Carsten Kempkensteffen

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.

The tumour-targeting human L19-IL2 immunocytokine: Preclinical safety studies, phase I clinical trial in patients with solid tumours and expansion into patients with advanced renal cell carcinoma

BACKGROUND L19-IL2, a tumour-targeting immunocytokine composed of the recombinant human antibody fragment L19 (specific to the alternatively-spliced EDB domain of fibronectin, a well characterised marker of tumour neo-vasculature) and of human IL2, has demonstrated strong therapeutic activity in animal cancer models. This phase I/II trial was performed to evaluate safety, tolerability, recommended phase II dose (RD) and early signs of activity of L19-IL2. PATIENTS AND METHODS Five cohorts of patients with progressive solid tumours (n=21) received an intravenous infusion of L19-IL2 (from 5 to 30 Mio IU IL2 equivalent dose) on days 1, 3 and 5 every 3 weeks. This treatment cycle was repeated up to six times. In the following expansion phase, patients with metastatic renal cell carcinoma (RCC) (n=12) were treated at the RD of L19-IL2. Clinical data and laboratory findings were analysed for safety, tolerability and activity. RESULTS Preclinical studies in rats and monkeys did not raise any safety concerns. The RD was defined to be 22.5 Mio IU IL2 equivalent. Pharmacokinetics of L19-IL2 was dose proportional over the tested range, with a terminal half-life of 2-3h. Toxicities were manageable and reversible with no treatment-related deaths. We observed stable disease in 17/33 patients (51%) and 15/18 with mRCC (83%) after two cycles. Median progression-free survival of RCC patients in the expansion phase of the study was 8 months (1.5-30.5). CONCLUSIONS L19-IL2 can be safely and repeatedly administered at the RD of 22.5 Mio IU IL2 equivalent in advanced solid tumours. Preliminary evaluation suggests clinical activity of L19-IL2 in patients with mRCC.

The Role of Positron Emission Tomography in the Evaluation of Residual Masses After Chemotherapy for Advanced Stage Seminoma

PURPOSE Treatment in patients with seminoma who have residual or recurrent masses following chemotherapy is still a matter of debate. Surgical resection is currently the most common recommendation for masses greater than 3 cm, resulting in overtreatment in up to 70% of those affected. We analyzed the accuracy of preoperative positron emission tomography for predicting viable tumor residuals in patients with seminoma. MATERIALS AND METHODS In a prospective, multicenter trial computerized tomography and FDG (2-(F-18)-fluoro-2-deoxy-D-glucose) positron emission tomography were performed before surgical resection for residual or recurrent masses in 20 patients who had undergone chemotherapy for stage IIb, IIc or III seminoma. Histopathological findings were directly correlated with positron emission tomography results. RESULTS Of the patients 18 presented with residual masses and 2 had recurrent masses following chemotherapy. Histopathological assessment revealed viable tumor in 3 patients and benign lesions in 17. All patients with viable tumor were identified correctly by positron emission tomography. No false-negative results were observed but 9 patients had false-positive positron emission tomography results. This resulted in a negative predictive value of 1 (95% CI 0.63-1) and a positive predictive value of 0.25 (95% CI 0.05-0.57) for FDG-positron emission tomography in our patient cohort. CONCLUSIONS Our data indicate that FDG-positron emission tomography is capable of excluding viable disease in residual masses, even those exceeding 3 cm. Therefore, it may be considered an additional tool to improve patient counseling. However, the decision to perform surgical resection of the residual mass should not be based exclusively on a positive positron emission tomography image since false-positive results appear to be common.

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.

Regularly methylated novel pro-apoptotic genes associated with recurrence in transitional cell carcinoma of the bladder

Epigenetic silencing of tumor suppressor genes by promoter hypermethylation has been shown for a variety of genes in bladder cancer. Various p53 target genes have been investigated, but only few demonstrated promoter hypermethylation when semiquantitative detection methods were applied. To address to the question whether promoter methylation of novel p53 effector genes is a common event in transitional cell carcinoma of the bladder, we selected the p53 target genes apoptotic protein‐activating factor (APAF‐1), Caspase 8 (CASP‐8), death‐associated protein kinase, (DAPK‐1) and insulin‐like growth‐factor‐binding protein‐3 (IGFBP‐3), performing quantitative methylation‐specific real‐time PCR. The individual level of methylation (normalized index of methylation) was correlated with clinicopathological features as well as the biological behavior of the superficial and muscleinvasive tumors. Tissue was obtained from 110 tumor patients and 20 patients without urological malignancy. The median follow‐up of the tumor patients was 52 months. Hypermethylation of the promoter region in tumor specimens was common for APAF‐1 (100%), DAPK‐1 (74%) and IGFBP‐3 (66%), but not for CASP‐8 (3.6%). It was seen less frequently and with undetectable or low methylation levels in the normal urothelium group. The APAF‐1 methylation levels significantly correlated with tumor stage and tumor grade. The APAF‐1 and IGFBP‐3 methylation levels were able to separate tumors with higher recurrence risk from low‐risk tumors in nonmuscleinvasive and muscleinvasive tumors. In multivariate analysis, APAF‐1 and IGFBP‐3 methylation levels were independent prognostic markers for recurrence in superficial bladder tumors. This study provides new insights into the role of promoter methylation of selected p53 target genes. The extent of promoter methylation of specific genes offers additional prognostical information and is associated with the outcome in patients with nonmuscleinvasive and muscleinvasive bladder cancer.

Promoter Hypermethylation Profile of Kidney Cancer with New Proapoptotic p53 Target Genes and Clinical Implications

Purpose: Risk stratification of renal cell carcinoma is based on the histopathologic classification. Promoter hypermethylation as a mechanism of gene inactivation in renal cell carcinoma has been shown for only a small number of genes. We examined the usefulness of quantitative methylation analysis with a new set of p53 target genes for determining the clinical outcome and aggressiveness of the tumor disease. Experimental Design: The genes selected were APAF-1, CASPASE-8, DAPK-1, IGFBP-3, and PML. The tissue samples analyzed were taken from tumor specimens obtained from 90 consecutive patients with clear cell renal carcinoma and from 20 normal kidney specimens. Quantitative methylation analysis of CpG sites in the promoter region was done by methylation-specific real-time PCR and the normalized index of methylation (NIM) was determined for each sample. Results: Hypermethylation of the promoter region was common for APAF-1 (97%) and DAPK-1 (41%) but not for IGFBP-3 (3%), PML (3%), or CASP-8 (0%). The tumor patients had a median follow-up of 55 months. A correlation was found between the methylation level of APAF-1 and tumor size and nodal status, but not for tumor stage, grade, and age of patient. Kaplan-Meier analysis was able to identify patients with a higher risk of recurrence and tumor-related death by using APAF-1 (≥56% NIM) and DAPK-1 (≥10% NIM) methylation levels. In multivariate analysis, APAF-1 and DAPK-1 methylation levels were independent prognostic markers for metastatic disease and death from renal cell carcinoma. Conclusions: Our findings indicate that promoter hypermethylation of APAF-1 and DAPK-1 is a marker of aggressive renal cell carcinoma and provides independent prognostic information on disease outcome.

Prostate cancer detection on transrectal ultrasonography-guided random biopsy despite negative real-time magnetic resonance imaging/ultrasonography fusion-guided targeted biopsy: reasons for targeted biopsy failure

To examine the value of additional transrectal ultrasonography (TRUS)‐guided random biopsy (RB) in patients with negative magnetic resonance imaging (MRI)/ultrasonography (US) fusion‐guided targeted biopsy (TB) and to identify possible reasons for TB failure.

Expression parameters of the polycomb group proteins BMI1, SUZ12, RING1 and CBX7 in urothelial carcinoma of the bladder and their prognostic relevance.

Background: The Polycomb group (PCG) proteins are epigenetic transcriptional repressors involved in the control of cellular proliferation and oncogenesis. This study aimed at examining whether mRNA tumor levels of the PCG family members BMI1, SUZ12, RING1, and CBX7 relate to histopathological parameters in urothelial carcinomas of the bladder and whether they may provide prognostic information following tumor resection. Methods: The relative gene expression of BMI1, SUZ12, RING1, and CBX7 was analyzed by real-time RT-PCR in tumor tissue obtained from 93 patients with urothelial carcinoma of the bladder undergoing surgical treatment. Expression data was correlated with pathological variables and outcome. Results: PCG family members BMI1, SUZ12, RING1, and CBX7 are commonly expressed in urothelial carcinomas of the bladder. The relative CBX7 mRNA expression levels gradually decreased from superficial (pTa) to invasive (pT1) and finally to muscle-invasive (≥pT2) tumors (p = 0.008). Furthermore, CBX7 expression was statistically significantly correlated with tumor grade (p = 0.04). No correlation of mRNA levels with histopathological tumor features or tumor recurrence was observed for the other PCG components investigated. Conclusion: Expression levels of CBX7 inversely correlate with the progression of tumor stage and grade in urothelial carcinomas of the bladder, suggesting that downregulation of CBX7 indicates aggressive urothelial carcinoma phenotype.

Diagnostic and prognostic potential of differentially expressed miRNAs between metastatic and non-metastatic renal cell carcinoma at the time of nephrectomy

BACKGROUND MicroRNAs are promising diagnostic and prognostic biomarkers in oncology. We aimed to evaluate the prognostic potential of selected microRNAs in primary clear cell renal cell carcinomas (ccRCC) as predictors of tumor recurrence after radical nephrectomy. METHODS miR-122, miR-141, miR-155, miR-184, miR-200c, miR-210, miR-224, and miR-514, validated as differentially expressed in a previous study, were measured by RT-PCR in matched malignant and non-malignant tumor samples after nephrectomy from 111 patients (89 without, 22 with metastases) and clinicopathological and outcome data were collected. Non-parametric statistical tests, receiver-operating characteristics, Kaplan-Meier-, and univariate as well as multivariate Cox regression analyses were performed. RESULTS Downregulation of miR-141/-184/-200c/-514 and upregulation of miR-122/-155/-210/-224 were not different between samples of non-metastatic and metastatic tumors except for miR-122 and miR-514. miR-514 was further downregulated in metastatic compared with non-metastatic tumors while the upregulation of miR-122 was significantly reduced in metastatic carcinomas. All miRNAs were suitable to discriminate malignant from non-malignant tissue. miR-122 and miR-514 were significantly related to the recurrence risk but only miR-514 provided independent prognostic information in the final model including relevant clinicopathological variables. CONCLUSIONS MiR-122 and miR-514 play a role in tumor recurrence after nephrectomy. Expression of miR-514 was particularly downregulated in primary metastatic tumor and those that recur and might be a suitable adjunct marker for predicting tumor recurrence.

Sequence Therapy in Patients with Metastatic Renal Cell Carcinoma: Comparison of Common Targeted Treatment Options Following Failure of Receptor Tyrosine Kinase Inhibitors

BACKGROUND The best sequence of targeted therapy in patients with metastatic renal cell carcinoma (mRCC) has not been sufficiently defined. OBJECTIVE To describe the efficacy and toxicity of sequential everolimus (EV) versus receptor tyrosine kinase inhibitor (rTKI) following failure of first rTKI treatment. DESIGN, SETTING, AND PARTICIPANTS Retrospective study of 108 patients receiving rTKI or EV after progression on rTKI therapy at two German academic centres. INTERVENTION Sequence of systemic targeted treatment with sunitinib (n=85) or sorafenib (n=23) followed by EV (n=62) or another rTKI (n=46; sorafenib, n=35; sunitinib, n=11). MEASUREMENTS We measured response rate (Response Evaluation Criteria in Solid Tumours 1.0) and toxicity. Survival analysis (Kaplan-Meier method and Cox regression) was conducted for progression-free survival (PFS) and overall survival (OS). RESULTS AND LIMITATIONS Main patient characteristics did not significantly differ by sequence of treatment groups (rTKI-rTKI vs rTKI-EV). Response rate following first rTKI failure was not significantly different between sequential therapies with a disease control rate of 51.6% (EV) and 43.5% (rTKI). The corresponding median PFS was 3.6 mo (95% confidence interval [CI], 1.8-5.4) for EV and 4.0 mo (3.2-4.9) for rTKI treatment. The estimated OS was longer for the rTKI-EV group (43 mo; 95% CI, 33.9-52.1) than for the rTKI-rTKI group (29 mo; 95% CI, 18.6-39.5; p=0.03), but this difference lost statistical significance in multivariable-adjusted analyses. Intrinsic rTKI resistance was independently associated with inferior subsequent PFS (hazard ratio [HR]: 1.79; 95% CI, 1.15-3.62; p=0.015) and OS (HR: 6.54; 95% CI, 3.01-14.20; p<0.001). Limitations are the retrospective design, limited numbers of cases, and residual confounding factors. CONCLUSIONS The sequence therapies rTKI-EV and rTKI-rTKI may be equally efficacious in terms of PFS and response rate, whereas a tendency towards superior survival was observed for the rTKI-EV sequence. These data, particularly the potential benefit of an early change of mode of action, need confirmation in randomised comparative trials.