Clinicopathological Significance of Overall Frequency of Allelic Loss (OFAL) in Lesions Derived from Thyroid Follicular Cell.

BACKGROUND: Loss of heterozygosity (LOH) and microsatellite instability (MSI) are frequent molecular events in thyroid tumor etiopathogenesis occurring in several chromosomal critical areas, including 3p12-25.3, 7q21-31, 10q22-24, and 15q11-13, with loci of tumor suppressor genes. OBJECTIVE: We evaluated the usefulness of LOH/MSI as a diagnostic/prognostic biomarker in lesions derived from thyroid follicular cells: follicular thyroid carcinoma (FTC); follicular adenoma (FA), papillary thyroid carcinoma (PTC), and nodular goiter (NG). METHODS: We performed allelotyping (GeneMapper Software v. 4.0.) of ten microsatellite markers linked to the 1p31.2, 3p21.3, 3p24.2, 9p21.3, 11p15.5, and 16q22.1 region on DNA from 93 primary thyroid lesions then evaluated the LOH/MSI frequency and overall frequency of allelic loss (OFAL). RESULTS: We found regions with significantly increased frequency of LOH/MSI for specific histotypes: the 3p24.2 region for FA and 1p31.2 for FTC. LOH/MSI in 3p21.3 was significantly elevated in PTC and FTC. LOH/MSI in 3p21.3 was increased for small size tumors (T1a + T1b), tumors with no regional lymph node involvement (N0 + Nx), American Joint Committee on Cancer (AJCC) stage I tumors, and tumor diameter (Td) < 10 mm; in 1p31.2 for T2-3, N1, stage II-IV, and Td 10-30 mm; in 11p15.5 for T2-3, N1, stage II-IV, and Td > 30 mm. OFAL values were significantly higher in younger patients (< 40 years), in men, in those with T2-3 stage tumors, in those with increased Td, and in FA and FTC compared with NG and PTC. CONCLUSIONS: We confirmed the occurrence of LOH/MSI in 3p21.3 at an early stage of tumorigenesis and mapped 1p31.2 and 11p15.5 as characteristic for advanced-stage tumors. The results of our study may enable consideration of OFAL, defined as LOH/MSI coincidence in various chromosomal regions, as a tumor progression marker. OFAL values were significantly higher in follicular neoplasms (FA and FTC) than in PTC or NG; hence, increased OFAL values can be regarded as a characteristic feature of the follicular phenotype.

A Strong Decrease in TIMP3 Expression Mediated by the Presence of miR-17 and 20a Enables Extracellular Matrix Remodeling in the NSCLC Lesion Surroundings.

Background: Lung cancer is one of the most common causes of death worldwide with a relatively high fatality rate and a mean 5-years survival of about 18%. One of the hallmarks of cancer is the extracellular matrix (ECM) remodeling, which is crucial for metastasis. This process may be regulated by miRs targeting metalloproteinases (MMPs) associated with the ECM breakdown and metastatic process or blocking the action of tissue inhibitors of metalloproteinases (TIMPs). Search for early biomarkers is essential in detecting non-small cell lung cancer (NSCLC) and distinguishing its subtypes: Adenocarcinoma (AC) from Squamous Cell Carcinoma (SCC), enabling targeted chemotherapy. Methods: MiR-17 and miR-20a targeting MMP2 and TIMP3 were selected by TCGA data analysis with further validation using miRTarBase and literature. The study group comprised 47 patients with primary NSCLC (AC and SCC subtypes). RNA was isolated from the tumor and normal-looking neighboring tissue (NLNT) free of cancer cells. MiRs from peripheral blood exosomes were extracted on admission and 5-7 days after surgery. Gene and miRs expression were assessed in qPCR using TaqMan probes. Results: The MMP2 has been expressed on a similar level in NLNT, as in cancer. While, TIMP3 expression was decreased both in cancer tissue and NLNT, with significantly lower expression in cancer. TIMP3 downregulation in NLNT and in SCC subtype correlated negatively with miR-20a. The preoperative miR-17 expression was significantly higher among patients with SCC compared to AC. Receiver operating characteristic (ROC) analysis of miR-17 as AC subtype classifier revealed 90% specificity and 48% sensitivity in optimal cut-off point with area under ROC curve (AUC): 0.71 (95%CI: 0.55-0.87). Within NSCLC subtypes: a strong negative correlation between pack-years (PY) and TIMP3 expression was observed for NLNT in the SCC group. Conclusion: The TIMP3 silencing observed in the NLNT and its negative correlation with presurgical expression of miR-20a (from serum exosomes), suggest that miRs can influence ECM remodeling at a distance from the center of the lesion. The miRs expression pattern in serum obtained before surgery significantly differs between AC and SCC subtypes. Moreover, decreased TIMP3 expression in NLNT (in SCC group) negatively correlates with the amount of tobacco smoked in a lifetime in PY.

The Expression Levels of IL-4/IL-13/STAT6 Signaling Pathway Genes and SOCS3 Could Help to Differentiate the Histopathological Subtypes of Non-Small Cell Lung Carcinoma.

BACKGROUND: The interleukin (IL)-4/IL-13/signal transducer and activator of transcription (STAT) 6 signaling pathway and the SOCS3 gene, one of its main regulators, constitute an important link between the inflammation process in the epithelial cells and inflammatory-related tumorigenesis. The present study is the first to evaluate IL-4, IL-13, STAT6, and SOCS3 mRNA expression in non-small cell lung carcinoma (NSCLC) histopathological subtypes. METHODS: Gene expression levels were assessed using TaqMan® probes by quantitative reverse transcription PCR (qRT-PCR) in lung tumor samples and unchanged lung tissue samples. RESULTS: Increased expression of IL-4, IL-13, and STAT6 was observed in all histopathological NSCLC subtypes (squamous cell carcinoma [SCC], adenocarcinoma [AC], and large cell carcinoma [LCC]). Significantly higher expression of IL-13 and STAT6 (p = 0.019 and p = 0.008, respectively) was found in SCC than in LCC. No statistically significant differences were found for IL-4. Significantly higher SOCS3 expression was found in LCC than in AC (p = 0.027). A negative correlation (rho = -0.519) was observed for the STAT6 and SOCS3 genes in SCC (p = 0.005). No associations were found between gene expression and tumor staging (post-operative Tumor Node Metastasis [pTNM], American Joint Committee on Cancer [AJCC]), patients' age, sex, or history of smoking. CONCLUSIONS: As the number of LCC cases in our study was quite low, the statistically significant results obtained should be confirmed in a larger group of patients, particularly as the relationships identified between increased IL-4, IL-13, and STAT6 mRNA expression and decreased SOCS3 expression suggest that these genes may serve as potential diagnostic markers for differentiating between NSCLC histopathological subtypes.

Immunoexpression analysis of selected JAK/STAT pathway molecules in patients with non- small-cell lung cancer.

INTRODUCTION    Signal transducer and activator of transcription (STAT) proteins are critically involved in tumorigenesis in various cancers, including lung cancer. OBJECTIVES    The aim of the study was to analyze the immunoexpression levels of 3 STAT proteins: STAT3, STAT5, and STAT6 in their phosphorylated forms (pSTATs), STAT inhibitors PIAS3 and SOCS3, and additionally cyclooxygenase 2 (COX­2), as potential diagnostic and prognostic markers in lung cancer. PATIENTS AND METHODS    The study included 71 patients diagnosed with non- small-cell lung cancer (NSCLC). The immunoexpression levels of the proteins were assessed in lung tissue samples, using an enzyme­linked immunosorbent assay. Tumors were staged using the postoperative TNM classification. RESULTS    All studied STATs were overexpressed in 54% to 55% of NSCLC specimens. Significantly higher STAT3 and STAT6 immunoexpression levels were observed in squamous cell carcinoma. Significant differences between NSCLC samples and controls were found for STAT5. Significantly higher STAT5 levels were observed in pT2 tumors. The COX­2 overexpression was observed in 55% of NSCLC specimens and was significantly higher in T2 tumors. STAT inhibitors were underexpressed in 56% to 58% of NSCLC specimens. The PIAS3 immunoexpression was significantly lower in non-squamous cell carcinoma. The SOCS3 level was significantly lower in smaller tumors (pT1). Negative correlations between STAT5 and PIAS3 levels, as well as between STAT6 and SOCS levels, and a positive correlation between STAT5 and COX-2 levels were observed. CONCLUSIONS    The deregulated expression of the studied pSTATs and their inhibitors may be involved in the development and progression of lung cancer. The observed differences between the histotypes suggest the potential usefulness of STAT proteins as diagnostic markers. Our results may contribute to the search for targets in lung cancer therapy.

Small suitability of the DLEC1, MLH1 and TUSC4 mRNA expression analysis as potential prognostic or differentiating markers for NSCLC patients in the Polish population.

According to the latest data, lung cancer is one of the most common cancer worldwide, men contributing nearly 21.2% and women 8.6% of all diagnosed cancers. Late detection of tumour drastically reduces the chance for a cure. Thus, it is important to search for candidate biomarkers for screening of early stage nonsmall cell lung carcinoma (NSCLC). Tumour suppressor genes, DLEC1, TUSC4 and MLH1, localized on 3p21 are recognized to play a role in NSCLC carcinogenesis. The aim of this study was to assess the relationship between the DLEC1, TUSC4 and MLH1 mRNA expression, and clinical features of NSCLC patients, tobacco addiction, and tumour histopathological characteristics. The DLEC1, TUSC4 and MLH1 expression was analysed in lung tumour tissue samples obtained from 69 patients diagnosed with NSCLC: squamous cell carcinoma (n = 34), adenocarcinoma (n = 24), large cell carcinoma (n = 5), carcinoma adenosquamosum (n = 5). A decreased gene expression (RQ < 0.7) was observed for DLEC1 in 60.9% of tumour samples, for MLH1 in 50.7% and for TUSC4 in 26% of NSCLC samples. DLEC1 was decreased in more aggressive subtypes: large cell carcinoma and adenocarcinoma-squamous cell carcinoma. The simultaneous downregulation of two of the studied genes, DLEC1 andMLH1,was observed in 30.4% of NSCLCsamples, highlighting the importance of these two genes in lung carcinogenesis. We found no correlation between the DLEC1, TUSC4 and MLH1 gene expression and NSCLC patient characteristics (gender, age and smoking) or cancer histopathology. No significant differences in the gene expression among NSCLC subtypes indicate the weakness of DLEC1, TUSC4 and MLH1 expression analysis as potential differentiating markers of NSCLC subtypes in the Polish population.

Triiodothyronine regulates cell growth and survival in renal cell cancer.

Triiodothyronine plays an important role in the regulation of kidney cell growth, differentiation and metabolism. Patients with renal cell cancer who develop hypothyreosis during tyrosine kinase inhibitor (TKI) treatment have statistically longer survival. In this study, we developed cell based model of triiodothyronine (T3) analysis in RCC and we show the different effects of T3 on renal cell cancer (RCC) cell growth response and expression of the thyroid hormone receptor in human renal cell cancer cell lines from primary and metastatic tumors along with human kidney cancer stem cells. Wild-type thyroid hormone receptor is ubiquitously expressed in human renal cancer cell lines, but normalized against healthy renal proximal tube cell expression its level is upregulated in Caki-2, RCC6, SKRC-42, SKRC-45 cell lines. On the contrary the mRNA level in the 769-P, ACHN, HKCSC, and HEK293 cells is significantly decreased. The TRß protein was abundant in the cytoplasm of the 786-O, Caki-2, RCC6, and SKRC-45 cells and in the nucleus of SKRC-42, ACHN, 769-P and cancer stem cells. T3 has promoting effect on the cell proliferation of HKCSC, Caki-2, ASE, ACHN, SK-RC-42, SMKT-R2, Caki-1, 786-0, and SK-RC-45 cells. Tyrosine kinase inhibitor, sunitinib, directly inhibits proliferation of RCC cells, while thyroid hormone receptor antagonist 1-850 (CAS 251310­57-3) has less significant inhibitory impact. T3 stimulation does not abrogate inhibitory effect of sunitinib. Renal cancer tumor cells hypostimulated with T3 may be more responsive to tyrosine kinase inhibition. Moreover, some tumors may be considered as T3-independent and present aggressive phenotype with thyroid hormone receptor activated independently from the ligand. On the contrary proliferation induced by deregulated VHL and or c-Met pathways may transgress normal T3 mediated regulation of the cell cycle.

FHIT promoter methylation status, low protein and high mRNA levels in patients with non-small cell lung cancer.

FHIT is a tumor suppressor gene that is frequently silenced in non-small cell lung cancer (NSCLC) and also in preneoplastic lesions. Promoter hypermethylation was previously observed in NSCLC, and its epigenetic silencing, observed on mRNA or protein level, was proposed to predict NSCLC outcome. In the present study we evaluated the relationship between FHIT expression on mRNA level and promoter methylation, or immunoexpression level. The aim of this study was to analyze the usefulness of FHIT as early differentiating biomarker in NSCLC patients. Lung tissue specimens were obtained from 59 patients with diagnosed NSCLC (SCC=34, AC=20, LCC=5). FHIT promoter methylation was assessed in methylation-specific PCR. Relative expression analysis of FHIT was performed in real-time PCR (qPCR) and protein immunoexpression by ELISA assay. Significant differences in FHIT expression between NSCLC histopathological groups (SCC, AC, LCC) were observed (p=0.000009), with the lowest level in SCC. FHIT expression was significantly higher (p=0.034) in men vs. women. Methylated FHIT alleles were present both in NSCLC and control specimens. Mean MI value was higher in control tissue vs. neoplasm, and in men vs. women and it increased with patient age. Significant increase in MI level was observed in N0 group vs. N1 and N2, according to the TNM staging (p=0.0073). Differences in FHIT expression levels between AC, LCC and SCC indicated the usefulness of this gene as a diagnostic marker for NSCLC subtype differentiation. FHIT promoter hypermethylation both in cancer and control tissue indicated the presence of epigenetic alterations in early stage of NSCLC development. Differences in gene promoter methylation between cancer patients with and without node infiltration might be considered as a prognostic marker. Significantly lower FHIT protein immunoexpression was revealed in the group with long and intense history of smoking assessed as PYs (PY<40 vs. PY≥40, p=0.01). These results suggest the need of further study on FHIT as a potential biomarker.

Expression level and methylation status of three tumor suppressor genes, DLEC1, ITGA9 and MLH1, in non-small cell lung cancer.

Despite therapeutic advances, lung cancer remains one of the most common causes of cancer-related death in the world. There is a need to develop biomarkers of diagnostic and/or prognostic value and to translate findings in basic science research to clinical application. Tumor suppressor genes (TSGs) represent potential useful markers for disease detection, progression and treatment target. We tried to elucidate the role of three 3p21.3 TSGs: DLEC1, ITGA9 and MLH1, in non-small cell lung cancer (NSCLC). We assessed their expression pattern by qPCR in 59 NSCLC tissues and in the matched macroscopically unchanged lung tissues. Additionally, we analyzed gene promoter methylation status by methylation-specific PCR in NSCLC samples. We did not find significant correlations between gene expression and methylation. In case of DLEC1 and ITGA9, expression levels were decreased in 71-78 % of tumor samples and significantly different between tumor and normal tissues (P = 0.0001). It could point to their diagnostic value. ITGA9 could also be regarded as a diagnostic marker differentiating NSCLC subtypes, as its expression level was significantly lower in squamous cell carcinoma (P = 0.001). The simultaneous down-regulation of DLEC1 and ITGA9 was observed in 52.5 % of NSCLCs. MSPs revealed high frequencies of gene promoter methylation in NSCLCs: 84 % for DLEC1 and MLH1 and 57 % for ITGA9. Methylation indexes reflected moderate gene methylation levels: 34 % for ITGA9, 27 % for MLH1 and 26 % for DLEC1. However, frequent simultaneous methylation of the studied genes in more than 50 % of NSCLCs suggests the possibility of consider them as a panel of epigenetic markers.

Altered Cyclooxygenase-2 Expression in Pulmonary Sarcoidosis is not Related to Clinical Classifications.

Elevated COX-2 activity is associated with the development of chronic lung diseases leading to bronchial obstruction, including sarcoidosis. The aim of the study was to examine expression pattern of COX-2 messenger RNA (mRNA). Expression was performed by q-PCR method in bronchoalveolar lavage (BAL) cells and peripheral blood (PB) lymphocytes in sarcoidosis patients (n = 61) and control group (n = 30). Analysis of COX-2 mRNA expression level in BAL fluid and PB revealed downregulation in sarcoidosis and control groups. In PB lymphocytes, the statistically significant difference between patients and controls was observed (P = 0.003, Mann-Whitney U test), with higher expression in patients. There were no statistically significant differences between patients without and with parenchymal involvement (stages I vs. II-IV), between patients with acute vs. insidious onset of disease and between patients with abnormal vs. normal spirometry (P > 0.05, Mann-Whitney U test). Results suggest that expression of COX-2 mRNA in patients with pulmonary sarcoidosis is not related to clinical classifications.

Assessment of the frequency of genetic alterations (LOH/MSI) in patients with intraepithelial cervical lesions with HPV infection: a pilot study.

In the present study, we analyzed (1) the type of HPV infection and (2) the frequency of loss of heterozygosity and microsatellite imbalance (LOH/MSI) in normal cytology and cervical intraepithelial neoplasia (CIN1-3). The cytological material included: low-grade squamous intraepithelial lesions (CIN1, n = 11), high-grade lesions (CIN2 and CIN3, n = 13), and cytologically normal cells from non-neoplastic cervical samples (n = 8). HPV genotyping was done using RealLine HPV 16/18 kit. We used 20 microsatellite markers from: 1p31.2, 3p14.3, 3p21.3, 3p22.2, 3p24.2, 3p25.3, 7q32.2, 9p21.3, 11p15.5, 12q23.2, and 16q22.1. LOH/MSI was correlated with clinicopathological parameters. The presence of HPV DNA was revealed in 78.13 % samples, including normal cytology. LOH/MSI was the most frequent for: 3p25.3 (39 %), 3p22.2 (20.83 %), 3p24.2 (20 %), and 3p14.3 (16.67 %). It was demonstrated that D3S1234 (FHIT; 3p14.3), D3S1611 (MLH1; 3p22.2), D3S1583 (RARB; 3p24.2), D3S1317 and D3S3611 (VHL; 3p25.3) could differentiate patients with CIN2/CIN3 versus CIN1, showing significantly higher frequency in CIN2/CIN3. LOH/MSI frequency for other than 3p markers was lower, 10-22.2 %. The simultaneous occurrence of LOH/MSI for several markers (OFAL) was higher in CIN2/CIN3. Significant differences in OFAL were found between samples with versus without HPV infection. In HPV-positive patients, significant differences in OFAL were found between normal cytology, CIN1 and CIN2/CIN3. HPV infection influences the increase in LOH/MSI frequency, especially in tumor suppressor gene loci. Several studied microsatellite markers seem to be useful for CIN grading. Hopefully, the obtained results, if confirmed on larger patient cohort, would allow creating a panel of markers supporting clinical diagnosis in patients with HPV infection.

Altered miRNA expression in pulmonary sarcoidosis.

BACKGROUND: miRNAs control important cellular functions including angiogenesis/angiostasis or fibrosis and reveal altered expression during pathological processes in the lung. METHODS: The aim of the study was to investigate the expression of selected miRNAs (miR-let7f, miR-15b, miR-16, miR-20a, miR-27b, miR-128a, miR-130a, miR-192 miR-221, miR-222) in patients with pulmonary sarcoidosis (n = 94) and controls (n = 50). The expression was assessed by q-PCR in BALF cells and peripheral blood lymphocytes (PB lymphocytes). For statistical analysis, the Kruskal-Wallis test, Mann-Whitney U- test, Neuman-Keuls' multiple comparison test, and Spearman's rank correlation were used. RESULTS: In BALF cells, significantly higher expression of miR-192 and miR-221 and lower expression of miR-15b were found in patients than controls. MiR-27b, miR-192 and miR-221 expression was significantly higher in patients without parenchymal involvement (stages I) than those at stages II-IV. Patients with acute disease demonstrated significantly higher miR-27b, miR-192 and miR-221 expression than those with insidious onset. For PB lymphocytes, patients demonstrated significantly greater miR-15b, miR-27b, miR-192, miR-221 and miR-222 expression, but lower miR-let7f and miR-130a expression, than controls. Stage I patients demonstrated significantly higher miR-16 and miR-15b expression than those in stages II-IV, and patients with the acute form demonstrated higher miR-130a and miR-15b expression. In BALF cells, miR-16 and miR-20a expression was significantly higher in patients with lung volume restriction, and miR-let7f was higher in the PB lymphocytes in patients with obturation. Several correlations were observed between the pattern of miRNA expression, lung function parameters and selected laboratory markers. CONCLUSION: The obtained results suggest that the studied miRNAs play a role in the pathogenesis of sarcoidosis, and that some of them might have negative prognostic value.

Immunoexpression of TGF-ß/Smad and VEGF-A proteins in serum and BAL fluid of sarcoidosis patients.

BACKGROUND: The chronic course of pulmonary sarcoidosis can lead to lung dysfunction due to fibrosis, in which the signalling pathways TGF-ß/Smad and VEGF-A may play a key role. METHODS: We evaluated immunoexpression of TGF-ß1, Smad2, 3, and 7, and VEGF-A in serum and bronchoalveolar lavage (BAL) fluid of patients (n = 57) classified according to the presence of lung parenchymal involvement (radiological stage I vs. II-III), acute vs. insidious onset, lung function test (LFT) results, calcium metabolism parameters, percentage of BAL lymphocytes (BAL-L%), BAL CD4(+)/CD8(+) ratio, age, and gender. Immunoexpression analysis of proteins was performed by ELISA. RESULTS: The immunoexpression of all studied proteins were higher in serum than in BAL fluid of patients (p >0.05). The serum levels of TGF-ß1 (p = 0.03), Smad2 (p = 0.01), and VEGF-A (p = 0.0002) were significantly higher in sarcoidosis patients compared to healthy controls. There were no differences within the sarcoidosis group between patients with vs. without parenchymal involvement, acute vs. insidious onset, or patients with normal vs. abnormal spirometry results. In patients with abnormal spirometry results a negative correlation was found between forced vital capacity (FVC) % predicted value and TGF-ß1 immunoexpression in BAL fluid, and positive correlations were observed between the intensity of lung parenchymal changes estimated by high-resolution computed tomography (HRCT scores) and Smad 2 level in serum. CONCLUSIONS: TGF-ß/Smad signalling pathway and VEGF-A participate in the pathogenesis of sarcoidosis. BAL TGF-ß1, and Smad 2 in serum seem to be promising biomarkers with negative prognostic value, but further studies are required to confirmed our observations.

Quantitative analysis of mRNA expression levels and DNA methylation profiles of three neighboring genes: FUS1, NPRL2/G21 and RASSF1A in non-small cell lung cancer patients.

BACKGROUND: Tumor suppressor gene (TSG) inactivation plays a crucial role in carcinogenesis. FUS1, NPRL2/G21 and RASSF1A are TSGs from LUCA region at 3p21.3, a critical chromosomal region in lung cancer development. The aim of the study was to analyze and compare the expression levels of these 3 TSGs in NSCLC, as well as in macroscopically unchanged lung tissue surrounding the primary lesion, and to look for the possible epigenetic mechanism of TSG inactivation via gene promoter methylation. METHODS: Expression levels of 3 TSGs and 2 DNA methyltransferases, DNMT1 and DNMT3B, were assessed using real-time PCR method (qPCR) in 59 primary non-small cell lung tumors and the matched macroscopically unchanged lung tissue samples. Promoter methylation status of TSGs was analyzed using methylation-specific PCRs (MSP method) and Methylation Index (MI) value was calculated for each gene. RESULTS: The expression of all three TSGs were significantly different between NSCLC subtypes: RASSF1A and FUS1 expression levels were significantly lower in squamous cell carcinoma (SCC), and NPRL2/G21 in adenocarcinoma (AC). RASSF1A showed significantly lower expression in tumors vs macroscopically unchanged lung tissues. Methylation frequency was 38-76%, depending on the gene. The highest MI value was found for RASSF1A (52%) and the lowest for NPRL2/G21 (5%). The simultaneous decreased expression and methylation of at least one RASSF1A allele was observed in 71% tumor samples. Inverse correlation between gene expression and promoter methylation was found for FUS1 (rs = -0.41) in SCC subtype. Expression levels of DNMTs were significantly increased in 75-92% NSCLCs and were significantly higher in tumors than in normal lung tissue. However, no correlation between mRNA expression levels of DNMTs and DNA methylation status of the studied TSGs was found. CONCLUSIONS: The results indicate the potential role of the studied TSGs in the differentiation of NSCLC histopathological subtypes. The significant differences in RASSF1A expression levels between NSCLC and macroscopically unchanged lung tissue highlight its possible diagnostic role in lung cancer in situ recognition. High percentage of lung tumor samples with simultaneous RASSF1A decreased expression and gene promoter methylation indicates its epigenetic silencing. However, DNMT overexpression doesn't seem to be a critical determinate of its promoter hypermethylation.

Expression of STAT5, COX-2 and PIAS3 in correlation with NSCLC histhopathological features.

Signal transducers and activators of transcription (STATs), their inhibitors and cyclooxygenase-2 (COX-2) participate in transformations of many various types of cancers. The aim of the present study was to evaluate the relationship between STAT5A/B, COX-2, and PIAS3 mRNA expression and tumor staging, metastasis status, and histopathological subtype in 71 patients with confirmed non-small cell lung cancer (NSCLC) diagnosis. Total RNA was isolated from NSCLC tissue samples and the expression of the studied genes was assessed using TaqMan probes in real-time PCR assay. The expression levels of STAT5A, STAT5B, and COX-2 genes were increased in 69%, 79%, and 71% NSCLC samples respectively, while PIAS3 expression was decreased in the majority (69%) of the studied tissues. Statistically significant differences were observed between STAT5 isoforms (P = 0.0008), with higher expression of STAT5B. We found statistically significant positive correlation between STAT5B and COX-2 (rho = 0.045), and significant negative correlation between STAT5B and PIAS3 (rho = -0.049). The negative correlation between STAT5B and PIAS3 (rho = -0.43) was also observed in T2a+T2b tumor group. Additionally, STAT5B and COX-2 expression levels were significantly different between T1a+T1b and T2a+T2b tumors (P = 0.002 and P = 0.041, respectively), with higher expression of both genes in T2 tumor stage. PIAS3 expression was significantly lower in NSCC subtype as compared with SCC subtype (P = 0.017). Also, STAT5A and STAT5B immunoexpression was assessed, and the results indicated significantly higher protein levels in NSCLC patients as compared with controls (P = 0.048 and P = 0.034, respectively). High STAT5B immunoexpression was positively correlated with STAT5B gene expression in tumors (rho = 0.755). STAT5B protein level was also significantly higher in T2a+T2b tumors, reflecting high STAT5B gene expression in this group. There was no statistically significant association between mRNA and protein expression levels of the studied genes and patients' characteristics: age, gender, smoking. The obtained results highlight the importance of the genes STAT5B and COX-2 in lung cancer progression.

HPV16 E6*II gene expression in intraepithelial cervical lesions as an indicator of neoplastic grade: a pilot study.

Integration of the HPV genome into a host cell DNA leads to the deregulated overexpression of the viral E6 and E7 oncoproteins, and this is a key factor for progression from low-grade cervical lesions to high-grade lesions and invasive cervical cancer. The aim of our study was to analyze the expression levels of HPV E6*I/E6*II and E7 genes in cervical neoplasia of different grades. The analysis involved 10 low-grade squamous intraepithelial lesions (CIN1), 15 high-grade lesions (CIN2 and CIN3), as well as normal cytology samples (n=10). HPV genotyping was done using RealLine HPV 16/18 kit. The expression analysis was performed in real-time PCR assay using gene-specific primers and SYBR Green. HPV16 DNA was found in 65.71% patients, including also normal cytology samples. The increased expression level of E6*I was observed in 12 (34.3%) patients. The expression of E6*II was increased in 10 (28.6%) samples, and E7 overexpression was found in 14 (40%) patients. Significant positive correlation was observed between the amount of HPV16 DNA and the levels of E6*I and E6*II expression. There were no statistically significant differences in expression levels of the studied genes between the groups (CIN1 vs. CIN2/CIN3 vs. normal cytology). Statistically significant differences were found in CIN2/CIN3 group, with the higher expression of E6*II as compared with E6*I. We suggest that the expression level of E6*II gene might be used as an indicator of cervical cancer severity, in patients with high-grade cervical neoplasia, but these observations need to be confirmed in a larger patient cohort.

Allelic imbalance in 1p, 7q, 9p, 11p, 12q and 16q regions in non-small cell lung carcinoma and its clinical association: a pilot study.

In lung cancer pathogenesis, genetic instability, i.e., loss of heterozygosity (LOH) and microsatellite instability (MSI) is a frequent molecular event, occurring at an early stage of cancerogenesis. The presence of LOH/MSI in non-small cell lung carcinoma (NSCLC) was found in many chromosomal regions, but exclusive of 3p their diagnostic value remains controversial. In this study we focused on other than 3p regions-1p31.2, 7q32.2, 9p21.3, 11p15.5, 12q23.2 and 16q22-the loci of many oncogenes and tumour suppressor genes. To analyze the potential role of LOH/MSI involved in NSCLC pathogenesis we allelotyped a panel of 13 microsatellite markers in a group of 56 cancer specimens. Our data demonstrate the presence of allelic loss for all (13) analyzed markers. Total LOH/MSI frequency in NSCLC was the highest for chromosomal region 11p15.5 (25.84 %), followed by 9p21.3 and 1p31.2 (19.87 and 16.67 % respectively). A statistically significant increase of total LOH/MSI frequency was detected for the 11p15.5 region (p = 0.0301; χ(2) test). The associations of total LOH/MSI frequency: 1) increase in 11p15.5 region (p = 0.047; χ(2) test) and 2) decrease in 7q32.2 region (p = 0.037; χ(2) test) have been statistically significant in AJCC III (American Joint Committee on Cancer Staging). In Fractional Allele Loss (FAL) index analysis, the correlation with cigarette addiction has been statistically significant. The increased amount of cigarettes smoked (pack years) in a lifetime correlates with increasing FAL (p = 0.024; Kruskal-Wallis test). These results demonstrate that LOH/MSI alternation in studied chromosomal regions is strongly influenced by tobacco smoking but do not seem to be pivotal NSCLC diagnostic marker with prognostic impact.