UNIVERSITY OF MEDICINE AND PHARMACY CRAIOVA DOCTORAL SCHOOL DOCTORATE THESIS BRIEF

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1 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

2 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. 2

3 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 nonproliferative, typical and atypical lesions and 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 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 3

4 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 pseudostratified 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. 4

5 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, in situ and invasive. 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 tumorhost 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 5

6 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 table: The prognostic and predictive factors in breast cancer are summarized in the following 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 6

7 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 SIGMA 1:1000 growth factor 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 Cytoplasmic protein of gene DAKO 1:40 bcl-2 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 7

8 Our study was multricenric and performed on the casuistry of the Emergency County Hospital Craiova spread over a period of four years ( ). 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. 8

9 The correlation between age and histological appearance Tumor Total Invasive ductal Invasive lobular Papillary Mucinous 1 1 Squamous cell Medullary Cribriform Apocrine tubular Unspecified infiltrating Paget's disease 2 2 Mixed Total The study group included patients ranging between 25 and 86 years old (average years old) and the age group with the highest number of lesions was the years old group

10 Fig. 1 Invasive ductal with osteoclastic differentiation. Col HE 100X Fig. 2 Invasive lobular. Col HE 100X Fig. 3 Papillary. Col HE 100X Fig. 4 Mixed breast, 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 (111 cases). The most common benign lesions associated with invasive mammary s were in situ, cystic mastoza, typical and atypical hyperplasia, sclerosing adenoza and papillary lesions. 10

11 Lesions associated with invasive mammary s Invasive s Associated lesions CDIS CLIS Atypical hyperplasia Typical hyperplasia Adenoza Papillary lesions Mastoza Apocrine metaplasia Ductal Lobular Papillary Squamous 1 2 Mucin 1 2 Tubular 1 Pithy 1 Cribriform Apocrine Infiltrative Mixed Paget Total 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. 11

12 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). 12

13 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-,Her ,48% ER-/PR-,Her ,90% Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 1 - Invasive papillary positive PR. Col IHC X100 Fig. 2 - Invasive papillary ER positive. Col IHC X100 Fig. 3 - HER2 positivity 3+. Col IHC X200 Fig. 4 - Intraductal papilloma alpha-actin positive. Col IHC X100 13

14 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 s. 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 by lobular. CEA (carcinoembryonic antigen) was studied in our casuistic in 20 cases, given its role in evaluating proliferative lesions to. 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. 14

15 GENERAL CONCLUSION Clinico-statistical study was conducted on a number of 338 cases of malignant breast tumors diagnosed within 4 years between 2008 and Of these, we selected cases of invasive breast accompanied by associated lesions (63.9%). Lesions associated to invasive breast s 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, in situ and invasive, most of the lesions being considered lesions accompanying an invasive. SELECTIVE BIBLIOGRAPHY 1. Abd El-Rehim DM, Pinder SE, Paish CE, et al. - Expression of luminal and basalcytokeratins in human breast. J Pathol (2004) 203: Arpino Grazia, Heidi Weiss, Adrian V LEE et al. - Estrogen receptor positive, progesterone receptor negative breast cancer: Association with Growth Factor Receptor Expression andtamoxifen Resistance. Journ of Nat Cancer Inst (2005) Vol 97. No 17. sept 3. Bharagva R., J. Striebel, A. Onisko, K. McManus, D. J. Dabbs - Ki-67 labeling index în breast : An immunohistochemical study with correlation to molecular subtypes. J Clin Oncol 26: 2008 (May 20 suppl; abstr 22107) 4. Cotran RS, Kumar V, Robbins SL - Cellular injury and adaptation. In Robbins Pathologic Basis of Disease. Edited by Cotran RS, Kumar V, Robbins SL. Philadelphia, London, Toronto, Montreal, Sydney, Tokyo: W.B. Saunders Company (1989) Di Tommaso L, Franchi G, Destro A, Broglia F, Minuti F, Rahal D, Roncalli M. - Toker cells of the breast. Morphological and immunohistochemical characterization of 40 cases. Hum Pathol. (2008) Sep;39(9): doi: /j.humpath Epub 2008 Jul 9 15

16 6. Greenlee Robert T., PhD; and Bickol N. Mukesh, PhD - Breast Cancer Subtypes Based on ER/PR and Her2 Expression: Comparison of Clinicopathologic Features and Survival Clinical Medicine & Research (2009) Volume 7, Number 1/2: Howard E M, Lau AK, et al. - Expression of E-cadherin in high risk breast cancer. J cancer res Clin Oncol (2005) 131: Jensen RA, Page DL, Dupont WD, Rogers LW. - Invasive breast cancer risk in women with sclerosing adenosis. Cancer. (1989) Nov 15;64(10): Joyce J. A. and Pollard J. W. - Microenvironmental regulation of metastasis, Nature Reviews Cancer, (2009) vol. 9, no. 4, pp Kollias J, Ellis IO, Elston CW, Blamey RW - Clinical and histological predictors of contralateral breast cancer. Eur JSurg Oncol (1999) 25: Pervez Shahid, Khan H - Infiltrating ductal breast with central necrosis closely mimicking ductal in situ (comedo type): a case series Journal of Medical Case Reports (2007), 1:83 doi: / Ribeiro-Silva A, Zamzelli Ramalho LN, Garcia SB, et al. - Is p63 reliable indetecting microinvasion in ductal in situ of the breast? Pathol Oncol Res 2003;9: Schnitt SJ, Connolly JL, Tavassoli FA, etal. - Interobserver reproducibility in the diagnosis of ductal proliferative breast lesions using standardized criteria. Am J Surg Pathol (1992) 16: Shaaban AM, Sloane JP, West CR, Moore FR, Jarvis C, Williams EM, Foster CS - Histopathologic types of benign breast lesions and the risk of breast cancer: case-control study. Am J Surg Pathol (2002) 26: Stål O, Pérez-Tenorio G, Akerberg L, Olsson B, Nordenskjöld B, Skoog L,Rutqvist LE. - Akt kinases in breast cancer and the results of adjuvanttherapy. Breast Cancer Res 2003, 5:R Tsubura A, Okada H, Senzaki H, et al. - Keratin expression in the normal breast and in breast. Histopathology (1991) 18:

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