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Purpose: Prostate cancer aggressiveness and appropriate therapy are routinely determined following biopsy sampling. Current clinical and pathologic parameters are insufficient for accurate risk prediction leading primarily to overtreatment and also missed opportunities for curative therapy.Experimental Design: An 8-biomarker proteomic assay for intact tissue biopsies predictive of prostate pathology was defined in a study of 381 patient biopsies with matched prostatectomy specimens. A second blinded study of 276 cases validated this assay's ability to distinguish “favorable” versus “nonfavorable” pathology independently and relative to current risk classification systems National Comprehensive Cancer Network (NCCN and D'Amico).Results: A favorable biomarker risk score of ≤0.33, and a nonfavorable risk score of >0.80 (possible range between 0 and 1) were defined on “false-negative” and “false-positive” rates of 10% and 5%, respectively. At a risk score ≤0.33, predictive values for favorable pathology in very low-risk and low-risk NCCN and low-risk D'Amico groups were 95%, 81.5%, and 87.2%, respectively, higher than for these current risk classification groups themselves (80.3%, 63.8%, and 70.6%, respectively). The predictive value for nonfavorable pathology was 76.9% at biomarker risk scores >0.8 across all risk groups. Increased biomarker risk scores correlated with decreased frequency of favorable cases across all risk groups. The validation study met its two coprimary endpoints, separating favorable from nonfavorable pathology (AUC, 0.68; P < 0.0001; OR, 20.9) and GS-6 versus non–GS-6 pathology (AUC, 0.65; P < 0.0001; OR, 12.95).Conclusions: The 8-biomarker assay provided individualized, independent prognostic information relative to current risk stratification systems, and may improve the precision of clinical decision making following prostate biopsy. Clin Cancer Res; 21(11); 2591–600. ©2015 AACR.

Supplementary Methods, Table S1, Figures S1-7. Methods for quantitative multiplex proteomics imaging (QMPI) Clinical studies: Statistical plan Table S1. Clinical Validation Study. Comparison of Predictive Value of the 8-Biomarker Assay for Favorable Pathology with D'Amico Risk Categories. Figure S1. Outline of all four quantitative multiplex immunofluorescence triplex assay formats Figure S2. Clinical validation study, full cohort (N=276): performance for "GS 6" pathology (surgical Gleason =3+3 and localized {less than or equal to}T3a). A) Sensitivity (P[risk score> threshold| "non-GS 6" pathology]) of the assay, as a function of medical decision level. Figure S3. Clinical validation study, full cohort (N=274): performance for prediction of favorable pathology (surgical Gleason {less than or equal to}3+4 and organ-confined {less than or equal to}T2). Figure S4. Clinical validation study, Subset of validation cohort that contained sufficient annotation for National Comprehensive Cancer Network (NCCN) and D'Amico categorization (N=256) Figure S5. Clinical validation study: performance for prediction of favorable pathology. Figure S6. Net Reclassification Index analysis illustrates how biomarker assay categories of favorable (risk score {less than or equal to}0·33) and non-favorable (risk score >0·8) may supplement NCCN Figure S7. Decision Curve Analysis provides another method for characterizing performance of different risk systems and at different cut points.

RNA-binding proteins are instrumental for post-transcriptional gene regulation, controlling all aspects throughout the lifecycle of RNA molecules. However, transcriptome-wide methods to profile RNA-protein interactions in vivo remain technically challenging and require large amounts of starting material. Herein, we present an improved library preparation strategy for crosslinking and immunoprecipitation (CLIP) that is based on tailing and ligation of cDNA molecules (TLC). TLC involves the generation of solid-phase cDNA, followed by ribotailing to increase the efficiency of subsequent adapter ligation. These modifications result in a streamlined, fully bead-based library preparation strategy, which eliminates time-consuming purification procedures and drastically reduces sample loss, allowing the profiling of RNA-protein interactions from as few as 1000 cells. In the accompanying manuscript, we have applied TLC-CLIP to four endogenous RNA-binding proteins, demonstrating its reproducibility and improved precision due to a higher number of crosslinking-induced deletions that serve as an intrinsic quality metric and increase both specificity and nucleotide-resolution.

Viruses rely on the reprogramming of cellular processes to enable efficient viral replication; this often requires subcompartmentalization within the host cell. Liquid-liquid phase separation (LLPS) has emerged as a fundamental principle to organize and subdivide cellular processes, and plays an important role in viral life cycles. Despite substantial advances in the field, elucidating the exact organization and function of these organelles remains a major challenge. In this review, we summarize the biochemical basis of condensate formation, the role of LLPS during viral infection, and interplay of LLPS with innate immune responses. Finally, we discuss possible strategies and molecules to modulate LLPS during viral infections.

RNA-binding proteins are instrumental for post-transcriptional gene regulation, yet transcriptomewide methods to profile RNA-protein interactions remain technically challenging. We present an improved library preparation strategy for crosslinking and immunoprecipitation (CLIP) that involves tailing and ligation of cDNA molecules (TLC) for increased sensitivity and efficiency. TLC-CLIP eliminates time-consumingpurifications, reduces sample loss and minimises experimental steps, allowing precise profiling of RNA-protein interactions from limited starting material at nucleotide resolution.

SUMMARYUnderstanding male fertility requires an in-depth characterisation of spermatogenesis, the developmental process by which male gametes are generated. Spermatogenesis occurs continuously throughout a male’s reproductive window and involves a complex sequence of developmental steps, both of which make this process difficult to decipher at the molecular level. To overcome this, we transcriptionally profiled single cells from multiple distinct stages during the first wave of spermatogenesis, where the most mature germ cell type is known. This naturally enriches for spermatogonia and somatic cell types present at very low frequencies in adult testes. Our atlas, available as a shiny app (https://marionilab.cruk.cam.ac.uk/SpermatoShiny), allowed us to reconstruct the three main processes of spermatogenesis: spermatogonial differentiation, meiosis, and spermiogenesis. Additionally, we profiled the chromatin changes associated with meiotic silencing of the X chromosome, revealing a set of genes specifically and strongly repressed by H3K9me3 in the spermatocyte stage, but which escape post-meiotic silencing in spermatids.

Structural variants can contribute to oncogenesis through a variety of mechanisms. Despite their importance, the identification of structural variants in cancer genomes remains challenging. Here, we present a framework that integrates optical mapping, high-throughput chromosome conformation capture (Hi-C), and whole genome sequencing to systematically detect SVs in a variety of normal or cancer samples and cell lines. We identify the unique strengths of each method and demonstrate that only integrative approaches can comprehensively identify structural variants in the genome. By combining Hi-C and optical mapping, we resolve complex SVs and phase multiple SV events to a single haplotype. Furthermore, we observe widespread structural variation events affecting the functions of non-coding sequences, including the deletion of distal regulatory sequences, alteration of DNA replication timing, and the creation of novel 3D chromatin structural domains. Our results indicate that non-coding structural variations may be underappreciated mutational drivers in cancer genomes.

Transposable elements (TEs) contribute to the large amount of repetitive sequences in mammalian genomes and have been linked to species-specific genome innovations by rewiring regulatory circuitries. However, organisms need to restrict TE activity to ensure genome integrity, especially in germline cells to protect the transmission of genetic information to the next generation. This review features our current understandings of mammalian PIWI-interacting RNAs (piRNAs) and their role in TE regulation in spermatogenesis. Here we discuss functional implication and explore additional molecular mechanisms that inhibit transposon activity and altogether illustrate the paradoxical arms race between genome evolution and stability., Transposable elements can be activated during germ cell maturation, potentially leading to genome instability and rewiring of the genetic circuitry. In this review, the authors discuss how the piRNA machinery suppresses these elements to ensure accurate spermatogenesis.

Genome stability relies on proper coordination of mitosis and cytokinesis, where dynamic microtubules capture and faithfully segregate chromosomes into daughter cells. With a high-content RNAi imaging screen targeting more than 2,000 human lncRNAs, we identify numerous lncRNAs involved in key steps of cell division such as chromosome segregation, mitotic duration and cytokinesis. Here, we provide evidence that the chromatin-associated lncRNA, linc00899, leads to robust mitotic delay upon its depletion in multiple cell types. We perform transcriptome analysis of linc00899-depleted cells and identify the neuronal microtubule-binding protein, TPPP/p25, as a target of linc00899. We further show that linc00899 binds TPPP/p25 and suppresses its transcription. In cells depleted of linc00899, upregulation of TPPP/p25 alters microtubule dynamics and delays mitosis. Overall, our comprehensive screen uncovers several lncRNAs involved in genome stability and reveals a lncRNA that controls microtubule behaviour with functional implications beyond cell division., Long noncoding RNAs (lncRNAs) regulate key steps of cell division. Here, the authors perform a comprehensive RNAi imaging screen targeting more than 2,000 human lncRNAs, and suggest a role of chromatin-associated linc00899 in regulation of cell division by suppressing the transcription of microtubule-binding protein TPPP/p25.

Male gametes are generated through a specialised differentiation pathway involving a series of developmental transitions that are poorly characterised at the molecular level. Here, we use droplet-based single-cell RNA-Sequencing to profile spermatogenesis in adult animals and at multiple stages during juvenile development. By exploiting the first wave of spermatogenesis, we both precisely stage germ cell development and enrich for rare somatic cell-types and spermatogonia. To capture the full complexity of spermatogenesis including cells that have low transcriptional activity, we apply a statistical tool that identifies previously uncharacterised populations of leptotene and zygotene spermatocytes. Focusing on post-meiotic events, we characterise the temporal dynamics of X chromosome re-activation and profile the associated chromatin state using CUT&RUN. This identifies a set of genes strongly repressed by H3K9me3 in spermatocytes, which then undergo extensive chromatin remodelling post-meiosis, thus acquiring an active chromatin state and spermatid-specific expression., The transcriptional regulation of murine spermatogenesis is not well understood. Here, the authors use single-cell and bulk RNA-Sequencing of juvenile and adult mice to characterise somatic and germ cell development, and chromatin profile the X chromosome to show that spermatid-specific genes are repressed by H3K9me3 during meiosis.

Background Mammalian species exhibit a wide range of lifespans. To date, a robust and dynamic molecular readout of these lifespan differences has not yet been identified. Recent studies have established the existence of ageing-associated differentially methylated positions (aDMPs) in human and mouse. These are CpG sites at which DNA methylation dynamics show significant correlations with age. We hypothesise that aDMPs are pan-mammalian and are a dynamic molecular readout of lifespan variation among different mammalian species. Results A large-scale integrated analysis of aDMPs in six different mammals reveals a strong negative relationship between rate of change of methylation levels at aDMPs and lifespan. This relationship also holds when comparing two different dog breeds with known differences in lifespans. In an ageing cohort of aneuploid mice carrying a complete copy of human chromosome 21, aDMPs accumulate far more rapidly than is seen in human tissues, revealing that DNA methylation at aDMP sites is largely shaped by the nuclear trans-environment and represents a robust molecular readout of the ageing cellular milieu. Conclusions Overall, we define the first dynamic molecular readout of lifespan differences among mammalian species and propose that aDMPs will be an invaluable molecular tool for future evolutionary and mechanistic studies aimed at understanding the biological factors that determine lifespan in mammals. Electronic supplementary material The online version of this article (10.1186/s13059-018-1397-1) contains supplementary material, which is available to authorized users.

Purpose: Prostate cancer aggressiveness and appropriate therapy are routinely determined following biopsy sampling. Current clinical and pathologic parameters are insufficient for accurate risk prediction leading primarily to overtreatment and also missed opportunities for curative therapy. Experimental Design: An 8-biomarker proteomic assay for intact tissue biopsies predictive of prostate pathology was defined in a study of 381 patient biopsies with matched prostatectomy specimens. A second blinded study of 276 cases validated this assay's ability to distinguish “favorable” versus “nonfavorable” pathology independently and relative to current risk classification systems National Comprehensive Cancer Network (NCCN and D'Amico). Results: A favorable biomarker risk score of ≤0.33, and a nonfavorable risk score of >0.80 (possible range between 0 and 1) were defined on “false-negative” and “false-positive” rates of 10% and 5%, respectively. At a risk score ≤0.33, predictive values for favorable pathology in very low-risk and low-risk NCCN and low-risk D'Amico groups were 95%, 81.5%, and 87.2%, respectively, higher than for these current risk classification groups themselves (80.3%, 63.8%, and 70.6%, respectively). The predictive value for nonfavorable pathology was 76.9% at biomarker risk scores >0.8 across all risk groups. Increased biomarker risk scores correlated with decreased frequency of favorable cases across all risk groups. The validation study met its two coprimary endpoints, separating favorable from nonfavorable pathology (AUC, 0.68; P < 0.0001; OR, 20.9) and GS-6 versus non–GS-6 pathology (AUC, 0.65; P < 0.0001; OR, 12.95). Conclusions: The 8-biomarker assay provided individualized, independent prognostic information relative to current risk stratification systems, and may improve the precision of clinical decision making following prostate biopsy. Clin Cancer Res; 21(11); 2591–600. ©2015 AACR.

Summary Topological domains are key architectural building blocks of chromosomes, but their functional importance and evolutionary dynamics are not well defined. We performed comparative high-throughput chromosome conformation capture (Hi-C) in four mammals and characterized the conservation and divergence of chromosomal contact insulation and the resulting domain architectures within distantly related genomes. We show that the modular organization of chromosomes is robustly conserved in syntenic regions and that this is compatible with conservation of the binding landscape of the insulator protein CTCF. Specifically, conserved CTCF sites are co-localized with cohesin, are enriched at strong topological domain borders, and bind to DNA motifs with orientations that define the directionality of CTCF’s long-range interactions. Conversely, divergent CTCF binding between species is correlated with divergence of internal domain structure, likely driven by local CTCF binding sequence changes, demonstrating how genome evolution can be linked to a continuous flux of local conformation changes. We also show that large-scale domains are reorganized during genome evolution as intact modules., Graphical Abstract, Highlights • Multi-species Hi-C comparisons reveal robust conservation of chromosome organization • CTCF binding has evolved under two regimes with different effects on structure • Oriented CTCF motifs determine the directionality of CTCF-mediated interactions, To explore the mechanisms underlying the evolution of chromosomal domain structures, Vietri Rudan et al. compare four mammalian species and reveal a direct link between insulator site divergence and the evolution of chromatin domain structure. Their data point to a direct role for CTCF/cohesin in driving structural change in the genome.

Structural variants can contribute to oncogenesis through a variety of mechanisms, yet, despite their importance, the identification of structural variants in cancer genomes remains challenging. Here, we present an integrative framework for comprehensively identifying structural variation in cancer genomes. For the first time, we apply next-generation optical mapping, high-throughput chromosome conformation capture (Hi-C), and whole genome sequencing to systematically detect SVs in a variety of cancer cells.Using this approach, we identify and characterize structural variants in up to 29 commonly used normal and cancer cell lines. We find that each method has unique strengths in identifying different classes of structural variants and at different scales, suggesting that integrative approaches are likely the only way to comprehensively identify structural variants in the genome. Studying the impact of the structural variants in cancer cell lines, we identify widespread structural variation events affecting the functions of non-coding sequences in the genome, including the deletion of distal regulatory sequences, alteration of DNA replication timing, and the creation of novel 3D chromatin structural domains.These results underscore the importance of comprehensive structural variant identification and indicate that non-coding structural variation may be an underappreciated mutational process in cancer genomes.

Background: Key challenges of biopsy-based determination of prostate cancer aggressiveness include tumour heterogeneity, biopsy-sampling error, and variations in biopsy interpretation. The resulting uncertainty in risk assessment leads to significant overtreatment, with associated costs and morbidity. We developed a performance-based strategy to identify protein biomarkers predictive of prostate cancer aggressiveness and lethality regardless of biopsy-sampling variation. Methods: Prostatectomy samples from a large patient cohort with long follow-up were blindly assessed by expert pathologists who identified the tissue regions with the highest and lowest Gleason grade from each patient. To simulate biopsy-sampling error, a core from a high- and a low-Gleason area from each patient sample was used to generate a ‘high' and a ‘low' tumour microarray, respectively. Results: Using a quantitative proteomics approach, we identified from 160 candidates 12 biomarkers that predicted prostate cancer aggressiveness (surgical Gleason and TNM stage) and lethal outcome robustly in both high- and low-Gleason areas. Conversely, a previously reported lethal outcome-predictive marker signature for prostatectomy tissue was unable to perform under circumstances of maximal sampling error. Conclusions: Our results have important implications for cancer biomarker discovery in general and development of a sampling error-resistant clinical biopsy test for prediction of prostate cancer aggressiveness.

Purpose The assessment of iron content in brain white matter (WM) is of high importance for studying neurodegenerative diseases. While R2* mapping and quantitative susceptibility mapping is suitable for iron mapping in gray matter, iron mapping in WM still remains an unsolved problem. We propose a new approach for iron mapping, independent of diamagnetic contributions of myelin by assessing the temperature dependency of the paramagnetic susceptibility. Theory and Methods We used unfixed human brain slices for relaxometry and calculated R2′ as a measure for microscopic susceptibility variations at several temperatures (4°C–37°C) at 3 Tesla. The temperature coefficient of R2′ (TcR2p) was calculated by linear regression and related to the iron concentration found by subsequent superconducting quantum interference device (SQUID) magnetometry and by inductively coupled plasma mass spectrometry. Results In line with SQUID measurements, R2′ mapping showed a linear temperature dependency of the bulk susceptibility with the highest slope in gray matter. Even in WM, TcR2p yielded a high linear correlation with the absolute iron concentration. Conclusion According to Curie's law, only paramagnetic matter exhibits a temperature dependency while the diamagnetism shows no effect. We have demonstrated that the temperature coefficient (TcR2p) can be used as a measure of the paramagnetic susceptibility despite of an unknown diamagnetic background. Magn Reson Med 73:1282–1288, 2015. © 2014 Wiley Periodicals, Inc.

The molecular mechanisms underlying the genesis of cholangiocarcinomas (CCs) are poorly understood. Epigenetic changes such as aberrant hypermethylation and subsequent atypical gene expression are characteristic features of most human cancers. In CC, data regarding global methylation changes are lacking so far. We performed a genome-wide analysis for aberrant promoter methylation in human CCs. We profiled 10 intrahepatic and 8 extrahepatic CCs in comparison to non-neoplastic biliary tissue specimens, using methyl-CpG immunoprecipitation (MCIp) combined with whole-genome CpG island arrays. DNA methylation was confirmed by quantitative mass spectrometric analysis and functional relevance of promoter hypermethylation was shown in demethylation experiments of two CC cell lines using 5-aza-2′deoxycytidine (DAC) treatment. Immunohistochemical staining of tissue microarrays (TMAs) from 223 biliary tract cancers (BTCs) was used to analyze candidate gene expression at the protein level. Differentially methylated, promoter-associated regions were nonrandomly distributed and enriched for genes involved in cancer-related pathways including Wnt, transforming growth factor beta (TGF-β), and PI3K signaling pathways. In CC cell lines, silencing of genes involved in Wnt signaling, such as SOX17, WNT3A, DKK2, SFRP1, SFRP2, and SFRP4 was reversed after DAC administration. Candidate protein SFRP2 was substantially down-regulated in neoplastic tissues of all BTC subtypes as compared to normal tissues. A significant inverse correlation of SFRP2 protein expression and pT status was found in BTC patients. Conclusion: We provide a comprehensive analysis to define the genome-wide methylation landscape of human CC. Several candidate genes of cancer-relevant signaling pathways were identified, and closer analysis of selected Wnt pathway genes confirmed the relevance of this pathway in CC. The presented global methylation data are the basis for future studies on epigenetic changes in cholangiocarcinogenesis. (Hepatology 2014;59:544–554)

The human LINE-1/L1 ORF2 protein is a multifunctional enzyme which plays a vital role in the life cycle of the human L1 retrotransposon. The protein consists of an endonuclease domain, followed by a central reverse transcriptase domain and a carboxy-terminal C-domain with unknown function. Here, we explore the nucleic acid binding properties of the 180-amino acid carboxy-terminal segment (CTS) of the human L1 ORF2p in vitro. In a series of experiments involving gel shift assay, we demonstrate that the CTS of L1 ORF2p binds RNA in non-sequence-specific manner. Finally, we report that mutations destroying the putative Zn-knuckle structure of the protein do not significantly affect the level of RNA binding and discuss the possible functional role of the CTS in L1 retrotransposition., Highlights • The 180-aa C-terminal segment (CTS) of human L1 ORF2p was expressed and purified from bacteria. • The nucleic acid binding properties of the CTS of L1ORF2p were examined in vitro. • The CTS of L1 ORF2p is an RNA binding domain. • The CTS of L1 ORF2p binds RNA in non-sequence-specific manner in the low nanomolar range. • Disruption of the putative Zn-knuckle structure does not significantly affect RNA binding.

Purpose Magnetic resonance relaxation times of most tissues are expected to depend on temperature, which can impact findings in postmortem magnetic resonance imaging or when using magnetic resonance imaging for relaxation-based thermometry. The purpose of this study was to investigate the exact temperature dependency of the relaxation times T1, T2, T2*, and the magnetization transfer ratio in different structures of the human brain. Methods To prevent fixation and autolysis effects, this study was performed with fresh postmortem brain tissues. Following autopsy, coronal brain slices from five deceased subjects were subjected to relaxometry at 3T in a temperature range between 4°C and 37°C. Heating of the tissue was achieved by flushing the vacuum packed brain slices with water at a predefined temperature. Results T1 showed a linear dependency on temperature with the highest temperature coefficient in the cortex (17.4 ms/°C) and the lowest in the white matter (3.4 ms/°C). T2 did not depend on temperature. T2* and magnetization transfer ratio scaled with temperature only in deep gray matter. Conclusion The temperature coefficient for T1 is higher than expected from previous reports and varies across brain structures. The coefficients obtained in this study can serve as reference for thermometry or for correcting quantitative postmortem magnetic resonance imaging. Magn Reson Med 71:1575–1580, 2014. © 2013 Wiley Periodicals, Inc.

Cholangiocarcinoma (CC) is a rare malignancy of the extrahepatic or intrahepatic biliary tract with an outstanding poor prognosis. Non-surgical therapeutic regimens result in minimally improved survival of CC patients. Global genomic analyses identified a few recurrently mutated genes, some of them in genes involved in epigenetic patterning. In a previous study, we demonstrated global DNA methylation changes in CC, indicating major contribution of epigenetic alterations to cholangiocarcinogenesis. Here, we aimed at the identification and characterization of CC-related, differentially methylated regions (DMRs) in potential microRNA promoters and of genes targeted by identified microRNAs. Twenty-seven hypermethylated and 13 hypomethylated potential promoter regions of microRNAs, known to be associated with cancer-related pathways like Wnt, ErbB, and PI3K-Akt signaling, were identified. Selected DMRs were confirmed in 2 independent patient cohorts. Inverse correlation between promoter methylation and expression suggested miR-129-2 and members of the miR-200 family (miR-200a, miR-200b, and miR-429) as novel tumor suppressors and oncomiRs, respectively, in CC. Tumor suppressor genes deleted in liver cancer 1 (DLC1), F-box/WD-repeat-containing protein 7 (FBXW7), and cadherin-6 (CDH6) were identified as presumed targets in CC. Tissue microarrays of a representative and well-characterized cohort of biliary tract cancers (n=212) displayed stepwise downregulation of CDH6 and association with poor patient outcome. Ectopic expression of CDH6 on the other hand, delayed growth in the CC cell lines EGI-1 and TFK-1, together suggesting a tumor suppressive function of CDH6. Our work represents a valuable repository for the study of epigenetically altered miRNAs in cholangiocarcinogenesis and novel putative, CC-related tumor suppressive miRNAs and oncomiRs.

Most human aneuploidies originate maternally, due in part to the presence of highly stringent checkpoints during male meiosis. Indeed, male sterility is common among aneuploid mice used to study chromosomal abnormalities, and male germline transmission of exogenous DNA has been rarely reported. Here we show that, despite aberrant testis architecture, males of the aneuploid Tc1 mouse strain produce viable sperm and transmit human chromosome 21 to create aneuploid offspring. In these offspring, we mapped transcription, transcriptional initiation, enhancer activity, non-methylated DNA, and transcription factor binding in adult tissues. Remarkably, when compared with mice derived from female passage of human chromosome 21, the chromatin condensation during spermatogenesis and the extensive epigenetic reprogramming specific to male germline transmission resulted in almost indistinguishable patterns of transcriptional deployment. Our results reveal an unexpected tolerance of aneuploidy during mammalian spermatogenesis, and the surprisingly robust ability of mouse developmental machinery to accurately deploy an exogenous chromosome, regardless of germline transmission. DOI: http://dx.doi.org/10.7554/eLife.20235.001

The eukaryotic translation initiation factor (eIF) 3a, the largest subunit of the eIF3 complex, is a key functional entity in ribosome establishment and translation initiation. In the past, aberrant eIF3a expression has been linked to the pathology of various cancer types but, so far, its expression has not been investigated in transitional cell carcinomas. Here, we investigated the impact of eIF3 expression on urinary bladder cancer (UBC) cell characteristics and UBC patient survival. eIF3a expression was reduced through inducible knockdown in the UBC-derived cell lines RT112, T24, 5637 and HT1197. As a consequence of eIF3a down-regulation, UBC cell proliferation, clonogenic potential and motility were found to be decreased and, concordantly, UBC tumour cell growth rates were found to be impaired in xenotransplanted mice. Polysomal profiling revealed that reduced eIF3a levels increased the abundance of 80S ribosomes, rather than impairing translation initiation. Microarray-based gene expression and ontology analyses revealed broad effects of eIF3a knockdown on the transcriptome. Analysis of eIF3a expression in primary formalin-fixed paraffin embedded UBC samples of 198 patients revealed that eIF3a up-regulation corresponds to tumour grade and that high eIF3a expression corresponds to longer overall survival rates of patients with low grade tumours. From our results we conclude that eIF3a expression may have a profound effect on the UBC phenotype and, in addition, may serve as a prognostic marker for low grade UBCs.

The assessment of iron content in brain white matter (WM) is of high importance for studying neurodegenerative diseases. While R2 * mapping and quantitative susceptibility mapping is suitable for iron mapping in gray matter, iron mapping in WM still remains an unsolved problem. We propose a new approach for iron mapping, independent of diamagnetic contributions of myelin by assessing the temperature dependency of the paramagnetic susceptibility.We used unfixed human brain slices for relaxometry and calculated R2 ' as a measure for microscopic susceptibility variations at several temperatures (4°C-37°C) at 3 Tesla. The temperature coefficient of R2 ' (TcR2p) was calculated by linear regression and related to the iron concentration found by subsequent superconducting quantum interference device (SQUID) magnetometry and by inductively coupled plasma mass spectrometry.In line with SQUID measurements, R2 ' mapping showed a linear temperature dependency of the bulk susceptibility with the highest slope in gray matter. Even in WM, TcR2p yielded a high linear correlation with the absolute iron concentration.According to Curie's law, only paramagnetic matter exhibits a temperature dependency while the diamagnetism shows no effect. We have demonstrated that the temperature coefficient (TcR2p) can be used as a measure of the paramagnetic susceptibility despite of an unknown diamagnetic background.

The molecular mechanisms underlying the genesis of cholangiocarcinomas (CCs) are poorly understood. Epigenetic changes such as aberrant hypermethylation and subsequent atypical gene expression are characteristic features of most human cancers. In CC, data regarding global methylation changes are lacking so far. We performed a genome-wide analysis for aberrant promoter methylation in human CCs. We profiled 10 intrahepatic and 8 extrahepatic CCs in comparison to non-neoplastic biliary tissue specimens, using methyl-CpG immunoprecipitation (MCIp) combined with whole-genome CpG island arrays. DNA methylation was confirmed by quantitative mass spectrometric analysis and functional relevance of promoter hypermethylation was shown in demethylation experiments of two CC cell lines using 5-aza-2'deoxycytidine (DAC) treatment. Immunohistochemical staining of tissue microarrays (TMAs) from 223 biliary tract cancers (BTCs) was used to analyze candidate gene expression at the protein level. Differentially methylated, promoter-associated regions were nonrandomly distributed and enriched for genes involved in cancer-related pathways including Wnt, transforming growth factor beta (TGF-β), and PI3K signaling pathways. In CC cell lines, silencing of genes involved in Wnt signaling, such as SOX17, WNT3A, DKK2, SFRP1, SFRP2, and SFRP4 was reversed after DAC administration. Candidate protein SFRP2 was substantially down-regulated in neoplastic tissues of all BTC subtypes as compared to normal tissues. A significant inverse correlation of SFRP2 protein expression and pT status was found in BTC patients.We provide a comprehensive analysis to define the genome-wide methylation landscape of human CC. Several candidate genes of cancer-relevant signaling pathways were identified, and closer analysis of selected Wnt pathway genes confirmed the relevance of this pathway in CC. The presented global methylation data are the basis for future studies on epigenetic changes in cholangiocarcinogenesis.

Eukaryotic gene expression is a complicated process primarily regulated at the levels of gene transcription and mRNA translation. The latter involves four main steps: initiation, elongation, termination and recycling. Translation regulation is primarily achieved during initiation which is orchestrated by 12 currently known eukaryotic initiation factors (eIFs). Here, we review the current state of eIF research and present a concise summary of the various eIF subunits. As eIFs turned out to be critically implicated in different oncogenic processes the various eIF members and their contribution to onset and progression of cancer are featured.

MR relaxation times of most tissues are ex- pected to depend on temperature which can impact find- ings in post mortem MRI or MRI based thermometry. The purpose of this study was to investigate the exact tem- perature dependency of the T1 relaxation in different structures in unfixed and fixed human brain samples. Following autopsy, coronal brain slices from five de- ceased subjects underwent MRI at 3T in a temperature range between 4°C and 37°C. T1 showed a linear depend- ency on temperature with the highest temperature coeffi- cient in the cortex (17 ms/°C) and the lowest in the white matter (3ms/°C). The fixation of the tissue lowered T1 and additionally the temperature coefficient in all regions investigated. The temperature coefficients obtained in this study serve as reference for thermometry and for correct- ing quantitative post mortem MRI.

Magnetic resonance relaxation times of most tissues are expected to depend on temperature, which can impact findings in postmortem magnetic resonance imaging or when using magnetic resonance imaging for relaxation-based thermometry. The purpose of this study was to investigate the exact temperature dependency of the relaxation times T(1), T(2), T(2) *, and the magnetization transfer ratio in different structures of the human brain.To prevent fixation and autolysis effects, this study was performed with fresh postmortem brain tissues. Following autopsy, coronal brain slices from five deceased subjects were subjected to relaxometry at 3T in a temperature range between 4°C and 37°C. Heating of the tissue was achieved by flushing the vacuum packed brain slices with water at a predefined temperature.T1 showed a linear dependency on temperature with the highest temperature coefficient in the cortex (17.4 ms/°C) and the lowest in the white matter (3.4 ms/°C). T(2) did not depend on temperature. T(2) * and magnetization transfer ratio scaled with temperature only in deep gray matter.The temperature coefficient for T(1) is higher than expected from previous reports and varies across brain structures. The coefficients obtained in this study can serve as reference for thermometry or for correcting quantitative postmortem magnetic resonance imaging.

The most common malignant brain tumor in adults is glioblastoma multiforme (GBM) showing a very heterogeneous, diffuse infiltrative and aggressive growth. The mean survival is between 8 and 18 months. The standard therapy for glioma consists of neurosurgery, radiation, and chemotherapy with temozolomide (SOC), but frequently resistance to temozolomide or irradiation will be developed. Translational research is focusing on the molecular mechanisms of glioma formation and development of resistance to identify new therapeutic targets. We have transplanted more than 50 glioma tissue samples to immunodeficient mice and were able to establish 13 new PDX models (engraftment rate 25%). Identical morphology of the s.c. growing PDX (pleomorphic cells with marked anisocytosis/ anisonucleosis; solid, picket similar growth, atypical mitoses) compared with the original glioma was proven by histological and immunohistochemical staining. Heterogeneity of the tumor cells in their size, shape and nuclear irregularities were observed. Glioma PDX models with stable growth were screened for sensitivity towards several targeted and cytotoxic drugs (everolimus, sorafenib, bevacicumab, irinotecan, salinomycin, temozolomide). Strong initial response to the treatment with bevacicumab, irinotecan, or temozolomide was observed. From our panel, (10/13) PDX were sensitive to temozolomide-treatment, (9/13) to irinotecan, and (7/13) to bevacicumab. The glioma PDX were resistant to most of the other targeted drugs, however in some single models sensitivity was observed. The frequency of common “onco-mutations” was analyzed using the Illumina TrueSeq Cancer panel sequencing. Although some frequent mutations were detected, i.e. in KDR, FGFR3, PIK3CA, PTEN, P53 and NOTCH1, no correlation with drug sensitivity have be identified. Extended correlation between drug sensitivity, gene expression profiles, and further mutations are still under analysis. The available data demonstrate, that our glioma PDX model panel has retained the original tumor biology and reflect the heterogeneity of the disease, ensuring a high similarity to the clinical situation. Our approach can not only be used for testing of established and new drugs, but also offers an individualized treatment of patients. For large scale screening the s.c. transplantation of glioma PDX is still the method of choice. However, for some drugs it may become important to implement a functional blood-brain barrier (BBB) in the model system. We have established a technology for orthotopic transplantation of glioma cells using the cell lines U373 and U87. Cells are transplanted into the left hemisphere of immunodeficient mice (NMRI nu/nu). Both cell lines showed good take rates and thus can be used for evaluation of systemic therapy approaches where an intact BBB is required. This technology has been further developed for orthotopic transplantation of glioma PDX and as surrogate models for brain metastases from breast and lung cancer (Ref: Hoffmann et al., Neurooncology, 2009). Summary: Our new clinically relevant glioma models enable a better understanding of tumor biology, allow correlations between biomarkers, growth and drug sensitivity and have an essential role to improve individual glioma therapy. Citation Format: Andrea Orthmann, Annika Hoffmann, Reiner Zeisig, Amin EL-Heliebi, Christina Ernst, Johannes Haybäck, Andreas Jödicke, Susanne Kuhn, Michael Linnebacher, Jens Hoffmann, Iduna Fichtner. Therapeutic response to bevacicumab, irinotecan, and temozolomide of PDX from human glioma cannot be correlated to common mutations as routinely identified by panel sequencing. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B24.

5090 Background: Current clinical and pathological parameters are insufficient for accurate prediction of progression risk for patients with biopsy Gleason grades 3+3 or 3+4. Reasons for this inclu...

Natalizumab is an effective treatment in patients with highly active relapsing-remitting multiple sclerosis (RRMS). However, its positive therapeutic effects have to be weighed against the potential serious adverse event of progressive multifocal leukoencephalopathy (PML). Thus, whenever patients with MS on natalizumab develop uncommon and progressive neurologic symptoms, the suspicion of PML has to be raised. The risk of PML becomes higher with increasing duration of natalizumab treatment, prior immunosuppressive treatment, and JC virus (JCV) antibody seropositivity.1 We here report a case of MS in which unusual symptoms in the context of 5 years of natalizumab treatment and seroconversion to JCV antibody positivity led to the initial suspicion of PML and a final diagnosis of Creutzfeldt-Jakob disease (CJD).

Background: Steatohepatitis (SH) and SH-associated hepatocellular carcinoma (HCC) are of considerable clinical significance. SH is morphologically characterized by steatosis, hepatocyte ballooning and cytoplasmic protein aggregates termed Mallory-Denk bodies (MDBs). MDBs are generally composed of misfolded keratin (K) 8, 18 and in part p62 and ubiquitin. It is well established that K aggregates are critical for MDB formation, thus Ks are associated with SH which is one of the main preconditions for the development of liver cirrhosis and hepatocellular carcinoma (HCC). Aims: In this study we aimed to clarify the cellular and molecular mechanisms of relative K8 excess over K18 on hepatocarcinogenesis in mice and its functional and clinical implication in human alcoholic and non-alcoholic SH (ASH/NASH)-induced liver cancer. Methods: 18 month-old krt18-deficient (krt18-/-129P2/OlaHsd background) mice were investigated for the incidence of SH and SH-induced liver tumors by radiology, macroscopy, histology, immunohistochemistry, gene expression, lipidome analysis, and immunoblotting. Results: Aged krt18-/- mice developed the entire morphological spectrum of SH whereas aged wild-type (wt) mice showed simple steatosis. Aminotransaminase levels were also elevated in aged krt18-/- mice. Interestingly, 64% of male and 25% of female 18 month-old krt18-/- mice developed liver tumors revealing morphological and genetic features of HCC whereas 45% of male and 16% of female krt18+/- mice and 42% of wt mice developed HCC. Conclusion: Aged krt18-/- mice represent a novel spontaneous SH and SH associated HCC model with significant gender differences revealing features related to human HCC. Therefore, variations of hepatocellular K18 seem to have an effect on the susceptibility regarding SH and SH-induced HCC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A38. Citation Format: Anita Kuldeep Mehta, Kira Bettermann, Eva M. Lederer, Christina Ernst, Sonja M. Kessler, Kensuke Kojima, Xintong Chen, Yujin Hoshida, Bryan C. Fuchs, Vendula Svendova, Michael G. Schimek, Monika Mach, Michael R. Speicher, Tatjana Stojakovic, Thomas M. Magin, Pavel Strnad, Michael Trauner, Alexandra K. Kiemer, Snorri S. Thorgeirsson, Michael Karin, Josep M. Llovet, Kurt Zatloukal, Helmut Denk, Carolin Lackner, Johannes Haybaeck. Steatohepatitis-associated hepatocellular carcinoma: Evidence of a keratin-based disease. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A38.