Stephan Kruck

DNA methylation is associated with downregulation of the organic cation transporter OCT1 (SLC22A1) in human hepatocellular carcinoma

BackgroundOrganic cation transporters (OCTs) determine not only physiological processes but are also involved in the cellular uptake of anticancer agents. Based on microarray analyses in hepatocellular carcinoma (HCC), SLC22A1/OCT1 mRNA seems to be downregulated, but systematic protein expression data are currently missing. Moreover, the underlying molecular mechanisms responsible for altered SLC22A1 expression in HCC are not fully understood. Therefore, we investigated the role of DNA methylation in the transcriptional regulation of the family members SLC22A1/OCT1, SLC22A2/OCT2 and SLC22A3/OCT3 in HCC.MethodsSemiquantitative immunohistochemistry of SLC22A1 protein expression was performed in paired HCC and histological normal adjacent liver tissues (n = 71) using tissue microarray analyses, and the results were correlated with clinicopathological features. DNA methylation, quantified by MALDI-TOF mass spectrometry and gene expression of SLC22A1, SLC22A2 and SLC22A3 were investigated using fresh-frozen HCC (n = 22) and non-tumor adjacent liver tissues as well as histologically normal liver samples (n = 120) from a large-scale liverbank.ResultsBased on tissue microarray analyses, we observed a significant downregulation of SLC22A1 protein expression in HCC compared to normal adjacent tissue (P < 0.0001). SLC22A1 expression was significantly inverse correlated with expression of the proliferation marker MIB1/Ki-67 (rs = -0.464, P < 0.0001). DNA methylation of SLC22A1 was significantly higher in HCC compared with non-tumor adjacent liver tissue and was lowest in histologically normal liver tissue. Methylation levels for SLC22A1 in combination with RASSF1A resulted in a specificity of > 90% and a sensitivity of 82% for discriminating HCC and tumor-free liver tissue.ConclusionsDNA methylation of SLC22A1 is associated with downregulation of SLC22A1 in HCC and might be a new biomarker for HCC diagnosis and prognosis. Moreover, targeting SLC22A1 methylation by demethylating agents may offer a novel strategy for anticancer therapy of HCC.

DNA Methylation of the SLC16A3 Promoter Regulates Expression of the Human Lactate Transporter MCT4 in Renal Cancer with Consequences for Clinical Outcome

Purpose: The monocarboxylate transporter 4 (MCT4) is a metabolic target in tumor biology because it mediates lactate transport across membranes resulting in antiapoptotic effects. Cell experiments support the importance of MCT4 in clear cell renal cell carcinoma (ccRCC). In this study, we assessed the prognostic potential of MCT4 expression in ccRCC and its epigenetic regulation by DNA methylation as novel predictive marker for patient outcome using independent ccRCC cohorts. Experimental Design: MCT4 protein expression was quantified in 207 ccRCC and corresponding nontumor tissues. Data of an independent ccRCC cohort from The Cancer Genome Atlas (TCGA) were analyzed on MCT4 mRNA (n = 482) and DNA methylation (n = 283) level. The findings on MCT4 expression and DNA methylation in the SLC16A3 promoter were validated in a third cohort (n = 64). Promoter activity assays were conducted in four RCC cell lines. Results: MCT4 protein expression was upregulated (P < 0.0001) in ccRCC and showed significant association with cancer-related death. Upregulation of MCT4 mRNA expression (P < 0.00001) was confirmed in the TCGA cohort. Single CpG sites correlated inversely with mRNA expression and were associated with overall survival in Kaplan–Meier analyses [HR = 0.39; 95% confidence interval (CI), 0.24–0.64; P[log-rank] = 1.23e−04]. Promoter activity studies confirmed MCT4 regulation by DNA methylation. The significant correlation between MCT4 protein and gene expression or DNA methylation at single CpG sites was validated in a third cohort. Again, higher methylation at individual CpG sites was associated with prolonged survival [HR = 0.05; 95% CI, 0.01–0.40; P[log-rank] = 6.91e−05]. Conclusion: We identified SLC16A3 promoter DNA methylation as a novel epigenetic mechanism for MCT4 regulation in ccRCC with first evidence of a biological rationale for prognosis and clinical outcome. Clin Cancer Res; 19(18); 5170–81. ©2013 AACR.

Activation of PI3K Is Associated with Reduced Survival in Renal Cell Carcinoma

Objectives: The epidermal growth factor receptor- (EGFR) activated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt) pathway is associated with tumorigenesis and progression. The aims of the present study were to determine the expression patterns of Akt pathway parameters PI3K, phosphatase and tensin homolog (PTEN), phosphor-Akt (p-Akt) and their combination, for their possible prognostic value in renal cell carcinoma (RCC). PTEN dephosphorylates the liquid product of PI3K. Methods: Tumor samples from 176 RCC patients were investigated for PTEN, p-Akt and PI3K expression by immunohistochemistry. Expression levels were correlated to clinical variables and postoperative outcome by uni- and multivariate statistical analysis. Results: The various expression levels within the tumor samples were independent of histological grade and tumor stage, due to different levels of activation of the PI3K/p-Akt pathway. The activation of PI3K protein was found to be significantly associated with reduced survival times (p = 0.0304, multivariate analysis). Analysis of combined biomarker expressions showed that decreased long-term survival was correlated with PTEN low/p-Akt high expression (p < 0.05). Conclusions: Activation of the PI3K pathway is significantly associated with adverse clinical outcome in RCC. Analysis of biomarker combinations might identify high-risk patients and a subsequent need to adapt treatment modalities. Molecular pathways regulating PI3K activation appear to be promising targets for drug development in the clinical management of RCC patients.

Clinical and Cost Effectiveness of Hexaminolevulinate-guided Blue-light Cystoscopy: Evidence Review and Updated Expert Recommendations

CONTEXT Non-muscle-invasive bladder cancer (NMIBC) is associated with a high recurrence risk, partly because of the persistence of lesions following transurethral resection of bladder tumour (TURBT) due to the presence of multiple lesions and the difficulty in identifying the exact extent and location of tumours using standard white-light cystoscopy (WLC). Hexaminolevulinate (HAL) is an optical-imaging agent used with blue-light cystoscopy (BLC) in NMIBC diagnosis. Increasing evidence from long-term follow-up confirms the benefits of BLC over WLC in terms of increased detection and reduced recurrence rates. OBJECTIVE To provide updated expert guidance on the optimal use of HAL-guided cystoscopy in clinical practice to improve management of patients with NMIBC, based on a review of the most recent data on clinical and cost effectiveness and expert input. EVIDENCE ACQUISITION PubMed and conference searches, supplemented by personal experience. EVIDENCE SYNTHESIS Based on published data, it is recommended that BLC be used for all patients at initial TURBT to increase lesion detection and improve resection quality, thereby reducing recurrence and improving outcomes for patients. BLC is particularly useful in patients with abnormal urine cytology but no evidence of lesions on WLC, as it can detect carcinoma in situ that is difficult to visualise on WLC. In addition, personal experience of the authors indicates that HAL-guided BLC can be used as part of routine inpatient cystoscopic assessment following initial TURBT to confirm the efficacy of treatment and to identify any previously missed or recurrent tumours. Health economic modelling indicates that the use of HAL to assist primary TURBT is no more expensive than WLC alone and will result in improved quality-adjusted life-years and reduced costs over time. CONCLUSIONS HAL-guided BLC is a clinically effective and cost-effective tool for improving NMIBC detection and management, thereby reducing the burden of disease for patients and the health care system. PATIENT SUMMARY Blue-light cystoscopy (BLC) helps the urologist identify bladder tumours that may be difficult to see using standard white-light cystoscopy (WLC). As a result, the amount of tumour that is surgically removed is increased, and the risk of tumour recurrence is reduced. Although use of BLC means that the initial operation costs more than it would if only WLC were used, over time the total costs of managing bladder cancer are reduced because patients do not need as many additional operations for recurrent tumours.

Thymidine kinase and cancer monitoring

Thymidine kinases (TK) have a key function in the synthesis of DNA. Two isoenzymes have been characterized: TK1 is cell cycle-dependent and present in the cytoplasm whereas TK2--located in mitochondria--is cell cycle-independent. The diagnostic and prognostic role of TK1 has recently been investigated. TK1 might be helpful for screening and monitoring of human malignancies. TK1 may also serve as a prognostic factor for progression. Herein, we summarize the status of TK1 for cancer monitoring and point out its use as a proliferation marker. A comprehensive overview about the association of TK-1 with various entities is given.

Small renal Oncocytomas: Differentiation with multiphase CT

OBJECTIVES To evaluate characteristic imaging findings of tumor attenuation in multiphase computed tomography (CT) between renal oncocytomas and clear-cell renal cell carcinoma (ccRCC) of small tumor size (≤5 cm). METHODS We retrospectively identified 20 patients with complete four-phase CT with either histologically confirmed small renal oncocytoma (N=10) or ccRCC (N=10) who underwent subsequent total or partial nephrectomy. Exclusion criteria for RCC were non-clear-cell components in histology and a tumor diameter>5 cm. The relative attenuation of solid renal lesions and normal renal cortex was determined in the unenhanced, corticomedullary, nephrographic and excretory phase. Statistical comparison was carried out by Wilcoxon Rank Sum Test. RESULTS Mean tumor size of renal oncocytomas was 2.8±0.4 cm (1.2-5) and of ccRCC 2.5±0.2 cm (1.7-4.4; p=0.57). All lesions were homogenous without extended areas of necroses. In the nephrographic phase, the difference of attenuation between renal cortex and tumor lesion was highest in both entities (oncocytoma, 48.1±5.2 HU; ccRCC, 67.5±12.1) but not between entities (p=0.30). In the corticomedullary phase, renal oncocytomas showed greater isodensity to the normal renal cortex (13.9±4.3 HU) compared to clear-cell RCC (51.5±5.0 HU; p=0.003). No further significant differences were found for the unenhanced and excretory phase. CONCLUSIONS In this study, the maximum tumor-to-kidney contrast coincided with the nephrographic phase which was thus the most reliable for the detection of a renal lesion<5 cm. For lesion characterization, the corticomedullary phase was most useful for differentiating both entities. This finding is particularly important for the preoperative planning of a partial nephrectomy.

Second-line systemic therapy for the treatment of metastatic renal cell cancer

The discovery of molecular mechanisms driving the progression of renal cell carcinoma (RCC) has led to the development of drugs that target RCC at the molecular level. Inhibition of VEGF-targeting pathways is successful as a front-line treatment in patients with metastatic RCC. In addition, bevacizumab/IFN-α, sunitinib and pazopanib are recommended for first-line use in good- or intermediate-risk patients, whereas temsirolimus is approved for poor-risk patients. Second-line options are valuable as these patients eventually progress. The present review addresses which drug is best in this second-line setting. Options for sequential therapy include tyrosine kinase inhibitor (TKI)–mTOR inhibitor or TKI–TKI sequences. We also address the question of whether sequential therapy with TKIs or the combination of VEGF followed by mTOR inhibition is the best choice for specific patients, and which sequence of TKIs is most beneficial.

Cisplatin Hypersensitivity of Testicular Germ Cell Tumors Is Determined by High Constitutive Noxa Levels Mediated by Oct-4

Testicular germ cell tumors (TGCT) are considered a paradigm of chemosensitive tumors. Embryonal carcinoma cells represent the pluripotent entity of TGCTs and are characterized by expression of Oct-4, a key regulator of pluripotency and a determinant of their inherent hypersensitivity to cisplatin. However, the mechanisms underlying this Oct-4-mediated sensitivity are poorly understood. We previously showed that p53 is a major player in cisplatin hypersensitivity and therefore investigated whether Oct-4 may directly affect p53 activity. Despite a significant decrease in sensitivity, depletion of Oct-4 neither did alter cisplatin-induced transactivation of p53 target genes nor its subcellular localization. These data indicate that, rather than directly modulating p53 activity, Oct-4 provides a cellular context that augments the proapoptotic activity of p53. As mitochondrial priming by the Bcl-2 family is a known determinant of chemosensitivity, we compared the constitutive levels of these proteins in Oct-4-positive and -depleted cells. We identified Noxa as the only Bcl-2 family protein to be highly correlated with Oct-4 status and cisplatin sensitivity. Compared with differentiated cells, constitutive Noxa levels were significantly higher in Oct-4-positive cell lines and cancer patient samples. Furthermore, RNA interference-mediated knockdown of Oct-4 resulted in reduced Noxa transcript, in an almost complete loss of constitutive Noxa protein and decreased cisplatin hypersensitivity to a similar extent as did Noxa depletion. In conclusion, our study indicates that Noxa is a central determinant of hypersensitivity to cisplatin. Oct-4-dependent high constitutive levels of this BH3-only protein prime embryonal carcinoma cells to undergo rapid and massive apoptosis in response to p53 activation.

Minimally Invasive Percutaneous Nephrolithotomy: A Comparative Study of the Management of Small and Large Renal Stones

OBJECTIVE To compare the safety and efficacy of minimally invasive percutaneous nephrolitholapaxy (MIP) between small (<2 cm) and large (>2 cm) renal calculi, because although MIP has proved its efficacy in small lower caliceal stones, the efficacy in large renal calculi has been questioned. MATERIALS AND METHODS The data from 191 consecutive minimally invasive percutaneous nephrolithotomy (MIP) procedures at a single institution from January 2007 to March 2011 were reviewed retrospectively. All stone sizes and complexity were included (98 were <2 cm and 93 were ≥ 2 cm). We performed a comparative analysis of procedures for calculi <2 cm and ≥ 2 cm regarding the stone-free rate, the need for auxiliary procedures, and complications. The Student t test for parametric continuous variables and the chi-square test or Fischers exact test for nominal variables were applied. RESULTS The primary stone-free rate was significantly lower for the large than for the small stones (76.3% vs 90.8%, P = .007), and the secondary stone-free rate after one auxiliary procedure (second-look percutaneous nephrolithotomy, ureterorenoscopy, or shock wave lithotripsy) was not significantly different between the 2 groups (94.6% vs 98.9%, P = .1). The total complication rate was not significantly different (26.9% vs 19.4%, P = .2) between the 2 groups either. Grade III complications occurred in 5.2% of all patients, and no grade IV or V complications were observed. CONCLUSION Using MIP, the total stone-free rate was greater for the small than for the large calculi; however, most patients could be rendered stone-free with the use of one auxiliary procedure. The high success rate and low rate of higher grade complications justify the application of MIP for large stones.

High cytoplasmic expression of p27(Kip1) is associated with a worse cancer-specific survival in clear cell renal cell carcinoma

Whats known on the subject? and What does the study add?