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Background MicroRNAs (miRNAs) are non-coding regulatory molecules 18-25 nucleotides in length that act as post-transcriptional regulators of gene expression. MiRNAs affect various biological processes including carcinogenesis. Deregulation of miRNAa expression has been described in a variety of tumors including papillary thyroid carcinoma (PTC). The aim of the present study was to investigate the role of selected miRNAs in PTC and find associations between miRNA expression and the BRAF (V600E) mutation. Materials and methods The study group comprised a total of 62 patients with surgically treated PTC. The control group consisted of 30 patients with nodular goitre that were surgically treated in the same time period. The expression status of miR-146b, miR-181a, miR-187, miR-221 and miR-222 was determined using quantitative real-time PCR. BRAF mutation analysis was performed by PCR with reverse hybridization. Results MiR-146b, miR-181a, miR-187, miR-221 and miR-222 were up-regulated in PTC compared to normal thyroid gland tissue of the same patient. MiR-146b, miR-187, miR-221 and miR-222 were also up-regulated in PTC compared to nodular goitre. The recurrent tumors were statistically significantly associated with up-regulation of miR-221. The mutation V600E of BRAF gene was significantly associated with up-regulation of miR-146b and with down-regulation of miR-187. Conclusion Over-expression of selected miRNAs in PTC compared to normal thyroid gland tissue and nodular goitre was found. Moreover, miR-221 may serve as a prognostic marker as its over-expression was significantly associated with recurrent tumors.

Background: Sjögren’s syndrome patients, like other patients with autoimmune disorders, display dysregulation in the function of their immune system. Fas and Fas Ligand (FasL) belong among dysregulated proteins. Although they have been long studied, there are still several unknown aspects of biological importance of Fas and FasL. Methods: In patients with Sjögren’s syndrome and age-matched healthy individuals, we studied Fas and FasL on IL-2Rα+ cells and in serum using flow cytometry and enzyme-linked immunosorbent assay, respectively. We also measured the expression of Bcl-2 and Bax by reverse transcription quantitative real-time PCR (RT-qPCR) and percentage of apoptotic and dead cells using Annexin V and 7-AAD staining in lymphocytes. Results: FasL was increased in patients’ T and B cells and serum while Fas was increased in patients’ monocytes, T and B cells. No signs of increased apoptosis were found. We suspect an effect of non-steroidal anti-inflammatory therapy on the percentage of Fas+ B cells which decreased with the therapy in our patients. In the healthy individuals, there was a noticeable pattern in the expression of Fas which was mutually correlated between populations of mononuclear cells; this correlation was absent in the patients with Sjögren’s syndrome. Conclusions: The increased expression of Fas in IL-2Rα+ lymphocytes does not increase the apoptosis of lymphocytes. These lymphocytes increase the expression of FasL and may induce apoptosis in other cells. The lack of correlation for the expression of FasL in Sjögren’s syndrome patients may suggest a loss of regulation.

Background The lack of effective biomarkers for the screening and early detection of ovarian cancer (OC) is one of the most pressing problems in oncogynecology. Because epigenetic alterations occur early in the cancer development, they provide great potential to serve as such biomarkers. In our study, we investigated a potential of a four-gene methylation panel (including CDH13, HNF1B, PCDH17 and GATA4 genes) for the early detection of high-grade serous ovarian carcinoma (HGSOC). Methods For methylation detection we used methylation sensitive high-resolution melting analysis and real-time methylation specific analysis. We also investigated the relation between gene hypermethylation and gene relative expression using the 2−ΔΔCt method. Results The sensitivity of the examined panel reached 88.5%. We were able to detect methylation in 85.7% (12/14) of early stage tumors and in 89.4% (42/47) of late stage tumors. The total efficiency of the panel was 94.4% and negative predictive value reached 90.0%. The specificity and positive predictive value achieved 100% rates. Our results showed lower gene expression in the tumor samples in comparison to control samples. The more pronounced downregulation was measured in the group of samples with detected methylation. Conclusions In our study we designed the four-gene panel for HGSOC detection in ovarian tissue with 100% specificity and sensitivity of 88.5%. The next challenge is translation of the findings to the less invasive source for biomarker examination, such as plasma. Our results indicate that combination of examined genes deserve consideration for further testing in clinical molecular diagnosis of HGSOC.

Introduction: The amniotic fluid nicotinamide phosphoribosyltransferase (NAMPT) levels have not been compared among women with preterm prelabor rupture of membranes (PPROM) comorbid with intra-amni...

BACKGROUND Goeckerman therapy (GT) of psoriasis involves dermal application of crude coal tar containing polycyclic aromatic hydrocarbons (PAHs) and exposure to ultraviolet radiation (UVR). Little is known about GT influence on DNA epigenetics. OBJECTIVE The study aim was to discover epigenetic mechanisms altered by the exposure related to the GT of psoriasis. METHODS Observed group of patients with plaque psoriasis (n = 23) was treated by GT with 3 % CCT. Before and after GT, we analyzed the levels of benzo[a]pyrene-7,8-diol-9,10-epoxide-DNA adducts (BPDE-DNA), p53 protein in serum, 5-methylcytosine (5-mC, global DNA methylation), and methylation in selected CpG sites of p53 gene. RESULTS We found a significant increase in the levels of BPDE-DNA (p < 0.01) and serum levels of p53 protein (p < 0.01) after GT, and an insignificant decrease in the percentage of 5-mC in peripheral blood DNA. Methylation of p53 CpG sites was affected neither by psoriasis nor by GT. The study confirmed good effectiveness of GT (significantly reduced psoriasis area and severity index; p < 0.001). CONCLUSION Our findings indicate that there is a significantly increased genotoxic hazard related to the exposure of PAHs and UV radiation after GT of psoriasis. However, global DNA methylation and p53 gene methylation evade the effect of GT, as they remained unchanged (Tab. 4, Fig. 3, Ref. 50).

Epigenetic aberrations are well known to play an important role in carcinogenesis, and also have a great potential to serve as biomarkers in many types of cancers, including ovarian cancer in which sensitive and specific biomarkers and detection methods are critically needed. The aim of this study was to investigate methylation of cadherin genes CDH10, CDH13 and CDH18 in ovarian cancer tissue by comparison with control tissue. The study group consisted of 38 patients with ovarian cancer and 25 control patients. For detection of epigenetic events we used next generation sequencing, the most important data were confirmed using high-resolution melting analysis and real-time PCR. We observed significantly higher methylation in CDH13, sporadic methylation in CDH10 and loss of methylation in CDH18 in the ovarian cancer group compared with the control group. These observations suggest that changes in methylation of cadherin genes may be one of the major mechanisms associated with ovarian cancer progression. In addition, because of the high frequency of methylation of the CDH13 gene in the early stages of ovarian cancer, the analyzed CpG sites might be good targets for next study of potential ovarian cancer screening biomarkers.

Background: Epigenetic modifications have been recognized as an important mechanism underlying carcinoma progression. DNA methylation plays an important role in cancer biology and represents potentially heritable changes in gene expression that do not involve DNA sequence. The aim of this study was to investigate promoter methylation of selected genes in sinonasal carcinoma by comparison with noncancerous sinonasal tissue. Methods: To search for epigenetic events (methylation in 25 tumor suppressor genes) we used MS-MLPA (Methylation-specific multiplex ligation-dependent probe amplification) to compare methylation status of 59 formalin fixed, paraffin embedded tissue samples of sinonasal carcinomas with 18 control samples. The most important changes in methylation were confirmed using MSP (Methylation specific PCR). Detected alterations in methylation were compared with clinicopathological characteristics. Results: Using a 20% cut-off for methylation (MS-MLPA), we found significantly higher methylation in GATA5 (P=0.0005), THSB1 (P=0.0002) and PAX5 (P=0.03) genes in the sinonasal cancer group compared to the control group. Methylation in five or more genes was associated with impaired overall survival (P=0.017). Conclusion: These findings provide evidence that alterations in methylation profile may be one of the major mechanisms in sinonasal carcinogenesis. In addition, changes in methylation could potentially be used as prognostic factors of sinonasal carcinoma and may have implications for future individualized therapy based on epigenetic changes.

Background Aberrant DNA methylation of protocadherins (PCDHs) has been associated with development and progression of various types of cancer. It could represent possible direction in the search for critically needed tumor biomarkers for ovarian cancer. Objective To investigate methylation of δ2 group of non-clustered PCDHs in high-grade serous ovarian carcinoma (HGSOC) tissue in comparison with control tissue. Methods We used next-generation sequencing for detecting regions with the most altered methylation. For further confirmation of discovered alterations we used methylation-sensitive high-resolution melting analysis. Results PCDH17 methylation was detected in almost 70% of HGSOC patients without any methylation in the group of control samples and was found both in the late stage tumors as well as in the early stage ones. Other selected PCDHs did not show any relevant changes in methylation. Subsequent gene expression analysis of PCDH17 revealed decreased expression in all of the tumor samples in comparison to the control ones. Statistically significant negative correlation was found between methylation and levels of expression suggesting potentially methylation-based silencing. Conclusions Methylation of PCDH17 could play an important role in development and progression of HGSOC and has potential to become a target in the search for new clinical biomarkers.

Psoriatic lesions are characterized by hyperproliferation, aberrant differentiation of keratinocytes resistant to apoptosis and inflammation. miR-31 plays pro-proliferative, pro-differentiative and pro-inflammatory roles and modulates apoptosis in psoriatic keratinocytes. Endothelin-1 (ET-1) is produced by psoriatic keratinocytes and suppresses apoptosis. Inflammation increases the production of ET-1, which in turn leads to the chronic stimulation of keratinocyte proliferation. The aim of this study was to identify the putative link between two potential biomarkers (miR-31 and ET-1) in patients with psoriasis. The study design included experimental group (29 patients with psoriasis), and the control group (22 blood donors). The PASI score evaluated the state of the disease (median: 18.6; interquartile range 14.5-20.9). Both, the serum level of ET-1 and the whole blood level of miR-31 were significantly increased (p

Purpose: Our goal was to develop two-tier strategy based on in house-designed methylation specific-duplex polymerase chain reactions (MS-PCRs) that could serve as a relatively simple, cost effective, time efficient approach for molecular screening of imprinted regions on chromosomes 15 and 14. Patients and methods: Patients were referred to examination during infancy due to hypotonia and motor development delay. Duplex MS-PCRs were performed that enabled detection of methylated/unmethylated DNA in NDN and MEG3 CpG islands via plurality of detection channels on PCR instrument Rotor Gene 6000. Results and discussion: Both, copy number variations as well as methylation changes, were revealed by our in house-designed methodology by focusing NDN gene. No imprinting aberrations were yet discovered in MEG3 gene. Clinical features of the patients were compared. In agree with literature no typical facial features were observed in PWS patient with imprinting defect and AS UPD patient seems to have a relatively better development and language ability in comparison to deletional form of the disease. Conclusion: In conclusion we were able to establish new, throughput and robust diagnostic approach to PWS/AS.

The aim of this study was a detailed clinicopathological investigation of sinonasal NUT midline carcinoma (NMC), including analysis of DNA methylation and microRNA (miRNA) expression. Three (5%) cases of NMC were detected among 56 sinonasal carcinomas using immunohistochemical screening and confirmed by fluorescence in situ hybridization. The series comprised 2 males and 1 female, aged 46, 60, and 65 years. Two tumors arose in the nasal cavity and one in the maxillary sinus. The neoplasms were staged pT1, pT3, and pT4a (all cN0M0). All patients were treated by radical resection with adjuvant radiotherapy. Two patients died 3 and 8 months after operation, but one patient (pT1 stage; R0 resection) experienced no evidence of disease at 108 months. Microscopically, all tumors consisted of infiltrating nests of polygonal cells with vesicular nuclei, prominent nucleoli and basophilic cytoplasm. Abrupt keratinization was present in only one case. Immunohistochemically, there was a diffuse expression of cytokeratin (CK) cocktail, CK7, p40, p63, and SMARCB1/INI1. All NMCs tested negative for EBV and HPV infection. Two NMCs showed methylation of RASSF1 gene. All other genes (APC, ATM, BRCA1, BRCA2, CADM1, CASP8, CD44, CDH13, CDKN1B, CDKN2A, CDKN2B, CHFR, DAPK1, ESR1, FHIT, GSTP1, HIC1, KLLN, MLH1a, MLH1b, RARB, TIMP3, and VHL) were unmethylated. All NMCs showed upregulation of miR-9 and downregulation of miR-99a and miR-145 and two cases featured also upregulation of miR-21, miR-143, and miR-484. In summary, we described three cases of sinonasal NMCs with novel findings on DNA methylation and miRNA expression, which might be important for new therapeutic strategies in the future.

Psoriasis is a multifactorial chronic inflammatory disease. We aimed to examine blood levels of nucleosomes derived from apoptotic cells, nucleosomal cell-free DNA (cfDNA) and immune-inflammatory biomarkers tumor necrosis factor-α (TNF-α), C-reactive protein (CRP), and interleukin 6 (IL-6) in psoriatic subjects. The study included 28 patients with exacerbated psoriasis vulgaris and 22 controls. The clinical and laboratory investigations included the determination of PASI score, BMI, cfDNA (by real-time PCR), nucleosomes, TNF-α, CRP, and IL-6. The range of PASI score in psoriatic patients was 10-34 (median 19). In the patients, we found significantly elevated levels (p

Background Tumors occurring in the sinonasal area are characterized by unfavorable outcome due to difficult diagnosis, treatment, and prognosis of the disease corresponding with the anatomic complexity of the area. Methods We used quantitative real-time polymerase chain reaction (PCR) to compare relative expression of miR-21, miR-141, and miR-200c in 70 formalin-fixed, paraffin-embedded samples of sinonasal carcinoma tissue (majority of squamous cell carcinoma [SCC] samples) with 17 control samples of sinonasal tissue. Results Our data showed significant upregulation of miR-21 in sinonasal cancer tissue. Expression levels of miR-141 and miR-200c were below detectable levels in both sinonasal cancer samples and healthy tissue. Kaplan-Meier analysis with log-rank survival showed that patients with SCC with high expression of miR-21 (highest quartile) had impaired survival close to reaching statistical significance (P = .0630). Conclusion Our results suggest that miR-21 upregulation is involved in tumorigenesis of sinonasal carcinoma and that it is associated with poor prognosis. Thus, miR-21 could be used as a valuable prognostic biomarker.

Goeckerman therapy (GT) for psoriasis combines the therapeutic effect of crude coal tar (CCT) and ultraviolet radiation (UVR). CCT contains polycyclic aromatic hydrocarbons, some of which can form DNA adducts that may induce mutations and contribute to carcinogenesis. The aim of our work was to evaluate the relationship between concentrations of benzo[a]pyrene-7,8-diol-9,10-epoxide-DNA adducts (BPDE-DNA adducts) and rs4646903 (CYP1A1 gene), rs1048943 (CYP1A1), rs1056836 (CYP1B1), rs1051740 (EPHX1), rs2234922 (EPHX1) and rs8175347 (UGT1A1) polymorphic sites, and GSTM1 null polymorphism in 46 patients with chronic stable plaque psoriasis who underwent GT. The level of BPDE-DNA adducts was determined using the OxiSelect BPDE-DNA Adduct ELISA Kit. Polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis (rs4646903, rs1048943, rs1051740, and rs2234922), fragment analysis (rs8175347), real-time PCR (rs1056836), and digital droplet PCR polymorphism (GSTM1) were used. CYP1B1*1/*1 wild-type subjects and CYP1B1*3/*1 heterozygotes for rs1056836 formed significantly higher amounts of BPDE-DNA adducts than CYP1B1*3/*3 homozygotes (p=0.031 and p=0.005, respectively). Regarding rs1051740, individuals with EPHX1*3/*1 heterozygosity revealed fewer adducts than EPHX1*1/*1 wild-type subjects (p=0.026). Our data suggest that CYP1B1/EPHX1 genotyping could help to predict the risk of DNA damage and to optimize doses of coal tar and UVR exposure in psoriatic patients in whom GT was applied.

The paper presents a prospective cohort study. Observed group was formed of children with plaque psoriasis (n=19) treated by Goeckerman therapy (GT). The study describes adverse (side) effects associated with application of GT (combined exposure of 3% crude coal tar ointment and UV radiation). After GT we found significantly increased markers of oxidative stress (8-hydroxy-2′-deoxyguanosine, 8-hydroxyguanosine, and 8-hydroxyguanine), significantly increased levels of benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) DNA adducts (BPDE-DNA), and significantly increased levels of total number of chromosomal aberrations in peripheral lymphocytes. We found significant relationship between (1) time of UV exposure and total number of aberrated cells and (2) daily topical application of 3% crude coal tar ointment (% of body surface) and level of BPDE-DNA adducts. The findings indicated increased hazard of oxidative stress and genotoxic effects related to the treatment. However, it must be noted that the oxidized guanine species and BPDE-DNA adducts also reflect individual variations in metabolic enzyme activity (different extent of bioactivation of benzo[a]pyrene to BPDE) and overall efficiency of DNA/RNA repair system. The study confirmed good effectiveness of the GT (significantly decreased PASI score).

BACKGROUND Epigenetic changes are considered to be a frequent event during tumor development. Various methylation changes have been identified and show promise as potential cancer biomarkers. The aim of this study was to investigate promoter methylation of GATA4 and TP53 genes in endometrioid carcinoma of endometrium. METHODS To search for promoter methylation of GATA4 and TP53 genes we used methylation-specific PCR (MSP) to compare the methylation status of 54 patients with endometrioid carcinoma of endometrium and 18 patients with normal endometrial tissue. RESULTS In our study MSP revealed GATA4 promoter methylation in 44 of 54 in the carcinoma group (81.5%), and in none of the control group. No methylation was observed in TP53 gene. CONCLUSIONS In conclusion, our study showed that there is significantly higher methylation in GATA4 gene in the endometrial cancer group compared with samples of non-neoplastic endometrium. The finding suggests the importance of hypermethylation of this gene in endometrial carcinogenesis and could have implications for future diagnostic and therapeutic strategies for endometrial cancer based on epigenetic changes.

Aims. Ovarian cancer is the most lethal gynecological malignancy, with typically late diagnosis. Altered DNA methylation of tumor suppressor gene promoters probably plays a relevant role in ovarian carcinogenesis and frequently occurs as an early event in the development of different types of cancer including ovarian carcinoma. GATA4 methylation has been reported in a variety of human cancers. The aim of this study was to investigate promoter methylation of the GATA4 gene in ovarian cancer by comparison with that in normal ovarian tissue. Methods. To search for promoter methylation of the GATA4 gene we used MSP (methylation-specific PCR) to compare the methylation status in 67 tissue samples of ovarian cancer with that in 40 control samples. Results. In our study, methylation-specific PCR revealed GATA4 promoter methylation in 21 of 67 specimens with ovarian cancer (31.3%), and in none of the control ovarian tissue samples. Conclusion. These results confirm that methylation in the GATA4 promoter region could play an important role in ovarian carcinogenesis, and show new loci which are highly methylated only in ovarian cancer samples and which are associated predominantly with the endometrioid type of ovarian carcinoma.

Background. Epigenetic changes are considered to be a frequent event during tumor development. Hypermethylation of promoter CpG islands represents an alternative mechanism for inactivation of tumor suppressor genes, DNA repair genes, cell cycle regulators and transcription factors. The aim of this study was to investigate promoter methylation of specific genes in endometrial cancer by comparison with normal endometrial tissue. Materials and Methods. We used MS–MLPA (Methylation-specific Multiplex ligation-dependent probe amplification) to compare the methylation status of 59 tissue samples of endometroid type of endometrial carcinoma with 20 control samples of non-neoplastic endometrium. Results. Using 15% cut-off for methylation, we observed significantly higher methylation in the CDH13 gene in endometrial cancer group. We observed significantly higher methylation in both WT1 and GATA5 genes in IB stage of endometroid carcinoma. We also observed significantly higher methylation in GATA5 gene in the group of poorly differentiated endometroid carcinoma. Conclusion. The findings suggest the importance of hypermethylation of CDH13, WT1 and GATA5 genes in endometrial carcinogenesis and could have implications for future diagnostic and therapeutic strategies of endometrial cancer based on epigenetic changes.