Aim: The aim of this study was to investigate the expression of Sox1 and Sox2 transcriptional factors in the subgranular zone (SGZ) of the hippocampus in 8 weeks old 5xFAD mice. Introduction: Transgenic 5xFAD mice represent a model of Alzheimer s disease (AD) characterized by an early deposition of amyloid plaques which disrupt the process of adult neurogenesis in the hippocampus. Certain transcriptional factors such as SoxB1 transcriptional factors are involved in the process of adult neurogenesis, but their roles in neurodegenerative disorders are not fully understood. Material and Methods: Transgenic mice of both genders and their respective non-transgenic controls (n=6 per group) were used in the study. Proliferating cells and immature neurons were detected by their immunohistochemical expression of Ki67 and doublecortin, while neuronal stem/precursor cells were identified by the expression of Sox1 and Sox2 proteins. Immunohistochemistry and counting were performed on hippocampal paraffin sections. Results: Immunohistochemical analysis showed that 5xFAD mice in the SGZ of the hippocampus have significantly lower numbers of Sox1 immunoreactive cells, while Sox2 immunoreactive cells were lower only in female 5xFAD mice. Furthermore, we have detected a decrease in the number of newly formed neurons in male transgenic mice, while the number of proliferating cells was unchanged when compared to non-transgenic controls. Conclusion: The results of our study show that early changes in the neurogenesis in 5xFAD model occur despite the preserved proliferative potential in the SGZ. Our results clearly indicate the importance of SoxB1 transcriptional factors in the early phases of AD.

Lung carcinoma is the leading cause of increases in the morbidity and mortality rates of malignant diseases worldwide. Adenocarcinoma has been the most common histological type in the last decades due to: changes in the tobacco industry, smoking habits and the use of immunohistochemistry. Among more than half of patients, lung adenocarcinoma is diagnosed in an advanced stage of the disease. The discovery of mutations in epidermal growth factor receptor (EGFR) in lung adenocarcinoma is a major advancement in molecular pathology and a new approach to the treatment of these patients. Patients with EGFR mutated lung adenocarcinoma receive a targeted therapy (Tyrosine Kinase Inhibitors-TKI) which leads to improvements in disease prognosis and quality of life. Real-time polymerase chain reaction (PCR) is the most widely used and most reliable method since it requires a minimum amount of starting material and allows the amplification of the desired DNA segment up to a billion times. In this way, deletions in exon 19 are detected in approximately 90% of cases, more often in women, non-smokers and in the territory of Asia. The following may be used for EGFR testing: fresh tissue, fast-frozen tissue, tissue molded into paraffin blocks after fixation in formalin and cytological material obtained by scraping from glass tiles. Tissue processed by decalcination, acid treated or heavy metal treated tissue should be avoided. Although surgical samples represent the golden standard in determining EGFR mutations, the results obtained are compatible with the results obtained by bronchoscopic biopsy and thus eliminate the need for invasive diagnostic procedures. Bronchoscopy is an invasive diagnostic method, whose objectives are to diagnose lung tumors, determine the endoscopic spread of the disease and assess tumor operability. The presence of a tumor may be indicated by a different bronchoscopic aspect of the endobronial mucosa. The sensitivity and specificity of this method depends on: bronchologist’s skills, endoscopic findings, the number of biopsy samples, the professional competence of pathologist-cytologist and the obtained tumor amount. The tumor amount is generally small and depends on the histological type, endoscopic findings, sampling technique and the presence of other cells. It is recommended to take three to five biopsy samples, used for diagnosing but also for molecular testing. Targeted therapy is applied based on the obtained results. Given that biopsy samples molded in paraffin are cut into multiple histological sections, and that the tumor amount decreases, it is necessary to minimize the “consumption”. The concentration of isolated DNA does not differ among patients with wt EGFR and mutated EGFR adenocarcinoma. To date, there has been no consensus regarding the number of tumor cells necessary to determine EGFR mutations, and it is recommended to take samples with a minimum of 200 to 400 tumor cells. Invalid results obtained by using the PCR method are most commonly the result of a small number of preserved tumor cells in a biopsy sample. Blood and necrosis may be limiting factors for molecular testing, but not exclusion factors for the same. Bronchoscopic biopsy sample is adequate for the determination of EGFR mutations because the majority of biopsy samples have more than 100 tumor cells, the difference between the concentration of isolated DNA in EGFR mutated and wt EGFR adenocarcinomas is not statistically significant, EGFR mutations are also detected in samples with a small number of tumor cells when using highly sensitive tests.

Aim: The aim of this study was to investigate the association of local tumor survivin expression and serum concentration with clinical and histopathological parameters in melanoma patients. Introduction: Survivin is a multifunctional protein abundantly expressed in tumors of various types, including melanoma. There are still sparse data regarding relationship of melanoma cell survivin expression with accepted histopathological characteristics as well as serum concentration. Material and Methods: The level of survivin expression was determined immunohistochemically in tumor tissue and with ELISA test in the serum of 84 melanoma patients with melanoma. Results: Survivin expression was significantly higher in the patients whose tumor had ulceration, higher mitotic index, higher Clark and Breslow stage, that made vascular invasion or spread through lymphatic vessels in primary tumor, and in the patients with metastatic disease. The patients with high survivin expression score had almost double shorter disease free interval DFI comparing to those with weak local survivin expression and a small number of survivin cells (9 - 7 vs 19 - 13 months, respectively). The degree of tumor infiltrating lymphocytes presence in tumor tissue was significantly inversely associated with serum survivin concentration. Conclusion: Conclusion Survivin expression in tumor tissue and its serum concetration significantly correlate with clinical and histopathological parameters. Serum levels could be important in initial follow-up as indicators of those patients that would have aggressive local tumor growth and spreading.

Aim: To examine the quality of tested lung carcinoma samples, frequency and type of EGFR mutations, and their correlation with patients clinical characteristics (gender, age, smoking habits, clinical stage). Introduction: Mutations in Epidermal Growth Factor Receptor (EGFR) have a role in lung carcinoma development and they are more prevalent in women and non-smokers. Evaluation of EGFR mutations in lung carcinomas in mandatory for targeted therapy with tyrosine kinase inhibitors. Test performance depends on the quality of tested samples and a test type. Material and Methods: We evaluated reports of EGFR mutation real-time PCR analyses in lung carcinoma samples performed from June 2017 till February 2018. Presence of mutations was correlated with clinical characteristics of lung carcinoma patients. Results: A total of 341 samples was received for testing, among which 40 (11.7%) was unsuitable for analysis due to a low tumor cell content (<5%). Three types of mutations were detected in a total of 24 (8%) cases: L858R in 12 (50%) cases, exon 19 deletion in 10 (41.7%) cases, and G719A/C/S in two cases (8.3%). Mutations were more prevalent in women (13.7%) then in men (4.3%) (p=0.004). Patients with EGFR mutated tumors were older (67,6ą9,4 years), compared to those with non-mutated tumors (62,3ą8,8 years) (p=0,003). Smoking habits and clinical stage were not associated with mutation status in lung carcinomas. Mutations were detected only in adenocarcinomas. Conclusion: Our results suggest the low frequency of EGFR mutations in tested patients, but they are more prevalent in women and older patients.

Aim: The aim of this research is to analyze the profile of Ezh2 expression in superficial urothelial bladder cancer, to investigate its correlation with clinicopathological parameters, as well as to determine the prognostic significance of Ezh2. Introduction: Superficial urothelial bladder cancer, without invasion of muscle layer, is associated with frequent recurrence, and represents significant burden for health care system. Ezh2 is epigenetic regulator with a major role in urothelial oncogenesis. Clinical investigations of Ezh2 inhibitors in treatment of solid cancers have already given encouraging results. Materials and Methods: Tumor samples from 410 patients with superficial bladder cancer (172 pTa, 238 pT1), obtained by transurethral resection, were incorporated in tissue microarrays, and analyzed immunohistochemically to Ezh2 expression. Correlation analysis with clinicopathological parameters was performed using SPSS 18.0. Results: High nuclear expression was found in 33.4% tumors, and it was significantly more frequent in pT1 (46.6%), compared to pTa tumors (15.1%) (p<0.001). Ezh2 expression was associated with high histologic grade, presence of carcinoma in situ, and cancer specific death (p<0.001, respectively). In Kaplan-Mayer survival analysis high Ezh2 expression was significantly associated with poor prognosis and shorter patients survival (p<0.001). There was no significant correlation between Ezh2 and recurrence of the disease, and recurrence free interval (p<0.05). Conclusion: Immunohistochemical expression of transcription repressor Ezh2 in superficial urothelial bladder cancer indicates aggressive behavior of the tumor, and poor prognosis. Ezh2 could be used as pronostic marker in selection of the patients that might require more intense clinical treatment, and as potential target of anticancer therapy.

Aim: Immunohistochemistry findings along with clinical features, are significantly important in differentiating the Extrapleural SFT in the neck, from other well-circumscribed mesenchymal neoplasms at this locations. Introduction: We present a rare case of a Extrapleural SFT in a 57 years old man in the neck, without significant past medical history. Material and Methods: The patient had a painless slow growing tumor, in right sight of the neck, diagnosed with physical examination. Total excision with local anesthesia was done, without previously biopsy of the tumor and other clinical investigations. Standard procedures for histology and immunohistochemical stains were done. Results: Tumor was well circumscribed, encapsulated measuring 5,5x4x4 cm. On section, the cut surface had a multinodular, whitish and firm appearance. On microscopic examination tumor was composed of alternating hypocellular and hypercellular areas separated from each other by thick bands of collagen and branching haemangiopericitoma like vessels. The tumor cells were round to spindle-shaped with little cytoplasm, indistinct borders, dispersed chromatin within vesicular nuclei. Area of myxoid change and subcapsular focus of hemorrhage was present, and 2 mitoses/10 HPF were found. Immunohistochemistry revealed diffuse positivity for CD34, Vimentin and BCL2, focal positivity for CD99, S100, SMA, and negativity for CKWS and EMA. Ki67 showed low proliferating index 3-5%. Conclusion: Although most cases of SFT are benign, there is no strict correlation between morphology and behavior, so patients with extrapleural solitary fibrous tumor have need of long-term post-resection follow-up. Further studies are needed to determine the optimal management of these neoplasms.

Aim: To present three cases and review literature of splenic myoid angioendothelioma (SMA) focusing on immunohistochemical features. Introduction: SMAs are very rare, mainly in middle-aged and elderly patients of both sexes and are characterized by a mixed proliferation of cord capillaries and myoid cells, distinguished from other splenic angioendotheliomas by additional myogenic/myofibroblast differentiation. Material and Methods: Three cases of SMA from the Department of Histopathology Registry of Clinical Centre of Serbia were detected during last 12 years (2006-2017) in one male and two female patients (42,5 ys average age). Histomorphological findings were revised by reviewing all serial HE sections, histochemical trichrome stains and immunohistochemical stainings for CD8, CD31, CD34, CD68, SMA, desmin and Ki-67. Results: All cases showed sharply demarcated non-encapsulated solitary tumors with diameters 42, 55 and 20 mm. Histologically there are dense network of capillary blood vessels intermingled with polygonal myoid stromal cells and at least focally expressed non-homogeneous cellularity of stromal and lymphoid cells with focal sclerosis. Neocapillaries show distinctive CD8- / CD31 / CD34 immunophenotype (differing them from splenic hamartomas) and characteristic mixture with myogenic elements (differing them from cord capillary hemangiomas): intense SMA (3/3), rare focal desmin (2/3) and focal CD68 (1/3) immunoexpression. Conclusion: SMA is underrecognized type of vascular neoplasia, which has a clinico-pathological differential diagnostic significance because it radiologically imitates splenic metastase. Cellular form of SMA must be distinguished from hamartoma, but also from hemangiopericytoma and well-differentiated angiosarcoma of the spleen

The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC), which attempts to standardize reporting and cytological criteria for fine-needle aspiration of thyroid nodules and was first introduced in 2009, has been updated. Although much of the original TBSRTC remains the same, several “enhancements” have been introduced in the 2017 version based on new data and developments in the field. The 2017 revision reaffirms that every thyroid FNA report should begin with one of six diagnostic categories, the names of which remain unchanged since they were first introduced: nondiagnostic or unsatisfactory; benign; atypia of undetermined significance (AUS) or follicular lesion of undetermined significance (FLUS); follicular neoplasm or suspicious for a follicular neoplasm; suspicious for malignancy; and malignant. In the frst edition of TBSRTC, the implied risk of malignancy (MOP) for each diagnostic category was calculated and provided as a range based on a review of the literature at that time: 0–3% for benign, ~5–15% for atypia of undetermined signifcance (AUS) or follicular lesion of undetermined signifcance (FLUS), 15–30% for follicular neoplasm or suspicious for follicular neoplasm, 60–75% for suspicious for malignancy, and 97–99% for the malignant category. In the second edition, these ranges have been revised, especially for the so-called “indeterminate” categories, representing estimates calculated primarily from studies of large case cohorts and meta-analyses of ultrasound-guided thyroid FNA published after 2007. Notably, the new document reinterprets the previous version in one major way, and that is TBSRTC’s careful accommodation of the new noncancer category of noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) tumors, which prior to April 2016 were categorized as thyroid cancer. NIFTP tumors have nuclear changes on cytologic evaluation that are identical to other forms of thyroid cancer, but on close long-term clinical follow-up they do not appear to recur or metastasize, and therefore, they do not behave clinically like thyroid cancer. NIFTP tumors typically fall into categories 3, 4, or 5, and can only be diagnosed as “not cancer” after a full surgical excision is performed and the entire tumor specimen is examined under a microscope. There have also been a number of other enhancements with the 2017 update: • The option of molecular testing in the standard management of AUS/FLUS and FN/SFN has been included. • The definition and diagnostic criteria for FN/SFN has been modified: cases demonstrating mild nuclear changes associated with papillary thyroid carcinoma are now included. The definition and diagnostic criteria for the papillary thyroid carcinoma subset of the malignant category now suggest limiting use to cases with “classical” features of papillary thyroid carcinoma. TBSRTC is now the most common classification worldwide for the reporting of thyroid FNA specimens. In view of this, ABSTRACTS 92 MATERIA MEDICA • Vol. 34 • Issue 1, suplement 1 • april 2018. new suggestions will be useful for various aspects of thyroid FNA including nomenclature, differential diagnosis, the potential impact of NIFTP on the indeterminate diagnostic categories, utility of molecular and IHC markers, and clinical management.

Breast cancer (BC) is the most prevalent cancer in the world among women and there are nearly 1.7 million new cases worldwide each year. Due to a number of remarkable advances made in both diagnosis and therapy, the survival rates for BC patients have increased in those regions with adequate medical facilities. According to contemporary recommendations, any pathological diagnosis of breast lesions, before any treatment, should be based on a Core Needle Biopsy (CNB), or on a Fine Needle Aspiration Cytology (FNAC), if CNB is not available. The prognosis of the newly diagnosed breast cancer patient depends on a number of factors, among which the most important is the extent of the spread of the disease to the axillary lymph nodes. Because any further treatment is influenced by the presence and number of axillary lymph nodes involved, a complete evaluation of the axillary lymph nodes is performed on every patient that is able to tolerate it, after a formal diagnosis of invasive carcinoma. At the very least an ultrasound with guided fine needle aspiration or core biopsy of suspicious lymph nodes should be undertaken.Although CNB is the main method employed in breast lesions diagnostics, FNAC still plays a significant role in the evaluation of pathological processes in the breast, a fact that has been well documented in the relevant literature in the last 20 years. The advantages of FNAC are: the sampling is quicker; the sampling technique usually does not require the use of anaesthetics; the trauma is small, and therefore more convenient for women using anticoagulant therapy; complications are rare; the availability of the results is within a few hours; skilled operators and pathologists regard this method as being highly sensitive in the detection of any malignant cells and the equipment is less expensive. The United Kingdom National Health Service Breast Screening Program (UK NHSBSP), began in 1988. Its guidelines have been published with regards to the mode of categorizing cell changes that may be seen in cytological samples obtained by needle aspiration. Five categories have been identified: C1 (unsatisfactory specimen - non-representative), C2 (benign), C3 (atypical - most likely benign), C4 (suspected - most likely malignant) and C5 (malignant). In 1996, the American National Cancer Institute (NCI) also suggested 5 categories for cytological diagnostics of breast lesions: benign, atypical, suspected, malignant and unsatisfactory. Patients with C3 and C4 categories, namely, atypical and suspected, which carry the risk of a malignant tumour, need to undergo further examination. C1 and C2 categories have to be correlated with the results of clinical and radiological examinations. C3 and C4 categories should not be represented in more than 5% of all analyzed aspirates. Currently, there is no individual morphological criterion that cytological diagnostics of malignant breast tumours could be based on. The most important cytological criteria that indicate whether it is a benign pathological process or a malignant tumor are: cellularity of the sample (a very important criterion, but it should be carefully interpreted), loss of cell cohesiveness (characteristic of malignant tumors), cellular arrangements, cell size, biphasicity in smear, the characteristics of the nucleus (size, contour, the appearance of chromatin, the appearance of nucleolus), characteristics of cytoplasm, nuclear-cytoplasmic ratio, APSTRAKTI 93 MATERIA MEDICA • Vol. 34 • Issue 1, suplement 1 • april 2018. mitotic figures, background appearance (necrosis, peripheral blood cells, mucus…) and the presence of inflammatory cells. It is also possible to perform immuno-histochemical staining on cytological samples, flow cytometry and molecular analyses. The FNAC treatment is characterised by solid sensitivity, specificity and predictive value. The sensitivity of FNAC ranges from 89% to 98% and the specificity is between 98% and 100%. Major shortcomings of this method are the impossibility of diagnosing in situ carcinoma and lesions followed by any abundant production of connective tissue. The CNB treatment has gained remarkable popularity since the 1980s and in many institutions has replaced FNAC. The limitations of both methods are; atypical ductal hyperplasia, fibroepithelial tumours, radial scarring and papillary lesions. In the diagnosis of breast lesions apart from aspiration cytology, other sampling techniques for cytological analysis are also applied. In the era of breast conservation therapy, breast tissue is most commonly sent for intraoperative consultation. A frozen section analysis is performed through freezing and sectioning the surgical specimen with subsequent staining, in order to obtain an extemporaneous assessment of the margins. Although this technique is extensively used by many surgeons to avoid the need for a postponed rescission, some pitfalls have been reported, such as the occurrence of artefacts due to the freezing and thawing of the adipose tissue in the specimen. A different intraoperative method for margins evaluation is imprint cytology, which consists of pressing each of the 6 faces of the specimen on 6 different slides, so that any malignant cell on any involved margin is theoretically present on the cytology of the respective slide, because of the tendency of tumour cells to adhere to glass as compared to adipocytes. Imprint cytology can also be used in assessing the representational value of the CNB samples. A significant number of authors suggest that the application of the imprint of cytology reduces the number of inadequate samples obtained by CNB and can also provide a preliminary diagnosis, especially in cases of adequately sampled malignant tumours. Nipple discharge (ND) accounts for approximately 5% of the breast-related symptoms and is the third most common reason women seek medical attention. Approximately 7% to 15% of unilateral NDs are caused by malignant lesions, primarily ductal carcinoma in-situ (DCIS). A cytological examination of the obtained content is significant in the final treatment decision. Cytological analysis, in particular FNAC, continues to play an important role in the diagnoses of breast cancer. Skilled professionals can determine breast cancer through an analysis of the cytological sample as a reliable and accurate method.

Pulmonary Langerhans cell histiocytosis (PLCH) is a rare disease of unknown etiology, which most commonly affects men, smokers, aged from 20 to 40. It is diagnosed by histological analysis of material obtained by lung biopsy, with immunohistochemical proving of Langerhans cells. The aim of this research is to determine pathohistological characteristics of PLCH and analyzing demographic, clinical and radiological parameters. Retrospective analysis of medical data for 13 patients, proven for PLCH at Institute for Pulmonary diseases of Vojvodina in period of fifteen years. PLCH was found at 9 (69.3%) women and at 4 (30.7%) men, average age 34.7 years. Main clinical symptoms were cough (76.9%) and chest pain (61.5%). Out of 13 patients, 11 (84.6%) were smokers. In most cases PLCH histologically corresponded to the cellular phase of the disease (46.1%), proliferative phase was present at 5 (38.4%), and the fibrotic phase at 2 (15.5%) patients. Immunohistochemically, Langerhans’ cells were positive for presence of CD1a and S-100 antigens in all 13 of analyzed cases, while CD68 antigen was positive in 6 patients. In 6 patients (46.2%) there was disease regression, and at 7 (53.8%) patients the disease progressed despite the applied therapy. In our research, PHLC was more common in younger females, smokers with cough and chest pain. At most of the patients, histologically disease was in the cellular phase. Langerhans cells were positive to presence of CD1a and S100 antigens in all 13 patients. At more than half of the patients the disease progresses despite the applied therapy.