Joerg Hennenlotter

Expansion and differentiation of neural progenitors derived from the human adult enteric nervous system.

BACKGROUND & AIMS Neural stem and progenitor cells from the enteric nervous system have been proposed for use in cell-based therapies against specific neurogastrointestinal disorders. Recently, enteric neural progenitors were generated from human neonatal and early postnatal (until 5 years after birth) gastrointestinal tract tissues. We investigated the proliferation and differentiation of enteric nervous system progenitors isolated from human adult gastrointestinal tract. METHODS Human enteric spheroids were generated from adult small and large intestine tissues and then expanded and differentiated, depending on the applied cell culture conditions. For implantation studies, spheres were grafted into fetal slice cultures and embryonic aganglionic hindgut explants from mice. Differentiating enteric neural progenitors were characterized by 5-bromo-2-deoxyuridine labeling, in situ hybridization, immunocytochemistry, quantitative real-time polymerase chain reaction, and electrophysiological studies. RESULTS The yield of human neurosphere-like bodies was increased by culture in conditional medium derived from fetal mouse enteric progenitors. We were able to generate proliferating enterospheres from adult human small or large intestine tissues; these enterospheres could be subcultured and maintained for several weeks in vitro. Spheroid-derived cells could be differentiated into a variety of neuronal subtypes and glial cells with characteristics of the enteric nervous system. Experiments involving implantation into organotypic intestinal cultures showed the differentiation capacity of neural progenitors in a 3-dimensional environment. CONCLUSIONS It is feasible to isolate and expand enteric progenitor cells from human adult tissue. These findings offer new strategies for enteric stem cell research and future cell-based therapies.

HYAL-1 Hyaluronidase: A Potential Prognostic Indicator for Progression to Muscle Invasion and Recurrence in Bladder Cancer

BACKGROUND For bladder cancer (BCa) patients undergoing bladder-sparing treatments, molecular markers may aid in accurately predicting progression to muscle invasion and recurrence. Hyaluronic acid (HA) is a glycosaminoglycan that promotes tumor metastasis. Hyaluronoglucosaminidase 1 (HYAL-1)-type hyaluronidase (HAase) promotes tumor growth, invasion, and angiogenesis. Urinary HA and HAase levels are diagnostic markers for BCa. OBJECTIVE We evaluated whether HA and HYAL-1 can predict progression to muscle invasion and recurrence among patients with non-muscle-invasive BCa. DESIGN, SETTING, AND PARTICIPANTS : Based on tissue availability, tissue microarrays were prepared from a cohort of 178 BCa specimens (144 non-muscle invasive, 34 muscle invasive). Follow-up information was available on 111 patients with non-muscle-invasive BCa (mean follow-up: 69.5 mo); 58 patients recurred and 25 progressed to muscle invasion (mean time to progress: 22.3 mo). MEASUREMENTS HA and HYAL-1 expression was evaluated by immunohistochemistry and graded for intensity and area of staining. Association of HA and HYAL-1 staining with BCa recurrence and muscle invasion was evaluated by univariate and multivariate models. RESULTS AND LIMITATIONS HA and HYAL-1 expression correlated with tumor grade, stage, and multifocality (p<0.05). In non-muscle-invasive BCa specimens, HYAL-1 staining was higher (234.3+/-52.2; 200.6+/-61.4) if patients experienced progression to muscle invasion or recurrence when compared with no progression or recurrence (164.1+/-48.2; 172.1+/-57; p<0.001). HA staining correlated with muscle invasion (p<0.001). In univariate analysis, age (p=0.014), multifocality (p=0.023), and HYAL-1 staining (p<0.001) correlated with muscle invasion, whereas only HYAL-1 correlated with recurrence (p=0.013). In multivariate analysis, HYAL-1 significantly associated with muscle invasion (p<0.001; 76.8% accuracy) and recurrence (p=0.01; 67.8% accuracy). CONCLUSIONS HYAL-1 is a potential prognostic marker for predicting progression to muscle invasion and recurrence.

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.

Can we still afford bladder cancer

Purpose of review Bladder cancer is the most expensive tumor with regard to both costs per patient per year and lifetime costs per patient. Nonmuscle invasive tumors account for the highest share of the overall costs for bladder cancer. Costs for follow-up (excluding costs for reinterventions) compared with transurethral resection and cystectomy account for a considerably lower portion of the overall costs. Recent findings From an economic standpoint, the biggest potential for savings could, therefore, be in the treatment and re-treatment of nonmuscle invasive tumors. Cost reductions have already been demonstrated with a reduction of hospital admission for such treatments, use of photodynamic diagnosis supported transurethral resection to avoid residual tumors, and adjuvant chemoinstillation and immunoinstillation to prolong or prevent tumor recurrences. Summary Further cost reductions can be achieved with a centralization of patients undergoing cystectomy by shifting these patients to high-volume surgeons in high-volume hospitals, thereby reducing complications and length of hospital stay. In advanced stages predictive markers may identify suitable subgroups for molecular therapies and, therefore, avoid costs of inappropriate treatments.

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.

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.

Immunocytology Is a Strong Predictor of Bladder Cancer Presence in Patients With Painless Hematuria: A Multicentre Study

BACKGROUND Although the performance of immunocytology has been established in the surveillance of patients with urothelial carcinoma of the bladder (UCB), its value in the initial detection of UCB in patients with painless hematuria remains unclear. OBJECTIVE To determine whether immunocytology improves our ability to predict the likelihood of UCB in patients with painless hematuria. Further, to test the clinical benefit of immunocytology in this setting using decision curve analysis. DESIGN, SETTING, AND PARTICIPANTS The subjects were 1182 consecutive patients without a history of UCB presenting with painless hematuria and were enrolled at three centres. INTERVENTION All patients underwent upper-tract imaging, cystourethroscopy, voided urine cytology, and immunocytology analysis. Bladder tumors were biopsied and histologically confirmed as UCB. MEASUREMENTS Multivariable regression models were developed. Area under the curve was measured and compared using the DeLong test. A nomogram was constructed from the full multivariable model. Decision curve analysis was performed to evaluate the clinical benefit associated with use of the multivariable models including immunocytology. RESULTS AND LIMITATIONS Immunocytology had the largest contribution to a multivariable model for the prediction of UCB (odds ratio: 18.3; p<0.0001), which achieved a 90.8% predictive accuracy. Decision curve analysis revealed that models incorporating immunocytology achieved the highest net benefit at all threshold probabilities. CONCLUSIONS Immunocytology is a strong predictor of the presence of UCB in patients who present with painless hematuria. Incorporation of immunocytology into predictive models improves diagnostic accuracy by a statistically and clinically significant margin. The use of immunocytology in the diagnostic workup of patients with hematuria appears promising and should be further evaluated.

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?

Correlation of bFGF expression in renal cell cancer with clinical and histopathological features by tissue microarray analysis and measurement of serum levels

The prognostic value of bFGF for surgically treated renal cell cancer (RCC) patients was evaluated by immunohistochemistry (IHC) and the tissue microarray technique (TMA). Additionally, preoperative serum bFGF levels were correlated to tumour stage and the presence of metastases at initial diagnosis. Serum levels of bFGF were measured by ELISA in 39 healthy volunteers, in 37 patients with benign urologic diseases and in 74 RCC patients, 26 of whom revealed lymph node or distant metastases. bFGF expression as detected by IHC was investigated in 777 tissue cores from 259 different RCC patients [median follow-up: 138 (36–240) months]. Eighty eight patients died from tumour progression. For each patient, the TMA slides contained a tissue core from the primary tumour, its invasion front and the normal renal parenchyma. bFGF serum levels were higher in RCC patients vs healthy volunteers (P<0.01) and vs patients with benign urologic diseases (P<0.01). Metastasized patients revealed higher bFGF serum levels than organ-confined specimens (P<0.01). As detected by IHC only increased bFGF expression in the invasion front tissue correlated with the patients’ long-term survival (log rank test) (P=0.03). In multivariate analysis regional LN metastases (P<0.01), the histological grading (P<0.01), and an increased bFGF expression in the invasion front (P=0.04) independently predicted the patients’ clinical prognosis. Not the expression of bFGF in the primary tumour but in its invasion front reflects the aggressiveness of RCC, hereby indicating a different biological potential within both areas. The value of bFGF serum levels as indicators of systemic tumour dissemination remains to be determined.

γH2AX assay in ex vivo irradiated tumour specimens: A novel method to determine tumour radiation sensitivity in patient-derived material

PURPOSE To establish a clinically applicable protocol for quantification of residual γH2AX foci in ex vivo irradiated tumour samples and to apply this method in a proof-of-concept feasibility study to patient-derived tumour specimens. MATERIAL AND METHODS Evaluation of γH2AX foci formation and disappearance in excised FaDu tumour specimens after (a) different incubation times in culture medium, 4Gy irradiation and fixation after 24h (cell recovery), (b) 10h medium incubation, 4Gy irradiation and fixation after various time points (double strand break repair kinetics), and (c) 10h medium incubation, irradiation with graded single radiation doses and fixation after 24h (dose-response). The optimised protocol was applied to patient-derived samples of seminoma, prostate cancer and glioblastoma multiforme. RESULTS Post excision or biopsy, tumour tissues showed stable radiation-induced γH2AX foci values in oxic cells after >6h of recovery in medium. Kinetics of foci disappearance indicated a plateau of residual foci after >12h following ex vivo irradiation. Fitting the dose-response of residual γH2AX foci yielded slopes comparable with in situ irradiation of FaDu tumours. Significant differences in the slopes of ex vivo irradiated patient-derived tumour samples were found. CONCLUSION A novel clinically applicable method to quantify residual γH2AX foci in ex vivo irradiated tumour samples was established. The first clinical results suggest that this method allows to distinguish between radiosensitive and radioresistant tumour types. These findings support further translational evaluation of this assay to individualise radiation therapy.