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1.  Piwi-interacting RNAs as novel prognostic markers in clear cell renal cell carcinomas 
Piwi-interacting RNAs (piRNAs) are small RNAs of 27–30 nucleotides mapping to transposons or clustering in repeat genomic regions. Preliminary studies suggest an important role in cancerogenesis. This study is the first one investigating their prognostic impact in clear cell renal cell cancer (ccRCC) patients.
Three piRNAs (piR-30924, piR-57125, and piR-38756) selected on the basis of initial piRNA microarray analyses were determined using RT-qPCR in non-metastatic (n = 76) and metastatic (n = 30) ccRCC tissue at the time of nephrectomy in comparison to normal renal tissue (n = 77) and tissue from distant ccRCC metastases (n = 13). Primary clinical end points were recurrence-free and overall survival.
piR-57125 showed lower expression in metastatic than in non-metastatic tumors, whereas the expression of piR-30924 and piR-38756 increased in metastatic tumors. The higher expression of piR-30924 and piR-38756 as well as the lower expression of piR-57125 in metastatic primary tumors were significantly associated with tumor recurrence and overall survival. Multivariate Cox regression analyses revealed both piR-30924 and piR-57125 as independent prognostic predictors. This impact was even more pronounced in non-metastatic patients.
This study demonstrates that the expression levels of these piRNAs in primary non-metastatic and metastatic ccRCC tissue can serve as potential prognostic biomarkers in combination with clinicopathological factors.
Electronic supplementary material
The online version of this article (doi:10.1186/s13046-015-0180-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4467205  PMID: 26071182
Clear cell renal cell carcinoma; piRNA; Prognosis; Metastasis; Recurrence; Overall survival
2.  Risk prediction models for biochemical recurrence after radical prostatectomy using prostate-specific antigen and Gleason score 
Asian Journal of Andrology  2014;16(6):897-901.
Many computer models for predicting the risk of prostate cancer have been developed including for prediction of biochemical recurrence (BCR). However, models for individual BCR free probability at individual time-points after a BCR free period are rare. Follow-up data from 1656 patients who underwent laparoscopic radical prostatectomy (LRP) were used to develop an artificial neural network (ANN) to predict BCR and to compare it with a logistic regression (LR) model using clinical and pathologic parameters, prostate-specific antigen (PSA), margin status (R0/1), pathological stage (pT), and Gleason Score (GS). For individual BCR prediction at any given time after operation, additional ANN, and LR models were calculated every 6 months for up to 7.5 years of follow-up. The areas under the receiver operating characteristic (ROC) curve (AUC) for the ANN (0.754) and LR models (0.755) calculated immediately following LRP, were larger than that for GS (AUC: 0.715; P = 0.0015 and 0.001), pT or PSA (AUC: 0.619; P always <0.0001) alone. The GS predicted the BCR better than PSA (P = 0.0001), but there was no difference between the ANN and LR models (P = 0.39). Our ANN and LR models predicted individual BCR risk from radical prostatectomy for up to 10 years postoperative. ANN and LR models equally and significantly improved the prediction of BCR compared with PSA and GS alone. When the GS and ANN output values are combined, a more accurate BCR prediction is possible, especially in high-risk patients with GS ≥7.
PMCID: PMC4236336  PMID: 25130472
artificial neural network; prostate cancer; recurrence
3.  Identification of a DNA methylation signature to predict disease-free survival in locally advanced rectal cancer 
Oncotarget  2014;5(18):8123-8135.
In locally advanced rectal cancer a preoperative predictive biomarker is necessary to adjust treatment specifically for those patients expected to suffer relapse. We applied whole genome methylation CpG island array analyses to an initial set of patients (n=11) to identify differentially methylated regions (DMRs) that separate a good from a bad prognosis group. Using a quantitative high-resolution approach, candidate DMRs were first validated in a set of 61 patients (test set) and then confirmed DMRs were further validated in additional independent patient cohorts (n=71, n=42). We identified twenty highly discriminative DMRs and validated them in the test set using the MassARRAY technique. Ten DMRs could be confirmed which allowed separation into prognosis groups (p=0.0207, HR=4.09). The classifier was validated in two additional cohorts (n=71, p=0.0345, HR=3.57 and n=42, p=0.0113, HR=3.78). Interestingly, six of the ten DMRs represented regions close to the transcriptional start sites of genes which are also marked by the Polycomb Repressor Complex component EZH2. In conclusion we present a classifier comprising 10 DMRs which predicts patient prognosis with a high degree of accuracy. These data may now help to discriminate between patients that may respond better to standard treatments from those that may require alternative modalities.
PMCID: PMC4226671  PMID: 25261372
4.  The Antiapoptotic Function of miR-96 in Prostate Cancer by Inhibition of FOXO1 
PLoS ONE  2013;8(11):e80807.
microRNAs (miRNAs) are small molecules that regulate gene expression posttranscriptionally. In a previous study, we identified miR-96 to be upregulated in prostate cancer specimens in comparison to normal adjacent tissue and to be an independent marker of biochemical relapse in a multivariate prediction model. Therefore, we investigated the functional role of miR-96 in prostate carcinogenesis. LNCaP and DU145 prostate cancer cells were transiently transfected with miR-96 precursors and phenotypic changes were analyzed. The miR-96 increased proliferation and impaired apoptosis induced by camptothecine in these cells. In silico target prediction analysis identified FOXO1 as potential pro-apoptotic miR-96 target. miR-96 was able to bind to both bindings sites in the FOXO1 3’ UTR in a luciferase reporter gene assay. Overexpression of miR-96 in LNCaP cells resulted in a reduced FOXO1 expression. Overexpression of FOXO1 induced a strong apoptotic phenotype that was partially rescued by coexpression of miR-96. RT-qPCR and immunohistochemistry of 69 prostate cancer specimens revealed a downregulation of FOXO1 and an inverse correlation of miR-96 and FOXO1 protein expression. In conclusion, we show that miR-96 can regulate apoptosis in prostate cancer, by inhibiting the FOXO1 transcription factor.
PMCID: PMC3834337  PMID: 24260486
5.  Renal cell neoplasias: reversion-inducing cysteine-rich protein with Kazal motifs discriminates tumor subtypes, while extracellular matrix metalloproteinase inducer indicates prognosis 
Matrix metalloproteinases can promote invasion and metastasis, which are very frequent in renal cell carcinoma even at the time of diagnosis. Knowing the reversion-inducing cysteine-rich protein with Kazal motifs (RECK) as an inhibitor of matrix metalloproteinases and the extracellular matrix metalloproteinase inducer (EMMPRIN) protein as inducer, we aimed to determine their expression, localization and possible antagonistic action in the pathogenesis and progression of renal cell tumors in a retrospective study.
Tumor and adjacent normal tissues of 395 nephrectomized patients were immunostained for RECK and EMMPRIN on a tissue microarray.
RECK strongly decreased in renal cell carcinoma compared to normal counterparts (Wilcoxon signed rank test, P < 0.001), and it discriminated tumor entities showing the highest expression in oncocytomas. EMMPRIN, however, could be significantly correlated to pT stage and Fuhrman grading (Spearman’s correlation coefficient rs = 0.289 and rs = 0.382, respectively). Higher expression of EMMPRIN was associated with decreased overall survival in Kaplan-Meier analysis (P < 0.001), and the EMMPRIN level could independently predict survival for cases without metastasis and involvement of lymph nodes. Decreased RECK expression was confirmed by Western blotting in tissue of eight normal/tumor matches of patients after radical nephrectomy, whereas the EMMPRIN pattern appeared to be heterogeneous.
We propose RECK down regulation in renal cell carcinoma to be an early event that facilitates tumor formation and progression. EMMPRIN, however, as a prognostic tumor marker, increases only when aggressiveness is proceeding and could add an additional step to invasive properties of renal cell carcinoma.
PMCID: PMC3853196  PMID: 24131772
RECK; EMMPRIN; Renal cell carcinoma; TMA
6.  A New Algorithm for Integrated Analysis of miRNA-mRNA Interactions Based on Individual Classification Reveals Insights into Bladder Cancer 
PLoS ONE  2013;8(5):e64543.
MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. It has been proposed that miRNAs play an important role in cancer development and progression. Their ability to affect multiple gene pathways by targeting various mRNAs makes them an interesting class of regulators.
Methodology/Principal Findings
We have developed an algorithm, Classification based Analysis of Paired Expression data of RNA (CAPE RNA), which is capable of identifying altered miRNA-mRNA regulation between tissues samples that assigns interaction states to each sample without preexisting stratification of groups. The distribution of the assigned interaction states compared to given experimental groups is used to assess the quality of a predicted interaction. We demonstrate the applicability of our approach by analyzing urothelial carcinoma and normal bladder tissue samples derived from 24 patients. Using our approach, normal and tumor tissue samples as well as different stages of tumor progression were successfully stratified. Also, our results suggest interesting differentially regulated miRNA-mRNA interactions associated with bladder tumor progression.
The need for tools that allow an integrative analysis of microRNA and mRNA expression data has been addressed. With this study, we provide an algorithm that emphasizes on the distribution of samples to rank differentially regulated miRNA-mRNA interactions. This is a new point of view compared to current approaches. From bootstrapping analysis, our ranking yields features that build strong classifiers. Further analysis reveals genes identified as differentially regulated by miRNAs to be enriched in cancer pathways, thus suggesting biologically interesting interactions.
PMCID: PMC3663800  PMID: 23717626
7.  Mutated KRAS Results in Overexpression of DUSP4, a MAP-Kinase Phosphatase, and SMYD3, a Histone Methyltransferase, in Rectal Carcinomas 
Genes, chromosomes & cancer  2010;49(11):1024-1034.
Mutations of the KRAS oncogene are predictive for resistance to treatment with antibodies against the epithelial growth factor receptor in patients with colorectal cancer. Overcoming this therapeutic dilemma could potentially be achieved by the introduction of drugs that inhibit signaling pathways that are activated by KRAS mutations. To identify comprehensively such signaling pathways we profiled pretreatment biopsies and normal mucosa from 65 patients with locally advanced rectal cancer - 30 of which carried mutated KRAS - using global gene expression microarrays. By comparing all tumor tissues exclusively to matched normal mucosa, we could improve assay sensitivity, and identified a total of 22,297 features that were differentially expressed (adjusted P-value <0.05) between normal mucosa and cancer, including several novel potential rectal cancer genes. We then used this comprehensive description of the rectal cancer transcriptome as the baseline for identifying KRAS-dependent alterations. The presence of activating KRAS mutations is significantly correlated to an upregulation of 13 genes (adjusted P-value <0.05), among them DUSP4, a MAP-kinase phosphatase, and SMYD3, a histone methyltransferase. Inhibition of the expression of both genes has previously been shown using the MEK1-inhibitor PD98059 and the antibacterial compound Novobiocin, respectively. These findings suggest a potential approach to overcome resistance to treatment with antibodies against the epithelial growth factor receptor in patients with KRAS-mutant rectal carcinomas.
PMCID: PMC3535184  PMID: 20725992
8.  Clinical judgment is the most important element in overhydration assessment of chronic hemodialysis patients 
The assessment of hydration status remains a challenging task in hemodialysis (HD) management. There are only limited data available on the relevance of clinical decisions in the estimation of dialysis overhydration (OH). The objective of this study was to examine the significance of clinical judgment in the assessment of pre-dialysis OH.
We compared the performance of three methods of OH assessment: (1) clinical judgment guided by a single clinical examination with (2) multifrequency bioimpedance analysis (BIA) and (3) complex systematic clinical approach. We additionally studied the associations of these methods with selected laboratory and imaging parameters.
Any of the single parameters alone reached a sufficient level of accuracy for reliable prediction of OH. Clinical judgment was the single most important factor in OH estimation, and also had the highest contribution when in combination with other parameters. BIA reliably measured extracellular fluid, but the automatically calculated OHBIA exhibited a substantial degree of inaccuracy that precludes the use of BIA as a standard at present. The combination of clinical judgment with additional clinical parameters had the highest prediction accuracy for OH. Among the parameters studied, vena cava collapsibility index and calf circumference showed the strongest association with OH. Echocardiography, cardiothoracic index, atrial natriuretic peptide levels and spirometry did not have acceptable sensitivity.
The systematic clinical approach combining physician and patient inputs, laboratory and imaging data enables an individualized decision and a superior accuracy in OH assessment.
Electronic supplementary material
The online version of this article (doi:10.1007/s10157-012-0745-9) contains supplementary material, which is available to authorized users.
PMCID: PMC3751418  PMID: 23192771
Hemodialysis; Overhydration; Clinical judgment; Bioimpedance
9.  Influence of tibial slope asymmetry on femoral rotation in patients with lateral patellar instability 
The geometry of the tibial plateau and its influence on the biomechanics of the tibiofemoral joint has gained increased significance. However, no quantitative data are available regarding the inclination of the medial and lateral tibial slope in patients with patellar instability. It was therefore the purpose of this study to evaluate tibial slope characteristics in patients with patellar dislocations and to assess the biomechanical effect of medial-to-lateral tibial slope asymmetry on lateral patellar instability.
Medial and lateral tibial slope was measured on knee magnetic resonance images in 107 patients and in 83 controls. The medial-to-lateral tibial slope asymmetry was assessed as the intra-individual difference between the medial and lateral tibial plateau inclination considering severity of trochlear dysplasia. The effect of tibial slope asymmetry on femoral rotation was calculated by means of radian measure.
Severity of trochlear dysplasia was significantly associated with an asymmetric inclination of the tibial plateau. Whereas the medial tibial slope showed identical values between controls and study patients (n.s.), lateral tibial plateau inclination becomes flatter with increasing severity of trochlear dysplasia (p < 0.01). Consequently, the intra-individual tibial slope asymmetry increased steadily (p < 0.01) and increased internal femoral rotation in 20° and 90° of knee flexion angles in patients with severe trochlear dysplasia (p < 0.01). In addition, the extreme values of internal femoral rotation were more pronounced in patients with patellar instability, whereas the extreme values of external femoral rotation were more pronounced in control subjects (p = 0.024).
Data of this study indicate an association between tibial plateau configuration and internal femoral rotation in patients with lateral patellar instability and underlying trochlear dysplasia. Thereby, medial-to-lateral tibial slope asymmetry increased internal femoral rotation during knee flexion and therefore might aggravate the effect of femoral antetorsion in patients with patellar instability.
Level of evidence
PMCID: PMC3751338  PMID: 23096490
Patellar instability; Tibial slope; Femoral rotation; Biomechanics
10.  Selenoprotein P Status Correlates to Cancer-Specific Mortality in Renal Cancer Patients 
PLoS ONE  2012;7(10):e46644.
Selenium (Se) is an essential trace element for selenoprotein biosynthesis. Selenoproteins have been implicated in cancer risk and tumor development. Selenoprotein P (SePP) serves as the major Se transport protein in blood and as reliable biomarker of Se status in marginally supplied individuals. Among the different malignancies, renal cancer is characterized by a high mortality rate. In this study, we aimed to analyze the Se status in renal cell cancer (RCC) patients and whether it correlates to cancer-specific mortality. To this end, serum samples of RCC patients (n = 41) and controls (n = 21) were retrospectively analyzed. Serum Se and SePP concentrations were measured by X-ray fluorescence and an immunoassay, respectively. Clinical and survival data were compared to serum Se and SePP concentrations as markers of Se status by receiver operating characteristic (ROC) curve and Kaplan-Meier and Cox regression analyses. In our patients, higher tumor grade and tumor stage at diagnosis correlated to lower SePP and Se concentrations. Kaplan-Meier analyses indicated that low Se status at diagnosis (SePP<2.4 mg/l, bottom tertile of patient group) was associated with a poor 5-year survival rate of 20% only. We conclude that SePP and Se concentrations are of prognostic value in RCC and may serve as additional diagnostic biomarkers identifying a Se deficit in kidney cancer patients potentially affecting therapy regimen. As poor Se status was indicative of high mortality odds, we speculate that an adjuvant Se supplementation of Se-deficient RCC patients might be beneficial in order to stabilize their selenoprotein expression hopefully prolonging their survival. However, this assumption needs to be rigorously tested in prospective clinical trials.
PMCID: PMC3467258  PMID: 23056383
11.  Predictors of Renal Replacement Therapy in Acute Kidney Injury 
Nephron Extra  2012;2(1):247-255.
Criteria that may guide early renal replacement therapy (RRT) initiation in patients with acute kidney injury (AKI) currently do not exist.
In 120 consecutive patients with AKI, clinical and laboratory data were analyzed on admittance. The prognostic power of those parameters which were significantly different between the two groups was analyzed by receiver operator characteristic curves and by leave-1-out cross validation.
Six parameters (urine albumin, plasma creatinine, blood urea nitrogen, daily urine output, fluid balance and plasma sodium) were combined in a logistic regression model that estimates the probability that a particular patient will need RRT. Additionally, a second model without daily urine output was established. Both models yielded a higher accuracy (89 and 88% correct classification rate, respectively) than the best single parameter, cystatin C (correct classification rate 74%).
The combined models may help to better predict the necessity of RRT using clinical and routine laboratory data in patients with AKI.
PMCID: PMC3567877  PMID: 23599703
Acute kidney injury; Dialysis; Logistic regression model; Renal failure; Renal replacement therapy
12.  Reference miRNAs for miRNAome Analysis of Urothelial Carcinomas 
PLoS ONE  2012;7(6):e39309.
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.
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.
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.
PMCID: PMC3380005  PMID: 22745731
13.  Detection of Simultaneous Group Effects in MicroRNA Expression and Related Target Gene Sets 
PLoS ONE  2012;7(6):e38365.
Expression levels of mRNAs are among other factors regulated by microRNAs. A particular microRNA can bind specifically to several target mRNAs and lead to their degradation. Expression levels of both, mRNAs and microRNAs, can be obtained by microarray experiments. In order to increase the power of detecting microRNAs that are differentially expressed between two different groups of samples, we incorporate expression levels of their related target gene sets. Group effects are determined individually for each microRNA, and by enrichment tests and global tests for target gene sets. The resulting lists of p-values from individual and set-wise testing are combined by means of meta analysis. We propose a new approach to connect microRNA-wise and gene set-wise information by means of p-value combination as often used in meta-analysis. In this context, we evaluate the usefulness of different approaches of gene set tests. In a simulation study we reveal that our combination approach is more powerful than microRNA-wise testing alone. Furthermore, we show that combining microRNA-wise results with ‘competitive’ gene set tests maintains a pre-specified false discovery rate. In contrast, a combination with ‘self-contained’ gene set tests can harm the false discovery rate, particularly when gene sets are not disjunct.
PMCID: PMC3378551  PMID: 22723856
14.  Immunoadsorption therapy in patients with multiple sclerosis with steroid-refractory optical neuritis 
In multiple sclerosis relapses refractory to intravenous corticosteroid therapy, plasma exchange is recommended. Immunoadsorption (IA) is regarded as an alternative therapy, but its efficacy and putative mechanism of action still needs to be established.
We prospectively treated 11 patients with multiple sclerosis who had optical neuritis and fulfilled the indications for apheresis therapy (Trial registration DE/CA25/00007080-00). In total, five IA treatments were performed using tryptophan-IA. Clinical activity (visual acuity, Expanded Disability Status Scale, Incapacity Status Scale), laboratory values and visual evoked potentials were measured before, during and after IA, with a follow-up of six months. Moreover, proteomic analyses were performed to analyze column-bound proteins as well as corresponding changes in patients’ sera.
After the third IA, we detected an improvement of vision in eight of eleven patients, whom we termed responders. Amongst these, the mean visual acuity improved from 0.15 ± 0.12 at baseline to 0.47 ± 0.32 after the third IA (P = 0.0252) up to 0.89 ± 0.15 (P < 0.0001) at day 180 ± 10 after IA. Soluble interleukin-2 receptor decreased in responders (P = 0.03), whereas in non-responders it did not. Proteomic analyses of proteins adsorbed to IA columns revealed that several significant immunological proteins as well as central nervous system protein fragments, including myelin basic protein, had been removed by IA.
IA was effective in the treatment of corticosteroid-refractory optic neuritis. IA influenced the humoral immune response. Strikingly, however, we found strong evidence that demyelination products and immunological mediators were also cleared from plasma by IA.
PMCID: PMC3418188  PMID: 22537481
Apheresis; Autoimmune diseases; Evoked potentials/visual; Immunoadsorption; Multiple sclerosis; Optic neuritis; Proteomics
15.  MiR-133b Targets Antiapoptotic Genes and Enhances Death Receptor-Induced Apoptosis 
PLoS ONE  2012;7(4):e35345.
Despite the importance of microRNAs (miRs) for regulation of the delicate balance between cell proliferation and death, evidence for their specific involvement during death receptor (DR)-mediated apoptosis is scarce. Transfection with miR-133b rendered resistant HeLa cells sensitive to tumor necrosis factor-alpha (TNFα)-induced cell death. Similarly, miR-133b caused exacerbated proapoptotic responses to TNF-related apoptosis-inducing ligand (TRAIL) or an activating antibody to Fas/CD95. Comprehensive analysis, encompassing global RNA or protein expression profiling performed by microarray experiments and pulsed stable isotope labeling with amino acids in cell culture (pSILAC), led to the discovery of the antiapoptotic protein Fas apoptosis inhibitory molecule (FAIM) as immediate miR-133b target. Moreover, miR-133b impaired the expression of the detoxifying protein glutathione-S-transferase pi (GSTP1). Expression of miR-133b in tumor specimens of prostate cancer patients was significantly downregulated in 75% of the cases, when compared with matched healthy tissue. Furthermore, introduction of synthetic miR-133b into an ex-vivo model of prostate cancer resulted in impaired proliferation and cellular metabolic activity. PC3 cells were also sensitized to apoptotic stimuli after transfection with miR-133b similar to HeLa cells. These data reveal the ability of a single miR to influence major apoptosis pathways, suggesting an essential role for this molecule during cellular transformation, tumorigenesis and tissue homeostasis.
PMCID: PMC3332114  PMID: 22532850
16.  Ultrasonic scalpel causes greater depth of soft tissue necrosis compared to monopolar electrocautery at standard power level settings in a pig model 
BMC Surgery  2012;12:3.
Ultrasonic scalpel (UC) and monopolar electrocautery (ME) are common tools for soft tissue dissection. However, morphological data on the related tissue alteration are discordant. We developed an automatic device for standardized sample excision and compared quality and depth of morphological changes caused by UC and ME in a pig model.
100 tissue samples (5 × 3 cm) of the abdominal wall were excised in 16 pigs. Excisions were randomly performed manually or by using the self-constructed automatic device at standard power levels (60 W cutting in ME, level 5 in UC) for abdominal surgery. Quality of tissue alteration and depth of coagulation necrosis were examined histopathologically. Device (UC vs. ME) and mode (manually vs. automatic) effects were studied by two-way analysis of variance at a significance level of 5%.
At the investigated power level settings UC and ME induced qualitatively similar coagulation necroses. Mean depth of necrosis was 450.4 ± 457.8 μm for manual UC and 553.5 ± 326.9 μm for automatic UC versus 149.0 ± 74.3 μm for manual ME and 257.6 ± 119.4 μm for automatic ME. Coagulation necrosis was significantly deeper (p < 0.01) when UC was used compared to ME. The mode of excision (manual versus automatic) did not influence the depth of necrosis (p = 0.85). There was no significant interaction between dissection tool and mode of excision (p = 0.93).
Thermal injury caused by UC and ME results in qualitatively similar coagulation necrosis. The depth of necrosis is significantly greater in UC compared to ME at investigated standard power levels.
PMCID: PMC3305372  PMID: 22361346
17.  Identification of Metastamirs as Metastasis-associated MicroRNAs in Clear Cell Renal Cell Carcinomas 
MicroRNAs (miRNAs) play a pivotal role in cancerogenesis and cancer progression, but their specific role in the metastasis of clear cell renal cell carcinomas (ccRCC) is still limited. Based on microRNA microarray analyses from normal and cancerous samples of ccRCC specimens and from bone metastases of ccRCC patients, we identified a set of 57 differentially expressed microRNAs between these three sample groups of ccRCC. A selected panel of 33 miRNAs was subsequently validated by RT-qPCR on total 57 samples. Then, 30 of the 33 examined miRNAs were confirmed to be deregulated. A stepwise down-regulation of miRNA expression from normal, over primary tumor to metastatic tissue samples, was found to be typical. A total of 23 miRNAs (miR-10b/-19a/-19b/-20a/-29a/-29b/-29c/-100/-101/-126/-127/-130/-141/-143/-145/-148a/-192/-194/-200c/-210/-215/-370/-514) were down-regulated in metastatic tissue samples compared with normal tissue. This down-regulated expression in metastatic tissue in comparison with primary tumor tissue was also present in 21 miRNAs. In cell culture experiments with 5-aza-2'-deoxycytidine and trichostatin A, epigenetic modifications were shown as one reason of this down-regulation. The altered miRNA profiles, comprising newly identified metastasis-associated miRNAs, termed metastamir and the predicted miRNA-target interactions together with the significant correlations of miRNAs that were either lost or newly appeared in the studied sample groups, afford a solid basis for further functional analyses of individual miRNAs in RCC metastatic progression.
PMCID: PMC3492794  PMID: 23139634
Renal cell carcinoma; Microarray; RT-qPCR; microRNAs; Metastasis.
18.  Central nervous system rather than immune cell-derived BDNF mediates axonal protective effects early in autoimmune demyelination 
Acta Neuropathologica  2011;123(2):247-258.
Brain-derived neurotrophic factor (BDNF) is involved in neuronal and glial development and survival. While neurons and astrocytes are its main cellular source in the central nervous system (CNS), bioactive BDNF is also expressed in immune cells and in lesions of multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). Previous data revealed that BDNF exerts neuroprotective effects in myelin oligodendrocyte glycoprotein-induced EAE. Using a conditional knock-out model with inducible deletion of BDNF, we here show that clinical symptoms and structural damage are increased when BDNF is absent during the initiation phase of clinical EAE. In contrast, deletion of BDNF later in the disease course of EAE did not result in significant changes, either in the disease course or in axonal integrity. Bone marrow chimeras revealed that the deletion of BDNF in the CNS alone, with no deletion of BDNF in the infiltrating immune cells, was sufficient for the observed effects. Finally, the therapeutic effect of glatiramer acetate, a well-characterized disease-modifying drug with the potential to modulate BDNF expression, was partially reversed in mice in which BDNF was deleted shortly before the onset of disease. In summary, our data argue for an early window of therapeutic opportunity where modulation of BDNF may exert neuroprotective effects in experimental autoimmune demyelination.
PMCID: PMC3259380  PMID: 22009304
Neurotrophins; BDNF; EAE; Conditional knock-out mice; Neuroimmunology
19.  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 
BMC Cancer  2011;11:302.
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.
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.
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.
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.
PMCID: PMC3199863  PMID: 21767383
20.  Reporting FDR analogous confidence intervals for the log fold change of differentially expressed genes 
BMC Bioinformatics  2011;12:288.
Gene expression experiments are common in molecular biology, for example in order to identify genes which play a certain role in a specified biological framework. For that purpose expression levels of several thousand genes are measured simultaneously using DNA microarrays. Comparing two distinct groups of tissue samples to detect those genes which are differentially expressed one statistical test per gene is performed, and resulting p-values are adjusted to control the false discovery rate. In addition, the expression change of each gene is quantified by some effect measure, typically the log fold change. In certain cases, however, a gene with a significant p-value can have a rather small fold change while in other cases a non-significant gene can have a rather large fold change. The biological relevance of the change of gene expression can be more intuitively judged by a fold change then merely by a p-value. Therefore, confidence intervals for the log fold change which accompany the adjusted p-values are desirable.
In a new approach, we employ an existing algorithm for adjusting confidence intervals in the case of high-dimensional data and apply it to a widely used linear model for microarray data. Furthermore, we adopt a concept of different relevance categories for effects in clinical trials to assess biological relevance of genes in microarray experiments. In a brief simulation study the properties of the adjusting algorithm are maintained when being combined with the linear model for microarray data. In two cancer data sets the adjusted confidence intervals can indicate significance of large fold changes and distinguish them from other large but non-significant fold changes. Adjusting of confidence intervals also corrects the assessment of biological relevance.
Our new combination approach and the categorization of fold changes facilitates the selection of genes in microarray experiments and helps to interpret their biological relevance.
PMCID: PMC3154206  PMID: 21756370
21.  Brain volume perfusion CT performed with 128-detector row CT system in patients with cerebral gliomas: A feasibility study 
European Radiology  2011;21(9):1811-1819.
Validation of the feasibility and efficacy of volume perfusion computed tomography (VPCT) in the preoperative assessment of cerebral gliomas by applying a 128-slice CT covering the entire tumour.
Forty-six patients (25 men, 21 women; mean age 52.8 years) with cerebral gliomas were evaluated with VPCT. Two readers independently evaluated VPCT data, drawing volumes of interest (VOIs) around the tumour according to maximum intensity projection volumes, which were mapped automatically onto the cerebral blood volume (CBV), flow (CBF) and permeability (Ktrans) perfusion datasets. As control, a second VOI was placed in the contralateral healthy cortex. Correlation among perfusion parameters, tumour grade, hemisphere and VOIs was assessed. The diagnostic power of perfusion parameters was analysed by receiver operating characteristics curve analyses.
VPCT was feasible in the assessment of the entire tumour extent. Mean values of Ktrans, CBV, CBF in high-grade gliomas were significantly higher compared with low-grade (p < 0.01). Ktrans demonstrated the highest diagnostic (97% sensitivity), positive (100%) and negative (94%) prognostic values.
VPCT was feasible in all subjects. All areas of different perfusion characteristics are depicted and quantified in colour-coded 3D maps. The derived parameters correlate well with tumour histopathology, differentiating low- from high-grade gliomas.
PMCID: PMC3151396  PMID: 21573969
Cone-beam computed tomography; Brain neoplasms; Glioma; Feasibility studies
22.  Sequential interim analyses of survival data in DNA microarray experiments 
BMC Bioinformatics  2011;12:127.
Discovery of biomarkers that are correlated with therapy response and thus with survival is an important goal of medical research on severe diseases, e.g. cancer. Frequently, microarray studies are performed to identify genes of which the expression levels in pretherapeutic tissue samples are correlated to survival times of patients. Typically, such a study can take several years until the full planned sample size is available.
Therefore, interim analyses are desirable, offering the possibility of stopping the study earlier, or of performing additional laboratory experiments to validate the role of the detected genes. While many methods correcting the multiple testing bias introduced by interim analyses have been proposed for studies of one single feature, there are still open questions about interim analyses of multiple features, particularly of high-dimensional microarray data, where the number of features clearly exceeds the number of samples. Therefore, we examine false discovery rates and power rates in microarray experiments performed during interim analyses of survival studies. In addition, the early stopping based on interim results of such studies is evaluated. As stop criterion we employ the achieved average power rate, i.e. the proportion of detected true positives, for which a new estimator is derived and compared to existing estimators.
In a simulation study, pre-specified levels of the false discovery rate are maintained in each interim analysis, where reduced levels as used in classical group sequential designs of one single feature are not necessary. Average power rates increase with each interim analysis, and many studies can be stopped prior to their planned end when a certain pre-specified power rate is achieved. The new estimator for the power rate slightly deviates from the true power rate but is comparable to other estimators.
Interim analyses of microarray experiments can provide evidence for early stopping of long-term survival studies. The developed simulation framework, which we also offer as a new R package 'SurvGenesInterim' available at, can be used for sample size planning of the evaluated study design.
PMCID: PMC3098786  PMID: 21527044
23.  Symptoms of the oral cavity and their association with local microbiological and clinical findings—a prospective survey in palliative care 
Supportive Care in Cancer  2011;20(3):531-537.
Symptoms of the oral cavity clearly encompass more than radiation or chemotherapy-induced mucositis. Still, the burden of oral symptoms in palliative care has hardly been addressed directly, and considerations towards underlying disease processes have often been extrapolated from oncology patients.
We therefore conducted a prospective explorative survey with pilot character on patients on a specialized palliative care unit, describing symptom pattern (self assessment), clinical signs, bacteriological, mycological and virological findings, and correlating features of clinical history.
Taste disturbances, dry mouth and the presence of Candida were found to be the most prevalent and correlating items. The broad spectrum of further symptoms, signs and findings did not show relevant correlations and did not permit any unilateral causal attributions.
Apart from the described focus on Candida colonisation/infections, a symptom-guided polypragmatic approach therefore seems to be justifiable for patients with oral symptoms in palliative care.
PMCID: PMC3271219  PMID: 21336528
Palliative care; Oral symptoms; Taste disturbances; Dry mouth; Candida
24.  Metabolic profiling reveals key metabolic features of renal cell carcinoma 
Recent evidence suggests that metabolic changes play a pivotal role in the biology of cancer and in particular renal cell carcinoma (RCC). Here, a global metabolite profiling approach was applied to characterize the metabolite pool of RCC and normal renal tissue. Advanced decision tree models were applied to characterize the metabolic signature of RCC and to explore features of metastasized tumours. The findings were validated in a second independent dataset. Vitamin E derivates and metabolites of glucose, fatty acid, and inositol phosphate metabolism determined the metabolic profile of RCC. α-tocopherol, hippuric acid, myoinositol, fructose-1-phosphate and glucose-1-phosphate contributed most to the tumour/normal discrimination and all showed pronounced concentration changes in RCC. The identified metabolic profile was characterized by a low recognition error of only 5% for tumour versus normal samples. Data on metastasized tumours suggested a key role for metabolic pathways involving arachidonic acid, free fatty acids, proline, uracil and the tricarboxylic acid cycle. These results illustrate the potential of mass spectroscopy based metabolomics in conjunction with sophisticated data analysis methods to uncover the metabolic phenotype of cancer. Differentially regulated metabolites, such as vitamin E compounds, hippuric acid and myoinositol, provide leads for the characterization of novel pathways in RCC.
PMCID: PMC3822498  PMID: 19845817
kidney cancer; metabolism; metabolomics; metastasis
25.  Suitable reference genes for relative quantification of miRNA expression in prostate cancer 
Experimental & Molecular Medicine  2010;42(11):749-758.
Real time quantitative PCR (qPCR) is the method of choice for miRNA expression studies. For relative quantification of miRNAs, normalization to proper reference genes is mandatory. Currently, no validated reference genes for miRNA qPCR in prostate cancer are available. In this study, the expression of four putative reference genes (hsa-miR-16, hsa-miR-130b, RNU6-2, SNORD7) was examined with regard to their use as normalizer. After SNORD7 was already shown an inappropriate reference gene in preliminary experiments using total RNA pools, we studied the expression of the putative reference genes in tissue and normal adjacent tissue sample pairs from 76 men with untreated prostate carcinoma collected after radical prostatectomy. hsa-miR-130b and RNU6-2 showed no significantly different expression between the matched malignant and non-malignant tissue samples, whereas hsa-miR-16 was significantly underexpressed in malignant tissue. Softwares geNorm and Normfinder predicted hsa-miR-130b and the geometric mean of hsa-miR-130b and RNU6-2 as the most stable reference genes. Normalization of the four miRNAs hsa-miR-96, hsa-miR-125b, hsa-miR-205, and hsa-miR-375, which were previously shown to be regulated, shows that normalization to hsa-mir-16 can lead to biased results. We recommend using hsa-miR-130b or the geometric mean of hsa-miR-130b and small RNA RNU6-2 for normalization in miRNA expression studies of prostate cancer.
PMCID: PMC2992854  PMID: 20890088
gene expression profiling; microRNA; polymerase chain reaction; prostate neoplasms

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