Hellmuth-Alexander Meyer

Reference miRNAs for miRNAome Analysis of Urothelial Carcinomas

Background/Objective Reverse transcription quantitative real-time PCR (RT-qPCR) is widely used in microRNA (miRNA) expression studies on cancer. To compensate for the analytical variability produced by the multiple steps of the method, relative quantification of the measured miRNAs is required, which is based on normalization to endogenous reference genes. No study has been performed so far on reference miRNAs for normalization of miRNA expression in urothelial carcinoma. The aim of this study was to identify suitable reference miRNAs for miRNA expression studies by RT-qPCR in urothelial carcinoma. Methods Candidate reference miRNAs were selected from 24 urothelial carcinoma and normal bladder tissue samples by miRNA microarrays. The usefulness of these candidate reference miRNAs together with the commonly for normalization purposes used small nuclear RNAs RNU6B, RNU48, and Z30 were thereafter validated by RT-qPCR in 58 tissue samples and analyzed by the algorithms geNorm, NormFinder, and BestKeeper. Principal Findings Based on the miRNA microarray data, a total of 16 miRNAs were identified as putative reference genes. After validation by RT-qPCR, miR-101, miR-125a-5p, miR-148b, miR-151-5p, miR-181a, miR-181b, miR-29c, miR-324-3p, miR-424, miR-874, RNU6B, RNU48, and Z30 were used for geNorm, NormFinder, and BestKeeper analyses that gave different combinations of recommended reference genes for normalization. Conclusions The present study provided the first systematic analysis for identifying suitable reference miRNAs for miRNA expression studies of urothelial carcinoma by RT-qPCR. Different combinations of reference genes resulted in reliable expression data for both strongly and less strongly altered miRNAs. Notably, RNU6B, which is the most frequently used reference gene for miRNA studies, gave inaccurate normalization. The combination of four (miR-101, miR-125a-5p, miR-148b, and miR-151-5p) or three (miR-148b, miR-181b, and miR-874,) reference miRNAs is recommended for normalization.

The FUSE binding proteins FBP1 and FBP3 are potential c-myc regulators in renal, but not in prostate and bladder cancer

BackgroundThe three far-upstream element (FUSE) binding proteins (FBP1, FBP2, and FBP3) belong to an ancient family of single-stranded DNA binding proteins which are required for proper regulation of the c-myc proto-oncogene. Whereas it is known that c-myc alterations play a completely different role in various carcinomas of the urogenital tract, the relevance of FBPs is unclear.MethodsFBP1, FBP3 and c-myc expression was studied in 105 renal cell, 95 prostate and 112 urinary bladder carcinomas by immunohistochemistry using tissue microarrays.ResultsHigh rates of FBP1 and FBP3 expression were observed in all cancer types. There was a concomitant up-regulation of FBP1 and FBP3 in renal cell and prostate carcinomas (p < 0.001 both). C-myc expression was detectable in 21% of prostate, 30% of renal and 34% of urothelial carcinomas. Interestingly, strong FBP1 and FBP3 expression was associated with c-myc up-regulation in clear cell renal cell carcinomas (p < 0.001 and 0.09 resp.), but not in bladder or prostate cancer.ConclusionThe correlation between FBP1/FBP3, c-myc and high proliferation rate in renal cell carcinoma provides strong in vivo support for the suggested role of FBP1 and FBP3 as activators of c-myc. The frequent up-regulation of FBP1 and FBP3 in urothelial and prostate carcinoma suggests that FBPs also have an important function in gene regulation of these tumors.

An artificial neural network for five different assay systems of prostate-specific antigen in prostate cancer diagnostics

To compare separate prostate‐specific antigen (PSA) assay‐specific artificial neural networks (ANN) for discrimination between patients with prostate cancer (PCa) and no evidence of malignancy (NEM).

Reduced Serum Selenoprotein P Concentrations in German Prostate Cancer Patients

Selenium (Se) is essentially needed for the biosynthesis of selenoproteins. Low Se intake causes reduced selenoprotein biosynthesis and constitutes a risk factor for tumorigenesis. Accordingly, some Se supplementation trials have proven effective to reduce prostate cancer risk, especially in poorly supplied individuals. Because Se metabolism is controlled by selenoprotein P (SEPP), we have tested whether circulating SEPP concentrations correlate to prostate cancer stage and grade. A total of 190 men with prostate cancer (n = 90) and “no evidence of malignancy” (NEM; n = 100) histologically confirmed by prostate biopsy were retrospectively analyzed for established tumor markers and for their Se and SEPP status. Prostate specific antigen (PSA), free PSA, total Se, and SEPP concentrations were determined from serum samples and compared with clinicopathologic parameters. The diagnostic performance was analyzed with receiver operating characteristic curves. Median Se and SEPP concentrations differed significantly (P < 0.001) between the groups. Median serum Se concentrations in the 25th to 75th percentile were 95.9 μg/L (82-117.9) in NEM patients and 81.4 μg/L (67.9-98.4) in prostate cancer patients. Corresponding serum SEPP concentrations were 3.4 mg/L (1.9-5.6) in NEM and 2.9 mg/L (1.1-5.5) in prostate cancer patients. The area under the curve (AUC) of a marker combination with age, PSA, and percent free PSA (%fPSA) in combination with the SEPP concentration, yielded the highest diagnostic value (AUC 0.80) compared with the marker combination without SEPP (AUC 0.77) or %fPSA (AUC 0.76). We conclude that decreased SEPP concentration in serum might represent an additional valuable marker for prostate cancer diagnostics.(Cancer Epidemiol Biomarkers Prev 2009;18(9):2386–90)

Different prostate-specific antigen assays give different results on the same blood sample: an obstacle to recommending uniform limits for prostate biopsies

To show the effect of different results for total prostate specific antigen (tPSA) and percentage free/total PSA (%fPSA) obtained with different assays for differentiating between benign and malignant prostate diseases.

Between-Method Differences in Prostate-Specific Antigen Assays Affect Prostate Cancer Risk Prediction by Nomograms

BACKGROUND To date, no published nomogram for prostate cancer (PCa) risk prediction has considered the between-method differences associated with estimating concentrations of prostate-specific antigen (PSA). METHODS Total PSA (tPSA) and free PSA were measured in 780 biopsy-referred men with 5 different assays. These data, together with other clinical parameters, were applied to 5 published nomograms that are used for PCa detection. Discrimination and calibration criteria were used to characterize the accuracy of the nomogram models under these conditions. RESULTS PCa was found in 455 men (58.3%), and 325 men had no evidence of malignancy. Median tPSA concentrations ranged from 5.5 μg/L to 7.04 μg/L, whereas the median percentage of free PSA ranged from 10.6% to 16.4%. Both the calibration and discrimination of the nomograms varied significantly across different types of PSA assays. Median PCa probabilities, which indicate PCa risk, ranged from 0.59 to 0.76 when different PSA assays were used within the same nomogram. On the other hand, various nomograms produced different PCa probabilities when the same PSA assay was used. Although the ROC curves had comparable areas under the ROC curve, considerable differences were observed among the 5 assays when the sensitivities and specificities at various PCa probability cutoffs were analyzed. CONCLUSIONS The accuracy of the PCa probabilities predicted according to different nomograms is limited by the lack of agreement between the different PSA assays. This difference between methods may lead to unacceptable variation in PCa risk prediction. A more cautious application of nomograms is recommended.

Improved prostate cancer detection with a human kallikrein 11 and percentage free PSA-based artificial neural network

Abstract Human kallikrein 11 (hK11) was evaluated in a percentage free PSA-based artificial neural network (ANN) to reduce unnecessary prostate biopsies. Serum samples from 357 patients with (n=132) and without (n=225) prostate cancer (PCa) were analyzed and ANN models were constructed and compared to all parameters. The discriminatory power of hK11 was lower than that of PSA, but receiver operator characteristic (ROC) analyses demonstrated significantly larger areas under the curves for the ANN compared to all other parameters. ANNs with hK11 may lead to a further reduction in unnecessary prostate biopsies, especially when analyzing patients with less than 15% free PSA.

Integrated microRNA and mRNA Signature Associated with the Transition from the Locally Confined to the Metastasized Clear Cell Renal Cell Carcinoma Exemplified by miR-146-5p

Background MicroRNAs (miRNAs) regulate gene expression by interfering translation or stability of target transcripts. This interplay between miRNA and their mRNA has been proposed as an important process in cancer development and progression. We have investigated molecular networks impacted by predicted mRNA targets of differentially expressed miRNAs in patients with clear cell renal cell carcinoma (ccRCC) diagnosed with or without metastasis. Material and Methods miRNA and mRNA microarray expression profiles derived from primary ccRCC from patients with (16 samples) or without diagnosed metastasis (22 samples) were used to identify anti-correlated miRNA-mRNA interaction in ccRCC. For this purpose, Ingenuity pathway analysis microRNA Target Filter, which enables prioritization of experimentally validated and predicted mRNA targets was used. By applying an expression pairing tool, the analysis was focused on targets exhibiting altered expression in our analysis, finding miRNAs and their target genes with opposite or same expression. The resulting identified interactions were revalidated by RT-qPCR in another cohort of ccRCC patients. A selection of the predicted miRNA-mRNA interactions was tested by functional analyses using miRNA knockdown and overexpression experiments in renal cancer cell lines. Results Among the significantly differentially expressed miRNAs, we have identified three miRNAs (miR-146a-5p, miR-128a-3p, and miR-17-5p) that were upregulated in primary tumors from patients without metastasis and downregulated in primary tumors from patients with metastasis. We have further identified mRNA targets, which expression were inversely correlated to these 3 miRNAs, and have been previously experimentally demonstrated in cancer setting in humans. Specifically, we showed that CXCL8/IL8, UHRF1, MCM10, and CDKN3 were downregulated and targeted by miR-146a-5p. The interaction between miR-146a-5p and their targets CXCL8 and UHRF1 was validated in cell culture experiments. Conclusions We identified novel target genes of dysregulated miRNAs, which are involved in the transition from primary RCC without metastases into tumors generating distant metastasis.

Effects of Systemic versus Local Administration of Corticosteroids on Mucosal Tolerance

Respiratory exposure to allergen induces T cell tolerance and protection against the development of airway hyperactivity in animal models of asthma. Whereas systemic administration of dexamethasone during the delivery of respiratory Ag has been suggested to prevent the development of mucosal tolerance, the effects of local administration of corticosteroids, first-line treatment for patients with bronchial asthma, on mucosal tolerance remain unknown. To analyze the effects of systemic versus local administration of different types of corticosteroids on the development of mucosal tolerance, mice were exposed to respiratory allergen to induce mucosal tolerance with or without systemic or intranasal application of different doses of dexamethasone or prednisolone. After the induction of mucosal tolerance, proliferation of T cells was inhibited in tolerized mice, whereas systemic applications of corticosteroids restored T cell proliferation and secretion of Th2 cytokines. In contrast, inhaled corticosteroids showed no effect on both T cell proliferation and cytokine secretion. In addition, mice systemically treated with corticosteroids showed an increased airway hyperactivity with a significant lung inflammation, but also an increased T effector cells/regulatory T cells ratio in the second lymphoid organs when compared with mice that receive corticosteroids by inhalation. These results demonstrate that local administration of corticosteroids has no effect on the development of immune tolerance in contrast to systemically applied corticosteroids. Furthermore, although different concentrations of corticosteroids are administered to patients, our results demonstrated that the route of administration rather than the doses affects the effect of corticosteroids on respiratory tolerance induction. Considering the broad application of corticosteroids in patients with allergic disease and asthma, the route of administration of steroid substances seems crucial in terms of treatment and potential side effects. These findings may help elucidate the apparently contradicting results of corticosteroid treatment in allergic diseases.

Value of Prostate Specific Antigen Density and Percent Free Prostate Specific Antigen for Prostate Cancer Prognosis

PURPOSE Limited data exist on the relationship of percent free prostate specific antigen and prostate specific antigen density with prostate cancer prognosis. Therefore, we compared percent free prostate specific antigen and prostate specific antigen density with prostate specific antigen, Gleason sum and stage to predict prostate cancer prognosis in a large cohort using a single prostate specific antigen and free prostate specific antigen assay. MATERIALS AND METHODS Between 1999 and 2007 a total of 1,656 patients with prostate cancer underwent laparoscopic radical prostatectomy at the Charité Berlin. There were 322 patients excluded from analysis for a variety of reasons. The final 1,334 patients had prostate specific antigen, free prostate specific antigen, prostate volume and complete pathological analysis available. RESULTS Median followup was 60.3 months (range 0.2 to 135). Median age (63 years, range 43 to 75) did not differ between the 1,092 patients without and the 242 with biochemical recurrence (p = 0.956), but prostate volume, prostate specific antigen and percent free prostate specific antigen differed significantly (p <0.0001). While prostate specific antigen and prostate specific antigen density increased significantly in patients with Gleason less than 7, 7 and greater than 7 tumors, percent free prostate specific antigen decreased significantly (p <0.0001). Prostate specific antigen, percent free prostate specific antigen and prostate specific antigen density differed significantly between pT2 and pT3 tumors, and between patients with vs without positive surgical margins. On univariate analysis Gleason sum, pathological stage, positive surgical margin, total prostate specific antigen, percent free prostate specific antigen and prostate specific antigen density were predictors of biochemical recurrence-free survival. Multivariate Cox regression analysis identified Gleason sum, pathological stage, positive surgical margin and prostate specific antigen density as independent predictors of biochemical recurrence-free survival, while percent free prostate specific antigen and total prostate specific antigen failed to be significant. CONCLUSIONS Few models for prostate cancer prognosis include prostate specific antigen density. There is substantial value in prostate specific antigen density but not in percent free prostate specific antigen for improving prostate cancer prognosis and biochemical recurrence prediction.