The Association of Death Receptors and TGF-ß1 Expression in Urothelial Bladder Cancer and Their Prognostic Significance.

Death receptor signalization that triggers the extrinsic apoptotic pathway and TGF-ß1 have important roles in urothelial carcinogenesis, with a complex interplay between them. The aim of this research was to assess the association of death receptors DR4, DR5, and FAS as well as TGF-ß1 immunohistochemical expression with the clinicopathological characteristics of urothelial bladder cancer (UBC) and to evaluate their prognostic significance. The decrease or loss of death receptors' expression was significantly associated with muscle-invasive tumors, while non-invasive UBC often retains the expression of death receptors, which are mutually strongly linked. High DR4 expression is a marker of low-grade tumors and UBC associated with exposition to known carcinogens. Conversely, TGF-ß1 was significantly associated with high tumor grade and advanced stage. High expression of DR4 and FAS indicates longer overall survival. High TGF-ß1 signifies an inferior outcome and is an independent predictor of adverse prognosis in UBC patients. This study reveals the expression profile of death receptors in UBC and their possible interconnection with TGF-ß1 and indicates independent prognostic significance of high FAS and TGF-ß1 expression in UBC, which may contribute to deciphering the enigma of UBC heterogeneity in light of the rapid development of novel and effective therapeutic approaches, including targeting of the TRAIL-induced apoptotic pathway.

Influence of Three Different Surgical Techniques on Microscopic Damage of Saphenous Vein Grafts-A Randomized Study.

Background and Objectives: The saphenous vein is one of the most common used grafts (SVG) for surgical revascularization. The mechanism of the SVGs occlusion is still unknown. Surgical preparation techniques have an important role in the early and late graft occlusion. Our study analyzed the influence of the three different surgical techniques on the histological and immunohistochemical characteristics of the vein grafts. Methods: Between June 2019 and December 2020, 83 patients who underwent surgical revascularization were prospectively randomly assigned to one of the three groups, according to saphenous vein graft harvesting (conventional (CVH), no-touch (NT) and endoscopic (EVH)) technique. The vein graft samples were sent on the histological (hematoxylin-eosin staining) and immunohistochemical (CD31, Factor VIII, Caveolin and eNOS) examinations. Results: The CVH, NT, and EVH groups included 27 patients (mean age 67.66 ± 5.6), 31 patients (mean age 66.5 ± 7.4) and 25 patients (mean age 66 ± 5.5), respectively. Hematoxylin-eosin staining revealed a lower grade of microstructural vein damage in the NT group (2, IQR 1-2) in comparison with CVH and EVH (3, IQR 2-4), (4, IQR 2-4) respectively (p < 0.001). Immunohistochemical examination revealed a high grade of staining in the NT group compared to the CVH and EVH group (CD 31 antibody p = 0.02, FVIII, p < 0.001, Caveolin, p = 0.001, and eNOS, p = 0.003). Conclusion: The best preservation of the structural vein integrity was in the NT group, while the lowest rate of leg wound complication was in the EVH group. These facts increase the interest in developing and implementing the endoscopic no-touch technique.

Developmental Characteristics of the Glomerular and Tubular Portion of the Nephron in the Human Foetal Kidney Cortex: Morphometrical and Immunohistochemical Analysis.

This study aimed to morphometrically examine the development of glomeruli and tubules in the kidney cortex of human foetuses at different gestational ages (GAs). We also investigated the expression of the proliferation marker Ki-67 and apoptosis-related markers Bcl-2 and Bax during nephrogenesis using immunohistochemistry. Kidney samples from 38 human foetuses of both sexes with GA ranging from 13 to 40 weeks were analysed. The samples were divided into 7 groups based on GA, each corresponding to 1 lunar month. Foetal kidneys showed a spatiotemporal gradient of nephron differentiation with the transient stages of nephron anlage located in the nephrogenic zone and immature nephrons located in the subjacent maturation zone. In the inner cortex, nephrons establish the morphological characteristics of definitive nephrons. The average area, perimeter, and Feret's diameter of the glomeruli formed within the kidney cortex gradually decreased up to a period of 29-32 weeks of gestation and subsequently increased until a period of 37-40 weeks. There was a weak negative correlation with GA. In contrast, the areal density of glomeruli increased up to a period of 21-24 weeks and then gradually decreased until a period of 37-40 weeks, showing a moderate negative correlation with GA. The average area of renal tubules slightly decreased until a period of 21-24 weeks of gestation and then gradually increased until a period of 36-40 weeks, showing a moderate positive correlation with GA. The average areal density of renal tubules increased significantly until a period of 21-24 weeks of gestation, remained relatively constant until a period of 33-36 weeks, and then increased significantly at 36-40 weeks. There was a strong positive correlation with GA. Our results showed that Ki-67 was expressed in numerous cells of the metanephric mesenchyme, pretubular aggregates, renal vesicles, comma-shaped bodies, and early S-shaped bodies. During subsequent development and the spatial expansion of nephrons towards the mature zone, the expression of Ki-67 was markedly reduced. Similarly, Bcl-2 was strongly expressed in induced nephrogenic progenitor cells, pretubular aggregates, renal vesicles, and comma-shaped bodies. As vascularisation and maturation of the nephron proceeded, Bcl-2 staining became less intense and limited to the parietal layer of the Bowman's capsule and renal tubules. Weak Bax expression was observed in individual scattered cells within segments of the nephrons at all developmental stages. In the mature zone, more intense Bax staining was observed in the renal tubules.

Bevacizumab with Chemotherapy as a First-Line Treatment for Advanced Ovarian Cancer in a Serbian Cohort.

Background and Objectives: For stage IIIb-IV ovarian cancer, bevacizumab-containing treatment is considered the standard of care. The purpose of this study was to evaluate the efficacy of bevacizumab in combination with carboplatin and paclitaxel as a first-line treatment for advanced ovarian cancer. Materials and Methods: Eligible patients had stage IIIc-IV ovarian cancer according to the International Federation of Gynecology and Obstetrics with no clinical signs or symptoms of gastrointestinal obstruction or a history of abdominal fistulae, gastrointestinal perforation, or intra-abdominal abscess or evidence of rectosigmoid involvement by pelvic examination, bowel involvement on computed tomography, or clinical symptoms of bowel obstruction in the previous 6 months. After debulking surgery, the patients received 175 mg/m2 paclitaxel and carboplatin (AUC 6) for the first six cycles and 7.5 mg/kg bevacizumab every three weeks up to 17 cycles until disease progression, unacceptable toxicity, or consent withdrawal. The primary endpoint was progression-free survival. The secondary endpoint was overall survival. Results: Between April 2017 and March 2020, 35 patients began study treatment. Bevacizumab was administered at 7.5 mg/kg in all the patients and for more than 7.5 months in 70% of them. The median progression-free survival was 20 months (95% CI: 16-23). The median overall survival was not reached. Conclusions: This was, to our knowledge, the first trial in Serbia to show progression-free survival and overall survival of combination regimens in advanced ovarian cancer. Based on the observed progression-free survival, bevacizumab combined with chemotherapy should be considered as a standard option in advanced ovarian cancer.

A case of mycotic keratitis due to Fusarium sp. with an undesirable outcome.

Fungal keratitis, an infective disease of the cornea, represents a serious diagnostic and therapeutic problem that, if not recognized on time, could lead to irreversible eye damage. Herein we report a case of fungal keratitis due to Fusarium spp. infection. The 60-year-old man was admitted to our clinic due to an atraumatic acute onset of the disease, with a decrease in the visual acuity, photophobia, redness, and severe pain in the right eye. Clinical observation revealed an ulcer that affected 1/3 of the cornea and a hypopion in the anterior chamber. After the first results of microbiological analyzes, local and systemic antifungal therapy was applied. Due to the fact that the patient voluntarily left the treatment, there was a drastic worsening of the local findings as a full thickness total corneal infiltrate with more intense anterior chamber reaction. Finally, evisceration was performed. Given the fact that fungal keratitis is more prevalent in developing countries, official protocols and available effective antifungals are crucial for adequate treatment and a favorable outcome of this infection.

The association between NOTCH3 expression and the clinical outcome in the urothelial bladder cancer patients.

Disrupted NOTCH activity is a driving event in urothelial bladder cancer (UBC). After activation by hypoxia, the NOTCH3 receptor participates in tumor cell proliferation, acquisition of the epithelial-mesenchymal transition phenotype, and angiogenesis. The aim was to analyze the association of NOTCH3 expression with histopathological and clinical parameters, and to determine its predictive impact on the clinical outcome in UBC patients. The present research included 614 UBC samples incorporated in paraffin tissue microarrays, evaluated by immunohistochemistry for NOTCH3 expression. The accrual period was four years, while the follow-up period was two years. The membranous expression was semi-quantified (0-3), and the mean degree was 1.81±0.94. Criteria for semi-quantification the NOTCH3 expression were the intensity of the staining and the percentage of positive cells. The samples with negative (0) and weak (1) NOTCH3 immunohistochemical (IHC) score were considered negative, while the samples that showed moderate (2) and strong (3) expression were considered positive. Higher degree of positivity was associated with higher risk of cancer-specific mortality (p<0.001). Independent predictors for cancer-specific mortality were NOTCH3 expression and high stage (p<0.001). NOTCH3 expression was not a statistically significant predictor of recurrence-free survival (p=0.816). This study indicated that NOTCH3 is a predictor of poor outcome, suggesting that the NOTCH3 could be potentially reliable IHC marker for selecting the UBC patients that would require more intensive follow-up, especially if they diagnosed in higher stage, with divergent differentiation in pathological report, and without recurrences which would lead them to more frequent medical assessments.

New cases of human dirofilariosis on the Balkan Peninsula - "Masked intruders" uncovered by a surgeon.

Dirofilaria spp. nematodes are accidental parasites of humans causing mild to serious, superficial or visceral infections. Superficial dirofilariosis is rather common in Europe and is typically manifested as subcutaneous form. Herein we report 46 new cases of human dirofilariosis (19 patients with subcutaneous, 18 patients with ocular, 4 patients wih genital, 2 patients with submucosal, 2 patients with pulmonary and 1 patient with intramuscular form of infection) that were recorded from the beginning of 2015 to May 2021 on the Balkan Peninsula with a goal to update the prevalence of this parasitosis and point out potential problems in diagnosis and treatment. Besides, given the high possibility of misinterpretation as tumor, our second aim was to encourage the inclusion of this pathogen in the differential diagnosis of subcutaneous nodules. Although quite common forms, subcutaneous and ocular dirofilariosis can be very often misdiagnosed in clinical practice due to the absence of specific clinical manifestations. Therefore, raising awareness of clinicians about this zoonosis is needed as well as closer collaboration between physicians and veterinarians.

Clinical and pathological significance of proliferation index and p53 expression in gastric adenocarcinoma.

PURPOSE: The purpose of our work was to investigate the association between proliferative index [proIDX] and expression index p53 (p53IDX) with the clinical and pathological characteristics of gastric adenocarcinoma. METHODS: The biopsy material of 90 patients operated on for gastric cancer was routinely processed in paraffin and archived. After the histopathological report was made, two study groups were formed, the first group (n=45) comprised biopsies with intestinal carcinoma and the second (n=45) biopsies of diffuse gastric cancer. In both cases, the control group consisted of biopsies of surrounding non-tumor tissue The routine Hematoxylin-Eosin and immunohistochemical ABC method with anti-Ki67 and anti-p53 antibodies was applied at sections 3-5 µm thick. The expression of Ki67 and p53 was quantified stereometrically. For statistical analysis SPSS (19.0) was used. RESULTS: Significantly higher Ki67 expression was found in both types of adenocarcinoma compared to the control group, as well as significant association of proIDX with most of testing parameters. Expression of p53 was significantly higher in the intestinal type compared to the diffuse type and the control group and was significantly associated with age and histological grade. Diffuse type particulary showed, significant association of p53IDX with most of the histological parameters tested. CONCLUSION: Our results point a highly significant correlation of the Ki67 and p53 expression with indicators of gastric adenocarcinoma progression, which may help to identify patients with an aggressive gastric adenocarcinoma phenotype.

Prognostic Value and Immunohistochemical Analysis of Mismatch Repair Deficiency in Patients with Stage II and III Colorectal Carcinoma-A Single-Center Study.

Background and objectives: Deficient mismatch repair (MMR) status is associated with good prognosis but poor therapeutic response to adjuvant chemotherapy in patients with colorectal cancer. However, there are some opposed arguments considering therapeutic outcomes in patients with evidenced MMR deficiency in colorectal cancer. The aim of the study was the investigation of prognostic value and immunohistochemical analysis of the MMR-deficiency tumors. Materials and Methods: The study enrolled 104 patients with resected stage II and III colorectal cancer samples from the period 2018-2019. Results: The tumors with deficient MMR status were significantly associated with age up to 50 years and right-sided localization (p < 0.001). During the follow-up period of 22.43 ± 6.66 months, 21 patients (20.2%) died, whereas 14 patients (13.5%) had relapses. The loss of mutL homologue 1/postmeiotic segregation increased 2 (MLH1/PMS2) expression, compared to proficient MMR tumors, was associated with shorter disease-free survival in patients with lymphovascular invasion (p < 0.05), perineural invasion (p < 0.01), stage III (p < 0.05) and high-grade tumor (p < 0.05). Conclusions: This retrospective pilot study of a single-center cohort of patients with stage II and III colorectal cancer highlights the clinical importance of using immunohistochemistry (IHC) analysis as a guide for diagnostic algorithm in a country with limited resources, but with a high prevalence of colorectal carcinoma in the young patients. MMR-deficiency tumors compared with proficient MMR colorectal cancer was not shown to be a significant predictor of disease-free and overall survival.

Eruptive Nevi on the Palms and Soles with no Association with an Underlying Disease or Medications.

Eruptive melanocytic nevi are an unusual phenomenon characterized by sudden onset of multiple melanocytic nevi on previously unaffected skin. The majority of case reports have linked this condition with blistering skin disease or immunosuppression. There are only three reports of eruptive nevi developing on the palms and /or soles in healthy individuals. Herein we present the clinical and dermoscopic features of two cases of eruptive acral nevi that developed in healthy individuals in the absence of any recognizable underlying disease and review the current literature of eruptive nevi.

Human dirofilariosis of buccal mucosa - First molecularly confirmed case and literature review.

When presented in unusual manner, human dirofilariosis is hard to recognize and often leads to misdiagnosis and unjustified use of various medications. Herein, we report a rare case of Dirofilaria infection localized in buccal mucosa. A 45-year-old male, residing in the Central Serbia, developed striking edema of buccal mucosa followed by leucocytosis, with both neutrophylia and eosinophylia. Thirty days after first symptoms occurred, nodule was formed along the lateral edge of right maxilla. Therefore, patient underwent surgical extirpation and subsequent histopathological analysis of the specimen revealed the presence of a nematode. Based on morphological characteristics of detected parasite, the diagnosis of submucosal Dirofilaria repens-like infection was made. The diagnosis was confirmed, being the first case in the world analyzed by molecular methods. So far, only 13 such cases have been reported worldwide and our paper brings attention to rare form of this zoonosis and updates the data about human dirofilariosis in buccal mucosa.

Prognostic Impact of Canonical TGF-ß Signaling in Urothelial Bladder Cancer.

Background and objectives: Dysregulation of TGF-ß signaling plays multiple roles in cancer development and progression. In the canonical TGF-ß pathway, TGF-ß regulates the expression of hundreds of target genes via interaction with Smads, signal transducers and transcriptional modulators. We evaluated the association of TGF-ß1, Smad2, and Smad4, the key components of canonical TGFß pathway, with clinicopathologic characteristics of urothelial bladder cancer, and assessed their prognostic value in prediction of patients' outcome. Materials and Methods: Immunohistochemical analysis of TGF-ß1, Smad2, and Smad4 expression was performed on 404 urothelial bladder cancer samples, incorporated in tissue microarrays. Expression status was correlated with clinicopathological and follow-up data. The median follow-up was 61 months. Results: High expression of TGF-ß1, Smad2, and Smad4 was detected in 68.1%, 31.7% and 45.2% of the tumors, respectively. TGF-ß1 overexpression was significantly associated with high tumor grade, and advanced pathologic stage (p < 0.001, respectively). Conversely, high Smad2 and Smad4 expression was linked to low tumor grade (p = 0,003, p = 0.048, respectively), and low tumor stage (p < 0.001, p = 0.003, respectively). Smad2 showed an inverse correlation with variant morphology and divergent differentiation of urothelial tumors (p = 0.014). High TGF-ß1 correlated directly, while Smad2 and Smad4 correlated inversely to cancer-specific death (p = 0.043, p = 0.003, and p = 0.022, respectively). There was a strong relationship between Smad2 and Smad4 expression (p < 0.001). Survival analyses showed that high Smad2 and Smad4 expression was associated with longer overall survival (p = 0.003, p = 0.034, respectively), while in multivariate regression analysis TGF-ß1 manifested as an independent predictor of poor outcome. Conclusions: Unraveling the complex roles and significance of TGF-ß signaling in urothelial bladder cancer might have important implications for therapy of this disease. Assessment of TGF-ß pathway status in patients with urothelial bladder cancer may provide useful prognostic information, and identify patients that could have the most benefit from therapy targeting TGF-ß signaling cascade.

Interplay between STAT3, Cell Adhesion Molecules and Angiogenesis-Related Parameters in Gastric Carcinoma. Does STAT3 Really Have a Prognostic Value?

Background and objectives: Gastric cancer (GC) is one of the deadliest malignancies, with the underlying pathophysiological mechanisms still not completely understood. In this study, we aimed to investigate the signal transducer and activator of transcription 3 (STAT3) moleculeconnection with the pathological features of GCs, and the expression of cell adhesive molecules (E-cadherin and ß-catenin) and angiogenesis-related factors (vascular endothelial growth factor (VEGF), HIF1α, and CD31)). Materials and Methods: This study comprised 136 cases of GCs with data related to the patients' demographic characteristics (age, gender) and pathological features (tumor location, gross type, Laurens' type of GC, histological differentiation, invasion depth, lymphovascular invasion and the presence of metastases) which were correlated with STAT3 expression. Additionally, STAT3 expression and the expression of adhesive molecules and angiogenesis-related factors were studied by immunohistochemical methods. Results: The expression of STAT3 was found to be significantly associated with the occurrence of poorly differentiated GCs in the lower portion of the stomach and with the presence of distant metastases. Interestingly, none of the investigated parameters related to cell adhesion or to angiogenesis were found to be related to the expression of STAT3. Conclusions: The lack of significant differences between the studied STAT3 expression and some of the molecules associated with different cancer features might be due to the characteristics of the studied population sample associated with the origin, heterogeneity, and cancer pathophysiological background. Nonetheless, the results of our study suggest that STAT3 could be a useful marker for the presence of distant GC metastases, which further indicates that STAT3 action might involve some other signaling molecules/pathways that warrant further elucidation.

T lymphocytes in the third trimester decidua in preeclampsia.

OBJECTIVE: The aim of study was to conduct immunohistochemical quantification of CD3+ and CD8+ decidual lymphocytes in preeclampsia. METHODS: A study group included 30 cases of preeclampsia and a control group included 20 healthy pregnant women, all delivered by Cesarean section. Samples of placental bed were analyzed after immunohistochemical staining of CD45+, CD3+ and CD8+ cells. RESULTS: The group with preeclampsia included a significantly higher number of CD3+ (p < 0.01) and CD8+ (p < 0.05) T lymphocytes. CONCLUSION: It is certain that thebalance dysregulation of T cell of the immune milieu of deciduais of importance in etiopathogenesis and manifestations of preeclampsia.

The Impact of MGMT Promoter Methylation and Temozolomide Treatment in Serbian Patients with Primary Glioblastoma.

Background and objective: Despite recent advances in treatment, glioblastoma (GBM) remains the most lethal and aggressive brain tumor. A continuous search for a reliable molecular marker establishes the methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) gene promoter as a key prognostic factor in primary glioblastoma. The aim of our study was to screen Serbian patients with primary glioblastoma for an MGMT promoter hypermethylation and to evaluate its associations with overall survival (OS) and sensitivity to temozolomide (TMZ) treatment. Materials and methods: A cohort of 30 Serbian primary glioblastoma patients treated with radiation therapy and chemotherapy were analyzed for MGMT promoter methylation and correlated with clinical data. Results: MGMT methylation status was determined in 25 out of 30 primary glioblastomas by methylation-specific PCR (MSP). MGMT promoter hypermethylation was detected in 12 out of 25 patients (48%). The level of MGMT promoter methylation did not correlate with patients' gender (p = 0.409), age (p = 0.536), and OS (p = 0.394). Treatment with TMZ significantly prolonged the median survival of a patient (from 5 to 15 months; p < 0.001). Conclusions: Due to a small cohort of primary GBM patients, our study is not sufficient for definitive conclusions regarding the prognostic value of MGMT methylation for the Serbian population. Our preliminary data suggest a lack of association between MGMT promoter methylation and overall survival and a significant correlation of TMZ treatment with overall survival. Further population-based studies are needed to assess the prognostic value of the MGMT promoter methylation status for patients with primary glioblastoma.

Clinical significance of Leptin receptor (LEPR) and Endoglin (CD105) expressions in colorectal adenocarcinoma.

PURPOSE: Carcinoma of the colon occurs quite more often in obese than in healthy people. The key molecule in the development of obesity is leptin, a product of Ob gene that expresses its effects through a specific receptor (LEPR), so our goal was to investigate the expression of LEPR in colorectal carcinoma and the association of their expression with neoangiogenesis, with local/regional and distant metastases and with tumor stage according to the Astler-Coller classification. METHODS: In the paraffin blocks taken from 75 patients treated for colorectal cancer, 3-4 µm thick cuts were made using routine hematoxylin-eosin (HE) and immunohistochemical ABC methods with anti-LEPR and anti-CD105 antibodies. After quantitative analysis of LEPR expression, the microvascular density per mm2 was calculated stereometrically. For the statistical processing, the SPSS software (version 13.0) was used. RESULTS: Pronounced expression of LEPR in stages B1 and B2 was present in 9.1% and in 16% of the cases. In the C2 and D stages, pronounced LEPR expression was found in 51.6%, i.e. 57.1% of the cases, which was significantly higher than in the stages B1 and B2. In the C2 and D stages, a high neoangiogenesis index was found in a significantly higher number of cases (67.7% and 100%) than in stages B1 and B2. LEPR expression had a highly significant correlation coefficient associated with tumor stage, neoangiogenesis index, metastases in the lymph nodes and with distant metastases. CONCLUSION: The increase of LEPR expression was accompanied by increased neoangiogenesis and an increase in the metastatic potential of colorectal cancer.

An appraisal of canine and human cases reveals an endemic status of dirofilariosis in parts of Serbia.

Dirofilaria immitis and D. repens are mosquito-borne nematodes that primarily infect canids, and can also cause mild to serious superficial or visceral infection in humans. In the present survey, peripheral blood from 150 asymptomatic dogs from Serbia were examined using the modified Knott's technique. Dirofilaria immitis, identified based on morphological and morphometric characteristics, was prevalent in dogs not receiving preventative treatment (in 44% and 60% of pound and pet dogs, respectively). These results, together with findings of autochthonous cases of subcutaneous D. repens infection in human patients from Southeastern Serbia emphasize the need for further investigations of this veterinary and public health problem.