UNIVERSITY OF MEDICINE AND PHARMACY "GRIGORE T. POPA-IASI" FACULTY OF DENTISTRY CLINICAL AND MORPHOLOGICAL PARTICULARITIES OF PAROTID GLAND TUMORS

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1 UNIVERSITY OF MEDICINE AND PHARMACY "GRIGORE T. POPA-IASI" FACULTY OF DENTISTRY CLINICAL AND MORPHOLOGICAL PARTICULARITIES OF PAROTID GLAND TUMORS PhD THESIS ABSTRACT SCIENTIFIC COORDINATOR: PROF. DR. EUGENIA POPESCU PhD STUDENT: LOREDANA-BEATRICE UNGUREANU IASI 2015

2 PHD THESIS TABLE OF CONTENTS TABLE OF CONTENTS... i ABBREVIATIONS... v UP-TO-DATE KNOWLEDGE... 1 CHAPTER PAROTID GLAND TUMORS: ANATOMY, HISTOLOGY AND HISTOPATOLOGY PAROTID GLAND ANATOMY The parotid gland Parotid gland innervation Parotid gland vascularization Head and neck lymph node stations PAROTID HISTOLOGY CLASSIFICATION OF SALIVARY GLAND TUMORS Histological classification of salivary gland tumors TNM classification HISTOPATHOLOGICAL CHARACTERISTICS OF MAIN PAROTID TUMORS Benign parotid tumors Pleomorphic adenoma Warthin Tumor Basal cell adenoma Mioepithelioma Malignant parotid tumors Mucoepidermoid carcinoma Adenocarcinoma NOS (Not Otherwise Specified) Adenoid cystic carcinoma Acinic cell carcinoma Carcinoma ex pleomorphic adenoma Epithelial-mioepithelial carcinoma Salivary duct carcinoma Polymorphous low grade adenocarcinoma Mioepithelial carcinoma Squamous cell carcinoma Intraparotid metastases Immunohistochemical aspects of parotid tumors Epithelial and myoepithelial markers Stromal markers Proliferation markers Growth factors and their ligands Cell cycle oncogenes Proteins involved in DNA repair Proteins involved in apoptosis Markers involved in cell adhesion, migration and epithelial mesenchymal transition Cyclooxygenase Cox Hormone receptor Mucins CHAPTER ELEMENTS OF CLINICAL AND PARACLINICAL INVESTIGATIONS, TREATMENT, COMPLICATIONS, PROGNOSTIC CLINICAL MANIFESTATIONS Clinical symptoms of parotid tumors

3 Clinical signs of malignancy PARACLINICAL INVESTIGATIONS Imaging paraclinical investigations Fine needle aspiration cytology Extemporaneous histological examination THERAPEUTIC APPROACH. COMPLICATIONS SURGICAL TREATMENT History Principles of surgical treatment Surgical indications Complications and sequelae of surgery Immediate complications Hemorrhage and hematoma Necrosis of skin flap Infection Salivar fistula Postsurgical sequelae Facial asymetry Frey sindrom Paresis or paralysis of facial nerve Tumor recurrence Sensory deficit in the great auricular nerve The syndrome of "first bite" ADJUVANT THERAPY Radiotherapy Complications of radiotherapy Chemotherapy PROGNOSTIC FACTORS Factors that depend on demographic characteristics of patients Factors that depend on clinicopathological characteristics of the tumor Factors that depend on treatment particularities Prognostic index PERSONAL CONTRIBUTIONS CHAPTER CLINICO-EPIDEMIOLOGICAL AND MORPHOLOGICAL STUDY OF PAROTID TUMORS INTRODUCTION MOTIVATION AND OBJECTIVES OF THE STUDY MATERIALS AND METHODS RESULTS Demographic and clinico-pathological characteristics of study group Demographic and clinico-pathological characteristics of benign tumors Pleomorphic adenoma Warthin tumor Demographic and clinico-pathological characteristics of primary malignant tumors Acinic cell carcinoma Squamous cell carcinoma Carcinoma ex pleomorphic adenoma Adenoid cystic carcinoma Adenocarcinoma NOS Lymphoma Demographic and clinico-pathological characteristics of secundary malignant tumors Intraparotid metastases

4 Clinico-morphologic characteristics of different types of parotid tumors DISCUSSIONS CONCLUSIONS CHAPTER MORPHOLOGICAL AND IMMUNOHISTOCHEMICAL STUDY OF PAROTID GLAND TUMORS INTRODUCTION MOTIVATION AND OBJECTIVES OF THE STUDY MATERIALS AND METHODS RESULTS Pleomorphic adenoma with predominant stromal component Pleomorphic adenoma with predominant epithelial component Pleomorphic adenoma with mixed component Warthin Tumor Acinic cell carcinoma Other parotid tumors DISCUSSION Ki67 protein Bcl-2 oncoprotein Bax protein Apoptotic index Correlation between the immunohistochemical markers CONCLUSIONS CHAPTER FINAL CONCLUSIONS PHD THESIS ASPECTS OF ORIGINALITY PERSPECTIVES OPENED BY PHD THESIS REFERENCES APPENDAGE 1. PUBLISHED OR ACCEPTED FOR PUBLISHING PAPERS DURING THE PHD STUDY 147 This thesis is illustrated with 130 figures and 69 tables and 324 references. In the summary only selected figures and tables from the whole of the thesis are included. Keywords: parotid tumors, clinical-epidemiological study, morphology 4

5 INTRODUCTION Knowing the anatomic particularities is essential for surgical practice in order to achieve a proper dissection with minimal consequences for the patient. Among the major salivary glands, the parotid gland is the largest. It is a pair gland located in the parotid lodge. The rapport of facial nerve with the parotid gland has great clinical importance because it divides, from surgical point of view, the parotid gland in a superficial lobe located lateral and a deep lobe medially located. From the histological point of view the parotid is a purely serous tubulo-acinar salivary gland composed from capsule, stroma and parenchyma. The parenchyma is composed from lobules that have two components: a secretory component constituted from serous acini and an excretory component represented by ducts (Wang et al., 2012, Treuting, Dintzis 2012, Badoual et al., 2013). The treatment of choice for parotid tumors is surgery associated or not with radiotherapy (Bjondal et al., 2011). The tumor excision followed by histopathological examination permit assessment of the lesion extent, the histological type of tumor and the identification of the elements of poor prognosis such as: perineural invasion, lymphatic and blood vessels invasion, the state of resection margins and the presence of multifocal tumors (Vander Poorten et al., 2010). Parotid tumors have a great histological variability due to multiple cell types that compose them. Parotid malignant neoplasms are rare (Mercante et al., 2014). Most of the parotid tumors are asymptomatic (Huang et al, 2007) and have an unpredictable development, so that a benign tumor could be very aggressive, while a malignant lesion could have a very slow development. In the immunohistochemical study of salivary gland tumors we used multiple classes of molecular markers. The markers of cell proliferation are represented by Ki67, PCNA and AgNOR. Among these Ki67 is the most commonly used, being considered a useful prognostic marker (Uro-Coste et al., 2009, Larsen et al., 2012). The proteins involved in apoptosis are represented by the family of B-cell lymphoma genes (Bcl-2) which contains pro-apoptotic proteins such as Bax, Bad, Bak and anti-apoptotic proteins such as Bcl-2, BCL-XL and survivin (Vander Poorten et al., 2010). Among these proteins bax and bcl-2 are the most widely used in different studies. Due to the small number of studies describing the epidemiology, the proliferative and the apoptotic potential of parotid tumors, new research programs are required to bring supplementary data regarding the diagnostic methods useful for determining the prognosis and therapeutic management. Lack of data on the immunostaining of those three markers (ki67, bcl-2, bax) in parotid tumors justifies this doctoral study that makes a characterization of the immunohistochemical aspects in the main parotid benign and malignant tumors. PERSONAL CONTRIBUTIONS CHAPTER 3 CLINICO-EPIDEMIOLOGICAL AND MORPHOLOGICAL STUDY OF PAROTID TUMORS 3.1. INTRODUCTION Parotid gland tumors are a heterogeneous group of tumors accounting for 85% of the salivary gland tumors, less than 10% of head and neck tumors and about 1% of all tumors. Most studies have described an incidence of salivary gland tumors of less than 15 cases per 100,000 inhabitants. Most salivary gland tumors originate in the parotid gland and are mostly benign (Barnes et al., 2005, Muenscher et al., 2009, Stenner et al., 2011). These tumors are 5

6 considered unique both because new histological types are added from one classification to another and due to the particularities of clinical behavior, so that some benign tumors have the capacity to relapse or to become malignant, while some malignant tumors have a long evolution which sometimes exceeds 20 years. Although salivary gland tumors and tumors of the parotid gland in particular have been extensively studied in terms of morphology and immunhistochemistry, data on the overall incidence of malignancies are still insufficient Motivation and objectives of the study The purpose of this study consisted of a five-year retrospective descriptive analysis ( ) of clinico-epidemiological and morphological aspects of parotid tumors treated in the Oral and Maxillofacial Surgery Clinic and diagnosed in the Pathology Laboratory from the Emergency University Hospital "St. Spiridon ". The research of literature has shown that there are few studies that describe a correlation between epidemiological and histopathological data for parotid tumors. Therefore, our study aims to bring new data on parotid tumors. The main objective of this study was to identify relationships between demographic, clinical and histological type of parotid gland tumor. The secondary objectives of the study were: 1. A descriptive analysis of demographic and morphological factors; 2. Establishing the relationships between demographic and clinical factors and between demographic and morphological factors MATERIAL AND METHOD The demographic and clinical data were obtained from medical reports of patients hospitalized in the Oral and Maxillofacial Surgery Clinic of the Emergency University Hospital "St. Spiridon" with the diagnosis of parotid tumor during Histopathological diagnoses were obtained from the records of Pathology Laboratory. To describe parotid tumors we used morphological classification of the World Health Organization 2005 (Barnes et al., 2005). Statistical analysis The variables considered in the study were represented by: demographic factors, clinical diagnosis and other clinical data, histopathological diagnosis. The obtained database was statistically processed using the programs Microsoft Office Excel 2007, SigmaPlot 12 and VassarStats. The numerical data from the study were analyzed using descriptive statistics; the results were expressed as mean ± SD. For univariate analysis we used the χ2 test, t-student test and Mann Whitney U test, for multivariate analysis we used Multiple Logistic Regression. For risk assessment Odd Ratio was used. Results were considered significant at a P value < RESULTS The study group comprised 162 cases of parotid tumors. The studied patients were aged between 13 and 90. From these tumors, 116 were benign and 46 were malignant Demographic, clinical and pathological characteristics of the study group The gender distribution of patients showed that tumors occurred predominantly in males. Most cases (85 cases - 52%) came from urban areas. Mean age of patients in the study was 55 years. Most cases were located in the right parotid gland (51%). The distribution by age and years is illustrated in fig. 3.3 and

7 Fig Distribution of patients on age groups Fig.3.4. Distribution of patients on years In case of benign tumors included in the study group the mean age was 50 ± 16 years, and the average size of tumors was 3.49 ± 1.80 cm. The distribution of benign tumors is illustrated in figure 3.6 and those of malignant tumors in Figure 3.7. In the case of malignant tumors, the average age was 67 ± 2 years, and the average size of the tumor was 4.36 ± 0.46 cm. Fig 3.6. Distribution of parotid benign tumors Fig Distribution of parotid malignant tumors Gender distribution of benign and malignant tumors is shown in fig. 3.8, and the distribution on age groups in fig Most benign tumors have a size between 2 and 4 cm, while the majority of malignant tumors were more than 4 cm in size, the difference being statistically significant (P = 0.040). The average size of benign tumors was 3.49 ± 1.80 and those of malignant tumors 4.36 ± of 3.14 cm. The average age of patients with benign tumors was 50 years and that of malignant tumors was 67 years. The difference between them was statistically significant (P = 0.039) Masculine Feminine Benign tumors Malignant tumors Fig Distribution of benign and malignant tumors on gender. Fig Distribution of benign and malignant tumors on age groups. 7

8 A higher percentage of benign tumors occurred in urban areas (58.62%), while in rural areas the malignant tumors were more frequent (63.04%), the difference being statistically significant (P = ) (fig. 3.13, tab.3.6). Benign tumors were more frequently located in the right parotid gland (52.58%) and the malignant tumors in the left parotid gland (52.17%), the difference not being statistically significant (P = 0.710) (fig.3.14). Fig Distribution of patients with parotid tumors regarding the place of residence Fig Distribution of benign and malignant tumors regarding the location Table 3.6. Statistical analysis of patients with parotid tumors regarding the place of residence Variable Benign Tumors N (%) Malignant tumors N P (%) Place of residence Rural 48 (41,37%) 29 (63,04%) 2 =5,361; GL=1; Urban 68 (58,62%) 17 (36,95%) p=0,021 The most commonly associated clinical conditions were hypertension, diabetes, obesity. Facial palsy was associated particularly with malignant tumors. In the study group 4 cases of Warthin tumor were associated with thyroid colloid goiter. In most cases of benign tumors from the study group a superficial parotidectomy with facial nerve preservation was performed with plasty the remaining cavity with a portion of sternocleidomastoid muscle. In case of malignant lesions a superficial or total parotidectomy was performed, the facial nerve sacrifice being necessary in less than 50% of cases. To most patients with malignant tumors, a neck dissection comprising the lymph nodes was performed. The plasty of surgical defect was performed in most cases using a sternocleidomastoid muscle flap. Fig Clinical conditions associated with parotid tumors Fig Types of parotidectomies used in benign and malignant tumors 8

9 Demographic and clinico-morphologic characteristics of benign tumors The most common benign tumor was pleomorphic adenoma (70%). Most cases of pleomorphic adenoma were observed in the age group years, the majority having a size between 2 and 4 cm (44.44%). The characteristics of variables related to age and size are shown in Table Tabel Descriptive statistic pleomorphic adenoma Average Standard Standard Range Mode (the most frequent Error (SE) Deviation (SD) value) Age (Years) 45,87 1,68 15, Size (cm) 3,65 0,21 1,89 0,5-10,5 4 The second most frequent benign tumor was Warthin tumor (21%). Most cases of Warthin tumor were recorded in the age group years. Most of the tumors had a size between 2 and 4 cm (48%). The characteristics of variables age and size are shown in Table Tabel Descriptive statistic - Warthin tumor Average SE SD Range Mode Age (years) 57,36 2,37 11, Size (cm) 3,02 0,31 1,58 0, Demographic and clinico-pathologic characteristics of primary malignant tumors From primary malignant tumors the acinic cell carcinoma represented 20.83% of the cases. Most cases aged between years. The size ranged between 2 and 4 cm. The characteristics of variables age and size are illustrated in Table Tabel Descriptive statistic acinic cell carcinoma Average SE SD Range Mode Age (years) 50,4 10,15 22, Size (cm) 3,68 0,66 1, Carcinoma ex pleomorphic adenoma was developed in 12.5% of cases. All cases were included in the age group years. Most cases were larger than 4 cm (66.66%). Characteristics of variables age and size are given in Table Tabel Descriptive statistic - carcinoma ex pleomorphic adenoma Average SE SD Range Mode Age (years) 61,33 0,66 1, Size (cm) 7,3 3,20 5,55 1, The adenoid cystic carcinoma represented 12.5% of cases. Most cases were larger than 4 cm (66.66%). Characteristics of variables age and size are given in Table Tabel Descriptive statistic - adenoid cystic carcinoma Average SE SD Range Mode Age (years) 66 10, Size (cm) 3 1,25 2,17 0,5 4,5-9

10 From all primary malignant tumors adenocarcinoma NOS represented 12.5% of the cases. Most cases were registered in the age group years. All cases were larger than 4 cm. Characteristics of variables age and size are given in Table Tabel Descriptive statistic - adenocarcinoma NOS Average SE SD Range Mode Age (years) 66,33 3,66 6, Size (cm) 5,83 1,16 2, Demographic and clinico-pathologic characteristics of secondary malignant tumors. Secondary malignant tumors were represented, in particular, by metastatic squamous cell carcinoma and metastatic melanoma, the cases most often occurring in the age group years old. Most of the tumors were less than 2 cm in size or greater than 4 cm. The characteristics of variables age and size are given in Table Tabel Descriptive statistic - Metastasis Average SE SD Range Mode Age (years) 69,47 3,41 14, Size (cm) 3,12 0,56 2,34 0, Clinico-morphologic characteristics of different types of parotid tumors By analyzing gender distribution of parotid tumors of different histological types we found that the only statistically significant difference regarding sex existed between pleomorphic adenoma and Warthin tumor (P <0.001). All three types of pleomorphic adenoma occurred in women with no significant difference between them (P = 0.291). It was found that the majority of malignant tumors were present in patients from rural areas, while benign tumors occurred in patients from urban areas. Most cases of malignant tumors were located in the left parotid gland and the benign tumors in the right parotid gland. All types of pleomorphic adenoma were observed in the right parotid gland with no statistically significant difference (P = 0.726). In the present study the analysis of risk factors showed that gender, age and place of residence can influence parotid tumor histology. In our study, the risk of developing pleomorphic adenoma was 7.40 times higher in women than in men (OR = 7.401, CI = , P <.0001). Men had a fold higher risk of developing Warthin tumor than women (OR = 16.47, CI = , P <.0001). The risk of developing malignancy was 7.14 times higher in patients older than 50 years (OR = 7.141, CI = , P <.0001). The risk of developing malignancy was 2.41 times higher in patients from rural areas (OR = 2.416, CI = , P = 0.012). Diabetes, obesity and systemic hypertension were not significant risk factors in our study. Due to incomplete data on smoking, this parameter could not be introduced in the study DISCUSSION In the literature there are many epidemiological studies about salivary gland tumors, some of them emphasizing the importance of parotid tumor pathology. Many of these are performed on a limited number of cases and there are only a limited number of cohort studies. The incidence of salivary gland tumors varies a lot, being generally less than 15 cases / 100,000 inhabitants (Ettl et al., 2008, Speight, Barret 2009, Atarbashi Moghadam et al, 2010, Gomes et al, 2012 Badou et al, 2013 Bradley, McGurk 2013). Our study could not 10

11 appreciate the true incidence of parotid tumors in the Moldova counties because of the small number of cases. In the research conducted by other authors the patients with benign tumors had an average age ranging from 47 and 51 years (Shehata, 2010, Azua-Romeo et al., 2014), while malignant tumors were within the age range years (Poorten et al., 2009). Similar with these studies, patients with benign tumors selected by us had a mean age of 50 years. Compared to the same studies the average age of patients with malignant tumors in our study was 67 years. Most researches indicates a predominance of parotid tumors in women (de Oliveira et al., 2009, Atarbashi Moghadam et al., 2010, Tadbir et al., 2012 Saghravanian et al., 2013). In contrast to these data we found, in our region, that most of parotid tumors occurred in males (55%). Similar to the study conducted by Lin et al. (2008) both benign and malignant tumors in the present study occurred in males. This difference might be due to a reduced number of malignant tumors in our study. Studies in the literature do not specify the distribution of tumors regarding the place of residence. We found in our research a predominance of benign tumors in urban areas (58.62%) and a predominance of malignant tumors in rural areas (63.04%), the difference being statistically significant (P = 0.012). This could be due to a low addressability to the doctor for patients from rural areas. The average size of tumors in our study was slightly higher than in other reports (Chan et al., 2010). Unlike other studies (Mercante et al., 2014), in our study, most of malignant tumors exceeded 4 cm, probably due to late addressability of patients to the doctor. Most benign tumors in our study had the size ranging between 2-4 cm, similar to other studies in the literature (Teymoortash et al., 2007, Vargas et al., 2008, Papadogeorgakis, 2011). Unlike the study conducted by Lin et al (2008), in which both malignant and benign tumors were located in the right parotid gland, our results showed that the majority of benign tumors affected the right parotid gland (52.58%) while malignant tumors were located on the left (53.33%). Consistent with other studies (de Oliveira et al., 2009, Fonseca et al., 2012) our data confirms that benign parotid tumors accounted for 71% of all tumors while the malignant tumors represented 29%. In our study, similar to other studies (Barnes et al., 2005, Upton et al., 2007, Lahoz Zamarro et al., 2008, Ali et al., 2010, Chan et al., 2010, Lukšić et al., 2012 ) the most frequent benign tumor was pleomorphic adenoma, followed by Warthin tumor. Among the most common malignant tumors there are: mucoepidermoid carcinoma, adenoid cystic carcinoma, adenocarcinoma NOS (Not Other Speciefied), acinic cell carcinoma, carcinoma ex pleomorphic adenomas (Barnes et al., 2005, Grădinaru et al., 2007, Boukheris et al., 2009, de Oliveira et al., 2009, Chan et al., 2010, Tian et al., 2010, Rose et al., 2010, Bjørndal et al., 2012, Fonseca et al., 2012 Lukšić et al., 2012 Badou et al., 2013, Bradley, McGurk 2013 Lawal et al., 2013 Saghravanian et al., 2013 Amana et al., 2014). In our study we observed that the most common malignancies were acinic cell carcinoma, squamous cell carcinoma and adenoid cystic carcinoma. The difference between rural and urban areas regarding the histological type of tumor was statistically significant (P = 0.017). Some studies have indicated that in the majority of parotid carcinomas a superficial parotidectomy was performed (Shang et al., 2012), while others have shown that in case of parotid carcinomas most situations imposed either total or extensive parotidectomy (Poorten et al., 2009). In our study, a superficial parotidectomy was performed in case of most patients with benign tumors; while in case of malignant tumors a superficial or total parotidectomy was performed more frequently. The etiological factors are not fully known. The role of smoking and alcohol, long regarded as etiologic factors, has not been clinically proven (To et al., 2012). Our study 11

12 showed that the risk factors were represented by female gender for pleomorphic adenoma, male gender and urban area for Warthin tumor and age over 50 years and rural areas for malignant tumors. Associated clinical disorders were not risk factors. CHAPTER 4 MORPHOLOGICAL AND IMMUNOHISTOCHEMICAL STUDY OF PAROTID TUMORS 4.1. INTRODUCTION Although in the literature there is a wide range of immunohistochemistry studies for parotid tumors, all failed to establish specific diagnostic markers for these tumors. It is known that in case of malignant tumors the proliferative capacity is enhanced. Tumor growth is the result of an imbalance between cell division and cell death (Melnick et al, 1993, Robbins et al, 2009). The most commonly used immunohistochemical marker for diagnosis of cell proliferation is the protein Ki67. Apoptosis is a programmed cell death and it has two important roles in tumors: induction of cell death for tumor regression and in case of tumors with high proliferation rate (Robbins et al, 2009). The commonly used immunohistochemical markers involved in apoptosis are protein bcl-2 with anti-apoptotic role and bax with a pro-apoptotic role MOTIVATION AND OBJECTIVES OF THE STUDY The purpose of this study consisted in an immunohistochemical analysis of benign and malignant tumors of salivary gland, diagnosed histologically in the Pathology Laboratory of Emergency University Hospital "St. Spiridon " between In the current literature there is still little information about the proliferative potential and physiological balance between pro-apoptotic and antiapoptotic factors for Warthin tumor or malignant tumors. The main objective of this study was to identify correlations between Ki67 and bcl2 and bax positivity in case of parotid pleomorphic adenoma, Warthin tumor and acinic cell carcinoma. Secondary objectives of the study were: 1. descriptive analysis of histopathological appearance, 2. analysis of immunostaining for Ki67, bcl2 and bax in pleomorphic adenoma, Warthin tumor and acinic cell carcinoma, 3. Establishing correlations between markers. 4.3 MATERIAL AND METHODS The study included a total of 41 cases of parotid tumors diagnosed in the Pathology Laboratory of Emergency Hospital "St. Spiridon Iasi ". The 41 cases studied were divided into 6 lots as follows: Lot 1-6 cases of pleomorphic adenoma with predominantly myxoid component, Lot 2-6 cases of pleomorphic adenoma with predominantly epithelial component, Lot 3-6 cases of pleomorphic adenoma with mixed component, Lot 4-6 cases of Warthin tumor, Lot 5-5 cases of acinic cell carcinoma, Lot 6 - other tumors. Lot 6 was used only for histopathological and/or immunohistochemical description For the histopathological and immunohistochemical exam the fragments harvested during surgery were fixed in 10% buffered formalin, and then all the required steps were carried out to achieve the necessary stains. 12

13 The immunohistochemical evaluation of molecular biology markers on histological sections (Ki67, bcl-2, bax) For quantification of immuno marking, four sections from each block were performed. One section was stained with hematoxylin-eosin and the other three with Ki67, Bcl-2 and Bax. Sections were processed according to standard immunohistochemical technique. Immunohistochemical reactions were performed in the Pathology Laboratory of Emergency Hospital St. Spiridon Iași Histological sections were examined using a three-step algorithm: 1. the presence of immuno marking (brown nuclear stain for Ki-67 and brown cytoplasmic stain for bcl 2 and bax); 2. the identification of cellular areas with positive immuno marking; 3. counting at least 1000 cells on multiple fields with high magnification (40X) and percent conversion of the obtained results. Immuno marking Ki67, bcl-2, bax was assessed using a semiquantitative score on 4 groups proposed by Alves et al. (2002): 0 - negative - 0-5% positive cells; 1 - weak positive % positive cells; 2 - moderately positive % positive cells; 3 - strongly positive - more than 50% positive cells. For statistical analysis we used in our study both descriptive statistics and analytical methods. The databases were processed using statistical functions corresponding to data set. To determine the strength of correlation between the three markers we used Pearson Product Moment correlation test RESULTS From the cases of pleomorphic adenoma we chose 3 different lots: pleomorphic adenoma with predominantly myxoid component, pleomorphic adenoma with predominantly epithelial component and pleomorphic adenoma with mixed component). The clinically presentation of patients was as a prominent tumor mass in the preauricular parotid lodge. Pleomorphic adenoma appeared macroscopically as a well defined round mass. On the cut section solid areas and cystic mucoid areas were highlighted. Fig Right parotid pleomorphic adenoma preoperator aspect (Prof.Dr. E. Popescu casuistry) Fig 4.2. Pleomorphic adenoma. Macroscopical aspect of surgical specimen (Prof.Dr. E. Popescu casuistry) In the case of salivary glands the score Ki-67 was 0 in all cases. In most cases immuno marking bcl-2 was weakly positive (score 1), while bax was moderately positive or negative (score 2). By comparing the percentage of immunostain between the three markers in the tumor it was observed that there is a statistically significant difference in all three types of pleomorphic adenoma. 13

14 Pleomorphic adenoma with predominant myxoid component Most cases of pleomorphic adenoma with myxoid component showed satellite nodules, thin capsule and capsule invasion. In 50% of cases stroma was chondro-myxoid. In the studied cases the immunhistochemical reaction for Ki67 in the tumor showed an average of 3.01% ± 2.32%, average score being Regarding the reaction of bcl-2, the mean value in the tumor was 81.15% ± 11.84, all cases having score 3. For bax, the value had an average of 80% ± 13.24%, all cases having score 3. Statistically significant differences were observed between Ki67 and Bcl2 (P <0.001) and between Ki67 and bax (P = <0.001). Between bax and bcl-2 there was not statistically significant difference (P = 0.843). Fig PA with predominant myxoid component - (HEx400). Fig PA with predominant myxoid component - (ki67x400). Fig PA with predominant myxoid component - (bcl-2x400). Fig PA with predominant myxoid component - (bax x40). Pleomorphic adenoma with predominantly epithelial component This variant of pleomorphic adenoma was associated more frequently with sclerohyaline stroma, capsule of uneven thickness, satellites nodules. In the studied cases the immunohistochemical reaction for Ki67 showed an average of 1.99% ± 0.83%, score 0. In case of bcl-2 reaction, the average was 73.32% ± 17.12, all cases having score 3. For bax, the value had an average of 64.95% ± 20.40%, most cases having score 3. Statistically significant differences were observed between Ki67 and Bcl2 (P <0.001) and between Ki67 and Bax (P = <0.001). Between Bcl-2 and Bax there was no significant difference (P = 0.362). 14

15 Fig PA with epithelial component intense cellular tumor (HEx100). Fig PA with epithelial component ki67 immunostain (Ki67x400). Fig PA with epithelial component Bcl-2 immunostain (bcl-2 x400). Fig PA with epithelial component Bax immunostain (bax x400). Pleomorphic adenoma with mixed component The majority of studied cases had a hyaline stroma and capsule invasion. In the studied cases the immunohistochemical reaction for Ki67 showed an average of 1.67%, the score being 0. The immunostain for bcl-2 was 73.13%, the majority of cases having score 3, and for bax was %, most cases having score 3. Statistically significant differences were observed between Ki67 and Bcl2 (P = 0.031) and between Ki67 and Bax (P = 0.008). Between Bcl-2 and Bax there was not a significant difference (P = 0.151). Fig PA with mixt component (HEx 200). Fig PA with mixt component - (ki67x400). 15

16 Fig PA with mixt component - (Bcl-2 x400). Fig PA with mixt component - (Bax x400). Warthin tumor From a clinical point of view, Warthin tumor is difficult to distinguish from pleomorphic adenoma being a prominent lesion located in the parotid lodge or preauricular. Macroscopically the tumor was round, well defined, encapsulated with a smooth surface. Fig Right parotid Warthin tumor preoperator aspect (Prof.Dr. E. Popescu casuistry) Fig Warthin tumor surgical specimen (Prof.Dr. E. Popescu casuistry) Most cases had an abundant lymphoid stroma with germinal center. In most cases the capsule was thin. Immunohistochemical reactions for ki67 at the level of epithelium showed an average of 5.16%, most cases with score 1. At the level of lymphoid tissue ki67 had an average of 10.07%, the score being also 1. At the level of epithelium positive immunostaining occurred primarily at the level of the basal layer cells. There were a greater number of positive cells in the lymphoid area. In the studied cases the immunhistochemical reaction for bcl 2 at the level of epithelium showed an average of 29.47%, the score being 2 in most cases. In the lymphoid tissue bcl-2 had an average of 72.98%, most cases having score 3. At the level of epithelium, positive immunostaining occurred primarily at the level of the basal layer cells. There were a greater number of positive cells in the lymphoid area. Immunohistochemical reaction for bax at the level of epithelium showed an average of 95.95%, the score being 3 in all cases. In the lymphoid tissue bax had an average of 45.76%, 50% of cases having score 3. Positive immunostaining was intense in epithelial cells lining the cystic spaces. In the lymphoid cells the positivity was lower. In the connective-vascular axis the cells were negative. By comparing the percentage of immunostaining between the three markers in the epithelium, we observed statistically significant differences between ki67 and Bcl-2 (P 16

17 <0.001), between Ki67 and bax (P <0.001) and between bcl-2 and bax (P <0.001). In the lymphoid tissue statistically significant differences were observed between Ki67 and Bcl 2 (P = 0.003) and between Ki67 and Bax (P = 0.048). Between Bcl-2 and Bax there was no statistically significant difference (P = 0.071). Fig Warthin tumor the lymphoid stroma contains sometime germinal centers (HEx100). Fig We observed a greater number of positive cells in the lymphoid area (ki67x200). Fig Warthin tumor - Bcl-2 pozitive immunostain (Bcl-2x400). Fig Warthin tumor. Pozitive Bax immunostain (Bax x400). Fig Comparing the ki67 immunostain in Warthin tumor Fig Comparing the bcl-2 immunostain in Warthin tumor 17

18 Fig Comparing the bax immunostain in Warthin tumor Parotid carcinomas were clinically characterised as imprecisely defined tumoral masses, unencapsulated, infiltrative, some of them with an ulcerated surface. Fig Ulcerative and infiltrative carcinoma preoperator aspect (Prof.Dr. E. Popescu casuistry) Fig Ulcerative and infiltrative carcinoma surgical specimen (Prof.Dr. E. Popescu casuistry) Acinic cell carcinoma Among the studied cases, 40% had a hyalin stroma, in 2 cases the tumor cells had PAS positive zymogen granules in the cytoplasm. The immunohistochemical reaction evidentiated a mean value of 8,52% for ki67, 68,53% for bcl-2 and 77,15% for bax. By comparing the percent of immunostain between the three markers at the level of the tumor we observed that between ki67 and bax there were statistically significant differences (P=0,008) and also between ki67 and bcl2 (P<0,001). Fig Acinic cell carcinoma - (HEx400). Fig Acinic cell carcinoma (ki67x400). 18

19 Fig Acinic cell carcinoma - (bcl-2 x 400). Fig Acinic cell carcinoma (Bax x 400). Carcinoma ex pleomorphic adenoma The immunostain for ki67, bcl-2 and bax was different in pleomorphic adenoma and carcinoma ex pleomorphic adenoma. Thus ki-67 index was higher in the carcinoma component than in adenoma. It was noted that there is no correlation between the three markers or between size and the values of immunohistochemical markers neither in pleomorphic adenoma with stromal, epithelial or mixed component, nor in Warthin tumor or acinic cell carcinoma. Statistically significant differences were recorded only between ki67 and bcl-2 and between ki67 and bax. Fig Carcinoma ex PA (HEx100). Fig Carcinoma ex PA (ki67x100). Fig Carcinoma ex PA. Bcl-2 pozitive immunostain in PA (bcl-2 x400). Fig Carcinoma ex PA. Bcl-2 pozitive immunostain in carcinoma (Bcl-2 x400). 19

20 Fig Carcinoma ex PA. Bax immunostain in PA (Bax x400). Fig Carcinoma ex PA. Bax immunostain in carcinoma (Bax x400) DISCUSSION Studies in the literature regarding the expression of Ki67, Bcl-2, Bax markers are small in number. Ferreira et al (2014) conducted a study on pleomorphic adenoma localized in minor salivary glands. The authors of that study used in their research the apoptotic index bcl- 2/bax, the study making a comparison between the histological variants of pleomorphic adenoma. Ki67 protein Ki67 index is very useful in clinical practice because it is considered that a positive intense immunostaining and a ki67 index of 3 are poor prognostic factors in salivary gland carcinomas (Cheuk, Chan 2007). Unlike other studies that have indicated a rate of ki67 immunostaining less than 1% in the normal salivary gland (Hirabayashi, 1999, Tadbir et al., 2012, Ferreira et al., 2014), we observed an immunostaining ki67 0 in most cases. Various studies in the literature have indicated a rate of immunostaining in pleomorphic adenoma with an average around 2.5%, the score being 0 (Hirabayashi, 1999, Sakamoto et al., 2004, Vargas et al., 2008, Ferreira et al., 2014). Similarly, in our study the score was also 0. In a study realised by Terada et al. (2008) the authors indicated an index ki67 of 3% in Warthin tumor. In our group the mean immunostaining was 5,16% in case of epithelium and 10,08% in case of lymphoid tissue. Similar to other studies that showed that the index ki67 was higher in case of carcinoma ex pleomorphic adenoma than in pleomorphic adenoma (Katori et al., 2007, Matsubayashi, Yoshihara 2007, deroche et al., 2008), our results showed a mean ki67 immunostain of 45% at the level of malignant component comparing with the pleomorphic adenoma component where it was of 0, 40%. Acinic cell carcinoma is the carcinoma with the slowest development. Some studies have found a low Ki67 index (at most 10%, the index being 1) (Vargas et al., 2008, Veličković et al., 2013). Similarly, the immunostain in our group had a mean Ki67 of 7.83%, the Ki-67 score being 1 in 50% of the cases. Bcl-2 oncoprotein The second marker often used in the research carried out on the salivary gland tumors is bcl-2, an anti-apoptotic oncoprotein. It was found that in the malignant tumors a strongly positive immunostain bcl-2 indicates a poor prognosis (Soini et al., 1998, Sakamoto et al., 2005). 20

21 Similar to other studies that indicated a positive immunostain only at the level of ductal structure in the normal salivary gland (Soini et al., 1998), the present research showed a reduced positivity only in some basal cells. The bcl-2 immunostain had a mean of 16,42%. In pleomorphic adenoma the bcl-2 positive immunostaining varied in different publications. In some of these the immunostaining was strongly positive, especially in the myoepithelial cells (Hellquist et al., 1996, Ihreler et al., 2009, Atarbashi et al., 2014, Ferreira et al., 2014) while in the others the immunostaining was negative (Gordon Nunez et al., 2008). Our research showed an intense immunostaining (score 3), with an average of 75.87% in most cases of pleomorphic adenoma. Similar to the study realised by Atarbashi et al., (2014) we did not find any correlation between tumor size and expression of bcl-2. These differences between studies could indicate the reduced role of bcl-2 in determining the biological behavior of pleomorphic adenoma. Unlike the study realised by Obtulovicova et al. (2014) in what the pleomorphic adenoma with myxoid component had a more intense immunostaining, in our study bcl-2 immunostaining was slightly higher in pleomorphic adenoma with predominant stromal component. In some research bcl-2 was tested in Warthin tumor and positive cytoplasmic immunostaining was observed in both epithelial layers, the surrounding stroma being negative (Soini et al., 1998, Abd-Elhamid, Elsafei 2009). In contrast, our results showed a positive epithelial immunostaining, located mainly in the basal layer, with an average of 29.47%, the score bcl-2 being 2 or 1. A study realised by Al-Rawi et al. (2010) indicated an intense positive expression of bcl-2 in malignant tumors. In our research bcl-2 was positive in the majority of cases, the positive immunostaining exceeding the percentage of 50% from tumoral cells. In most studies bcl-2 positivity in carcinoma ex pleomorphic adenoma was reduced in luminal and myoepithelial cells, the expression being stronger in peripheral areas of carcinoma (Freitas et al., 2005, Heiduschka et al., 2011). We noticed that bcl-2 immunostaining was more intense in areas of carcinoma (score 3) rather than in pleomorphic adenoma. Given the fact that an increased bcl-2 index indicates a poor prognosis, these tumors may have a poor outcome. Most studies showed a moderate positive bcl-2 immunostaining in acinic cell carcinoma (Hellquist et al., 1996, Heiduschka et al., 2011). In our study of bcl-2, all 5 cases from study group with acinic cell carcinoma had an intense bcl-2 immunostaining with an average of 66.52% (score 3). Bax protein Although it is known to be a pro-apoptotic protein (Gomes et al., 2012) and that a low bax index indicates a poor prognosis (Freitas et al., 2005), however this protein is still little studied in salivary gland pathology. Similar to the study of Soini et al. (1998) our results showed a strong expression of bax in the ductal cells from normal salivary gland. Unlike literature studies in which the Bax immunostaining in pleomorphic adenoma was stronger in the epithelial component, the score Bax being 2 or 3 and the average 22% (Soini et al., 1998, Ferreira et al., 2014), we observed an average immunostaining of 66.74% the average percentage being higher in pleomorphic adenoma with predominant stromal component, indicating a higher degree of apoptosis in this version. Our study aimed to research this marker in Warthin tumor. According to our results we observed a strongly positive immunostaining in epithelial cells and moderately positive in the stroma. In the epithelial tissue the average immunostaining was 95.95%, all tumors presenting a score 3. In the lymphoid tissue, the immunostain was less intense, with an average of 45.76%, the score being 3 in 50% of cases. 21

22 Because there are no data in the literature on bax immunostaining in acinic cell carcinoma and carcinoma ex pleomorphic adenoma, our study group included 5 cases of the first type of tumor and 3 cases from the second. In acinic cell carcinoma the immunostain was also strongly positive with an average of 79.3%, the majority of cases having the Bax index 3. Unlike the study realised by Soini et al. (1998) that showed that in the malignant tumors Bax immunostaining is intensely positive, in carcinoma ex pleomorphic adenoma from our study group the Bax immunostaining was weakly positive indicating a possible adverse outcome of these tumors. Apoptotic index Most authors recommend the use of Bcl-2/Bax ratio in assessing the apoptotic potential of the tumor (de Siqueira et al., 2014, Ferreira et al., 2014). Similarly the study realised by Ferreira et al. (2014), in our study the index Bcl-2/Bax was supraunitar in the majority of pleomorphic adenoma. CHAPTER 5 CONCLUSIONS In the studied territory we observed a growing trend in the number of parotid tumors between comparative with the period The most important statistically significant differences were found between benign and malignant tumors regarding the place of residence. Both the age of 50 years old and rural areas could be considered important risk factors for malignant tumors. By the immunohistochemical study of Ki67, bcl-2 and bax markers we observed that there was a balance between pro-apoptotic and anti-apoptotic factors in pleomorphic adenomas, Warthin tumor and acinic cell carcinoma explaining the favorable development of these tumors. The only tumors in which proliferative and apoptotic index may be predictive are the malignant tumors, but their number is reduced. ORIGINAL ASPECTS OF THE DOCTORAL THESIS It highlights the following original contributions of this thesis: An epidemiological study on the Moldavian Region correlated with histopathological suntypes of tumors located in the parotid gland was conducted. It was the first study that analyzed the expression of markers ki67, bcl-2 and bax on the same specimen in Warthin tumor, acinic cell carcinoma and carcinoma ex pleomorphic adenoma. PERSPECTIVES THAT THE PhD THESIS OPENS The PhD thesis opens the following perspectives: The possibility of achieving a larger study, possibly a multicenter one, regarding the incidence and demographic characteristics of parotid tumors The possibility of carrying out a study on more cases showing the immunostaining for markers of proliferation and apoptosis in malignant tumors and Warthin tumor. 22

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