Comparable Toxicity of Surface-Modified TiO2 Nanoparticles: An In Vivo Experimental Study on Reproductive Toxicity in Rats.

Nanoparticles (NPs), a distinct class of particles ranging in size from 1 to 100 nm, are one of the most promising technologies of the 21st century, and titanium dioxide NPs (TiO2 NPs) are among the most widely produced and used NPs globally. The increased application of TiO2 NPs raises concerns regarding their global safety and risks of exposure. Many animal studies have reported the accumulation of TiO2 NPs in female reproductive organs; however, evidence of the resultant toxicity remains ambiguous. Since the surface area and chemical modifications of NPs can significantly change their cytotoxicity, we aimed to compare the toxic effects of pristine TiO2 powder with surface-modified TiO2 powders with salicylic acid (TiO2/SA) and 5-aminosalicylic acid (TiO2/5-ASA) on the ovaries, oviducts, and uterus on the 14th day following acute oral treatment. The results, based on alterations in food and water intake, body mass, organ-to-body mass ratio, hormonal status, histological features of tissues of interest, and antioxidant parameters, suggest that the modification with 5-ASA can mitigate some of the observed toxic effects of TiO2 powder and encourage future investigations to create NPs that can potentially reduce the harmful effects of TiO2 NPs while preserving their positive impacts.

SLUG and SNAIL as Potential Immunohistochemical Biomarkers for Renal Cancer Staging and Survival.

Renal cell carcinoma (RCC) is the deadliest urological neoplasm. Up to date, no validated biomarkers are included in clinical guidelines for the screening and follow up of patients suffering from RCC. Slug (Snail2) and Snail (Snail1) belong to the Snail superfamily of zinc finger transcriptional factors that take part in the epithelial-mesenchymal transition, a process important during embryogenesis but also involved in tumor progression. We examined Slug and Snail immunohistochemical expression in patients with different stages of renal cell carcinomas with the aim to investigate their potential role as staging and prognostic factors. A total of 166 samples of malignant renal cell neoplasms were analyzed using tissue microarray and immunohistochemistry. Slug and Snail expressions were evaluated qualitatively (presence or absence), in nuclear and cytoplasmic cell compartments and compared in relation to clinical parameters. The Kaplan-Meier survival analysis showed the impact of the sarcomatoid component and Slug expression on the survival longevity. Cox regression analysis separated Slug as the only independent prognostic factor (p = 0.046). The expression of Snail was associated with higher stages of the disease (p = 0.004), especially observing nuclear Snail expression (p < 0.001). All of the tumors that had metastasized showed nuclear immunoreactivity (p < 0.001). In clear cell RCC, we showed a significant relationship between a high nuclear grade and nuclear Snail expression (p = 0.039). Our results suggest that Slug and Snail could be useful immunohistochemical markers for staging and prognosis in patients suffering from various RCCs, representing potential targets for further therapy strategies of renal cancer.

The Utility of Mitochondrial Detection Methods Applied as an Additional Tool for the Differentiation of Renal Cell Tumors.

The precise differentiation of renal cell tumors (RCTs) is sometimes hard to achieve using standard imaging and histopathological methods, especially for those with eosinophilic features. It has been suggested that the vast overabundance of mitochondria, as a well-known hallmark of eosinophilic cytoplasm, and could be a characteristic of distinct tumor types with opposing clinical outcomes. Thus, we intended to explore the associations between mitochondrial distribution patterns in different RCTs, including 43 cell renal cell carcinomas (ccRCCs), 15 papillary renal cell carcinomas (pRCCs), 20 chromophobe renal cell carcinomas (chRCCs), and 18 renal oncocytomas (ROs). Tumor samples were stained with two anti-mitochondrial antibodies (mitochondrial antibody Ab-2, clone MTC02; prohibitin, II-14-10, MA5-12858), applying immunohistochemistry and immunofluorescence to define mitochondrial distribution patterns (coarse scanty, moderate granular, and diffuse granular). Our results revealed significantly different expression patterns among the investigated RCTs (p < 0.001). The majority of ccRCCs exhibited coarse scanty mitochondrial staining, while all chRCCs had moderate granular expression. Nevertheless, all ROs, all pRCCs, and two cases of ccRCC presenting with higher nuclear grade and eosinophilic cytoplasm had diffuse granular mitochondrial expression. Moreover, with increased distribution of mitochondria, the intensity of staining was higher (p < 0.001). Here we present a strategy that utilizes fast and easy mitochondrial detection to differentiate RO from chRCC, as well as other eosinophilic variants of RCC with high accuracy.

Immunohistochemical Pattern of Histone H2A Variant Expression in an Experimental Model of Ischemia-Reperfusion-Induced Acute Kidney Injury.

Ischemia-reperfusion injury (IRI) is a frequent cause of AKI, resulting in vasoconstriction, cellular dysfunction, inflammation and the induction of oxidative stress. DNA damage, including physical DNA strand breaks, is also a potential consequence of renal IRI. The histone H2A variants, primary H2AX and H2AZ participate in DNA damage response pathways to promote genome stability. The aim of this study was to evaluate the immunohistochemical pattern of histone H2A variants' (H2AX, γH2AX(S139), H2AXY142ph and H2AZ) expression in an experimental model of ischemia-reperfusion-induced acute kidney injury in spontaneously hypertensive rats. Comparing the immunohistochemical nuclear expression of γH2AX(S139) and H2AXY142ph in AKI, we observed that there is an inverse ratio of these two histone H2AX variants. If we follow different regions from the subcapsular structures to the medulla, there is an increasing extent gradient in the nuclear expression of H2AXY142ph, accompanied by a decreasing nuclear expression of γH2AX. In addition, we observed that different structures dominated when γH2AX and H2AXY142ph expression levels were compared. γH2AX was expressed only in the proximal tubule, with the exception of when they were dilated. In the medulla, H2AXY142ph is predominantly expressed in the loop of Henle and the collecting ducts. Our results show moderate sporadic nuclear H2AZ expression mainly in the cells of the distal tubules and the collecting ducts that were surrounded by dilated tubules with PAS (periodic acid-Schiff stain)-positive casts. These findings may indicate the degree of DNA damage, followed by postischemic AKI, with potential clinical and prognostic implications regarding this condition.

Matrix Metalloproteinases 2 and 9 and Their Tissue Inhibitors in the Diagnostics of Medullary Thyroid Carcinoma.

Medullary Thyroid Carcinoma (MTC) is a tumor of the neuroendocrine system. In recent years, the need to assess the MTC diagnostic-related parameters has emerged with the aim to elucidate the mechanisms involved in this pathology. The objective of this study was to evaluate the role of Matrix Metalloproteinases (MMPs) 2 and 9, their tissue inhibitors of matrix metalloproteinases (TIMPs), S100 protein, and amyloid in the diagnostic of MTC. Thirty-two samples with MTC (72% women) were included in this cross-sectional study and divided by groups: T category 1 (T1)≤20 mm and T category 2 (T2) 20 to 40 mm of tumor size. MMPs 2 and 9, TIMPs 2 and 1, S100 protein, and calcitonin in tissues were obtained by immunohistochemical techniques. The presence of amyloid in tissue sections was detected on Thioflavin T-stained slides under fluorescent microscope. Percentage of positive cells (P) observed for MMP-2 was higher in those samples presenting T2 MTC with respect to those with T1 MTC ( P <0.05). Moreover, P-MMP-2 showed a direct correlation with higher T category of MTC (Rho=0.439, P < 0.001), whereas P-MPP-9 was directly correlated with S100 protein and the intensity of calcitonin in tissues (Rho=0.419, P =0.017; Rho=0.422, P =0.016, respectively. Therefore, MMPs were directly correlated with some traditional biomarkers of MTC. In this regard, P-MMP-2 was more expressed in type 2 MTC. Combining the analysis of traditional and other useful biomarkers of MTC as MMPs 2 and 9 could be a useful strategy in the diagnostic of MTC.

Clinicopathological Relevance of PAX8 Expression Patterns in Acute Kidney Injury and Chronic Kidney Diseases.

Transcription factor PAX8, expressed during embryonic kidney development, has been previously detected in various kidney tumors. In order to investigate expression of PAX8 transcription factor in acute kidney injury (AKI) and chronic kidney diseases (CKD), immunohistochemical analysis was performed. Presence, location and extent of PAX8 expression were analyzed among 31 human kidney samples of AKI (25 autopsy cases, 5 kidney biopsies with unknown etiology and 1 AKI with confirmed myoglobin cast nephropathy), as well as in animals with induced postischemic AKI. Additionally, expression pattern was analyzed in 20 kidney biopsy samples of CKD. Our study demonstrates that various kidney diseases with chronic disease course that results in the formation of tubular atrophy and interstitial fibrosis, lead to PAX8 expression in the nuclei of proximal tubules. Furthermore, patients with PAX8 detected within the damaged proximal tubuli would be carefully monitored, since deterioration in kidney function was observed during follow-up. We also showed that myoglobin provoked acute kidney injury followed with large extent of renal damage, was associated with strong nuclear expression of PAX8 in proximal tubular cells. These results were supported and followed by data obtained in experimental model of induced postischemic acute kidney injury. Considering these findings, we can assume that PAX8 protein might be involved in regeneration process and recovery after acute kidney injury. Thus, accordingly, all investigation concerning PAX8 immunolabeling should be performed on biopsy samples of the living individuals.

Effects of Automation on Sustainability of Immunohistochemistry Laboratory.

The COVID-19 pandemic that hit the world recently caused numerous changes affecting the health system in every department. Reduced staff numbers, mostly due to illness, led to an increase in automation at every stage of laboratory work. The immunohistochemistry (IHC) laboratory conducts a high volume of slide staining every day. Therefore, we analyzed time and total costs required to obtain IHC slides in both the manual and automated way, comparing their efficiency by processing the same sample volume (48 microscope slides-the maximum capacity that an automated immunostainer-DAKO, Autostainer Link 48, Part No AS48030-can process over a single cycle). The total IHC procedure time to run 48 slides manually by one technician was 460 min, while the automated process finished a cycle within 390 min (15.22% less time). The final cost of a single manual IHC slide was 12.26 EUR and 7.69 EUR for slides labeled in the automated immunostainer, which reduced final costs by 37.27%. Thus, automation of the IHC procedure reduces the time and costs of the IHC process, contributing significantly to the sustainability of the healthcare system during the COVID-19 pandemic, overcoming insufficient human resources.

The Fractal and GLCM Textural Parameters of Chromatin May Be Potential Biomarkers of Papillary Thyroid Carcinoma in Hashimoto's Thyroiditis Specimens.

Occasionally, Hashimoto's thyroiditis (HT) and papillary thyroid carcinoma (PTC) share similar nuclear features. The current study aims to quantify the differences between the investigated specimens of HT-associated PTC versus the HT alone, to reduce the subjective experience of an observer, by the use of fractal parameters as well as gray-level co-occurrence matrix (GLCM) textural parameters. We have analyzed 250 segmented nuclei per group (nn = 25 per patient and np = 10 patients per group) using the ImageJ software (NIH, Bethesda, MD, USA) as well as an in-house written code for the GLCM analysis. The mean values of parameters were calculated for each patient. The results demonstrated that the malignant cells from the HT-associated PTC specimens showed lower chromatin fractal dimension (p = 0.0321) and higher lacunarity (p = 0.0038) compared with the corresponding cells from the HT specimens. Also, there was a statistically significant difference between the investigated specimens, in the contrast, correlation, angular second moment, and homogeneity, of the GLCM corresponding to the visual texture of follicular cell chromatin. The differences in chromatin fractal and GLCM parameters could be integrated with other diagnostic methods for the improved evaluation of distinctive features of the HT-associated PTC versus the HT in cytology and surgical pathology specimens.

The effect of antioxidant status on overall survival in renal cell carcinoma.

INTRODUCTION: The oxidative stress contributes to all three phases of carcinogenesis and represents a concomitant condition in renal cell carcinoma (RCC). RCC is the most common type of neoplasm of the kidney, and despite numerous studies the set of predictive and prognostic markers of survival are still unknown. The aim of our study was to examine the relation between antioxidant (AO) status and overall survival (OS) in RCC patients. MATERIAL AND METHODS: Our study included 95 patients with RCC, who underwent radical nephrectomy. We analysed the prognostic role of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, glutathione reductase, glutathione, and malondialdehyde) and other clinicopathological factors (size, grade, stage, and histological subtype) on the OS of RCC patients. RESULTS: The 5-year OS was 54.6%. The survival analysis related to AO parameters showed no significant difference in survival of RCC patients. The concentration of malondialdehyde, an indicator of lipid peroxidation, also had no significant effect on the survival rate of RCC patients. Univariate and multivariate analysis confirmed the significance of clinicopathological parameters (size, p < 0.001; Fuhrman grade, p = 0.001, and stage, p < 0.001) for patients' survival. CONCLUSIONS: In our cohort of patients, different antioxidant parameters were not found to be predictors for OS of patients with RCC, who underwent radical nephrectomy.

Sex specific associations in genome wide association analysis of renal cell carcinoma.

Renal cell carcinoma (RCC) has an undisputed genetic component and a stable 2:1 male to female sex ratio in its incidence across populations, suggesting possible sexual dimorphism in its genetic susceptibility. We conducted the first sex-specific genome-wide association analysis of RCC for men (3227 cases, 4916 controls) and women (1992 cases, 3095 controls) of European ancestry from two RCC genome-wide scans and replicated the top findings using an additional series of men (2261 cases, 5852 controls) and women (1399 cases, 1575 controls) from two independent cohorts of European origin. Our study confirmed sex-specific associations for two known RCC risk loci at 14q24.2 (DPF3) and 2p21(EPAS1). We also identified two additional suggestive male-specific loci at 6q24.3 (SAMD5, male odds ratio (ORmale) = 0.83 [95% CI = 0.78-0.89], Pmale = 1.71 × 10-8 compared with female odds ratio (ORfemale) = 0.98 [95% CI = 0.90-1.07], Pfemale = 0.68) and 12q23.3 (intergenic, ORmale = 0.75 [95% CI = 0.68-0.83], Pmale = 1.59 × 10-8 compared with ORfemale = 0.93 [95% CI = 0.82-1.06], Pfemale = 0.21) that attained genome-wide significance in the joint meta-analysis. Herein, we provide evidence of sex-specific associations in RCC genetic susceptibility and advocate the necessity of larger genetic and genomic studies to unravel the endogenous causes of sex bias in sexually dimorphic traits and diseases like RCC.

Cyclin D1 and p57 expression in relation to clinicopathological characteristics and overall survival in patients with renal cell carcinoma.

PURPOSE: There is a need for identifying molecular prognostic biomarkers to better predict clinical outcomes in patients with renal cell carcinoma (RCC). This study investigated the pattern of cyclin D1 and p57 expression in RCC patients and evaluated their relation with clinicopathological characteristics and overall survival (OS). METHODS: Immunohistochemistry was applied to paraffin-embedded tissue sections of 74 RCC patients. Two cut-off groups were defined by the fraction of positive cells as follows: ≤10% and >10% positive cells for cyclin D1, and ≤5% and >5% positive cells for p57. RESULTS: Cyclin D1 expression in >10% of positive cells was observed mostly in the clear cell RCC, while p57 expression in ≤5% of positive cells was found in 86% of chromophobe RCC specimens. The higher expression of cyclin D1 and lower expression of p57 were more frequent in grade I-II tumors. OS was associated with unfavorable clinicopathological characteristics. However, cyclin D1/p57 expression did not influence the survival rates. CONCLUSION: Although cyclin D1 and p57 expression did not affect survival rates in RCC patients, proper validation and establishment of the qualitative cut-off point are needed for these tumors.

Immunohistochemical analysis of cyclin A expression in Wilms tumor.

BACKGROUND: Cyclin A overexpression is found in a variety of human tumors and correlates with unfavorable outcome. We analyzed immunohistochemical expression of cyclin A in Wilms tumor (WT) in relation to clinicopathological characteristics, preoperative chemotherapy (PrOpChTh), and overall survival (OS). METHODS: This retrospective study involved 43 patients who underwent nephrectomy from January 1996 to October 2010. Tumor stage and histological subtype were determined by revised Societé International d'Oncologie Pediatrique protocol, based on histological components/alterations caused by PrOpChTh, within the prognostic group of low, intermediate and high risk, and with criteria for anaplasia. The regressive/necrotic changes in total tumor mass of primary tumor and the proportion of epithelial, blastemal, and stromal components in the remaining viable tumor tissue were also determined. Cyclin A expression was evaluated by immunohistochemistry using a polyclonal rabbit, antihuman antibody (H-432). RESULTS: Cyclin A overexpression was found in 34.3% of WTs, with higher frequency in tumors with epithelial (31.3%) and blastemal (37.1%) components than those with stromal component (17.7%). Regarding histological type, cyclin A overexpression was found most often in focal anaplasia (100%), stromal (60%), and diffuse anaplastic (66.7) WTs. The overexpression was also more frequent in stages 3 and 4 (77.8% and 66.7%, respectively) compared to tumors in stages 1 and 2 (13.3% and 12.5%, respectively; p = 0.004) in all components, as well as in blastemal component in stages 3 and 4 (77.8% and 66.7%, respectively) vs. stages 1 and 2 (13.3% and 25%, respectively, p = 0.009). Cyclin A overexpression in all components was 66.7% in WTs with metastasis and 31.3% in WTs without metastasis (p = 0.265, Fisher test). Log-rank testing revealed differences of OS regarding stage (p = 0.000), prognostic groups (p = 0.001), and cyclin A expression in blastemal component (p = 0.025). After univariate analysis, tumor stage (p = 0.001), prognostic group (p = 0.004), and cyclin A expression in blastemal component (p = 0.042) were significant prognostic factors for OS; however, after multivariate analysis, none of these factors were confirmed as independent predictors of survival. DISCUSSION: This study showed that cyclin A overexpression might be associated with the development and progression of WT with anaplasia. Also, cyclin A overexpression was more often observed in advanced stages (3 and 4) of WT, in the group of high-risk WTs, and in focal and diffuse anaplasia WTs. There was no relation of cyclin A overexpression and metastatic ability of WT. Although this study has not confirmed the prognostic value of cyclin A overexpression, its association with unfavorable prognosis should be further evaluated.

The influence of obesity-related factors in the etiology of renal cell carcinoma-A mendelian randomization study.

BACKGROUND: Several obesity-related factors have been associated with renal cell carcinoma (RCC), but it is unclear which individual factors directly influence risk. We addressed this question using genetic markers as proxies for putative risk factors and evaluated their relation to RCC risk in a mendelian randomization (MR) framework. This methodology limits bias due to confounding and is not affected by reverse causation. METHODS AND FINDINGS: Genetic markers associated with obesity measures, blood pressure, lipids, type 2 diabetes, insulin, and glucose were initially identified as instrumental variables, and their association with RCC risk was subsequently evaluated in a genome-wide association study (GWAS) of 10,784 RCC patients and 20,406 control participants in a 2-sample MR framework. The effect on RCC risk was estimated by calculating odds ratios (ORSD) for a standard deviation (SD) increment in each risk factor. The MR analysis indicated that higher body mass index increases the risk of RCC (ORSD: 1.56, 95% confidence interval [CI] 1.44-1.70), with comparable results for waist-to-hip ratio (ORSD: 1.63, 95% CI 1.40-1.90) and body fat percentage (ORSD: 1.66, 95% CI 1.44-1.90). This analysis further indicated that higher fasting insulin (ORSD: 1.82, 95% CI 1.30-2.55) and diastolic blood pressure (DBP; ORSD: 1.28, 95% CI 1.11-1.47), but not systolic blood pressure (ORSD: 0.98, 95% CI 0.84-1.14), increase the risk for RCC. No association with RCC risk was seen for lipids, overall type 2 diabetes, or fasting glucose. CONCLUSIONS: This study provides novel evidence for an etiological role of insulin in RCC, as well as confirmatory evidence that obesity and DBP influence RCC risk.

Immunohistochemical expression of proliferative markers in renal cell carcinoma.

PURPOSE: The purpose of this study was to investigate into the expression of cyclin A and telomerase in renal cell carcinoma (RCC) and to analyze the relationship between expression and the clinicopathological characteristics of the tumor and their impact on survival. METHODS: The overall material included 74 samples of RCC and 4 of normal renal tissue. Primary cyclin A antibody from Santa Cruz Biotechnology and TERT MA5-16034 antibody from Thermo Fisher Scientific Inc were used. Staining was performed by streptavidin-biotin technique using DAKO LSAB+ kit. Statistical analyses were performed using of SPSS 23 Statistics software from IBM. RESULTS: No differences in cyclin A and telomerase expression among gender and age groups were found, nor did the tumor dimensions have any significant impact on expression. Also, tumor grades and stages did not differ. However, histological types differed in favor of the papillary type. A significant positive correlation between both markers, as well as between the expression and tumor stage and grade was noticed. Only the tumor stage had negative impact on survival. CONCLUSIONS: Although not affecting survival, the expression of cyclin A and telomerase increased with tumor stage and grade, suggesting that cyclin A and telomerase could be potential proliferative immunohistochemical markers of RCC.

Concomitance of Polymorphisms in Glutathione Transferase Omega Genes Is Associated with Risk of Clear Cell Renal Cell Carcinoma.

Glutathione S-transferases (GSTs), a superfamily of multifunctional enzymes, play an important role in the onset and progression of renal cell carcinoma (RCC). However, novel GST omega class (GSTO), consisting of GSTO1-1 and GSTO2-2 isoenzymes, has not been studied in RCC yet. Two coding single nucleotide polymorphisms (SNPs) supposedly affect their functions: GSTO1*C419A (rs4925) causing alanine to aspartate substitution (*A140D) and GSTO2*A424G (rs156697) causing asparagine to aspartate substitution (*N142D), and have been associated with several neurodegenerative diseases and cancers. Functional relevance of yet another GSTO2 polymorphism, identified at the 5' untranslated (5'UTR) gene region (GSTO2*A183G, rs2297235), has not been clearly discerned so far. Therefore, we aimed to assess the effect of specific GSTO1 and GSTO2 gene variants, independently and in interaction with established risk factors (smoking, obesity and hypertension) on the risk for the most aggressive RCC subtype, the clear cell RCC (ccRCC). Genotyping was performed in 239 ccRCC patients and 350 matched controls, while plasma levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker of oxidative DNA damage, were determined by ELISA. As a result, combined effect of all three variant genotypes exhibited almost 3-fold risk of RCC development. Additionally, this association was confirmed at the haplotype level [variant GSTO1*A/GSTO2*G (rs156697)/GSTO2*G (rs2297235) haplotype], suggesting a potential role of those variants in propensity to RCC. Regarding the gene-environment interactions, variant GSTO2*G (rs156697) homozygous smokers are at higher ccRCC risk. Association in terms of oxidative DNA damage was found for GSTO2 polymorphism in 5'UTR and 8-OHdG. In conclusion, the concomitance of GSTO polymorphisms may influence ccRCC risk.

HTERT promoter methylation and single nucleotide polymorphism (-245 T>C) affect renal cell carcinoma behavior in Serbian population.

PURPOSE: Renal cell carcinoma (RCC) is the most common renal cancer in adults and includes several subtypes that may be distinguished by their histology, genetic background, clinical course and responses to treatment. Human telomerase reverse transcriptase (hTERT), a crucial enzyme for telomere maintenance, has been linked to RCC development. The purpose of this study was to search for genetic and epigenetic alterations in hTERT (promoter mutations and methylation and gene amplification), and to establish a possible association between molecular and clinico-pathological characteristics of RCC. METHODS: DNA was extracted from 31 formalin-fixed, paraffin-embedded tumor samples and 23 blood samples from 54 patients with RCC. Polymerase chain reaction (PCR) products were sequenced and analyzed using the Sequencher software. HTERT amplification was determined by quantitative PCR, while the promoter methylation status was assessed by methylation specific PCR. Statistical analysis was performed using SPSS. RESULTS: No mutations could be detected in the hTERT promoter but only a single nucleotide polymorphism (SNP) (-245 T>C). In 54 analyzed RCC cases, the variant allele C was present in homozygous or heterozygous form in 48% of the patients. The C allele was significantly more frequent in low grade tumors (p=0.046). Gene amplification was detected in 19.4% of the 31 RCCs and hTERT methylation in 54.8% of the 31 samples. An association was established between methylation and histological type of RCC (p=0.047). CONCLUSIONS: HTERT seems to be implicated in RCC pathogenesis since the promoter polymorphism exerts a modulation effect on tumor behavior. In addition, hTERT promoter methylation status is related to RCC histology.

Immunohistochemical study of cyclin A and p16 expression in patients with renal cell carcinoma.

PURPOSE: Renal cell carcinoma (RCC) is the most common malignant kidney tumor in adults. Dysregulation of the cell cycle can lead to cancer development. In this study, the mitosis-associated cyclin A and p16, a negative controller, were investigated as potential key points in the RCC development. METHODS: This retrospective study included 74 patients with RCC. The expression of cyclin A and p16 and their correlation to histopathological parameters (TNM stage, histological subtype, nuclear grade, tumor size), gender, age, and clinical outcome were studied and analyzed. RESULTS: The highest median value for cyclin A (40%; range 0-70)) and for p16 (57.5%); range 35-80) were found in the papillary histological subtype. Survival analysis showed that in the group of patients that had died before September 2015, the median value for cyclin A was 20% (range 0-60), which was significantly higher than 5% (range 0-70), found in the group of patients that survived (p=0.019). CONCLUSIONS: In relation to the histological subtype, the papillary type of RCC was associated with a significantly higher expression of cyclin A and p16 compared to other subtypes of RCC. High expression of cyclin A indicated worse prognosis, therefore cyclin A could be considered to be a significant prognostic marker.

Postnatal Developmental Changes in Fractal Complexity of Giemsa-Stained Chromatin in Mice Spleen Follicular Cells.

Although there are numerous recent works focusing on fractal properties of DNA and chromatin, many issues regarding changes in chromatin fractality during physiological aging remain unclear. In this study, we present results indicating that in mice, there is an age-related reduction of chromatin fractal complexity in a population of spleen follicular cells (SFCs). Spleen tissue was obtained from 16 mice and fixated in Carnoy solution. The youngest animal was newborn, and each animal was exactly 1 month older than the previous. We performed fractal analysis of SFC chromatin structure, stained using Giemsa technique. Fractal analysis was done in a plugin algorithm of ImageJ software. We also performed gray-level co-occurrence matrix (GLCM) analysis of all chromatin structures with the calculation of parameters such as angular second moment and inverse difference moment. Giemsa-stained SFC chromatin exhibited an age-dependent reduction of fractal dimension with statistically significant (p<0.01) linear trend. Moreover, there was a statistically significant increase of SFC chromatin lacunarity. The chromatin GLCM parameters did not significantly change. To our knowledge, this is the first study to perform fractal and GLCM analyses of SFC chromatin and to investigate potential changes of fractal parameters during postnatal development.

Polymorphisms of survivin -31 G/C gene are associated with risk of urothelial carcinoma in Serbian population.

PURPOSE: Survivin is thought to play an important role in carcinogenesis and is found to be associated with poor clinical outcome in various malignancies. Gene -31 G/C polymorphism has been identified as a risk factor for the development of several types of tumors. The purpose of this study was to investigate the association between survivin gene promoter -31C/G polymorphism and urothelial carcinoma (UC) risk in Serbian population and to compare the different expressions of survivin in UC of different disease stages, histological grades and tumor location in the upper or lower urinary tract. METHODS: DNA from 94 patients with primary UC and from 82 healthy subjects was subjected to PCR restriction fragment length polymorphism analysis (PCR-RFLP) to identify individual genotypes. UC samples were subjected to immunohistochemical analysis to assess survivin expression in these lesions. RESULTS: It was observed that the frequency of G/G genotype was greater in patients with UC (58.7%) than in controls (32%). Compared with study subjects carrying the C/G or C/C genotypes, significantly increased UC risk was found for individuals carrying the G/G genotype. Those carrying the G/G genotype had a significantly increased UC risk compared with those with C/G or C/C genotypes. Patients with UC carrying the G/G genotype had a greater prevalence of muscle-invading (stage T2-T4), high-grade (G2) tumor and immunohistochemicaly overexpressed survivin compared with those carrying the C/G or C/C genotypes. CONCLUSIONS: G/G genotype of the -31C/G polymorphism might be a risk factor for UC development.

GSTM1 genotype is an independent prognostic factor in clear cell renal cell carcinoma.

PURPOSE: Owing to dual functionality of cytosolic glutathione S-transferases (GSTs), they might affect both the development and the progression of renal cell carcinoma (RCC). However, the data on the prognostic value of GST polymorphism in patients with RCC are scarce. Hence, we evaluated the effect of GST gene variants on both the risk of RCC development and the postoperative prognosis in patients with clear cell RCC (ccRCC). METHODS: GST genotypes were determined in 305 patients with RCC and 326 matched controls, whereas the overall survival was evaluated in patients with ccRCC only. The presence of GSTM1:ASK1 protein-protein interaction in ccRCC tissue samples was analyzed by methods of immunoprecipitation and immunoblot. RESULTS: We noted an increased risk of RCC development in carriers of GSTM1-null and GSTP1-variant genotype (P<0.05). On the contrary, survival analysis indicated shorter overall survival for patients with ccRCC with GSTM1-active genotype (P = 0.026). Furthermore, patients with ccRCC with GSTM1-active genotype had significantly higher hazard ratio (P<0.05), in analyzed regression models, compared with the carriers of GSTM1-null genotype. Finally, the presence of GSTM1:ASK1 protein-protein interaction was found in all RCC tissue samples studied. CONCLUSIONS: Carriers of GSTM1-null and GSTP1-variant genotypes are in increased risk of RCC development. On the contrary, GSTM1-null genotype is associated with favorable postoperative prognosis in ccRCC. The possible molecular mechanism underlying the role of GSTM1 protein in RCC progression might be the presence of GSTM1:ASK1 protein-protein interaction. Hence, determination of GSTM1-genotype might serve as a valuable indicator in both RCC risk assessment and postoperative prognosis.