UNIVERSITY OF MEDICINE AND PHARMACY CRAIOVA

DOCTORAL SCHOOL

DOCTORATE THESIS

BRIEF

Histopathology aspects and protein

expression profiles of invasive breast

tumors and lesions associated to them

SCIENTIFIC COORDINATOR

Professor Ştefania Crăiţoiu MD, PhD

PhD STUDENT

Irina-Anca Eremia

CRAIOVA -2013

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CONTENT

INTRODUCTION……………………………………………………….....................pag. 3

KNOWLEDGE STAGE…………..........……………………………....................................

CHAPTER I - EMBRIOLOGY, ANATOMY AND HISTOLOGY OF MAMMARY

GLAND………………....................................................................................................pag. 3

CHAPTER II – EPIDEMIOLOGY AND RISK FACTORS IN INVASIVE BREAST

TUMORS ........................................................................................................................pag. 4

CHAPTER III – A HISTOLOGIC CLASSIFICATION OF BREAST TUMORS

...........................................................................................................................................pag. 5

CHAPTER IV – PROGNOSTIC AND PREDICTIVE FACTORS OF BREAST

CANCER

...........................................................................................................................................pag. 6

PERSONAL CONTRIBUTION .............................................................................................

CHAPTER V – MATERIAL AND METHOD............................................................pag. 7

CHAPTER VI – HISTOPATHOLOGICAL RESULTS ............................................pag. 8

CHAPTER VII – IMMNUOHISTOCHEMICAL RESULTS ................................ pag. 12

VIII - GENERAL CONCLUSIONS ...........................................................................pag. 15

SELECTIVE BIBLIOGRAPHY ................................................................................pag. 15

KEY WORDS: Invasive breast tumors, lesions associated with breast tumors,

histopathological types, immunohistochemical markers.

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INTRODUCTION

Breast cancer is one of the most common malignant tumors diagnosed in more than a

quarter of the cases of malignant tumors found in women, constantly increasing mortality

and morbidity.

The conventional histopathological diagnosis reveals many microscopic subtypes of

malignant tumors and lesions associated with these tumors. These lesions are non-

proliferative, typical and atypical lesions and carcinoma in situ.

Following the evolution of these lesions (clinical, imaging, cytological,

histopathological) is important in early detection of breast cancer.

Breast cancer is a heterogeneous multifactorial disease, which is reflected in the

existence of a wide spectrum of phenotypic subsets of tumors with varied degree of

aggressiveness. Genomics and proteomics research confirmed this heterogeneity also on

molecular level.

Based on the gene expression and the immunohistochemical profile of some cell

proliferation markers or with role in mammary carcinogenesis, has been made a

classification of breast cancer in molecular subtypes, introduced since 2001 and accepted in

2004.

KNOWLEDGE STAGE

CHAPTER I

EMBRIOLOGY, ANATOMY AND HISTOLOGY OF MAMMARY GLAND

Form, function and pathology of the mammary gland are major issues both medical

and social, as we define as mammals, by breastfeeding function. Breast cancer continues to

be a topical issue because the disease frequency is maintained at a high level (for women

ranks first in the incidence of the disease) and in later stages the disease evolution is usually

serious.

Responsible for the genesis of mammary gland are the ectoderm and the

mesenchyme. From the ectoderm will be formed ducts and alveoli, and from the mesoderm

will be formed the connective tissue and vascular structures.

Mammary glands develop from the ectoderm foils on the ventral surface of the

embryo. On the ventral portion of the body, during the fourth week of gestation, two

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ectodermal lanes (milk line) develop. At the mid-gestation (20-32 weeks gestation), under

the influence of placental hormones in mammary buds canalicular system is developed,

initially in the shape of full cords. In the last two months of gestation, the epithelial cords

sanitation is met and also, the development of lobulo-alveolar channels of glandular

structures.

The mammary gland is composed of three major structures: skin, fat subcutaneous

tissue and mammary tissue (parenchyma and stroma). Mammary gland is influenced by

hormone and depending on the hormonal status has a certain histologic appearance. The idle

mammary gland (at rest) is represented by acini covered by cylinder-cubical epithelium with

round nucleus and by a basal layer composed of myoepithelial cells. Lactating mammary

gland has a considerable multiplication of glandular acini and a considerable reduction of the

stroma.

Histologically, the mammary gland is a tubulo-acini gland composed of 15/20

individual glands, each with a galactofor channel that opens on the nipple area. Each gland

consists of lobules, and the lobules from gandular structures are arranged in groups between

which is connective tissue forming stroma.

At the level of mammary ducts, the epithelium is initially cylindrical pseudo-

stratified and then double-stratified, with a layer of flattened cells, myoepithelial cells, and a

layer of cube-shaped cells. The connective tissue surrounding the lobules contains

lymphocytes and plasma cells.

Immunohistochemical, at the mammary acini level there is found the following panel

of antibodies: the basal membrane is positive on collagen IV, luminal epithelial cells express

cytokeratins CK8-18, CK14, CK7, EMA apical in active secretory regions and hormonal

markers ER, PR, the myoepithelial cells express smooth muscle alpha-actin, CK5-6.CK17,

S100, intranuclear p63.

CHAPTER II

EPIDEMIOLOGY AND RISK FACTORS

Breast cancer remains the most common cancer in women, it is estimated that in the

United States were diagnosed 192,370 cases of breast cancer, in 2009, accounting for 27% of

all cancers in women. There are several deaths from cancer each year attributable to breast

cancer, the second leading cause of death after lung cancer. The incidence of breast cancer

increases rapidly with age.

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Factors with a higher level of risk are: history of breast cancer, genetic predisposition

(BRCA1 and BRCA2 genes are autosomal dominant genes and are involved in most cases of

family cancer), breast cancer precursor lesions.

Factors with moderate levels of risk are increased alcohol consumption, and low risk

factors are nulliparity, postmenopausal obesity and replacement hormone therapy.

CHAPTER III

A HISTOLOGIC CLASSIFICATION OF BREAST TUMORS

Breast cancer, one of the most common injuries encountered in women, may be

accompanied by benign lesions. It is important to correctly diagnose benign lesions,

carcinoma in situ and invasive carcinoma.

Most women with breast symptoms will have a benign etiology, only 1 in 10 women

has breast cancer. After establishing a firm diagnosis is necessary both benign reassurance

and an appropriate plan of managing the disease.

The classification of breast tumors is made by various schemes derived from the

histopathological appearance, tumor grade, tumor stage and the protein and genic expression.

The histopathological classification is performed by the descriptive criteria, and currently the

most widely used classification is the one proposed by OMS (Tavassoli F. 2003). This

classification has both diagnostic and prognostic role, various entities described in

association with variable evolution.

Histological grading is an important indicator of prognosis in breast neoplasia. The

majority of grading tumor system uses three major components: the nuclear grade, the

formation of tubules and mitotic index, which is usually marked on a scale of 1-3. The

accurate quantification criteria are specific of each system.

Staging of mammary tumors (T=tumor, N=regional lymph nodes, M=distant

metastases) is important for the patients classification in groups with therapeutic and

prognostic significance. This staging is necessary to determine the type of tumor and tumor-

host relationship. Histopathological classification (pTNM) has prognostic and treatment

recommendation value.

Immunohistochemical classification is based on the expression of estrogen receptor

(ER), progesterone (PR) and Her2/neu protein, these three markers representing the gold

standard in practice. Initially, the classification was done by dividing mammary tumors by

identifying the estrogen receptors: positive and negative. In 2000, Perou et al. suggested that

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there are at least four molecular classes of breast cancer: luminal-like, basal-like, Her2

positive and unclassifiable.

CHAPTER IV

PROGNOSTIC AND PREDICTIVE FACTORS OF BREAST CANCER

The prognostic and predictive factors in breast cancer are summarized in the following

table:

The prognostic and predictive factors in breast cancer

Prognostic factors

Axillary node status

Tumour size

Age

Vascular and lymphatic invasion

Histological grade

Histological subtype

Response to adjuvant therapy

Hormone receptor status

Her2 new expression

Predictive factors

Hormone receptor status

Her2 new expression

Additional potential prognostic / predictive factors

Profile of genic expression

uPAI / PAI expression

Micro marrow metastases

Analysis of p53

The cathepsin D level

Microvascular density

Breast cancer is one of the most common malignancy in women and is the second

cause of death after lung cancer in the United States (Jemal A, Siegel R, Ward E et al.,

2007). In the last two decades, the mortality rate has decreased significantly, primarily due to

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the early use of adjuvant systemic therapy, and because early detection of tumors using

screening methods.

PERSONAL CONTRIBUTION

CHAPTER V

MATERIAL AND METHOD

Used antibodies

Antigen Clone Specificity Manufacturer Dilution

ER 1D5 Nuclear receptor for estrogen Neomarkers 1:100

PgR 1A6 Nuclear receptors for progesterone Neomarkers 1:25

Her2-neu poli Membrane protein of gene Her2/neu DAKO 1:250

CK5/6 D5/16B4 Cytokeratin 5 and 6 DAKO 1:100

CK7 Cytokeratin 7 DAKO 1:100

CK14 LL002 Cytokeratin 14 Novocastra 1:20

CK HMW 34βE12 Cytokeratin 1,5,10 and 14 DAKO 1:50

EGFR 2911 Membrane receptors for epidermal

growth factor

SIGMA 1:1000

SMA 1A4 Smooth muscle Alpha actin SIGMA 1:1500

CD10 56C6 Myoepithelial precursors Novocastra 1:10

P63 4A4 Myoepithelial precursors Santa Cruz 1:500

VIM V9 Myoepithelial precursors DAKO 1:100

Ki-67 MIB1 Nuclear factor of cell proliferation DAKO 1:50

PCNA PC10 Nuclear factor of cell proliferation DAKO 1:200

P53 DO7 Protein of gene p53 Neomarkers 1:50

Bcl-2 124 Cytoplasmic protein of gene

bcl-2

DAKO 1:40

AKT poli Akt 1,2,3 isoforms DAKO 1:1000

CEA DAKO

CD4 OPD4 Helper T lymphocytes DAKO 1:100

CD8 144B Suppressor T lymphocytes DAKO 1:25

CD20 L26 B lymphocytes DAKO 1:400

CD45RO UCHL1 T lymphocytes DAKO 1:100

CD34 QBEnd 10 Endothelial cells DAKO 1:25

UBI poli Ubicuitina Abcam 1:100

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Our study was multricenric and performed on the casuistry of the Emergency County

Hospital Craiova spread over a period of four years (2008-2011). The final studied cases

consisted of 216 tumors, which were selected of 394 mammary tumors histopathologically

examined.

For grading the studied breast tumors we have used Nottingham grading system. The

obtained data were recorded in the examination protocol of mammary tumors used in the

Morphopathology Laboratory. Immunohistochemical reactions were performed on 4 micron

sections obtained from the blocks included in paraffin, which were spread on glass slides

pre-treated with polylysine or electrically charged, and used a high panel of antibodies.

CHAPTER VI

HISTOPATHOLOGICAL RESULTS

We have analyzed 394 cases of mammary tumors by examining the electronic

records of the results, the type and histological grade, the stage, the lesions associated to

invasive breast tumors and immunohistochemical expression.

Of these, there were selected the cases that meet the selection criteria.

Afetr this review, 216 patients remained in the study and the following parameters were

evaluated: age, macroscopic examination, tumor size and histological appearance,

association with in situ component and other associated lesions, the tumor differentiation

degree, presence of lymph node metastasis or distant metastasis and pTNM determination.

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The correlation between age and histological appearance

Tumor 20-29 30-39 40-49 50-59 60-69 70-79 80-89 Total

Invasive ductal

carcinoma

1 4 19 34 31 16 6 111

Invasive lobular

carcinoma

2 11 5 4 2 24

Papillary

carcinoma

1 1 3 7 3 15

Mucinous

carcinoma

1 1

Squamous cell

carcinoma

1 2 3 4 10

Medullary

carcinoma

1 1 2

Cribriform

carcinoma

1 1 2

Apocrine

carcinoma

1 1 2

tubular

carcinoma

1 1 2

Unspecified

infiltrating

carcinoma

5 6 5 4 1 21

Paget's disease 2 2

Mixed

carcinoma

1 2 6 6 6 2 23

Total 2 8 30 60 51 44 21 216

The study group included patients ranging between 25 and 86 years old (average

55.47 years old) and the age group with the highest number of lesions was the 50-59 years

old group.

1 2

10

3 4

5 6

Fig. 1 – Invasive ductal carcinoma with osteoclastic differentiation. Col HE 100X

Fig. 2 – Invasive lobular carcinoma. Col HE 100X

Fig. 3 – Papillary carcinoma. Col HE 100X

Fig. 4 – Mixed breast carcinoma, ductal and lobular. Col HE 40X

Fig. 5 – Carcinoma in situ, comedocarcinom type. Col HE 200X

Fig.6 – Association of papillary hyperplasia, solid and cribriform ductal hyperplasia,

cystic modification with cylindrical cells. Col HE x100

Of the 216 studied cases, the most common microscopic form was the invasive ductal

carcinoma (111 cases).

The most common benign lesions associated with invasive mammary carcinomas

were carcinoma in situ, cystic mastoza, typical and atypical hyperplasia, sclerosing adenoza

and papillary lesions.

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Lesions associated with invasive mammary carcinomas

Invasive

carcinomas

Associated lesions

CDIS CLIS Atypical

hyperplasia

Typical

hyperplasia

Adenoza Papillary

lesions

Mastoza Apocrine

metaplasia

Ductal 126 11 37 27 15 62 5

Lobular 4 2 8 3 2 9 1

Papillary 4 2 3 1

Squamous 1 2

Mucin 1 2

Tubular 1

Pithy 1

Cribriform 2 1 1

Apocrine 2 1 1

Infiltrative 6 2 11 6 2 9 1

Mixed 9 7 13 5 4 11 1

Paget 2 2 1

Total 154 4 22 72 43 26 99 10

Other studied morphological aspects were appearance of the tumor stroma, the

presence and the quantity assessing of the inflammatory infiltrate, the presence and amount

of necrosis, the appearance of tumor margins and the presence of lymphovascular neural

invasion and the presence of microcalcifications.

Desmoplazic stromal response is characterized by the activity of fibroblasts,

extracellular matrix remodeling, angiogenesis and presence of the inflammatory infiltrate.

Tumor stroma with a dense fibrocollagen structure, unequal represented in most

cases (179), it was sometimes reduced (28 cases) and rich in rare cases (9 cases).

Intra-and peritumoral inflammatory infiltrate was generally composed of mature

lymphocytes and rarely of plasma cells. Inflammatory infiltrate was more abundant in the

neighborhood of necrosis areas.

Inflammatory infiltrate was subjectively assessed and marked as follows: absent in

32 cases, reduced in 45 cases, moderate in 102 cases and intense in 27 cases.

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The presence of necrosis was recorded in 126 cases and had focal aspect. The

quantification of the necrosis ranged from focal, where there were involved a small number

of tumor cells, and marked in large areas of necrosis where there were dispersed tumor

islands.

Tumor staging was done using the TNM classification system (2002), based on

tumor size, number of lymph node metastases and distant metastases. Depending on the

tumor size, we obtained the following results:

Tis 1 case, T1b 4 cases, T1c 44 cases of injury with maximum diameter ≤ 2 cm (T1),

120 lesions with diameter of > 2 cm and ≤ 5 cm (T2), lesions with diameter of 31> 5 cm

(T3) and 17 any size lesions, but with the direct extension to the chest wall, tegument (T4).

The presence of lymph node metastases and their quantification was necessary to

establish pTNM classification.

Regarding the evaluation of regional lymph nodes, the following data were recorded:

N0 - 98 cases, N1 - 49 cases, N2 - 28 cases, N3 - 14 cases and Nx (lymph nodes absent or

insufficient) -23 cases.

Distant metastasis (M1) were found in 6 cases, presenting the following location:

bone metastases-1 case, 1 case of bowel metastasis, liver metastasis-1 case, 1 case of

pulmonary metastasis, gingival metastasis-1 case and 1 case of ovarian metastases.

There were studied: the invasion of adipose tissue, invasion of striated muscle tissue,

skin invasion, vascular invasion, and perineural invasion. Invasion of adipose tissue was

present in 211 cases presented two aspects. Most commonly, in 158 cases, at the optical

microscopy examination was observed the presence of isolated tumor cells arranged in

islands in adipose tissue beyond the tumor-stroma interface. The invasion of the tegument

was observed in 9 cases and was accompanied by ulceration.

CHAPTER VII

IMMNUOHISTOCHEMICAL RESULTS

Immunohistochemical study objectives were: the study of hormone receptors (ER,

PG, Her2), markers to identify tumor phenotype (CK, E-cadherin, actin, p63), markers of

cell proliferation (Ki67, p53, PCNA), tumor angiogenesis markers (EGFR ) and markers of

cell apoptosis (BCL2, Akt and ubiquitin).

In this study, hormone receptors were analyzed and grouped into four

immunophenotypes according to their expression (positive or negative).

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Hormone receptors ER and PR is an important predictive factor in breast cancer

therapy. We considered positive score nuclear immunostaining in more than 10% of tumor

cells.

Study immunophenotypes hormone receptors

Hormonal markers No. of cases %

ER+/PR+,Her2+; 68 31,48%

ER+/PR+,Her2-; 91 42,12%

ER-/PR-,Her2+ 14 6,48%

ER-/PR-,Her2- 43 19,90%

Fig. 1 Fig. 2

Fig. 3 Fig. 4

Fig. 1 - Invasive papillary carcinoma positive PR. Col IHC X100

Fig. 2 - Invasive papillary carcinoma ER positive. Col IHC X100

Fig. 3 - HER2 positivity 3+. Col IHC X200

Fig. 4 - Intraductal papilloma alpha-actin positive. Col IHC X100

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For diagnostic, there were used markers such as: cytokeratins, p63, alpha actin to

identify myoepithelial cells.

In our study we used basal cytokeratin (CK5 / 6, CK7 and CK14 34βE12). CK5 / 6

are cytokeratin with high molecular weight marking external myoepithelial cells and are

used mainly in papillary carcinomas.

34βE12 has low specificity, showing immunoreactivity both in myoepithelial cells

and in the luminal epithelial cells.

Alpha-actin of the smooth muscle (SMA) marked the cytoplasm of tumor cells,

normal myoepithelial cells and vascular wall.

P63 has constantly marked the myoepithelial cell nuclei from the normal structures

level and around in situ component, and there were also observed dispersed positive

epithelial malignant cells.

We used E-cadherin in 54 cases to distinguish an invasive ductal carcinoma by

lobular carcinoma.

CEA (carcinoembryonic antigen) was studied in our casuistic in 20 cases, given its

role in evaluating proliferative lesions to carcinoma.

Impairment of the normal regulation of the cell cycle resulting in an increase in the

mitotic activity that can be identified by immunohistochemistry, using anti-proliferation

factors as antibodies. We used antibodies to Ki67, PCNA and p53.

Ki 67 and PCNA were positive in the nuclear level in all cases. The marking intensity

was variable and we have noticed the low levels of intratumoral heterogeneity.

Immunohistochemical detection of p53 protein gene is an important prognostic

marker, correlated with increased histological grade, increased mitotic activity and

aggressive behavior of the tumor.

The most common molecular pathways involved in mammary carcinogenesis, as

described in the literature, mainly cell cycle regulation, apoptosis, angiogenesis, cell

adhesion, maintenance of a malignant phenotype, and resistance to drug therapy.

Angiogenesis study is important because of its clinical significance in the early stages

of tumor growth and angiogenesis markers are used as predictive factors of of tumor

progression and metastasis. There also have been used EGFR and CD34 to highlight the

tumor emboli.

Apoptosis study was conducted on a sample of 30 cases and there have been used the

following markers: Bcl2 protooncogene, Akt and ubiquitin.

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GENERAL CONCLUSION

Clinico-statistical study was conducted on a number of 338 cases of malignant breast

tumors diagnosed within 4 years between 2008 and 2011. Of these, we selected cases of

invasive breast carcinoma accompanied by associated lesions (63.9%).

Lesions associated to invasive breast carcinomas were: cystic mastoza in 45.8% of

cases, the typical hyperplasia in 33% of cases, the atypical hyperplasia in 10% of cases,

sclerosing adenosis in 19.9% of cases, papillary lesions in 12% of cases, apocrine metaplasia

in 4.6% of cases.

Immunohistochemical study contains several aspects, such as: the study of hormone

receptors (ER, PR, Her2), study of used markers to diagnose the tumor subtypes

(cytokeratins, E-cadherin, alpha-actin, p63, CEA), cell proliferation markers (Ki67, p53,

PCNA), tumor angiogenesis markers (EGFR CD34), markers of cell apoptosis (bcl2 Akt and

ubiquitin).

In our study we found that only a small proportion of associated lesions can be

considered precursor lesions, in which we observed lineage of the atypical lesion, carcinoma

in situ and invasive carcinoma, most of the lesions being considered lesions accompanying

an invasive carcinoma.

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