Digital morphology network for effective teaching of cytology, histology and histopathology for medical and biology curriculum. VM3.0 Erasmus+ project.

INTRODUCTION: Digital microscopy transformation, the basis for the virtual microscopy applications, is a challenge but also a requirement in modern Medical Education. This paper presents the scope, background, methods, and results of the project "Digital Transformation of Histology and Histopathology by Virtual Microscopy (VM) for an Innovative Medical School Curriculum", VM3.0, funded by the European Union under the Erasmus+ framework (ref.no.2022-1-RO01-KA220-HED-000089017). The project was initiated at Grigore T. Popa University of Medicine and Pharmacy, Iași, Romania, with the support of Euroed Foundation, Iași, and cooperation of University partners from Gdansk (Poland), Plovdiv (Bulgaria), Alicante (Spain), and Patras (Greece) aimed to implement digital histology and histopathology teaching in a common network. MATERIALS AND METHODS: The backbone of the project was the development of a Digital Slide Platform based on the scans of histological slides collected from all the partners of the participating universities and the creation of a simple and fast digital/internet communication tool that could be used to improve histology and histopathology teaching of medical and natural sciences students. The construction of a Virtual Microscopy Library (VML) has been based on the acquisition of whole scans of high-quality histological slides stained by hematoxylin and eosin (H&E) and other classical staining methods and description of the details in English as well as respective languages of the project's partners. The VML can be used for different approches, both for students' instruction in classes as well as for individual students' work and self-testing. Universities from other countries could use the modal structure of the developed VML system on the condition that more slides are provided and the implementation of national language(s) is implemented. CONCLUSIONS: The combined efforts of all university partners allowed to establish the dynamic low-cost virtual microscopy educational system. The VM system could help unify the standards of cytology, histology, and histopathology teaching in a quest for the digital transformation of the European educational system.

The immunoreactivity of GLI1 and VEGFA is a potential prognostic factor in kidney renal clear cell carcinoma.

Kidney renal clear cell carcinoma (KIRC) is the most common type of kidney cancer and its pathogenesis is strongly associated with VHL-HIF-VEGF signaling. SHH ligand is the upstream SHH pathway regulator, while GLI1 is its major effector that stimulates as a transcription factor, i.a. expression of VEGFA gene. The aim of present study was to assess the prognostic significance of SHH, GLI1 and VEGFA immunoreactivity in KIRC tissues. The analysis included paired tumor and normal samples from 34 patients with KIRC. The immunoreactivity of SHH, GLI1 and VEGFA proteins was determined by immunohistochemical (IHC) renal tissues staining. The IHC staining results were assessed using the immunoreactive score (IRS) method which takes into account the number of cells showing a positive reaction and the intensity of the reaction. Increased GLI1 protein immunoreactivity was observed in KIRC tissues, especially in early-stage tumors, according to the TNM classification. Elevated expression of the VEGFA protein was noted primarily in high-grade KIRC samples according to the Fuhrman/WHO/ISUP scale. Moreover, a directly proportional correlation was observed between SHH and VEGFA immunoreactivity in TNM 3 + 4 and Fuhrman/ISUP/WHO 3 + 4 tumor tissues as well as in samples of patients with shorter survival. We also observed an association between shorter patient survival as well as increased and decreased immunoreactivity, of the VEGFA and GLI1, respectively. The aforementioned findings suggest that the expression pattern of SHH, GLI1 and VEGFA demonstrates prognostic potential in KIRC.

Pro-Inflammatory Cytokine Gene Expression in Penile Cancer: Preliminary Studies.

Background and Objectives: Penile cancer is a rare neoplasm in developed countries with an incidence of 0.8/100,000 per male inhabitant. Despite the development of personalized medicine and multimodal treatment, the outcome of penile cancer treatment is insufficient. Our study aimed to assess the levels of pro-inflammatory cytokines' mRNA such as interleukin 1-A (encoded by IL1A gene, alias IL-1A), interleukin 1-B (IL1B, IL-1B), interleukin 1 receptor antagonist (IL1RN, IL-1RN), interleukin 6 (IL6, IL-6), transforming growth factor ß1 (TGFB1, TGFß-1), and Interferon-gamma (INFG, INF-γ) in penile cancer tissue and associate them with tumor progression and patient survival. Material and Methods: Skin biopsies from patients suffering from penile cancer (n = 6) and unchanged foreskin from 13 healthy adult males undergoing circumcision due to a short frenulum were obtained. Pro-inflammatory cytokine mRNA levels were quantified through qPCR. Results: We observed higher expression of pro-inflammatory cytokine genes (IL-1A, IL-1B, IL-6, INF-γ, TGF-ß) in penile cancer tissue. The average follow-up period was 48 months (range: 38-54 months), during which only one penile tumor progression was observed However, this was without association with the nature of tumor (patient refused radical treatment). Conclusions: This is the first study to show increased expression of cytokines such as IL-1A, IL-1B, IL-6, INF-γ, and TGF-ß in penile cancer with positive correlation between TNM staging and INF-γ levels in tumor samples (rs = 0.672, p = 0.045), which may be associated with the immunosuppressive role of the tumor environment.

Age-related effects of fenofibrate on the hepatic expression of sirtuin 1, sirtuin 3, and lipid metabolism-related genes.

BACKGROUND: Sirtuin 1 (Sirt1) and sirtuin 3 (Sirt3) participate in the regulation of lipid metabolism. Our aim was to investigate the effects of the hypolipemic drug fenofibrate (FN) on hepatic Sirt1 and Sirt3 expression, in relation to the expression of lipid metabolism-related genes and in the context of aging. METHODS AND RESULTS: Young and old male Wistar rats were fed standard chow or supplemented with 0.1% or 0.5% FN for 30 days (n=7-10 in each group). In young rats, 0.1% FN did not affect Sirt1 expression, however, 0.5% FN decreased Sirt1 and both doses reduced Sirt3 protein levels. In old rats, 0.5% FN decreased hepatic Sirt1 mRNA and both doses reduced Sirt1 protein levels, but not Sirt3 expression. Although hepatic Pparα protein levels did not change, FN treatment of young rats induced Cpt1b expression, whereas Lcad, Acox1, Pmp70, and Hmgcs2 expression increased only after 0.1% FN, and Fas2 expression decreased after 0.5% FN. In the liver of old rats, both doses increased Cpt1b and Lcad expression. Only 0.1% FN increased Pmp70 and Hmgcs2 expression, and only 0.5% FN increased Acox1 and Fas2 mRNA levels. CONCLUSIONS: Treatment with fenofibrate at low or high doses may downregulate the expression of Sirt1 and Sirt3 proteins in the rat liver. The dosage of FN affects molecular changes, and aging alters the response to 0.5% FN.

Altered mRNA Expression of NFKB1 and NFKB2 Genes in Penile Lichen Sclerosus, Penile Cancer and Zoon Balanitis.

BACKGROUND: The nuclear factor-κB transcription factors 1 and 2 (NFKB1 and NFKB2) are key components of the NF-κB pathway, which responds to inflammatory signals. Since the NFKB1/2 factors are activated via different inflammatory molecules, we aimed to check their expression levels in penile cancer (PC), penile dermatoses: lichen sclerosus (PLS) and zoon balanitis (ZB). METHODS: Skin biopsies from altered and healthy looking foreskin were obtained from 59 (49 LS; early PLS: 13, moderate PLS: 32, severe PLS: 4; 6 PC; 4 ZB) and unchanged foreskin from 13 healthy control adult males undergoing circumcision. NFKB1/2 mRNA levels were quantified by qPCR. RESULTS: The highest levels of NFKB1 and NFKB2 were observed in PC, ca. 22 and 3.5 times higher than in control, respectively. NFKB1 expression was correlated with PLS progression (rs = 0.667) and was ca. 20 times higher in advanced PLS than in controls and early PLS. Occurrence of micro-incontinence was associated with elevated NFKB1 levels in PLS. CONCLUSION: This is the first study regarding gene profiles of NFKB1/2 in PC and penile dermatoses. New drugs targeting modulation of canonical-activated NF-κB pathway should be studied and introduced to the treatment of PLS and PC apart from other treatments.

Aging and short-term calorie restriction differently affect the cardiac and skeletal muscle expression of genes regulating energy substrate utilization in male rats.

Aging affects the energy metabolism differently in the cardiac and skeletal muscles. The study aim was to assess the effects of short-term calorie restriction (SCR) and refeeding on the expression of genes involved in the control of cardiac and skeletal muscle energy metabolism in old vs. young male rats. Young (4 mo) and old (24 mo) rats were subjected to 60% SCR for 30 days, and refed ad libitum for 2 or 4 days. In the cardiac (CM) and skeletal muscles (SM) we compared the gene expression (qPCR) of carnitine palmitoyltransferase-I (Cpt-I), peroxisome proliferator-activated receptor beta/delta (Ppar-ß/δ), glucose transporter 4 (Glut4), peroxisome proliferator-activated receptor-γ coactivator-1α (Pgc-1α), and sirtuin 3 (Sirt3). In CM, aging increased Cpt-I expression but did not affect the other genes. In SM, Cpt-I, Glut4, Pgc-1α, and Sirt3 mRNA levels were lower in old than young rats. In CM of only young rats SCR increased Cpt-I expression which remained elevated after refeeding. Upon SCR, the expression of Ppar-ß/δ, Glut4, Pgc-1α, and Sirt3 in CM increased in young but not old rats, and refeeding re-established control levels. In SM of young rats SCR increased Ppar-ß/δ and Pgc-1α, and decreased Sirt3 expression, whereas refeeding generally decreased these mRNA levels. In SM of old rats SCR decreased only Pgc-1α expression. The adaptive response to SCR and subsequent refeeding is muscle tissue-specific and differs in young and old male rats. SCR appears to increase the efficiency of glucose and fatty acid utilization in the cardiac muscle of young, but not old male rats.

Expression levels of sonic hedgehog pathway genes and their targets are upregulated in early clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive subtype of kidney cancer, with high mortality rates worldwide. The sonic hedgehog (SHH) molecular cascade is altered in various malignancies in tumorigenesis, and several SHH pathway inhibitors have been considered as potential anticancer drugs. The aim of the present study was to determine the expression profile of SHH signaling components and their target genes in ccRCC. Additionally, the present study examined the effects of SHH pathway inhibitory drugs (RU­SKI43, cyclopamine and GLI­antagonist 61) on cell viability, cell cycle progression, expression levels of SHH target genes and migration ability in 786­O, ACHN and HK2 cells. The study also included paired tumor and normal samples from 62 patients with ccRCC. The mRNA levels in clinical samples and cell lines were measured via reverse transcription­quantitative PCR. Cell viability was examined using a sulforhodamine B assay. Flow cytometry was used to investigate cell cycle progression and the migratory rate of cells was assessed using a wound healing assay. High mRNA levels of SHH, smoothened (SMO), glioma­associated zinc finger protein (GLI)1­3, BCL2 apoptosis regulator (BCL2), MYC proto­oncogene (MYC), vascular endothelial growth factor A (VEGFA) and cyclin D1 (CCND1) were observed in the tumor tissues, especially in early ccRCC, according to the TNM stage or World Health Organization/International Society of Urological Pathology (ISUP) grade. High expression levels of VEGFA, as well as low CCND1 mRNA expression, were associated with short overall survival, and increased VEGFA expression was an independent prognostic factor of a poor outcome in patients with advanced ISUP grade (Cox hazard ratio test). Cyclopamine treatment was found to arrest 786­O cells in the G2/M phase and decreased the expression levels of GLI1, BCL2, VEGFA and CCND1. RU­SKI43 inhibited cell migration and decreased the expression levels of BCL2, MYC and CCND1 in ACHN cells. Overall, the results of the present study suggested that SHH signaling may be involved in the early development of ccRCC, and the expression levels of CCND1 and VEGFA may serve as prognostic factors of this disease. Cyclopamine and RU­SKI43 appear to be potential anti­renal cell carcinoma drugs; however, this hypothesis requires verification by further in vivo studies.

Sonic Hedgehog signaling pathway in gynecological and genitourinary cancer (Review).

Cancers of the urinary tract, as well as those of the female and male reproductive systems, account for a large percentage of malignancies worldwide. Mortality is frequently affected by late diagnosis or therapeutic difficulties. The Sonic Hedgehog (SHH) pathway is an evolutionary conserved molecular cascade, which is mainly associated with the development of the central nervous system in fetal life. The present review aimed to provide an in­depth summary of the SHH signaling pathway, including the characterization of its major components, the mechanism of its upstream regulation and non­canonical activation, as well as its interactions with other cellular pathways. In addition, the three possible mechanisms of the cellular SHH cascade in cancer tissue are discussed. The aim of the present review was to summarize significant findings with regards to the expression of the SHH pathway components in kidney, bladder, ovarian, cervical and prostate cancer. Reports associated with common deficits and de­regulations of the SHH pathway were summarized, despite the differences in molecular and histological patterns among these malignancies. However, currently, neither are SHH pathway elements included in panels of prognostic/therapeutic molecular patterns in any of the discussed cancers, nor have the drugs targeting SMO or GLIs been approved for therapy. The findings of the present review may support future studies on the treatment of and/or molecular targets for gynecological and genitourinary cancers.

The protective impact of Trans-Cinnamaldehyde (TCA) against the IL-1b induced inflammation in in vitro osteoarthritis model by regulating PI3K/AKT pathways.

INTRODUCTION: Osteoarthritis (OA) is a severe joint degeneration disease in elderly people described by the advanced degradation of articular cartilage, which ultimately leads to chronic pain. Trans-cinnamaldehyde (TCA) exerted its anti-inflammatory function in numerous disease syndromes; however, its role in the pathogenesis of OA remains unknown. The current research aimed to explore the potential protective impact of TCA in the progression of osteoarthritis in vitro. MATERIAL AND METHODS: Human knee articular chondrocytes were treated with 10 ng/ml IL-1b alone for 24 h or in a combination in a pretreatment with TCA at different concentrations (2, 5, 10 µg/mL, 24 h). The viability and cell apoptosis were determined by CCK-8 assay and flow cytometry methods. The protein levels of IL-8, PGE2, and TNF-a and the levels of phosphorylated AKT and PI3K were evaluated using ELISA assay. Moreover, RT-qPCR was used to measure the relative mRNA expression of MMP-13, iNOS, COX-2, and ADAMTS-5 in IL-1b-induced chondrocytes. RESULTS: Our results revealed that the treatment with TCA had no effect on chondrocytes' proliferation and apoptosis. Moreover, the protein levels of IL-8, TNF-a, and PGE2 were considerably reduced in IL-1b-induced chondrocytes treated with different concentrations of TCA. Furthermore, the mRNA expression of MMP-13, iNOS, COX-2, and ADAMTS-5 and the phosphorylation of AKT and PI3K were markedly reduced in IL-1b-induced chondrocytes with the increase in the concentration of TCA. CONCLUSIONS: Trans-cinnamaldehyde inhibited the inflammation induced by IL-1b in chondrocytes through the PI3K/AKT pathway, which suggests that TCA might serve as a potential therapeutic agent for osteoarthritis treatment.

Fenofibrate impairs liver function and structure more pronounced in old than young rats.

INTRODUCTION: Since old animals are known to accumulate lipids in some organs, we compared effects of fenofibrate (FN) on systemic lipid metabolism, activity of liver marker enzymes and structure in young and old rats. MATERIAL AND METHODS: Young and old rats were fed chow supplemented with 0.1 % or 0.5 % FN. After 30 days, intraperitoneal glucose tolerance test (IPGTT) was performed, and blood and liver samples were collected. RESULTS: In young rats, 0.1 % FN, but not 0.5 % FN, decreased serum Chol by 74 %, and did not affect TG levels at either doses. In old rats, 0.5 % FN, but not 0.1 % FN, decreased Chol and TG level by 56 % and 49 %, respectively. In young rats, 0.1 % and 0.5 % FN increased serum activity of ALP by 227 % and 260 %, respectively, and did not affect AST and ALT activities. In old rats, only 0.5 % FN increased serum ALP activity by 150 %, respectively. In old rats, neither dose of FN affected serum AST activity, and only 0.5 % FN increased serum ALT activity by 200 %. The histological examination of liver structure revealed that both doses of FN impaired lobular architecture, expansion of bile canaliculi, and degeneration of parenchymal cells with the presence of cells containing fat droplets; administration of FN increased area occupied by collagen fibers. CONCLUSIONS: Although 0.5 % FN decreased serum Chol concentration, it increased serum ALP activity and impaired liver structure in both in both age groups of rats. Thus, FN treatment should be under the control of liver function, especially in older patients.

Expression of HTRA Genes and Its Association with Microsatellite Instability and Survival of Patients with Colorectal Cancer.

HtrA proteases regulate cellular homeostasis and cell death. Their dysfunctions have been correlated with oncogenesis and response to therapeutic treatment. We investigated the relation between HtrA1-3 expression and clinicopathological, and survival data, as well as the microsatellite status of tumors. Sixty-five colorectal cancer patients were included in the study. The expression of HTRA1-3 was estimated at the mRNA and protein levels by quantitative PCR and immunoblotting. Microsatellite status was determined by high-resolution-melting PCR. We found that the HTRA1 mRNA level was higher in colorectal cancer tissue as compared to the unchanged mucosa, specifically in primary lesions of metastasizing cancer. The levels of HtrA1 and HtrA2 proteins were reduced in tumor tissue when compared to unchanged mucosa, specifically in primary lesions of metastasizing disease. Moreover, a decrease in HTRA1 and HTRA2 transcripts' levels in cancers with a high level of microsatellite instability compared to microsatellite stable ones has been observed. A low level of HtrA1 or/and HtrA2 in cancer tissue correlated with poorer patient survival. The expression of HTRA1 and HTRA2 changes during colorectal carcinogenesis and microsatellite instability may be, at least partially, associated with these changes. The alterations in the HTRA1/2 genes' expression are connected with metastatic potential of colorectal cancer and may affect patient survival.

Expression of the Sonic Hedgehog pathway components in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common and the most aggressive histopathological subtype of kidney cancer, with patients exhibiting high mortality rates for metastatic tumors. The Sonic Hedgehog (SHH) pathway serves a crucial role in embryonic development. The abnormal activity of SHH signaling is observed in a broad range of malignancies. However, its role in ccRCC is still undetermined. The aim of the present study was to assess the expression of the SHH pathway genes in ccRCC. Neoplastic and morphologically unchanged kidney tissues were obtained during radical nephrectomy from 37 patients with ccRCC. The SHH, PTCH1, SMO and GLI1 mRNA levels were assessed using the reverse transcription-quantitative PCR. Western blot analysis was used to assess the full-length and C-terminal SHH protein level. The mRNA levels of SHH, SMO and GLI1 were approximately 2-, 2,5- and 7-fold higher in ccRCC tissue compared with control kidney tissue, respectively. Correlational analysis between the mRNA levels of SHH pathway genes and patients' clinicopathological factors revealed decreased and increased mRNA levels of PTCH1 and SMO respectively, in tumor samples derived from older patients (age >62). Furthermore, the level of C-terminal SHH protein in ccRCC samples was significantly lower in a group of males compared with females. No correlation was exhibited between molecular data and patient survival. Western blot analysis indicated a ~3-fold higher level of SHH full-length protein, and a 4-fold lower level of the C-terminal SHH protein domain, in ccRCC tumor tissues compared with normal kidney samples. The current study indicated an involvement of the SHH pathway in ccRCC development.

In vitro evaluation of electroporated gold nanoparticles and extremely-low frequency electromagnetic field anticancer activity against Hep-2 laryngeal cancer cells.

INTRODUCTION: The extremely-low frequency electromagnetic field (ELFEMF) has been proposed for use in cancer therapy since it was found that magnetic waves interfere with many biological processes. Gold nanoparticles (Au-NPs) have been widely used for drug delivery during cancer in vitro studies due to their low cytotoxity and high biocompatibility. The electroporation of cancer cells in a presence of Au-NPs (EP Au-NPs) can induce cell apoptosis, alterations of cell cycle profile and morphological changes. The impact of ELFEMF and EP Au-NPs on morphology, cell cycle and activation of apoptosis-associated genes on Hep-2 laryngeal cancer cell line has not been studied yet. MATERIALS AND METHODS: ELFEMF on Hep-2 cells were carried out using four different conditions: 25/50 mT at 15/30 min, while Au-NPs were used as direct contact (DC) or with electroporation (EP, 10 pulses at 200V, equal time intervals of 4 sec). MTT assay was used to check the toxicity of DC Au-NPs. Expression of CASP3, P53, BAX and BCL2 genes was quantified using qPCR. Cell cycle was analyzed by flow cytometry. Hematoxylin and eosin (HE) staining was used to observe cell morphology. RESULTS: Calculated IC50 of DC Au-NPs 24.36 µM (4.79 µg/ml) and such concentration was used for further DC and EP AuNPs experiments. The up-regulation of pro-apoptotic genes (CASP3, P53, BAX) and decreased expression of BCL2, respectively, was observed for all analyzed conditions with the highest differences for EP AuNPs and ELFEMF 50 mT/30 min in comparison to control cells. The highest content of cells arrested in G2/M phase was observed in ELFEMF-treated cells for 30 min both at 25 or 50 mT, while the cells treated with EP AuNPs or ELFEMF 50 mT/15 min showed highest ratios of apoptotic cells. HE staining of electroporated cells and cells exposed to ELFEMF's low and higher frequencies for different times showed nuclear pleomorphic cells. Numerous apoptotic bodies were observed in the irregular cell membrane of neoplastic and necrotic cells with mixed euchromatin and heterochromatin. CONCLUSIONS: Our observations indicate that treatment of Hep-2 laryngeal cancer cells with ELFEMF for 30 min at 25-50 mT and EP Au-NPs can cause cell damage inducing apoptosis and cell cycle arrest.

Prognostic significance of VHL, HIF1A, HIF2A, VEGFA and p53 expression in patients with clear­cell renal cell carcinoma treated with sunitinib as first­line treatment.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell cancer, characterized by the highest mortality rate among other RCC subtypes due to the occurrence of metastasis and drug resistance following surgery. The Von Hippel­Lindau tumor suppressor (VHL)­hypoxia­inducible factor 1 subunit α (HIF1A)/hypoxia­inducible factor 2α (HIF2A)­vascular endothelial growth factor A (VEGFA) protein axis is involved in the development and progression of ccRCC, whereas sunitinib, a tyrosine kinase inhibitor, blocks the binding of VEGFA to its receptor. The aim of the present study was to examine the possible association of the gene expression of VHL, HIF1A, HIF2A, VEGFA and tumor protein P53 (P53) in cancer tissue with the outcome of ccRCC patients who were treated with sunitinib as first­line therapy following nephrectomy. A total of 36 ccRCC patients were enrolled, 11 of whom were administered sunitinib post­operatively. Tumor and control samples were collected, and mRNA and protein levels were assessed by reverse transcription­quantitative polymerase chain reaction and western blot analysis, respectively. High mRNA and protein expression levels of HIF2A and VEGFA were found to be associated with shorter overall survival (OS) and progression­free survival (PFS) rates, as well as with unfavorable risk factors of cancer recurrence and mortality. Resistance to sunitinib was also observed; the OS and PFS rates were shorter (median OS and PFS: 12 and 6 months, respectively, vs. undetermined). Sunitinib resistance was associated with high HIF2A and VEGFA protein levels (b=0.57 and b=0.69 for OS and PFS, respectively; P<0.001). Taken together, the findings of this study suggest that the protein levels of HIF2A and VEGFA in tumor tissue may serve as independent prognostic factors in ccRCC. ccRCC patients with increased intratumoral HIF2A and VEGFA protein levels, and unaltered VHL protein levels, are not likely to benefit from sunitinib treatment following nephrectomy; however, this hypothesis requires verification by large­scale replication studies.

WNT5A gene and protein expression in endometrial cancer.

INTRODUCTION: WNT5A (Wnt family member 5A) belongs to the WNT family of secreted signaling glycoproteins that play essential role in developmental, physiological and pathological processes. WNT5A was shown to take part in carcinogenesis process playing both oncogenic and suppressor functions in various types of human malignancies. This study aimed to assess the expression of the WNT5A gene at the mRNA and protein levels in the specimens derived from endometrial cancer (EC) or unchanged control endometrium. The associations between the WNT5A expression levels and clinicopathological characteristics and survival of EC patients were evaluated. MATERIALS AND METHODS: Total RNA was isolated in order to assess the relative amounts of WNT5A mRNA by quantitative polymerase chain reaction (QPCR) in samples of unchanged endometrial control (n = 8) and tumor samples of EC patients (n = 28). Immunohistochemistry (IHC) was used to determine the presence of WNT5A protein in the sections of formalin-fixed, paraffin-embedded tissue specimens derived from unchanged endome-trial controls (n = 6) and EC tumors (n = 19). Significance of differences in WNT5A expression levels between the studied groups of EC patients and correlations between the WNT5A and demographic data, pathological features, hematological parameters and overall survival of the patients were evaluated by statistical analysis. RESULTS: The level of WNT5A mRNA was decreased in EC in comparison to unchanged endometrium. WNT5A expression was associated with primary tumor invasion status exhibiting reduced level of transcripts in EC that involved organs beyond the uterus when compared to the uterus-confined cancers. WNT5A immunoreactivity was visualized in the cytoplasm and nuclei of EC cells as well as in the luminal and glandular epithelial cells of unchanged endometrium. WNT5A mRNA expression levels correlated negatively with cytoplasmic, and positively with nuclear immunoreactivity of the WNT5A protein in the EC cells. In addition, the relationships between blood leucocyte count (in particular granulocytes and lymphocytes) of patients with EC and their WNT5A mRNA and protein expression levels were established. A positive correlation between the nuclear immunoexpression of WNT5A protein in the cancer cells in cell nuclei and mean platelet volume in blood was also found. CONCLUSIONS: The results of the first study of WNT5A expression at the transcript and protein levels indicate that it could be considered as a potential marker of molecular changes that take place during EC development.

Distinct Expression Patterns of Two Tumor Necrosis Factor Superfamily Member 15 Gene Isoforms in Human Colon Cancer.

BACKGROUND: Tumor necrosis factor superfamily member 15 (TNFSF15) gene is involved in development of several cancers. It encodes two proteins: tumor necrosis factor ligand-related molecule 1A (TL1A) and vascular endothelial growth inhibitor 192 (VEGI-192). The main receptor for TL1A is death receptor 3 (DR3). AIMS: We investigated expression of TL1A, VEGI-192, and DR3 transcripts in different stages of colon cancer and compared them with survival of patients. We also aimed to reveal possible effects of microsatellite instability (MSI) and selected TNFSF15 single-nucleotide polymorphisms (SNPs) on expression of this gene. METHODS: Forty-five healthy individuals and 95 colon cancer patients were included in the study. Expression of VEGI-192, TL1A, and DR3 was measured by quantitative PCR. SNP and MSI analyses were performed on DNA isolated from normal or cancer tissue. RESULTS: Expression of VEGI-192 and TL1A was elevated in colon cancer, although the level of VEGI-192 decreased, while the level of TL1A increased with the progression of cancer. Patients with low expression of TL1A and/or high expression of VEGI-192 in tumor-transformed tissue showed longer survival. DR3 expression was decreased in the cancer, but it did not change with the tumor progression. Alleles T of rs6478108 and G of rs6478109 SNPs were associated with elevated expression of the TNFSF15 gene. There was no relation between the MSI status and TNFSF15 expression levels. CONCLUSIONS: Expression of the TNFSF15 gene isoforms was associated with the progression of colon cancer. Levels of TL1A and VEGI-192 transcripts can be considered as independent prognostic factors for colon cancer.

Presence of Chlamydophila pneumoniae DNA in blood cells is a frequent event in patients with the late stage of primary cutaneous lymphomas and with atopic dermatitis.

INTRODUCTION: Microbial infection and associated super antigens have been implicated in the pathogenesis of cutaneous T-cell lymphoma (CTCL), and many patients die from complicating bacterial infections. It has been postulated that Chlamydophila pneumoniae (C. pneumoniae) infection may be involved in the pathogenesis of Mycosis fungoides (MF) but published data are limited and controversial. AIM: To analyze the frequency of (C. pneumoniae) DNA presence in blood samples of lymphoma cases. MATERIAL AND METHODS: Using Q-PCR method we analyzed the presence of DNA in the blood samples obtained from 57 patients with CTCL (55 - mycosis fungoides (MF)/Sézary syndrome (SS), one primary cutaneous anaplastic large cell lymphoma (CD30+) and one NKT cell lymphoma) and 3 patients with cutaneous B-cell lymphomas, and 120 individuals from control groups (40 patients with psoriasis, 40 patients with atopic dermatitis and 40 healthy controls). RESULTS: Chlamydophila pneumoniae DNA was identified in 13 of 55 cases in the MF/SS group (23.6%), in 1 patient with CD30+ large cell lymphoma and in 1 of 3 patients with B-cell lymphoma. The presence of C. pneumoniae was confirmed in 1 of 40 psoriatic patients (2.5%), in 5 of 40 patients with atopic dermatitis (12.5%) and in none of 40 healthy individuals. Presence of C. pneumoniae DNA in MF patients was strongly associated with disease progression; rs = 0.756; p = 0.0123 for groups IA → IVB, and was noted more frequently in advanced (III + IV) stages than in early (I-II) stages (p = 0.0139). There are no differences in the mean age of MF/SS patients with and without infection. CONCLUSIONS: The presence of C. pneumoniae DNA in the blood cells is a frequent event in late stages of MF/SS and may be explained by Th2 shift and suppression of the immune system during the course of the disease.

FasR and FasL in colorectal cancer.

Colorectal cancer (CRC) is one of the most common solid organ cancers prevalent worldwide causing, in spite of advancing therapeutic methodology, high rate of patient mortality, especially due to metastasis development. The cancer stem cell (CSC) theory of tumor growth indicates that CSCs within the tumor mass have great capacity to initiate and sustain tumor growth. Following the suggestion that Fas signaling can be engaged in apoptosis, tumor maintenance, senescence or DICE (death induced by CD95 or CD95L elimination), the attempts to broaden the knowledge concerning the relationships between CSCs features and FasR/FasL appeared to be necessary. The most important advantage of our study was the simultaneously analysis of CSCs from commonly used CRC lines (HCT116 and HT29) and tumor fragments collected from CRC patients. Moreover, the sphere-promoting expansion of CRC lines brought a specific three-dimensional specific environment for CSC exploration. We further investigated the function of Fas signaling in CRC lines depending on the culture mode as we incubated HCT116 and HT29 cells with anti-FasR agonistic antibodies. It appeared to act in a line-dependent and culture mode-dependent manner and influenced some particular features of CSCs such as spherogenicity, proliferation and phenotype. Additionally, the analysis of mRNA level showed that disease progression is associated with significantly increased expression of FasR and/or FasL. In conclusion, our observation seems to confirm that spherical model of cancer lines is more reliable for some sophisticated analysis because of their greater resemblance to the CSCs from human CRC samples in comparison to commonly used adherent cells, at least according to aspects of their biology analyzed in this study. That can be extended to the resemblance of in vitro sphere forming conditions to the in vivo environment. However, the greatest difference concerns the level of apoptosis, thus, this issue require further experiments.

Overexpression of the YAP1 oncogene in clear cell renal cell carcinoma is associated with poor outcome.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of RCC (70-80%). Yes-associated protein 1 (YAP1) protein is a nuclear effector of the Hippo pathway and acts as a transcriptional co-activator of genes involved in the processes of growth and development of tissues. Hippo signaling, with its key kinases, MST2 and large tumor suppressor kinase 1 (LATS1), plays a significant role in the negative regulation of the amount and activity of YAP1 protein. Components of the Hippo pathway and YAP1 levels are frequently dysregulated in a variety of tumors, suggestive of their possible involvement in carcinogenesis. Our aim was to evaluate gene and protein expression profiles of YAP1, MST2 and LATS1 and the methylation status of MST2 and LATS1 promoters in ccRCC. mRNA levels of MST2, LATS1 and YAP1 genes were assessed in the tumor and matched normal kidney tissues of 86 patients, and in 12 samples of local metastases by quantitative PCR (qPCR). Proteins were semi-quantified in 58 patient samples by western blotting. Hypermethylation of LATS1 and MST2 promoters was measured by methylation­specific high­resolution-melting qPCR. We found that LATS1 promoter hypermethylation, decreased LATS1 mRNA/protein and increased YAP1 mRNA/protein levels in tumor samples were associated with higher TNM and Fuhrman's stages and patient survival. Higher YAP1 mRNA levels were associated with poor outcome (HR=4.03, p=0.036). No changes in MST2 (promoter/mRNA/protein) were found. We propose that deregulation of LATS1 and YAP1 expression is associated with ccRCC progression and poor patient survival. Measurement of YAP1 mRNA levels in paired tumor-normal kidney tissue samples may serve as a new prognostic factor in ccRCC.

Expression and Prognostic Significance of EP300, TP53 and BAX in Clear Cell Renal Cell Carcinoma.

BACKGROUND: Histone acetyltransferase E1A-binding protein p300 (EP300), tumor protein p53 (TP53) and B-cell lymphoma-2-associated X protein (BAX) contribute to the regulation of the cell cycle and apoptosis, cellular processes that are often impaired in cancer cells. The aim of this study was to determine the expression levels of EP300, TP53 and BAX genes and their respective proteins in clear cell renal cell carcinoma (ccRCC) and evaluate the value of these factors as prognostic factors. MATERIALS AND METHODS: EP300, TP53 and BAX expression at the transcript and protein levels were determined by quantitative polymerase-chain reaction (QPCR) and immunohistochemistry (IHC) in paired tumor and kidney specimens from 31 patients with ccRCC. RESULTS: Levels of EP300, TP53 and BAX transcripts were found increased in tumor tissues. Immunoreactivity for TP53 was elevated in cancer cells when compared to unchanged kidney, while EP300 and BAX immunoexpression in ccRCC did not differ from that of normal renal tissue. Immunoreactivity for TP53 was positively associated with larger tumor size. In contrast, stronger BAX immunoexpression correlated with smaller tumor diameters. The average immunoreactivity for BAX was higher in localized, kidney-confined tumor than in advanced/recurrent tumors. None of the analyzed transcripts or proteins correlated with the overall survival of patients. CONCLUSION: Although TP53 and BAX immunoreactivity levels were associated with some clinicopathological parameters of the patients, the expression of EP300, TP53 and BAX did not reveal any prognostic significance in ccRCC.