression from the G1 to S phase of the cell cycle while
abnormal DNA is repaired. Cells with the inactivated or mutant p53 protein cannot, and thus
the replication of abnormal DNA is not prevented. Consequently, inactivation of wild type p53
gene product represents the most common genetic alteration in human carcinogenesis including
breast cancer.9
Knowledge about the p53 gene is important because breast carcinoma is a genetically diverse
and heterogeneous disease, and clinical course varies greatly according to these genetic
variations.9
Some studies have indicated that breast cancers with p53 mutations, were associated with a
higher tumour grade, negative oestrogen and progesterone receptor (ER/PR) status and the
more aggressive basal phenotype.10 In contrast, other authors have reported that a p53 mutation
does not impact on the outcome of early breast cancer and that the evidence is not strong
enough for p53 status to be recommended as a routine marker in clinical practice.11
Besides genetic factors, environmental factors also play a prominent role in the development of
breast cancer.8 This includes reproductive factors like age at menarche and menopause, age at
delivery of first full-term baby, nulliparity, breast feeding, and oral contraceptives. Other
18
environmental factors include cigarette smoking, alcohol consumption, obesity, exposure to
ionizing radiation during breast development, breast augmentation surgery and dietary factors.8
Some studies have been able to identify genetic composition, hormonal influence and
environmental factors as major predictors of breast cancer incidence and outcome of
management.12 The discovery of oestrogen and progesterone receptors on breast cancer brought
a new dimension to the management and prognostication of breast cancer with hormonal
therapy introduced as an adjuvant therapy. Tamoxifen, letrozole, anastrole, estemestane,
toremifen, fulvestant, mogesterol acetate and androgen are drugs that have been used as
hormonal therapy in breast cancer. Ovarian ablation as well as luteinizing hormone releasing
hormone have also been tried.13
However, the expression of oestrogen and progesterone receptors in breast cancers varies from
race to race and this has been correlated with other prognostic indices in different parts of the
world.4 Breast cancer genes mutation, which was initially thought to be rare among the blacks,
has been shown to be quite common by recent studies.14
Few studies on the level of ER, PR, HER-2 and p53 protein expression have been carried out in
Nigeria.14 These studies just as those conducted outside of Nigeria have been important
reference points in subsequent management of patients with breast cancer. 14
This study was conceived with a view to improving the management of our patients in the
teaching hospital. There is paucity of information on such studies in Nigeria and Africa. Many
centres in Nigeria have not been using immunohistochemical profiles of primary invasive
carcinoma of the female breast in order to improve management of our patients in the teaching
hospitals most especially in the areas of targeted therapy. This study will also help in the
grading, prognostication, and monitoring of the recovery of patients with primary invasive
19
breast carcinoma. Besides, this study will also provide a data base of the immunohistochemical
profiles of the primary carcinoma of the breast in this environment.
1.2 RATIONALE FOR THE STUDY
Although there is an increase in the literature on histopathological features of breast carcinoma
globally, a review of literature has shown that less work has been done in Nigeria in
comparison to other parts of the world. Only few studies have been conducted on the molecular
aspects of this disease of global public health importance in Nigeria. Of note are previous
papers published by Oluwole et al21, Adeniji et al 27, Adelusola et al26and Adesunkanmi et
al30on breast cancer in Ile-Ife, Ibadan, Calabar, etc. These papers have failed to address most of
the issues on the molecular features of breast cancer in our environment. There have been
advances in the study of breast cancers in the area of histological grade, lymph node stage,
stratification of tumour sizes into groups and histological variants. Also,
immunohistochemistry was not available as at the time the previous studies were done. This
study therefore seeks to apply the recent advances in breast cancer study and use
Immunohistochemistry to document in detail the pathological features of primary invasive
breast carcinoma as seen in our patients.
20
With our increasing knowledge of breast cancer, the continual relevance of the prognostic
indices in our histopathological reports needs to be evaluated from time to time. This study also
seeks to look at these prognostic indices and their continual relevance.
1.3 AIM AND OBJECTIVES
1.3.1 Aim
The aim of this study is to determine the immunohistochemical profile of primary invasive
carcinomas of the female breast seen at the LAUTECH Teaching Hospitals in Osogbo and
Ogbomoso over a ten year period.
1.3.2 Specific Objectives
1. To classify primary invasive carcinomas of the breast in LAUTECH Teaching Hospitals
using the WHO classification.
2. To grade these carcinomas using the Nottingham histologic grading system.
3. To determine the frequency of immunohistochemical markers ER, PR, HER-2/neu and p53
in primary invasive carcinomas of the female breast.
21
4. To classify primary invasive carcinomas of the breast into molecular groups using these
immunohistochemical characteristics
5. To correlate the ER, PR, HER-2/neu and p53 expression with the histological grades of the
breast cancers
CHAPTER TWO
REVIEW OF LITERATURE
2.1 HISTORICAL BACKGROUND AND EPIDEMIOLOGY
The earliest recorded history of breast tumour came from ancient Egypt in 1600BC in a
papyrus obtained by Edwin Smith (1822-1906).18 Since then through the classical Greek
period, the Medieval period and the Renaissance period, different efforts were made to manage
the entity now called breast cancer.18 By the eighteenth century breast exclusion surgery had
become a mode of management for breast cancer.19 The safety of this surgery was further
improved in the nineteenth century. By the twentieth century it had become obvious that
significant higher rate of cure were not to be anticipated by surgery alone. This stimulated
scientific inquiry through epidemiological studies, laboratory research, and statistical analysis
of practical experiences with surgery in the various pathological stages. Radiotherapy,
22
hormones, chemotherapy and immune response became an adjuvant therapy in the
management of breast cancer.19
The breast is a modified sweat gland that is rudimentary in males. The breast lies on the
anterior chest wall over the pectoralis major muscle and extends from the second to the sixth
rib in the vertical axis and from the sternal edge to the mid axillary line in the horizontal axis.1
The adult female breast consists of a series of ducts, ductules and lobular acinar units
embedded within the stroma which is composed of varying amount of fibrous and adipose
tissue.1
Breast cancer is the leading female malignancy worldwide.4 It continues to be a major cause of
death in both premenopausal and postmenopausal women in our society.15 The increase in the
incidence of breast cancers could be due to a change in socioeconomic factors and lifestyle
similar to that of the Western world.16, 17 Also cases of breast cancers are expected to increase
in the developing countries in the nearest future because of the increase in life expectancy since
older women are more likely to develop the disease.17 Mortality from breast cancer is quite
high.17
Chidozie20 studied 116 Nigeria women with breast cancer in Benin and found the peak age of
presentation in Nigeria to be 40-49years, which was a decade earlier than findings from studies
conducted on Caucasians in Europe and America. Majority of breast cancer cases were of high
grade and carried a very poor prognosis because of the late presentation of patients to the
hospital. Oluwole et al21 reviewed cases of breast diseases seen in Ile-Ife between 1977 and
1986. The findings showed 26% had breast cancer. The peak age incidence, histological grade
and the stage of presentation of breast cancer in this study were similar to the findings of
Chidozie.20 Of interest is the finding of Otu et al22 in Calabar who found two peak age
incidences, an early peak made up of patients in the 26-36years group and a late and even
higher peak, consisting of older patients in the 46-50 years group. However other findings in
23
the study were similar to the previous studies by Chidozie and Oluwole et al.21 Follow up study
by Chidozie had attributed the poor prognosis to rapidly progressing breast cancer seen in one
third of patients in his study.23 Hassan et al24 also found an early age incidence in breast cancer
patients in Zaria, with infiltrating ductal carcinoma being the commonest malignant breast
disease. The patients also presented late at stage III and stage IV.24 These findings are in
agreement with a study conducted by Ihekweba in Ibadan who looked at the cases of breast
cancer in Ibadan between 1971 and 1980.25
Adelusola et al26 studied 236 cases of histologically diagnosed breast cancer in Ile-Ife, Osun
state, Nigeria. He also recorded two peak age incidences, which are 40-49years and 60-69
years. One hundred and ninety four cases had the diagnosis of infiltrating ductal carcinoma
(NOS). This finding is in agreement with the previous study by Oluwole et al.21 In 1997,
Adeniji et al27 analyzed cases of breast cancer in Ile-Ife over a period of nineteen years and
only found ten cases occurring in men giving an incidence rate of 1.9% and the remaining
98.1% in females. All the cases were infiltrating ductal carcinoma. This agreed with the general
global knowledge that breast cancer is rare in men.
The study of Muguti in Zimbabwe is similar to the study of Otu et al22 in Calabar with a
bimodal pattern of age peaks which was an early peak between 35 and 40 years and a late peak
between 60 and 65 years.28 The study conducted by Omar in Egypt showed that breast cancer
was the commonest malignancy in Egypt, age presentation, histological picture and mode of
presentation similar to Nigeria cases.29 This is in agreement with the findings of many other
subsequent workers in Nigeria including the study by Adesunkanmi et al30 in Ile-Ife which
shows that the pattern of breast cancer in Nigeria had not changed much over the years.
2.1.1 Incidence of primary invasive breast carcinoma
Breast cancer is the leading female malignancy in the world.29 Globally, the incidence of breast
cancer is increasing, however the increasing incidence varies in the developed and developing
24
countries by up to six fold.29 For example, the incidence of breast cancer is significantly lower
in Japan, Thailand, Nigeria, and India compared to Denmark, New Zealand, United Kingdom
and the United States.29 Some other studies in correlation to this also found incidence rates to
be high in North America, North Europe and Oceania; intermediate in Central and South
America, and South and East Europe; and low in Africa and Asia.31 Interestingly immigrants
from low incidence countries tend to acquire the rate of their new environments.16
Over the past decades the incidence of breast cancer from developing countries has increased
by one to two percent.17 Incidence rate in the developing countries could not be estimated due
to non-availability of data, giving the impression that incidence in these areas is low, however
data from various cancer registries in developing countries suggest that age standardized
incidence rates are rising even more rapidly in low incidence regions such as Africa and Asia.32
The increase in the incidence in these areas has been attributed to change in social economic
factors and change in lifestyle e.g. late child bearing, dietary changes and associated changes in
menstrual cycles.16 Also more cases of breast cancer are expected in the developing countries
in the nearest future because of the increase in life expectancy since older women are far more
likely to develop the disease.16
Mortality of breast cancer is quite high. Breast cancer is the leading cause of cancer death in
women.16 In the year 2000 breast cancer resulted in 189,000 deaths in developed countries and
184,000 deaths in developing countries accounting for 16 and 12 percent of all cancer deaths
respectively.16
2.1.2 Age at diagnosis of primary invasive breast carcinoma
The peak age distribution of breast cancer in the western world is between 45 and 55 years.
This is different in African-Americans where breast cancer tends to occur earlier.32 Some
25
studies from Nigeria even show a more complex age distribution as discussed earlier in other
parts of this literature review.21, 22, 23, 24, 25, 26
In United States of America it was noted that African-American women under the age of 45
years have a greater incidence of breast cancer than Caucasian-American women in this young
age range.32 However these rates equalize during the fifth decade of life, and for women over
the age of 50 years. Pathak et al33 have proposed a plausible and interesting explanation for the
younger age distribution of African-American breast cancer patients. Those investigators
correlated the short-term increase in breast cancer risk that occurs in the postpartum period
with premenopausal breast cancer risk. They hypothesized that the higher prevalence of early
childbearing that is observed among African-American compared with Caucasian-American
women accounts for the higher incidence of early-onset of breast cancer in the former. Palmer
et al34 reported supporting data for this concept in an analysis of The Black Women’s Health
Study. These investigators demonstrated a dual effect of pregnancy on breast cancer risk:
multiparity increased breast cancer risk prior to the age of 45 years but was protective against
breast cancer risk after age 45. Postmenopausal obesity is an established risk factor for breast
cancer because of the higher circulating oestrogen levels that result from fatty tissue
metabolism of adrenal gland steroids in the absence of ovarian function.35 Flegal et al36
analyzed the Third National Health and Nutrition Examination Survey (NHANESIII) and
found that more than half the African-American women over the age of 40 years were obese
and more than 80% were overweight. However contrary to this and as previously noted, breast
cancer incidence rates are significantly lower for African-American women in the
postmenopausal age range.37
Other NHANES findings have implicated physical inactivity and inadequate intake of
micronutrients, as well as other dietary components, as factors contributing to pre- and
postmenopausal breast cancer risk among African-American women.37 The extent to which
26
dietary fat contributes to breast cancer incidence among African-American women is unclear at
present.37
Some studies have related age of patients with prognosis in breast cancer. Most authors have
reported a poorer prognosis in terms of recurrence rate and risk of metastasis in women less
than 35 years in comparison to the older age group.38 Cancers in these younger patients were
also observed more commonly to show factors associated with a worse prognosis which
include grade-3 histology, lymphatic vessel invasion, presence of areas of necrosis and
oestrogen receptor negativity. Similar findings were reported also by Brightmore et al39 as
well as Briks et al40 in different studies.
2.1.3. Sex distribution, racial influence and socio-economic status of primary invasive
breast carcinoma
Breast cancer is relatively uncommon in males. The risk of development of breast cancer is
increased in females compared to males at a ratio of 100:1. Men who are carriers of BRCA2
mutation have higher risk of development of breast cancer.32 A male carrying BRCA2 mutation
has about 6 percent chance of developing breast cancer during his lifetime.32
Caucasian women have overall higher risk of development of breast cancer compared to
African-Americans. This difference is not very apparent until the menopausal age. Breast
cancer incidence in Caucasian women is about twice compared to American Asian, or Hispanic
women. Breast cancer risk is very low in native Americans.41
As previously mentioned, although the incidence of breast cancer is lower in African American
women compared to the Caucasian population, the African American population has a higher
27
breast cancer death rate (31.0 per 100,000) compared to Caucasian women or in fact, compared
to any other racial or ethnic population in the United States.41 Difference in biologic behaviour
and genetic differences including genetic mutations specific to African American women, the
presence of risk factors, access to health care system, health behaviours and relatively later
stage at the time of diagnosis are factors that have contributed to decreased survival of African
American women with breast cancer.41 The incidence of breast cancer is greater in women of
higher socio-economic background while the mortality is higher in women of low socio-
economic background.41
2.2 Carcinogenesis of primary invasive breast carcinoma
The various risk factors in breast cancer can actually be linked to genetic, hormonal and
environmental influences. Acquisition of breast cancer can either be familial or sporadic. These
two categories have different age incidences and behaviours.42 Sporadic cases of breast cancer
are commoner. Risk factors of sporadic breast cancer include estrogen and progesterone
receptors positivity, reproductive behaviour, exposure to external oestrogen stimulation, diet
and environmental radiation whereas in familial cancer, inheritance of mutant BRCA1 and
BRCA2 plays a vital role.42
Breast cancer tumorigenesis can be described as a multi-step process in which each step is
thought to correlate with one or more distinct mutations in major regulatory genes. The
question to be addressed is how far a multi-step progression model for sporadic breast cancer
would differ from that for hereditary breast cancer. Hereditary breast cancer is characterized
28
by an inherited susceptibility to breast cancer on basis of an identified germline mutation in one
allele of a high penetrance susceptibility gene (such as BRCA1, BRCA2, CHEK 2, TP53 or
PTEN).43 Inactivation of the second allele of these tumour suppressor genes would be an early
event in this oncogenic pathway (Knudson's "two-hit" model). Sporadic breast cancers result
from a serial stepwise accumulation of acquired and uncorrected mutations in somatic genes,
without any germline mutation playing a role. Mutational activation of oncogenes, often
coupled with non-mutational inactivation of tumour suppressor genes, is probably an early
event in sporadic tumours, followed by more, independent mutations in at least four or five
other genes, the chronological order of which is likely less important.43
Oncogenes that have been reported to play an early role in sporadic breast cancer are MYC,
CCND1 (Cyclin D1) and ERBB2 (HER2/neu). In sporadic breast cancer, mutational
inactivation of BRCA1/2 is rare, as inactivation requires both gene copies to be mutated or
totally deleted. However, non-mutational functional suppression could result from various
mechanisms, such as hypermethylation of the BRCA1 promoter or binding of BRCA2 by
EMSY. In sporadic breast tumorigenesis, at least three different pathway-specific mechanisms
of tumour progression are recognizable, with breast carcinogenesis being different in ductal
versus lobular carcinoma, and in well differentiated versus poorly differentiated ductal
cancers.43
Thus, different breast cancer pathways emerge early in the process of carcinogenesis,
ultimately leading to clinically different tumor types.43 As mutations acquired early during
tumorigenesis will be present in all later stages, large-scale gene expression profiling using
DNA microarray analysis techniques can help to classify breast cancers into clinically relevant
subtypes.43
2.3 Traditional pathological features of primary invasive breast carcinoma
29
The traditional histopathological features that have been considered in breast cancer and would
be discussed in this chapter are the site and size of the tumour, axillary lymph node
involvement by breast cancer, histological variants of breast cancer, histological grading of
breast cancer, the role of inflammation in breast cancer, oestrogen and progesterone receptors
and breast cancer genes.
2.3.1 Site and size of the primary tumour
Left breast tend to be more involved in breast cancer compared to the right breast. The most
common location of breast cancer is the upper outer quadrant.42
The size of the carcinoma is the second most important factor in prognosticating breast cancer
and it is independent of lymph node metastasis. Size therefore forms part of the basis for the
traditional tumour, nodes, metastasis (TNM) clinical prognostic classification developed by the
International Union Against Cancer.42 The diameter of the tumour has been used as an
objective measurement of tumour size in different studies. By convention and for comparative
purposes, primary tumour sizes are divided into three groups: tumors equal to or less than 2 cm,
tumors 2cm to 5cm, and tumors over 5cm in diameter.42
A previous study conducted by Russo et al44 at the Michigan Cancer Foundation, Detroit, on
the characteristics of primary breast carcinomas of female patients shows the size distribution
of the primary breast carcinomas as follows: < 2cm was 37.3%, 2-5cm was 52.0%, and >5cm
was 10.7%. The relative risk of recurrence in this study for each unit increase in tumour size
was 1.54 for tumors between 2 and 5cm, and 2.45 for tumors over 5cm in diameter and the
relative risk of death for each unit increase in tumour size was 1.57 for tumors 2-5cm and 3.19
for tumors over 5cm in diameter. Bhalla and Chattree in their study found the average
recurrence interval of 49 months, 23 months and 14 months respectively in tumour size
category of 2cm, 2-5cm and >5cm.45
30
Observation from both studies described shows a decline in prognosis with increase in size of
the primary tumour. This is further confirmed by other studies conducted by Nemoto et al46,
Fisher B et al47 and McGuire WL.48
2.3.2 Axillary lymph node involvement
Breast cancer often metastasizes through lymphatic and vascular channels. The clinical picture
seen in breast cancer is often due to this dissemination. Over the years axillary lymph node
metastasis is known to be the most important prognostic factor in invasive carcinomas when
there is no distant metastasis. Data from the US National Surgical Adjuvant Breast Project
(NSABP) shows that women with axillary lymph node involvement have a 40-60% risk of
relapse at 5 years and a 75-80% risk at 10 years. Their overall survival was about 60% at 5
years and <40% at 10 years.46, 47 National surveys of the American College of Surgeon indicate
that every additionally involved lymph node adds to the risk of relapse. It was also found that
the 5 year disease-free survival ranged from 81% in patients with no involved axillary nodes to
18% in patients with 21 or more involved nodes.46
Russo et al44 using the conventional categorization of no axillary node involvement (LN0);
those with one to three involved lymph nodes (LN1-3); and those with four or more involved
nodes (LN4+) found the five year disease-free survival in the different category to be LN0 as
48.6%, LN 1-3 – as 28.6%, and LN 4+ as 22.8%. The proportion free of recurrent disease at 60
months post-operatively was reported as 72%, 60%, and 43% respectively. Bhalla and
Chattree in their study found a decline in survival with increase in the number of lymph nodes
involved.45 Similar decreases in survival with increasing number of involved axillary lymph
nodes have also been reported by Fisher et al.47
2.3.3 Histological classification and histological grading of primary epithelial breast
cancer
31
The World Health Organization classification of breast cancer of 2013 (see appendix I) is used
for the classification of primary epithelial malignant neoplasm of the breast.49
Infiltrating ductal carcinoma is the most frequently encountered malignant tumour of the
breast, accounting for 65 to 80% of all mammary cancers.12 It is termed ‘not otherwise
specified because it is not classified into any of the other categories of invasive mammary
carcinoma. The relative frequency of some of the other types of breast carcinoma is Intraductal
carcinoma 20-30%, Medullary carcinoma 1-5% and Invasive lobular carcinoma 5-10%. Katchy
et al 50 in a study reported that infiltrating ductal carcinoma, NST represented 65.7% of breast
carcinomas while papillary carcinoma, Medullary carcinoma, and tubular carcinoma
represented 9%, 4.5% and 4.5% respectively.
Survival generally, has been related to specific histological types of breast cancer and it was
also noted that the relatively uncommon histological types of breast cancer: Medullary,
Colloid, Tubular and Adenoid cystic carcinomas tend to be prognostically more favourable.15,
47,51
Histological grade provides prognostic information in many tumors including breast cancers.
Two main methods have evolved, based either on nuclear factors or a combination of cellular
features (nuclear, cytological and architectural).52 The latter method is the most widely
accepted for grading breast cancer and has been refined with the stricter definition of more
objective criteria. As a result the reproducibility of histological grading of breast cancer,
previously questioned has been improved; and recent studies have confirmed that good
correlations between pathologists could be obtained if strict criteria are used.52
The currently acceptable grading method is the Nottingham breast cancer grading methods (See
Appendix II), which takes into cognisance tubule formation, nuclear size/pleomorphism and
mitotic count.52 This is an improvement on Greenough who described a system of grading
mammary carcinomas based on three histological components of the tumour cells: the degree
32
of tubule formation; the size, shape and chromatism of the nuclei; and the frequency of
mitoses.53 Studies by Black and associates, as well as Bloom and Richardson show a
relationship between the tumour grade and the survival of patients.54, 55 Other investigators have
confirmed similar close relationship between breast tumour grades and tumour recurrence as
well as overall survival of patients with breast cancer.56
Histological grade, assessed by this method is a strong indicator of patient survival.56 Patients
with grade 1 carcinoma have 85% chances of surviving for 10 years compared with less than
45% for patients with grade 3 tumors.56 A similar method is currently being applied to
cytological preparation with the aim of obtaining prognostic information preoperatively.
2.3.4 The role of inflammation in primary invasive breast carcinoma
The role of inflammation in breast cancer is controversial. Various studies have shown that
inflammation occurring in breast cancer may be an indication of better prognosis, worse
prognosis and may have no effect on prognosis.57, 58 Normally inflammation in breast cancer
suggests a form of immune response. In actual fact the host immune response is known to be
impaired in malignancies. For a tumour cell to elicit an adaptive immune response, the tumour
cells must produce antigens that are recognizable by the immune system and also stimulate the
activation and proliferation of the relevant immune cells. Tumour cells mediate the impaired
immune response by loss or reduction in its antigenicity or by causing defect in its activation
process.58
Breast cancers express tumour associated antigens including oncofetal antigen, c-erbB-2,
MUC-1 and a reduction or absence of class 1 histocompatibility complex molecule in 50% of
cases.59
33
Inflammatory infiltrate as seen in breast cancer is composed of lymphocytes, macrophages and
dendritic cells.58 The lymphocytes express low level of TH1 cytokines consistent with impaired
cytotoxic activity against autologous tumour. The dendritic cells which are also present in the
stroma of invasive carcinoma also show reduced ability to stimulate the allogeneic T cells. In
tumors in which these cells are active there is a better prognosis.60 Non-immune effect of
inflammation is the promotion of growth of cancer cells by producing proteolytic enzymes that
stimulate angiogenesis.
Three distinct patterns of inflammation are seen in breast cancers. The most common is a
diffuse infiltrate of T lymphocytes and macrophages. The other two patterns are perivascular
and perilobular infiltrate of T lymphocytes and macrophages.60
2.3.5 Oestrogen and progesterone receptors in primary invasive breast carcinoma
Oestrogen receptors (ER) are cellular proteins that bind oestrogens with a high affinity and
specificity. They are a necessary component for oestrogen-mediated cellular activity. The
presence of progesterone receptors (PR) demonstrates an active ER mechanism for the
induction of PR expression. Immunohistochemical staining permits the detection and
localization of ER/PR within sections from formalin-fixed, paraffin-embedded tissues. Some
advances in the production of monoclonal antibodies and in antigen retrieval methods have
greatly improved the ability to detect ER/PR in paraffin-embedded tissues.61 Because of such
improvements, ER/PR testing of archival paraffin materials demonstrates good concordance
with biochemical and immunocytochemical assays in frozen tissues.62
Approximately two thirds of postmenopausal breast cancer patients have hormone dependent
breast cancer that requires oestrogen for tumour growth. It is well established that oestrogens
enhance growth and proliferation of certain target cells such as breast epithelial cells and
oestrogen dependent mammary carcinoma cells.63 In postmenopausal women, oestradiol does
34
not appear to function as a circulating hormone; it is biosynthesized from androgens by the
cytochrome P450 enzyme complex called aromatase, which is a product of the CYP19 gene,
with the highest levels of this enzyme present in the peripheral adipose tissues of
postmenopausal women.64 Oestrogen acts mainly at a local level as a paracrine or intracrine
factor. Aromatase has been found and measured in the stromal cell component of the normal
breast as well as in breast tumour. Also, the enzyme has been detected in breast epithelial cells
in vitro.65 Furthermore, expression of aromatase is highest in or near breast tumour sites.66 It
has been observed that aromatase activity and expression is highest in the breast quadrant
containing the tumour, such expression in the tumour containing quadrant is equal to that in the
tumour itself, but double that in a quadrant of the same breast which does not contain a tumour,
which in turn is double the expression in the cancer free breast.67 Evidence that postmenopausal
obesity and weight gain are positively associated with postmenopausal breast cancer risk has
been substantiated especially in women who never used hormone replacement therapy.68
Expression of ER/PR is race-dependent; however, this difference is only established in
postmenopausal women.69 A multiracial study in California USA shows that there was no
difference in steroid hormone receptor status detected in premenopausal breast cancer patients
of different races. In postmenopausal women, 65% of Whites were found to have tumours
positive for cytoplasmic oestrogen receptors (CER) compared with 58, 52, and 41% in women
of mixed race, Blacks, and Asians, respectively. The proportions of tumours that contained a
full complement of receptors (CER, nuclear oestrogen receptors, and cytoplasmic progesterone
receptors) were similar in Blacks, Whites, and Asians in premenopausal group.69 In
postmenopausal patients, significantly fewer White women had tumours devoid of all
receptors, while having a higher incidence of tumours with an abnormal or defective receptor
distribution.
35
The stage of the disease and the degree of nodal involvement seems not to affect receptor status
in any population group, but very large tumours had fewer receptors. White patients with large
neoplasms had a significantly higher incidence of CER than Blacks or Asians. Indications are
that receptor status is inherent to the natural history of the disease and is not influenced by
clinical features.69
Studies have shown that ER/PR receptor status of breast cancer cannot be used to predict the
cancer that will eventually metastasize. It also could not be used to predict survival after
metastasis.70 Also, breast weight directly correlates with ER/PR receptor positivity in the
postmenopausal women. There is evidence that local oestrogen synthesis by breast aromatase
contributes to mammary carcinogenesis. The higher the breast weight, the higher the ER/PR
activity. Also there is an increase in aromatase activity in the peripheral tissue
postmenopausally.69
There is a relationship between ER/PR receptor status and 5 year survival rate.70 Predicting the
clinical outcome in management of patient with tamoxifen shows that proliferating activity
individualises patient with 5 year survival rate.70 This is less with ER positive receptor while
PR positivity alone does not correlate with survival rate in tamoxifen therapy. Mehrdad Nadji
et al71 in an appraisal of ER/PR receptors and correlation with prognosis found that breast
cancer belonging to certain histologic variants with a better prognosis also correspond with the
variants that show high positivity for ER/PR receptor. All pure tubular, colloid, and infiltrating
lobular carcinomas were ER positive. All medullary, apocrine, and metaplastic and most high-
nuclear-grade carcinomas were ER negative.71
Nikos Tsakountakis et al72 in a study in Greece of the relationship of hormonal risk factor of
breast cancer most especially, ER/PR expression with HER2/neu was unable to establish a
concrete link. This was attributed to the small sample size and it was believed that a larger
36
sample size would give a better result that might show a concrete link of expression of ER/PR
with Her2/neu in this study.
Finally, diet seems not to have a direct influence on expression of ER/PR receptors. This is
expressed in studies that have shown that fatty diet does not contribute to the expression of
ER/PR, the same way that it has not been seen to contribute to the risk of developing breast
cancer.73
2.3.6 HER-2/neu in primary invasive breast carcinoma
HER-2 is a member of the epidermal growth factor receptor (GFR/ ERBB) family. ERBB2, a
known proto-oncogene, is located at the long arm of human chromosome 17(17q12). HER2 is
so named because it has a similar structure to human epidermal growth factor receptor or
HER1. Neu is so named because it was derived from a rodent glioblastoma cell line, a type of
neural tumour. ErbB was so named for its similarity to ErbB (avian erythroblastosis oncogene
B), the oncongene later found to code for EGFR.74 HER-2 regulates signaling through several
pathways such as mitogen-activated protein kinase (MAPK); phosphoinositide 3-kinase
(P13K/Akt): Phospholipase C; Protein Kinase C (PKC); signal transducer and activator of
transcription (STAT).75 Signaling through the ErbB family of receptors promotes cell
proliferation and opposes apoptosis. Amplification or over-expression of the ERBB2 gene
occurs in approximately 15-30% of breast cancers.76, 77 It is strongly associated with increased
disease recurrence and a poor prognosis.78 Over-expression is also known to occur in ovarian,
stomach, endometrial carcinoma.79 HER2 proteins have been shown to form clusters in cell
membranes that may play a role in tumorigenesis.80
HER-2 is the target of the monoclonal antibody trastuzumab (Herceptin). Trastuzumab is
effective only in cancers where HER-2 is over-expressed. An important downstream effect of
trastuzumab binding to HER-2 is an increase in P27, a protein that halts cell proliferation.81 It
has been found that in patients with ER + (oestrogen receptor positive)/HER2+ tumours
37
compared with those with ER-/HER2+tumours signaling is through oestrogen receptors.
Normally, oestradiol and tamoxifen acting through the oestrogen receptor down – regulate the
expression of HER2.81
Immunohistochemistry is used to measure the amount of HER2 protein present in breast biopsy
samples obtained by fine-needle aspiration, core needle biopsy, vacuum-assisted breast biopsy,
or surgical excision. Alternatively, fluorescence in situ hybridization (FISH) can be used to
measure the number of copies of the gene which are present.81
HER-2-overexpressed tumours are regarded as biologically aggressive neoplasms. HER2 over
expression is associated with partial resistance to endocrine treatment.82 The complex cross-talk
between ER and HER2 pathways might be an underlying cause of resistance.83
In a study conducted in Ile-Ife by Titiloye et al84 out of 73 cases of breast cancers analyzed for
ER positivity, 45 cases (61.6%) were absolutely negative. Seventy-nine percent were PR
negative (53 out of 67 cases) and only 3 (4.1%) out of 73 cases showed HER 2 / neu
overexpression. Out of 68 cases with complete immunohistochemical data, 50% were triple
negative.84
In another study in South India, where 321 female breast cancer cases were analyzed, ER, PR
and HER-2/neu expression was seen in 59, 51 and 27% of cases respectively. Triple-negative
breast cancers constituted 25% of the cases.85
2.3.7 p53 protein in primary invasive breast carcinoma
The p53 tumour suppressor protein, encoded by the TP53 gene located on chromosome
17p13.1, is a transcription factor that when activated as part of the cellular stress response,
regulates genes involved in cellular processes including the cell cycle, angiogenesis,
replication, repair, apoptosis and senescence.86 The importance of p53 as a tumour suppressor
and sequence-specific transcription factor in human cells is highlighted by the occurrence of
p53 mutations in the majority of cancers.87 Majority of p53 mutations are single amino acid
38
changes that result in missense mutation.88 At the molecular level, p53 mutations found in
breast cancers are usually associated with loss of the ability to maintain proper cell cycle
checkpoints, suppress transformation caused by oncogenes, induce apoptosis and maintain the
integrity of the genome.89 Inheritance of one mutant p53 allele predisposes individuals to
develop malignant tumours such as sarcomas, breast cancer, leukemia, brain tumours and
carcinomas of the adrenal gland. Such individuals are said to have the Li-Fraumeni
syndrome.90
Mutation in p53 is associated with 25% of sporadic cases of breast cancer. However, sporadic
mutations occur at much higher frequencies in BRCA 1or BRCA2 germline-associated breast
cancers due to a decreased efficiency to repair damage.90 BRCA and p53 are involved in
maintaining genome stability by controlling aspects of homologous recombination and repair,
centrosome regulation, cell cycle check points and transcription, where loss of either increases
the possibility of cancer.91 BRCA-1 associated cancers have an altered spectrum of mutations
that may reflect changes in mutagenesis.90 Whereas BRCA-1 mutations are absent in somatic
breast tumours, silencing of the gene through hypermethylation has been reported in sporadic
cases.92 Such epigenetic changes have been reported to associate with oestrogen receptor-
negative (ER-) tumours and occur concomitantly with p53 mutations.92
Based on recent population-based-studies, these subtypes were prevalent among African
American and/or premenopausal women and correlated with a more aggressive disease and
shortened survival, irrespective of lymph node status.93 Regardless of subtype, p53 status (Wild
type or mutant) also displays a signature expression profile of breast tumours, which is a
prognostic indicator of patient survival, where WT p53 associates with a more favourable
outcome.94
However, p53 mutations in breast cancer have been associated with poor prognosis, earlier
onset, increased aggressiveness of tumours, aneuploidy, and adverse responses to
39
chemotherapy.94 The p53 mutations are frequent in the hormone receptor-negative subtypes
(HER2+/ER-) and the basal-like subtypes (ER-, progesterone receptor negative PR-, HER2-,
Cytokeratin 5/6+).94
In some studies of breast cancers with somatic p53 missense mutations where HER2, ER, PR
status were assessed, functional missense mutations were more common in HER 2 negative
tumours (7 of 25, 28%) compared with HER 2 positive tumours (2 of 15, 13%). Among triple-
negative tumours with p53 missense mutations, 5 of 16 (31%) carried functional mutations.94
There are higher frequencies of functional/total missense mutations among Caucasians and
Asians (7 of 29 and 2 of 3, respectively) than among African American patients (2 of 14,
14%).94 Functional mutations are associated with good prognostic factors. Patients carrying
nonfunctional mutations were more likely to be stage III at diagnosis (53% versus 27%), have
high grade tumours (59% versus 35%), and to relapse in distant sites (34% versus 10%) as well
as local sites (12 versus 0%).94 However, women with nonfunctional mutations were more
likely to die (36% versus 20%) at 3years after treatment than those with functional mutations.
Tumours with p53 mutations are also chemosensitive because breast cancer subtypes that
usually have a higher proportion of p53 mutations are more highly proliferative.94
In a study done by Temmin et al95 in Kuwait, 82 cases of breast cancers in young women were
analyzed. About 65% of them displayed positive immunostaining for HER2. About 63.7% of
breast tumours with p53 over expression were aneuploid. About 64.8% of the p53 positive
tumours were node positive. About 93.5% of the p53 immunopositive carcinomas were ER-
negative, and in 95.7% of this subclass of patients no progesterone receptor (PR) could be
detected. The association of negative hormone receptor status and positivity for
p53immunostaining points to tumour aggressiveness.95
In another study done by Gukas et al96 in Jos, Nigeria, where 178 breast cancer cases were
analyzed by immunohistochemistry, 25% and 27.8% of cases expressed ER and PR
40
respectively. The HER-2 and CCNDI antigens were expressed in 25% and 5.7% of cases
respectively. The p53 protein was the most frequently expressed (47.2% of cases). High grade
tumours were significantly more likely to be ER and PR negative.96
In a similar study done by Agboola et al 97 in Shagamu, Nigeria where there was correlation of
p53 expression between Nigeria and UK breast cancer cases. 308 Nigerian cases of breast
cancer were analyzed, and p53 positive expression was significantly correlated with a negative
expression of the steroid hormone receptors ER and PR, p53 was highly expressed and
accounted for 63.3% in Nigerian breast cancer cases, while in UK breast tumours, p53
expression was observed in 25.8%.100 In those breast tumours showing positive p53, a
significant proportion of breast cancers from the Nigerian series were from patients that were
premenopausal and diagnosed before 50 years. Also, the tumours were significantly larger in
size with evidence of metastasis into lymph node and vascular invasion compared with the UK
series.97
In addition, a greater proportion of the breast tumours expressing p53 were associated with
basal phenotype compared to UK. On the contrary, majority of the UK series were positively
correlated with luminal phenotype compared with tumour expressing p53 arising from Nigerian
women.97 Porter et al98 also reported high expression of p53 in African-American compared
with Caucasian women. Similar results were also reported by Jones et al98 observing
differences in p53 expression between African American and Caucasian women. p53
expression was associated with unfavourable tumour characteristics in Nigerian compared with
British counterparts and this is similar to findings regarding p53 expression in breast cancer.100
2.3.8 Recent advances in the diagnosis of primary invasive breast carcinoma
Recent studies in breast cancer point to the fact that breast cancer might not actually be a single
entity. Therefore, there is a need to modify the classification of breast cancer in line with the
41
current thought. The current histological classification schemes are descriptive and relatively
subjective with reliance on assessment by experienced Histopathologists. Furthermore the
histological appearance of the tumours cannot fully reveal the underlying complex genetic
alterations and the biological events involved in their development and progression. The
proposed new classification is based on key molecular events involved in the process of
carcinogenesis providing a molecular explanation for the different morphological phenotypes
and behaviour. Recent high-throughput genomic studies have offered the opportunity to
challenge the molecular complexity of breast cancer and have provided evidence for an
alternative method for classifying breast cancer into biologically and clinically distinct groups
based on gene expression patterns.101 Such new molecular taxonomies have identified many
genes, some of which are being proposed as candidate genes for sub-grouping breast cancer.101
Such studies have been applied on a relatively small number of tumours and require validation
in large series and comparison with traditional classification systems prior to acceptance in
clinical practice. This has partly been achieved using high throughput tissue screening tissue
microarray (TMA) technology. These studies have examined expression of proteins known to
be of relevance in breast cancer and have resulted in recognition of classes of breast cancer
broadly similar to those identified by gene expression studies.101
Perou et al102 were the first to provide a classification system based on gene expression
analysis, and this consisted of four major molecular classes of breast cancer; luminal type,
basal like, normal-like and HER-2 positive. Subsequent studies suggested the existence of more
molecular classes and this ultimately led to addition of a fifth and sixth category with the
molecular spectrum now expanding to luminal A (LUMA), luminal B (LUMB), HER2 over
expressing, basal like, normal-like and claudin-low.103, 105
The luminal A group is the largest group of invasive breast carcinoma (NST). It is most
common subtype representing 40 to 55% of breast cancers. It is characterized by the expression
42
of genes activated by the ER transcription factor that are typically expressed in the luminal
epithelium lining the mammary ducts. It also presents a low expression of genes related to cell
proliferation.102 The luminal A Immunohistochemistry (IHC) profile is characterized by the
expression of ER, PGR, Bcl-2 and cytokeratin CK8/18, an absence of HER2/neu, a low rate of
proliferation measured by Ki67 a low histological grade. Patients with this subtype of cancer
have a good prognosis; the relapse rate is 27.8% being significantly lower than that for other
subtypes.104 In addition, survival from the time of relapse is also longer (median 2.2 years).
They have a distinct pattern of recurrence with higher incidence of bone metastases (18.7%)
and with respect to other localizations such as central nervous system, liver and lung which
represent less than 10%. The majority are well- to moderately differentiated, and most occur in
postmenopausal women. These cancers are slow growing and have good response to hormonal
therapy. The histologic correlation includes tubular carcinoma, cribriform carcinoma, mucinous
carcinoma, endocrine carcinoma, and classic lobular carcinoma.105 The treatment of this
subgroup of breast cancer is mainly based on third-generation hormonal aromatase inhibitors
(AI) in postmenopausal patients, selective oestrogen receptor modulators (SERMs) like
tamoxifen and pure selective regulators of ER like fulvestrant.106
The luminal B group constitutes 15% to 20% of invasive breast carcinoma (NST). While they
express oestrogen receptors, luminal B cancers do not show a corresponding expression of
oestrogen-regulated genes, and may therefore rely upon alternative pathways for growth.107
They occasionally are referred to as triple-positive cancers. They respond to chemo and
hormonal therapy. The histologic correlation include: invasive ductal carcinoma (NST) and
micropapillary carcinoma.105 Compared to the luminal A, they have a more aggressive
phenotype, higher histological grade and proliferative index and worse prognosis. The pattern
of distant relapse also differs, and although the bone is still the most common site of recurrence
(30%), this subtype has a higher recurrence rate in sites such as the liver (13.8%). Additionally,
43
the survival from time of relapse is lower (1.6 years).107 Luminal A and B both express ER, but,
since luminal B’s prognosis is very different, a strong effort to find biomarkers that distinguish
between these two subtypes has been made. The main biological difference between the two
subtypes is an increased expression of proliferation genes, such as MKI67 and cyclin B1 in the
luminal B subtype which also often expresses EGFR and HER-2. At the molecular level,
luminal B cancers appear dramatically distinct from luminal A cancers, at the levels of gene
expression, gene copy, somatic mutation, and DNA methylation; luminal B cancers are also
genetically and genomically altered to a greater extent than luminal A cancers.105
Normal breast-like carcinomas account for 6% to 10% of invasive breast carcinoma (NST).108
They are poorly characterized and have been grouped into the classification of intrinsic
subtypes with fibroadenomas and normal breast samples.102 They express genes characteristic
of adipose tissue, presenting an intermediate prognosis between luminal and basal-like and
usually do not respond to neo-adjuvant chemotherapy. They lack the expression of ER, HER2
and PGR, so these tumors can also be classified as triple negative (TN), without being
considered basal-like as they are negative for CK5 and EGFR. The clinical significance of
these tumours remains to be determined and due to their rarity there are few studies on this
subtype. There are doubts about their real existence and some researchers believe they could be
a technical artifact from high contamination with normal tissue during the microarrays.109
The basal subtype accounts for 5-15% of ductal NST carcinomas.110 These tumours express
proteins normally found in the outer, contractile myoepithelial (or basal) layer of normal breast
ducts and acini, including P-cadherin, p63 or laminin, CK5/6, CK14, CK17 and smooth muscle
markers such as smooth muscle actin (SMA), caldesmon and calponin. They are usually grade
3 and ‘triple negative’(ER, PR and HER2) for not expressing ER, PR or HER-2, with a poorer
prognosis than NST carcinomas with a luminal immunophenotype. These cancers have been
associated with TP53 mutation and BRCA 1 mutation. They are high grade and have high
44
proliferation rates. They are associated with an aggressive course, frequent metastasis to
viscera, bone, brain, liver and lungs. They have poor prognosis.102, 105 Fulford and colleagues
have now characterized specific morphological features which are strongly associated with the
basal phenotype and can be used in routine diagnostic practice to identify this important
subset.110 The main features are the presence of a central scar, tumour necrosis, presence of
spindle cells or squamous metaplasia, high mitotic count and high nuclear- cytoplasmic ratio.
Members of this group are medullary carcinomas, metaplastic carcinomas, adenoid cystic
carcinoma, secretory carcinoma and carcinoma with a central fibrotic focus.110
Her-2 positive cancers represent 7% to 12% of invasive breast cancers (NST). This group
comprises PR/ER-negative carcinomas that overexpress HER 2/neu protein. They have high
proliferation rate with TP53 mutation. The overexpression of HER 2 / neu is due to
amplification of the segment of DNA on 17q21 that includes the HER 2/neu gene and varying
numbers of adjacent genes. This amplicon dominates the gene signature of this group. These
cancers are usually poorly differentiated and are associated with metastasis to the brain, bone,
liver, and lung. They are more likely to have multicentric disease, multiple positive nodes and
local recurrence. They usually affect younger people. They respond to trastuzumab (Herceptin),
anthracycline based chemotherapy, and HER 2 targeted therapy, although they have poor a
prognosis.102, 105 The poor prognosis of HER 2 originates in its high risk of early relapse.105
Claudin-low breast cancer: after the initial molecular classification into subtypes of breast
cancer, a new intrinsic subtype was identified in 2007.111 It is characterized by a low expression
of genes involved in tight junctions and intercellular adhesion, including claudin-3, -4, -7
cingulin, ocludin, and E-cadherin hence the name claudin-low. This subtype is located in the
hierarchical clustering near the basal-like tumors, suggesting that both subtypes share some
characteristic gene expression such as low expression of HER-2 and luminal gene cluster. In
contrast to the basal-like subtype, this new group over-expresses a set of 40 genes related to
45
immune response indicating a high infiltration of tumors immune system cells.112 Claudin-low
tumours have a poor prognosis, albeit presenting a low expression of genes related to cell
proliferation. Otherwise, they overexpress a subset of genes closely linked to mesenchymal
differentiation and epithelial–mesenchymal transition. These features are associated with the
acquisition of a cancer stem cell (CSC) phenotype. It is a relatively rare subset of tumors (12–
14%) clinically corresponding to high grade infiltrating ductal carcinomas, that can present
metaplastic or medullary differentiation.113 Immunohistochemically, they are normally TN; but,
like with the basal-like tumors, the concordance TN/claudin-low is not 100% and about 20% of
claudin-low tumors are positive for hormone receptors.114 These tumors show poor long-term
prognosis and an insufficient response to neoadjuvant chemotherapy with intermediate values
between basal and luminal tumors.112, 114
Infiltrating lobular carcinoma is a distinct entity characterized by its discohesive phenotype, its
lack of expression of E Cadherin and HER 2 and frequent expression of ER protein.115 It also
carries a slightly better prognosis than ductal NST carcinoma and exhibits a predilection for
unusual metastatic sites such as retroperitoneum and serosal surfaces.116
Until recently the tubulo-lobular variant has remained controversial, with debate concerning its
assignment as a lobular subtype. It now appears, following a study from Marchio and
colleagues, using an elegant 3-D modelling technique that tubulo-lobular carcinomas have
similar E-cadherin positivity to pure tubular carcinomas and share the same architectural and
growth patterns.117 They are therefore best classified as variants of tubular rather than lobular
carcinoma.
2.3.9 Invasive lobular carcinoma morphological diversity and variants
Up to 14% of all invasive breast cancers are invasive lobular carcinoma.118 Invasive lobular
carcinoma shows diversity in grade as well as morphology with a number of distinct variants.
46
Some show a very sparse distribution of neoplastic cells for which the term sparse cell variant
is occasionally used, a term not widely recognized. It remains conceivable therefore that
further subtyping of invasive lobular carcinoma should occur based on integrated clinical,
morphological, cytogenetic and molecular studies.118
The established subtypes of invasive lobular carcinoma are; classic, solid, alveolar,
pleomorphic and tubulolobular. These types may exist with each other or with other variants of
breast cancer and in these circumstances the term lobular mixed type has been proposed by the
National Health services breast screening programme (NHSBSP).119 As a group the variant
lesions have a worse prognosis than the classic lesion. For these variants mixed patterns are
commonplace, in particular for solid and alveolar subtypes, where a pleomorphic element is
typical.
2.3.10 Molecular pathology and Cytogenetics
The molecular profile of classic lobular neoplasms is distinctive, though not entirely unique.
The tumours are typically E-cadherin, EGFR and HER2 negative, cytokeratins 1, 5, 10 and 14,
ER and PR positive.120 Some ductal carcinomas share this profile and that pleomorphic lobular
carcinomas can be ER and PR negative and HER2 positive (and HER2 gene amplified).121
Some rare families have been detected whose kindred have a heritable defect in the E-cadherin
gene and who develop either lobular carcinoma or diffuse gastric carcinoma or
both.122 Cytogenetic studies support a continuum between in situ lobular neoplasia and invasive
lobular carcinoma. Classic lobular carcinomas have relatively low numbers of changes
compared to cohorts of breast cancers with loss of 16q and gain of 1p, resembling grade I
ductal carcinomas.123 This and the relatively common observation of tumors with mixed ductal
grade I and lobular phenotypes have led to the conclusion that these lesions are closely related.
47
2.3.11 Sentinel node biopsy
Sentinel node biopsy has been shown to be a valid technique in breast cancer management, but
several questions remain unanswered, including its cost-effectiveness compared with
conventional axillary staging methods such as sampling and the significance of the detection of
so-called micro-metastatic disease. The latter is yet unresolved, partly because it has not proved
possible to reach a consensus on the definition of the term micro-metastasis and partly because
most published studies are too small to be of statistical significance. The International Union
against Cancer (UICC) and the American Joint Committee on Cancer (AJCC) have taken the
pragmatic decision to designate metastases measuring <0.2mm as ‘isolated tumour cells’ and
include this category in pNO; metastases measuring between 0.2 and 2 mm are designated as
micrometastases and categorized as pN1M1.124
In view of these uncertainties the National Health Service Breast Screening Programme
(NHSBSP) Pathology Guidelines state that sentinel node biopsy should still be regarded as a
research area and there is no justification for the routine use of additional techniques such as
immunohistochemistry in the evaluation of lymph nodes since they simply reveal
micrometastases of doubtful clinical significance.125 This view is supported by a recent study
by Rutledge et al who found that macro-metastases (metastases of greater than 2mm) were
found in non-sentinel nodes in 38 out of 60 cases where the sentinel node contained a macro-
metastasis (63%) compared with only 1 out of 29 cases where the sentinel node was the site of
a micro-metastasis (3%).126
2.3.12 Lymphovascular invasion
It is now accepted that the presence of lymphovascular invasion (LVI) is related to nodal status,
local and distant recurrence and overall survival in breast carcinoma. LVI provides little
additional prognostic information in lymph node positive patients but a recent study from
48
Nottingham, using multivariate analysis, has confirmed it to be an independent factor in lymph
node negative patients.127 Even in patients receiving adjuvant hormone or cytotoxic therapy the
presence of LVI increases the relative risk of death by a factor of 1.7.127
2.3.13 Role of cytology in the evaluation of prognostic markers
Material obtained by aspiration techniques can be used to evaluate the expression of receptors
such as oestrogen receptor and progesterone receptor, as well as the levels of expression of
other markers such as E cadherin and p53. Cyto-centrifuged material is better with respect to
yield of tumour cells and in terms of antigen preservation.128 Encouraging results in evaluating
the expression of HER-2 by fluorescent in situ hybridizations and immunocytochemistry using
aspiration material were recently reported.129
2.3.14 Nottingham prognostic index
The NPI, based on assessment of tumour size, histological grade and lymph node stage is
established as the most useful means of stratifying patients with invasive breast carcinoma for
therapeutic management. The Index has been used to demonstrate the significant overall
improvement in survival, which has occurred since the application of systemic adjuvant
therapy based on stratification, by prognostic group, from 55% to 77% at 10 years follow-up.130
One potential drawback of the stratification into prognostic groups is the fact that each group
contains individuals with potential survivals that differ by as much as 10%. This has now been
rectified by a new calculation (-3.0079 x NPI2 + 12.295 x NPI + 83.84) which gives greater
accuracy in individual survival prediction than is obtained from group survivals.130
Although traditional histological factors are currently regarded as the ‘gold standard’ in breast
carcinoma, indices based on molecular and genetic markers are being developed in the hope
that they provide a more objective estimation of prognosis. It is interesting in this context to
49
note the theoretical study based on one such index carried out by Eden and colleagues. They
found that that the NPI had similar prognostic power to the gene-expression profile developed
by Van’t Veer et al.131 It is doubtful if this gene expression could actually replace routine
histological appraisal of breast cancer.
Breast cancer- a stem cell disorder?
The latest trend is engineered towards establishing breast cancer as a stem cell disorder. Many
questions are yet to be answered on this but it is suspected that the breast tissue might have its
own stem cell with the potential of transforming into a malignant cell.132 It is hoped that further
study in this area will be a major breakthrough in breast cancer management.
CHAPTER THREE
MATERIALS AND METHODS
3.1 DESCRIPTION OF THE STUDY AREA
This study was carried out in LAUTECH Teaching Hospitals in Osogbo and Ogbomoso.
Ogbomoso is located on Latitude 8o 08’ 00” East and Longitude of 4o 16’ 00” North of the
Equator. Ogbomoso, the second largest city in Oyo state after Ibadan, which is the capital of
Oyo state, lies within the savannah region and it is a gateway to the northern part of Nigeria
from the West.139
The Osogbo metropolis, the Osun state capital lies between longitude 4° 34’E and latitude 7°
46’N. Osogbo city seats the Headquarters of both Osogbo Local Government Area (situated at
Oke Baale Area of the city) and Olorunda Local Government Area (situated at Igbonna Area of
the city). 140
50
The Osogbo metropolis has the tiers of hospital levels; Ladoke Akintola University of
Technology Teaching Hospital, State comprehensive hospital and Local Health Centres.
3.2. Study Design
This is a hospital based retrospective study, which involve the retrieval of archival records,
paraffin wax blocks and surgical specimens of cases histologically diagnosed as breast cancer
in the histopathology Department of LAUTECH Teaching Hospital between January 2005 and
December 2014.
3.2.1 INCLUSION CRITERIA
All histologically-diagnosed cases of breast cancer from core needle biopsy, lumpectomies and
mastectomies received at the histopathological laboratory of the Department of histopathology
of the LAUTECH Teaching Hospital between 2005 and 2014 with traceable archival slides or
tissue blocks as well as clinical data including age, and site of biopsy were included in the
study.
3.2.2 EXCLUSION CRITERIA
Cases with incomplete bio-data as well as those with missing blocks (where the slides are faded
or cannot be retrieved) were excluded from the study sample. All cases of breast cancer in the
males were excluded.
Previously diagnosed cases of breast carcinoma through lumpectomy or needle biopsy with
same diagnosis on mastectomy specimen were looked out for, to guide against duplication of
data, in such cases only information from the mastectomy specimen which is expected to be
more detailed were used.
3.3. ETHICAL CONSIDERATION
51
Ethical approval has been obtained from the research and Ethics Committee of LAUTECH
Teaching Hospital Ogbomoso, Oyo state. The permission of the heads of Histopathology
department of LAUTECH Teaching Hospitals Osogbo and Ogbomoso has been sought.
There was no risk of this study to the patients, their relatives or to the community as the study
was carried out on archival tissue samples and patients’ clinical records and data generated
during the course of this study were accessible to the investigator only.
All information was coded by number and no name was recorded. All data was transferred to a
password-protected personal computer. Furthermore, all published articles arising from this
research will bear no information revealing the identity of any patient.
3.4. DATA COLLECTION
The materials for this study consist of paraffin-wax blocks of all histologically diagnosed
female breast carcinoma seen at the Department of Histopathology, LAUTECH Teaching
Hospital during the study period.
The original request cards were retrieved, studied and essential clinical details which include
age, and the side (right or left) of the breast tumour were extracted.
Tissue blocks were retrieved and fresh sections about 3-5µm cut from formalin fixed and
paraffin embedded blocks and stained with Hematoxylin and Eosin dyes for histologic
analyses.
3.5. Materials:
Microtome
Sialinized glass slides (positively charged)
Xylene
Ethanol
Hydrogen peroxide
Distilled Deionized (DD) water
Pressure cooker
Plastic slide rack/lids
52
Plastic, microwaveable rack containers
Humid chamber
Counterstains, i.e., hematoxylin
The primary antibody specific to ER (ER6F11 (Dako); for PR, (Dako); for HER-2 is
(ERBB2) (Dako) and for p53, Do-7 (Santacruz).
Buffers:
Tris Buffer pH 7.6
0.05M Tris HCL
0.15M NaCl
0.01% Triton X‐100
Citrate Buffer pH 6.0
10 mMSodium Citrate
0.1% Tween 20
Tris/Saline Buffer pH 7.6
0.05 M TrisHCl
0.015 M NaCl
Peroxide Buffer (optional)
3% peroxide in dd water
3.6. METHODS
PRINCIPLE:
Formalin fixation plus tissue processing to paraffin as well as oven dehydration with ethanol
can “mask” many antigenic sites and hinder antibody binding. In the past, protease treatments
were the classical method of exposing these sites, however, new information has led to
methods that expose the sites better and perhaps are more easily controlled. These methods
involve exposure of the paraffin sections to high heat in while bathed in various solutions with
controlled pH. These conditions can be easily achieved with the help of a microwave oven or
pressure cooker. The procedures for immunohistochemical staining was performed at the
Breast Cancer Laboratory Medical Genetic and Bioethics Research Unit, Institute for
Advanced Medical Research and Training (IMRAT), College of Medicine, University of
Ibadan, Oyo state.
PROCEDURE:
53
Immunohistochemical studies were done by the indirect immunoperoxidase method on the
formalin fixed paraffin-embedded sections (FFPE).
1. Breast specimens were routinely fixed in 10% formalin, embedded in paraffin wax,
paraffin sections were cut at 2- 5 μm, mounted on sialinized slides, and melted at
60oC in a pressure cooker for 2 hours to aid in attachment of sections to glass slides.
2. Slides were dipped into xylene (three times) for 5 minute each to remove the
paraffin wax.
3. The tissues were re‐hydrated by dipping the slides into absolute ethanol (100%),
then 95%ethanol, and finally 70% ethanol.
4. The slides were transferred from 70% ethanol to the Tris buffer and soaked for one
hour.
5. Endogenous peroxidase was quenched by dipping slides into a fresh aqueous
solution of 3% peroxide for 3 min
6. Slides were rinsed with Tris buffer for 3 min
7. Prepared, deparaffinized/rehydrated slides were placed into two full racks and
added to the pressure cooker.
8. The pressure cooker was placed into a 700‐900 Watt microwave oven and cooked
on high power for 40 min
9. Slides were placed in Tris/saline buffer
10. The sections were exposed to the primary antibody (dilution of 1:60 for ER and PR;
1:350 for HER-2 and 1:50 for p53 for one hour).
11. Primary antibody was washed from the slide with Tris buffer
12. Sections were soaked in Tris buffer for 10 minutes (2X 5 min washes).
13. The slides were covered and incubated in a humid chamber for 5 minutes.
14. Sections were rinsed for 10 min (2X 5 min washes) in Tris buffer.
15. A solution of chromogen, 3,3′‐diaminobenzidine (DAB) at 1 mg/ml in
Tris buffer with 0.016% fresh H2O2 was added to the slides and incubated for
approximately 8 minutes.
16. The DAB was washed from the slides with tap water.
17. Slides were dipped in a solution of haematoxylin that is diluted 1:1 in distilled water
and stained for one minute to produce a very light nuclear counterstain.
18. Slides were washed for 1 min in dd water
54
19. Slides were dehydrated by dipping in 95% ethanol for 1 min, then 100% ethanol for
1 min
20. Slides were washed 3 times in xylene
21. Slides were covered with coverslip and viewed. The expression of ER, PR, HER-2
and p53 by the tumour cells was determined using the colometric method.
22. Immunostains was interpreted as positive or negative, and where positive, were
categorised as nuclear, membranous and cytoplasmic by the investigator under the
guidance of the supervising consultants.
The scoring was performed using the modified histochemical score (H-score), a
semiquantitative assessment. Nuclear staining intensity was scored from 0, 1, 2 to 3
in combination with the proportion of cells involved in order to get a range of 0-7 as
the final score for ER and PR positivity as depicted in appendix III. For Her-2
expression, only membrane staining pattern was scored from 0, 1+, 2+, to 3+ where
0/1+ indicates negative; 2+ stands for equivocal and 3+ means positive as depicted
in appendix IV. For p53 expression, nuclear staining pattern was scored from 0, 1+,
2+ to 3+ where 0/1+ indicates negative and 2+/3+ indicates positive as depicted in
appendix v.
Negative and positive controls were performed by omitting the primary antibody
and including control tissues was specified by the antibody supplier respectively.
Photomicrographs of some selected slides are also presented.
Nottingham breast cancer grading scheme was based on three criteria such as tubule
formation, nuclear size with pleomorphism and mitotic count. Each of this criterion
was scored from 1 to 3 while the final score was calculated to get the grade of the
breast tumour ranging from 1 to 3 as depicted in appendix II.
The molecular classification was based on positivity and negativity of ER, PR and
HER-2. Those breast cancers that are negative for the three antibodies are grouped
as basal-like whereas those that are positive for Her-2 and negative for ER and PR
are grouped as HER-2 positive. The luminal groups are those with ER and PR
positive breast cancers.
55
3.7. METHOD OF ANALYSIS
Data obtained was analyzed using both Microsoft Excel and EPI Info statistical software
[version 3.5.4] and statistical package for social sciences 20 (SPSS version 20). The
information obtained was reported using frequencies and percentages. Quantitative data was
presented as mean ± SD [Standard Deviation]. Qualitative data was presented using Chi-square
tests which was used to determine/establish statistical relationship between histological and
immunohistochemical diagnoses. A 95% confidence interval was used in this study and a P
value of ≤ 0.05 was considered statistically significant.
CHAPTER FOUR
RESULTS
Three hundred and forty three cases of female breast cancer were seen during the study period.
Out of these, 205 cases had immunohistochemistry done on them. Breast cancer occurred
slightly higher in the left breast with 172 cases (50.1%) recorded while 171 cases (49.9%) were
on the right breast.
4.1 Age
Two hundred and eighty nine cases were analyzed for age. This is because some of the
histology request cards and the records did not indicate the age of some patients with breast
cancer.
The age range was 20 to 89 years (mean=49.70 years). The peak age incidence is the 6th decade
(50-59years).
56
Majority of the cases occurred between the 4th and 7th decade (30-69 years). More than 50%
occurred over the age of 50 years. (Figure 1 shows age distribution of the female breast cancer
patients).
4.2 Histological type
The commonest histological variant of female breast cancer seen was infiltrating ductal
carcinoma with 305 (88.9%) of the cases. Sixteen cases (4.7%) were invasive lobular
carcinoma while only three cases (0.9%) each were metaplastic carcinoma and carcinosarcoma.
There were 2 cases (0.6%) each recorded for malignant phyllodes and poorly differentiated
carcinoma. There was only 1 case (0.3%) each of apocrine and tubular carcinoma. There were
4 (1.2%) cases of mucinous carcinoma. (Table 1)
4.3 Tumour size
All the 343 breast cancer cases had specified tumour size. In the documented cases, the tumour
size ranged from 1-22 centimeters in the widest diameter (mean=6.2cm). Thirty one (9.0%)
cases had tumour size of less than or equal to 2.0 cm and was classified as tumour size stage 1,
178 (51.9%) had tumour size of 2-5cm and was classified as tumour size stage 2 and 134
(39.1%) had tumour size of greater than 5cm and was classified as stage 3. (Table 2)
4.4 Nottingham grade
Nottingham histological grade using tubule formation, pleomorphism and mitosis was used to
score 343 cases.
Seventy five (22.0%) cases were high grade (Grade 3), 244 (71.0%) cases were intermediate
grade (Grade 2) and 24 (7.0%) cases were low grade (Grade 1) as depicted in table 3.
57
4.5 Lymph node metastasis
Table 4 shows breast cancer positivity in lymph nodes.
Lymph node biopsy was checked in 343 cases. Eighty two cases (23.9%) of the biopsied lymph
nodes showed lymph nodes metastasis while 261 cases (76.1%) were negative for the
malignancy.
Two hundred and sixty one cases (76.1%) had no metastasis seen in the lymph node (lymph
node stage 0), 67 cases (19.5%), 3 cases (0.9%) and 7 cases (2.0%) had 1, 2 and 3 positive
lymph nodes respectively. Seventy seven cases (22.4%) were in this second category of lymph
node stage 2 (1-3 positive lymph nodes). Five cases (1.5%) had 6 positive lymph nodes. This is
in the third category of stage 3 (lymph node positivity of greater than 4). There was no four,
five and more than six lymph nodes involvement.
4.6 Immunohistochemistry
Two hundred and five female breast cancer cases were processed and stained for oestrogen
receptor, progesterone receptor, Her-2/neu antigen and p53 positivity.
Oestrogen and progesterone receptors
Two hundred and five female breast cancer cases were analyzed for oestrogen and
progesterone receptor stains. One hundred and twenty seven cases (62.0%) were positive for
oestrogen receptor while 78 cases (38.0%) were negative. One hundred and forty five cases
(70.7%) were positive for progesterone receptor while 60 cases (29.3%) were negative. (Table
5 and 6).
Her-2/neu
58
Two hundred and five cases were analyzed for Her-2/neu stain. Seventy cases (34.2%) were
positive for the stain while eighty one cases (39.5%) were negative. Fifty four cases (26.3%)
were equivocal. The intensity score for Her-2/neu is as seen in Table 7
p53
Two hundred and five cases were analyzed for p53 stain. One hundred and fifty six cases
(76.1%) were positive while 49 cases (23.9%) were negative. (Table 8)
The two hundred and five cases had a complete result from the four immunohistochemical
markers. Forty eight cases (23.9%) were negative for ER, PR, Her-2/neu. (Triple negative)
4.7 Molecular Classification
The breast cancer cases are classified into four different groups: luminal A, luminal B. Basal-
like and Her-2 positive.
Out of two hundred and five cases, 123 (60%) cases constitute the luminal groups while 49
(23.9%) cases made up of basal-like group and Her-2 positive group constitutes 33 (16.1%).
(Figure 2)
There are some selected photomicrographs of some variants of female breast cancers seen in
our centres as depicted in Figure 3-15.
4.8 Nottingham Prognostic Index
59
The Nottingham Prognostic Index (NPI) combines nodal status, tumour size and histological
grade. NPI can define 3 subsets of patients with different probabilities of dying from breast
cancer; good (≤3.4), moderate (3.41 - 5.4), and poor (> 5.4) prognosis groups.
Out of three hundred and forty three cases, 30.6% show good prognosis while 48.7% show
moderate prognosis and 20.7% show poor prognosis.
Table 1: Frequency distribution of histologic types of breast cancers
Type Frequency (%)
60
Invasive Ductal carcinoma NST
Invasive Lobular Carcinoma
Medullary carcinoma
Metaplastic carcinoma
Mucinous carcinoma
Malignant Phyllodes tumour
Apocrine carcinoma
Carcinosarcoma
Tubular carcinoma
Poorly differentiated carcinoma
Total
305 (88.9)
16 (4.7)
6 (1.7)
3 (0.9)
4 (1.2)
2 (0.6)
1 (0.3)
3 (0.9)
1 (0.3)
2 (0.6)
343 (100.0)
Table 2: Distribution of Breast Cancer in Ogbomoso and Osogbo Based on Tumour Size
61
GRADE TUMOUR SIZE NUMBER PERCENTAGE (%)
1 ≤ 2 31 9.0
2 2-5 178 51.9
3 > 5cm
TOTAL
134
343
39.1
100
Table 3: Nottingham histologic grade of Breast cancer
NHG Frequency (%)
62
Grade I
Grade II
Grade III
Total
24 (7)
244 (71)
75 (22)
343 (100)
63
Table 4: Distribution of cases of breast cancer according to lymph node positivity
No of Positive Lymph Nodes Number of Cases Percentage (%) of Cases
0 261 76.1
1 67 19.5
2 3 0.9
3 7 2.0
6
TOTAL
5
343
1.5
100
64
Table 5: Frequency Distribution according to ER Expression Status
ER Score Frequency (%) Interpretation
Zero
2
3
4
5
6
7
Total
8 (3.9)
70 (34.1)
43 (20.9)
27 (13.2)
25 (12.2)
14 (6.8)
18 (8.9)
205 (100)
Negative
Negative
Positive
Positive
Positive
Positive
Positive
ER - Oestrogen receptor
65
Table 6: Frequency Distribution according to PR Expression Status
PR Score Frequency (%) Interpretation
Zero
2
3
4
5
6
7
Total
15 (7.3)
45 (22.0)
61 (29.8)
13 (6.3)
55 (26.8)
12 (5.9)
4 (1.9)
205 (100)
Negative
Negative
Positive
Positive
Positive
Positive
Positive
PR - Progesterone receptor
66
Table 7: Frequency Distribution according to HER-2 Expression Status
HER-2 Score Frequency (%) Interpretation
Zero
1
2
3
Total
21 (10.2)
60 (29.3)
54 (26.3)
70 (34.2)
205 (100)
Negative
Negative
Equivocal
Positive
HER-2 – Human Epidermal Growth Factor Receptor-2
67
Table 8: Frequency Distribution according to p53 Expression Status
p53 Score Frequency (%) Interpretation
Zero
1+
2+
3+
Total
13 (6.3)
36 (17.6)
85 (41.5)
71 (34.6)
205 (100)
Negative
Negative
Positive
Positive
68
Table 9: Comparison of Nottingham grade and ER, PR, Her2/neu and p53 Positivity
Nottingham
grade
ER positive (%) PR positive (%) Her2/neu
positive (%)
p53 positive
(%)
Grade I
Grade II
Grade III
Total
10 (7.9)
103 (81.1)
14 (11.0)
127 (100)
8 (5.6)
121 (83.4)
16 (11.0)
145 (100)
4 (5.7)
46 (65.7)
20 (28.6)
70 (100)
8 (5.2)
118 (75.6)
30 (19.2)
156 (100)
Chi-square: 15.261
df: 6
There was statistically significant association between the Nottingham grade of the
tumour and ER, PR, HER-2 and p53 status (p-value=0.018321)
69
Table 10.1 Nottingham Prognostic Index (NPI) Components
Nottingham Grade Tumour Size Lymph Node Status
24 (7%)
244 (71%)
75 (22%)
31 (9.0%)
178 (51.9%)
134 (39.1%)
261 (76.1%)
77 (22.4%)
5 (1.5%)
Table 10.2 Nottingham Prognostic Index (NPI)
NPI Score Number of Cases (%) Interpretation
< 3.4
3.4 - 5.4
> 5.4
105 (30.6)
167 (48.7)
71 (20.7)
Good Prognosis
Moderate Prognosis
Poor Prognosis
70
Figure 1
Age distribution of female breast cancer in Ogbomoso and Osogbo
0
10
20
30
40
50
60
70
80
90
100
20-29 30-39 40-49 50-59 60-69 70-79 80-89
Fre
qu
ency
Age group in decades
71
Figure 2: Molecular classification of primary invasive breast carcinoma
86
37
49
33
0
10
20
30
40
50
60
70
80
90
100
LUMINAL A LUMINAL B BASAL-LIKE HER-2 POSITIVE
72
Figure 3
Photomicrograph of Invasive ductal carcinoma (NST). ER positive X40
Note that the tumour cells pick up the stain in the nucleus. The score in this case was 7.
73
Figure 4
Photomicrograph of Invasive ductal carcinoma (NST). PR positive x40.
Note that the Score in this case was 7. It shows nuclear positivity to Progesterone receptor
74
Figure 5
Photomicrograph of Invasive ductal carcinoma (NST). Her-2/neu positive x40
Note that intensity score for this case was 3. Her-2/neu stains the membrane in
comparison to ER/PR which stains the nucleus.
75
Figure 6
Photomicrograph of Invasive ductal carcinoma (NST). p53 positive x40.
Note that intensity score for this case was 3. p53 stains the nucleus like ER/PR
76
Figure 7
Photomicrograph of Invasive ductal carcinoma (NST) H & E x40.
This case was Nottingham grade 3. Note the number of tubule formation, high
pleomorphism and mitotic figure on the picture.
77
Figure 8
Photomicrograph of Invasive lobular carcinoma H & Ex40
Note the column of malignant epithelial cells arranged in Indian file pattern.
78
Figure 9
Photomicrograph of Mucinous Carcinoma H& E x 40
Note the clusters of malignant cells within the pools of extracellular mucin
79
Figure 10
Photomicrograph of Tubular Carcinoma H& E x40
Note the presence of well-formed tubules lined by single layer of cells with small uniform
nuclei. The tumour cells lack myoepithelial cell layers placing the tumour cells in direct
contact with the fibrous stroma.
80
Figure 11
Photomicrograph of Apocrine Carcinoma H&E x40
Note the presence of tubules lined by tumour cells that exhibit apocrine metaplasia.
81
Figure 12
Photomicrograph of Medullary carcinoma H&E x40
Note the presence of solid, syncytial-like sheets of large cells with vesicular, pleomorphic
nuclei with prominent nucleoli. There are frequent mitotic figures. There is moderate to
marked lymphoplasmacytic infilterate surrounding the tumour.
82
Figure 13
Photomicrograph of Metaplastic carcinoma H&E x40
Note the presence of matrix-producing tumour and squamous cell carcinoma.
83
Figure 14
Photomicrograph of Malignant Phyllodes H&E x40
Note the presence of nodules of proliferating stroma covered by epithelium. The tumour
has infiltrative borders.
84
Figure 15
Photomicrograph of Carcinosarcoma H&E x40
Note the presence angulated tubules and mesenchymal cells. The lining tumour cells have
vesicular nuclei and clear cytoplasm. There is high mitotic rate.
85
CHAPTER FIVE
Discussion
The total number of female breast cancer cases in this study was three hundred and forty three
(343) which is more than the number and percentage of cases done in Ile-Ife, Osun state by
Adelusola and Titiloye et al84 which is 66.7% and majority of other centres in Nigeria such as
Benin, Calabar and Zaria with 77%, 74.3% and 64% respectively.20,22,24
The Ladoke Akintola University of Technology Teaching Hospitals Ogbomoso and Osogbo
where this study was carried out are tertiary health institution in Oyo and Osun states of
Nigeria.
In this study, female breast cancer occurrence is slightly more in the left side of the breast than
the right side in 50.1% and 49.9% of cases respectively. This is not different from known
pattern of breast cancer occurring slightly more commonly in the left breast for reasons not
known. Adesunkanmi and Oluwole et al 21 found that breast cancer occurred slightly more on
the left side of the breast in 53.3% and 52.5% respectively of the cases studied.30
In this study female breast cancer was observed to occur between 20-89 years with a mean of
49.70 years. This is in agreement with Adesunkanmi et al30 in a previous study at Ile-Ife, Osun
state which put the age range at 23-85 years and a mean of 48 years. Ihekwaba also recorded a
mean age of 48 years.25 Otu et al22 however recorded a lower age range of 21 to 70 years and a
mean age of 40 years and this was similar to result from Adelusola et al26 in a previous study at
Ile-Ife, Osun state which recorded a mean age of 49 years. Adelusola et al26 also found two
peak age groups of 40-49 years and 60-69 years among the different age groups. However, the
peak age group in the current study was in the 6th decade (50-59) years. This is a decade or two
later than the studies already mentioned from other parts of the country. The study of Otu et
al22 is of interest as it recorded earlier peak age group occurrence, an early age group of 26-35
years and later age of 46-50 years.22 Gukas et al96 in a comparison of pattern of occurrence of
breast cancer in Nigeria and British women noted that the age group seen in African breast
cancer is 1 or 2 decades less than that seen in the Caucasians. The age group seen in this study
tends towards the pattern of the Caucasians. The reason for this might be attributed to increase
in awareness of breast cancer in comparison to what obtained 10 to 15 years ago. Also standard
of living and life expectancy are improving in our society.96
86
In this study, out of the primary invasive carcinoma of the female breast, the commonest
histological variant seen was infiltrating ductal carcinoma, not specified type with 305 cases
(88.9%). The commonest histological variant of breast cancer globally was infiltrating ductal
carcinoma, none otherwise specified which other nomenclature put as no special type. Tubular
carcinoma and mucinous carcinoma have been documented to have better prognosis out of the
invasive breast cancers. Mucinous carcinoma was seen in 1.2 % of cases in this study while
tubular carcinoma was seen in only 1 case. This is in agreement with an earlier study by
Titiloye et al84 in Ile-Ife. This is probably because the social lifestyle of the patients could be
similar since Osogbo and Ile-Ife are located within the same Osun state.
The smallest tumour size seen in this study was 1cm. Only 31 cases (9.0%) had a tumour size
of 2.0 cm or less. This means that the early detection of tumour in Ogbomoso and Osogbo was
poor. Most tumours of less than 2.0 cm would require ancillary investigation which include
mammogram and breast ultrasound before they could be detected. Our centres currently lack
this ancillary method and our screening method has been mass education on self-breast
examination. The bulk of the cases 178 cases (51.9 %) present at stage 2 of the tumour (greater
than 2cm and not more than 5 cm). At this stage, tumour would be palpably enlarged and
patients eventually presented to the hospital. One hundred and thirty four cases (39.1%)
presented at tumour size stage 3 (tumour size greater than 5 cm). This study has shown that
majority of cases of breast cancer in Ogbomoso and Osogbo had tumour size within stage 2 and
3. This is in agreement with Adesunkanmi et al30 that isolated poor awareness, poverty, socio-
cultural behaviour and lack of instituted screening program using ancillary investigative tools
as the cause of late presentation of patients with breast cancer in Osun state. This is also in
agreement with other studies from the country and is in disparity with what is obtained from
industrialized countries where proper screening program has been instituted.23, 25, 44
Some previous studies from Nigeria only looked at the lymph node in the context of either
positive for malignancy or negative for malignancy without actually counting the number of
positive lymph nodes. This has precluded further study in this area related to survival rate and
use of Nottingham prognostic indices. This study therefore looked at the number of lymph
node biopsied and the number that were positive for breast cancer. Different categories of
lymph node stage has been found to correspond with survival rate in breast cancer patients in
previous studies done by Russo et al44 and a study by Bhalla and Chantree.45 A conclusion
could not be drawn from this study because it could not be ascertained whether both
pathologists and surgeons that originally managed the patients actually looked at the lymph
87
node as a case of absent or present for breast cancer. The large number of cases with only one
lymph node reported is in support of this notion.
The overall grading of female breast cancer showed that a grade 2 cancer occurs in 71.0% of
the cases. This grading is not in agreement with Ikpat et al 133 in a study of 300 patients in
Calabar, Nigeria where he found 44 to be grade 1, 119 were grade 2 and 137 were grade 3.
Studies conducted on blacks in America showed similarity to this study.134
There are no studies from our centres that have used immunohistochemistry markers as a
prognostic index in management of breast cancer. In this study, ER, PR, Her-2/neu and p53
immunohistochemical markers were used to categorize breast cancer. The frequency of
positivity of breast cancer to oestrogen receptor, progesterone receptor, Her-2/neu; and p53 as
seen in this study were 62.0%, 70.7%, 61.5%, 76.1%. This result showed a high positivity for
the four immunohistochemical stains. Some studies obtained from African American have
shown that breast cancer cases in this group of people showed a reduction in the positivity for
oestrogen and progesterone receptor than the Caucasians also living in America though this
difference was only seen in the post-menopausal age group.69 Age recorded to less than 50
years and above 50 years did not show a significant difference in ER and PR positivity in this
study. Various studies have also shown that stage of the disease and degree of nodal
involvement seems not to affect receptor status in any population group, but cases with very
large tumour sizes tends to have fewer receptors.135 The tumours seen in this study are
predominantly large in size and this could have resulted in high positivity to oestrogen and
progesterone receptors.
Most of the tumours in this study were Her-2/neu positive. Only 38.5% showed Her-2 /neu
negativity. There is needed to start looking at the possibility of using Herceptin in the
management of this category of patients.
In the assessment of all the cases that had complete ER, PR and Her-2 results, 24.0% were
triple negative. The findings in this study is not far from the assertion by Rheis-Filho and Tutt
of preferential affectation of young and African American women by triple negative cancers.136
The concept of the triple negative cancer today globally is that they most likely belong to group
of basal-like breast cancer described in a new classification based immunohistochemistry
positivity and response to treatment. African Americans have their origin from Africa and are
thought to have similar genetic composition with native Africans like the cohort in this study.
The luminal groups of breast cancer show positivity for ER/PR and has been shown to have a
88
better prognosis. Her-2/neu positive breast cancers belong to a different group because they
respond well to Herceptin. The basal-like breast cancers shows positivity to myoepithelial
markers, have high histological grade, large tumour size and carries the worst prognosis in the
new classification of breast cancer. This behaviour is similar to the findings of the breast cancer
cases seen in this study. However, a larger sample size and further works using myoepithelial
markers would be needed to confirm these findings.
In this study, expressions of the p53 regulatory proteins in 205 breast cancer cases were also
evaluated. Although, there is paucity of information on the p53 expression in Nigerian breast
cancer, the results from this study showed high positivity (76.1%) to p53. This is in agreement
with other studies on p53 protein expression of diagnosis in black women. Porter et al98
reported high expression of p53 in African-American compared with Caucasian women.
Similar results were also obtained from Jones et al99 observing differences in p53 expression
between African-American and Caucasian women. Consistent with the tumours of aggressive
behaviour (i.e diagnosed earlier in life, premenopausal, tumours were larger than 2cm,
metastasis into lymph node and lymphatic vessels), p53 expression was associated with
unfavourable tumour characteristics in Nigerian compared with British counterparts and this is
similar to the findings in this study.100 Studies also suggest that p53 dysfunction contributes to
the modulation of the efficacy of the chemotherapy and therefore considered as prime factor for
chemotherapy failure.137
Conclusion
The pathological features of primary epithelial breast malignancies seen in Ogbomoso and
Osogbo show that large proportion of the cases were characterized by large tumour sizes, high
histological grade, presence of lymph node and high percentage of oestrogen and progesterone
receptors; Her-2/neu and p53 positivity. The features seen in the previous studies have not
really changed over the years. The findings in this study are consistent with the gloomy outlook
of breast cancer as documented by previous studies obtained in Nigeria. The large proportion
of the triple negative and p53 positive cases is a pointer to the poor prognosis of primary
epithelial female breast malignancy in our hospitals and it calls for urgent attention.
Our laboratory must adhere strictly to the use of minimum data set for breast cancer diagnosis,
and the country should adopt a screening method with the use of ancillary investigation
accessible to the patients in addition to the current mass education in self-breast examination.
Immunohistochemistry should be used routinely in the diagnosis and management of breast
89
cancer; multidisciplinary team approach should be used for breast cancer diagnosis and
management. More studies should be undertaken to enhance a better understanding of
biological behaviour of primary epithelial breast malignancies in Nigeria.
The use of stem cell obtained from tumour site for experimental purposes has brought a new
phase to the study of pathogenesis, progression and drug management in cancers. Stem cells
have been noted in breast cancer. A further study using stem cell from Nigerian breast cancer
cases will throw more light on the trends seen in breast cancer cases in Nigeria.
90
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APPENDIX I
WHO HISTOLOGICAL CLASSIFICATION OF PRIMARY EPITHELIAL BREAST
CANCER 2013
EPITHELIAL TUMOURS
Invasive breast carcinoma
Invasive ductal carcinoma of nospecial type (NST)
Pleomorphic carcinoma
Carcinoma with osteoclast-like stromal giant cells
Carcinoma with choriocarcinomatous features
Carcinoma with melanotic features
Invasive lobular carcinoma
Classic lobular carcinoma
Solid lobular carcinoma
Alveolar lobular carcinoma
Pleomorphic lobular carcinoma
Tubulolobular carcinoma
Mixed lobular carcinoma
Tubular carcinoma
Cribiform carcinoma
Mucinous carcinoma
Carcinoma with medullary features
Medullary Carcinoma
Atypical Medullary Carcinoma
Invasive carcinoma NST with medullary features
Carcinoma with apocrine differentiation
Carcinoma with signet-ring-cell differentiation
Invasive micropapillary carcinoma
Metaplastic carcinoma of no special type
Low-grade adenosquamous carcinoma
Fibromatosis-like metaplastic carcinoma
Squamous cell carcinoma
Metaplastic carcinoma with
mesenchymal differentiation
Chondroid differentiation
Osseous differentiation
Other types of mesenchymal differentiation
Mixed metaplastic carcinoma
Myoepithelial carcinoma
Rare types
Carcinoma with neuroendocrine features
Neuroendocrine tumour, well-differentiated
Neuroendocrine carcinoma, poorly differentiated (small cell carcinoma)
Carcinoma with neuroendocrine differentiation
Secretory carcinoma
Invasive papillary carcinoma
Acinic cell carcinoma
105
Mucoepidermoid carcinoma
Polymorphous carcinoma
Oncocytic carcinoma
Lipid rich carcinoma
Glycogen-rich clear cell carcinoma
Sebaceous carcinoma
Salivary glands/skin adnexal type tumours
Cylindroma
Clear cell hidradenoma
106
APPENDIX II
NOTTINGHAM BREAST CANCER GRADING SCHEME TABULATED
TUBULE
FORMATION
NUCLEAR SIZE AND
PLEOMORPHISM
MITOTIC COUNT
(MITOSIS at 40x
objective with diameter of
0.63mm=area of
0.312mm2)
SCORE 1 More than 75% of the
cancer cells are
forming tubules
Tumours with small
uniformly sized nuclei
0-11 mitoses seen
SCORE 2 Between 10% and
75% of the cancer cells
are forming tubules.
Tumours with
moderate variability
12-22 mitoses seen
SCORE 3 Less than 10% of the
cancer cells are
forming tubules
Tumours with marked
irregularities and high
pleormorphism
23 and above mitoses seen
FINAL SCORE.
Grade 1 = Score of 3, 4, and 5
Grade 2 = Score of 6 or 7
Grade 3 = Score of 8 or 9.
107
Appendix III
Quick Score for ER and PR
Proportion score Observation Intensity score Observation
Zero
1
2
3
4
Zero staining
1-25%
26-50%
51-75%
76-100%
Zero
1
2
3
No staining of any
nuclei even at high
magnification
Weak staining(only
visible at high
magnification)
Moderate staining
(Readily visible at
low magnification)
Strong staining
(strikingly positive
even at low
magnification)
The score for intensity is then added to the score for proportion, giving a range of 0-7.
Chances of benefit from Hormonal therapy
0-1—No effect
2-3 —Small (20%) chance of benefit
4-6 —Moderate (50%) chance of benefit
7—Good (75%) chance of benefit
108
Appendix IV
Her 2neu expression scoring method
Membrane Staining pattern Score
< 10% of tumour cells
>10% of tumour cells showing faint/barely
perceptible membrane stain
Weak to moderate complete membrane
staining in >10% of tumour cells or <30%
with strong staining
Strong complete membrane staining in
>30% of tumour cells
0
1+
2+
3+
Interpretation:
0/1+: Negative
2+: Equivocal
3+: Positive
109
Appendix V
p53 expression scoring method.
Nuclear Staining pattern Score
< 10% of tumour cells
>10% of tumour cells showing faint/barely
perceptible nuclear stain
Weak to moderate complete nuclear
staining in >10% of tumour cells or <30%
with strong staining
Strong complete nuclear staining in >30%
of tumour cells
0
1+
2+
3+
Interpretation:
0/1+: Negative
2+: Positive
3+: Positive