Radoslaw Charkiewicz

MicroRNAs expression profiling of eutopic proliferative endometrium in women with ovarian endometriosis

BackgroundThe eutopic endometrium of women with endometriosis, compared with disease-free individuals, contains certain molecular alterations, including the differential expression of microRNA (miRNA). The aim of the study was to compare the expression of the most relevant miRNAs in the eutopic endometrium of women with and without ovarian endometriosis.MethodsA total of 46 regularly menstruating patients, 21 patients with ovarian endometriosis and 25 controls, underwent surgery in the proliferative phase of the cycle. The eutopic endometrium was collected through aspirating biopsy prior to laparoscopy. Only patients with advanced (stage III and IV) histopathologically confirmed ovarian endometriosis were included. TaqMan MicroRNA Array Cards were applied to examine the expression of 667 human miRNAs in 10 patients with endometriosis and 10 controls. Custom-made, low-density real-time PCR arrays were used to confirm the expression of 15 selected molecules in 21 endometriosis patients and 25 disease-free individuals.ResultsOf 667 miRNAs, 2 were highly likely to be upregulated and 13 were downregulated in the eutopic endometrium of patients with endometriosis compared with the controls. Validation using real-time PCR showed that hsa-miR-483-5p (p = 0.012) and hsa-miR-629* (p = 0.02) are significantly downregulated in patients with endometriosis.ConclusionsChanges in the expression of select miRNAs might lead to or be a consequence of an early defect in the physiological activity of the proliferative endometrium, ultimately resulting in the overgrowth of this tissue outside the uterus.

Overexpression of epidermal growth factor receptor as a prognostic factor in colorectal cancer on the basis of the Allred scoring system

Background Overexpression of epidermal growth factor receptor (EGFR) is found in many types of neoplasms. The aim of the study was to evaluate EGFR expression in colorectal cancer (CRC) specimens and to determine whether EGFR expression correlates with clinicopathological data and overall survival. Patients and methods Tissue specimens from 181 consecutive CRC patients treated at the Military Institute of Medicine in 2006–2010 were collected and examined for EGFR expression, by immunohistochemistry staining. The staining intensity and percentage of cells with membranous EGFR expression were scored and then grouped according to the parameters of the Allred Scoring system. Cutoff values were subjected to further statistical analysis. Univariate tests and a multivariate Cox proportional hazards model were used in data analysis. Results EGFR was overexpressed in 96 of 181 CRC specimens (53%). EGFR expression was not correlated with other clinicopathological variables. On univariate analysis, overexpression of EGFR, determined by PS (percentage score) (>3) and total score (sum of PS and intensity score) (>4), was associated with poor overall survival. On multivariate analysis, EGFR overexpression (PS > 3) was an independent adverse prognostic factor (hazard ratio [HR] 1.62; 95% confidence interval [CI]: 1.03–2.53). Elevated carcinoembryonic antigen (CEA) serum concentration before treatment, performance status (Word Health Organization [WHO]-2), and tumor localized in colon and liver metastases were also independent unfavorable prognostic factors. Conclusion EGFR overexpression (PS > 3) in a CRC patient population was an independent adverse prognostic factor. Implementation of the Allred Scoring system criteria into clinical practice might facilitate treatment decisions in CRC patients.

Determination of cathepsins B, D and G concentration in eutopic proliferative endometrium of women with endometriosis by the surface plasmon resonance imaging (SPRI) technique

OBJECTIVE To determine the concentrations of cathepsins B, D and G in proliferative eutopic endometrium of patients with and without endometriosis, by use of the surface plasmon resonance imaging (SPRI) technique. STUDY DESIGN A total of 55 patients were recruited in the study: 31 patients with endometriosis (stages I-IV) and 24 controls. Endometrial samples were obtained in the first phase of the menstrual cycle from regularly menstruating premenopausal women, prior to laparoscopy, by the use of aspiration biopsy. Endometriosis was appropriately classified according to the Revised American Fertility Society classification and confirmed by histopathology in every case. The SPRI technique was used to determine the concentration of cathepsins B, D and G. To compare the two groups for quantitative data, Mann-Whitney-Wilcoxons test was used due to the non-normal distribution of the tested variables and normality of distribution was assessed using Shapiro-Wilk W test. RESULTS The concentration of the three examined cathepsins was higher in the proliferative eutopic endometrium of patients with endometriosis, especially in advanced stages, e.g. III and IV, when compared to healthy individuals. Corresponding median values were, for cathepsin B: [7.93 pmol/mg (min-max 2.82-15.71) vs 1.2 pmol/mg (min-max 0.7-15.49) p=0.0014], for cathepsin D: [1.86 pmol/mg (min-max 0.51-5.4) vs 1.03 pmol/mg (min-max 0.4-2.72) p=0.00041] and for cathepsin G: [0.6 pmol/mg (min-max 0.33-2.51) vs 0.3 pmol/mg (min-max 0.16-1.29) p=0.00051]. CONCLUSIONS Increased concentrations of cathepsins B, D and G in the proliferative eutopic endometrium may play a role in the implantation of endometrial tissue outside the uterine cavity.

Profiling of selected angiogenesis-related genes in proliferative eutopic endometrium of women with endometriosis

OBJECTIVE To compare the expression level of the most relevant angiogenesis-related genes in the eutopic endometrium of women with and without endometriosis. STUDY DESIGN 32 regularly menstruating patients (18 with endometriosis and 14 controls) underwent surgery in the proliferative phase of the cycle. Eutopic endometrium was collected by the use of aspirating biopsy prior to laparoscopy. Only patients with advanced (stage III and IV) histopathologically confirmed ovarian endometriosis were studied. Real-time PCR gene arrays were applied to examine the expression of 84 human angiogenesis-connected genes. Western-blot and enzyme-linked immunosorbent assays (ELISA) were used to confirm the expression of selected proteins. RESULTS We found significantly higher levels of AKT1 (p=0.003), TYMP (p=0.02), JAG1 (p=0.007), LAMA5 (p=0.005) and TIMP-1 (p=0.03) in eutopic endometrium of patients with endometriosis as compared with controls. By the use of Western blot we found clearly positive expression of AKT1 whereas ELISA assays confirmed expression of AKT1, TYMP, JAG1, LAMA5 and TIMP1. CONCLUSION Changes in the expression of selected genes might lead to or be a consequence of an early defect in the physiological activity of proliferative endometrium ultimately resulting in its overgrowth outside the uterine cavity.

Lipid mediators involved in the oxidative stress and antioxidant defence of human lung cancer cells.

Background The oxidative modifications of bioactive macromolecules have important roles in carcinogenesis. Of particular interest are lipid peroxidation products, which are involved in the activation of Nrf2 and endocannabinoids that affect cancer progression. Methods In lung cancer tissues (squamous cell lung carcinoma - SCC and adenocarcinoma - AC), the glutathione peroxidase and catalase activity and glutathione level, together with the expression of Nrf2 and its activators/inhibitors were estimated. The oxidative modifications of DNA (8-hydroxy-2′-deoxyguanosine and N7-methylguanine), endocannabinoids (anandamide and 2- arachidonylglyceriol), their receptors (CB1/2, TRV1, GPR55), phospholipid fatty acids (arachidonic, linoleic and docosahexaenoic), and reactive aldehydes (4-hydroxynonenal, 4-oxononenal and malondialdehyde) were determined. Results Tumour tissues showed lower antioxidant capacity than healthy tissues, which was accompanied by lower levels of fatty acids and higher levels of reactive aldehydes. Disturbances in antioxidant capacity and enhanced DNA oxidative modifications were observed in 88% of AC patients and 81% of SCC patients. The 4-hydroxynonenal-Histidine adducts were detected in the necrotic and stromal cells in all tumours. These findings were associated with the enhanced Nrf2 activity, especially in AC. The strong difference between the cancer subtypes was evident in the levels of endocannabinoids, with an increase in 89% of SCC and a decrease in 85% of AC patients being observed. Additionally, the increase in the expression of CB1/2 receptors was observed only in 82% of AC, while the expression of VR1 and GPR55 was enhanced in 79% of SCC and 82% of AC patients. Conclusions This study shows significant differences in the redox status, Nrf2 pathway and endocannabinoid system between SCC and AC tissues. Understanding the relation between the various lipid mediators and antioxidants in different lung cancer subtypes may be beginning for further research on the effective anticancer therapy.

Selective gene expression profiling of mTOR-associated tumor suppressor and oncogenes in ovarian cancer

The aim of this study was to selectively profile the activation status of mammalian target of rapamycin (mTOR)-associated oncogenes and tumor suppressor genes (TSGs) in ovarian cancer specimens, healthy ovaries and benign ovarian tumors, including endometrial cysts. We used a novel type of microfluidic gene array to examine the expression of 15 human tumor suppressors and oncogenes in ovarian cancer specimens of 53 patients, benign ovarian cysts of 29 women (endometrial and simple) and 11 healthy ovaries of individuals in whom the material was obtained during total hysterectomies performed because of fibroid changes. The array was custom-designed to include the following genes: NF1, RHEB, mTOR1, AKT-1, PTEN, TSC1, TSC2, KRAS, RPS6KB1, 4EBP1, TP53, EIF4E, STK11, PIK3CA and BECN1. Confirmatory immunohistochemical detection was performed for a group of selected proteins. Particularly significant differences were observed as to the expression of PTEN (p < 0.0001), TP53 (p = 0.0003), PIK3CA (p = 0.0003) and BECN1 (p = 0.0014) which were shown to be downregulated in cancer patients when compared to healthy ovaries and benign ovarian cysts (endometrial and simple). These markers did not show association with grade or stage of the tumor. Immunohistochemistry showed that PTEN, TP53, PIK3CA and BECN1 proteins are expressed in ovarian cancer. Our results indicate that there are significant differences in the expression of some of the mTOR-related tumor suppressors and oncogenes which could be associated with the pathogenesis of ovarian cancer.

Maternal plasma and amniotic fluid chemokines screening in fetal Down syndrome.

Objective. Chemokines exert different inflammatory responses which can potentially be related to certain fetal chromosomal abnormalities. The aim of the study was to determine the concentration of selected chemokines in plasma and amniotic fluid of women with fetal Down syndrome. Method. Out of 171 amniocentesis, we had 7 patients with confirmed fetal Down syndrome (15th–18th weeks of gestation). For the purpose of our control, we chose 14 women without confirmed chromosomal aberration. To assess the concentration of chemokines in the blood plasma and amniotic fluid, we used a protein macroarray, which allows the simultaneous determination of 40 chemokines per sample. Results. We showed significant decrease in the concentration of 4 chemokines, HCC-4, IL-28A, IL-31, and MCP-2, and increase in the concentration of CXCL7 (NAP-2) in plasma of women with fetal Down syndrome. Furthermore, we showed decrease in concentration of 3 chemokines, ITAC, MCP-3, MIF, and increase in concentration of 4 chemokines, IP-10, MPIF-1, CXCL7, and 6Ckine, in amniotic fluid of women with fetal Down syndrome. Conclusion. On the basis of our findings, our hypothesis is that the chemokines may play role in the pathogenesis of Down syndrome. Defining their potential as biochemical markers of Down syndrome requires further investigation on larger group of patients.

Validation for histology-driven diagnosis in non-small cell lung cancer using hsa-miR-205 and hsa-miR-21 expression by two different normalization strategies.

Targeted therapy of non‐small cell lung cancer (NSCLC) demands a more accurate tumor classification that is crucial for patient selection in personalized treatment. MicroRNAs constitute a promising class of biomarkers and a helpful tool for the distinction between lung adenocarcinoma (AC) and squamous cell lung carcinoma (SCC). The aim of this study was to evaluate the impact of two different normalization strategies, using U6 snRNA and hsa‐miR‐103 as reference genes, on hsa‐miR‐205 and hsa‐miR‐21 expression levels, in terms of the classification of subtypes of NSCLC. By means of a quantitative real‐time polymerase chain reaction (qRT‐PCR) microRNA expression levels were evaluated in a classification set of 98 surgically resected NSCLC fresh‐frozen samples, and validated findings in an independent set of 42 NSCLC samples. The microRNA expression levels were exploited to develop a diagnostic test using two data normalization strategies. The performance of microRNA profiling in different normalization methods was compared. We revealed the microRNA‐based qRT‐PCR tests to be appropriate measures for distinguishing between AC and SCC (the concordance of histologic diagnoses and molecular methods greater than 88%). Performance evaluation of microRNA tests, based on the two normalization strategies, showed that the procedure using hsa‐miR‐103 as reference target has a slight advantage (sensitivity 83.33 and 100% in classification and validation set, respectively) compared to U6 snRNA. Molecular tests based on microRNA expression allow a reliable classification of subtypes for NSCLC and can constitute a useful diagnostic strategy in patient selection for targeted therapy.

K-Ras gene mutation status as a prognostic and predictive factor in patients with colorectal cancer undergoing irinotecan- or oxaliplatin-based chemotherapy

Background: CRC caused more than 600,000 estimated deaths in 2008. Dysregulated signaling through the RAS/RAF/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway due to mutations in K-Ras and B-Raf are common events in CRC. Methods: Incidence of mutations in codons 12 and 13 of K-Ras and exons 11 and 15 of B-Raf were analyzed in amplified PCR products from primary tumors of 273 patients with CRC, and their prognostic and predictive significance was assessed. The prognostic role of clinical and pathological factors was also examined. Results: K-Ras mutations were present in 89 patients (32.6%), of whom 76 (85.4%) had mutations in codon 12 and 10 (11.2%) had mutations in codon 13. B-Raf gene mutations were present in 17 patients (6.9%), of whom 6 (35.3%) had mutations in exon 15. Multivariate analysis revealed a predictive significance for K-Ras mutations with respect to time to progression in patients treated with irinotecan and oxaliplatin as first-line chemotherapy. There was no predictive significance for B-Raf gene mutation status in these patients. The following risk factors were found to affect overall survival (OS) rates: primary tumor location, lymph node involvement grade, carcinoembryonic antigen (CEA) level before treatment, and performance status according to WHO criteria. Conclusions: Based on the results of this study, K-Ras mutation status may be a suitable indicator of patient eligibility and a prognostic indicator for responsiveness to anti-EGFR therapy alone, or in combination with chemotherapy. Also, K-Ras mutation status may predict time to progression in patients treated with irinotecan and oxaliplatin.

Molecular differences in the KRAS gene mutation between a primary tumor and related metastatic sites - case report and a literature review

In recent years the the set of diagnostic tools in colorectal cancers has been extended by the assessment of the KRAS gene status. Currently it is a necessary step in order to qualify patients for the targeted therapy. The results of the analysis of several studies revealed a high rate of compliance of the KRAS gene mutational status in primary and metastatic tumors. In this paper we present a rare case of incompatibility of the KRAS mutations in the primary tumor located in the colon and metastatic changes in the liver.