Clinical value of microRNA-19a-3p and microRNA-99a-5p in bladder cancer.

Introduction: MicroRNAs (miRNA) are small (approximately 17 to 25 nucleotides in length), single stranded, non-coding RNAs that play an important role in the control of gene expression at the post-transcriptional stage, by inhibiting protein translation or promoting mRNA degradation. The main aim of the study was to evaluate the clinical utility of the tested markers (miRNAs 19a-3p and 99a-5p), which might be important in the diagnostics of non-invasive bladder cancer (BC). Material and methods: The study involved a group of 60 patients suffering from BC (histopathologically confirmed), in which 20 patients were diagnosed with muscle invasive BC (INBC) and 40 patients with non-muscle invasive BC (NINBC). The control group consisted of 20 samples of normal urothelium, which did not show any cancerous changes during histopathological examination. We assessed the expression of microRNA, using real-time PCR and the miRCURY LNA Universal RT microRNA PCR Kit by Exiqon, Denmark. Results: Reduced expression of both analyzed markers was observed in most cases: miR-19a-3p in 51.8% and miR-99a-5p in 65.5% (as follows Mann-Whitney U test p < 0.000001 and Student's t test p = 0.034262). Moreover, miR-19a-3p in our tested group was useful to differentiate between low and high grade disease in non-invasive stages (t test p = 0.0315435). Furthermore, miR-19a-3p and miR-99a-5p were able to discriminate patients in low grade for groups with or without recurrence. Conclusions: Our data indicated that miR-19a-3p and miR-99a-5p were significantly altered in bladder cancer samples and useful as diagnostic markers.

G870A Polymorphic Variants of CCND1 Gene and Cyclin D1 Protein Expression as Prognostic Markers in Laryngeal Lesions.

CCND1 gene encodes Cyclin D1 protein, the alternations and overexpression of which are commonly observed in human cancers. Cyclin D1 controls G1-S transition in the cell cycle. The aim of the study was to assess utility of the genotyping and protein expression in predicting the susceptibility of transformation from normal tissue to precancerous laryngeal lesions (PLLs) and finally to laryngeal cancer (LC). Four hundred and thirty-five patients (101 with LC, 100 with PLLs and 234 healthy volunteers) were enrolled in the study. Cyclin D1 expression was examined by immunohistochemistry and G870A polymorphism of gene CCND1 by PCR-RFLP technique. We confirmed association between the A allele and risk of developing LC from healthy mucosa (p = 0.006). Significantly higher expression of Cyclin D1 was observed in LC compering with PLLs (p < 0.0001) and we found that it could be a predictive marker of shorter survival time. To sum up, in the study population CCND1 gene polymorphism A870G and Cyclin D1 expression have a significant impact on the risk of developing PLLs and LC, and, therefore, Cyclin D1 could be a useful marker for the prediction of survival time in LC, whereas CCND1 gene polymorphism does not have a direct impact on patients' outcome.

Detection of PIK3CA Gene Mutation in Head and Neck Squamous Cell Carcinoma Using Droplet Digital PCR and RT-qPCR.

Head and neck squamous cell carcinomas (HNSCC) are the seventh cause of human malignancy with low survival rate due to late diagnosis and treatment. Its etiology is diverse; however genetic factors are significant. The most common mutations in HNSCC were found in the genes: PIK3CA (10-12%), BRCA1 (6%), and BRCA2 (7-9%). In some cases, these biomarkers correlate with recurrences or survival showing a potential of prognostic and predictive value. A total of 113 formalin-fixed paraffin embedded (FFPE) tumor samples were collected from patients with HNSCC (oral cavity: 35 (31.0%); oropharynx: 30 (26.0%); larynx: 48 (43.0%)). We examined PIK3CA H1047R mutation by Real Time PCR (RT-qPCR) and droplet digital PCR (ddPCR). BRCA1 and BRCA2 mutations were analyzed by RT-qPCR while p16 protein expression was assessed by immunohistochemistry. Finally, we identified HPV infection by RT-qPCR. The relationships between genomic alterations and clinical parameters were assessed using the Yates' corrected Chi-squared test or Fisher's exact test for nominal variables. Kaplan Meier plots were applied for survival analysis. Our results revealed 9 PIK3CA H1047R mutations detected by ddPCR: 8 of them were negative in RT-qPCR. Due to the use of different methods to test the presence of the PIK3CA gene mutation, different treatment decisions might be made. That is why it is so important to use the most sensitive methods available. We confirmed the usefulness of ddPCR in the PIK3CA mutation assessment in FFPE samples.

Expression profile of microRNAs (106b-3p, 130b-3, 145-3p, 199a-5p) in urine and serum samples from patients with the diagnosis of bladder cancer.

MicroRNAs (miRNA) are short, single stranded, non-coding RNAs that play an important role in controlling gene expression at the posttranscriptional stage. There is no bladder cancer marker that has been approved as an alternative for diagnostic cystoscopy and urine cytology so far, thus research for alternative, more sensitive, and less invasive methods of bladder cancer detection are being made. AIM: The aim of the study was to compare the relative expression levels of miRNAs in patients with bladder cancer. MATERIALS AND METHODS: Urine and serum samples were collected from patients with the diagnosis of bladder cancer (NMIBC 71%, MIBC 29%). We assessed expression of 4 miRNAs (106b-3p, 130b-3, 145- 3p and 199a-5p) using real-time PCR and double delta (ΔΔCt) method. The analysis was performed with the Mann-Whitney U test. RESULTS: miRNA 145-3p was significantly underexpressed in urine (p=0,0111) compared with control group, whereas in serum we did not find relevant differences between groups (p=0,0903). Overexpression was observed for miRNA 199a-5p tested in urine (p=0,0262) and for miRNA 106b-3p for both urine and serum (p=0,0262 and p=0,0149 respectively). For miR-130b-3 we did not find statistically significant differences neither for urine (p=0,6335) nor serum (p=0,2443). CONCLUSIONS: A correlation between the relative levels of expression for miRNA 106b-3p, 199a-5p and miRNA 145-3p was detected. We also observed differences between the results obtained for urine and serum. In the context of urinary cancers diagnosis urine seems to be more useful material than serum. We plan to continue our studies assessing expression levels of miRNA 106b-3 and miRNA 145-3p.

Molecular subtyping of bladder cancer using Kohonen self-organizing maps.

Kohonen self-organizing maps (SOMs) are unsupervised Artificial Neural Networks (ANNs) that are good for low-density data visualization. They easily deal with complex and nonlinear relationships between variables. We evaluated molecular events that characterize high- and low-grade BC pathways in the tumors from 104 patients. We compared the ability of statistical clustering with a SOM to stratify tumors according to the risk of progression to more advanced disease. In univariable analysis, tumor stage (log rank P = 0.006) and grade (P < 0.001), HPV DNA (P < 0.004), Chromosome 9 loss (P = 0.04) and the A148T polymorphism (rs 3731249) in CDKN2A (P = 0.02) were associated with progression. Multivariable analysis of these parameters identified that tumor grade (Cox regression, P = 0.001, OR.2.9 (95% CI 1.6-5.2)) and the presence of HPV DNA (P = 0.017, OR 3.8 (95% CI 1.3-11.4)) were the only independent predictors of progression. Unsupervised hierarchical clustering grouped the tumors into discreet branches but did not stratify according to progression free survival (log rank P = 0.39). These genetic variables were presented to SOM input neurons. SOMs are suitable for complex data integration, allow easy visualization of outcomes, and may stratify BC progression more robustly than hierarchical clustering.

EORTC risk tables - their usefulness in the assessment of recurrence and progression risk in non-muscle-invasive bladder cancer in Polish patients.

INTRODUCTION: The assessment of risk of recurrence and progression of bladder cancer (BC) is still rather difficult. We decided to check the rates of the changes mentioned above in the group of the Polish patients after a year-long observation and next to compare them with the results calculated in the European Organisation of Research and Treatment of Cancer (EORTC) risk tables. METHODS: The tested group consisted of 91 patients who underwent transurethral resection of bladder tumour (TURBT). When being diagnosed, 60 cases were in the pTa clinical stage, whereas 30 cases were in T1. The coexisting carcinoma in situ (CIS) was observed in four cases. On the basis of the scores obtained from the EORTC tables, the patients were divided into the groups of low, intermediate or high risk of disease recurrence and progression. RESULTS: Recurrence was noticed in 23 patients (25%), while progression was observed in 11 patients (12.1%). The rate of the observed recurrences proved to be lower than it had been predicted in all the groups, except for one of the intermediate-risk group (score 1- 4). Moreover, the rate of the progressions predicted according to the EORTC risk tables was higher in all the risk groups. CONCLUSIONS: It can be noticed that the rate of real recurrences is lower than expected, whereas the rate of the observed progressions is overestimated. Partly, it could be the result of using a relatively small group of patients for observation and applying a different method of treatment.