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Summary: We have developed a laboratory information management system (LIMS) for a next-generation sequencing (NGS) laboratory within the existing Galaxy platform. The system provides lab technicians standard and customizable sample information forms, barcoded submission forms, tracking of input sample quality, multiplex-capable automatic flow cell design and automatically generated sample sheets to aid physical flow cell preparation. In addition, the platform provides the researcher with a user-friendly interface to create a request, submit accompanying samples, upload sample quality measurements and access to the sequencing results. As the LIMS is within the Galaxy platform, the researcher has access to all Galaxy analysis tools and workflows. The system reports requests and associated information to a message queuing system, such that information can be posted and stored in external systems, such as a wiki. Through an API, raw sequencing results can be automatically pre-processed and uploaded to the appropriate request folder. Developed for the Illumina HiSeq 2000 instrument, many features are directly applicable to other instruments.Availability and implementation: The code and documentation are available at http://tron-mainz.de/tron-facilities/computational-medicine/galaxy-lims/Contact: jelle.scholtalbers@tron-mainz.de [ABSTRACT FROM PUBLISHER]

Motivation: Next-generation sequencing technology enables an entirely new perspective for clinical research and will speed up personalized medicine. In contrast to microarray-based approaches, RNA-Seq analysis provides a much more comprehensive and unbiased view of gene expression. Although the perspective is clear and the long-term success of this new technology obvious, bioinformatics resources making these data easily available especially to the biomedical research community are still evolving.Results: We have generated RNA-Seq Atlas, a web-based repository of RNA-Seq gene expression profiles and query tools. The website offers open and easy access to RNA-Seq gene expression profiles and tools to both compare tissues and find genes with specific expression patterns. To enlarge the scope of the RNA-Seq Atlas, the data were linked to common functional and genetic databases, in particular offering information on the respective gene, signaling pathway analysis and evaluation of biological functions by means of gene ontologies. Additionally, data were linked to several microarray gene profiles, including BioGPS normal tissue profiles and NCI60 cancer cell line expression data. Our data search interface allows an integrative detailed comparison between our RNA-Seq data and the microarray information. This is the first database providing data mining tools and open access to large scale RNA-Seq expression profiles. Its applications will be versatile, as it will be beneficial in identifying tissue specific genes and expression profiles, comparison of gene expression profiles among diverse tissues, but also systems biology approaches linking tissue function to gene expression changes.Availability and implementation: http://medicalgenomics.org/rna_seq_atlasContact: kruppm@uni-mainz.de; teufel@uni-mainz.deSupplementary information: Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

The article focuses on the development of a sustainable project for improving the storage at the historic site Winterthur Museum, Garden and Library created by Henry Francis du Port. Topics include the history of the museum building acquired by the du Pont family in 1810, the architectural features of the museum building, and the initiatives for the preservation of the Winterthur Museum, Garden and Library. It offers information on the application of the Collection Policy for the preservation of the historic site.

Rett syndrome (RTT) is a postnatal neurodevelopmental disorder characterized by the loss of acquired motor and language skills, autistic features, and unusual stereotyped movements. RTT is caused by mutations in the X-linked gene encoding methyl-CpG binding protein 2 (MeCP2). Mutations in MECP2 cause a variety of neurodevelopmental disorders including X-linked mental retardation, psychiatric disorders, and some cases of autism. Although MeCP2 was identified as a methylation-dependent transcriptional repressor, transcriptional profiling of RNAs from mice lacking MeCP2 did not reveal significant gene expression changes, suggesting that MeCP2 does not simply function as a global repressor. Changes in expression of a few genes have been observed, but these alterations do not explain the full spectrum of Rett-like phenotypes, raising the possibility that additional MeCP2 functions play a role in pathogenesis. In this study, we show that MeCP2 interacts with the RNA-binding protein V box-binding protein I and regulates splicing of reporter minigenes. Importantly, we found aberrant alternative splicing patterns in a mouse model of RU. Thus, we uncovered a previously uncharacterized function of MeCP2 that involves regulation of splicing, in addition to its role as a transcriptional repressor. [ABSTRACT FROM AUTHOR]

There is a mass of detailed data concerning technical risk assessment methods and practices for underground work. But there is very little advice or guidance on the broad apportionment of the total risk between the various phases of an underground project or general advice on how risk might be managed. The Working Group has produced a generic Risk Budget covering five typical phases of an underground works project, which illustrates the heavy bias of risk towards the early phases. Using a practical example the report illustrates how project risk can be managed in a structured manner. [Copyright &y& Elsevier]

Background: Tumor stroma, of which fibroblasts are the most abundant cell, resembles a non‐healing wound, where a procoagulant environment creates a permissive milieu for cancer growth. We aimed to determine if tumor expression of coagulation factors (procoagulant phenotype), and systemic hypercoagulability, occur at the preinvasive (ductal carcinoma in situ; DCIS) stage and correlate with breast cancer subtype, disease‐free survival (DFS), and overall survival (OS). Methods: In a prospective cohort of early breast cancer (DCIS, n = 76; invasive, n = 248) tumor, normal breast and plasma were examined. Fibroblast and epithelial expression of Tissue Factor (TF), thrombin, PAR1, PAR2, and plasma thrombin‐antithrombin (TAT) and D‐dimer were correlated with clinicopathological data, and 5‐year survival. Results: Fibroblast expression of TF, thrombin, and PAR1 was increased in DCIS and invasive cancer compared to normal breast fibroblasts (P ≤.003, all). Fibroblast TF, thrombin, PAR1, and PAR2 was increased in cancers with high Ki67, high grade, ER‐ (vs ER+), and HER2+ (vs HER2‐) (all P <.05). On univariate analysis, fibroblast TF expression was inversely associated with DFS (P =.04) and OS (P =.02). D‐dimer was higher in node positive (507 (CI: 411‐625) ng/mL, n = 68) vs negative patients (428 (CI: 387‐472) ng/mL, n = 171, P =.004) and inversely associated with OS (P =.047). On multivariate analysis, plasma TAT was associated with reduced OS (HR 3.26, CI 1.16‐3.1, P =.02), with a high plasma TAT (≥3.2 ng/mL) associated with > 3‐fold mortality risk compared to low TAT. Conclusion: This demonstrates procoagulant phenotypic changes occur in fibroblasts at the preinvasive stage. Fibroblast procoagulant phenotype is associated with aggressive breast cancer subtypes and reduced survival. Coagulation may be a therapeutic target in breast cancer. [ABSTRACT FROM AUTHOR]

Background: Angiosarcoma is an uncommon endothelial malignancy and a highly aggressive soft tissue sarcoma. Due to its infiltrative nature, successful management of localized angiosarcoma is often challenging. Systemic chemotherapy is used in the metastatic setting and occasionally in patients with high-risk localized disease in neoadjuvant or adjuvant settings. However, responses tend to be short-lived and most patients succumb to metastatic disease. Novel therapies are needed for patients with angiosarcomas. Methods: We performed a retrospective analysis of patients with locally advanced or metastatic angiosarcoma, who were treated with checkpoint inhibitors at our institution. We collected their clinical information and outcome measurements. In one patient with achieved complete response, we analyzed circulating and infiltrating T cells within peripheral blood and tumor tissue. Results: We have treated seven angiosarcoma (AS) patients with checkpoint inhibitors either in the context of clinical trials or off label [Pembrolizumab + Axitinib (NCT02636725; n = 1), AGEN1884, a CTLA-4 inhibitor (NCT02694822; n = 2), Pembrolizumab (n = 4)]. Five patients had cutaneous angiosarcoma, one primary breast angiosarcoma and one radiation-associated breast angiosarcoma. At 12 weeks, 5/7 patients (71%) had partial response of their lesions either on imaging and/or clinical exam and two (29%) had progressive disease. 6/7 patients are alive to date and, thus far, 3/7 patients (43%) have progressed (median 3.4 months)- one achieved partial response after pembrolizumab was switched to ongoing Nivolumab/Ipilimumab, one died of progressive disease at 31 weeks (primary breast angiosarcoma) and one was placed on pazopanib. One patient had a complete response (CR) following extended treatment with monotherapy AGEN1884. No patient experienced any ≥ grade 2 toxicities. Conclusions: This case series underscores the value of targeted immunotherapy in treating angiosarcoma. It also identifies genetic heterogeneity of cutaneous angiosarcomas and discusses specific genetic findings that may explain reported benefits from immunotherapy. [ABSTRACT FROM AUTHOR]

Background: The presentation of HLA peptide complexes to T cells is a highly regulated and tissue specific process involving multiple transcriptionally controlled cellular components. The extensive polymorphism of HLA genes and the complex composition of the proteasome make it difficult to map their expression profiles across tissues. Methods: Here we applied a tailored gene quantification pipeline to 4323 publicly available RNA-Seq datasets representing 55 normal tissues and cell types to examine expression profiles of (classical and non-classical) HLA class I, class II and proteasomal genes. Results: We generated the first comprehensive expression atlas of antigen presenting-related genes across 56 normal tissues and cell types, including immune cells, pancreatic islets, platelets and hematopoietic stem cells. We found a surprisingly heterogeneous HLA expression pattern with up to 100-fold difference in intra-tissue median HLA abundances. Cells of the immune system and lymphatic organs expressed the highest levels of classical HLA class I (HLA-A,-B,-C), class II (HLA-DQA1,-DQB1,-DPA1,-DPB1,-DRA,-DRB1) and non-classical HLA class I (HLA-E,-F) molecules, whereas retina, brain, muscle, megakaryocytes and erythroblasts showed the lowest abundance. In contrast, we identified a distinct and highly tissue-restricted expression pattern of the non-classical class I gene HLA-G in placenta, pancreatic islets, pituitary gland and testis. While the constitutive proteasome showed relatively constant expression across all tissues, we found the immunoproteasome to be enriched in lymphatic organs and almost absent in immune privileged tissues. Conclusions: Here, we not only provide a reference catalog of tissue and cell type specific HLA expression, but also highlight extremely variable expression of the basic components of antigen processing and presentation in different cell types. Our findings indicate that low expression of classical HLA class I molecules together with lack of immunoproteasome components as well as upregulation of HLA-G may be of key relevance to maintain tolerance in immune privileged tissues. [ABSTRACT FROM AUTHOR]

cAMP/PKA signalling is compartmentalised with tight spatial and temporal control of signal propagation underpinning specificity of response. The cAMP-degrading enzymes, phosphodiesterases (PDEs), localise to specific subcellular domains within which they control local cAMP levels and are key regulators of signal compartmentalisation. Several components of the cAMP/PKA cascade are located to different mitochondrial sub-compartments, suggesting the presence of multiple cAMP/PKA signalling domains within the organelle. The function and regulation of these domains remain largely unknown. Here, we describe a novel cAMP/PKA signalling domain localised at mitochondrial membranes and regulated by PDE2A2. Using pharmacological and genetic approaches combined with real-time FRET imaging and high resolution microscopy, we demonstrate that in rat cardiac myocytes and other cell types mitochondrial PDE2A2 regulates local cAMP levels and PKA-dependent phosphorylation of Drp1. We further demonstrate that inhibition of PDE2A, by enhancing the hormone-dependent cAMP response locally, affects mitochondria dynamics and protects from apoptotic cell death. [ABSTRACT FROM AUTHOR]

Tumour-specific mutations are ideal targets for cancer immunotherapy as they lack expression in healthy tissues and can potentially be recognized as neo-antigens by the mature T-cell repertoire. Their systematic targeting by vaccine approaches, however, has been hampered by the fact that every patient's tumour possesses a unique set of mutations ('the mutanome') that must first be identified. Recently, we proposed a personalized immunotherapy approach to target the full spectrum of a patient's individual tumour-specific mutations. Here we show in three independent murine tumour models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that, unexpectedly, the majority of the immunogenic mutanome is recognized by CD4+ T cells. Vaccination with such CD4+ immunogenic mutations confers strong antitumour activity. Encouraged by these findings, we established a process by which mutations identified by exome sequencing could be selected as vaccine targets solely through bioinformatic prioritization on the basis of their expression levels and major histocompatibility complex (MHC) class II-binding capacity for rapid production as synthetic poly-neo-epitope messenger RNA vaccines. We show that vaccination with such polytope mRNA vaccines induces potent tumour control and complete rejection of established aggressively growing tumours in mice. Moreover, we demonstrate that CD4+ T cell neo-epitope vaccination reshapes the tumour microenvironment and induces cytotoxic T lymphocyte responses against an independent immunodominant antigen in mice, indicating orchestration of antigen spread. Finally, we demonstrate an abundance of mutations predicted to bind to MHC class II in human cancers as well by employing the same predictive algorithm on corresponding human cancer types. Thus, the tailored immunotherapy approach introduced here may be regarded as a universally applicable blueprint for comprehensive exploitation of the substantial neo-epitope target repertoire of cancers, enabling the effective targeting of every patient's tumour with vaccines produced 'just in time'. [ABSTRACT FROM AUTHOR]

Following inconspicuous preclinical testing, the superagonistic anti-CD28 mAb TGN1412 was applied to six study participants who all developed a devastating cytokine storm.We verified that TGN1412 treatment of fresh PBMCs induced only moderate responses, whereas restoration of tissue-like conditions by high-density preculture (HDC) allowed vigorous cytokine production. TGN1412 treatment of T cells isolated from HDC-PBMCs induced moderate cytokine responses, which upon additional anti-IgG crosslinking were significantly boosted. Moreover, coincubation of TGN1412-treated T cells with B cells expressing the intermediate affinity Fcγ receptor IIB (CD32B), or coincubation with CD32B+ transfectants, resulted in robust T cell activation. This was surprising because TGN1412 was expressed as an Ig of the subclass 4 (IgG4), which was shown before to exhibit only minor affinity to FcγRs. Transcriptome analysis of TGN1412-treated T cells revealed that similar gene signatures were induced irrespective of whether T cells derived from fresh or HDC-PBMCs were studied. Collectively, these data indicate that HDC-PBMCs and HDC-PBMC- derived T cells mount rapid TGN1412 responses, which are massively boosted by FcγR crosslinking, in particular by CD32- expressing B cells. These results qualify HDC-PBMCs as a valuable in vitro test system for the analysis of complex mAb functions. [ABSTRACT FROM AUTHOR]

An abstract of the article "Defining the regulatory network of the tissue-specific splicing factors Fox-1 and Fox-2," by Chaolin Zhang, Zuo Zhang, John Castle, Shuying Sun, Jason Johnson, Adrian R. Krainer and Michael is presented.

Malaria caused by Plasmodium falciparum results in approximately 1 million annual deaths worldwide, with young children and pregnant mothers at highest risk. Disease severity might be related to parasite virulence factors, but expression profiling studies of parasites to test this hypothesis have been hindered by extensive sequence variation in putative virulence genes and a preponderance of host RNA in clinical samples. We report here the application of RNA sequencing to clinical isolates of P. falciparum, using not-so-random (NSR) primers to successfully exclude human ribosomal RNA and globin transcripts and enrich for parasite transcripts. Using NSR-seq, we confirmed earlier microarray studies showing upregulation of a distinct subset of genes in parasites infecting pregnant women, including that encoding the well-established pregnancy malaria vaccine candidate var2csa. We also describe a subset of parasite transcripts that distinguished parasites infecting children from those infecting pregnant women and confirmed this observation using quantitative real-time PCR and mass spectrometry proteomic analyses. Based on their putative functional properties, we propose that these proteins could have a role in childhood malaria pathogenesis. Our study provides proof of principle that NSR-seq represents an approach that can be used to study clinical isolates of parasites causing severe malaria syndromes as well other blood-borne pathogens and blood-related diseases. [ABSTRACT FROM AUTHOR]

From a large-scale screen using splicing microarrays and RT-PCR, we identified 63 alternative splicing (AS) events that are coordinated in 3 distinct temporal patterns during mouse heart development. More than half of these splicing transitions are evolutionarily conserved between mouse and chicken. Computational analysis of the introns flanking these splicing events identified enriched and conserved motifs including binding sites for CUGBP and ETR-3-like factors (CELF), muscleblind-like (MBNL) and Fox proteins. We show that CELF proteins are down-regulated >10-fold during heart development, and MBNL1 protein is concomitantly up-regulated nearly 4-fold. Using transgenic and knockout mice, we show that reproducing the embryonic expression patterns for CUGBP1 and MBNL1 in adult heart induces the embryonic splicing patterns for more than half of the developmentally regulated AS transitions. These findings indicate that CELF and MBNL proteins are determinative for a large subset of splicing transitions that occur during postnatal heart development. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are a class of noncoding RNAs that post-transcriptionally regulate gene expression in plants and animals. To investigate the influence of miRNAs on transcript levels, we transfected miRNAs into human cells and used microarrays to examine changes in the messenger RNA profile. Here we show that delivering miR-124 causes the expression profile to shift towards that of brain, the organ in which miR-124 is preferentially expressed, whereas delivering miR-1 shifts the profile towards that of muscle, where miR-1 is preferentially expressed. In each case, about 100 messages were downregulated after 12?h. The 3'untranslated regions of these messages had a significant propensity to pair to the 5'region of the miRNA, as expected if many of these messages are the direct targets of the miRNAs. Our results suggest that metazoan miRNAs can reduce the levels of many of their target transcripts, not just the amount of protein deriving from these transcripts. Moreover, miR-1 and miR-124, and presumably other tissue-specific miRNAs, seem to downregulate a far greater number of targets than previously appreciated, thereby helping to define tissue-specific gene expression in humans. [ABSTRACT FROM AUTHOR]

Following publication of the original article [1], the authors have reported that the following sentence "While of the same IgG1 class as ipilimumab, preclinical data suggests this molecule may have enhanced activity against T regulatory cells". [ABSTRACT FROM AUTHOR]

Background/aims: Clinical fellows support the hospital workforce while gaining experience in different specialities, research, leadership and teaching. The authors aimed to assess the impact of clinical fellow programmes in an acute teaching hospital trust., Methods: An anonymous electronic service evaluation was sent to clinical fellows to investigate their views on whether the programme had improved patient safety, doctors' clinical performance, training and wellbeing. Thematic analysis was used to analyse the free-text responses., Results: A total of 95 out of 144 clinical fellows responded to the evaluation survey. The clinical fellows believed that the programme had improved patient safety, clinical performance (time to manage acute patients), foundation and internal medicine training, undergraduate teaching and junior doctors' wellbeing. Four similar themes emerged from the free-text responses: career development, patient safety, training and doctors' wellbeing., Conclusions: Clinical fellow programmes may improve patient safety, clinical performance, training, undergraduate education and doctors' wellbeing.

Background/aim: Clotting factors promote cancer development. We investigated if coagulation proteins promote proliferation and migration in colorectal cancer (CRC) cell lines and whether their direct inhibitors can attenuate these effects., Materials and Methods: DLD-1 and SW620 cells were treated with tissue factor (0, 50, 100 and 500 pg/mL ± 10 μg/mL 10H10 [anti-tissue factor antibody]), thrombin (0.0, 0.1, 1.0 and 10.0 U/mL ± 0.5 μM dabigatran [thrombin inhibitor]) and Factor Xa, FXa (0.0, 0.1, 1.0 and 10.0 U/mL ± 100 ng/mL rivaroxaban [FXa inhibitor]) and their effects on proliferation and migration were quantified using the PrestoBlue® and transwell migration assays, respectively., Results: Thrombin increased proliferation from 48 h treatment compared to its control (48 h 6.57 ± 1.36 u vs. 2.42 ± 0.13 u, p = 0.001, 72 h 9.50 ± 1.54 u vs. 4.50 ± 0.47 u, p = 0.004 and 96 h 10.77 ± 1.72 u vs. 5.57 ± 0.25 u, p = 0.008). This increase in proliferation was attenuated by dabigatran at 72 h (2.23 ± 0.16 u vs. 3.26 ± 0.43 u, p = 0.04). Tissue factor (0 pg/mL 20.7 ± 1.6 cells/view vs. 50 pg/mL 32.4 ± 1.9 cells/view, p = 0.0002), FXa (0.0 U/mL 8.9 ± 1.1 cells/view vs. 10.0 U/mL 17.7 ± 1.7 cells/view, p < 0.0001) and thrombin (0.0 U/mL 8.9 ± 1.3 cells/view vs. 10.0 U/mL 20.2 ± 2.0 cells/view, p < 0.0001) all increased migration compared to their controls. However, their direct inhibitors did not attenuate these increases., Conclusion: Thrombin, FXa and TF all increase migration in CRC in vitro. Thrombin induced increase in proliferation is abrogated by dabigatran. Dabigatran may have potential as an anti-cancer therapy in CRC., (© 2023 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: Breast cancer results in a three- to four-fold increased risk of venous thromboembolism (VTE), which is associated with reduced patient survival. Despite this, the mechanisms underpinning breast cancer-associated thrombosis remain poorly defined. Tumor cells can trigger endothelial cell (EC) activation resulting in increased von Willebrand factor (VWF) secretion. Importantly, elevated plasma VWF levels constitute an independent biomarker for VTE risk. Moreover, in a model of melanoma, treatment with low molecular weight heparin (LMWH) negatively regulated VWF secretion and attenuated tumor metastasis., Objective: To investigate the role of VWF in breast cancer metastasis and examine the effect of LMWH in modulating EC activation and breast tumor transmigration., Methods: von Willebrand factor levels were measured by ELISA. Primary ECs were used to assess tumor-induced activation, angiogenesis, tumor adhesion, and transendothelial migration., Results and Conclusion: Patients with metastatic breast cancer have markedly elevated plasma VWF:Ag levels that also correlate with poorer survival. MDA-MB-231 and MCF-7 breast cancer cells induce secretion of VWF, angiopoietin-2, and osteoprotegerin from ECs, which is further enhanced by the presence of platelets. Vascular endothelial growth factor-A (VEGF-A) plays an important role in modulating breast cancer-induced VWF release. Moreover, VEGF-A from breast tumor cells also contributes to a pro-angiogenic effect on ECs. VWF multimers secreted from ECs, in response to tumor-VEGF-A, mediate adhesion of breast tumor cells along the endothelium. LMWH inhibits VWF-breast tumor adhesion and transendothelial migration. Our findings highlight the significant crosstalk between tumor cells and the endothelium including increased VWF secretion which may contribute to tumor metastasis., (© 2022 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Background: Cancer-associated neoantigens (neoAg) derived from tumor genomic sequencing and predictive algorithms for mutated peptides are a promising basis for therapeutic vaccines under investigation. Although these are generally designed to bind major histocompatibility complex class I and induce CD8 cytolytic T lymphocyte (CTL) activity, results from preclinical and clinical studies demonstrate that the majority of neoAg vaccines efficiently induce CD4 T helper (Th) responses but not CTL. Despite this, these vaccines have demonstrated clinical efficacy. Therefore, understanding the mechanisms of CD4 + T cell-mediated tumor protection is critical to optimizing this immunotherapeutic strategy., Methods: We investigated this phenomenon in the mineral oil-induced plasmacytoma (MOPC).315.BM (MOPC315) mouse model of multiple myeloma, a malignancy of plasma cells. MOPC315 cells express in their lambda chain a unique tumor-specific neoAg, an idiotypic (Id) peptide. We generated a vaccine formulated with this Id peptide fused to a heat shock protein HSC70 binding (HSB) motif co-delivered with poly (I:C). The immunogenicity of the Id-vaccine was measured in splenocytes by ELISpot. Mice were challenged with MOPC315 cells and antitumor immunity was assessed by co-incubating splenocytes and bone marrow mononuclear cells derived from vaccinated mice and controls, with the Id antigen and irradiated MOPC315 cells. The frequency of activated CD4 and CD8 T cells and their phenotype were characterized by flow cytometry., Results: Id-vaccine efficiently induced antigen-specific CD4 Th activity and antitumor immunity, protecting mice from MOPC315 tumor growth. CD4 cytolytic activity was not detected under these conditions. Polyfunctional CD8 T cells homed to the bone marrow microenvironment of protected mice and preferentially expanded only when restimulated ex vivo with both Id peptide and MOPC315 cells. Protective activity was abrogated by depletion of either CD4 or CD8 lymphocytes., Conclusion: These results demonstrate that Id-HSB +poly (I:C) vaccine protects against MOPC315 growth by priming Id-specific CD4 Th cells that confer protection against tumor but are not directly cytotoxic. These data indicate that activation of CD8 CTL against MOPC315-associated antigens not present in the vaccine is one of the major mechanisms of tumor immunity., Competing Interests: Competing interests: DL is an employee of Agenus and JC was an employee of Agenus at the time the research was being conducted. All other authors do not have relevant competing interests., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: Here, we assess the usage of high throughput sequencing (HTS) in rheumatic research and the availability of public HTS data of rheumatic samples., Methods: We performed a semiautomated literature review on PubMed, consisting of an R-script and manual curation as well as a manual search on the Sequence Read Archive for public available HTS data., Results: Of the 699 identified articles, rheumatoid arthritis (n=182 publications, 26%), systemic lupus erythematous (n=161, 23%) and osteoarthritis (n=152, 22%) are among the rheumatic diseases with the most reported use of HTS assays. The most represented assay is RNA-Seq (n=457, 65%) for the identification of biomarkers in blood or synovial tissue. We also find, that the quality of accompanying clinical characterisation of the sequenced patients differs dramatically and we propose a minimal set of clinical data necessary to accompany rheumatological-relevant HTS data., Conclusion: HTS allows the analysis of a broad spectrum of molecular features in many samples at the same time. It offers enormous potential in novel personalised diagnosis and treatment strategies for patients with rheumatic diseases. Being established in cancer research and in the field of Mendelian diseases, rheumatic diseases are about to become the third disease domain for HTS, especially the RNA-Seq assay. However, we need to start a discussion about reporting of clinical characterisation accompany rheumatological-relevant HTS data to make clinical meaningful use of this data., Competing Interests: Competing interests: SB and JCC have nothing to declare. AS has received speaker fees (less than US$10 000) and grant/research support by AbbVie, Novartis, Roche and GSK., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

Background: Breast cancer patients are at a four-fold increased risk of developing a venous thromboembolism (VTE), a major cause of death in this group. Conversely, coagulation factors promote tumour growth and metastasis. This has been evidenced in preclinical models, with an inhibitory effect of anticoagulants on cancer growth through proliferative, angiogenic, apoptotic, cancer stem cell and metastatic processes. The extrinsic clotting pathway is also more upregulated in patients in the relatively poorer prognosis oestrogen receptor (ER)-negative breast cancer subgroup, with increased tumour stromal expression of the coagulation factors Tissue Factor and thrombin. Rivaroxaban (Xarelto®, Bayer AG, Leverkusen, Germany) is a direct oral anticoagulant (DOAC). It is a Factor Xa inhibitor that is routinely prescribed for the prevention of stroke in non-valvular atrial fibrillation and for both VTE prophylaxis and treatment. This trial will assess the anti-proliferative and other anti-cancer progression mechanisms of Rivaroxaban in ER-negative early breast cancer patients., Methods: This UK-based preoperative window-of-opportunity phase II randomised control trial will randomise 88 treatment-naïve early breast cancer patients to receive 20 mg OD Rivaroxaban treatment for 11 to 17 days or no treatment. Treatment will be stopped 24 h (range 18-36 h) prior to surgery or repeat core biopsy. All patients will be followed up for 2 weeks following surgery or repeat core biopsy. The primary endpoint is change in tumour Ki67. Secondary outcome measures include tumour markers of apoptosis and angiogenesis, extrinsic clotting pathway activation and systemic markers of metastasis, tumour load and coagulation., Discussion: Laboratory evidence supports an anti-cancer role for anticoagulants; however, this has failed to translate into survival benefit when trialled in patients with metastatic disease or poor prognosis cancers, such as lung cancer. Subgroup analysis supported a potential survival benefit in better prognosis advanced disease patients. This is the first study to investigate the anti-cancer effects of anticoagulants in early breast cancer., Trial Registration: UK National Research Ethics Service (NRES) approval 15/NW/0406, MHRA Clinical Trials Authorisation 48380/0003/001-0001. The sponsor is Manchester University NHS Foundation Trust, and the trial is co-ordinated by Cancer Research UK Liverpool Cancer Trials Unit (LCTU). EudraCT 2014-004909-33 , registered 27 July 2015. ISRCTN14785273 .

Background: XPAND II was a prospective, multicenter, single-arm, open-label, continued-access study designed to confirm the results from the XPAND study, a multicenter, prospective, randomized study for breast reconstruction. The AeroForm device received clearance from the U.S. Food and Drug Administration in December 2016 based on the results of the pivotal XPAND trial, which compared the AeroForm to saline expanders., Methods: Fifty women were treated in the XPAND II study and implanted with the AeroForm device (86 devices). The study endpoint was successful completion of the second-stage surgery, and secondary endpoints were days to complete expansion and reconstruction, and patient/physician satisfaction. Following implantation, women were administered 10-cc doses of carbon dioxide at home up to three times daily. When adequate expansion was achieved, the expanders were exchanged for standard breast implants., Results: The primary endpoint (successful exchange to standard breast implant, precluding non-device-related failures) is 100 percent. All-cause interim success is 95 percent, with three subjects (four breasts) failing primary exchange because of non-device-related reasons. Median time to complete expansion was 21 days (range, 5 to 117 days). Median time to complete the reconstruction was 112 days (range, 55 to 329 days). Ninety-six percent of the subjects were very or moderately satisfied with the AeroForm expansion process., Conclusions: Results of the XPAND II continued access study confirm and improve on previous results from the randomized trial (XPAND). These results validate that the AeroForm patient-controlled, needle-free carbon dioxide tissue expander is safe and effective for two-stage breast reconstruction., Clinical Question/level of Evidence: Therapeutic, IV.

Adoptive T cell therapy using patient T cells redirected to recognize tumor-specific antigens by expressing genetically engineered high-affinity T-cell receptors (TCRs) has therapeutic potential for melanoma and other solid tumors. Clinical trials implementing genetically modified TCRs in melanoma patients have raised concerns regarding off-target toxicities resulting in lethal destruction of healthy tissue, highlighting the urgency of assessing which off-target peptides can be recognized by a TCR. As a model system we used the clinically efficacious NY-ESO-1-specific TCR C 259 , which recognizes the peptide epitope SLLMWITQC presented by HLA-A * 02:01. We investigated which amino acids at each position enable a TCR interaction by sequentially replacing every amino acid position outside of anchor positions 2 and 9 with all 19 possible alternative amino acids, resulting in 134 peptides (133 altered peptides plus epitope peptide). Each peptide was individually evaluated using three different in vitro assays: binding of the NY-ESO c259 TCR to the peptide, peptide-dependent activation of TCR-expressing cells, and killing of peptide-presenting target cells. To represent the TCR recognition kernel, we defined Position Weight Matrices (PWMs) for each assay by assigning normalized measurements to each of the 20 amino acids in each position. To predict potential off-target peptides, we applied a novel algorithm projecting the PWM-defined kernel into the human proteome, scoring NY-ESO c259 TCR recognition of 336,921 predicted human HLA-A * 02:01 binding 9-mer peptides. Of the 12 peptides with high predicted score, we confirmed 7 (including NY-ESO-1 antigen SLLMWITQC) strongly activate human primary NY-ESO c259 -expressing T cells. These off-target peptides include peptides with up to 7 amino acid changes (of 9 possible), which could not be predicted using the recognition motif as determined by alanine scans. Thus, this replacement scan assay determines the "TCR fingerprint" and, when coupled with the algorithm applied to the database of human 9-mer peptides binding to HLA-A * 02:01, enables the identification of potential off-target antigens and the tissues where they are expressed. This platform enables both screening of multiple TCRs to identify the best candidate for clinical development and identification of TCR-specific cross-reactive peptide recognition and constitutes an improved methodology for the identification of potential off-target peptides presented on MHC class I molecules., (Copyright © 2019 Karapetyan, Chaipan, Winkelbach, Wimberger, Jeong, Joshi, Stein, Underwood, Castle, van Dijk and Seibert.)

Mutation-derived neoantigens distinguish tumor from normal cells. T cells can sense the HLA-presented mutations, recognize tumor cells as non-self and destroy them. Therapeutically, immunotherapy antibodies can increase the virulence of the immune system by increasing T-cell cytotoxicity targeted toward neoantigens. Neoantigen vaccines act through antigen-presenting cells, such as dendritic cells, to activate patient-endogenous T cells that recognize vaccine-encoded mutations. Infusion of mutation-targeting T cells by adoptive cell therapy (ACT) directly increases the number and frequency of cytotoxic T cells recognizing and killing tumor cells. At the same time, publicly-funded consortia have profiled tumor genomes across many indications, identifying mutations in each tumor. For example, we find basal and HER2 positive tumors contain more mutated proteins and more TP53 mutations than luminal A/B breast tumors. HPV negative tumors have more mutated proteins than HPV positive head and neck tumors and in agreement with the hypothesis that HPV activity interferes with p53 activity, only 14% of the HPV positive mutations have TP53 mutations vs. 86% of the HPV negative tumors. Lung adenocarcinomas in smokers have over four times more mutated proteins relative to those in never smokers (median 248 vs. 61, respectively). With an eye toward immunotherapy applications, we review the spectrum of mutations in multiple indications, show variations in indication sub-types, and examine intra- and inter-indication prevalence of re-occurring mutation neoantigens that could be used for warehouse vaccines and ACT.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor with poor prognosis to most patients. Immunotherapy of GBM is a potentially beneficial treatment option, whose optimal implementation may depend on familiarity with tumor specific antigens, presented as HLA peptides by the GBM cells. Further, early detection of GBM, such as by a routine blood test, may improve survival, even with the current treatment modalities. This study includes large-scale analyses of the HLA peptidome (immunopeptidome) of the plasma-soluble HLA molecules (sHLA) of 142 plasma samples, and the membranal HLA of GBM tumors of 10 of these patients' tumor samples. Tumor samples were fresh-frozen immediately after surgery and the plasma samples were collected before, and at multiple visits after surgery. In total, this HLA peptidome analysis involved 52 different HLA allotypes and resulted in the identification of more than 35,000 different HLA peptides. Strong correlations were observed in the signal intensities and in the repertoires of identified peptides between the tumors and plasma-soluble HLA peptidomes of the individual patients, whereas low correlations were observed between these HLA peptidomes and the tumors' proteomes. HLA peptides derived from Cancer/Testis Antigens (CTAs) were selected based on their presence among the HLA peptidomes of the patients and absence of expression of their source genes from any healthy and essential human tissues, except from immune-privileged sites. Additionally, peptides were selected as potential biomarkers if their levels in the plasma-sHLA peptidome were significantly reduced after the removal of tumor mass. The CTAs identified among the analyzed HLA peptidomes provide new opportunities for personalized immunotherapy and for early diagnosis of GBM., (© 2019 Shraibman et al.)

Patients with glioblastoma currently do not sufficiently benefit from recent breakthroughs in cancer treatment that use checkpoint inhibitors 1,2 . For treatments using checkpoint inhibitors to be successful, a high mutational load and responses to neoepitopes are thought to be essential 3 . There is limited intratumoural infiltration of immune cells 4 in glioblastoma and these tumours contain only 30-50 non-synonymous mutations 5 . Exploitation of the full repertoire of tumour antigens-that is, both unmutated antigens and neoepitopes-may offer more effective immunotherapies, especially for tumours with a low mutational load. Here, in the phase I trial GAPVAC-101 of the Glioma Actively Personalized Vaccine Consortium (GAPVAC), we integrated highly individualized vaccinations with both types of tumour antigens into standard care to optimally exploit the limited target space for patients with newly diagnosed glioblastoma. Fifteen patients with glioblastomas positive for human leukocyte antigen (HLA)-A*02:01 or HLA-A*24:02 were treated with a vaccine (APVAC1) derived from a premanufactured library of unmutated antigens followed by treatment with APVAC2, which preferentially targeted neoepitopes. Personalization was based on mutations and analyses of the transcriptomes and immunopeptidomes of the individual tumours. The GAPVAC approach was feasible and vaccines that had poly-ICLC (polyriboinosinic-polyribocytidylic acid-poly-L-lysine carboxymethylcellulose) and granulocyte-macrophage colony-stimulating factor as adjuvants displayed favourable safety and strong immunogenicity. Unmutated APVAC1 antigens elicited sustained responses of central memory CD8 + T cells. APVAC2 induced predominantly CD4 + T cell responses of T helper 1 type against predicted neoepitopes.

Tumor cells accumulate aberrations not present in normal cells, leading to presentation of neoantigens on MHC molecules on their surface. These non-self neoantigens distinguish tumor cells from normal cells to the immune system and are thus targets for cancer immunotherapy. The rapid development of molecular profiling platforms, such as next-generation sequencing, has enabled the generation of large datasets characterizing tumor cells. The simultaneous development of algorithms has enabled rapid and accurate processing of these data. Bioinformatic software tools encoding the algorithms can be strung together in a workflow to identify neoantigens. Here, with a focus on high-throughput sequencing, we review state-of-the art bioinformatic tools along with the steps and challenges involved in neoantigen identification and recognition., (© 2019 Elsevier Inc. All rights reserved.)

Glioblastoma multiforme (GBM) is the most aggressive brain tumor with poor prognosis to most patients. Immunotherapy of GBM is a potentially beneficial treatment option, whose optimal implementation may depend on familiarity with tumor specific antigens, presented as HLA peptides by the GBM cells. Furthermore, early detection of GBM, such as by a routine blood test, may improve survival, even with the current treatment modalities. This study includes large-scale analyses of the HLA peptidome (immunopeptidome) of the plasma-soluble HLA molecules (sHLA) of 142 plasma samples, and the membranal HLA of GBM tumors of 10 of these patients' tumor samples. Tumor samples were fresh-frozen immediately after surgery and the plasma samples were collected before, and at multiple visits after surgery. In total, this HLA peptidome analysis involved 52 different HLA allotypes and resulted in the identification of more than 35,000 different HLA peptides. Strong correlations were observed in the signal intensities and in the repertoires of identified peptides between the tumors and plasma-soluble HLA peptidomes of the individual patients, whereas low correlations were observed between these HLA peptidomes and the tumors' proteomes. HLA peptides derived from Cancer/Testis Antigens (CTAs) were selected based on their presence among the HLA peptidomes of the patients and absence of expression of their source genes from any healthy and essential human tissues, except from immune-privileged sites. Additionally, peptides were selected as potential biomarkers if their levels in the plasma-sHLA peptidome were significantly reduced after the removal of tumor mass. The CTAs identified among the analyzed HLA peptidomes provide new opportunities for personalized immunotherapy and for early diagnosis of GBM., (© 2018 Shraibman et al.)

Mass spectrometry (MS)-based immunopeptidomics investigates the repertoire of peptides presented at the cell surface by major histocompatibility complex (MHC) molecules. The broad clinical relevance of MHC-associated peptides, e.g. in precision medicine, provides a strong rationale for the large-scale generation of immunopeptidomic datasets and recent developments in MS-based peptide analysis technologies now support the generation of the required data. Importantly, the availability of diverse immunopeptidomic datasets has resulted in an increasing need to standardize, store and exchange this type of data to enable better collaborations among researchers, to advance the field more efficiently and to establish quality measures required for the meaningful comparison of datasets. Here we present the SysteMHC Atlas (https://systemhcatlas.org), a public database that aims at collecting, organizing, sharing, visualizing and exploring immunopeptidomic data generated by MS. The Atlas includes raw mass spectrometer output files collected from several laboratories around the globe, a catalog of context-specific datasets of MHC class I and class II peptides, standardized MHC allele-specific peptide spectral libraries consisting of consensus spectra calculated from repeat measurements of the same peptide sequence, and links to other proteomics and immunology databases. The SysteMHC Atlas project was created and will be further expanded using a uniform and open computational pipeline that controls the quality of peptide identifications and peptide annotations. Thus, the SysteMHC Atlas disseminates quality controlled immunopeptidomic information to the public domain and serves as a community resource toward the generation of a high-quality comprehensive map of the human immunopeptidome and the support of consistent measurement of immunopeptidomic sample cohorts., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Next-Generation Sequencing (NGS) enables the rapid generation of billions of short nucleic acid sequence fragments (i.e., "sequencing reads"). Especially, the adoption of gene expression profiling using whole transcriptome sequencing (i.e., "RNA-Seq") has been rapid. Here, we describe an in silico method, seq2HLA, that takes standard RNA-Seq reads as input and determines a sample's (classical and non-classical) HLA class I and class II types as well as HLA expression. We demonstrate the application of seq2HLA using publicly available RNA-Seq data from the Burkitt's lymphoma cell line DAUDI and the choriocarcinoma cell line JEG-3.

The B16F10 murine melanoma cell line displays a low expression of MHC class I molecules favoring immune evasion and metastases in immunocompetent C57 BL/6 wild-type mice. Here, we generated metastases to the liver, an organ that is skewed towards immune tolerance, by intrasplenic injection of B16F10 cells in syngeneic C57 BL/6 compared to allogeneic Balb/c mice. Surprisingly, Balb/c mice, which usually display a pronounced M2 macrophage and Th2 T cell polarization, were ∼3 times more susceptible to metastasis than C57 BL/6 mice, despite a much higher M1 and Th1 T cell immune response. The anti-metastatic advantage of C57 BL/6 mice could be attributed to a more potent NK-cell mediated cytotoxicity against B16F10 cells. Our findings highlight the role of NK cells in innate anti-tumor immunity in the context of the liver - particularly against highly aggressive MHC I-deficient cancer cells. Moreover, the B16F10 model of melanoma liver metastasis is suited for developing novel therapies targeting innate NK cell related immunity in liver metastases and liver cancer.

Background: People are frequently confronted with untrustworthy claims about the effects of treatments. Uncritical acceptance of these claims can lead to poor, and sometimes dangerous, treatment decisions, and wasted time and money. Resources to help people learn to think critically about treatment claims are scarce, and they are widely scattered. Furthermore, very few learning-resources have been assessed to see if they improve knowledge and behavior., Objectives: Our objectives were to develop the Critical thinking and Appraisal Resource Library (CARL). This library was to be in the form of a database containing learning resources for those who are responsible for encouraging critical thinking about treatment claims, and was to be made available online. We wished to include resources for groups we identified as 'intermediaries' of knowledge, i.e. teachers of schoolchildren, undergraduates and graduates, for example those teaching evidence-based medicine, or those communicating treatment claims to the public. In selecting resources, we wished to draw particular attention to those resources that had been formally evaluated, for example, by the creators of the resource or independent research groups., Methods: CARL was populated with learning-resources identified from a variety of sources-two previously developed but unmaintained inventories; systematic reviews of learning-interventions; online and database searches; and recommendations by members of the project group and its advisors. The learning-resources in CARL were organised by 'Key Concepts' needed to judge the trustworthiness of treatment claims, and were made available online by the James Lind Initiative in Testing Treatments interactive (TTi) English (www.testingtreatments.org/category/learning-resources).TTi English also incorporated the database of Key Concepts and the Claim Evaluation Tools developed through the Informed Healthcare Choices (IHC) project (informedhealthchoices.org)., Results: We have created a database of resources called CARL, which currently contains over 500 open-access learning-resources in a variety of formats: text, audio, video, webpages, cartoons, and lesson materials. These are aimed primarily at 'Intermediaries', that is, 'teachers', 'communicators', 'advisors', 'researchers', as well as for independent 'learners'. The resources included in CARL are currently accessible at www.testingtreatments.org/category/learning-resources., Conclusions: We hope that ready access to CARL will help to promote the critical thinking about treatment claims, needed to help improve healthcare choices.

Introduction: Given the current postulated plasticity between epithelial and mesenchymal states of migratory cancer cells the detection of non-epithelial CTCs is an important scientific and clinical goal., Methods: We used the filtration-based ISET technology to enrich circulating tumour cells (CTCs) in early breast cancer blood samples and identify them using a morphology-based immunocytochemistry (ICC) approach., Results: We found greater numbers of putative CTCs by this approach than by the cytokeratin-based CellSearch technology, but a high number of CTC false positives were identified in healthy volunteer samples which were not reduced in successive blood draws. Preliminary work using an oestrogen receptor (ER)-based multiplex ICC method in metastatic breast cancer ISET samples indicated a low number of ER+ CTCs even at this advanced stage., Conclusions: This work highlights the challenges in enumerating CTCs without conventional epithelial markers.