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.

7

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)

11

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

12

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

15

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