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Abstract Background Lung cancer is a very frequent and lethal tumor with an identifiable risk population. Cytological analysis and chest X-ray failed to reduce mortality, and CT screenings are still controversially discussed. Recent studies provided first evidence for the potential usefulness of autoantigens as markers for lung cancer. Methods We used extended panels of arrayed antigens and determined autoantibody signatures of sera from patients with different kinds of lung cancer, different common non-tumor lung pathologies, and controls without any lung disease by a newly developed computer aided image analysis procedure. The resulting signatures were classified using linear kernel Support Vector Machines and 10-fold cross-validation. Results The novel approach allowed for discriminating lung cancer patients from controls without any lung disease with a specificity of 97.0%, a sensitivity of 97.9%, and an accuracy of 97.6%. The classification of stage IA/IB tumors and controls yielded a specificity of 97.6%, a sensitivity of 75.9%, and an accuracy of 92.9%. The discrimination of lung cancer patients from patients with non-tumor lung pathologies reached an accuracy of 88.5%. Conclusion We were able to separate lung cancer patients from subjects without any lung disease with high accuracy. Furthermore, lung cancer patients could be seprated from patients with other non-tumor lung diseases. These results provide clear evidence that blood-based tests open new avenues for the early diagnosis of lung cancer.

Abstract Background Chronic obstructive pulmonary disease (COPD) is a respiratory inflammatory condition with autoimmune features including IgG autoantibodies. In this study we analyze the complexity of the autoantibody response and reveal the nature of the antigens that are recognized by autoantibodies in COPD patients. Methods An array of 1827 gridded immunogenic peptide clones was established and screened with 17 sera of COPD patients and 60 healthy controls. Protein arrays were evaluated both by visual inspection and a recently developed computer aided image analysis technique. By this computer aided image analysis technique we computed the intensity values for each peptide clone and each serum and calculated the area under the receiver operator characteristics curve (AUC) for each clone and the separation COPD sera versus control sera. Results By visual evaluation we detected 381 peptide clones that reacted with autoantibodies of COPD patients including 17 clones that reacted with more than 60% of the COPD sera and seven clones that reacted with more than 90% of the COPD sera. The comparison of COPD sera and controls by the automated image analysis system identified 212 peptide clones with informative AUC values. By in silico sequence analysis we found an enrichment of sequence motives previously associated with immunogenicity. Conclusion The identification of a rather complex humoral immune response in COPD patients supports the idea of COPD as a disease with strong autoimmune features. The identification of novel immunogenic antigens is a first step towards a better understanding of the autoimmune component of COPD.

Objective: Investigate the feasibility of detecting early treatment-induced tumor tissue changes in patients with advanced lung adenocarcinoma using diffusion-weighted MRI-derived radiomics features., Methods: This prospective observational study included 144 patients receiving either tyrosine kinase inhibitors (TKI, n = 64) or platinum-based chemotherapy (PBC, n = 80) for the treatment of pulmonary adenocarcinoma. Patients underwent diffusion-weighted MRI the day prior to therapy (baseline, all patients), as well as either + 1 (PBC) or + 7 and + 14 (TKI) days after treatment initiation. One hundred ninety-seven radiomics features were extracted from manually delineated tumor volumes. Feature changes over time were analyzed for correlation with treatment response (TR) according to CT-derived RECIST after 2 months and progression-free survival (PFS)., Results: Out of 14 selected delta-radiomics features, 6 showed significant correlations with PFS or TR. Most significant correlations were found after 14 days. Features quantifying ROI heterogeneity, such as short-run emphasis (p = 0.04 (pfs) /0.005 (tr) ), gradient short-run emphasis (p = 0.06 (pfs) /0.01 (tr) ), and zone percentage (p = 0.02 (pfs) /0.01 (tr) ) increased in patients with overall better TR whereas patients with worse overall response showed an increase in features quantifying ROI homogeneity, such as normalized inverse difference (p = 0.01 (pfs) /0.04 (tr) ). Clustering of these features allows stratification of patients into groups of longer and shorter survival., Conclusion: Two weeks after initiation of treatment, diffusion MRI of lung adenocarcinoma reveals quantifiable tissue-level insights that correlate well with future treatment (non-)response. Diffusion MRI-derived radiomics thus shows promise as an early, radiation-free decision-support to predict efficacy and potentially alter the treatment course early., Critical Relevance Statement: Delta-Radiomics texture features derived from diffusion-weighted MRI of lung adenocarcinoma, acquired as early as 2 weeks after initiation of treatment, are significantly correlated with RECIST TR and PFS as obtained through later morphological imaging., Key Points: Morphological imaging takes time to detect TR in lung cancer, diffusion-weighted MRI might identify response earlier. Several radiomics features are significantly correlated with TR and PFS. Radiomics of diffusion-weighted MRI may facilitate patient stratification and management., (© 2024. The Author(s).)

The identification of targetomes remains a challenge given the pleiotropic effect of miRNAs, the limited effects of miRNAs on individual targets, and the sheer number of estimated miRNA-target gene interactions (MTIs), which is around 44,571,700. Currently, targetome identification for single miRNAs relies on computational evidence and functional studies covering smaller numbers of targets. To ensure that the targetome analysis could be experimentally verified by functional assays, we employed a systematic approach and explored the targetomes of four miRNAs (miR-129-5p, miR-129-1-3p, miR-133b, and miR-873-5p) by analyzing 410 predicted target genes, both of which were previously associated with Parkinson's disease (PD). After performing 13,536 transfections, we validated 442 of the 705 putative MTIs (62,7%) through dual luciferase reporter assays. These analyses increased the number of validated MTIs by at least 2.1-fold for miR-133b and by a maximum of 24.3-fold for miR-873-5p. Our study contributes to the experimental capture of miRNA targetomes by addressing i) the ratio of experimentally verified MTIs to predicted MTIs, ii) the sizes of disease-related miRNA targetomes, and iii) the density of MTI networks. A web service to support the analyses on the MTI level is available online ( https://ccb-web.cs.uni-saarland.de/utr-seremato ), and all the data have been added to the miRATBase database ( https://ccb-web.cs.uni-saarland.de/miratbase )., (© 2024. The Author(s).)

Background: Although a pivotal role of microRNA (miRNA, miR) in the pathogenesis of Huntington's disease (HD) is increasingly recognized, the molecular functions of miRNAs in the pathomechanisms of HD await further elucidation. One of the miRNAs that have been associated with HD is miR-34a-5p, which was deregulated in the mouse R6/2 model and in human HD brain tissues., Methods: The aim of our study was to demonstrate interactions between miR-34a-5p and HD associated genes. By computational means we predicted 12 801 potential target genes of miR-34a-5p. An in-silico pathway analysis revealed 22 potential miR-34a-5p target genes in the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway "Huntington's disease"., Results: Using our high-throughput miRNA interaction reporter assay (HiTmIR) we identified NDUFA9, TAF4B, NRF1, POLR2J2, DNALI1, HIP1, TGM2 and POLR2G as direct miR-34a-5p target genes. Direct binding of miR-34a-5p to target sites in the 3'UTRs of TAF4B, NDUFA9, HIP1 and NRF1 was verified by a mutagenesis HiTmIR assay and by determining endogenous protein levels for HIP1 and NDUFA9. STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis identified protein-protein interaction networks associated with HD like "Glutamine Receptor Signaling Pathway" and "Calcium Ion Transmembrane Import Into Cytosol"., Conclusion: Our study demonstrates multiple interactions between miR-34a-5p and HD associated target genes and thereby lays the ground for future therapeutic interventions using this miRNA., (© 2023. The Author(s).)

Background: To explore the prognostic value of serial dynamic contrast-enhanced (DCE) MRI in patients with advanced pulmonary adenocarcinoma undergoing first-line therapy with either tyrosine-kinase inhibitors (TKI) or platinum-based chemotherapy (PBC)., Methods: Patients underwent baseline (day 0, n = 98), and post-therapeutic DCE MRI (PBC: day + 1, n = 52); TKI: day + 7, n = 46) at 1.5T. Perfusion curves were acquired at 10, 40, and 70 s after contrast application and analysed semiquantitatively. Treatment response was evaluated at 6 weeks by CT (RECIST 1.1); progression-free survival (PFS) and overall survival  were analysed with respect to clinical and perfusion parameters. Relative uptake was defined as signal difference between contrast and non-contrast images, divided by the non-contrast signal. Predictors of survival were selected using Cox regression analysis. Median follow-up was 825 days., Results: In pre-therapeutic and early post-therapeutic MRI, treatment responders (n = 27) showed significantly higher relative contrast uptake within the tumor at 70 s after application as compared to non-responders (n = 71, p ≤ 0.02), response defined as PR by RECIST 1.1 at 6 weeks. There was no significant change of perfusion at early MRI after treatment. In multivariate regression analysis of selected parameters, the strongest association with PFS were relative uptake at 40 s in the early post-treatment MRI and pre-treatment clinical data (presence of liver metastases, ECOG performance status)., Conclusion: Higher contrast uptake within the tumor at pre-treatment and early post-treatment MRI was associated with treatment response and better prognosis. DCE MRI of pulmonary adenocarcinoma may provide important prognostic information., (© 2022. The Author(s).)

Background: Liquid rebiopsies can detect resistance mutations to guide therapy of anaplastic lymphoma kinase-rearranged (ALK + ) non-small-cell lung cancer (NSCLC) failing tyrosine kinase inhibitors (TKI). Here, we analyze how their results relate to the anatomical pattern of disease progression and patient outcome., Methods: Clinical, molecular, and radiologic characteristics of consecutive TKI-treated ALK + NSCLC patients were analyzed using prospectively collected plasma samples and the 17-gene targeted AVENIO kit, which covers oncogenic drivers and all TP53 exons., Results: In 56 patients, 139 instances of radiologic changes were analyzed, of which 133 corresponded to disease progression. Circulating tumor DNA (ctDNA) alterations were identified in most instances of extracranial progression (58/94 or 62%), especially if concomitant intracranial progression was also present (89%, P<0.001), but rarely in case of isolated central nervous system (CNS) progression (8/39 or 21%, P<0.001). ctDNA detectability correlated with presence of "short" echinoderm microtubule-associated protein-like 4 ( EML4 ) -ALK fusion variants (mainly V3, E6:A20) and/or TP53 mutations (P<0.05), and presented therapeutic opportunities in <50% of cases. Patients with extracranial progression and positive liquid biopsies had shorter survival from the start of palliative treatment (mean 52 vs. 69 months, P=0.002), regardless of previous and subsequent therapy and initial ECOG performance status. Furthermore, for patients with extracranial progression, ctDNA detectability was associated with shorter next-line progression-free survival (PFS) (3 vs. 13 months, P=0.003) if they were switched to another systemic therapy (49/86 samples), and with shorter time-to-next-treatment (TNT) (3 vs. 8 months, P=0.004) if they were continued on the same treatment due to oligoprogression (37/86). In contrast, ctDNA detectability was not associated with the outcome of patients showing CNS-only progression. In 6/6 cases with suspicion of non-neoplastic radiologic lung changes (mainly infection or pneumonitis), ctDNA results remained negative., Conclusions: Positive blood-based liquid rebiopsies in ALK + NSCLC characterize biologically more aggressive disease and are common with extracranial, but rare with CNS-only progression or benign radiologic changes. These results reconcile the increased detection of ALK resistance mutations with other features of the high-risk EML4-ALK V3-associated phenotype. Conversely, most oligoprogressive patients with negative liquid biopsies have a more indolent course without need for early change of systemic treatment., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tlcr-21-32). PC reports grants and personal fees from Novartis, grants and personal fees from Roche, grants and personal fees from AstraZeneca, personal fees from Pfizer, grants and personal fees from Takeda, personal fees from Chugai, personal fees from Boehringer, outside the submitted work; SD reports personal fees from Roche, outside the submitted work; DK reports personal fees from Pfizer, personal fees from BMS, personal fees from AstraZeneca, outside the submitted work; AV reports personal fees from AstraZeneca, outside the submitted work; MR reports personal fees from Amgen, personal fees from AstraZeneca, personal fees from BMS, personal fees from Boehringer, personal fees from Lilly, personal fees from Merck, personal fees from MSD, personal fees from Novartis, personal fees from Pfizer, personal fees from Roche, personal fees from Samsung, outside the submitted work; AS reports personal fees from AstraZeneca, personal fees from Lilly, personal fees from Bayer, personal fees from BMS, personal fees from Illumina, personal fees from Janssen, personal fees from MSD, personal fees from Novartis, personal fees from Pfizer, personal fees from Roche, personal fees from Seattle Genomics, outside the submitted work; MT reports grants from AstraZeneca, personal fees from Lilly, personal fees from BMS, personal fees from MSD, personal fees from Novartis, personal fees from Roche, personal fees from Boehringer, personal fees from Celgene, personal fees from Takeda, personal fees from AbbVie, outside the submitted work; HS reports grants and personal fees from Roche, outside the submitted work. The other authors have no conflicts of interest to declare. AS serves as an unpaid editorial board member of Translational Lung Cancer Research from Sep 2019 to Sep 2021. The other authors have no conflicts of interest to declare., (2021 Translational Lung Cancer Research. All rights reserved.)

MicroRNAs are regulators of gene expression. A wide-spread, yet not validated, assumption is that the targetome of miRNAs is non-randomly distributed across the transcriptome and that targets share functional pathways. We developed a computational and experimental strategy termed high-throughput miRNA interaction reporter assay (HiTmIR) to facilitate the validation of target pathways. First, targets and target pathways are predicted and prioritized by computational means to increase the specificity and positive predictive value. Second, the novel webtool miRTaH facilitates guided designs of reporter assay constructs at scale. Third, automated and standardized reporter assays are performed. We evaluated HiTmIR using miR-34a-5p, for which TNF- and TGFB-signaling, and Parkinson's Disease (PD)-related categories were identified and repeated the pipeline for miR-7-5p. HiTmIR validated 58.9% of the target genes for miR-34a-5p and 46.7% for miR-7-5p. We confirmed the targeting by measuring the endogenous protein levels of targets in a neuronal cell model. The standardized positive and negative targets are collected in the new miRATBase database, representing a resource for training, or benchmarking new target predictors. Applied to 88 target predictors with different confidence scores, TargetScan 7.2 and miRanda outperformed other tools. Our experiments demonstrate the efficiency of HiTmIR and provide evidence for an orchestrated miRNA-gene targeting., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: In 2016 the first-in-human phase I study of a miRNA-based cancer therapy with a liposomal mimic of microRNA-34a-5p (miR-34a-5p) was closed due to five immune related serious adverse events (SAEs) resulting in four patient deaths. For future applications of miRNA mimics in cancer therapy it is mandatory to unravel the miRNA effects both on the tumor tissue and on immune cells. Here, we set out to analyze the impact of miR-34a-5p over-expression on the CXCL10/CXCL11/CXCR3 axis, which is central for the development of an effective cancer control., Methods: We performed a whole genome expression analysis of miR-34a-5p transfected M1 macrophages followed by an over-representation and a protein-protein network analysis. In-silico miRNA target prediction and dual luciferase assays were used for target identification and verification. Target genes involved in chemokine signaling were functionally analyzed in M1 macrophages, CD4 + and CD8 + T cells., Results: A whole genome expression analysis of M1 macrophages with induced miR-34a-5p over-expression revealed an interaction network of downregulated target mRNAs including CXCL10 and CXCL11. In-silico target prediction in combination with dual luciferase assays identified direct binding of miR-34a-5p to the 3'UTRs of CXCL10 and CXCL11 . Decreased CXCL10 and CXCL11 secretion was shown on the endogenous protein level and in the supernatant of miR-34a-5p transfected and activated M1 macrophages. To complete the analysis of the CXCL10/CXCL11/CXCR3 axis, we activated miR-34a-5p transfected CD4 + and CD8 + T cells by PMA/Ionomycin and found reduced levels of endogenous CXCR3 and CXCR3 on the cell surface., Conclusions: MiR-34a-5p mimic administered by intravenous administration will likely not only be up-taken by the tumor cells but also by the immune cells. Our results indicate that miR-34a-5p over-expression leads in M1 macrophages to a reduced secretion of CXCL10 and CXCL11 chemokines and in CD4 + and CD8 + T cells to a reduced expression of CXCR3. As a result, less immune cells will be attracted to the tumor site. Furthermore, high levels of miR-34a-5p in naive CD4 + T cells can in turn hinder Th1 cell polarization through the downregulation of CXCR3 leading to a less pronounced activation of cytotoxic T lymphocytes, natural killer, and natural killer T cells and possibly contributing to lymphocytopenia., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

T cells are central to the immune response against various pathogens and cancer cells. Complex networks of transcriptional and post-transcriptional regulators, including microRNAs (miRNAs), coordinate the T cell activation process. Available miRNA datasets, however, do not sufficiently dissolve the dynamic changes of miRNA controlled networks upon T cell activation. Here, we established a quantitative and time-resolved expression pattern for the entire miRNome over a period of 24 h upon human T-cell activation. Based on our time-resolved datasets, we identified central miRNAs and specified common miRNA expression profiles. We found the most prominent quantitative expression changes for miR-155-5p with a range from initially 40 molecules/cell to 1600 molecules/cell upon T-cell activation. We established a comprehensive dynamic regulatory network of both the up- and downstream regulation of miR-155. Upstream, we highlight IRF4 and its complexes with SPI1 and BATF as central for the transcriptional regulation of miR-155. Downstream of miR-155-5p, we verified 17 of its target genes by the time-resolved data recorded after T cell activation. Our data provide comprehensive insights into the range of stimulus induced miRNA abundance changes and lay the ground to identify efficient points of intervention for modifying the T cell response., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objectives: To quantitatively and qualitatively evaluate image quality in dual-layer CT (DLCT) compared to single-layer CT (SLCT) in the thorax, abdomen, and pelvis in a reduced-dose setting., Methods: Intraindividual, retrospective comparisons were performed in 25 patients who received at least one acquisition of all three acquisition protocols SLCT low (100 kVp), DLCT high (120 kVp), and DLCT low (120 kVp), all covering the venous-phase thorax, abdomen, and pelvis with matched CTDI vol between SLCT low and DLCT low . Reconstruction parameters were identical between all scans. Image quality was assessed quantitatively at 10 measurement locations in the thorax, abdomen, and pelvis by two independent observers, and subjectively with an intraindividual forced choice test between the three acquisitions. Dose-length product (DLP) and CTDI vol were extracted for dose comparison., Results: Despite matched CTDI vol in acquisition protocols, CTDI vol and DLP were lower for SLCT low compared to DLCT low and DLCT high (DLP 408.58, 444.68, 647.08 mGy·cm, respectively; p < 0.0004), as automated tube current modulation for DLCT low reached the lower limit in the thorax (mean 66.1 mAs vs limit 65 mAs). Noise and CNR were comparable between SLCT low and DLCT low (p values, 0.29-0.51 and 0.05-0.20), but CT numbers were significantly higher for organs and vessels in the upper abdomen for SLCT low compared to DLCT low . DLCT high had significantly better image quality (Noise and CNR). Subjective image quality was superior for DLCT high , but no difference was found between SLCT low and DLCT low ., Conclusions: DLCT low showed comparable image quality to SLCT low , with the additional possibility of spectral post-processing. Further dose reduction seems possible by decreasing the lower limit of the tube current for the thorax., Key Points: • Clinical use of reduced-dose DLCT is feasible despite the required higher tube potential. • DLCT with reduced dose shows comparable objective and subjective image quality to reduced-dose SLCT. • Further dose reduction in the thorax might be possible by adjusting mAs thresholds.

Neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD) are characterized by the accumulation of misfolded proteins in the endoplasmic reticulum (ER) and the unfolded protein response (UPR). Modulating the UPR is one of the major challenges to counteract the development of neurodegenerative disorders and other diseases with affected UPR. Here, we show that miR-34a-5p directly targets the IRE1α branch of the UPR, including the genes BIP , IRE1α , and XBP1 . Upon induction of ER stress in neuronal cells, miR-34a-5p overexpression impacts the resulting UPR via a significant reduction in IRE1α and XBP1s that in turn leads to decreased viability, increased cytotoxicity and caspase activity. The possibility to modify the UPR signaling pathway by a single miRNA that targets central genes of the IRE1α branch offers new perspectives for future therapeutic approaches against neurodegeneration.

Oligoprogression (OPD) of non-small-cell lung cancer (NSCLC) occurs in approximately half of patients under targeted compounds (TKI) and facilitates use of regional therapies that can prolong survival. In order to characterize OPD in immunotherapy (IO)-treated NSCLC, we analyzed the failure pattern under PD-1/PD-L1 inhibitors ( n = 297) or chemoimmunotherapy ( n = 75). Under IO monotherapy, OPD was more frequent (20% vs. 10%, p < 0.05), occurred later (median 11 vs. 5 months, p < 0.01), affected fewer sites (mean 1.1 vs. 1.5, p < 0.05), and involved fewer lesions (1.4 vs. 2.3, p < 0.05) in the first compared to later lines. Lymph nodes (42%, mainly mediastinal) and the brain (39%) were mostly affected, followed by the lung (24%) and other organs. Compared to multifocal progression, OPD occurred later (11 vs. 4 months, p < 0.001) and was associated with longer survival (26 vs. 13 months, p < 0.001) and higher tumor PD-L1 expression ( p < 0.001). Chemoimmunotherapy showed a similar incidence of OPD as IO monotherapy (13% vs. 11% at 2 years). Local treatments were applied regularly for brain but only in 50% for extracranial lesions. Thus, NSCLC oligoprogression is less common under IO than under TKI, but also favorable. Since its frequency drops later in the disease, regular restaging and multidisciplinary evaluation are essential in order to exploit the full therapeutic potential.

Computed tomography (CT) scans are the gold standard to measure treatment success of non-small cell lung cancer (NSCLC) therapies. Here, we investigated the very early tumor response of patients receiving chemotherapy or targeted therapies using a panel of already established and explorative liquid biomarkers. Blood samples from 50 patients were taken at baseline and at three early time points after therapy initiation. DNA mutations, a panel of 17 microRNAs, glycodelin, glutathione disulfide, glutathione, soluble caspase-cleaved cytokeratin 18 (M30 antigen), and soluble cytokeratin 18 (M65 antigen) were measured in serum and plasma samples. Baseline and first follow-up CT scans were evaluated and correlated with biomarker data. The detection rate of the individual biomarkers was between 56% and 100%. While only keratin 18 correlated with the tumor load at baseline, we found several individual markers correlating with the tumor response to treatment for each of the three time points of blood draws. A combination of the five best markers at each time point resulted in highly significant marker panels indicating therapeutic response (R 2 = 0.78, R 2 = 0.71, and R 2 = 0.71). Our study demonstrates that an early measurement of biomarkers immediately after therapy start can assess tumor response to treatment and might support an adaptation of treatment to improve patients' outcome.

Background: Micro(mi)RNAs are increasingly recognized as central regulators of immune cell function. While it has been predicted that miRNAs have multiple targets, the majority of these predictions still await experimental confirmation. Here, miR-34a, a well-known tumor suppressor, is analyzed for targeting genes involved in immune system processes of leucocytes., Methods: Using an in-silico approach, we combined miRNA target prediction with GeneTrail2, a web tool for Multi-omics enrichment analysis, to identify miR-34a target genes, which are involved in the immune system process subcategory of Gene Ontology., Results: Out of the 193 predicted target genes in this subcategory we experimentally tested 22 target genes and confirmed binding of miR-34a to 14 target genes including VAMP2, IKBKE, MYH9, MARCH8, KLRK1, CD11A, TRAFD1, CCR1, PYDC1, PRF1, PIK3R2, PIK3CD, AP1B1, and ADAM10 by dual luciferase assays. By transfecting Jurkat, primary CD4 + and CD8 + T cells with miR-34a, we demonstrated that ectopic expression of miR-34a leads to reduced levels of endogenous VAMP2 and CD11A, which are central to the analyzed subcategories. Functional downstream analysis of miR-34a over-expression in activated CD8 + T cells exhibits a distinct decrease of PRF1 secretion., Conclusions: By simultaneous targeting of 14 mRNAs miR-34a acts as major hub of T cell regulatory networks suggesting to utilize miR-34a as target of intervention towards a modulation of the immune responsiveness of T-cells in a broad tumor context.

NF-κB functions as modulator of T cell receptor-mediated signaling and transcriptional regulator of miR-34a. Our in silico analysis revealed that miR-34a impacts the NF-κB signalosome with miR-34a binding sites in 14 key members of the NF-κB signaling pathway. Functional analysis identified five target genes of miR-34a including PLCG1, CD3E, PIK3CB, TAB2, and NFΚBIA. Overexpression of miR-34a in CD4 + and CD8 + T cells led to a significant decrease of NFΚBIA as the most downstream cytoplasmic NF-κB member, a reduced cell surface abundance of TCRA and CD3E, and to a reduction of T cell killing capacity. Inhibition of miR-34a caused an increase of NFΚBIA, TCRA, and CD3E. Notably, activation of CD4 + and CD8 + T cells entrails a gradual increase of miR-34a. Our results lend further support to a model with miR-34a as a central NF-κB regulator in T cells.

Adjusting intracellular calcium signaling is an important feature in the regulation of immune cell function and survival. Here we show that miR-34a-5p, a small non-coding RNA that is deregulated in many common diseases, is a regulator of store-operated Ca 2+ entry (SOCE) and calcineurin signaling. Upon miR-34a-5p overexpression, we observed both a decreased depletion of ER calcium content and a decreased Ca 2+ influx through Ca 2+ release-activated Ca 2+ channels. Based on an in silico target prediction we identified multiple miR-34a-5p target genes within both pathways that are implicated in the balance between T-cell activation and apoptosis including ITPR2, CAMLG, STIM1, ORAI3, RCAN1, PPP3R1, and NFATC4. Functional analysis revealed a decrease in Ca 2+ activated calcineurin pathway activity measured by a reduced IL-2 secretion due to miR-34a-5p overexpression. Impacting SOCE and/or downstream calcineurin/NFAT signaling by miR-34a-5p offers a possible future approach to manipulate immune cells for clinical interventions.

MiRNAs play a central role in physiological and pathological processes. Both for the biological understanding and for their clinical application, it is essential to understand the interaction of miRNAs and their targets. Target identification largely hinges on in-silico prediction, which requires a complete consideration of miRNA binding sites within the UTRs of target genes. Here, we show that 5-mer sites might also play an essential role for human miRNA-target binding. We implemented and employed an algorithm to all pairs of 2,588 human miRNAs annotated in miRBase and the 3' UTRs of 16725 genes (>43 million combinations). Our in-silico analysis showed a highly significant enrichment (p = 1.4 × 10 -69 ) of 5-mer binding sites in 3' UTRs across all experimentally validated miRNA-target gene pairs. We next confirmed the central role of 5-mer binding sites by reporter assays and demonstrated that two non-canonical 5-mer sites of miR-34a in the 3' UTR of T-cell receptor alpha (TCRA) have a significantly stronger influence on its posttranscriptional regulation than the canonical binding sites. These observations indicate an essential role of 5-mer binding sites for the miRNA targeting in human cells.

Purpose: To evaluate the safety, efficacy and outcome of percutaneous balloon-expandable covered stent graft placement for uretero-iliac artery fistula (UAF) treatment., Methods: This retrospective study evaluated the single-center experience of percutaneous balloon-expandable covered stent graft placement (ADVANTA™, Atrium Hudson, NH, USA) in UAF. Data were obtained from a prospective institutional database. Patient follow-up included complications, symptoms recurrence and mortality rate., Results: Ten UAFs in eight patients (3 males; 5 females) with a mean age of 64.5 (35-77) years were identified. All patients had a history pelvic malignancy, extirpative surgery (n = 6), long-term ureteral stenting (n = 7) and pelvic radiation (n = 5). All procedures were completed successfully without complications. Thirty-day mortality rate was zero. At a median follow-up of 6 (1-60) months, one patient suffered recurrent hematuria requiring a secondary stent graft placement 26 months after the initial treatment. During follow-up, five patients died of the underlying disease (43, 66, 105, and 183 and 274 days after the last procedure)., Conclusion: Percutaneous balloon-expandable stent graft placement in UAF is a safe and effective treatment option. Implantation of stent grafts should be considered as treatment of choice in UAF.

Although microRNAs have been recognized as central cellular regulators, there is an evident lack of knowledge about their targets. Here, we analyzed potential target genes for miR-148a functioning in Ras signaling in B cells, including SOS1 and SOS2. A dual-luciferase reporter assay showed significantly decreased luciferase activity upon ectopic overexpression of miR-148a in HEK-293T cells that were co-transfected with the 3'UTR of either SOS1 or SOS2. Each of the 3'UTRs of SOS1 and SOS2 contained two binding sites for miR-148a both of which were necessary for the decreased luciferase activity. MiR-148a overexpression in HEK-293T lead to significantly reduced levels of both endogenous SOS1 and SOS2 proteins. Likewise, reduced levels of SOS proteins were found in two B cell lines that were transfected with miR-148a. The level of ERK1/2 phosphorylation as one of the most relevant downstream members of the Ras/ERK signaling pathway was also reduced in cells with miR-148a overexpression. The data show that miR-148a impairs the Ras/ERK signaling pathway via SOS1 and SOS2 proteins in B cells., Competing Interests: CONFLICTS OF INTEREST The authors declare no competing financial interests.

Since the benefit of prostate-specific antigen (PSA) screening remains controversial, new non-invasive biomarkers for prostate carcinoma (PCa) are still required. There is evidence that microRNAs (miRNAs) in whole peripheral blood can separate patients with localized prostate cancer from healthy individuals. However, the potential of blood-based miRNAs for the differential diagnosis of PCa and benign prostatic hyperplasia (BPH) has not been tested. We compared the miRNome from blood of PCa and BPH patients and further investigated the influence of the tumor volume, tumor-node-metastasis (TNM) classification, Gleason score, pretreatment risk status, and the pretreatment PSA value on the miRNA pattern. By microarray approach, we identified seven miRNAs that were significantly deregulated in PCa patients compared to BPH patients. Using quantitative real time PCR (qRT-PCR), we confirmed downregulation of hsa-miR-221* (now hsa-miR-221-5p) and hsa-miR-708* (now hsa-miR-708-3p) in PCa compared to BPH. Clinical parameters like PSA level, Gleason score, or TNM status seem to have only limited impact on the overall abundance of miRNAs in patients' blood, suggesting a no influence of these factors on the expression of deregulated miRNAs.

Background: The dependency of miRNA abundance from physiological processes such as exercises remains partially understood. We set out to analyze the effect of physical exercises on miRNA profiles in blood and plasma of endurance and strength athletes in a systematic manner and correlated differentially abundant miRNAs in athletes to disease miRNAs biomarkers towards a better understanding of how physical exercise may confound disease diagnosis by miRNAs., Methods: We profiled blood and plasma of 29 athletes before and after exercise. With four samples analyzed for each individual we analyzed 116 full miRNomes. The study set-up enabled paired analyses of individuals. Affected miRNAs were investigated for known disease associations using network analysis., Results: MiRNA patterns in blood and plasma of endurance and strength athletes vary significantly with differences in blood outreaching variations in plasma. We found only moderate differences between the miRNA levels before training and the RNA levels after training as compared to the more obvious variations found between strength athletes and endurance athletes. We observed significant variations in the abundance of miR-140-3p that is a known circulating disease markers (raw and adjusted p value of 5 × 10(-12) and 4 × 10(-7)). Similarly, the levels of miR-140-5p and miR-650, both of which have been reported as makers for a wide range of human pathologies significantly depend on the training mode. Among the most affected disease categories we found acute myocardial infarction. MiRNAs, which are up-regulated in endurance athletes inhibit VEGFA as shown by systems biology analysis of experimentally validated target genes., Conclusion: We provide evidence that the mode and the extent of training are important confounding factors for a miRNA based disease diagnosis.

Circulating miRNAs have been associated with numerous human diseases. The lack of understanding the functional roles of blood-born miRNAs limits, however, largely their value as disease marker. In a systems biology analysis we identified miR-34a as strongly associated with pathogenesis. Genome-wide analysis of miRNAs in blood cell fractions highlighted miR-34a as most significantly up-regulated in CD3+ cells of lung cancer patients. By our in silico analysis members of the protein kinase C family (PKC) were indicated as miR-34a target genes. Using a luciferase assay, we confirmed binding of miR-34a-5p to target sequences within the 3'UTRs of five PKC family members. To verify the biological effect, we transfected HEK 293T and Jurkat cells with miR-34a-5p causing reduced endogenous protein levels of PKC isozymes. By combining bioinformatics approaches with experimental validation, we demonstrate that one of the most relevant disease associated miRNAs has the ability to control the expression of a gene family., Competing Interests: The authors have no conflicts of interest.

We present a human miRNA tissue atlas by determining the abundance of 1997 miRNAs in 61 tissue biopsies of different organs from two individuals collected post-mortem. One thousand three hundred sixty-four miRNAs were discovered in at least one tissue, 143 were present in each tissue. To define the distribution of miRNAs, we utilized a tissue specificity index (TSI). The majority of miRNAs (82.9%) fell in a middle TSI range i.e. were neither specific for single tissues (TSI > 0.85) nor housekeeping miRNAs (TSI < 0.5). Nonetheless, we observed many different miRNAs and miRNA families that were predominantly expressed in certain tissues. Clustering of miRNA abundances revealed that tissues like several areas of the brain clustered together. Considering -3p and -5p mature forms we observed miR-150 with different tissue specificity. Analysis of additional lung and prostate biopsies indicated that inter-organism variability was significantly lower than inter-organ variability. Tissue-specific differences between the miRNA patterns appeared not to be significantly altered by storage as shown for heart and lung tissue. MiRNAs TSI values of human tissues were significantly (P = 10(-8)) correlated with those of rats; miRNAs that were highly abundant in certain human tissues were likewise abundant in according rat tissues. We implemented a web-based repository enabling scientists to access and browse the data (https://ccb-web.cs.uni-saarland.de/tissueatlas)., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Purpose: Right atrial volume (RAV) is a prognostic factor of the right heart function. This retrospective study evaluates the comparability of computed tomography (CT) and magnetic resonance imaging (MRI) for right atrial volumetry with conventional planimetric and diametric methods., Material and Methods: 29 retrospectively included patients (18 male, 47±12years) underwent CT and 1.5 Tesla MRI within 17±20days. RAV was measured using biplane and ellipsoid method (MRI and CT) and Simpson's method (CT). For interobserver comparison measurements were carried out by two observers. Pearson's correlation, Lin's concordance coefficient, and Bland-Altman statistics were calculated., Results: There is a correlation of RAV between CT and MRI, r [ellipsoid]=0.65; r [biplane]=0.64 (p<0.001). MRI volumes were significantly lower than CT volumes, [mean±SD] 43±19ml versus 27±9ml, (p<0.002). There was a close interobserver correlation (CT: r=0.83, MRI: r=0.73; p<0.001) but a relatively wide range in Bland-Altman analysis; limits of agreement from ±13ml up to ±29ml., Conclusions: Compared to left atrial volumes, a higher variability was found for RAV values both in interobserver statistiscs and in intermodality comparison. Complex shape of the right atrium, artifacts due to contrast material (CT), high venous return in inspiration (CT), high flow contrast media administration (CT) and increased heart rate (MRI) might cause these clinically relevant variations., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Circulating microRNAs (miRNAs) from blood are increasingly recognized as biomarker candidates for human diseases. Clinical routine settings frequently include blood sampling in tubes with EDTA as anticoagulant without considering the influence of phlebotomy on the overall miRNA expression pattern. We collected blood samples from six healthy individuals each in an EDTA blood collection tube. Subsequently, the blood was transferred into PAXgeneTM tubes at three different time points, i.e. directly (0 min), 10 min, and 2 h after phlebotomy. As control blood was also directly collected in PAXgeneTM blood RNA tubes that contain a reagent to directly lyse blood cells and stabilize their content. For all six blood donors at the four conditions (24 samples) we analyzed the abundance of 1,205 miRNAs by human Agilent miRNA V16 microarrays., Results: While we found generally a homogenous pattern of the miRNA abundance in all 24 samples, the duration of the EDTA treatment appears to influence the miRNA abundance of specific miRNAs. The most significant changes are observed after longer EDTA exposition. Overall, the impact of the different blood sample conditions on the miRNA pattern was substantially lower than intra-individual variations. While samples belonging to one of the six individuals mostly cluster together, there was no comparable clustering for any of the four tested blood sampling conditions. The most affected miRNA was miR-769-3p that was not detected in any of the six PAXgene blood samples, but in all EDTA 2h samples. Accordingly, hsa-miR-769-3p was also the only miRNA that showed a significantly different abundance between the 4 blood sample conditions by an ANOVA analysis (Benjamini-Hochberg adjusted p-value of 0.003). Validation by qRT-PCR confirmed this finding., Conclusion: The pattern of blood-borne miRNA abundance is rather homogenous between the four tested blood sample conditions of six blood donors. There was a clustering between the miRNA profiles that belong to a specific blood donor, but not between any of the four tested blood sampling conditions. The results show a limited overall impact of the blood sampling conditions on the miRNA pattern. Notwithstanding, the abundance of single miRNAs can be significantly altered by different blood sampling conditions.

There is an urgent need of comprehensive longitudinal analyses of circulating miRNA patterns to identify dynamic changes of miRNAs in cancer patients after surgery. Here we provide longitudinal analysis of 1,205 miRNAs in plasma samples of 26 patients after lung cancer resection at 8 time points over a period of 18 months and compare them to 12 control patients. First, we report longitudinal changes with respect to the number of detected miRNAs over time and identified a significantly increased number of miRNAs in patients developing metastases (p = 0.0096). A quantitative analysis with respect to the expression level of the detected miRNAs revealed more significant changes in the miRNA levels in samples from patients without metastases compared to the non-cancer control patients. This analysis provided further evidence of miRNA plasma levels that are changing over time after tumor resection and correlate to patient outcome. Especially hsa-miR-197 could be validated by qRT-PCR as prognostic marker. Also for this miRNA, patients developing metastases had levels close to that of controls while patients that did not develop metastases showed a significant up-regulation.In conclusion, our data indicate that the overall miRNome of a patient that later develops metastases is less affected by surgery than the miRNome of a patient who does not show metastases. The relationship between altered plasma levels of specific miRNAs with the development of metastases would partially have gone undetected by an analysis at a single time point only.

Human glioblastomas are characterized by frequent DNA amplifications most often at chromosome regions 7p11.2 and 12q13-15. Although amplification is a well-known hallmark of glioblastoma genetics the function of most amplified genes in glioblastoma biology is not understood. Previously, we cloned Ku70-binding protein 3 (KUB3) from the amplified domain at 12q13-15. Here, we report that glioblastoma cell cultures with endogenous KUB3 gene amplification and with elevated KUB3 protein expression show an efficient double-strand break (DSB) repair after being irradiated with 1 Gy. A significantly less efficient DSB repair was found in glioma cell cultures without KUB3 amplification and expression. Furthermore, we found that a siRNA-mediated reduction of the endogenous KUB3 expression in glioblastoma cells resulted in a reduction of the repair efficiency. HeLa cells transfected with KUB3 showed an increased DSB repair in comparison to untreated HeLa cells. In addition, KUB3 seems to influence DSB efficiency via the DNA-PK-dependent repair pathway as shown by simultaneous inhibition of KUB3 and DNA-PK. The data provide the first evidence for a link between the level of KUB3 amplification and expression in glioma and DSB repair efficiency.

There is growing evidence that simultaneous analysis of multiple autoantibody reactions can be utilized for diagnosis of neoplasms. Using a set of 57 meningioma-associated antigens, we recently separated meningioma patients from individuals without known disease with an accuracy of 90.3%. Here, we ask whether a largely increased set of immunogenic antigens can further improve this discrimination. We used an array with 1,827 human recombinant clones and measured reactivity of serum autoantibodies against the clones by a novel automated image analysis procedure. We were able to separate meningioma sera from sera of healthy controls with a specificity of 95.62%, a sensitivity of 91.83% and an accuracy of 93.84%. Of the analyzed clones, 23 in-frame clones were highly informative for the classification of meningioma vs. normal sera as shown by their AUC values. These results demonstrate that the accuracy of a serum-based diagnostic can be readily and considerably improved by screening extended sets of proteins., (Copyright © 2010 UICC.)

Background: Lung cancer is a very frequent and lethal tumor with an identifiable risk population. Cytological analysis and chest X-ray failed to reduce mortality, and CT screenings are still controversially discussed. Recent studies provided first evidence for the potential usefulness of autoantigens as markers for lung cancer., Methods: We used extended panels of arrayed antigens and determined autoantibody signatures of sera from patients with different kinds of lung cancer, different common non-tumor lung pathologies, and controls without any lung disease by a newly developed computer aided image analysis procedure. The resulting signatures were classified using linear kernel Support Vector Machines and 10-fold cross-validation., Results: The novel approach allowed for discriminating lung cancer patients from controls without any lung disease with a specificity of 97.0%, a sensitivity of 97.9%, and an accuracy of 97.6%. The classification of stage IA/IB tumors and controls yielded a specificity of 97.6%, a sensitivity of 75.9%, and an accuracy of 92.9%. The discrimination of lung cancer patients from patients with non-tumor lung pathologies reached an accuracy of 88.5%., Conclusion: We were able to separate lung cancer patients from subjects without any lung disease with high accuracy. Furthermore, lung cancer patients could be separated from patients with other non-tumor lung diseases. These results provide clear evidence that blood-based tests open new avenues for the early diagnosis of lung cancer.

Seroreactivity profiling emerges as valuable technique for minimal invasive cancer detection. Recently, we provided first evidence for the applicability of serum profiling of glioma using a limited number of immunogenic antigens. Here, we screened 57 glioma and 60 healthy sera for autoantibodies against 1827 Escherichia coli expressed clones, including 509 in-frame peptide sequences. By a linear support vector machine approach, we calculated mean specificity, sensitivity, and accuracy of 100 repetitive classifications. We were able to differentiate glioma sera from sera of the healthy controls with a specificity of 90.28%, a sensitivity of 87.31% and an accuracy of 88.84%. We were also able to differentiate World Health Organization grade IV glioma sera from healthy sera with a specificity of 98.45%, a sensitivity of 80.93%, and an accuracy of 92.88%. To rank the antigens according to their information content, we computed the area under the receiver operator characteristic curve value for each clone. Altogether, we found 46 immunogenic clones including 16 in-frame clones that were informative for the classification of glioma sera versus healthy sera. For the separation of glioblastoma versus healthy sera, we found 91 informative clones including 26 in-frame clones. The best-suited in-frame clone for the classification glioma sera versus healthy sera corresponded to the vimentin gene (VIM) that was previously associated with glioma. In the future, autoantibody signatures in glioma not only may prove useful for diagnosis but also offer the prospect for a personalized immune-based therapy.

Background: Chronic obstructive pulmonary disease (COPD) is a respiratory inflammatory condition with autoimmune features including IgG autoantibodies. In this study we analyze the complexity of the autoantibody response and reveal the nature of the antigens that are recognized by autoantibodies in COPD patients., Methods: An array of 1827 gridded immunogenic peptide clones was established and screened with 17 sera of COPD patients and 60 healthy controls. Protein arrays were evaluated both by visual inspection and a recently developed computer aided image analysis technique. By this computer aided image analysis technique we computed the intensity values for each peptide clone and each serum and calculated the area under the receiver operator characteristics curve (AUC) for each clone and the separation COPD sera versus control sera., Results: By visual evaluation we detected 381 peptide clones that reacted with autoantibodies of COPD patients including 17 clones that reacted with more than 60% of the COPD sera and seven clones that reacted with more than 90% of the COPD sera. The comparison of COPD sera and controls by the automated image analysis system identified 212 peptide clones with informative AUC values. By in silico sequence analysis we found an enrichment of sequence motives previously associated with immunogenicity., Conclusion: The identification of a rather complex humoral immune response in COPD patients supports the idea of COPD as a disease with strong autoimmune features. The identification of novel immunogenic antigens is a first step towards a better understanding of the autoimmune component of COPD.

Serum-based diagnosis offers the prospect of early lung carcinoma detection and of differentiation between benign and malignant nodules identified by CT. One major challenge toward a future blood-based diagnostic consists in showing that seroreactivity patterns allow for discriminating lung cancer patients not only from normal controls but also from patients with non-tumor lung pathologies. We addressed this question for squamous cell lung cancer, one of the most common lung tumor types. Using a panel of 82 phage-peptide clones, which express potential autoantigens, we performed serological spot assay. We screened 108 sera, including 39 sera from squamous cell lung cancer patients, 29 sera from patients with other non-tumor lung pathologies, and 40 sera from volunteers without known disease. To classify the serum groups, we employed the standard Naïve Bayesian method combined with a subset selection approach. We were able to separate squamous cell lung carcinoma and normal sera with an accuracy of 93%. Low-grade squamous cell lung carcinoma were separated from normal sera with an accuracy of 92.9%. We were able to distinguish squamous cell lung carcinoma from non-tumor lung pathologies with an accuracy of 83%. Three phage-peptide clones with sequence homology to ROCK1, PRKCB1 and KIAA0376 reacted with more than 15% of the cancer sera, but neither with normal nor with non-tumor lung pathology sera. Our study demonstrates that seroreactivity profiles combined with statistical classification methods have great potential for discriminating patients with squamous cell lung carcinoma not only from normal controls but also from patients with non-tumor lung pathologies.

Purpose: Recent studies impressively showed the diagnostic potential of seroreactivity patterns for different tumor types, offering the prospect for low-cost screening of numerous tumor types simultaneously. One of the major challenges toward this goal is to prove that seroreactivity profiles do not only allow for identifying a tumor but also allow for distinguishing tumors from other pathologies of the same organ., Experimental Design: We chose glioma as a model system and tested 325 sera (88 glioma, 95 intracranial tumors, 60 other brain pathologies, and 82 healthy controls) for seroreactivity on a panel of 35 antigens., Results: We were able to discriminate between glioma and all other sera with cross-validated specificity of 86.1%, sensitivity of 85.2%, and accuracy of 85.8%. We obtained comparably good results for the separation of glioma versus nontumor brain pathologies and glioma versus other intracranial tumors., Conclusion: Our study provides first evidence that seroreactivity patterns allow for an accurate discrimination between a tumor and pathologies of the same organ even between different tumor types of the same organ.