[Implications of TCGA Network Data on 2nd Generation Immunotherapy Concepts Based on PD-L1 and PD-1 Target Structures]. / Implikationen der TCGA Netzwerkdaten für Zweit-Generations-Immuntherapiekonzepte auf Basis der PD-L1 und PD-1 Zielstrukturen.

The era of cytokines, given to patients with metastatic renal cell carcinoma (mRCC) as part of an unspecific immunomodulatory treatment concept, seems to have ended with the introduction of targeted therapies. However, preliminary data from studies on treatment with checkpoint inhibitors (e. g. anti-PD-1 and anti-PD-L1) may point the way to second-generation immunotherapy. The rationale of such immunomodulatory treatment is to stop or interrupt the tumour from "escaping" the body's immune defence. Thompson et al. report that increased protein expression of PD-L1 (CD274/ B7-H1) in tumour cells and tumour-infiltrating immune cells (TILs; lymphocytes and histiocytes) is associated with unfavourable clinical pathological parameters as well as poor survival. In small pilot groups of mRCC patients it was found that increased PD-L1 protein expression in tumours and TILs may be correlated with the objective response to anti-PD-1 treatment. Sometimes, however, a very wide variety of response rates was observed, which raises the question if this can be explained by individual expression levels of PD-L1 (CD 274) or PD-1 (PDCD1).Recently published data from the Cancer Genome Atlas (TCGA) Kidney Renal Clear Cell Carcinoma (KIRC) Network now provide a genome-wide data base that allows us to review or validate the molecular results obtained in clear cell renal cell carcinomas (ccRCC) to date.In this study, we analysed the TCGA KIRC mRNA expression data for PD-L1 and PD-1 for a possible association with clinical pathological parameters and the survival of 417 ccRCC patients.The mRNA expression of PD-L1 in primary nephrectomy specimens revealed no significant association with unfavourable clinical parameters. Interestingly, though, a positive correlation with patient survival was found (HR=0,59, p=0,006).These results, which partly contradict the concept applied to date, point out the necessity to ascertain the characteristics of PD-L1 and PD-1 expression at mRNA and protein level in an appropriately sized patient population and evaluate the clinical significance.

Hsa-mir-124-3 CpG island methylation is associated with advanced tumours and disease recurrence of patients with clear cell renal cell carcinoma.

BACKGROUND: Whether methylation of the microRNA (mir)-124-3 CpG island is of relevance for the clinical course of a solid cancer and whether it shows association with clinicopathology or survival of patients with renal cell cancer (RCC) is not known as yet. METHODS: In a cross-sectional study, relative methylation of mir-124-3 was measured in 111 RCC samples and 77 paired normal appearing tissues using quantitative methyl-specific PCR. Results were statistically compared with tumour histology, clinicopathological parameters and disease recurrence. RESULTS: We found tumour-specific hypermethylation of mir-124-3 in samples of RCCs with clear cell histology (ccRCC) compared with paired normal appearing tissues (P<0.0001). Methylation was significantly increased in tumours with state of advanced disease (P<0.0001). Higher relative methylation was associated with worse recurrence-free survival in both univariate (hazard ratio=9.37; P=0.0005) as well as bivariate Cox regression analyses considering age, sex, diameter of tumours and state of advanced disease, metastasis and lymph node metastases as covariates (hazard ratios=5.9-18.2; P-values of 0.0003-0.008). CONCLUSION: We identified mir-124-3 CpG islands (CGI) methylation as a relevant epigenetic mark for ccRCC thus underlining the need for functional studies of potentially affected signalling pathways in kidney tumour models. Methylation of mir-124-3 is suggested as an independent prognosticator for ccRCC.

Fibronectin 1 protein expression in clear cell renal cell carcinoma.

Fibronectin 1 (FN1) is a glycoprotein that is involved in cell adhesion and migration processes including embryogenesis, wound healing, blood coagulation, host defenses and metastasis. The aim of this study was to elucidate the FN1 protein expression in renal cell carcinoma (RCC) and to determine its potential prognostic relevance. A total of 270 clear cell RCC tissue specimens were collected from patients undergoing surgery for renal tumors. Biomarker expression was determined by immunohistochemistry and correlated with clinical variables. Survival analysis was carried out for 153 patients with complete follow-up data and pathologically proven clear cell carcinoma of the kidney. The follow-up group had a mean follow-up period of 83.8 months (IQR 26.2-136.2 months). The calculated median 5-year overall and tumor-specific survival rate of all 153 evaluable patients was 66.6 and 71.0%, respectively. A higher disease-related mortality rate was observed among patients with cytoplasmic FN1 expression (41.3 vs. 24.7%, p=0.039, Fisher's exact test). No significant correlation was found between FN1 staining and patient characteristics such as age, gender, tumor differentiation and visceral metastasis. However, there was a trend for FN1 expression and correlation with tumor stage and lymph node metastasis (p=0.085 and p=0.203; respectively). The Kaplan-Meier analysis revealed significant differences in the 5-year tumor-specific survival for patients with and without cytoplasmic FN1 expression (64.8 vs. 77.7%; p=0.035, log-rank test). However, results of the multivariate Cox regression analysis showed that FN1 expression was not an independent marker of either overall or tumor-specific survival. In conclusion, FN1 protein expression in RCC is associated with a higher disease-related mortality rate, indicating a possible role in RCC progression. Therefore, our data on FN1 encourage further investigations to determine the role of FN1 in RCC.

Prognostic and diagnostic relevance of hypermethylated in cancer 1 (HIC1) CpG island methylation in renal cell carcinoma.

The tumour suppressor gene hypermethylated in cancer 1 (HIC1) is a transcriptional repressor, which functionally cooperates with p53. Loss of HIC1 function is associated with the development of various tumor entities. The aim of this study was to elucidate the relevance of CpG island (CGI) methylation of HIC1 in renal cell carcinoma (RCC). DNA methylation of HIC1 was analysed in a total of 98 tumor and 70 tumor adjacent normal specimens. After conducting bisulfite conversion, relative methylation levels were quantitated using pyrosequencing. Relative methylation values were compared for paired tumor and normal specimen and for correlation with clinico-pathologic and follow-up data of patients. Tumor-specific hypermethylation could not be detected for the subregion of the HIC1 - CGI analyzed in this study. Comparing the level of methylation in tumors to clinicopathological data solely, patients without lymph node metastases demonstrated a higher level of methylation compared to patients with lymph node metastases (p=0.030). Patients recurrence-free survival (p=0.0074) both in univariate as well as bivariate cox regression analysis. This study identifies HIC1 hypermethylation in tumors as an independent predictor of reduced recurrence-free survival, which fits into our current understanding of hypermethylated HIC1 being a marker for poor prognosis. Therefore, HIC1 - CGI methylation could be a candidate marker to improve individualized therapy and risk stratification.

[Galectin expression in urological cancer. Diagnostic, prognostic and therapeutic potential]. / Galectin-Expression an urologischen Tumoren. Diagnostisches, prognostisches und therapeutisches Potential.

Galectins are a family of 15 proteins. They interact with glycoproteins in both the extracellular and intracellular milieu and regulate various biological cycles including cell growth, cell differentiation, cell adhesion and apoptosis. In biomolecular studies certain patterns of expression showed a correlation with metastasis and apoptosis. Therefore, galectins could be used as potential markers for early tumour recognition, long-term prognosis and a better-founded choice of therapy. Acknowledging these possibilities this review points out the standing of galectins with all currently available data in the development and progression of renal, bladder and prostatic tumours. The expression patterns of galectin-1 and -3 have been researched extensively. For example, several studies could show a decreased expression of galectin-3 in clear renal cell carcinoma, which correlated with a poor clinical prognosis. On the contrary, patients with a bladder tumour showed an elevated serum level of galectin-3. Furthermore, in analysis of prostatic tumour tissue galectin-1 was found to be an independent predictor of a PSA relapse. In addition, pathological patterns of galectin expression could be detected in non-urological malignancies such as breast cancer. Though all findings indicate a future significance of galectins as markers of urological malignancies with clinical relevance, more research will be necessary to particularly assess the evolutional-dependent function of galectins in the process of tumourigenesis.

[Expression of corticotropin releasing factor receptor 2 (CRFR2) in the human prostate. A new potential target for medical therapy of benign prostatic hyperplasia]. / Expression des "Corticotropin releasing faktor rezeptors 2" (CRFR2) in der humanen Prostata. Ein möglicher Ansatzpunkt bei der Therapie der benignen Prostatahyperplasie.

BACKGROUND: Expression of urocortin (Ucn), a 40-amino-acid neuropeptide, was demonstrated in the prostatic tissue of patients with benign prostatic hyperplasia (BPH). Ucn showed a significant role in the regulation of local inflammation, proliferation, and relaxation of smooth muscle tone in different organs through activation of corticotropin releasing factor receptor 2 (CRFR2). However, CRFR2 expression in human benign prostatic tissue remains unknown. Our study therefore aimed to investigate CRFR2 expression in prostatic tissue. METHODS: CRFR2 expression was evaluated in tissue samples of human prostate (n=8) by means of reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS: mRNA of CRFR2 was abundantly present in RT-PCR of prostate lysates. Immunohistochemistry revealed CRFR2 expression in the cytoplasm of basal and luminal epithelial cells as well as in cystic glands. Smooth muscle components of the stroma and vascular endothelial cells also showed extensive staining for CRFR2. CONCLUSIONS: Our study showed for the first time that human prostatic tissue expresses CRFR2. Pharmacological CRFR2 modulation might be a potential medical treatment for clinical BPH.

[Methylation of the RASSF1A tumor suppressor gene promoter. Risk factor for carcinogenesis of urological tumors]. / Die Methylierung des RASSF1A-Tumorsuppressorgenpromotors. Risikofaktor für die Karzinogenese urologischer Tumoren.

Molecular targets of known risk factors for the development of urological tumors, such as age, smoking, and adiposity, have not yet been elucidated. Hypermethylation of CpG islands in promoters can lead to silencing of gene expression and has frequently been detected in tumors. Age-dependent accumulation of methylation of gene promoters has been observed in various normal tissues and is discussed as a common risk factor for carcinogenesis.Here we describe the RASSF1A tumor suppressor gene as exhibiting an age-dependent promoter methylation in normal kidney tissue, which is additionally affected by the risk factors of anthracosis and adiposity. Furthermore, we found significantly increased methylation of the RASSF1A promoter when comparing peripheral versus central zone prostatic tissue samples.Preliminary expression analysis indicates that RASSF1A could be involved in early tumorigenesis. Our results support the hypothesis that age and other lifestyle-dependent factors may influence promoter methylation of specific genes, possibly serving as future individual tumor risk markers.

Heterogeneous p27(Kip1) expression within primary renal cell cancers, their invasive margins and peritumoral renal parenchyma correlation with pathological and prognostic features.

INTRODUCTION: The expression of the negative cell cycle regulator p27(Kip1) is frequently found to be deregulated in various human cancer types. Whether expression of p27(Kip1) can be used as prognostically relevant biological variables for renal cell cancer patients still remains to be clarified. Therefore, in the present investigation the expression within different tissue areas obtained from renal cell carcinomas was determined. PATIENTS AND METHODS: For analysis of p27(Kip1) in 420 tumor nephrectomy specimens obtained from 420 consecutively included patients, tissue microarrays were used comprising of 1,260 tissue samples each obtained from the tumor itself, the invasive front as well as non-malignant surrounding parenchyma. A sufficient follow-up after surgical therapy was available in 251 cases. RESULTS: In univariate survival analysis, decreased expression of p27(Kip1) within tissue cores obtained from the invasion front was significantly correlated with the patients' disease-specific long-term survival (p = 0.02, log-rank test). In contrast, expression of p27(Kip1) protein within the primary tumors was not identified to reveal any prognostically important information. In Cox regression analysis, histological stage and grade (p < 0.01), the presence of regional lymph node (p < 0.01) or distant metastases at the time of surgery (p < 0.01) as well as decreased expression of p27(Kip1) (p = 0.04) within the invasion front tissue samples independently predicted the disease-specific long-term survival following surgery. CONCLUSION: Our analysis demonstrated that p27(Kip1) is heterogeneously expressed in renal cell carcinomas. Moreover, the result of the present study supports the prognostic value of p27(Kip1) protein expression for patients diagnosed with renal cell carcinoma.

Positron emission tomography (PET) in the urooncological evaluation of the small pelvis.

Positron emission tomography (PET) with the use of ((18)F)2-fluoro-D: -2-desoxyglucose (FDG) has been investigated to be a highly sensitive and specific imaging modality in the diagnostic of primary and recurrent tumors and in the control of therapies in numerous non-urologic cancers. The aim of this review is to validate the significance of PET as a diagnostic tool in malignant urological tumors of the small pelvis. A systematic review of the current literature concerning the role of PET for malignant prostate, testicular and bladder tumors was carried out. The data indicate no additional role for PET in comparison with conventional imaging in tumor detection and local staging for prostate, bladder or testicular cancer. Tumor recurrence in prostate cancer seems to be more effectively identified with acetate and choline than with FDG, but this effect is more pronounced with higher PSA values. The value of PET in the identification of metastatic disease in either tumor entity can not be finally outlined as the clinical data are partly missing, controversial or in the process of evaluation. FDG-PET can be regarded as accepted imaging modality in the restaging of seminomatous germ cell tumors after chemotherapy.

Tissue microarrays: applications in urological cancer research.

Tissue microarrays (TMAs) are used to simultaneously study the expression of proteins in hundreds of tissue samples, offering the important advantage to screen large tissue banks for biomarker expression and to simultaneously examine serial sections obtained from the same tumor specimen by a time- and cost-effective analytical approach. This review article presents an overview of the importance and the impact of this technique in cancer research with increasing number of investigations using TMAs in its eighth anniversary. Its application, limitations, and selected previous study results regarding urologic malignancies are presented and discussed.

Reduced Expression of the Cell Cycle Regulator p27Kip1 within the Invasion Front of Renal Cell Carcinomas Proved to be a Significant Marker for Disease-specific Survival.

BACKGROUND: The expression of the negative cell cycle regulator p27Kip1 is frequently found to be deregulated in various human cancer types. Whether the expression of p27Kip1 can be used as a prognostic marker for renal cell cancer patients still remains to be clarified. Therefore, in the present investigation the expression of protein within different tissue areas obtained from renal cell carcinomas, their invasion front and corresponding histologically benign renal parenchyma was determined and statistically correlated with several tumor and patient characteristics including the disease-specific long-term survival following surgical treatment. PATIENTS AND METHODS: For analysis of p27Kip1 expression in 420 tumor nephrectomy specimens obtained from 420 consecutively included patients, tissue microarrays were used comprising 1260 tissue samples each obtained from the tumor itself, the invasive front as well as the non-malignant surrounding parenchyma. A sufficient follow-up after surgical therapy was available in 251 cases In total, 88 out of 251 patients (35%) had died from tumor progression after a median follow-up of 138 (36-240) months. RESULTS: In univariate survival analysis, decreased expression of p27Kip1 within tissue cores obtained from the invasion front was significantly correlated with the patients' disease-specific long-term survival (p=0.02, log rank test). In contrast, expression of p27Kip1 within the primary tumors was not identified to reveal any prognostically important information. In Cox regression analysis, histological stage and grade (p<0.01), the presence of regional lymph node (p<0.01) or distant metastases at the time of surgery (p<0.01) as well as decreased expression of p27Kip1 (p=0.04) within the invasion front tissue samples independently predicted the disease-specific long-term survival following surgery. CONCLUSION: Our analysis demonstrated that p27Kip1 is heterogeneously expressed in renal cell carcinomas. Moreover, the results of the present study supports the prognostic value of p27Kip1 protein expression for patients diagnosed with renal cell carcinoma.

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.

Predictive value of altered p27Kip1 and p21WAF/Cip1 protein expression for the clinical prognosis of patients with localized prostate cancer.

The p21WAF/Cip and the p27Kip1 genes have been identified as inductors of cell cycle arrest at the G1-checkpoint. Alterations of both genes have been suggested to be involved in the development of a variety of human malignancies due to a loss of critical antiproliferative mechanisms. To evaluate the prognostic importance of these alterations for patients with clinically localized prostate cancer, in 86 specimens (T1-T4) from 86 patients undergoing radical prostatectomy at the Department of Urology at Hannover University Medical School, were investigated. The immunohistochemical expression of the p27Kip1 and p21WAF/Cip protein was correlated to recurrence-free and long-term survival, age, depth of tumour infiltration, histological grade and lymph node status in these patients. After a median follow-up of 71 months (1-198 months), 14 of 20 (70%) patients (Group 1) with loss of p27Kip1 protein expression or a relative amount of < 10% of positively stained tumour cells developed recurrent disease in contrast to 18 of 66 (27%) patients (Group 2) with retained p27Kip1 protein expression (> or = 10% of positively stained tumour cells). The median recurrence-free survival times were 39 (4-134) months and 67 (4-198) months for patients in Groups 1 and 2 (p < 0.01), respectively. In multivariate analysis, loss of p27Kip1 protein expression was identified as the only independent prognostic parameter for recurrence-free survival. Univariate analysis (log-rank test) identified histological grading (p < 0.01) and reactivity for p27Kip1 (p = 0.046) (> or = 10% positivity) as prognostic factors for disease-specific long-term survival. However, during multivariate analysis none of the biological variables investigated retained independent prognostic importance regarding overall survival. Neither a low or a high expression of p21Waf/Cip could be correlated with the clinical prognosis of the patients following radical prostatectomy. This study confirms the independent prognostic value of decreased p27Kip1 protein expression in patients with localized prostate cancer, while a prognostic importance of p21Waf/Cip in addition to established patients' and tumour characteristics like tumour stage and histological grading appears rather unlikely.

Expression of the p53 and Maspin protein in primary prostate cancer: correlation with clinical features.

The serine protease inhibitor Maspin has been reported to inhibit the invasiveness and motility of prostate cancer tumor cells. Additionally, a p53-dependent regulatory pathway of Maspin in prostate cancer cell lines has been indicated. The first aim of our study was to determine the prognostic value of Maspin protein expression for the recurrence-free survival of patients undergoing radical prostatectomy for the treatment of clinically localized prostate cancer. Secondly, Maspin expression was correlated to p53 protein expression in order to gain additional information on a possible and previously suggested regulatory influence of the wild-type p53 protein on the Maspin protein expression. Tumor specimens obtained from 84 patients undergoing radical prostatectomy for localized prostate cancer were investigated for the expression of the Maspin and p53 protein by an immunohistochemic approach. Maspin protein expression was correlated with further patients' and tumor characteristics such as tumor stage, histologic grading, regional lymph node status, p53 protein expression and recurrence-free survival of the patients following radical prostatectomy. After a median follow-up of 64 months (24-197 months), 23 of 40 patients (58%) with a negative or decreased Maspin expression (group 1) developed local recurrence or systemic tumor progression in contrast to 8 of 44 patients (18%) with a retained expression of the Maspin protein (group 2) (p = 0.02; log-rank test). The median recurrence-free survival following radical prostatectomy was 26 months (12-37 months) for group 1 patients and 41 months (5-134 months) for patients from group 2 (p = 0.04). A positive immunohistochemic staining reaction for the p53 protein was significantly correlated with a decreased expression of the Maspin protein (p = 0.015; Spearman correlation coefficient). Additionally, loss of Maspin protein expression was correlated to higher tumor stages (p = 0.002) and an increasing histologic dedifferentiation (p = 0.03). This is the first study to indicate that Maspin protein possibly functions as a clinically relevant inhibitor of tumor progression, preventing the local invasiveness and further systemic progression of prostate cancer. Our investigation delivers first hints for a p53-dependent regulatory pathway of the Maspin protein in human prostate cancer.

Quantitation of DNA extracted after micropreparation of cells from frozen and formalin-fixed tissue sections.

Quantitation of DNA from microdissected fresh-frozen or paraffin-embedded tissue sections would be not only a valuable tool for ensuring optimum reaction conditions for many types of qualitative polymerase chain reaction (PCR) analyses, but also a prerequisite for any kind of subsequently performed genetic analyses aimed at the absolute quantitation of target sequences. The present study describes the quantitation of DNA after microdissection and extraction of cells with the PicoGreen fluorescence method. The limits of detection and of quantitative determination, respectively, have been determined by measuring dilutional series of three different DNA extractions, using either a medium-scale preparation from a solid tissue specimen or a known number of leukocytes or microdissected cells from frozen tumor sections. As corresponding limits of detection, 26, 24, and about 40 diploid genomes, and as limits of quantitative determination, 80, 73, and about 120 diploid genomes were obtained. Furthermore, it was shown that formalin fixation as well as hematoxylin staining of frozen sections with Delafield's and Mayer's alum or Weigert's iron hematoxylin before microdissection significantly diminishes the amount of extractable DNA and may lead to less reliable results, even of qualitative PCR analysis. In conclusion, the PicoGreen method allows precise quantitation of DNA corresponding to a minimum of about 120 diploid cells. It provides the basis for reliable qualitative analyses as well as the precondition for further quantitative genetic measurements from microdissected frozen or formalin-fixed and paraffin-embedded tissue sections.

Predictive value of decreased p27Kip1 protein expression for the recurrence-free and long-term survival of prostate cancer patients.

The p27Kip1 gene has been identified as inductor of cell cycle arrest at the G1 checkpoint to prevent entry of somatic cells into the S phase of the cell cycle when substantial DNA damage has occurred. It has been suggested that decreased expression of the p27Kip1 protein may contribute to the development of human malignancies due to loss of critical antiproliferative mechanisms. In the present study, 95 specimens (T1-T4) from 95 randomly selected patients undergoing radical prostatectomy at the Urological Department of Hannover University (82 patients) as well as in the Josef Hospital Regensburg (13 patients) between 1981 and 1992 for whom tissue blocks for immunohistochemical investigation were available, were investigated for different biological and clinical characteristics as possible predictors for recurrence-free and long-term survival: age, depth of tumour infiltration, histological grade, lymph node status, as well as decreased expression of the p27Kip1 protein. After a median follow-up up of 56 months (24-151 months), seven of 21 (33%) patients (Group 1) with loss of p27Kip1 protein expression or a relative amount of <10% of positively stained tumour cells developed recurrent disease in contrast to 17 of 74 (23%) patients (Group 2) with retained p27Kip1 protein expression (> or =10% of positively stained tumour cells). The median recurrence-free survival was 14 months (5-40 months) for patients from Group 1 and 31 months (7-133 months) for Group 2 patients (P = 0.02). In multivariate analysis, loss of p27Kip1 protein expression was identified as the only independent prognostic parameter for recurrence-free survival. In contrast, neither the univariate nor the multivariate analysis showed a correlation between loss of p27Kip1 protein expression and the long-term survival of the patients. Prospective studies are urgently needed to confirm the independent prognostic value of decreased p27Kip1 protein expression together with overexpression of the p53 tumour suppressor protein in patients with localized prostate cancer. The availability of more refined prognostically important biological variables in addition to established prognostic factors like tumour stage or Gleason score might help decision making in patients at high risk for the development of local recurrence or systemic tumour progression.