Limited utility of qPCR-based detection of tumor-specific circulating mRNAs in whole blood from clear cell renal cell carcinoma patients.

BACKGROUND: RNA sequencing data is providing abundant information about the levels of dysregulation of genes in various tumors. These data, as well as data based on older microarray technologies have enabled the identification of many genes which are upregulated in clear cell renal cell carcinoma (ccRCC) compared to matched normal tissue. Here we use RNA sequencing data in order to construct a panel of highly overexpressed genes in ccRCC so as to evaluate their RNA levels in whole blood and determine any diagnostic potential of these levels for renal cell carcinoma patients. METHODS: A bioinformatics analysis with Python was performed using TCGA, GEO and other databases to identify genes which are upregulated in ccRCC while being absent in the blood of healthy individuals. Quantitative Real Time PCR (RT-qPCR) was subsequently used to measure the levels of candidate genes in whole blood (PAX gene) of 16 ccRCC patients versus 11 healthy individuals. PCR results were processed in qBase and GraphPadPrism and statistics was done with Mann-Whitney U test. RESULTS: While most analyzed genes were either undetectable or did not show any dysregulated expression, two genes, CDK18 and CCND1, were paradoxically downregulated in the blood of ccRCC patients compared to healthy controls. Furthermore, LOX showed a tendency towards upregulation in metastatic ccRCC samples compared to non-metastatic. CONCLUSIONS: This analysis illustrates the difficulty of detecting tumor regulated genes in blood and the possible influence of interference from expression in blood cells even for genes conditionally absent in normal blood. Testing in plasma samples indicated that tumor specific mRNAs were not detectable. While CDK18, CCND1 and LOX mRNAs might carry biomarker potential, this would require validation in an independent, larger patient cohort.

Circular RNAs and Their Linear Transcripts as Diagnostic and Prognostic Tissue Biomarkers in Prostate Cancer after Prostatectomy in Combination with Clinicopathological Factors.

As new biomarkers, circular RNAs (circRNAs) have been largely unexplored in prostate cancer (PCa). Using an integrative approach, we aimed to evaluate the potential of circRNAs and their linear transcripts (linRNAs) to act as (i) diagnostic biomarkers for differentiation between normal and tumor tissue and (ii) prognostic biomarkers for the prediction of biochemical recurrence (BCR) after radical prostatectomy. In a first step, eight circRNAs (circATXN10, circCRIM1, circCSNK1G3, circGUCY1A2, circLPP, circNEAT1, circRHOBTB3, and circSTIL) were identified as differentially expressed via a genome-wide circRNA-based microarray analysis of six PCa samples. Additional bioinformatics and literature data were applied for this selection process. In total, 115 malignant PCa and 79 adjacent normal tissue samples were examined using robust RT-qPCR assays specifically established for the circRNAs and their linear counterparts. Their diagnostic and prognostic potential was evaluated using receiver operating characteristic curves, Cox regressions, decision curve analyses, and C-statistic calculations of prognostic indices. The combination of circATXN10 and linSTIL showed a high discriminative ability between malignant and adjacent normal tissue PCa. The combination of linGUCY1A2, linNEAT1, and linSTIL proved to be the best predictive RNA-signature for BCR. The combination of this RNA signature with five established reference models based on only clinicopathological factors resulted in an improved predictive accuracy for BCR in these models. This is an encouraging study for PCa to evaluate circRNAs and their linRNAs in an integrative approach, and the results showed their clinical potential in combination with standard clinicopathological variables.

Tissue-Based MicroRNAs as Predictors of Biochemical Recurrence after Radical Prostatectomy: What Can We Learn from Past Studies?

With the increasing understanding of the molecular mechanism of the microRNAs (miRNAs) in prostate cancer (PCa), the predictive potential of miRNAs has received more attention by clinicians and laboratory scientists. Compared with the traditional prognostic tools based on clinicopathological variables, including the prostate-specific antigen, miRNAs may be helpful novel molecular biomarkers of biochemical recurrence for a more accurate risk stratification of PCa patients after radical prostatectomy and may contribute to personalized treatment. Tissue samples from prostatectomy specimens are easily available for miRNA isolation. Numerous studies from different countries have investigated the role of tissue-miRNAs as independent predictors of disease recurrence, either alone or in combination with other clinicopathological factors. For this purpose, a PubMed search was performed for articles published between 2008 and 2017. We compiled a profile of dysregulated miRNAs as potential predictors of biochemical recurrence and discussed their current clinical relevance. Because of differences in analytics, insufficient power and the heterogeneity of studies, and different statistical evaluation methods, limited consistency in results was obvious. Prospective multi-institutional studies with larger sample sizes, harmonized analytics, well-structured external validations, and reasonable study designs are necessary to assess the real prognostic information of miRNAs, in combination with conventional clinicopathological factors, as predictors of biochemical recurrence.

Urinary miR-183 and miR-205 do not surpass PCA3 in urine as predictive markers for prostate biopsy outcome despite their highly dysregulated expression in prostate cancer tissue.

BACKGROUND: MicroRNAs (miRNAs) have shown to be promising novel biomarkers in various cancers. We aimed to translate the results of an own previous tissue-based miRNA profile of prostate carcinoma (PCa) with upregulated miR-183 and downregulated miR-205 into a urine-based testing procedure for diagnosis of PCa. METHODS: Urine sediments were prepared from urine samples collected after a standardized digital-rectal examination (DRE) of patients undergoing prostate biopsy with PSA (prostate-specific antigen) values <20 µg/L in consecutive order. According to the sample-size calculation (α=0.05, power=0.95), 38 patients each with PCa and without PCa were randomly enrolled in this study. PCA3 (prostate cancer associated 3) in urine as Food and Drug Administration-approved assay was determined as reference standard for comparison. The miRNAs were measured by RT-qPCR using TaqMan assays and normalized using different approaches. RESULTS: Both miRNAs were correlated to the mRNA PSA concentrations in the sediments indicating a relationship to the released prostate cells after DRE. However, they had no discriminating capacity between patients with and without PCa. In contrast, PCA3 clearly differentiated between these two patients groups. There was also no significant correlation between miRNAs and standard clinicopathologic variables like Gleason score and serum PSA. CONCLUSIONS: The data of our study show that miR-183 and miR-205 failed to detect early and aggressive PCa despite their highly dysregulated expression in cancer tissue. Our results and the critical evaluation of the few data of other studies raise serious doubts concerning the capability of urinary miRNAs to replace or improve PCA3 as predictive marker for prostate biopsy outcome.

Fatty acid binding proteins (FABPs) in prostate, bladder and kidney cancer cell lines and the use of IL-FABP as survival predictor in patients with renal cell carcinoma.

BACKGROUND: Fatty acid binding proteins (FABP) play an important role in carcinogenesis. Modified FABP expression patterns were described for prostate, bladder and for renal cell carcinoma. Studies on metabolic relationships and interactions in permanent cell lines allow a deeper insight into molecular processes. The aim of this study is therefore a systematic overview on mRNA and protein expressions of seven FABPs in frequently used urological cell lines. METHODS: Nine cell lines of renal carcinomas, seven of urinary bladder carcinomas, and five of prostate carcinomas were investigated. Quantitative RT-qPCR and western blotting were used to determine different FABPs. In addition, 46 paired cancerous and noncancerous tissue samples from nephrectomy specimen with renal cell carcinomas were investigated regarding the ileum FABP mRNA expression level and associated with survival outcome. RESULTS: General characteristics of all urological carcinoma cell lines were the expression of E-and IL-FABP on mRNA and protein level, while the expressions differed between the cell lines. The protein expression was not always congruent with the mRNA expression. Renal cell carcinoma cell lines showed expressions of L-, H- and B-FABP mRNA in addition to the general FABP expression in five out of the eight investigated cell lines. In bladder cancer cell lines, we additionally found the expression of A-FABP mRNA in six cell lines, while H-FABP was present only in three cell lines. In prostate cancer cell lines, a strong reduction of A- and E- FABP mRNA was observed. The expression of B-FABP mRNA and protein was observed only in the 22 RV-1 cells. IL-FABP mRNA was over-expressed in renal tumour tissue. The IL-FABP ratio was identified as an independent indicator of survival outcome. CONCLUSIONS: Distinctly different FABP expression patterns were observed not only between the cell lines derived from the three cancer types, but also between the cell lines from the same cancer. The FABP patterns in the cell lines do not always reflect the real situation in the tumours. These facts have to be considered in functional studies concerning the different FABPs.

Reference genes for the relative quantification of microRNAs in renal cell carcinomas and their metastases.

To obtain accurate results in miRNA expression changes between different sample sets using real-time quantitative polymerase chain reaction (RT-qPCR) analyses, normalization to reference genes that are stably expressed across the sample sets is generally used. A literature search of miRNA expression studies in renal cell carcinoma (RCC) proved that non-miRNAs such as small RNAs or mRNAs have most frequently been used without preceding validation of their suitability. In this study, the most stably expressed miRNAs were ascertained from microarray-based data of miRNA expression in nonmalignant and malignant samples from clear cell RCC and from corresponding distant RCC metastases using the geNorm and NormFinder algorithms. Validation experiments with RT-qPCR were performed for the four best-ranked miRNAs (miR-28, miR-103, miR-106a, miR-151) together with the small RNU6B, RNU44, and RNU48 mostly described in literature. miR-28, miR-103, miR-106a, and RNU48 were proved as the most stably expressed genes. miR-28 is recommended as normalizer if only a single reference gene can be used, while the combinations of miR-28 and miR-103 or of miR-28, miR-103, and miR-106a, respectively, are preferred. RNU6B most frequently used as normalizer in miRNA expression studies should be abandoned in order to avoid misleading results.

Diagnostic and prognostic implications of microRNA profiling in prostate carcinoma.

This study aimed to investigate the microRNA (miRNA) profile in prostate carcinoma tissue by microarray analysis and RT-qPCR, to clarify associations of miRNA expression with clinicopathologic data and to evaluate the potential of miRNAs as diagnostic and prognostic markers. Matched tumor and adjacent normal tissues were obtained from 76 radical prostatectomy specimens. Twenty-four tissue pairs were analyzed using human miRNA microarrays for 470 human miRNAs. Differentially expressed miRNAs were validated by TaqMan RT-qPCR using all 76 tissue pairs. The diagnostic potential of miRNAs was calculated by receiver operating characteristics analyses. The prognostic value was assessed in terms of biochemical recurrence using Kaplan-Meier and Cox regression analyses. Fifteen differentially expressed miRNAs were identified with concordant fold-changes by microarray and RT-qPCR analyses. Ten microRNAs (hsa-miR-16, hsa-miR-31, hsa-miR-125b, hsa-miR-145, hsa-miR-149, hsa-miR-181b, hsa-miR-184, hsa-miR-205, hsa-miR-221, hsa-miR-222) were downregulated and 5 miRNAs (hsa-miR-96, hsa-miR-182, hsa-miR-182, hsa-miR-183, hsa-375) were upregulated. Expression of 5 miRNAs correlated with Gleason score or pathological tumor stage. Already 2 microRNAs classified up to 84% of malignant and nonmalignant samples correctly. Expression of hsa-miR-96 was associated with cancer recurrence after radical prostatectomy and that prognostic information was confirmed by an independent tumor sample set from 79 patients. That was shown with hsa-miR-96 and the Gleason score as final variables in the Cox models build in the 2 patient sets investigated. Thus, differential miRNAs in prostate cancer are useful diagnostic and prognostic indicators. This study provides a solid basis for further functional analyses of miRNAs in prostate cancer.

KLK15 is a prognostic marker for progression-free survival in patients with radical prostatectomy.

In search of biomarkers for prostate cancer, we evaluated the expression of the human kallikrein-related peptidase KLK15 in samples of prostatic adenocarcinomas from radical prostatectomies. Twenty-five pairs of cancerous and adjacent normal prostatic tissue were selected by laser capture microdissection. The tissue was used for quantification of KLK15 mRNA by reverse-transcriptase polymerase chain reaction. Immunohistochemical expression of the KLK15 protein in 193 samples of prostatic adenocarcinoma was analysed in relation to clinicopathological parameters of the patients and disease progression. Expression of KLK15 correlated with the pathological tumour stage and Gleason score of the cases, both at mRNA and at protein level. While mRNA expression in the tumour was elevated, the protein level of KLK15 was reduced compared with adjacent normal tissue and to prostatic intraepithelial neoplasia. Univariate Kaplan-Meier analysis showed a significant association of dichotomised KLK15 levels with disease progression defined by prostate-specific antigen relapse (p = 0.001). Multivariate analysis according to the Cox proportional hazards regression model identified dichotomised KLK15 expression, corrected for the patient parameters age, preoperative prostate-specific antigen level, pathological tumour stage, Gleason score and surgical margin status, as an independent prognostic factor for poor outcome (inclusion model, hazard ratio 1.802, 95% confidence interval 1.037-3.132, p = 0.037). We suggest KLK15 as a new independent tumour marker for patients at risk for disease progression after radical prostatectomy.

Robust microRNA stability in degraded RNA preparations from human tissue and cell samples.

BACKGROUND: RNA integrity is the essential factor that determines the accuracy of mRNA transcript measurements obtained with quantitative real-time reverse-transcription PCR (RT-qPCR), but evidence is clearly lacking on whether this conclusion also applies to microRNAs (miRNAs). We evaluated this issue by comparative analysis of the dependence of miRNA and mRNA measurements on RNA integrity in renal and prostate samples, under both model and clinical conditions. METHODS: Samples of total RNA isolated from human renal tissue and Caki-2 cells, as well as from prostate tissue and LNCaP cells, were incubated at 80 degrees C for 5-240 min. We subsequently determined the RNA integrity number (RIN) and used RT-qPCR to measure various miRNAs (miR-141, miR-155, miR-200c, and miR-210 in renal samples, and miR-96, miR-130b, miR-149, miR-205, and miR-222 in prostate samples). We similarly measured mRNAs encoded by CDH16 (cadherin 16, KSP-cadherin), PPIA [peptidylprolyl isomerase A (cycophilin A)], and TBP (TATA box binding protein) in renal samples, and HIF1A [hypoxia-inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor)], HPRT1 (hypoxanthine phosphoribosyltransferase 1), and KLK3 (kallikrein-related peptidase 3; also known as PSA) in prostate samples. Additionally, we quantified selected miRNAs and mRNAs in samples of RNAs with different RIN values that we isolated from clinical samples. The effect of RIN on the miRNA and mRNA data was assessed by linear regression analysis and group comparison. RESULTS: The heat-incubation experiments of cell line and tissue RNAs showed that RIN values had negligible or no effect on miRNA results, whereas all mRNAs gradually decreased with decreasing RIN values. These findings were corroborated by our findings with clinical samples. CONCLUSIONS: Our results suggest the stability of miRNAs to be generally robust, which makes feasible accurate miRNA measurements with RT-qPCR, even in degraded RNA preparations for which reliable mRNA analyses are commonly inapplicable.

Instability of circular RNAs in clinical tissue samples impairs their reliable expression analysis using RT-qPCR: from the myth of their advantage as biomarkers to reality.

Background: Circular RNAs (circRNAs) are a new class of RNAs with medical significance. Compared to that of linear mRNA transcripts, the stability of circRNAs against degradation owing to their circular structure is considered advantageous for their use as biomarkers. As systematic studies on the stability of circRNAs depending on the RNA integrity, determined as RNA integrity number (RIN), in clinical tissue samples are lacking, we have investigated this aspect in the present study under model and clinical conditions. Methods: Total RNA isolated from kidney cancer tissue and cell lines (A-498 and HEK-293) with different RIN after thermal degradation was used in model experiments. Further, RNA isolated from kidney cancer and prostate cancer tissue collected under routine surgical conditions, representing clinical samples with RIN ranging from 2 to 9, were examined. Quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) analysis of several circRNAs (circEGLN3, circRHOBTB3, circCSNK1G3, circRNA4, and circRNA9), their corresponding linear counterparts, tissue-specific reference genes, and three microRNAs (as controls) was performed. The quantification cycles were converted into relative quantities and normalized to the expression of specific reference genes for the corresponding tissue. The effect of RIN on the expression of different RNA entities was determined using linear regression analysis, and clinical samples were classified into two groups based on RIN greater or lesser than 6. Results: The results of model experiments and clinical sample analyses showed that all relative circRNA expression gradually decreased with reduction in RIN values. The adverse effect of RIN was partially compensated after normalizing the data and limiting the samples to only those with RIN values > 6. Conclusions: Our results suggested that circRNAs are not stable in clinical tissue samples, but are subjected to degradative processes similar to mRNAs. This has not been investigated extensively in circRNA expression studies, and hence must be considered in future for obtaining reliable circRNA expression data. This can be achieved by applying the principles commonly used in mRNA expression studies.

MicroRNA profiling of clear cell renal cell cancer identifies a robust signature to define renal malignancy.

MicroRNAs are short single-stranded RNAs that are associated with gene regulation at the transcriptional and translational level. Changes in their expression were found in a variety of human cancers. Only few data are available on microRNAs in clear cell renal cell carcinoma (ccRCC). We performed genome-wide expression profiling of microRNAs using microarray analysis and quantification of specific microRNAs by TaqMan real-time RT-PCR. Matched malignant and non-malignant tissue samples from two independent sets of 12 and 72 ccRCC were profiled. The microarray-based experiments identified 13 over-expressed and 20 down-regulated microRNAs in malignant samples. Expression in ccRCC tissue samples compared with matched non-malignant samples measured by RT-PCR was increased on average by 2.7- to 23-fold for the hsa-miR-16, -452*, -224, -155 and -210, but decreased by 4.8- to 138-fold for hsa-miR-200b, -363, -429, -200c, -514 and -141. No significant associations between these differentially expressed microRNAs and the clinico-pathological factors tumour stage, tumour grade and survival rate were found. Nevertheless, malignant and non-malignant tissue could clearly be differentiated by their microRNA profile. A combination of miR-141 and miR-155 resulted in a 97% overall correct classification of samples. The presented differential microRNA pattern provides a solid basis for further validation, including functional studies.

Brain-type and liver-type fatty acid-binding proteins: new tumor markers for renal cancer?

BACKGROUND: Renal cell carcinoma (RCC) is the most common renal neoplasm. Cancer tissue is often characterized by altered energy regulation. Fatty acid-binding proteins (FABP) are involved in the intracellular transport of fatty acids (FA). We examined the level of brain-type (B) and liver-type (L) FABP mRNA and the protein expression profiles of both FABPs in renal cell carcinoma. METHODS: Paired tissue samples of cancerous and noncancerous kidney parts were investigated. Quantitative RT-PCR, immunohistochemistry and western blotting were used to determine B- and L-FABP in tumor and normal tissues. The tissue microarray (TMA) contained 272 clinico-pathologically characterized renal cell carcinomas of the clear cell, papillary and chromophobe subtype. SPSS 17.0 was used to apply crosstables (chi2-test), correlations and survival analyses. RESULTS: B-FABP mRNA was significantly up-regulated in renal cell carcinoma. In normal tissue B-FABP mRNA was very low or often not detectable. RCC with a high tumor grading (G3 + G4) showed significantly lower B-FABP mRNA compared with those with a low grading (G1 + G2). Western blotting analysis detected B-FABP in 78% of the cases with a very strong band but in the corresponding normal tissue it was weak or not detectable. L-FABP showed an inverse relationship for mRNA quantification and western blotting. A strong B-FABP staining was present in 52% of the tumor tissues contained in the TMA. In normal renal tissue, L-FABP showed a moderate to strong immunoreactivity in proximal tubuli. L-FABP was expressed at lower rates compared with the normal tissues in 30.5% of all tumors. There was no correlation between patient survival times and the staining intensity of both FABPs. CONCLUSION: While B-FABP is over expressed in renal cell carcinoma in comparison to normal renal tissues L-FABP appears to be reduced in tumor tissue. Although the expression behavior was not related to the survival outcome of the RCC patients, it can be assumed that these changes indicate fundamental alterations in the fatty metabolism in the RCC carcinogenesis. Further studies should identify the role of both FABPs in carcinogenesis, progression and with regard to a potential target in RCC.

A Novel Predictor Tool of Biochemical Recurrence after Radical Prostatectomy Based on a Five-MicroRNA Tissue Signature.

Within five to ten years after radical prostatectomy (RP), approximately 15-34% of prostate cancer (PCa) patients experience biochemical recurrence (BCR), which is defined as recurrence of serum levels of prostate-specific antigen >0.2 µg/L, indicating probable cancer recurrence. Models using clinicopathological variables for predicting this risk for patients lack accuracy. There is hope that new molecular biomarkers, like microRNAs (miRNAs), could be potential candidates to improve risk prediction. Therefore, we evaluated the BCR prognostic capability of 20 miRNAs, which were selected by a systematic literature review. MiRNA expressions were measured in formalin-fixed, paraffin-embedded (FFPE) tissue RP samples of 206 PCa patients by RT-qPCR. Univariate and multivariate Cox regression analyses were performed, to assess the independent prognostic potential of miRNAs. Internal validation was performed, using bootstrapping and the split-sample method. Five miRNAs (miR-30c-5p/31-5p/141-3p/148a-3p/miR-221-3p) were finally validated as independent prognostic biomarkers. Their prognostic ability and accuracy were evaluated using C-statistics of the obtained prognostic indices in the Cox regression, time-dependent receiver-operating characteristics, and decision curve analyses. Models of miRNAs, combined with relevant clinicopathological factors, were built. The five-miRNA-panel outperformed clinically established BCR scoring systems, while their combination significantly improved predictive power, based on clinicopathological factors alone. We conclude that this miRNA-based-predictor panel will be worth to be including in future studies.

Circular RNAs in Clear Cell Renal Cell Carcinoma: Their Microarray-Based Identification, Analytical Validation, and Potential Use in a Clinico-Genomic Model to Improve Prognostic Accuracy.

Circular RNAs (circRNAs) may act as novel cancer biomarkers. However, a genome-wide evaluation of circRNAs in clear cell renal cell carcinoma (ccRCC) has yet to be conducted. Therefore, the objective of this study was to identify and validate circRNAs in ccRCC tissue with a focus to evaluate their potential as prognostic biomarkers. A genome-wide identification of circRNAs in total RNA extracted from ccRCC tissue samples was performed using microarray analysis. Three relevant differentially expressed circRNAs were selected (circEGLN3, circNOX4, and circRHOBTB3), their circular nature was experimentally confirmed, and their expression-along with that of their linear counterparts-was measured in 99 malignant and 85 adjacent normal tissue samples using specifically established RT-qPCR assays. The capacity of circRNAs to discriminate between malignant and adjacent normal tissue samples and their prognostic potential (with the endpoints cancer-specific, recurrence-free, and overall survival) after surgery were estimated by C-statistics, Kaplan-Meier method, univariate and multivariate Cox regression analysis, decision curve analysis, and Akaike and Bayesian information criteria. CircEGLN3 discriminated malignant from normal tissue with 97% accuracy. We generated a prognostic for the three endpoints by multivariate Cox regression analysis that included circEGLN3, circRHOBT3 and linRHOBTB3. The predictive outcome accuracy of the clinical models based on clinicopathological factors was improved in combination with this circRNA-based signature. Bootstrapping as well as Akaike and Bayesian information criteria confirmed the statistical significance and robustness of the combined models. Limitations of this study include its retrospective nature and the lack of external validation. The study demonstrated the promising potential of circRNAs as diagnostic and particularly prognostic biomarkers in ccRCC patients.

High expression of KLK14 in prostatic adenocarcinoma is associated with elevated risk of prostate-specific antigen relapse.

OBJECTIVE: Reliable prognostic tools for prostate cancer are still needed and KLK14, a young member of the growing family of human kallikrein-related peptidases, has been estimated to become a new significant marker. It is the aim of this study to analyze the clinical value of immunohistochemical expression of KLK14 in prostate cancer tissue samples. METHODS: Protein expression of KLK14 was assessed immunohistochemically in 186 tissue samples from radical prostatectomies. Areas of normal prostatic tissue, of prostatic epithelial neoplasia and of prostatic adenocarcinoma were checked in relation to clinicopathological parameters of the patients. Expression of KLK14 mRNA was quantified in 25 matches of normal and cancerous prostatic tissue, collected by laser capture microdissection. Real-time reverse transcriptase polymerase chain reaction analysis was used to supplement the immunohistochemical data. RESULTS: Expression of the KLK14 protein correlated with the pathological tumor status in prostate cancer and was associated with disease progression defined by prostate-specific antigen relapse in univariate Kaplan-Meier analysis. The multivariate Cox proportional hazards regression model proved KLK14 to be an independent prognostic factor in prostate cancer. CONCLUSION: In conclusion, we consider KLK14 to be a suitable prognostic marker for the detection of cases at risk of disease progression after radical prostatectomy.

ADAM9 is highly expressed in renal cell cancer and is associated with tumour progression.

BACKGROUND: A Disintegrin And Metalloprotease (ADAM) 9 has been implicated in tumour progression of various solid tumours, however, little is known about its role in renal cell carcinoma. We evaluated the expression of ADAM9 on protein and transcript level in a clinico-pathologically characterized renal cell cancer cohort. METHODS: 108 renal cancer cases were immunostained for ADAM9 on a tissue-micro-array. For 30 additional cases, ADAM9 mRNA of microdissected tumour and normal tissue was analyzed via quantitative RT-PCR. SPSS 14.0 was used to apply crosstables (Fisher's exact test and chi2-test), correlations and univariate as well as multivariate survival analyses. RESULTS: ADAM9 was significantly up-regulated in renal cancer in comparison to the adjacent normal tissue on mRNA level. On protein level, ADAM9 was significantly associated with higher tumour grade, positive nodal status and distant metastasis. Furthermore, ADAM9 protein expression was significantly associated with shortened patient survival in the univariate analysis. CONCLUSION: ADAM9 is strongly expressed in a large proportion of renal cell cancers, concordant with findings in other tumour entities. Additionally, ADAM9 expression is significantly associated with markers of unfavourable prognosis. Whether the demonstrated prognostic value of ADAM9 is independent from other tumour parameters will have to be verified in larger study cohorts.

Prevention of tick bites and protection against tick-borne diseases in south-eastern Poland.

The objective of this survey was to obtain information on the level of knowledge of prophylactic behaviour as a protection against fundamental tick-borne diseases among inhabitants of south-eastern Poland, and to determine whether the professions that increase the probability of tick contact, as well as previous contact with ticks, influenced the knowledge on tick-borne diseases and prophylactic behaviour of the respondents. The questionnaire included questions about gender of respondents, their place of residence and preventive behaviour against tick bite. The respondents were asked about the occupational hazard of working in tick infested areas, history of tick bites, awareness of diseases transmitted by ticks and the sources of their knowledge. The research was carried out in 2005. The most frequent preventive measures used were tick repellents (38 % ) and wearing protective clothing (35 % ). According to the questionnaire survey, 21 % of respondents did not take any preventive measures. The practice and form of prophylaxis was associated with the place of residence. In contrast to individuals from rural areas, a relatively low percentage of urban inhabitants used simple methods for protecting themselves against tick bites. Our survey indicates that the awareness of the Lublin region inhabitants concerning methods of tick bite prevention is still low and greater efforts are needed in promoting basic methods of protection and disseminating knowledge about these arthropods.

miR-9-5p in Nephrectomy Specimens is a Potential Predictor of Primary Resistance to First-Line Treatment with Tyrosine Kinase Inhibitors in Patients with Metastatic Renal Cell Carcinoma.

Approximately 20⁻30% of patients with metastatic renal cell carcinoma (mRCC) in first-line treatment with tyrosine kinase inhibitors (TKIs) do not respond due to primary resistance to this drug. At present, suitable robust biomarkers for prediction of a response are not available. Therefore, the aim of this study was to evaluate a panel of microRNAs (miRNAs) in nephrectomy specimens for use as predictive biomarkers for TKI resistance. Archived formalin-fixed, paraffin embedded nephrectomy samples from 60 mRCC patients treated with first-line TKIs (sunitinib, n = 51; pazopanib, n = 6; sorafenib, n = 3) were categorized into responders and non-responders. Using the standard Response Evaluation Criteria in Solid Tumors, patients with progressive disease within 3 months after the start of treatment with TKI were considered as non-responders and those patients with stable disease and complete or partial response under the TKI treatment for at least 6 months as responders. Based on a miRNA microarray expression profile in the two stratified groups of patients, seven differentially expressed miRNAs were validated using droplet digital reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) assays in the two groups. Receiver operating characteristic curve analysis and binary logistic regression of response prediction were performed. MiR-9-5p and miR-489-3p were able to discriminate between the two groups. MiR-9-5p, as the most significant miRNA, improved the correct prediction of primary resistance against TKIs in comparison to that of conventional clinicopathological variables. The results of the decision curve analyses, Kaplan-Meier analyses and Cox regression analyses confirmed the potential of miR-9-5p in the prediction of response to TKIs and the prediction of progression-free survival after the initiation of TKI treatment.

In search of suitable reference genes for gene expression studies of human renal cell carcinoma by real-time PCR.

BACKGROUND: Housekeeping genes are commonly used as endogenous reference genes for the relative quantification of target genes in gene expression studies. No conclusive systematic study comparing the suitability of different candidate reference genes in clear cell renal cell carcinoma has been published to date. To remedy this situation, 10 housekeeping genes for normalizing purposes of RT-PCR measurements already recommended in various studies were examined with regard to their usefulness as reference genes. RESULTS: The expression of the potential reference genes was examined in matched malignant and non-malignant tissue specimens from 25 patients with clear cell renal cell carcinoma. Quality assessment of isolated RNA performed with a 2100 Agilent Bioanalyzer showed a mean RNA integrity number of 8.7 for all samples. The between-run variations related to the crossing points of PCR reactions of a control material ranged from 0.17% to 0.38%. The expression of all genes did not depend on age, sex, and tumour stage. Except the genes TATA box binding protein (TBP) and peptidylprolyl isomerase A (PPIA), all genes showed significant differences in expression between malignant and non-malignant pairs. The expression stability of the candidate reference genes was additionally controlled using the software programs geNorm and NormFinder. TBP and PPIA were validated as suitable reference genes by normalizing the target gene ADAM9 using these two most stably expressed genes in comparison with up- and down-regulated housekeeping genes of the panel. CONCLUSION: Our study demonstrated the suitability of the two housekeeping genes PPIA and TBP as endogenous reference genes when comparing malignant tissue samples with adjacent normal tissue samples from clear cell renal cell carcinoma. Both genes are recommended as reference genes for relative gene quantification in gene profiling studies either as single gene or preferably in combination.

ADAM8 expression in prostate cancer is associated with parameters of unfavorable prognosis.

Gene products of the A disintegrin and metalloprotease (ADAM) family are critically involved in carcinogenesis and tumor progression of various solid tumors. Little is known about ADAM8 in prostate cancer. In our quest for novel diagnostic tissue markers of prostate cancer, we aimed to evaluate the expression of ADAM8 in prostate cancer and to correlate it with clinicopathological parameters. One hundred twenty-eight clinicopathologically characterized prostate cancer patients, with available follow-up data, were immunostained for ADAM8. Additionally, ADAM8 mRNA expression was quantified by real-time reverse transcription polymerase chain reaction (n = 59). ADAM8 protein expression was significantly associated with higher pT status, positive nodal status, and higher Gleason scores. Still, a significant prognostic value for the prostate-specific antigen relapse-free survival of ADAM8 could not be demonstrated. The differentiality of ADAM8 expression on protein and on mRNA level was low and partially inconclusive. Therefore, despite of its significant association with conventional parameters of an unfavorable prognosis, ADAM8 adds only limited information to the conventional histopathological assessment of prostate cancer.