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It remains unclear to what extent tumor heterogeneity impacts on protein biomarker discovery. Here, we quantified proteome intra-tissue heterogeneity (ITH) based on a multi-region analysis of prostate tissues using pressure cycling technology and Sequential Windowed Acquisition of all THeoretical fragment ion mass spectrometry. We quantified 6,873 proteins and analyzed the ITH of 3,700 proteins. The level of ITH varied depending on proteins and tissue types. Benign tissues exhibited more complex ITH patterns than malignant tissues. Spatial variability of 10 prostate biomarkers was validated by immunohistochemistry in an independent cohort (n = 83) using tissue microarrays. Prostate-specific antigen was preferentially variable in benign prostatic hyperplasia, whereas growth/differentiation factor 15 substantially varied in prostate adenocarcinomas. Furthermore, we found that DNA repair pathways exhibited a high degree of variability in tumorous tissues, which may contribute to the genetic heterogeneity of tumors. This study conceptually adds a new perspective to protein biomarker discovery: it suggests that recent technological progress should be exploited to quantify and account for spatial proteome variation to complement biomarker identification and utilization.

Urothelial carcinoma (UC), the most common cancer of the urinary bladder causes severe morbidity and mortality, e.g. about 40.000 deaths in the EU annually, and incurs considerable costs for the health system due to the need for prolonged treatments and long-term monitoring. Extensive aberrant DNA methylation is described to prevail in urothelial carcinoma and is thought to contribute to genetic instability, altered gene expression and tumor progression. However, it is unknown how this epigenetic alteration arises during carcinogenesis. Intact methyl group metabolism is required to ensure maintenance of cell-type specific methylomes and thereby genetic integrity and proper cellular function. Here, using two independent techniques for detecting DNA methylation, we observed DNA hypermethylation of the 5'-regulatory regions of the key methyl group metabolism genes ODC1, AHCY and MTHFR in early urothelial carcinoma. These hypermethylation events are associated with genome-wide DNA hypomethylation which is commonly associated with genetic instability. We therefore infer that hypermethylation of methyl group metabolism genes acts in a feed-forward cycle to promote additional DNA methylation changes and suggest a new hypothesis on the molecular etiology of urothelial carcinoma.

Background: We aimed to analyze the frequency and distribution of PD-L1 expression in specimens from prostate cancer (PC) patients using two different anti-PD-L1 antibodies (E1L3N, SP263). Materials and Methods: PD-L1 immunohistochemistry was performed in a tissue microarray consisting of 82 castration-resistant prostate cancer (CRPC) specimens, 70 benign prostate hyperplasia (BPH) specimens, 96 localized PC cases, and 3 PC cell lines, using two different antibodies (clones E1L3N, and SP263). Staining images for CD4, CD8, PD-L1, and PanCK of a single PD-L1 positive case were compared, using a newly developed dot-wise correlation method for digital images to objectively test for co-expression. Results: Depending on the antibody used, in tumor cells (TC) only five (E1L3N: 6%) and three (SP263: 3.7%) samples were positive. In infiltrating immune cells (IC) 12 (SP263: 14.6%) and 8 (E1L3N: 9.9%) specimens showed PD-L1 expression. Two PC cell lines (PC3, LnCaP) also displayed membranous immunoreactivity. All localized PCs or BPH samples tested were negative. Dot-wise digital correlation of expression patterns revealed a moderate positive correlation between PD-L1 and PanCK expression, whereas both PanCK and PD-L1 showed a weak negative Pearson correlation coefficient between CD4 and CD8. Conclusions: PD-L1 was not expressed in localized PC or BPH, and was only found in a minority of CRPC tumors and infiltrating immune cells. Protein expression maps and systematic dot-wise comparison could be a useful objective way to describe the relationship between immuno- and tumor-related proteins in the future, without the need to develop multiplex staining methods.

BACKGROUND Autophagy is a catabolic cellular process used for degradation of cytoplasmic organelles and preservation of cell viability. In this study we aimed to analyse the level of autophagy markers in benign and malignant prostate tissue and to evaluate the prognostic properties for patients with prostate cancer (PCa). RESULTS LC3b expression was significantly upregulated in PCa, especially in metastatic and castration-resistant PCa samples compared to benign prostate tissue (p<0.001). Evaluation of expression in malignant radical prostatectomy specimens revealed an inverse association with preoperative serum PSA levels (p=0.02) and Gleason Score (p=0.07). LC3b immunoreactivity was identified as a novel predictor of PCa specific death after radical prostatectomy, independent of Gleason score, tumour stage, and surgical margin status in a multivariable cox regression analysis (hazard ratio 0.09, 95% confidence interval 0.01-0.69, p=0.021). A significant association of ATG-5 and Beclin 1 with LC3b expression could be noticed (p<0.001), but no link with other clincopathologic parameters was observed. METHODS A Tissue microarray containing 468 formalin-fixed, paraffin-embedded prostate tissue cores was stained immunohistochemically for major autophagy proteins LC3b, ATG5 and Beclin 1. Immunoreactivity was semiquantitatively scored and correlated with pathologic and clinical parameters, including tumour stage, Gleason score, preoperative PSA level, biochemical recurrence rate and survival. The median clinical follow-up was 132 months. CONCLUSION LC3b was significantly overexpressed in malignant compared to benign prostate tissue. However, positive LC3b immunoreactivity in PCa, as a marker of increased autophagy, was independently associated with a reduced disease-specific mortality.

Several lymphangiogenic factors, such as vascular endothelial growth factors (VEGFs), have been found to drive the development of lymphatic metastasis in bladder cancer (BCa). Here, we have analyzed the gene expression of lymphangiogenic factors in tissue specimens from 12 non-muscle invasive bladder cancers (NMIBC) and 11 muscle invasive bladder cancers (MIBC), considering tumor and tumor-adjacent normal bladder areas obtained from the same organs. We then compared the results observed in patients with those obtained after treating human primary bladder microvascular endothelial cells (MEC) with either direct stimulation with VEGF-A or VEGF-C or by co-culturing (trans-well assay) MEC with bladder cancer cell lines varying in VEGF-A and VEGF-C production based on tumor grade. The genes of three markers of lymphatic endothelial commitment and development (PDPN, LYVE-1 and SLP-76) were significantly overexpressed in tissues of MIBC patients showing positive lymphovascular invasion (LVI+), lymph node metastasis (Ln+) and tumor progression. Their expression was also significantly enhanced either after direct stimulation of MEC by VEGF-A and VEGF-C or in the trans-well assay with each bladder cancer cell line. SLP-76 showed the highest gene expression. Both VEGF-A and VEGF-C also enhanced the expression of SLP-76 protein in MEC. However, a correlation between increase of SLP-76 gene expression and the ability of MEC to migrate could only be seen after induction by VEGF-C. The significant expression of SLP-76 in LVI+/Ln+ progressive MIBC and its overexpression in MEC after VEGF-A and VEGF-C stimulation suggest the need to develop this regulator of developmental lymphangiogenesis as a diagnostic tool in BCa.

BACKGROUND: Intratumoral hypoxia plays an important role with regard to tumor biology and susceptibility to radio- and chemotherapy. For further investigation of hypoxia-related changes, areas of certain hypoxia must be reliably detected within cancer tissues. Pimonidazole, a 2-nitroimindazole, accumulates in hypoxic tissue and can be easily visualized using immunohistochemistry. MATERIALS AND METHODS: To improve detection of highly hypoxic versus normoxic areas in prostate cancer, immunoreactivity of pimonidazole and a combination of known hypoxia-related proteins was used to create computational oxygen supply maps of prostate cancer. Pimonidazole was intravenously administered before radical prostatectomy in n = 15 patients, using the da Vinci robot-assisted surgical system. Prostatectomy specimens were immediately transferred into buffered formaldehyde, fixed overnight, and completely embedded in paraffin. Pimonidazole accumulation and hypoxia-related protein expression were visualized by immunohistochemistry. Oxygen supply maps were created using the normalized information from pimonidazole and hypoxia-related proteins. RESULTS: Based on pimonidazole staining and other hypoxia.related proteins (osteopontin, hypoxia-inducible factor 1-alpha, and glucose transporter member 1) oxygen supply maps in prostate cancer were created. Overall, oxygen supply maps consisting of information from all hypoxia-related proteins showed high correlation and mutual information to the golden standard of pimonidazole. Here, we describe an improved computer-based ex vivo model for an accurate detection of oxygen supply in human prostate cancer tissue. CONCLUSIONS: This platform can be used for precise colocalization of novel candidate hypoxia-related proteins in a representative number of prostate cancer cases, and improve issues of single marker correlations. Furthermore, this study provides a source for further in situ tests and biochemical investigations.

Infectious agents, including the BK polyomavirus (BKPyV), have been proposed as important inflammatory pathogens in prostate cancer. Here, we evaluated whether the preoperative antibody response to BKPyV large T antigen (LTag) and viral capsid protein 1 (VP1) was associated with the risk of biochemical recurrence in 226 patients undergoing radical prostatectomy for primary prostate cancer. Essentially, the multivariate Cox regression analysis revealed that preoperative seropositivity to BKPyV LTag significantly reduced the risk of biochemical recurrence, independently of established predictors of biochemical recurrence such as tumor stage, Gleason score and surgical margin status. The predictive accuracy of the regression model was denotatively increased by the inclusion of the BKPyV LTag serostatus. In contrast, the VP1 serostatus was of no prognostic value. Finally, the BKPyV LTag serostatus was associated with a peculiar cytokine gene expression profile upon assessment of the cellular immune response elicited by LTag. Taken together, our findings suggest that the BKPyV LTag serology may serve as a prognostic factor in prostate cancer. If validated in additional studies, this biomarker may allow for better treatment decisions after radical prostatectomy. Finally, the favorable outcome of LTag seropositive patients may provide a potential opportunity for novel therapeutic approaches targeting a viral antigen.

Tumor-associated blood vessels differ from normal vessels and proteins present only on tumor vessels may serve as biomarkers or targets for anti-angiogenic therapy in cancer. Comparing the transcriptional profiles of blood vascular endothelium from human invasive bladder cancer with normal bladder tissue, we found that the endothelial cell-specific molecule endocan (ESM1) was highly elevated on tumor vessels. Endocan was associated with filopodia of angiogenic endothelial tip cells in invasive bladder cancer. Notably, endocan expression on tumor vessels correlated strongly with staging and invasiveness, predicting a shorter recurrence-free survival time in non-invasive bladder cancers. Both endocan and VEGF-A levels were higher in plasma of patients with invasive bladder cancer than healthy individuals. Mechanistic investigations in cultured blood vascular endothelial cells or transgenic mice revealed that endocan expression was stimulated by VEGF-A through the phosphorylation and activation of VEGFR-2, which was required to promote cell migration and tube formation by VEGF-A. Taken together, our findings suggest that disrupting endocan interaction with VEGFR-2 or VEGF-A could offer a novel rational strategy to inhibit tumor angiogenesis. Further, they suggest that endocan might serve as a useful biomarker to monitor disease progression and the efficacy of VEGF-A-targeting therapies in patients with bladder cancer.