Kamil Torres

Diagnostic and prognostic significance of miRNA signatures in tissues and plasma of endometrioid endometrial carcinoma patients

The aim of our study was to define tissue and plasma miRNA signatures, which could potentially serve as diagnostic and prognostic markers in endometrioid endometrial cancer (EEC) and to investigate miRNA profiles in regard to clinicopathological characteristics. Tissue and plasma samples were collected from 122 women (77 EEC and 45 controls). Expression profiling of 866 human miRNAs and 89 human viral miRNAs was performed in 24 samples and was followed by qPCR validation in 104 patients. Expression of 16 miRNAs was analyzed in 48 plasma samples. Microarray study revealed regulation of 21 miRNAs in EEC tissues comparing to normal endometrium. Altered expression of 17 miRNAs was confirmed by qPCR performed in 104 tissue samples. Seven miRNAs were upregulated and two were downregulated in EEC plasma samples. Expression of a number of miRNAs was associated with International Federation of Gynecology and Obstetrics stage, grade, relapse and nodal metastases. Two miRNA signatures: miR‐92a/miR‐410 and miR‐92a/miR‐205/miR‐410 classified tumor tissues with higher accuracy in comparison to single miRNAs (AUC: 0.977, 95% CI: 0.927–0.996 and 0.984, 95% CI: 0.938–0.999, respectively). miRNA signature composed of miR‐205 and miR‐200a predicted relapse with AUC of 0.854 (95% CI: 0.691–0.951). Tissue miRNA signatures were independent prognostic markers of overall (miR‐1228/miR‐200c/miR‐429, HR: 2.98) and progression‐free survival (miR‐1228/miR‐429, HR: 2.453). Plasma miRNA signatures: miR‐9/miR‐1228 and miR‐9/miR‐92a, classified EEC plasma samples with high accuracy yielding AUCs of 0.909 (95% CI: 0.789–973) and 0.913 (95% CI: 0.794–0.976), respectively. We conclude that miRNA signatures hold a great promise to become noninvasive biomarkers for early EEC detection and prognosis.

Deregulation of miR-100, miR-99a and miR-199b in tissues and plasma coexists with increased expression of mTOR kinase in endometrioid endometrial carcinoma

BackgroundAlterations of mTOR gene expression have been implicated in the pathogenesis of endometrioid endometrial cancer however only few studies explored the cause of increased mTOR activation in this malignancy. miRNAs are small, noncoding RNAs, which were proven to regulated gene expression at the posttranscriptional level. The study aimed to explore deregulation of miRNAs targeting mTOR kinase (miR-99a, miR-100 and miR-199b) as a possible cause of its altered expression in EEC tissues. In addition expression of the three miRNAs was investigated in plasma of EEC patients and was assessed in terms of diagnostic and prognostic utility.MethodsWe investigated expression of mTOR kinase transcripts in 46 fresh tissue samples. Expression of miR-99a, miR-100 and miR-199b was investigated in the same group of fresh samples, and in additional 58 FFPE sections as well as in 48 plasma samples using qPCR. Relative quantification was performed using experimentally validated endogenous controls.ResultsmTOR kinase expression was increased in EEC tissues and was accompanied by decreased expression of all three miRNAs. Down-regulation of the investigated miRNAs was discovered in plasma of EEC patients and miRNA signatures classified EEC tissues (miR-99a/miR-100/miR-199b) and plasma (miR-99a/miR-199b) samples with higher accuracy in comparison to single miRNAs. We also revealed that miR-100 was an independent prognostic marker of overall survival.ConclusionsWe conclude that increased expression of mTOR kinase coexists with down-regulation of its targeting miRNAs, which could suggest a new mechanism of mTOR pathway alterations in EEC. In addition, our findings implicate that miRNA signatures can be considered promising biomarkers for early detection and prognosis of endometrioid endometrial carcinoma.

Selection and validation of endogenous controls for microRNA expression studies in endometrioid endometrial cancer tissues.

OBJECTIVES microRNAs comprise a family of small, non-coding RNAs, which regulate gene expression at the posttranscriptional level. Multiple studies implicated important roles of microRNAs in various malignancies including endometrioid endometrial carcinoma (EEC). qPCR is widely used in the studies investigating microRNA expression. Relative quantification of microRNA expression requires proper normalization methods and endogenous controls are widely used for this purpose. The aim of this study was experimental identification of stable endogenous controls for normalization of microRNA qPCR expression studies in EEC. METHODS Expression of twelve candidate endogenous controls (miR-16, miR-26b, miR-92a, RNU44, RNU48, U75, U54, U6, U49, RNU6B, RNU38B, U18A) was investigated in tissue samples obtained from 45 patients (30 EEC, 15 normal endometrium) using qPCR. Stability of candidate endogenous controls was evaluated using NormFinder, geNorm, BestKeeper and equivalency test. The results were then validated using larger group of samples. RESULTS RNU48, U75 and RNU44 were identified as stably and equivalently expressed between malignant and normal tissues. Both NormFinder and geNorm indicated that those three snRNAs were optimal for qPCR data normalization in EEC tissues. CONCLUSIONS In conclusion, we suggest that average expression of those snoRNAs could be used as a reliable endogenous control in microRNA qPCR studies in endometrioid endometrial cancer. In addition to identifying suitable endogenous controls in EEC, our study presents an appropriate strategy for validation of candidate reference genes for any microRNA qPCR study.

Major regulators of microRNAs biogenesis Dicer and Drosha are down-regulated in endometrial cancer

Alterations in microRNAs expression have been proposed to play role in endometrial cancer pathogenesis. Dicer and Drosha are main regulators of microRNA biogenesis and deregulation of their expression has been indicated as a possible cause of microRNAs alterations observed in various cancers. The objective of this study was to investigate Dicer and Drosha genes expression in endometrial cancer and to analyze the impact of clinicopathological characteristics on their expression. Fresh tissue samples were collected from 44 patients (26 endometroid endometrial carcinoma and 18 controls). Clinical and pathological data were acquired from medical documentation. Dicer and Drosha genes expressions were assessed by qRT-PCR using validated reference genes. Dicer and Drosha expression levels were significantly lower in endometrial cancer samples comparing to controls. Dicer was down-regulated by the factor of 1.54 (p = 0.009) and Drosha gene mean expression value was 1.4 times lower in endometrial cancer group versus control group (p = 0.008). Down-regulation of Dicer significantly correlated with decreased expression of Drosha (coefficient value 0.75). Decreased expression of Drosha correlated with higher histological grade and was influenced by BMI. Lower Dicer expression was found in nulli- and uniparous females comparing to multiparous individuals (p = 0.002). Neither the FIGO stage nor the menstrual status had significant influence on the expression of studied genes. This study revealed for the first time that expression alterations of main regulators of microRNAs biogenesis are present in endometrial cancer tissue and could be potentially responsible for altered microRNAs profiles observed in this malignancy.

Cytokine response in the postoperative period after surgical treatment of benign adnexal masses : comparison between laparoscopy and laparotomy

BackgroundCytokines are the main mediators of the inflammation and the response to trauma. The purpose of the present study was the comparative assessment in sera of patients with benign adnexal masses treated by laparoscopy or laparotomy of the following proinflammatory and anti-inflammatory cytokines: interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor-alpha (TNF-α), and IL-10 in the early postoperative period.MethodsA total of 40 patients with benign adnexal masses were studied; 25 of whom underwent laparoscopy and 15, laparotomy. Blood serum concentration of IL-1β, IL-6, IL-8, TNF-α, and IL-10 were measured by commercially available ELISA assays before and 4 h, 24 h, and 48 h after the operation.ResultsConcentrations of IL-6 were significantly increased in both groups at 4 h, 24 h, and 48 h after the surgery; levels of IL-10 showed a significant increase 4 h and 24 h after the operation; an increase in IL-1β levels was observed only after laparotomy; no significant variations were observed in serum levels of IL-8; the postoperative increase of IL-1β, IL-6, and IL-10 levels was more pronounced in patients undergoing laparotomy than in those treated laparoscopically; length of the surgical procedure, amount of CO2 used, tumor diameter, age, and body mass index (BMI) of the patients did not influence the postoperative patterns of the studied cytokines.ConclusionsSystemic cytokine response after operations for benign adnexal masses depends on the degree of the surgical trauma, and is less pronounced in patients undergoing laparoscopy.

MicroRNAs and their role in gynecological tumors

There have been only few events in the history of molecular biology that could be compared to the discovery of microRNAs and their role in cell physiology and pathology. MicroRNAs are small, single‐stranded, noncoding RNAs composed of 19–25 nucleotides (∼22 nt), which have been proven to regulate gene expression at the posttranscriptional level. The regulatory function of microRNAs was demonstrated in normal and diseased conditions. In particular, it has been linked to cell cycle regulation, cell proliferation and differentiation, inflammatory response, and apoptosis. Altered expression profiles of microRNA have been observed in many pathologies, including diabetes, rheumatoid arthritis, and several cancers. To date, more than 700 human microRNAs have been identified and in silico‐based analyses estimate at least 500 more to be identified. The purpose of this review is to present the current perspective on microRNAs structure and biogenesis as well as their contribution to the etiopathogenesis of gynecological tumors. We discuss results of the recent publications that indicate possibilities of microRNAs use as novel markers for tumors screening, early diagnosis, and treatment monitoring. The possible utilization of microRNAs as prognostic factors and specific therapy targets is also reviewed.

Highly increased maspin expression corresponds with up-regulation of miR-21 in endometrial cancer: a preliminary report.

Background: Maspin and programmed cell death 4 (Pdcd4) are tumor suppressor genes, and miR-21 is overexpressed in many solid tumors and was proven to negatively regulate a number of tumor suppressor genes including maspin and Pdcd4. The purpose of this study was to investigate the expression of maspin, Pdcd4, and miR-21 and their interrelations with clinicopathologic features in endometrial cancer using a quantitative approach. Methods: Maspin, Pdcd4, and miR-21 expressions were evaluated by a real-time polymerase chain reaction in 20 endometrial cancer and 10 normal endometrium samples. Results: Maspin showed a significantly increased expression in endometrial cancer samples compared with the control group and was up-regulated by a mean factor of 46.54 (SE range, 2.367-1160.26; 95% confidence interval, 0.515-15001, P < 0.0001). Expression of miR-21 was found significantly up-regulated in the sample group in comparison to control group by a mean factor of 2.312 (SE range, 0.741-7.778; 95% confidence interval 0.191-15.0, P = 0.028). No significant differences were present in the expression level of Pdcd4 between endometrial cancer and control groups. Comparison between IA and more advanced International Federation of Gynecology and Obstetrics stages of endometrial cancer in regard to expression levels of maspin, Pdcd4, and miR-21 did not reveal any significant differences. Similarly, no differences were encountered when histopathologic grading, myometrial invasion, age, body mass index, and parity were taken into consideration. Conclusions: Association between increased maspin expression and up-regulation of miR-21 in endometrial cancer suggests distinct and tissue-specific relationships of the 2 molecules in this type of malignancy and requires further studies that would reveal its clinical relevance.

Changes of procalcitonin level in multiple trauma patients.

BACKGROUND Some aspects of the pathophysiology of complications in multiple-trauma patients still remain unclear. Mediators of inflammation have been postulated as playing a key role in being responsible for life threatening complications of multiple trauma patients. The objective of this study was to evaluate the prognostic value of procalcitonin (PCT) level in multiple trauma patients. METHODS A prospective study took place including patients with multiple trauma hospitalised in several hospital units. PCT level was measured in blood from 45 patients, aged 18-70 years using enzyme-linked immunoassay. The patients were divided into three groups: group I - individuals with multiple trauma with central nervous system injury; group II - those with multiple trauma without CNS injury; and group III - patients with isolated central nervous system injury. RESULTS Initial PCT levels were below 0.5 ng mL(-1) regardless of the cause of trauma. In the 24th hour of observation, a statistically significant increase of PCT concentration vs. initial levels was recorded in all groups of patients. Then PCT levels decreased significantly at the 3rd measurement point in all groups, and they remained unchanged until the last measurement. The highest levels of PCT were observed in multiple trauma patients without CNS injury (group II). In this group of patients, a significantly longer duration of surgery in the post-trauma period affected PCT levels. PCT concentrations in patients who died were significantly greater than in survivors. CONCLUSIONS A long lasting elevated concentration of procalcitonin in the post-traumatic period, or its repeated increase, is a good marker of developing complications observed earlier than clinical manifestations.

Simulation techniques in the anatomy curriculum: review of literature.

Modern medical education faces a problem of combining the latest technology, procedures and information with classic teaching methods. Simulation is a technique, which replaces or amplifies doctor-patient experiences in controlled conditions and therefore evokes or replicates substantial aspects of the real world in a fully interactive manner. The basic course of anatomy in medical education could be recognised as the best example of implementing new educational techniques such as simulation, into the traditional medical curriculum. The PubMed database was searched using specific key words. Finally 72 articles were accepted and were divided into 3 basic categories of teaching methods: Category 1 - cadaveric dissection, Category 2 - simulator based education and Category 3 - other. A state of the art anatomical curriculum offers numerous possibilities and solutions including the oldest like cadaveric dissection and newest like simulators. Different simulation techniques are used with different intensity; however cadaveric dissection is still the most popular method. The second most frequent method is simulation-based training, in which North America is the leading country. The identification of anatomical structures during virtual surgical procedures or laparoscopic robotic procedures can be integrated into the traditional anatomy course. New technologies are supportive and beneficial in anatomy teaching however each excitement of new technologies sometimes should be tempered and evaluated for its usefulness in making the learning process constructive for students and their future practice.

Locked nucleic acid-inhibitor of miR-205 decreases endometrial cancer cells proliferation in vitro and in vivo

Pathogenesis of endometrial cancer has been connected with alterations of microRNA expression and in particular miR-205 up–regulation was consistently reported in this carcinoma. Presented study aimed to investigate if inhibition of miR-205 expression using LNA-modified-nucleotide would attenuate endometrial cancer cells proliferation in vitro and in vivo. In the course of the study we found that the proliferation of endometrial cancer cells (HEC-1-B, RL-95, KLE, Ishikawa) transfected with LNA-miR-205-inhibitor and evaluated using real time cell monitoring as well as standard cell proliferation assay, was significantly decreased. Next, LNA-miR-205-inhibitor was used to assess the in vivo effects of miR-205 inhibition of endometrial cancer growth. Cby.Cg-Foxn1/cmdb mice bearing endometrial cancer xenografts were intraperitoneally injected with nine dosages of 25mg/kg of miR-205-LNA-inhibitor or scramble control or phosphatase buffered saline and were observed for 32 days. We found that systemic administration of miR-205-LNA-inhibitor was technically possible, and exerted inhibitory effect on endometrial cancer xenograft growth in vivo with only mild toxic effects in treated animals. In conclusion our results suggest that systemic delivery of miR-205-LNA-inhibitor is feasible, devoid of significant toxicity, and could be a promising treatment strategy for endometrial cancer. Therefore it warrants further studies in other animal models.