Aim To evaluate the relationship between numerical and categorical immunohistochemical score of Ki-67 and human epidermal growth factor of receptor 2 (HER2) with clinicopathological parameters of breast cancer (BC). Methods The study included 311 patients with invasive BC diagnosed at the Department of Pathology, School of Medicine in Sarajevo, Bosnia and Herzegovina, during the period 2015-2019. The expression level of Ki-67 and HER2 was detected by immunohistochemical analysis. Results The expression of Ki-67, as a numerical variable correlated significantly with tumour grade (p=0.025), progesterone receptor (PR) (p=0.034) and categorical score of HER2 (p=0.028). When Ki-67 was categorized into high (>14%) and low (≤14%) level groups, a statistically significant association was found between Ki-67 level groups and HER2 status (categorical and numerical) (p=0.001 and p=0.043, respectively), as well as significant negative linear correlation with PR (p=0.037). The expression of HER2, as a numerical variable, showed a statistically significant correlation with tumour grade (p=0.038), PR (p=0.025) and categorical Ki-67 (p=0.043). Categorical score of HER2 correlated significantly with age (p=0.025), histologic type (p=0.039), tumour grade (p=0.016), estrogen receptor (ER), (p=0.002) progesterone receptor (PR) (p=0.0001), and categorical and numerical value of Ki-67 (p=0.0001 and p=0.0001, respectively). Conclusion The results demonstrated that the categorical immunohistochemical score of HER2 provided a greater association with clinicopathological parameters than numerical score of BC. Furthermore, a slightly better correlation with clinicopathological parameters was shown by the numerical value than by the categorical score of Ki-67 by applying a cut-off value of 14%.

Aim To investigate the impact of pre-treatment serum total prostate-specific antigen (PSA) level on prevalence of prostate carcinoma detection in prostate core needle biopsy, and its correlation with established prognostic factors. Methods Prostate needle biopsy samples of 115 patients with available pre-treatment serum total PSA (tPSA) level were analysed. For all cases where morphology alone was insufficient, immunohistochemistry was performed using p63, CKHMW and AMACR antibody panel in order to confirm or exclude the existence of prostate carcinoma. Results Statistically significant positive correlation between serum total PSA values and prevalence of finding prostate carcinoma in needle biopsy specimens was found (p=0.011), as well as in the case when the patients were classified into groups according to tPSA levels (p=0.028). Serum total PSA values and levels (level groups) showed significant positive correlation with Gleason score (p=0.029 and p=0.036, respectively) and Grade Group of prostate carcinomas (p=0.044 and p=0.046, respectively). Sensitivity of the screening test by using 4 ng/mL as cut off value for tPSA was 94.12% (CI: 80.32-99.28%), specificity 8.64% (CI: 3.55-17.00%), positive predictive value 30.19% (CI: 21.65-39.87%) and negative predictive value 77.78% (CI: 39.99-97.19%). Conclusion The increase of serum tPSA value increases the likelihood of finding prostate cancer on needle biopsy specimens. Due to such findings and its positive correlation with a grade of prostate cancer, our study indicates that tPSA can still be considered as a useful tool both in detecting and predicting aggressiveness of prostate cancer.

Introduction: Tumor microenvironment plays a significant role in tumor progression. Tumor stroma is one of the strongest modifiers of tumor cell response, cancer behavior, and cancer progression. This study aimed to investigate the correlation of matrix metalloproteinase-9 (MMP-9) expression and tumor-stroma ratio (TSR) with standard clinicopathological parameters in different molecular subtypes of breast cancer.Methods: Ninety biopsy samples of primary breast cancer diagnosed at the Department of Pathology, School of Medicine, Sarajevo, were selected for this study. The molecular subtype was determined based on the immunohistochemical expression of estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, and Ki-67. Stromal and tumoral MMP-9 immunohistochemical expression and the TSR were determined for each tumor.Results: Tumoral MMP-9 expression correlated positively with the presence of lymphovascular invasion (p= 0.016). TSR showed significant association and correlation with tumor grade (G) (p= 0.031; p= 0.049) and tumor size (pT) (p = 0.049;p= 0.021, respectively). Stromal MMP-9 expression correlated with histologic type, histologic grade of tumor, and lymphocytic inflammatory infiltrate (p= 0.021;p= 0.047, p= 0.038, respectively). A higher percentage of stromal MMP-9 expression correlated with the strongest lymphocytic response (p = 0.007). Significant correlation was observed between molecular subtypes and histologic grade of the tumor (p= 0.032).Conclusion: Our results, to some extent, confirm the significance of the tumor microenvironment in breast cancer, especially when it is about stromal MMP-9 expression. Although we observed significant association, without linear correlation, we found no significant correlation between molecular subtypes of breast cancer and MMP-9 expression.

Objective: To investigate a possible association between peritumoral lymphatic vessel density (LVD) and adipose tissue invasion (ATI) of cancer cells at the tumor margin and their correlation with other prognostic parameters in breast cancer, including lymph node status. Material and methods: Data of 75 patients with breast carcinoma were evaluated through combination of peritumoral LVD and ATI at the tumor margin and compared with clinicopathological parameters. Peritumoral LVD was assessed by immunostaining for D2-40 using the Chalkley counting method. Marginal ATI was defined as either the presence of more than 20 cancer cells in direct contact with the adipose tissue or as the presence of cancer cells in the adipose tissue. Cases were evaluated concerning to patient’s age, tumor size using the TNM staging system, histological type, histological grade (Nottingham histological score, Elston and Ellis), lymphovascular invasion, lymph node metastasis, estrogen receptor (ER), progesterone receptor (PR), HER2/neu status and Ki-67. Results: The relationship was identified between LVD and marginal ATI but without statistical significancy (r = -0,207; p=0,113). There was a significant correlation of the marginal ATI with Ki-67 expression (r=0,250; p =0.03). The regression model (all variables according to ATI) showed a significant total effect (p <0.05), where Ki-67 was an only independent indicator of ATI. Conclusion: The present study suggests that adipose tissue invasion of cancer cells at the tumor margin can be a better predictive biologic indicator of aggressiveness than peritumoral lymphatic vessel density in breast carcinoma. Keywords: breast carcinoma, peritumoral lymphatic vessel density, marginal adipose tissue invasion.

Introduction: The aims of this study were to investigate the immunohistochemical expression of cyclooxygenase-2 (COX- 2) and vascular endothelial growth factor-C (VEGF-C), their correlation with lymphangiogenesis, angiogenesis and clinicopathological significance in human gastric cancer. Material and methods: Tissue samples of gastric cancer of 60 patients, who underwent Billroth II resection, were analyzed. The expression of COX-2 and VEGF-C proteins was calculated using a semi-quantitative immunoreactive score method. Quantitative analysis of lymphangiogenesis and angiogenesis was performed according to the method described by Weidner. Lymphangiogenesis was evaluated by immunostaining with D2-40. Angiogenesis was assessed by CD105 immunostaining. Results: There was a statistically significant difference in the mean values of COX-2 (p< 0.01) and VEGF-C (p< 0.05) between gastric cancer samples and in control samples. Angiogenesis was significantly higher in neoplastic tissue then in control group (p<0.001). Expression of COX-2 showed a significant positive linear correlation with angiogenesis (p<0.05). However, COX-2 did not correlate with VEGF-C or lymphangiogenesis. There was an association between VEGF-C and lymphangiogenesis, but without statistical significance. Lymphangiogenesis significantly correlated with lymph node metastasis (p=0.007). Expression of COX-2 showed significant correlation with type of Bormann’s classification (p=0.019) and depth of invasion (p=0.03). Conclusions: The tumor cells are the major source of COX-2 and VEGF-C in gastric carcinomas. Their correlation did not show that COX-2 overexpression promotes tumor lymphangiogenesis through augmentation of VEGF-C. The results of this study suggest that neoangiogenesis is a dominant process during tumor progression, whereas lymphangiogenesis plays an important role in lymph node metastasis. Keywords: angiogenesis, CD105, COX-2, D2-40, gastric cancer, lymphangiogenesis, VEGF-C

Tussilago farfara (coltsfoot) and Verbascum thapsus (mullein) have been used as folk remedies for treating respiratory disorders. The aim of this study was to test the toxicity of the water extracts of T. farfara and V. thapsus in vivo in zebrafish and in vitro in BEAS 2B epithelial bronchial cells. To the best of our knowledge, this is the first study to investigate the antioxidative properties of T. farfara and V. thapsus extracts in cell culture. Our results show that the T. farfara leaf extract does not produce toxic effects on zebrafish embryos or BEAS 2B cells, and that it has a protective effect in BEAS 2B after induction of oxidative stress. The water extract from V. thapsus displayed pronounced toxic effects on zebrafish embryos and BEAS 2B cells and did not exhibit a significant antioxidative effect on BEAS 2B cells exposed to oxidative stress. Our results suggest that the use of T. farfara water leaf extract is potentially safe and effective in treating respiratory disorders, whereas the use of V. thapsus needs further investigation. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. ON173008]

Objectives The aim of this paper is to present a case of an IVF pregnancy of a 43-year old women being treated for recurrent endometrial cancer stage IA, grade I. Methods Our patient was initially treated five years ago when hysteroscopic ablation of endometrial polyp was performed. Pathologic examination of biopsy specimen reported a diagnosis of atypical polypoid adenomyoma. Two years later, patient was evaluated for recurrent local disease. At that time, pathologic examination revealed typical polypoid adenomyoma with presence of few glands with characteristics of well differentiated endometrioid adenocarcinoma stage IA, grade I. Results After a complete evaluation of cancer staging and a detailed explanation, patient rejected conventional surgical treatment and fertility preserving treatment was started. Progestin therapy via an intrauterine device (IUD) was administered. One year later, IUD was removed and patient underwent IVF cycle with a transfer of 3 embryos without success. At the age of 43, pregnancy was achieved in IVF cycle with administration of hCG. Conclusions Although there are existing evidence of effectiveness for using IUD progestin therapy in endometrial hyperplasia, studies for its use in treatment of endometrial cancer are lacking. We report a case of successful use of leveonorgestrel - IUD alone in fertility preserving treatment followed by achieved pregnancy.

Objectives According to the World Cancer Research Fund, over 380,000 new cases of endometrial cancer have been reported in 2018. After the identification of disease, histologic diagnosis is the main standard for managing further treatment approaches. The following analysis is aimed to examine cost-effectiveness of two preoperative endometrial sampling methods - hysteroscopy and curettage; comparing tumor localization, grading and staging in patients with endometrial cancer. Methods The differences in the specifications of 64 patients that underwent hysteroscopy or curettage in tertiary care center in Croatia were tested. According to five different guidelines, all cases of endometrial cancer were classified into groups of low risk, intermediate, high-intermediate or high risk. Statistical test used for analysis were Kolmogorov-Smirnov test, T-test ant the Z-test. Given data of 36 women who underwent hysteroscopy and 28 women who underwent curettage were analysed. Results Occurrence rates of Grade 1 and Grade 2 of endometrial cancer were higher in patients who underwent hysteroscopy (p=0.03572, p=0.03846) (picture 1). According to the classification of risk factors, no differences in incidence were found between two groups (picture 2). Conclusions In Croatia, the cervical dilation and curettage is a less-frequently used method despite being 62% more affordable than the hysteroscopy, while on the other hand, there is statistically significant difference in preferred choice of the two methods given the histologic grade of cancer and age at the time of diagnosis.

Objectives : The main objective of the study was to determine whether the distribution fsaand th Gastric cancer progression in correlation with distribution and density of T-lymphocytes e level of fsa: The main objective of the study was to determine whether the distribution and the level of density of tumor infiltrating CD4+ and CD8+ T-lymphocytes correlates with standard prognostic factors for gastric cancer and whether it has impact on tumor progression. Methods: The study included 60 tissue samples of operable gastric carcinomas of known regional lymph node status, stained by standard hematoxylin eosin and immunohi Objectives: The main objective of the study was to determine whether the distribution and the level of density of tumor infiltrating CD4+ and CD8+ T-lymphocytes correlates with standard prognostic factors for gastric cancer and whether it has impact on tumor progression. Methods: The study included 60 tissue samples of operable gastric carcinomas of known regional lymph node status, stained by standard hematoxylin eosin and immunohistochemical method in order to determine standard pathologic prognostic factors for gastric cancer and to evaluate distribution and density of tumor infiltrating T- lymphocytes. Results: CD8+ T lymphocytes were predominantly distributed along the margin of carcinoma infiltration, while inside the cancer tissue there were generally few. CD4+ lymphocytes were few in almost all three analyzed zones (margin of carcinoma infiltration, cancer stroma and cancer tissue). The density of CD8+ showed significant positive correlation with CD8+ T lymphocytes within the cancer’s stroma. There was statistically significant difference in density of CD4+ T lymphocytes distributed along the margin of carcinoma infiltration and histological tumor grade, as well as in tumor grade according to Goseki. Conclusion: CD8+ T lymphocytes are densely arranged along the margin of carcinoma infiltration and they correlate with histological grade of gastric carcinoma. Keywords: gastric cancer, tumor microenvironment, T- lymphocytes stochemical method in order to determine standard pathologic prognostic factors for gastric cancer and to evaluate distribution and density of tumor infiltrating T- lymphocytes. Results: CD8+ T lymphocytes were predominantly distributed along the margin of carcinoma infiltration, Gastric cancer progression in correlation with distribution and density of T-lymphocytes while inside the cancer tissue there were generally few. CD4+ lymphocytes were few in almost all three analyzed zones (margin of carcinoma infiltration, cancer stroma and cancer tissue). The density of CD8+ showed significant positive correlation with CD8+ T lymphocytes within the cancer’s stroma. There was statistically significant difference in density of CD4+ T lymphocytes distributed along the margin of carcinoma infiltration and histological tumor grade, as well as in tumor grade according to Goseki. Conclusion: CD8+ T lymphocytes are densely arranged along the margin of carcinoma infiltration and they correlate with histological grade of gastric carcinoma. Keywords: gastric cancer, tumor microenvironment, T- lymphocytes density of tumor infiltrating CD4+ and CD8+ T-lymphocytes correlates with standard prognostic factors for gastric cancer and whether it has impact on tumor progression. Methods : The study included 60 tissue samples of operable gastric carcinomas of known regional lymph node status, stained by standard hematoxylin eosin and immunohistochemical method in order to determine standard pathologic prognostic factors for gastric cancer and to evaluate distribution and density of tumor infiltrating T- lymphocytes. Results : CD8+ T lymphocytes were predominantly distributed along the margin of carcinoma infiltration, while inside the cancer tissue there were generally few. CD4+ lymphocytes were few in almost all three analyzed zones (margin of carcinoma infiltration, cancer stroma and cancer tissue). The density of CD8+ showed significant positive correlation with CD8+ T lymphocytes within the cancer’s stroma. There was statistically significant difference in density of CD4+ T lymphocytes distributed along the margin of carcinoma infiltration and histological tumor grade, as well as in tumor grade according to Goseki. Conclusion : CD8+ T lymphocytes are densely arranged along the margin of carcinoma infiltration and they correlate with histological grade of gastric carcinoma. Keywords : gastric cancer, tumor microenvironment, T- lymphocytes

Introduction: Tumor-associated macrophages (TAM) are the most common inflammatory cells in the tumor microenvironment (TM). As a response to microenvironmental signals, they polarize into tumor resisting M1 or promoting M2 macrophages. TAM and tumor-associated dendritic cells (TADC) can either promote tumor growth and tissue invasiveness or have anti-tumor activity. The aim of the study was the examination of prognostic value in the individual cell population in TM and their correlation with clinicopathological parameters of gastric cancer. Materials and Methods: The study analyzed 60 samples of gastric cancer, known status of regional lymph nodes and without dissemination at the time of diagnosis. The control group was normal gastric tissue samples. Traditional parameters of biological aggressiveness, tumor size, histological grade, and lymphovascular invasion, are determined after standard hematoxylin-eosin staining. TAM and TADS have been evaluated using the immunohistochemical method with CD68 (TAM), TNFa (TAM-M1), CD163 (TAM-M2), and S100 (TADC) antibodies. Expression evaluation of the tissue antigen was carried out by semiquantitative methods. Results: There were statistically significant differences of TAM density (P 0.05). Conclusion: TAMs and TADC have shown potential as biomarkers for evaluating the gastric cancer staging and progression. They showed promising prediction in depth of invasion, histological grade of tumor and tumor size. Keywords: gastric cancer, tumor microenvironment, TAM, TAM-M1/M2, TADC

Objective : The aim of this study was to investigate the impact of inflammation on expression of MMP-2 and MMP-9, as well as to identify the cellular sources of these enzymes in human dental pulps using immunohistochemistry. Methods : Fifty-four irreversibly inflamed samples of dental pulp were used as the experimental group. Fifty-one healthy pulps, obtained from teeth extracted for orthodontic reasons, were used as the control group. The tissue samples were formalin-fixed, paraffin-embedded, and cut into sections at 3- 4 μm. An immunohistochemical study was performed using monoclonal antibodies against MMP-2 and MMP-9. Evaluation of the immunohistochemical expression was determined by the semi-quantitative method and scored as follows: no staining (score 0), less than 10% of stained cells (score 1), less than 30% of weakly stained or strongly but incompletely stained cells (score 2), and more than 30% of strongly and completely stained cells (score 3). Results : Immunohistochemical analysis revealed a significantly greater expression of MMP-9 in inflamed than in healthy dental pulps (Mann–Whitney U, p=0.0001). In contrast, there was no difference in the expression of MMP-2 between these two groups (Mann–Whitney U, p=0.907). MMP-2 and MMP-9 immunoreactivity was detected the most frequently in endothelial cells. Conclusions : MMP-9 is highly overexpressed in inflamed dental pulps. There are no differences in the expression of MMP-2 between healthy and inflammed dental pulps. Endothelial cells represent the major cellular source of MMP-9, as well as MMP-2, in healthy and inflamed dental pulps. Keywords : dental pulp, inflammation, MMP-2, MMP-9, immunohistochemistry

Introduction: Cancer of the prostate (PCa) is the second most common cancer-related cause of death among men and the most common non-cutaneous malignancy in Western countries. Numerous papers have been published on the topic of various aspects of this disease; however, rather little has been written on the diagnostic and prognostic value of the prostate cancer obtained from needle biopsy. Aim: To examine the utility of Pixel Prostate software in determining the volume and topographic distribution cancer of the prostate (PCa), and to analyze it with other variables that are characteristic for PCa. Methods: retrospectively, 75 patients data and postoperative prostate specimens were analyzed, after determining topographic distribution and cancer volume (PCa), using PixelProstate software. Results: Mean VPCa was 6.99 cm3 (0.14-29.7; median 4.51), and mean percentage cancer volume relative to prostate volume (%VPCa) was 16% (0.1-67.2%; median 13%). 71% of the patients had T2 stage, while the rest had T3 stage. Apex involvement was present in 65% of the patients, while central zone involvement and extraprostatic extension were present in 23.5% and 22.7% of the patients, respectively. Preoperative Gleason score undergrading was present in 27 (36%) patients, while bilateral PCa finding was increased from 51% to 87%, postoperatively. The most discriminant variable according to the prediction of %VPCa>10% had preoperative bilateral needle biopsy findings, with AUC of 0.75 (<.001), with sensitivity and specificity of 84% and 70%, respectively; (+LR 2,8; PPV of 74%; NPV of 82%). %VPCa showed good correlation with prostate specific antigen (PSA) and PSA-density. Conclusion: A possibility of precise spatial orientation and volume characterization of the PCa by PixelProstate software was shown. Simultaneously, with time, a clinician, experienced by PP software feedback, gets better insight for the planning of future prostate biopsy, as an important factor in clinical decision making.

Introduction: to evaluate the frequency and significance of immunohistochemistry-based molecular subtypes of breast cancer and investigate their association with traditional pathological features for breast cancer among Bosnian women. Materials and methods: this study included 100 female patients with primary invasive breast cancer. Immunohistochemical analyses for estrogen receptor (ER), progesterone receptor (PR), HER-2 and Ki-67 were performed to define four biological subtypes: luminal A, luminal B, HER-2-positive and triple-negative. Results: the frequency of luminal A, luminal B, HER2-positive and triple-negative subtypes of breast cancer was 44%, 39%, 11% and 6%, respectively. Molecular subtypes of breast cancer among Bosnian women showed to be independent of traditional pathological features (p>0.05). Ki-67 showed significant di erence regarding luminal B tumor type, where high (≥14%) Ki-67 score was predominantly represented in 36 (92.3%) cases (p<0.001). Conclusion: immunohistochemistry-based molecular subtypes of breast cancer in Bosnian women somehow vary in pathological features, i.e. luminal A subtype in this sample comprised mostly ductal histological type, moderate di erentiation with the involvement of lymph nodes, known as worse prognostic factors, although with no statistical significance.