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1 UNIVERSITY OF MEDICINE AND PHARMACY CRAIOVA FACULTY OF MEDICINE DOCTORAL THESIS SUMMARY CLINICO-IMAGING STUDY OF INVASIVE DUCTAL BREAST CARCINOMAS CORRELATED TO HORMONAL RECEPTORS AND HER2/NEU ONCOPROTEIN STATUS Ph.D. SUPERVISOR Cristiana-Eugenia Siminonescu Ph.D. STUDENT Dana-Elena Enache CRAIOVA
2 CONTENTS THESIS INTRODUCTION... 6 CURRENT STAGE OF KNOWLEDGE CHAPTER I. EPIDEMIOLOGY AND RISK FACTORS IN BREAST CANCER I.A. BREAST CANCER EPIDEMIOLOGY I.B. BREAST CANCER RISK FACTORS CHAPTER II. MAMMARY CARCINOGENESIS DATA II.A. STEROID HORMONES ROLE IN MAMMARY CARCINOGENESIS II.B. GROWTH FACTORS ROLE IN MAMMARY CARCINOGENESIS II.C. ADHESION MOLECULES ROLE IN MAMMARY CARCINOGENESIS CHAPTER III. CLINICAL AND IMAGING DIAGNOSIS CRITERIA IN BREAST CARCINOMAS III.A. CLINICAL DIAGNOSIS CRITERIA IN BREAST CARCINOMAS III.B. IMAGING DIAGNOSIS CRITERIA IN BREAST CARCINOMAS CHAPTER IV. PROGNOSIS AND PREDICTIVE FACTORS IN BREAST CARCINOMA PERSONAL RESEARCH GOAL AND OBJECTIVES CHAPTER V. MATERIAL AND METHODS V.A. STUDIED MATERIAL V.B. USED METHODS CHAPTER VI. RESULTS VI.A. CLINICO-EPIDEMIOLOGICAL, IMAGING AND MACROSCOPIC STUDY OF INVASIVE DUCTAL BREAST CARCINOMAS VI.A.1 CLINICO-EPIDEMIOLOGICAL STUDY PARAMETERS OF INVASIVE DUCTAL BREAST CARCINOMAS VI.A.2. IMAGING STUDY PARAMETERS OF INVASIVE DUCTAL BREAST CARCINOMAS
3 VI.A.3. MACROSCOPIC MORPHOLOGICAL STUDY PARAMETERS OF INVASIVE DUCTAL BREAST CARCINOMAS VI.B. HISTOPATHOLOGICAL STUDY OF INVASIVE DUCTAL BREAST CARCINOMAS VI.C. IMMUNOHISTOCHEMICAL STUDY OF INVASIVE DUCTAL BREAST CARCINOMAS VI.C.1. HORMONE RECEPTORS (ER, PR) EXPRESSION ASSESSMENT, AND THEIR CORRELATION TO CLINICO-MORPHOLOGICAL PARAMETERS AND MAMMOGRAPHIC CALCIFICATIONS VI.C.2. HER2/NEU STATUS ASSESSMENT AND ITS CORRELATION TO CLINICO- MORPHOLOGICAL PARAMETERS, HORMONAL IMMUNOPHENOTYPE AND MAMMOGRAPHIC CALCIFICATIONS CHAPTER VII. DISCUSSIONS VII.A. CLINICO-EPIDEMIOLOGICAL, IMAGING AND MACROSCOPIC STUDIES RESULTS ANALYSIS OF DUCTAL BREAST CARCINOMAS VII.A.1. CLINICO-EPIDEMIOLOGICAL STUDY RESULTS ANALYSIS OF INVASIVE DUCTAL BREAST CARCINOMAS VII.A.2. IMAGING STUDY RESULTS ANALYSIS OF INVASIVE DUCTAL BREAST CARCINOMAS VI.A.3. MACROSCOPIC MORPHOLOGICAL STUDY RESULTS ANALYSIS OF INVASIVE DUCTAL BREAST CARCINOMAS VII.B. HISTOPATHOLOGICAL STUDY RESULTS ANALYSIS OF INVASIVE DUCTAL BREAST CARCINOMAS VII.C. IMMUNOHISTOCHEMICAL STUDY RESULTS ANALYSIS OF INVASIVE DUCTAL BREAST CARCINOMAS VII.C.1. ESTROGEN AND PROGESTERONE RECEPTORS STATUS ANALYSIS VII.C.2. HER2/NEU ONCOPROTEIN STATUS ANALYSIS CHAPTER VIII. CONCLUSIONS REFERENCES
4 KEYWORDS: carcinogenesis, Her2, hormone receptors (ER, PR), risk factors, predictors, metastasis, tumor grade, carcinoma, calcification, immunophenotype. INTRODUCTION Breast cancer is one of the most common malignancies in women, with fatal outcome, accounting for 15% of deaths by cancer. Due to the high incidence, this form of cancer captured the attention of researchers, currently being published numerous studies that examine various parameters: clinical, radiological, morphological and immunohistochemical. Although many epidemiological risk factors have been identified, breast cancer cause can not be specified individually. In other words, epidemiological research gives information about the patterns of breast cancer incidence among population, but not individually. Aim of this paper is to deepen knowledge regarding primitive breast carcinomas, both in terms of clinico-imaging characteristics, histopathological characteristics focusing on factors with prognosis value and immunohistochemical characteristics with emphasis on factors with predictive value. OBJECTIVES Completing this retrospective study on a group of 317 patients diagnosed with invasive ductal breast carcinomas involved achieving the following objectives: -Identification of epidemiological risk factors and pursuing employment in groups known to be at risk or presenting etiopathogenic features; - Identification of imaging characteristic changes and their classification in epidemiological risk groups; - Identification of histopathological types and major architectural patterns of breast carcinomas; - Determining the grade of histological differentiation and grade of tumor progression; - Assessing association with breast carcinoma known precursor lesions; - Immunohistochemical profiling of breast carcinoma; - Establishing relations between certain clinico-morphological and immunohistochemical parameters, degree of aggressiveness and biological behavior of breast carcinoma, with the identification of predictive value markers. 4
5 MATERIAL AND METHODS The studied material was collected from a total of 317 patients diagnosed with invasive ductal breast carcinoma, diagnosis established after surgery treatment performed in the general surgery clinics of the Emergency County Hospital Craiova, in a period between January 2009 and December The number of cases with this diagnosis showed an upward trend, the lowest number of cases were registered in 2009 (95 cases) and highest in 2011 (115 cases). Working methods, described in detail in the thesis, pursued the analysis of cases studied for assessing a series of clinico-epidemiological, imaging and morphological macroscopic parameters and also the possible correlations between them or with histopathological and molecular parameters investigated. Statistical data analysis sought to evaluate correlations between clinico-pathological and immunohistochemical parameters, on the basis of calculating the correlation coefficients (Pearson coefficient) by obtaining straight or curved correlation lines. RESULTS Regarding gender distribution of invasive ductal breast carcinomas analyzed were found both women and men, the vast majority of cases were diagnosed in women. Thus, we found 3 cases in males (0.95%) compared to 314 cases in females (99.05%). Women/men ratio was approximately 104.3:1. Histopathological study of cases was performed in the Laboratory of Pathology of Clinical Emergency County Hospital of Craiova and the IHC in the Department of Pathology of UMF Craiova. Casuistry analysis by age groups showed that the tumors were seen in patients aged between 25 and 83 years, presenting increased incidence with age. The highest incidence was found in the age group years (40.37%), followed by group years (24.60%) and age group years (18.61% ). One can say that 86.41% of cases were older than 50 years. For the rest of casuistry number of patients was much lower, namely 34 cases for age group years (10.72%), 9 cases under the age of 40 years (2.84%) and other 9 cases over 80 years. We observed that reproductive factors were most frequently found in the history of patients with breast cancer as risk factors, present in 76 of the analyzed cases. They were represented by: menarche under 12 years, present in the stories of 14 patients, menopausal 5
6 women aged over 55 present in 39 cases, the first delivery after 30 years reported by 12 patients and hormonal treatments identified in 11 cases. Subjective symptoms of onset was often nonspecific, especially in early stages when most subjects had accused a local embarrassment, then installing the other local or regional changes that have prompted the request for medical advice. Before the clinical examination of the breast focused on two components, inspection and palpation, a detailed medical anamnesis regarding any changes in the mammary gland should be performed. An important aspect in the detection of malignant lesions of the breast and posttreatment monitoring is represented by the imaging investigations that are currently vital in diagnosing and sometimes treating breast tumors. From imaging, mammography, which has a sensitivity of 90%, imposed as a method of screening that can detect breast cancer at an early stage faster than a routine clinical examination or self-examination. Mammographic screening widely used nowadays found that microcalcifications may be a marker of non-symptomatic breast cancers. Analysis of tumor distribution incidence in both mammary glands showed a slight predominance in the right mammary gland where I found 171 cases (53.94%) compared to the left mammary gland were I found 146 cases (46.05%). Morphological macroscopic parameters analysis allowed the estimation of tumor form, their color and consistency, the macroscopic subsidiary changes, and tumor size. Breast cancer is not a uniform entity, it consists of several different molecular profiles of tumor subtypes, biologic behavior and different risk profile, which is a challenge for clinical management. Clinical prognosis factors (regional lymph node involvement, tumor size, and absence or presence away) and histological (histological type, histological grade, mitotic index) plays a significant role in disease recurrence and patient survival. Prognosis factors such as tumor size, tumor grade, proliferation factors and the presence or absence of hormone receptors, are mainly used to detect a subset of patients likely to have a favorable outcome without adjuvant treatment. Tumor size is one of the strongest predictors of breast cancer behavior. Tumor size correlates with the presence and number of involved axillary lymph nodes and is an independent prognostic factor. Lymph node status proved to be the most important predictor of disease-free survival and overall survival in breast cancer. Morphological evaluation of histological grade in breast cancer provides useful information on prognosis. In breast cancer the presence of ER and PR is both a prognosis and a prediction factor. Assessment of ER and PR is determined by procedures in the routine management of patients with breast cancer, primarily as predictive factors for response to 6
7 adjuvant treatment. Their predictive power is mainly based on studies conducted over 2 decades ago, but only recently the immunohistochemical method has become the preferred method for determining estrogen and progesterone receptors in breast cancer. About 75% of breast cancers are ER+ and about 65% of ER+ cases are PR+. Women with positive hormone receptor tumors have a better prognosis because these cells grow more slowly than the tumors that are negative for these receptors. In addition, women with positive hormone receptor tumors have more treatment options. Therefore, immunohistochemical analysis of hormone receptors should be carried out systematically for all primary breast carcinomas. Pathological report must include the percentage or proportion of cells expressing the antigen to avoid using the terms "positive" and "negative." Prognosis significance of estrogen or progesterone receptor is limited, its use being a predictive factor for adjuvant treatment with tamoxifen. ErbB-2 gene amplification (Her2/neu) is present in approximately one third of breast cancers. Her2/neu overexpression is a prognosis factor associated with more aggressive tumors. Altered ErbB-2 is associated with high histological grade, low survival, hormone receptor modulation by tamoxifen and greater responsiveness to doxorubicin therapy. Diagnosis of invasive breast carcinoma followed the current WHO classification of lesions, associated premalignant lesions were specified (eg CDIS, CLIS) and also benign lesions (eg fibrocystic mastosis, intraductal papilloma), perineural invasion, lymphovascular invasion, resection margins status, presence of inflammatory infiltrate, presence of microcalcifications, skin invasion. DISCUSSIONS Breast cancer is the second most fatal cancer in women after lung cancer. The general approach in assessing breast cancer is characterized by a triple assessment: clinical examination, imaging examination (mammography and/or ultrasound/other explorations) and biopsy. Analysis of tumor distribution incidence in both mammary glands showed a slight predominance in the right breast (right/left 171:146). A comprehensive statistical study on breast cancer in the population of California, over the site of breast cancer indicates similar issues, but with a slight predominance in the left breast to the right, i.e. 50.8% versus 49.2%. Breast imaging reporting investigations using Data System (BI-RADS), developed by the American College of Radiology after 1993, is a standard classification of mammographic studies, in order to improve communication between specialties (radiologists, family 7
8 physicians, surgeons) and reduce confusion on mammographic interpretations. At first BI- RADS was initially used only for mammography, but was later adapted for ultrasound and MRI. In the last decade, histological classification of breast cancer has become widely accepted as a strong indicator of prognosis. The most used classification is Nottingham (Elston-Ellis). In medical literature the frequency of vascular invasion varies greatly in different studies between 5% to 50%. Lymphovascular and perineural invasion was present in 159 cases, of which tumor emboli in thin-walled vessels were found in 92 cases (57.8%) and perineural in 67 cases (42.2%). The only predictors that are associated with targeted therapy are receptors for estrogen (ER) and HER2 protein, but other factors play an important role in guiding treatment and prognosis. Anti-estrogen therapy (usually tamoxifen) is effective only in the subgroup of patients with breast cancer expressing estrogen receptors in the tumor. CONCLUSIONS The study included a total of 317 cases of invasive ductal breast carcinomas selected in a period of 3 years (January December 2011) and revealed the following aspects: Clinico-epidemiological data analysis showed uneven distribution of cases in the three years, with an upward trend, the lowest number of cases being registered in 2009 (95 cases) and highest in 2011 (115 cases). Gender distribution of analyzed invasive ductal breast carcinomas showed that the vast majority of cases were diagnosed in women (314 cases), the women/men ratio being 104.3:1. Casuistry analysis by age groups showed that the tumors were seen in patients aged between 25 and 83 years with increased incidence in elders, the highest incidence was achieved in the age group years (40.37%), followed by group years (24.60%) and years age group (18.61%). Of the 317 cases clinically investigated a total of 288 undertook imaging explorations, mammography (288 cases) and ultrasound (65 cases), which revealed BI-RADS following categories: 1.5. category for 64 cases, 2.0. category in 28 cases, 2.4. category in 44 cases, the 2.5. category in 134 cases and 3.5. category in only 18 cases. The histopathological study on invasive ductal carcinomas showed 248 cases of poorly differentiated tumors GIII, followed by moderately differentiated tumors, with a number of 57 cases and the well-differentiated GI, with only 12 cases. 8
9 Clinico-morphological staging of the 290 cases that could achieve compliance by ptnm system showed: stage I in 38 cases, stage IIA for 66 cases, stage IIB for 35 cases, stage IIIA for 51 cases, stage IIIC for 72 cases and stage IV for 28 cases. Tumor classification according to hormone immunophenotype (ER / PR) found in 40 cases (66.66%) both hormone receptor positivity (ER+/PR+), in 12 (22%) both hormone receptor negativity (ER-/PR- ), immunophenotypes with only one positive hormone receptor containing the fewest cases, respectively 5 cases (14.6%) for ER+PR- immunophenotype and 3 cases (5%) for ER-PR+ immunophenotype. According to the score Her2 score breast carcinomas were grouped as follows: score 0: 13 cases (21.67%), score 1+: 30 cases (50%), score 2+: 10 cases (16.67%) and score 3+: 7 cases (11.67%). Presence of mammographic calcifications in general is not suggestive for the Her2 status, however the presence of pleomorphic type calcifications is associated with Her2 positive tumors. ER immunostaining analysis showed the presence of estrogen receptors in 45 of the 60 cases studied (75%), the remaining 15 cases (25%) were ER negative. Regarding the progesterone receptor (PR) immunostaining I found that 43 cases (71.66%) showed PR positivity and 17 cases (28.34% of cases) were PR-. The results obtained in our study did not show significant differences between the two hormone receptor types positivity rates of (75% vs 71.66%) and correlates with the values published in studies on larger groups, although variations can sometimes be more obvious between the two receptors positivity rates. 9
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