Comparative genomic profiling of glandular bladder tumours.

Primary glandular bladder tumours (bladder adenocarcinoma [BAC], urachal adenocarcinoma [UAC], urothelial carcinoma with glandular differentiation [UCg]) are rare malignancies with histological resemblance to colorectal adenocarcinoma (CORAD) in the majority of this subgroup. Definite case numbers are very low, molecular data are limited and the pathogenesis remains poorly understood. Therefore, this study was designed to complement current knowledge by in depth analysis of BAC (n = 12), UAC (n = 13), UCg (n = 11) and non-invasive glandular lesions (n = 19). In BAC, in addition to known alterations in TP53, Wnt, MAP kinase and MTOR pathway, mutations in SMAD4, ARID1A and BRAF were identified. Compared to published data on muscle invasive bladder cancer (BLCA) and CORAD, UCg exhibited frequent "urothelial" like alterations while BAC and UAC were characterised by a more "colorectal" like mutational pattern. Immunohistochemically, there was no evidence of DNA mismatch repair deficiency or PD-L1 tumour cell positivity in any sample. Depending on the used antibody 0-45% of BAC, 0-30% of UCg and 0% UAC cases exhibited PD-L1 expressing tumour associated immune cells. A single BAC (9%, 1/11) showed evidence of ARID1A protein loss, and two cases of UCg (20%, 2/10) showed loss of SMARCA1 and PBRM1, respectively. Taken together, our data suggest at least in part involvement of similar pathways driving tumourigenesis of adenocarcinomas like BAC, UAC and CORAD independent of their tissue origin. Alterations of TERT and FBXW7 in single cases of intestinal metaplasia further point towards a possible precancerous character in line with previous reports.

Meta-Analysis of Genome-Wide Association Studies and Network Analysis-Based Integration with Gene Expression Data Identify New Suggestive Loci and Unravel a Wnt-Centric Network Associated with Dupuytren's Disease.

Dupuytren´s disease, a fibromatosis of the connective tissue in the palm, is a common complex disease with a strong genetic component. Up to date nine genetic loci have been found to be associated with the disease. Six of these loci contain genes that code for Wnt signalling proteins. In spite of this striking first insight into the genetic factors in Dupuytren´s disease, much of the inherited risk in Dupuytren´s disease still needs to be discovered. The already identified loci jointly explain ~1% of the heritability in this disease. To further elucidate the genetic basis of Dupuytren´s disease, we performed a genome-wide meta-analysis combining three genome-wide association study (GWAS) data sets, comprising 1,580 cases and 4,480 controls. We corroborated all nine previously identified loci, six of these with genome-wide significance (p-value < 5x10-8). In addition, we identified 14 new suggestive loci (p-value < 10-5). Intriguingly, several of these new loci contain genes associated with Wnt signalling and therefore represent excellent candidates for replication. Next, we compared whole-transcriptome data between patient- and control-derived tissue samples and found the Wnt/ß-catenin pathway to be the top deregulated pathway in patient samples. We then conducted network and pathway analyses in order to identify protein networks that are enriched for genes highlighted in the GWAS meta-analysis and expression data sets. We found further evidence that the Wnt signalling pathways in conjunction with other pathways may play a critical role in Dupuytren´s disease.

Differential diagnosis of bladder versus colorectal adenocarcinoma: keratin 7 and GATA3 positivity in nuclear ß-catenin-negative glandular tumours defines adenocarcinoma of the bladder.

AIMS: This study evaluates immunohistochemical markers for the differential diagnosis of primary bladder adenocarcinoma (BAC) from secondarily involving colorectal adenocarcinoma (CAC). Additional staining of putative precursor lesions (cystitis cystica et glandularis (CC) and intestinal metaplasia (IM)) supports insights into metaplastic cell development and aberrant differentiation in tumours. METHODS: Tissue microarray sections of formalin-fixed, paraffin-embedded tissues from clinically verified 11 BAC, 11 CAC, 18 invasive urothelial carcinomas (UCs), 22 normal urothelium samples, 25 CC and 15 IM were stained for keratin 7, 5/6, 5/14 and 20, ß-catenin, e-cadherin, cadherin 17, cdx2, uroplakin II and III, CD10, androgen receptor (AR), S100P, MUC2, MUC5AC and GATA3 expression. Data were analysed using Kruskal-Wallis/Dunn's multiple comparison test and Fisher's exact test. RESULTS: Significant difference (p<0.05) between all three tumour groups was observed for keratin 7 only. Further significant difference between BAC and CAC was found for GATA3 and nuclear ß-catenin staining. BAC-positive/CAC-negative markers without significance were: p63, keratin 5/6, 5/14, uroplakins II/III and AR. CC showed a urothelial phenotype (p63+, GATA3+, S100P+, uroplakin+ in single cells) with initial signs of intestinal differentiation (single cells cdx2+ or cadherin 17+). IM displayed a full intestinal phenotype (p63-, all urothelial markers-, cdx2/MUC2/MUC5AC+, cadherin17+). CONCLUSIONS: Differential diagnosis of BAC and CAC remains difficult, but positive staining for keratin 7 in nuclear ß-catenin-negative tumours argues for BAC. Additional markers like GATA3 and p63 may be added, as positivity in some cases may be helpful. However, for reliable histological diagnosis, knowledge of comprehensive clinical data is still essential.

Comparison of structural genetics of non-schistosoma-associated squamous cell carcinoma of the urinary bladder.

Little is known about genetic changes in squamous differentiation of non-schistosomiasis-associated bladder cancer. Therefore, we investigated pure squamous cell carcinomas (SqCC), squamous parts of mixed urothelial carcinomas with squamous differentiation (MIX) and mere urothelial cancers (UC) for structural genetic differences. Tissue microarray slides (n = 29 SqCC, n = 35 MIX and n = 23 UC) were analyzed by ZytoLight SPEC p16/CEN3/7/17 Quadruple Color Probe fluorescence-in-situ-hybridization (FISH) and DNA was investigated by comparative genomic hybridization (CGH) (n = 35 SqCCs, n = 40 MIX and n = 36 UC). By FISH the mean number of polysomic cells was lowest in SqCC (CEN3 P = 0.0498, CEN17 P = 0.0009). A slight tendency of lower copy numbers of chromosomes 3, 7 and 17 and higher numbers of the p16-locus in SqCC (P = 0.45) indicated less aneuploid tumor cells in SqCC compared to MIX and UC. In CGH SqCC showed the lowest mean number of aberrations per tumor (SqCC 5.37 changes, MIX 6.75 and UC 7.64; P = 0.1754). Significant differences between the three groups were found for loss of chromosome 3p (P = 0.004), 6q (P = 0.028), 11p (P = 0.024) and gains of 5p (P = 0.020). Loss of 3p was more frequent in SqCC (51.4%) than in MIX (37.5%) or UC (13.9%). To conclude, SqCCs show less polysomy and genetic alterations than MIX and UC. Loss of 3p is more frequent in SqCC but there are no absolute specific alterations for each tumor group. Squamous parts of mixed tumors show similar alterations than UC and should be considered as further development of UC, while pure SqCC seem to be a separate tumor group.

Mutations Causing Complex Disease May under Certain Circumstances Be Protective in an Epidemiological Sense.

Guided by the practice of classical epidemiology, research into the genetic basis of complex disease has usually taken for granted the dictum that causative mutations are invariably over-represented among clinically affected as compared to unaffected individuals. However, we show that this supposition is not true and that a mutation contributing to the etiology of a complex disease can, under certain circumstances, be depleted among patients. Populations with defined disease prevalence were repeatedly simulated under a Wright-Fisher model, assuming various types of population history and genotype-phenotype relationship. For each simulation, the resulting mutation-specific population frequencies and odds ratios (ORs) were evaluated. In addition, the relationship between mutation frequency and OR was studied using real data from the NIH GWAS catalogue of reported phenotype associations of single-nucleotide polymorphisms (SNPs). While rare diseases (prevalence <1%) were found to be consistently caused by rare mutations with ORs>1, up to 20% of mutations causing a pandemic disease (prevalence 10-20%) had ORs<1, and their population frequency ranged from 0% to 100%. Moreover, simulation-based ORs exhibited a wide distribution, irrespective of mutation frequency. In conclusion, a substantial proportion of mutations causing common complex diseases may appear 'protective' in genetic epidemiological studies and hence would normally tend to be excluded, albeit erroneously, from further study. This apparently paradoxical result is explicable in terms of mutual confounding of the respective genotype-phenotype relationships due to a negative correlation between causal mutations induced by their common gene genealogy. As would be predicted by our findings, a significant negative correlation became apparent in published genome-wide association studies between the OR of genetic variants associated with a particular disease and the prevalence of that disease.

Shannon's equivocation for forensic Y-STR marker selection.

Short tandem repeat (STR) markers are widely and continuously used in forensic applications. However, past research has demonstrated substantial allelic association between STR markers on both autosomes and the X chromosome, leading to partially redundant information that these markers can provide. Here, we quantify the allelic association between Y-chromosomal STR markers that are part of established forensic panels, separately for three different continental groups. We further propose a sequential marker selection procedure that is based on Shannon's equivocation and that accounts for allelic association between STR markers, leading to a maximal gain in independent information. In application to three real-world data sets, we demonstrate the procedure's superior performance when compared to single-locus diversity selection strategies, resulting in the optimal marker set for a given data set in the majority of marker subsets. Noting the inferior performance of the established Y-STR marker panels in a retrospective investigation, we suggest that future forensic marker selection should be guided, besides by other technical selection criteria, by an equivocation-based approach to obtain maximally discriminatory marker sets at minimal cost.

Comparison of two exploratory dietary patterns in association with the metabolic syndrome in a Northern German population.

Diet is related to many chronic disease conditions such as the metabolic syndrome (MetS). We set out to compare behaviour-related with disease-related patterns and their association with the MetS in a German cross-sectional study. A total of 905 participants of a Northern German cohort (aged 25-82 years) completed a FFQ, underwent anthropometric assessments and provided a blood sample. Dietary patterns were derived by principal component analysis (PCA) and reduced-rank regression (RRR) from forty-two food groups. Components of the MetS were used as response variables for the RRR analysis. Simplified patterns comprising ten food groups were generated. Logistic regression analysis was performed to evaluate the likelihood of having the MetS across the quartiles of simplified pattern scores. We identified two similar dietary patterns derived by PCA and RRR characterised by high intakes of potatoes, various vegetables, red and processed meat, fats, sauce and bouillon. Comparing simplified patterns, an increased RRR pattern score was associated with a higher OR (2·18, 95% CI 1·25, 3·81) of having the MetS than an increased PCA pattern score (OR 1·92, 95% CI 1·21, 3·03). Comparing concordant food groups by both dietary pattern methods, a diet high in legumes, beef, processed meat and bouillon was also positively associated with the prevalence of the MetS after adjustment for potential confounders (OR 1·71, 95% CI 1·04, 2·79). We identified a behaviour-related pattern that was positively associated with the MetS. The application of both dietary pattern methods may be advantageous to obtain information for designing and realising dietary guidelines. Prospective studies are needed to confirm the results.

Postdiagnosis body mass index and risk of mortality in colorectal cancer survivors: a prospective study and meta-analysis.

PURPOSE: Aim of this study was to investigate the association between postdiagnosis body mass index (BMI) and all-cause mortality in colorectal cancer (CRC) survivors in a prospective study and meta-analysis. METHODS: We conducted a prospective cohort study on 2,143 CRC survivors in Germany. Participants were recruited to the study on average 4 years after diagnosis, and postdiagnosis BMI was assessed at recruitment using a self-administered questionnaire. CRC survivors were followed up for a mean time of 3.5 years. The association between BMI and all-cause mortality was investigated using multivariable Cox proportional hazards models. Additionally, we performed a meta-analysis of studies on postdiagnosis BMI and all-cause mortality (n = 5, including this study) by applying random-effects models. RESULTS: In the prospective analysis, 349 participants died. BMI was not statistically significantly associated with all-cause mortality. Compared to normal weight survivors, the hazard ratios (HRs) [95% confidence interval (CI)] for all-cause mortality in underweight, overweight and obese survivors were 1.65 (0.79-3.45), 0.80 (0.62-1.03) and 0.84 (0.62-1.14), respectively. In the meta-analysis, individuals with underweight were at increased risk for all-cause mortality [HR (95% CI) 1.72 (1.18-2.49)], whereas individuals with overweight had a lower risk [HR (95% CI) 0.79 (0.71-0.88)], compared to normal weight subjects. For obesity, the risk of mortality was also reduced with only borderline significance [HR (95% CI) 0.88 (0.77-1.00)]. CONCLUSIONS: While the present study as well as single previously published studies showed that overweight was associated with a non-significant reduced risk for all-cause mortality, our meta-analysis indicated a decreased mortality risk among overweight CRC survivors.

A global analysis of Y-chromosomal haplotype diversity for 23 STR loci.

In a worldwide collaborative effort, 19,630 Y-chromosomes were sampled from 129 different populations in 51 countries. These chromosomes were typed for 23 short-tandem repeat (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643) and using the PowerPlex Y23 System (PPY23, Promega Corporation, Madison, WI). Locus-specific allelic spectra of these markers were determined and a consistently high level of allelic diversity was observed. A considerable number of null, duplicate and off-ladder alleles were revealed. Standard single-locus and haplotype-based parameters were calculated and compared between subsets of Y-STR markers established for forensic casework. The PPY23 marker set provides substantially stronger discriminatory power than other available kits but at the same time reveals the same general patterns of population structure as other marker sets. A strong correlation was observed between the number of Y-STRs included in a marker set and some of the forensic parameters under study. Interestingly a weak but consistent trend toward smaller genetic distances resulting from larger numbers of markers became apparent.

Dietary patterns associated with magnetic resonance imaging-determined liver fat content in a general population study.

BACKGROUND: The association between diet and fatty liver disease (FLD) has predominantly been analyzed for single nutrients or foods, and findings have been inconsistent. OBJECTIVE: We aimed to compare associations of hypothesis-driven and exploratory dietary pattern scores with liver fat content. DESIGN: Liver fat was measured by using magnetic resonance imaging as liver signal intensity (LSI) in a population-based, cross-sectional study that included 354 individuals. We applied partial least-squares regression to derive an exploratory dietary pattern score that explained variation in both the intake of 38 food groups, which were assessed by using a food-frequency questionnaire, and LSI. The hypothesis-driven score was calculated on the basis of published studies. Multivariable linear or logistic regression was used to investigate associations between dietary pattern scores and LSI or FLD. RESULTS: A higher percentage of LSI variation was explained by the exploratory (12.6%) compared with the hypothesis-driven (2.2%) dietary pattern. Of the 13 most important food groups of the exploratory dietary pattern, intakes of green and black tea, soups, and beer were also individually associated with LSI values. A 1-unit increase in the exploratory dietary pattern score was positively associated with FLD (OR: 1.56; 95% CI: 1.29, 1.88). Furthermore, a 1-unit increase in the hypothesis-driven dietary pattern score, which consisted of alcohol, soft drinks, meat, coffee, and tea, was positively associated with FLD (OR: 1.25; 95% CI: 1.10, 1.43). CONCLUSION: We defined a hypothesis-driven dietary pattern and derived an exploratory dietary pattern, both of which included alcohol, meat (poultry), and tea, associated with liver fat content independent from confounders, which should be explored in prospective studies.

Diagnosing fatty liver disease: a comparative evaluation of metabolic markers, phenotypes, genotypes and established biomarkers.

BACKGROUND: To date, liver biopsy is the only means of reliable diagnosis for fatty liver disease (FLD). Owing to the inevitable biopsy-associated health risks, however, the development of valid noninvasive diagnostic tools for FLD is well warranted. AIM: We evaluated a particular metabolic profile with regard to its ability to diagnose FLD and compared its performance to that of established phenotypes, conventional biomarkers and disease-associated genotypes. METHODS: The study population comprised 115 patients with ultrasound-diagnosed FLD and 115 sex- and age-matched controls for whom the serum concentration was measured of 138 different metabolites, including acylcarnitines, amino acids, biogenic amines, hexose, phosphatidylcholines (PCs), lyso-PCs and sphingomyelins. Established phenotypes, biomarkers, disease-associated genotypes and metabolite data were included in diagnostic models for FLD using logistic regression and partial least-squares discriminant analysis. The discriminative power of the ensuing models was compared with respect to area under curve (AUC), integrated discrimination improvement (IDI) and by way of cross-validation (CV). RESULTS: Use of metabolic markers for predicting FLD showed the best performance among all considered types of markers, yielding an AUC of 0.8993. Additional information on phenotypes, conventional biomarkers or genotypes did not significantly improve this performance. Phospholipids and branched-chain amino acids were most informative for predicting FLD. CONCLUSION: We show that the inclusion of metabolite data may substantially increase the power to diagnose FLD over that of models based solely upon phenotypes and conventional biomarkers.

Genome-wide investigation of gene-environment interactions in colorectal cancer.

Colorectal cancer (CRC), one of the most frequent neoplasias worldwide, has both genetic and environmental causes. As yet, however, gene-environment (G × E) interactions in CRC have been studied mostly for a small number of candidate genes only. Therefore, we investigated the possible interaction, in CRC etiology, between single-nucleotide polymorphisms (SNPs) on the one hand, and overweight, smoking and alcohol consumption on the other, at a genome-wide level. To this end, we adopted a two-tiered approach comprising a case-only screening stage I (314 cases) and a case-control validation stage II (259 cases, 1,002 controls). Interactions with the smallest p value in stage I were verified in stage II using multiple logistic regression analysis adjusted for sex and age. In addition, we specifically studied known CRC-associated SNPs for possible G × E interactions. Upon adjustment for sex and age, and after allowing for multiple testing, however, only a single SNP (rs1944511) was found to be involved in a statistically significant interaction, namely with overweight (multiplicity-corrected p = 0.042 in stage II). Several other G × E interactions were nominally significant but failed correction for multiple testing, including a previously reported interaction between rs9929218 and alcohol consumption that also emerged in our candidate SNP study (nominal p = 0.008). Notably, none of the interactions identified in our genome-wide analysis was with a previously reported CRC-associated SNP. Our study therefore highlights the potential of an "agnostic" genome-wide approach to G × E analysis.

Prognostic value of perinodal lymphovascular invasion following radical cystectomy for lymph node-positive urothelial carcinoma.

BACKGROUND: Metastasis of urothelial carcinoma of the bladder (UCB) into regional lymph nodes (LNs) is a key prognosticator for cancer-specific survival (CSS) after radical cystectomy (RC). Perinodal lymphovascular invasion (pnLVI) has not yet been defined. OBJECTIVE: To assess the prognostic value of histopathologic prognostic factors, especially pnLVI, on survival. DESIGN, SETTING, AND PARTICIPANTS: A total of 598 patients were included in a prospective multicentre study after RC for UCB without distant metastasis and neoadjuvant and/or adjuvant chemotherapy. En bloc resection and histopathologic evaluation of regional LNs were performed based on a prospective protocol. The final study group comprised 158 patients with positive LNs (26.4%). INTERVENTION: Histopathologic analysis was performed based on prospectively defined morphologic criteria of LN metastases. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Multivariable Cox proportional hazard regression models determined prognostic impact of clinical and histopathologic variables (age, gender, tumour stage, surgical margin status, pN, diameter of LN metastasis, LN density [LND], extranodal extension [ENE], pnLVI) on CSS. The median follow-up was 20 mo (interquartile range: 11-38). RESULTS AND LIMITATIONS: Thirty-one percent of patients were staged pN1, and 69% were staged pN2/3. ENE and pnLVI was present in 52% and 39%, respectively. CSS rates after 1 yr, 3 yr, and 5 yr were 77%, 44%, and 27%, respectively. Five-year CSS rates in patients with and without pnLVI were 16% and 34% (p<0.001), respectively. PN stage, maximum diameter of LN metastasis, LND, and ENE had no independent influence on CSS. In the multivariable Cox model, the only parameters that were significant for CSS were pnLVI (hazard ratio: 2.47; p=0.003) and pT stage. However, pnLVI demonstrated only a minimal gain in predictive accuracy (0.1%; p=0.856), and the incremental accuracy of prediction is of uncertain clinical value. CONCLUSIONS: We present the first explorative study on the prognostic impact of pnLVI. In contrast to other parameters that show the extent of LN metastasis, pnLVI is an independent prognosticator for CSS.

Dual energy CT in patients with polycystic kidney disease.

OBJECTIVES: To evaluate the diagnostic efficacy of dual source-dual energy CT (DECT) in the detection of neoplasia in patients with polycystic kidney disease (PKD). METHODS: A total of 21 patients with PKD underwent DECT on a dual source system, using kVp settings of Sn140/100 or 140/80. Colour-coded iodine maps and virtual unenhanced images were used to determine enhancement within cysts and to differentiate haemorrhagic from simple cysts. A cut-off of 15 HU was used as a threshold for malignancy. In patients with malignancy, histopathology was the gold standard; otherwise, patients underwent follow-up imaging for 150-908 days. RESULTS: On the basis of measured enhancement, 13 enhancing masses were seen in 4 patients (12 renal cell cancers and 1 adenoma); follow-up imaging showed no malignancy in 18 patients. Cysts did not enhance by more than 15 HU, whereas masses showed a mean enhancement of 45 (25-123) HU. Average radiation exposure was 9.6 mSv for the biphasic protocol and 5.8 mSv for DECT only. CONCLUSION: DECT greatly facilitates the detection of malignancy in patients with polycystic kidney disease, at the same time reducing radiation exposure by omission of a true unenhanced phase. KEY POINTS : • Identification of tumours within polycystic kidneys can be difficult. • Dual energy computed tomography (DECT) provides two separate sets of images. • Iodine maps and virtual non-enhanced (VNE) images can then be calculated. • DECT facilitates screening for potential renal tumours in polycystic kidneys.

Detection of inguinal lymph node involvement in penile squamous cell carcinoma by 18F-fluorodeoxyglucose PET/CT: a prospective single-center study.

BACKGROUND: The extent of lymph node involvement is the most relevant prognostic factor in patients with penile cancer. OBJECTIVE: To prospectively analyze the diagnostic accuracy of 18F-FDG-PET/CT-scan in the assessment of inguinal lymph node involvement in patients with invasive penile carcinoma. PATIENTS AND METHODS: Thirty-five patients with invasive penile carcinoma were staged prospectively by 18F-FDG-PET/CT-scan, and blindly evaluated by 2 nuclear medicine physicians. In total, lymph node involvement was assessed in 70 inguinal groins. Reference standard was either histology or clinical follow-up with a minimum of 31 months (mean: 48.4 months; range: 31-68 months). RESULTS: 18-FDG-PET/CT showed a sensitivity of 88.2% and a specificity of 98.1%. Positive predictive value (PPV) was 93.8%, while negative predictive value (NPV) was 96.3%. In two groins, metastasis of 5 and 7 mm were missed by PET/CT scan. CONCLUSION: 18F-FDG-PET/CT is a promising staging tool in assessing the inguinal lymph node involvement of patients with penile carcinoma. Integration of PET/CT scanning into preoperative staging algorithms may avoid surgical staging in selected patients.

Transcript signature predicts tissue NK cell content and defines renal cell carcinoma subgroups independent of TNM staging.

Clear cell renal cell carcinoma (ccRCC) is an aggressive and difficult to manage cancer. Immunotherapy has the potential to induce long-lasting regression in a small group of patients. However, severe side effects limit broad application which highlights the need for a marker to distinguish responder from nonresponder. TNMG staging, referring to tumor size, lymph node involvement, presence of metastasis, and grade of tumor differentiation, represents an important prognostic system but is not useful for predicting responders to immunotherapy. NK cells are potent antitumor effector cells, and a role as prognostic marker in some solid tumors has been suggested. As NK cells are responsive to various immune modifiers, they may be important mediators of patient response to immunotherapies, in particular those including IL-2. We report that the NK cell percentage within RCC-infiltrating lymphocytes, as determined by flow cytometry, allows ccRCC subgrouping in NK(high)/NK(low) tissues independent of TNMG classification. Quantitative reverse transcriptase polymerase chain reaction using whole-tissue RNA identified four markers (NKp46, perforin, CX(3)CL1, and CX(3)CR1) whose transcript levels reproduced the NK(high)/NK(low) tissue distinction identified by flow cytometry with high selectivity and specificity. Combined in a multiplex profile and analyzed using neural network, the accuracy of predicting the NK(high)/NK(low) groups was 87.8%, surpassing that of each single marker. The tissue transcript signature, based on a robust high-throughput methodology, is easily amenable to archive material and clinical translation. This now allows the analysis of large patient cohorts to substantiate a role of NK cells in cancer progression or response to immunotherapy.

Outcome of laser-assisted laparoscopic partial nephrectomy without ischaemia for peripheral renal tumours.

OBJECTIVES: Evaluation of clamp-off laser-assisted laparoscopic partial nephrectomy technique (LLPN) compared to the clamp-off laparoscopic (LPN) and open (OPN) techniques. PATIENTS AND METHODS: Between September 2008 and July 2011, 36 patients suffering from small peripheral renal tumours (RT) underwent LLPN (n = 12), LPN (n = 12) and OPN (n = 12) in a prospective single-centre study. RT were excised with laser, Sonosurg or monopolar scissors during LLPN, LPN and OPN, respectively. Blood vessels are identified and sutured before opening them; alternatively, laser energy was used to coagulate them (LLPN). Early and late postoperative complications were assessed. Follow-up was done according to EAU-guidelines. RESULTS: Mean age was 64.9 years. Mean operative time was 135.8 min (100-180) versus 144.2 (85-255) versus 113.6 (50-170) for LLPN versus LPN versus OPN, respectively. Median estimated blood loss (EBL) was 170.8 ml (50-600) versus 245.2 (50-700) versus 425.8 (100-900) for LLPN versus LPN versus OPN, respectively. Tumours (19 right and 17 left) were located in upper (11), midparenchyma (13) and lower pole (12). Mean tumour size was 2.7 cm (1.2-5.5). There were no reported perioperative complications/conversions. There were no positive margins. Histological evaluations were not compromised in any LLPN-case. Compared to LPN, LLPN offered significant lower EBL, shorter operative time, otherwise, comparable results. Follow-up was uneventful without tumour recurrences. CONCLUSION: Current prospective comparative study shows that LLPN is a reproducible efficient alternative to LPN/OPN. Besides the absence of renal ischaemia, LLPN offered lower EBL, good haemostasis and minimal parenchyma damage. Surgical and oncological outcomes are comparable to LPN and OPN.

Human renal cell carcinoma induces a dendritic cell subset that uses T-cell crosstalk for tumor-permissive milieu alterations.

Tissue dendritic cells (DCs) may influence the progression of renal cell carcinoma (RCC) by regulating the functional capacity of antitumor effector cells. DCs and their interaction with T cells were analyzed in human RCC and control kidney tissues. The frequency of CD209(+) DCs in RCCs was found to be associated with an unfavorable T(H)1 cell balance in the tissue and advanced tumor stages. The CD209(+) DCs in RCC were unusual because most of them co-expressed macrophage markers (CD14, CD163). The phenotype of these enriched-in-renal-carcinoma DCs (ercDCs) could be reiterated in vitro by carcinoma-secreted factors (CXCL8/IL-8, IL-6, and vascular endothelial growth factor). ErcDCs resembled conventional DCs in costimulatory molecule expression and antigen cross-presentation. They did not suppress cognate cytotoxic T-lymphocyte function and did not cause CD3ζ down-regulation, FOXP3 induction, or T-cell apoptosis in situ or in vitro; thus, they are different from classic myeloid-derived suppressor cells. ErcDCs secreted high levels of metalloproteinase 9 and used T-cell crosstalk to increase tumor-promoting tumor necrosis factor α and reduce chemokines relevant for T(H)1-polarized lymphocyte recruitment. This modulation of the tumor environment exerted by ercDCs suggests an immunologic mechanism by which tumor control can fail without involving cytotoxic T-lymphocyte inhibition. Pharmacologic targeting of the deviated DC differentiation could improve the efficacy of immunotherapy against RCC.

APOE ε4 is associated with higher vitamin D levels in targeted replacement mice and humans.

The allele ε4 of apolipoprotein E (APOE), which is a key regulator of lipid metabolism, represents a risk factor for cardiovascular diseases and Alzheimer's disease. Despite its adverse effects, the allele is common and shows a nonrandom global distribution that is thought to be the result of evolutionary adaptation. One hypothesis proposes that the APOE ε4 allele protects against vitamin D deficiency. Here we present, for the first time, experimental and epidemiological evidence that the APOE ε4 allele is indeed associated with higher serum vitamin D [25(OH)D] levels. In APOE4 targeted replacement mice, significantly higher 25(OH)D levels were found compared with those in APOE2 and APOE3 mice (70.9 vs. 41.8 and 27.8 nM, P<0.05). Furthermore, multivariate adjusted models show a positive association of the APOE ε4 allele with 25(OH)D levels in a small collective of human subjects (n=93; P=0.072) and a general population sample (n=699; P=0.003). The novel link suggests ε4 as a modulator of vitamin D status. Although this result agrees well with evolutionary aspects, it appears contradictory with regard to chronic diseases, especially cardiovascular disease. Large prospective cohort studies are now needed to investigate the potential implications of this finding for chronic disease risks.

DNA hypermethylation in papillary renal cell carcinoma.

OBJECTIVE: To investigate the pattern of DNA CpG island hypermethylation in papillary renal cell carcinoma (pRCC). MATERIAL AND METHODS: DNA from pRCC (n= 32) and adjacent normal tissue (n= 15) was isolated. A quantitative methylation-specific PCR was performed to analyse the methylation pattern at APC (actin beta), CDH1 (E-cadherin), GSTP1 (glutathione S-transferase pi 1), RASSF1A (Ras association domain family member 1A) and TIMP3 (TIMP metallopeptidase inhibitor 3); a sequence of ACTB without CpG was used to normalize for DNA input and to calculate the relative amount of methylated DNA (normalized index of methylation, NIM). RESULTS: RASSF1A hypermethylation was observed in most pRCC and normal samples (100 vs 94.4%), but the median NIM was significantly higher in pRCC samples (2.11 vs 0.61; P < 0.001). RASSF1A hypermethylation allowed discrimination of pRCC and normal tissue with a sensitivity of 87.5% and a specificity of 73.3% as determined via receiver operator characteristic analysis (area under curve = 0.814). Hypermethylation at APC (3.0 vs 6.7%), CDH1 (15.6 vs 0%), GSTP1 (21.9 vs 6.7%) and TIMP3 (6.3 vs 0%) was infrequent in pRCC and normal tissue. CDH1 was significantly correlated with pathological stage (P= 0.015), and patients with methylated CDH1 methylation showed a trend towards shorter recurrence-free survival (log-rank P= 0.057). The number of methylated gene sites was correlated with pathological stage (P= 0.007) and lymph node metastasis (P= 0.008). CONCLUSIONS: DNA hypermethylation at RASSF1A is common in pRCC tissue irrespective of the histological subtype, but also frequently seen at lower levels in normal adjacent tissue. Aberrant hypermethylation could be a prognostic marker for pRCC.