Complete lymph node removal through conventional axillary dissection (ALND) has been standard treatment for breast cancer patients for almost a century. In the 1990s, however, and in parallel with the advent of the sentinel lymph node (SLN) procedure, ALND came under increasing scrutiny due to its association with significant patient morbidity. Several studies have since provided evidence to suggest omission of ALND, often in favor of axillary radiation, in selected clinically node-negative, SLN-positive patients, thus supporting the current trend in clinical practice. Clinically node-positive patients, by contrast, continue to undergo ALND in many cases, if only for the lack of studies re-assessing the indication for ALND in these patients. Hence, there is a need for a clinical trial to evaluate the optimal treatment for clinically node-positive breast cancer patients in terms of surgery and radiotherapy. The TAXIS trial is designed to fill this gap by examining in particular the value of tailored axillary surgery (TAS), a new technique for selectively removing positive lymph nodes.In this international, multicenter, phase-III, non-inferiority, randomized controlled trial (RCT), including 34 study sites from four different countries, we plan to randomize 1500 patients to either receive TAS followed by ALND and regional nodal irradiation excluding the dissected axilla, or receive TAS followed by regional nodal irradiation including the full axilla. All patients undergo adjuvant whole-breast irradiation after breast-conserving surgery and chest-wall irradiation after mastectomy. The main objective of the trial is to test the hypothesis that treatment with TAS and axillary radiotherapy is non-inferior to ALND in terms of disease-free survival of clinically node-positive breast cancer patients in the era of effective systemic therapy and extended regional nodal irradiation. The trial was activated on 31 July 2018 and the first patient was randomized on 7 August 2018.Designed to test the hypothesis that TAS is non-inferior to ALND in terms of curing patients and preventing recurrences, yet is significantly superior in reducing patient morbidity, this trial may establish a new worldwide treatment standard in breast cancer surgery. If found to be non-inferior to standard treatment, TAS may significantly contribute to reduce morbidity in breast cancer patients by avoiding surgical overtreatment.ClinicalTrials.gov, ID: NCT03513614. Registered on 1 May 2018.www.kofam.ch, ID: NCT03513614. Registered on 17 June 2018.EudraCT No.: 2018–000372-14.The online version of this article (10.1186/s13063-018-3021-9) contains supplementary material, which is available to authorized users. Worldwide more than 2 million patients are diagnosed with breast cancer every year . It accoOmission of any surgical axillary staging in selected patients –6,Omission of axillary dissection in all patients with negative sentinel lymph node (SLN) procedures –9Omission of axillary dissection in selected patients with limited nodal disease in the SLN –12Axillary radiation vs. axillary dissection –15 or obALND has traditionally been considered standard care for all patients with breast cancer. Due to the morbidity of the procedure  and decThe trial findings supported the current trend in clinical practice toward decreased rates of axillary dissection in patients with non-palpable axillary lymph node metastases , 18 and The important landmark trial ACOSOG Z0011 has been criticized for several reasons. One limitation of ACOSOG Z0011 was the lack of standardization and detailed documentation of adjuvant radiation fields and the angles of the tangents, which makes it impossible to know how much of the axilla was irradiated . Since tIn parallel with the trend toward less axillary surgery, radiation oncologists have been broadening the indication for extended regional lymph node irradiation based on evidence from two large phase-III trials showing improved disease-free survival (DFS) –23 and aTwo surgical trials have been initiated to provide evidence for the safety of omitting any axillary surgery in selected clinically node-negative patients .POSNOC allows axillary RT as an alternative to ALND in the control arm. Similarly, BOOG 2013–07 allows axillary RT or ALND to complete axillary treatment in the control arm, but includes patients with one to three positive SLN undergoing mastectomy . Since tThe most progressive ongoing clinical trial on axillary management, partially overlapping with the proposed protocol, is Alliance A011202. It compares ALND with axillary radiation in patients with residual disease after chemotherapy, which was an exclusion criterion in both the AMAROS and the Z0011 trials. The trial tests the hypothesis that the AMAROS protocol in combination with extended regional nodal irradiation works in these patients without ALND to the extent that chemotherapy-resistant lymph node metastases are as radiosensitive as chemotherapy-naive disease.The optimal treatment of the regional lymph nodes in clinically node-positive patients is currently unclear, with less surgery, more RT or a combination thereof being under discussion. In the era of increasing post-mastectomy and extended regional node irradiation as well as effective systemic therapy, the indication for ALND in patients with clinically positive nodes and confirmed disease at the time of surgery has to be investigated in a clinical trial. Most of the above-mentioned trials excluded patients with residual metastases after neoadjuvant chemotherapy, and the omission of axillary dissection in patients with imaging-detected lymph node metastases is controversial since they may predict a larger volume of axillary disease –32.TAXIS is designed to determine whether ALND may be no longer necessary for confirmed nodal disease at first diagnosis of breast cancer given the prevalence of extended regional nodal irradiation in clinically node-positive patients in the adjuvant setting or incomplete response of nodal disease in the neoadjuvant setting. In many patients undergoing ALND, the number of negative lymph nodes removed exceeds the number of tumor-affected nodes. Removing multiple unaffected lymph nodes increases morbidity with no therapeutic benefit. It is a consequence of the commitment of radical surgery to the principle of complete tissue removal within the anatomical borders of the axilla. Benefits and harms of this approach must be re-assessed. TAXIS investigates tailored axillary surgery (TAS), a tailored approach focusing on the removal of tumor-affected lymph nodes while limiting the extent of surgery to minimize the number of negative nodes removed. TAS may offer non-inferior oncological outcomes with less morbidity than ALND in patients with clinically positive nodes at first presentation and confirmed nodal disease at surgery with or without neoadjuvant therapy.http://sakk.ch/en/sakk-provides/our-trials/breast-cancer/sakk-2316-taxis/). Enrollment of patients started in July 2018 and will stop after the randomization of the targeted total of 1500 patients, which is expected in Q4 2023. End-of-trial treatment is expected for Q3 2024. All patients will be followed up for 20 years after randomization of the last patient. The trial will be terminated after the last visit of the last patient, which is expected to be in 2043. See Fig. TAXIS is an international, multicenter, phase-III, non-inferiority randomized controlled trial with 34 study sites from four different countries currently planning to participate (Switzerland (Schweizerische Arbeitsgemeinschaft für Klinische Krebsforschung (SAKK)): 17; Austria (ABCSG): 10; Italy (IBCSG): 6; Hungary (IBCSG): (1). A list of sites and investigators participating in the trial can be downloaded from the public section of the SAKK website: The main objective of the trial is to test the hypothesis that treatment with TAS and axillary radiotherapy (RT) is non-inferior to axillary lymph node dissection (ALND) in terms of DFS of breast cancer patients with positive nodes at first presentation in the era of effective systemic therapy and extended regional nodal irradiation.Prior to enrollment, eligible patients are informed about the aims, procedures and possible risks of the trial, as well as its confidentiality policy regarding patient data, and are given sufficient opportunity and time to consider whether or not to participate. All patients are made aware that participation is voluntary and that they are allowed to refuse further participation in the trial whenever they want. Written informed consent is obtained from each patient before enrollment and prior to any trial-specific procedures.Eligible for inclusion in this trial are female and male patients aged ≥ 18 years if they meet the following criteria:Node-positivity detected by imaging (iN+) and confirmed by pathology or detected by palpation (cN1–2) and confirmed by pathologyNode-positivity initially detected by imaging  and residual disease intraoperatively confirmed by pathology (in SLN or non-SLN during surgery) in case of neoadjuvant treatmentNode-positivity initially detected by palpation and residual disease intraoperatively confirmed by pathology in case of neoadjuvant treatmentEligible for primary ALND or SLN procedure with frozen section and either (1) newly diagnosed or (2) isolated in-breast recurrence or second ipsilateral breast cancer Most suspicious axillary lymph node clippedAdequate condition for general anesthesia and breast cancer surgeryAbility to understand and complete the quality of life (QoL) questionnaireWHO performance status 0–2Node-positive breast cancer  American Joint Committee on Cancer/Union for International Cancer Control (AJCC/UICC)  stage IIStage IV breast cancerClinical N3 breast cancerClinical N2 breast cancer Contralateral breast cancerPrior axillary surgery (except prior sentinel node procedure in case of in-breast recurrence)Prior regional RTHistory of hematological or primary solid tumor malignancy, unless in remission for at least 5 years from pre-registration with the exception of adequately treated cervical carcinoma in situ or localized non-melanoma skin cancerConcurrent treatment with any experimental drug within 30 days of pre-registrationConcomitant use of other anticancer drugs or RTPatients are excluded on any of the following grounds:Patients are, furthermore, excluded if they have any serious underlying medical, psychiatric, psychological, familial or geographical condition, which, in the judgment of the investigator, may interfere with the planned staging, treatment, and follow-up, affect patient compliance or place the patient at high risk from treatment-related complications.Responsible surgeon○ Node-positivity detected by imaging (iN+) and confirmed by pathology○ Node-positivity detected by palpation (cN1–2) and confirmed by pathology○ Node-positivity initially detected by imaging  and residual disease confirmed by pathology (in SLN or non-SLN during surgery) after neoadjuvant treatment○ Node-positivity initially detected by palpation and residual disease confirmed by pathology after neoadjuvant treatmentType of positive node detection:Newly diagnosed vs. isolated in-breast recurrence or second ipsilateral breast cancer Normofractionated vs. hypofractionated RTMale / female patientA total of 1500 patients will be randomized at a ratio of 1:1 (750 patients per each treatment arm) to either receive TAS followed by ALND and regional nodal irradiation excluding the dissected axilla as a target volume (arm A), or receive TAS followed by regional nodal irradiation including the full axilla (arm B). Randomization is performed using the minimization method  with 80%Randomization is usually performed during the breast surgery as soon as the operating surgeon has certified that all inclusion criteria are met, including in particular the pathological confirmation of node-positivity and residual disease for patients who had received neoadjuvant treatment. If node-positivity cannot be confirmed on frozen section during primary surgery, the randomization takes place as soon as possible after the positive pathology results are available. Randomization is exclusively done online by using the centralized electronic data capture system secuTrial®. Patients who are not randomized will not enter into the trial and no further data will be collected except retrospective data for the two subprojects described in section “The investigational trial treatment consists of tailored axillary surgery (TAS), which is defined by the SLN procedure in combination with the selective removal of all palpable disease and documentation of the removal of the initially biopsy-proven and clipped lymph node metastasis by specimen radiography. If the clip is not documented in the specimen radiography, the patient is excluded and undergoes ALND.All patients undergo adjuvant whole-breast irradiation after breast-conserving surgery and chest-wall irradiation after mastectomy. While patients allocated to arm A (control arm) receive regional nodal irradiation excluding the dissected axilla as a target volume (levels (II)/III; medial supraclavicular; internal mammary lymph nodes), patients allocated to arm B  receive regional nodal irradiation including the full axilla .If indicated, patients may undergo adjuvant systemic treatment.A video of the intervention has been uploaded as Additional file 1.Additional file 1: Video S1. (MP4 383974 kb).All patients undergo breast-conserving surgery or mastectomy and the SLN procedure. The SLN procedure should preferably be performed by dual mapping, which includes all nodes that are either blue (blue dye), hot (technetium Tc-99 m), fluorescent (indocyanine green) or magnetic (superparamagnetic iron oxide particles), according to local SLN procedure practices. In addition to the removal of the sentinel nodes, all palpably suspicious nodes, defined as either hardened or irregular or very large or a combination thereof, are removed.Surgical evaluation of the levels I–III by palpation is mandatory to ensure that there is no palpably suspicious disease left behind in the axilla after TAS. The length of the incision and the opening of the clavipectoral fascia has to be chosen accordingly. Therefore, a minimum incision length of 2–3 cm is recommended. In case of palpably suspicious nodes left behind in the axilla after TAS, the patient must be excluded und typically undergoes ALND according to the decision of the treating surgeon.Surgeons who perform the SLN procedure as part of their clinical routine are allowed to perform TAS. TAS is feasible for all surgeons who master the SLN procedure since the only difference is the selective removal of palpable disease, which we expect to be a frequent step in the clinically node-positive patient population of this trial. However, thorough palpation of the axilla is also mandatory during the regular SLN procedure in current clinical practice to remove suspicious findings and minimize the false-negative rate. The difference is that during the regular SLN procedure that is performed as a staging procedure, this step is rarely necessary since it is only performed in clinically node-negative patients.The selective removal of the clipped lymph node by image-guided localization, a procedure increasingly referred to as targeted axillary dissection (TAD), is encouraged to increase the chances of successful clip removal. However, TAD is not a mandatory part of TAS, since it is technically challenging and, therefore, quite controversial, and the clipped lymph node corresponds to one of the SLNs in the majority of patients. TAD has recently emerged as an effective strategy to reduce the false-negative rate of the SLN procedure in patients with initially confirmed node-positive breast cancer that showed a complete clinical response in the nodes after neoadjuvant treatment. The metastatic node is marked with a clip during biopsy, or shortly after the lymph node metastasis has been confirmed by pathology or cytology, and is then selectively localized and removed during the procedure –37. Any Importantly, residual suspicious lymph nodes detected by imaging before the end of adjuvant treatment does neither demand nor prohibit take-back surgery for completion ALND or selective removal of these nodes or an additional RT boost. The study group is fully aware that some patients will have non-palpable residual disease in the axilla after TAS. The hypothesis of TAXIS is that this residual disease does not progress to recurrence. The clinical scenario of residual disease after axillary surgery detected by imaging, i.e., performed for RT treatment planning or staging, to rule out distant disease, has become more frequent after implementation of the ACOSOG Z0011 and the EORTC-AMAROS protocols . HistoloAfter randomization, the patients in arm A will be treated according to the current standard of care by ALND. ALND is defined by the intention of the surgeon to radically remove the entire soft tissue within the anatomical borders of the axilla. To ensure applicability of the findings of the present study to clinical practice, we purposefully refrained from further defining the exact technique or number of removed lymph nodes. One common way to perform ALND is described here. Standard ALND clears levels I and II. A clip may be applied by the surgeon to mark the medial border of dissection, commonly between levels II and III, and documented in the surgical report. A full level-III dissection above the pectoralis minor muscle is carried out when there is gross nodal disease. The latissimus dorsi muscle is identified and followed until it is crossed by the axillary vein. The surface of the vein is then cleared of fat. Dissection inferior to the vein is carried out, dividing the fat and controlling the branches of the axillary vein entering the specimen. The thoracodorsal bundle is the deep lateral branch. Once identified, the pectoralis minor is retracted and the level-III nodes are dissected – if indicated – from the space below the axillary vein. The long thoracic nerve is identified against the chest wall and dissected free from the specimen. The fat between the thoracodorsal and long thoracic nerves is encircled with a clip or bipolar vessel-sealing system, divided, and bluntly swept inferiorly. Branches of the thoracodorsal vessels entering the specimen are dissected, and the specimen is freed from its remaining attachments to the inferior chest wall. A closed suction drain is standard care for most surgeons. The dermis is re-approximated and the skin is closed.All patients undergo adjuvant whole-breast irradiation after breast-conserving surgery or chest-wall irradiation after mastectomy, as well as regional nodal irradiation, excluding the dissected axilla as a target volume in arm A (levels (II)/III; medial supraclavicular; internal mammary lymph nodes) and including the full axilla  as a target volume in arm B. In case of extensive nodular involvement in arm A, the target volume includes the area at risk of the dissected levels. Inclusion of internal mammary nodes is recommended in this high-risk group of patients irrespective of treatment arm . RT shouFor a detailed description of RT, see Where adjuvant systemic anticancer treatment is indicated, all drugs to be used are locally chosen according to international guidelines, including the sequence of systemic therapy in relation to surgery (neoadjuvant vs. adjuvant setting) , 44. HanFor a description of all evaluations performed before, during, and after the trial treatment, see Local recurrence, regional recurrence, distant recurrenceSecond breast cancerDeath from any causeThe primary endpoint of the trial is disease-free survival (DFS), defined as time from randomization until one of the following events, whichever comes first:Patients not experiencing an event will be censored at the date of the last available assessment.QoLThe secondary endpoints of the trial are:The primary objective is to compare patient-reported arm problems in the short (after 9 months), intermediate (after 24 months), and long term (after 60 and 120 months) in breast cancer patients with clinically positive nodal disease in the era of extended regional nodal irradiation who are randomized to TAS followed by axillary radiation vs. TAS followed by ALND. The primary QoL endpoint is the change in the ARM subscale of the FACT-B + 4 from baseline to 24 months after randomization , 46.Secondary objectives include the comparison of (1) short-, intermediate-, and long-term QoL and of (2) short-, intermediate-, and long-term effect of arm problems on daily and social activities between randomized groups.Overall survivalBreast cancer-specific survivalTime to local recurrenceTime to regional recurrenceTime to distant recurrence○ Lymphedema: the change in ipsilateral upper-extremity circumference, corrected for any change in the contralateral upper extremity, will be calculated using the following formula:Physician-reported morbidity outcomesFor a detailed description of the QoL assessment procedure, see I indicates ipsilateral upper-extremity circumference, C indicates contralateral upper-extremity circumference, a indicates assessments during trial treatment and follow-up, and b indicates baseline assessment. L will be calculated for both upper arm and forearm, and lymphedema is defined as present if L > 2 cm for either location. The proportion of patients with lymphedema will be calculated at each assessmentwhere ○ Decreased range of shoulder motion: the change in shoulder motion  assessed by goniometric measurement of arm movement  on both Adverse events (AEs) according to National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) v4.03Late RT-related AEsClipping-related AEs and specific AEs related to the surgical procedure and RT will be assessed according to NCI CTCAE v4.03Surgical site infections (SSI)Late AEs related to the RT will be assessed according to the Late Effects in Normal Tissues—Subjective, Objective, Management, and Analytic (LENT-SOMA) scaleSSI will be assessed according to the Centers for Disease Control and Prevention Surgical Site Infection Classification System.Immune profile of axillary lymph nodesEvaluation of the prognostic value of multigene testsPerformance characteristics of TASPatterns of use of neoadjuvant systemic treatmentImpact of TAS on adjuvant systemic treatment decisionsQuality assessment of delineation and dose planning and impact on treatment outcomes in adjuvant RTImpact of a bolus on outcome and RT-related AEs in patients with mastectomyThe following seven subprojects have been pre-specified:For a detailed description of the subprojects, see The sample size is based on the primary endpoint DFS. With a type I error of 5% and a power of 80%, 385 events will be needed to show non-inferiority of TAS and axillary RT in comparison to ALND with a non-inferiority hazard ratio of 1.289 (corresponding to a DFS at 5 years of 80% in the ALND arm and 75% in the TAS and axillary RT arm). The sample size needed is 1500 patients (750 per arm). One interim analysis for efficacy/futility is foreseen after 20% of the required events have occurred. A report including the interim efficacy results as well as patient characteristics, treatment administration and safety results  will be presented to an independent Data Monitoring Committee appointed by the SAKK Board. The SAKK Board will decide on the continuation/modification/early stopping of the trial based on the recommendations of the committee. All efficacy endpoints will be analyzed based on the per-protocol set. For the primary endpoint, DFS, the hazard ratio and one-sided 95% confidence interval will be calculated using a Cox regression model with the treatment arm as independent variable and the stratification factors as strata. The median DFS and the corresponding 95% confidence intervals using the Kaplan-Meier method will be presented for each treatment arm.Type of positive node detection at first diagnosis: cN1 vs. iN1Neoadjuvant vs. adjuvant systemic treatment vs. bothycN1 vs. ycN02 vs ≥ 25 kg/m2BMI < 25 kg/mNormofractionation vs. hypofractionationBreast-conserving surgery vs. mastectomyTriple-negative vs. HER2+ vs. ER- and/or PR-positive, and HER2-Planned subgroup analyses for the primary endpoint:No imputation of missing data will be performed. A row denoted “Missing” will be included in count tabulations if necessary to account for drop-outs and missing values. For continuous variables a column with the number of available observations will be added. Patients lost to follow-up before reaching the primary endpoint will not be replaced.Any AE related to the clipping of the most suspicious axillary lymph node andSpecific AEs related to the trial surgical procedure or the RT  as pre-defined in the original trial protocolPatients will be instructed by the investigator to report the occurrence of all AEs. The following events are reported by the investigator within the trial:The investigator assesses and records the AEs observed during the AE-reporting period: from the date of patient consent signature up to 20 years after randomization of the last patient. However, no AEs related to neoadjuvant and/or adjuvant systemic therapy will be reported.For a description of the study management and administration, see For a description of the study documentation, see Designed to test the hypothesis that TAS in combination with RT is non-inferior to ALND in terms of curing patients and preventing recurrences, yet is significantly superior in terms of patient morbidity, this trial has the potential to establish a new worldwide treatment standard in breast cancer surgery. If found to be non-inferior to standard treatment, TAS may contribute significantly to reduce morbidity in breast cancer patients by avoiding surgical overtreatment. For the SPIRIT checklist applicable to this study protocol, see Additional file Protocol no: SAKK 23/16 / IBCSG 57–18 / ABCSG-53Protocol version: final version 1.0, 6 April 2018EudraCT no: 2018–000372-14Trial type: clinical trial with other health interventionCategorization: risk category B according to the Swiss Human Research Act and its ordinance KlinV/OclinTrial sponsor: SAKK , Effingerstrasse 33, 3008 Bern, Switzerlandwww.kofam.ch/de/studienportal/suche/38529/studie/42266, on 17 June 2018, under NCT03513614.TAXIS was registered at ClinicalTrials.gov, on 1 May 2018, under NCT03513614 and on the Swiss National Clinical Trials Portal (SNCTP) at The trial was activated on 31 July 2018. The first patient was randomized on 7 August 2018.Additional file 2:SPIRIT checklist. (DOCX 58 kb)Additional file 3:QoL questionnaire. (PDF 514 kb)


MicroRNAs play important roles in many biological processes. Their aberrant expression can have oncogenic or tumor suppressor function directly participating to carcinogenesis, malignant transformation, invasiveness and metastasis. Indeed, miRNA profiles can distinguish not only between normal and cancerous tissue but they can also successfully classify different subtypes of a particular cancer. Here, we focus on a particular class of transcripts encoding polycistronic miRNA genes that yields multiple miRNA components. We describe ‘clustered MiRNA Master Regulator Analysis (ClustMMRA)’, a fully redesigned release of the MMRA computational pipeline , developed to search for clustered miRNAs potentially driving cancer molecular subtyping. Genomically clustered miRNAs are frequently co-expressed to target different components of pro-tumorigenic signaling pathways. By applying ClustMMRA to breast cancer patient data, we identified key miRNA clusters driving the phenotype of different tumor subgroups. The pipeline was applied to two independent breast cancer datasets, providing statistically concordant results between the two analyses. We validated in cell lines the miR-199/miR-214 as a novel cluster of miRNAs promoting the triple negative breast cancer (TNBC) phenotype through its control of proliferation and EMT. MicroRNAs (miRNAs) are small RNA molecules emerged as important regulators of gene expression at the post-transcriptional level. They have been shown to be involved in the regulation of all essential functions of the cells from differentiation and proliferation to apoptosis . Each miMultiple studies demonstrated the importance of miRNAs in all the cancer hallmarks defined by Hanahan and Weinberg  and indiIn this work, we focused our attention on a particular class of transcripts encoding polycistronic miRNA genes that yields multiple miRNA components. A famous example of this class of transcripts is the mir-17/92 polycistronic oncogene that plays a role in the development of various cancer types, especially in their most aggressive form . GenomicSince >30% of annotated human miRNAs are organized in genomic clusters, we can expect to find other oncogenic/tumor suppressor polycistronic miRNAs that are co-expressed to jointly regulate molecular pathways involved in cancer malignancy. Existing computational approaches for the identification of master miRNA regulators involved in cancer onset and subtyping are typically designed to detect the effect of a single miRNA (see review in ). HoweveHere, we present Clustered MiRNA Master Regulator Analysis (ClustMMRA), a fully redesigned release of the MiRNA Master Regulator Analysis (MMRA) ,28 pipelClustMMRA extends MMRA to a model in which multiple miRNAs belonging to the same genomic cluster coordinately target functionally related genes driving the phenotype of a particular cancer subtype. As the MMRA pipeline, ClustMMRA is a multi-step workflow that requires in input miRNA/mRNA expression profiles from matched tumor samples classified in different subtypes according to subtype-specific gene signatures. The final output of ClustMMRA provides key miRNA clusters contributing to the regulation of particular subtypes of the disease.We tested this novel pipeline to search for oncogenic/tumor suppressor polycistronic miRNAs driving breast cancer subtypes. ClustMMRA was applied to two independent breast cancer datasets whose samples were previously classified into four subtypes . We obtained statistically concordant results between the two analyses, identifying five clusters of miRNAs with aberrant expression in a specific subtype of both datasets. Among them, miR-199a/214 on chromosome 1 was found to be down-regulated in the triple negative subtype and associated to EMT regulation. Functional validation in cell lines confirms the regulatory effect of this cluster in shaping the triple negative subtype phenotype through its control of proliferation and EMT. Overall, our computational pipeline and experimental validations characterize a new genomic cluster of miRNAs implicated in the TNBC phenotype that might be further explored in diagnosis and therapeutic strategies. In addition, we evinced a cooperative mechanism for the regulatory activity of genomically clustered miRNAs.miRNA genes located within 10 kb of distance on the same chromosome and in the same strand.The genomic locations of miRNAs were retrieved from miRBase v18 . As in ,31, co-cBreast cancer (BRCA) RNA-seq and miRNA-seq Level 3 expression profiles were downloaded from The Cancer Genome Atlas (TCGA) in January 2016. Only those primary tumors profiled for both mRNA and miRNA expression were included in the analysis, obtaining a total of 397 samples. Two expression matrices (one for mRNAs and the second for miRNAs) were normalized obtaining the paired mRNA/miRNA expression dataset here referred to as TCGA. The Curie dataset was generated with microarray technologies (Agilent miRNA microarray kit V3 for miRNAs and Affymetrix U133plus2 for mRNA) and pre-processed following the procedure described in . Experimt-test and log fold change cutoff  > 0.5). Moreover, to increase the predictive power of the constructed signatures, those genes associated to more than one class according to the previous criteria, or having a difference between the first and second highest absolute(log fold changes) lower than 0.2 were discarded. The choice of thresholds was optimized to maximize the gene association to a unique subtype and the number of genes included in each signature (on average 117 genes per signature). For each subtype, two separated signatures were defined (‘down' and ‘up'), based on the sign of the expression change of their genes. The .gmt file of the constructed signatures is available at https://github.com/lcan88/Supplementary_miRNA_cluster/blob/master/signature_Breast.gmt. The reliability of these signatures were tested in two ways. First, their classification performances were validated on TCGA data. We classified the TCGA samples using our signatures with the Nearest Template Prediction (NTP) method  method , as done) method ,37. Only) method  plus a s) method . The prohttps://github.com/lcan88/clustMMRA.The MMRA pipeline is here extended to search for genomically co-clustered miRNAs potentially driving cancer subtyping. Similar to MMRA, the workflow of ClustMMRA , the subtype-specific expression of each miRNA is assessed by Kolmogorov–Smirnov (KS) statistical test and fold change cutoff. Clusters having at least two miRNAs with subtype-specific expression change in the same direction (both up-regulated or down-regulated) are selected for step (ii).microRNA.org 2010, PITA 2007) plus an experimental one (miRTarBase 2.5) and requiring the prediction by at least two of them. Changes in the results of the pipeline when the correlation between the expression profiles of the predicted miRNA-target interactions is also used are reported in Supplementary Text. The set of targets of a cluster has been defined as the union of the targets of individual miRNAs. To control the number of false positive results we defined a threshold for the significance of the overlap between the targets of the cluster and the genes present in the signature based on a null model. For 1000 times random set of genes of the same size of the set of targets are generated and tested for their intersection with the gene signature. The 95th percentile of the obtained distribution is used as threshold. The objective of step (i) and (ii) is to identify co-clustered and co-expressed miRNAs potentially regulating a gene expression signature in a joint manner, without necessarily having a high overlap in terms of target genes (regulons). Unlike the results of the MMRA pipeline, in which regulons can include only one miRNA, the ones identified by the ClustMMRA pipeline contain multiple miRNAs of the genomic cluster. Interference of indirect interactions may introduce links between miRNAs and spurious genes in the regulon. A Fisher's exact test has been performed to evaluate the statistical significance of the overlap between the genes included in each regulon and the gene signature of the associated subtype.In step (ii), we extract miRNA clusters having their predicted targets enriched for the gene signature of the corresponding subtype. Only miRNAs of the cluster classified as differentially expressed in step (i) are considered in step (ii). The targets of individual miRNAs have been predicted using four different databases . These cell lines were chosen as they represent a model of luminal A and TNBC cell lines, respectively (2 at 37°C in DMEM (for T47D cell line) or advanced DMEM (for MDA-MB-231 cell line) cell culture medium , with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 2 mM glutamine . Dulbecco phosphate-buffered saline (D-PBS), trypsin, and all the media additives were obtained by Lonza (Euroclone).For The sense (S) oligonucleotide sequence of each miRNA of the cluster has been designed following the sequences indicated in miRbase database . S oligoTo obtain the upregulation of each miRNA, S oligonucleotides, resuspended in water, were added three times a day for 3 days directly to the culture medium of the cells (<50% confluency) at a final concentration of 100 nM/day . The celn = 9).Tumor cell proliferation was assessed by following the protocol described in . BrieflyFor extracellular matrix (ECM) staining, cells were grown for 3 days before fixation with 100% cold methanol at –20°C (Sigma). Then, cells were permeabilised with 0.1% Triton X-100  in PBS 1× before staining with anti-collagen I . Anti rabbit 488  secondary antibody was used for the staining. Images were acquired using a Leica DMIL microscope  with Leica ICC50W camera. Images were quantified with ‘corrected total cell fluorescence (CTCF) method’, where the integrated density was subtracted with the area of selected cells multiplied for the mean fluorescence of the background readings .Total RNA was isolated using TRIzol reagent (Life Technologies) following the manufacturer's recommendations. To obtain cDNA from total RNA for gene expression analysis, two micrograms of total RNA were reverse transcribed using oligo dT primers in combination with High Capacity cDNA Reverse Transcription kit (Applied Biosystem), following the manufacturer's protocol.For miRNA analysis, one microgram of total RNA was reverse transcribed using MystiCq microRNA cDNA synthesis kit (Sigma), following the manufacturer's protocol, in order to reverse transcribe polyA-tailed miRNA into cDNA.RT-PCR analysis was performed using Power Up Sybr Green Master mix  in an Eco RT-PCR machine (Illumina). All the primers for human mRNA and miRNA amplification were home-made and are described below . miRNA a(−ΔΔCT) method (n = 9). A t test was calculated.The relative expression of miRNAs and genes was calculated for both T47D and MDA-MB-231 cell lines with the 2) method . Experimhttps://github.com/lcan88/clustMMRA) to identify polycistronic miRNAs underlying breast cancer molecular subtypes. For this study, two independent datasets were used, a first paired miRNA/mRNA expression dataset from a in-house cohort of 129 breast carcinoma tumour samples , revealing a significantly concordant expression pattern of co-clustered miRNAs. Among these co-clustered and co-expressed miRNAs, some are differentially expressed in multiple subtypes (18 and 37 clusters for Curie and TCGA respectively), with 15 out of 18 and 21 out of 37 differentially expressed in TNBC and luminal A with opposite sign.Expression data required for running ClustMMRA were pre-processed as described in Methods and the signatures for breast cancer subtypes were defined using the approach proposed in  see Met. Of thesP-value < 1e–05). In the step (iii) of ClustMMRA, a regulon for each miRNA cluster selected in step (ii) was constructed. As expected, in the constructed regulons, the miRNA targets tend to have a significantly higher Mutual Information with the miRNA in respect to the other non-target genes . The significant overlap between results obtained from the analysis of two independent datasets with ClustMMRA supports the high reproducibility across independent datasets of this approach. Notably, the results have an intersection with increasing statistical significance at each step of the pipeline. This trend confirms the accuracy of the proposed pipeline in selecting candidate clusters underlying cancer subtypes.In step (ii), 10 out of 28 (Curie) and 16 out of 47 (TCGA) subtype-specific miRNA clusters were found to have their predicted targets enriched in genes belonging to the corresponding gene signature. The output of step (ii) see  has an ienes see . The regSome results obtained with ClustMMRA in the breast cancer study have already been validated in the literature. MiR-493/136 and miR-379/656 clusters in the chromosomal region 14q32 have been reported as tumor suppressors in different types of human cancer , includiMultiple studies have identified miRNAs associated with tumor development and progression in most major cancer types . To testWe then tested the overlap between the output of ClustMMRA in CRC and in breast cancer. Approximately 50% of the clusters output of clustMMRA in breast are also present in CRC (four out of nine in TCGA and three out of seven in Curie) indicating that many of the identified clusters of miRNAs have a regulatory role across multiple cancer types.We compared the results of ClustMMRA in breast and colorectal cancer with those obtained by applying to the same datasets the MMRA pipeline for the identification of single master miRNA regulators. The goal is to investigate if the regulatory effect of a cluster can be detected by studying the effect of individual miRNAs belonging to the same cluster.We applied MMRA to the Curie and colorectal cancer (TCGA) datasets, using in each step the same thresholds employed for ClustMMRA. If at least two miRNAs of a given cluster are included in the output of MMRA, we consider this cluster as detected in the single-miRNA pipeline. Interestingly, four out of seven clusters detected by ClustMMRA  were not detected by MMRA in Curie. Concerning CRC, seven out of nine miRNA clusters detected by clustMMRA are lost in MMRA.This difference between the outputs of the two pipelines is given by the target enrichment analysis in step (ii) and the network analysis in step (iii). In fact, the four clusters missing in the final output of MMRA in Curie are included in the output of step (i), since they have at least two differentially expressed miRNA genes. They are filtered out in step (ii) since no miRNA gene in these clusters, when analyzed individually, reaches a significant enrichment of signatures genes in its targets for a certain subtype. This observation supports the hypothesis that co-clustered miRNAs participate in regulating the gene expression signature of a given cancer subtype without necessarily having a high overlap in terms of common target genes.regulons associated to each cluster, the nodes present in both TCGA and Curie datasets were kept, obtaining a network for each regulon with size of about 100 nodes. Then, biological processes and pathways associated to these regulons were identified through Fisher's exact enrichment test, using MSigDB . Invasive and mesenchymal state signatures confirm the association of these clusters to the TNBC subtype. Other general processes were found enriched in the regulons of these clusters: EMT (including the ‘HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION’ signature and multiple GO terms related to the extracellular matrix), stemness , cell cycle (‘IGLESIAS_E2F_TARGETS_UP’) and angiogenesis . Finally, the regulon of cluster miR-532/502 was found enriched in some breast cancer specific signatures clearly linking it to the TNBC subtype . Also, it was observed to be strongly associated to proliferation signatures .As summarized in Figure in silico results we considered miR-199a/miR-214 cluster, predicted to regulate TNBC according to clustMMRA. Considering the technical difficulty in producing the over-expression of multiple miRNAs in cell lines, we chose this cluster being the smallest of our output. Given that miR-199a/miR-214 is underexpressed in TNBC, its overexpression in a TNBC cell line should make the cell shift away from the TNBC subtype. We used as a measure to evaluate such a shift proliferation, in fact TNBCs are known to have a higher proliferation index than the other breast cancer subtypes . First, we examined by quantitative RT-PCR the expression of the individual mature miRNAs belonging to this cluster in T47D and MDA-MB-231 cells, which are luminal A and TNBC cells respectively .To test whether the deregulation of miR-199a/miR-214 cluster was sufficient to impact TNBC cells phenotype, MDA-MB-231 cells were treated with sense (S) oligonucleotides encoding for all the three miRNAs of the cluster  or scramble negative controls. We checked the overexpression of each miRNA of the cluster after transfection by RT-PCR analysis, shown in We then focused on the regulation of EMT predicted by clustMMRA . We thus performed an ECM assay in cells overexpressing miR-214, miR-199a-5p, miR-199a-3p alone or the cluster. This assay consists in the analysis of the effect of miRNA overexpression on ECM deposition by immunofluorescence of one component of the extracellular matrix . Some representative pictures for each sample are shown in Figure Over the last two decades there has been an explosion of research focused on miRNAs involvement in cancer initiation and progression, pointing out the potential of these small RNAs as biomarkers for diagnosis, prognosis and response to treatment. However, the majority of computational and experimental approaches for the identification of master miRNA regulators involved in cancer onset and subtyping are typically designed to detect the regulatory effect of a single miRNA. This can be a limitation in identifying regulation by multiple miRNA species acting cooperatively on cellular pathways and pathological changes.The computational pipeline here described, ClustMMRA, was specifically designed to search for genomically clustered miRNAs potentially driving cancer subtyping. ClustMMRA provides a computational framework to systematically investigate polycistronic miRNA transcripts involved in cancer subtyping or possibly in other biological contexts. In practice, the use of ClustMMRA can be generalized in order to study other classes of cooperatively acting miRNAs than the case of genomic clusters, such as co-expressed miRNAs from different genomic locations.In our study, ClustMMRA was applied to search for oncogenic / tumour suppressor polycistronic miRNAs driving breast cancer subtypes, pointing out five novel miRNA clusters whose regulatory effect is potentially associated to the triple negative subtype phenotype. Among them, the miR-199/miR-214 is identified as acting on EMT in TNBC subtype. Our computational and experimental validation of the regulatory effect of miR-199/miR-214 show that the down-regulation of this genomic cluster is associated to appearance of EMT-like phenotype in the TNBC cells. The upregulation of individual miRNAs belonging to the cluster or the entire cluster decreases the expression of a marker of mesenchymal phenotype (i.e. Slug) and increases the expression of epithelial markers (E-cadherin and beta-catenin). The effects on EMT obtained by the overexpression of miR-199/miR-214 cluster are further confirmed by its control on the deposition of the ECM proteins. Our results suggest that this cluster of miRNAs is possibly involved in the maintenance of more aggressive phenotypes of breast cancer, by regulating EMT target genes, and cell proliferation. Finally, our study supports the hypothesis of miRNA cooperativity from a polycistronic transcript as a possible mechanism of joint targeting to act on molecular pathways involved in cancer malignancy and subtyping. More accurate measurements and quantitative study might improve the understanding of these cooperative effects.Supplementary DataClick here for additional data file.


Volume-of-interest-based quantification of standardized-uptake-values was conducted in different parkinsonian syndrome-related target regions. PET results were subjected to multinomial logistic regression to create a prediction model discriminating among groups. Furthermore, we correlated tracer uptake with clinical findings. Elevated [18F]-THK5351 uptake in midbrain and diencephalon differentiated PSP patients from PD and MSA-C. MSA-C patients were distinguishable by high tracer uptake in the pons and cerebellar deep white matter when compared to PSP and PD patients, whereas MSA-P patients tended to show higher tracer uptake in the lentiform nucleus. A multinomial logistic regression classified 33/34 patients into the correct clinical diagnosis group. Tracer uptake in the pons, cerebellar deep white matter, and striatum was closely associated with the presence of cerebellar and parkinsonian symptoms of MSA patients. The current study demonstrates that combined MAO-B and tau binding of THK5351 facilitates differential diagnosis of parkinsonian syndromes. Furthermore, our data indicate a correlation of MSA phenotype with [18F]-THK5351 uptake in certain brain regions, illustrating their relevance for the emergence of clinical symptoms and underlining the potential of THK5351 PET as a biomarker that correlates with pathological changes as well as with disease stage.Neurodegenerative parkinsonian syndromes comprise a number of disorders that are characterized by similar clinical features but are separated on the basis of different pathologies, i.e., aggregates of α-synuclein or tau protein. Due to the overlap of signs and symptoms a precise differentiation is often difficult, especially early in the disease course. Enormous efforts have been taken to develop tau-selective PET imaging agents, but strong off-target binding to monoamine oxidase B (MAO-B) has been observed across first generation ligands. Nonetheless, astrogliosis-related MAO-B elevation is a common histopathological known feature of all parkinsonian syndromes and might be itself an interesting imaging target. Therefore, this study aimed to investigate the performance of [ Neurodegenerative parkinsonian syndromes comprise a number of disorders that are grouped together due to similar clinical features but are separated on the basis of different pathologies. Depending on the type of abnormal protein depositions, these disorders can histologically be divided into two groups: synucleinopathies and tauopathies . ParkinsEspecially in the early course of the disease, a precise differentiation of parkinsonian syndromes is often difficult based on clinical features alone and misdiagnosis is a frequent problem, with the most common misdiagnosis of PSP being PD and MSA . This is18F]-THK5351  has focused on the development of tau-selective imaging agents that allow regional quantification of tau burden. While molecular imaging of tau deposits by PET initially focused on Alzheimer’s disease, the most common tauopathy, an increasing number of post-mortem and human studies are being conducted in 4R-tau positive parkinsonian syndromes . However-THK5351 . Therefo disease . A signi18F]-THK5351 as a tool for differential diagnosis of neurodegenerative parkinsonian syndromes, i.e., PD, MSA, and PSP, in vivo. Furthermore, we sought to correlate individual regional tracer uptake with clinical phenotype and to compare PET results with magnetic resonance imaging.The aim of this study was to investigate the potential of combined MAO-B and tau binding of -THK5351 tracer retention in these brain areas may correlate to the amount of parkinsonian and cerebellar features.MSA patients can present with a combination of both parkinsonian and cerebellar features. As clinical phenotypes have been shown to be associated with predominant neurodegeneration and glial cytoplasmic inclusion pathology in the striatonigral and olivopontocerebellar system, respectively  we hypotThe unified MSA rating scale (UMSARS) is the most commonly used disease-specific rating instrument in MSA and was developed to rate functional impairment independent of the underlying motor disorder . To asse18F]-THK5351 was performed on a Raytest® SynChrom R&D single reactor synthesizer as reported previously  space. All individual PET images were spatially normalized to the previously established [18F]-THK5351 template using the PMOD FUSION tool . Global mean intensity scaling was used for image normalization.The PNEURO data processing pipeline of PMOD Version 3.5  was used for spatial normalization of all -THK5351 uptake in the midbrain and pons as predictor variables was still able to correctly classify 29/34 (85.3%) patients . A model using the MRI midbrain-to-pons area ratio as predictor, however, was able to accurately classify 17/26 (65.4%) patients .The multinomial logistic regression model was able to accurately classify 33/34 (97.1%) patients using the [18F]-THK5351 as a biomarker of disease stage and progression, a correlation analysis of tracer uptake and clinical parameters as well as with MRI atrophy was conducted. We observed a significant positive correlation of H&Y stage with [18F]-THK5351 uptake in the lentiform nucleus , the midbrain , and the diencephalon  and a negative correlation of the SEADL with [18F]-THK5351 uptake in the lentiform nucleus  and pons . There was no significant correlation of tracer uptake with disease duration.In order to assess the potential of [18F]-THK5351 tracer uptake and the amount of parkinsonian and cerebellar features measured by the UMSARS-P/C was performed. The UMSARS-P/C was positively correlated with tracer uptake in the pons  and cerebellar deep white matter  and negatively correlated with tracer uptake in the striatum   . Figure 18F]-THK5351 uptake in the pons  and cerebellar deep white matter  and a negative correlation with tracer uptake in the diencephalon  and midbrain .The MRI midbrain-to-pons area ratio showed a positive correlation with [18F]-THK5351 in the diencephalon, midbrain, pons, and cerebellar deep white matter are significant predictors of clinical diagnosis. Furthermore, our data indicate a strong association between region-specific tracer uptake and the amount of parkinsonian and cerebellar symptoms in MSA.We present the first study demonstrating the value of a combined MAO-B and tau radioligand for the differential diagnosis of neurodegenerative parkinsonian syndromes including primary tauopathies (PSP) and α-synucleinopathies . Tracer uptake levels of [18F]-AV1451 and [18F]-THK5351 have shown in PSP patients high tracer uptake in regions that are known from histological examination post mortem to be severely affected by tau pathology in PSP  and α-synucleinopathies (astrogliosis) can be assessed by a positive contrast. In particular a pure tau tracer would not be able to detect MSA or PD patients, whereas a pure α-synuclein ligand would blind to PSP. Second, the varying topology of neuropathology allows to discriminate clinically similar parkinsonian syndromes with different underlying proteinopathies. Third, astrogliosis has been shown to be an early biomarker in the time course of Alzheimer’s disease . In contrast, no significant group differences concerning tracer uptake could be detected in the striatum. In the lentiform nucleus, that is composed of putamen and globus pallidus, tracer uptake was highest in MSA-P patients, followed by MSA-C and PSP patients, but did not survive the significance threshold for multiple comparisons. This is probably due to the combination of elevated MAO-B levels and tau pathology in the putamen of PSP patients and the fact that MAO-B levels have been shown to be highly increased in MSA patients . Furthermore, tracer uptake in the cerebellar deep white matter of MSA-C patients was increased in comparison to PSP patients and showed a trend toward significance in comparison to PD (p = 0.010) and MSA-P (p = 0.077) patients. Both regions are known to be early foci of α-synuclein pathology in MSA-C , German Parkinson’s Disease Foundation (DPG), German PSP Association (PSP Gesellschaft), German Research Foundation (DFG) and the German Ministry of Education and Research (BMBF), International Parkinson’s Fonds (IPF); and has received institutional support from the German Center for Neurodegenerative Diseases (DZNE). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Left ventricular (LV) strain by speckle-tracking echocardiography (STE) is a comparatively new prognostic marker. Meta-analyses relating LV strain by STE to outcomes have been conducted in selected patient-based populations with established or suspected cardiovascular (CV) diseases. However, the evidence related to population-based studies of community-dwelling individuals is uncertain. The aim of this study is to provide a comprehensive systematic review and analysis of the current available literature regarding LV strain by STE as a predictor of adverse outcomes in population-based studies.Thesaurus and text-word searching will be used to search two online databases (MEDLINE and EMBASE) and additional sources will be identified from citation metrics and reference lists’ search. Dual search results’ screening, data extraction and quality assessment will be performed. Cohort studies of community/population-based samples who have had STE and followed up longitudinally for mortal and morbid events, and published in English and peer-reviewed journals will be included. Primary outcome will be all-cause mortality whereas secondary outcomes will be composite cardiac and CV end points. Risk of bias will be assessed using Newcastle-Ottawa Quality Assessment Scale of cohort studies that will be modified as appropriate. Any arising discrepancies will be discussed and resolved through consensus.Ethical approval is not required as this is a protocol for a systematic review. The findings of this study will be presented at scientific conferences and published in a peer-reviewed journal. Any amendments to the protocol will be documented and updated in the PROSPERO registry.CRD42018090302. This systematic review will evaluate the evidence related to the incremental prognostic value of left ventricular (LV) strain by speckle-tracking echocardiography (STE) in relation to mortality and cardiovascular events in community-dwelling individuals or population-based samples known to be at low risk relative to selected diseased populations.The results of this systematic review will add to the existing evidence on the utility of STE-based LV strain as a measure of cardiac function and risk, and influence its use in longitudinal population-based studies.Because of the anticipated heterogeneity in results/presentation of results and/or study design across a limited number of existing studies, there may be a limited scope for meta-analysis. When meta-analysis is possible, heterogeneity in associations between studies may be difficult to explain.Cardiovascular disease (CVD) is the leading cause of mortality worldwide and accounts for 31% of deaths.70, where ΔL=change in length and L0=original length.15LV strain imaging, tissue Doppler based and speckle-tracking based, has emerged as a powerful and sensitive tool allowing an accurate quantification of myocardial mechanics including longitudinal, circumferential and radial shortening/lengthening, and torsion.STE-based LV strain imaging has been shown to have an independent and additive prognostic value over conventional echocardiographic measures in a number of studies of diseased populations. These included patients with chronicPrevious systematic reviews and meta-analyses relating LV strain by STE to outcomes have been conducted in selected patient-based samples with establishedThis systematic review will be conducted and reported in adherence with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement39The inclusion criteria which relate to the type of study design, population and measurement procedure are summarised in 40The primary outcome will be all-cause mortality. Secondary outcomes will be (1) composite CV end points, including any combination of CV mortality, coronary heart disease (CHD) events , HF hospitalisation, new-onset atrial fibrillation (AF), life-threatening arrhythmia, recorded automatic implantable cardioverter defibrillator (AICD) shocks, stroke, transit ischaemic attack or peripheral arterial disease with arterial revascularisation procedure or (2) composite cardiac end point, including any combination of CV mortality, CHD events , HF hospitalisation, new-onset AF, life-threatening arrhythmia, recorded AICD shocks. Tertiary outcomes will be any individual secondary end point included in the composite cardiac or CV end point.The exclusion criteria will be as follows: (1) studies not meeting the inclusion criteria; (2) case–control studies, cross-sectional studies or randomised controlled trials which lack a placebo limb; (3) studies in unrepresentative samples of the general population  or patients with established/known CVD ; (4) LV strain measured by TDI or by another imaging modality ; (5) studies with end points that do not match those specified in this review  or (6) abstracts, reviews, conference proceedings or letters to the editor.This literature search will be performed systematically in the following online databases: MEDLINE (1946 to present) and EMBASE Classic + EMBASE (1947 to present) via OvidSP interface. Research has suggested that these databases are likely to be sufficient to identify relevant studies.10.1136/bmjopen-2018-023346.supp1Supplementary file 1Other sources will be identified by thoroughly searching the reference lists of all relevant articles. We will also search the citation metrics of those articles using Web of Science Core Collection for additional studies.Patients and the public were not involved in designing the protocol of this systematic review.The search results from each database will be retrieved and merged using reference manger software . Duplicates will be identified using the command ‘Find Duplicates’ and will be removed before screening is commenced. Titles and abstracts will then be screened for eligibility by two researchers working independently and irrelevant articles will be dismissed. Full texts of potentially eligible articles will be retrieved and double screened. A predefined eligibility form 10.1136/bmjopen-2018-023346.supp3Supplementary file 3This systematic review does not study a treatment comparison, and hence quality of evidence assessment based on strict GRADE guidelines  is not directly applicable. However, we will employ relevant components of a GRADE evaluation, namely quality of study, consistency and effect size in evaluating the strength of evidence; the issue of generalisability is dealt with through the inclusion/exclusion criteria.Descriptive summary of the characteristics and findings of the included studies will be provided in tables. We anticipate that there may be a limited scope for meta-analysis due to differences in the method of analysis and the reporting of results across studies. If the included studies are sufficiently homogeneous and report the same statistics , a quantitative synthesis using random effect meta-analysis will be used to pool the results of the association of interest. Based on our expectation, we will likely to use the HR and 95% CI as an effect estimate for presenting the results and will present the data graphically in forest plots.2 test47The degree of heterogeneity will be assessed using Higgins Thompson ISTE is increasingly and widely used in clinical practice. However, this technology suffers from inherent limitations, being influenced by technical as well as clinical factors.13 48This systematic review will identify and synthesise evidence regarding STE-derived measures as prognostic indicators of mortality and CV events in population-based/community-dwelling individuals. The restriction to studies not selected on the basis of disease or clinical status should minimise potential collider bias due to index case selection. The review will also potentially highlight known limitations of STE. This analysis will add to evidence on the utility of STE as a measure of cardiac function and risk and influence its use (or otherwise) in longitudinal population-based studies and community-dwelling samples.This protocol is for conducting a systematic review and hence no ethical approval is required. The findings of this review will be presented at scientific conferences and published in a peer-reviewed journal.


An overview is given of the SwissFEL soft X-ray free-electron laser (FEL) beamline, called Athos, and its numerous operation modes. In particular, several key hardware components are described, which enable these modes. The main FEL parameters and expected performance figures are also reported. The SwissFEL soft X-ray free-electron laser (FEL) beamline Athos will be ready for user operation in 2021. Its design includes a novel layout of alternating magnetic chicanes and short undulator segments. Together with the APPLE X architecture of undulators, the Athos branch can be operated in different modes producing FEL beams with unique characteristics ranging from attosecond pulse length to high-power modes. Further space has been reserved for upgrades including modulators and an external seeding laser for better timing control. All of these schemes rely on state-of-the-art technologies described in this overview. The optical transport line distributing the FEL beam to the experimental stations was designed with the whole range of beam parameters in mind. Currently two experimental stations, one for condensed matter and quantum materials research and a second one for atomic, molecular and optical physics, chemical sciences and ultrafast single-particle imaging, are being laid out such that they can profit from the unique soft X-ray pulses produced in the Athos branch in an optimal way. They can be part of catalytic systems to produce plastics or purify gases or synthesize fuels, ultrafast electronic switches and high-capacity magnetic storage media in information technology, or molecular complexes which govern cellular function and could cause hereditary disease. The cogs in such functional molecules and materials are the valence electrons, which, due to their electric charge, light mass and moderate binding energy, determine the physical, chemical and biological properties of matter. With soft X-ray spectroscopy, however, one can use well-defined resonant atomic transitions to specifically address particular electron orbitals. For this reason, a variety of soft X-ray spectroscopy methods are now ‘bread and butter’ for users of many synchrotrons in operation worldwide. In order to study and quantify the functionality of matter, it is necessary to follow the dynamics of valence electrons on their natural time scale. Only a soft X-ray free-electron laser (FEL), such as Athos at SwissFEL, offers the combination of peak intensity, wavelength tunability and femtosecond pulse length required to perform dynamic soft X-ray spectroscopy.et al., 2012et al., 2017et al., 2017etc.). A new undulator geometry, APPLE X  macropulse, are accelerated up to a fast kicker, which will deflect one bunch into the Athos dogleg at an energy of 3.15 GeV Fig. 1 while thThe optical layout will ultimately serve three experimental stations, but only the atomic, molecular and optical station (AMO) and the condensed matter station (CM or Furka) are currently under design. The commissioning of the undulator line should be completed in 2020 with user operation to follow in 2021.2.et al., 2017K value without shifting the magnet arrays longitudinally, that is to say without changing polarization. When the four magnet arrays are positioned in an asymmetric way with different left and right gaps, then a transverse field gradient can be generated for all polarizations: the so-called transverse-gradient undulator (TGU) configuration  operation to offer novel modes of operation to the users  The operation of the APPLE X undulator with a chicanes between all undulator modules.(ii) The utilization of small delaying external laser before the injection into the main undulator beamline.(iii) The manipulation of the electron beam with an monochromator (not in the baseline design).(iv) The spectral filtering of the SASE signal in between the FEL amplification process by means of a high-resolving The interplay of these modes gives unprecedented control of the spectral coherence, pulse length and radiation power independently. The schematic view in Fig. 22.1.In the TGU configuration the undulator field strength has a linear dependence on transverse position. The strength and direction of the gradient can be controlled with the position of the four magnet arrays. For Athos, this unique undulator field property allows for two modes. The first is a large-bandwidth mode, where a spatially tilted beam is injected into the undulator lattice without any external focusing  since a step-wise taper per undulator segment is not efficient enough.2.2.et al., 1978et al., 2013The most important difference of Athos in comparison with other FEL beamlines is the inclusion of small delaying chicanes between each undulator segment. One of its key benefits is not directly visible to the user, namely the reduction of the saturation length by a distributed optical klystron  , the beam is delayed and realigned with the undulator axis so that a new fresh slice will continue the FEL amplification. While this principle has been demonstrated at LCLS .The mode-locked lasing   and a linear tapering (tapering within the undulator segment thanks to the TGU configuration) opens the door to ‘slicing’ techniques   Yu, 1991 is not set al., 2014All laser manipulations affecting only a subsection of the bunch offer the additional benefit of an FEL signal that is passively locked to an optical laser. If both signals can be transported in parallel down to the user station, the jitter of pump–probe experiments can be reduced significantly. This has been demonstrated at FERMI with relative fluctuations of 6–9 fs  to install a monochromator as was done at LCLS , three compensating dipoles and a septum magnet, shown in Fig. 3The two kicker magnets (K1 and K2) separate the bunches vertically by ‘kicking’ one bunch up and the other one down. Compensating dipoles  bring the down-kicked bunch back to the straight-through machine axis and deflect further up the up-kicked one. The septum is positioned 8 m downstream of the separation point. The deflected bunch enters the septum dipole field with 10 mm separation from the straight-through bunch and is deflected 35 mrad towards the Athos beamline; the lower beam passes through the zero-field region of the septum to continue straight on to the Aramis beamline  is established in the kicker, electron bunches can be deflected alternately up and down. The kickers’ resonators, coaxial cables and electronics require stable water cooling.Resonant deflection is used to give moderate field strength but very stable operation. An RF driver excites a sine-wave current in the high-3.1.2.The DC Lambertson septum magnet , but they are two to three times less sensitive to temperature fluctuations. In addition, the permeability of the axial magnet is closer to unity, thus reducing non-linearity (hysteresis) effects when shifting the magnet arrays. Radial magnets have been magnetized under an inhomogeneous field such that the field is larger near the apex of the magnet while reducing the overall magnetic load. The magnetic field measurements of the individual magnet pairs  confirmed that they meet the quality required for undulators.The individual magnet geometry follows from the symmetry of the APPLE X topology. The undulator period is 38 mm with samarium cobalt (SmCo) magnets. Two different grades of SmCo materials are used: SmFour periods (16 magnets) are positioned in a block keeper as shown in Fig. 7−8 mbar is expected with ion getter pumps situated in the inter-undulator sections. The assembly of the prototype undulator UE38 was completed in 2018.When the gap and slit are at minimum, an aperture of only 6.5 mm is left open for the vacuum chamber. For this reason, a round copper vacuum chamber with 5 mm inner diameter and a wall thickness of only 0.2 mm was chosen. A vacuum level of around 103.4.et al., 2014et al., 2006et al., 2001et al., 2015et al., 2015et al., 2013et al., 2018Electron beam diagnostics based on a transverse deflection structure (TDS) placed downstream of the undulators (post-undulator TDS) in conjunction with an electron beam energy spectrometer can indirectly measure the pulse duration of ultrashort FEL pulses by analysing the induced energy spread on the electron bunch due to the FEL process , thus utilizing reflective optics only.The Athos beamline distributes the FEL radiation to three independent experimental stations with sufficient space in-between. A first horizontally deflecting mirror is located in the optics hutch at 54 m from the end of the undulator. It separates the FEL radiation from the After the grating chamber the beamline splits into three branches by introducing horizontally deflecting mirrors for Athos 1 and Athos 3 while Athos 2 is operated without such a mirror. Each branch has a separate exit slit and a dedicated Kirkpatrick–Baez (KB) mirror chamber for refocusing the beam into the experimental stations  and one for atomic, molecular and optical sciences (AMO), will be ready for the first experiments in 2021, while the third station (Athos 1 branch) is not yet defined and not yet financed either.The Athos soft X-ray experimental area, shown in Fig. 115.1.The AMO experimental station at the Athos 2 branch will be designed as a highly versatile tool for AMO physics, chemical sciences, soft X-ray imaging, and novel approaches in non-linear X-ray spectroscopy. The X-ray pulses from the Athos undulator will be delivered with the minimum required three bounces from a single offset mirror and two KB mirrors. This optical layout will minimize transport losses and preserve the pulse wavefront. A short focal length of 1.5 m from the downstream KB mirror is chosen to achieve a micrometre-sized focus and therefore sufficient fluence for multi-photon excitations of the targets while maintaining enough space for laser incoupling elements and differential pumping between the last optical element and the experimental station.A specific characteristic of the Athos AMO station will be the combination of an X-ray FEL with an infrared laser-driven attosecond high-harmonic generation (HHG) source. The combination of the two sources with their specific strengths will provide new tools for unravelling and controlling ultrafast chemical dynamics in gases, clusters and liquids from an entirely new point of view. In particular, electronic and nuclear dynamics could be measured in real time in isolated gas-phase molecules and clusters, solvated molecules, transition-metal complexes and nanoparticles in their natural environment. The femtosecond to sub-femtosecond intense X-ray pulses from the Athos undulator and the HHG source will be optimally exploited in combination with the element specificity of soft X-ray spectroscopy and the nanoscale resolution of X-ray imaging.e.g. the K shells of carbon (290 eV), nitrogen (410 eV), oxygen (540 eV) and fluorine (700 eV), but also the 3d transition-metal L shells of all elements between titanium (460 eV) and copper (950 eV). The transient absorption setup will be able to probe the X-ray-induced dynamics over the complete spectral range of the HHG supercontinuum in a single shot. Further, a variety of spectrometer and detector options, capable of detecting all reaction products, will be implemented, i.e. full 3D momenta of ions, high-resolution electron spectra, X-ray diffraction images and transient absorption spectra. Generally speaking, electron imaging spectrometers perform best for lower electron kinetic energies and are therefore not ideally suited for all X-ray energies. Therefore, we will complement the imaging spectrometers with a hemispherical analyser for electron detection. Another unique capability of the proposed experimental station will be the Mönch photon area detector, currently under development at the Paul Scherrer Institute, with dynamic gain switching. We aim at megapixel detectors but the exact pixel number and geometry will be the result of an optimization process of the Athos station, currently ongoing. A final and equally important aspect of the proposed station is sample handling and delivery. As sample delivery options at the AMO station we will provide supersonic jet sources for atomic, molecular and cluster beams. In addition, we aim at incorporating a flat liquid jet and an aerosol injector. The flat liquid jet will enable frontier experiments in chemical and biochemical research in the liquid phase, which is a particularly attractive combination with the X-ray-based transient absorption setup. The aerosol injector will allow us to extend the ultrafast imaging capabilities to a wide spectrum of chemical and biological systems.Fig. 12In summary, the highly versatile AMO experimental station at SwissFEL will include a variety of particle and photon spectrometers, imaging capabilities and sample delivery systems. This station, in combination with ultrafast optical laser driven sources and the unique pulse characteristics from the Athos undulator, will enable new approaches in ultrafast and non-linear X-ray sciences.5.2.et al., 2017The experimental station for condensed matter and quantum materials at the Athos beamline, named Furka, will be dedicated to time-resolved resonant inelastic and elastic X-ray scattering (tr-RIXS and tr-REXS) as well as soft X-ray diffraction (tr-SXD) to study ultrafast dynamics in correlated materials and, more generally, in quantum matter. Fig. 13In the future, a second chamber is foreseen with the aim of investigating non-linear optical effects on solid materials as well as imaging techniques. Possible extensions to time-resolved X-ray magnetic circular dichroism (tr-XMCD) could be explored as well by taking advantage of the circular polarized radiation provided by Athos in combination with an externally applied magnetic field.


Rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) disproportionately affect women during and following childbearing years. Antihuman leucocyte antigen (HLA) alloantibody responses are common in healthy parous women, and as these diseases are both linked with HLA and immune dysregulation, we sought to evaluate anti-HLA antibodies in RA and SLE.Anti-HLA antibodies were measured among parous SLE cases (n=224), parous RA cases (n=202) and healthy parous controls (n=239) and compared with each other as well as with nulliparous female and male controls. Antibody specificities were identified and compared against subject HLA types to determine autoreactivity versus alloreactivity. The association of anti-HLA antibodies with clinical outcomes was evaluated.Levels and frequencies of anti-HLA antibodies were significantly higher among parous females with SLE (52%) or RA (46%) compared with controls (26%), and anti-HLA antibodies were also found among nulliparous females and males with SLE and RA. Autoreactive anti-HLA antibodies were observed among SLE and RA antibody-positive subjects, but not healthy controls, with the highest frequency of autoreactive anti-HLA antibodies found in the SLE subjects. Higher levels of anti-HLA antibodies were associated with nephritis among the nulliparous SLE cases (p<0.01). The presence of anti-class I HLA antibodies was associated with younger age at diagnosis among both the RA and SLE nulliparous cases.Both autoreactive and alloreactive anti-HLA antibodies were found at high levels in RA and SLE subjects. These occurred even in the absence of alloexposure, particularly among SLE subjects and may be linked with disease severity. Human leucocyte antigens (HLA) are potent alloantigens, and anti-HLA antibody responses are common following alloexposures such as solid organ transplant, transfusion or pregnancy. In transplant or transfusion recipients, host-generated alloantibodies can contribute to transplant rejection or refractoriness to platelet transfusion.For pregnant women, these alloantibodies are generally considered benign, though some studies have shown associations with recurrent miscarriage and preterm labour.Specific HLA alleles have been associated with several autoimmune diseases, including rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). DRB1*0301 and DRB1*1501 are associated with increased risk of SLE, with the former specifically linked to anti-La and anti-Ro antibody positive cases and the latter to anti-Sm antibodies.Pregnancy can also have important implications for both SLE and RA. Women are at much higher risk of developing these diseases, particularly during and following their childbearing years, with peak incidence of SLE among young women and incidence of RA peaking later in life.Given the importance of the HLA region to SLE and RA, the prevalence of these diseases among women, and the immune dysregulation associated with SLE and RA, we evaluated the frequency of anti-HLA antibodies among parous patients with RA and SLE compared with healthy controls with similar alloexposures. We also identified the specificities of these anti-HLA antibodies to determine whether any of them were autoreactive and looked for associations of these anti-HLA antibodies with clinical outcomes. We hypothesised that higher frequencies of HLA antibodies would be found among parous women with SLE and RA than healthy parous controls and that these antibodies would be linked with more severe clinical outcomes.Parous female SLE and RA cases were drawn from the University of California San Francisco (UCSF) Mother-Child Immunogenetic Study.Overall, antibodies against class I and class II HLA antigens were measured using the One Lambda LabScreen Mixed Luminex assay , a bead-based multiplexing system. Serum or plasma samples were screened according to the manufacturer’s instructions. This kit uses multiantigen coated beads and reports results as a normalised to background (NBG) ratio for each bead. We used the highest reported value for each sample. Cut-off values for positive antibody results were set as previously described to be three SD above the mean value measured in a large panel of unexposed healthy subjects, with a NBG ratio greater than 10.8 for class I or 6.9 for class II considered antibody positive.Antibody specificities were evaluated using the One Lambda LabScreen Single Antigen HLA class I and class II assays and analysed using HLA Fusion V.3.0 (One Lambda).2<0.3) were excluded. Previous work comparing typed and imputed DRB1 demonstrated that individually, ≥98% of alleles were accurately called. To validate the current imputation data, imputed DRB1 types were compared against direct genotyping results for 47 of the parous female SLE and RA subjects  who were screened for antibody specificity. An exact match at the genotype level was found for 93%, with the remaining 7% remaining accurate at the one-field level of resolution. None of the errors in the imputed DRB1 types resulted in any changes to the auto-determinations versus allo-determinations for typed antibodies. Specificities of anti-HLA antibodies were compared against imputed subject HLA types to identify any autoreactive specificities. Individuals were considered positive for autoreactive anti-HLA antibodies if one or more of their specificities were against their own class I or class II HLA alleles. Three increasingly stringent cut-offs were evaluated for the antibody specificity assay with X2 the least stringent and X6 the most stringent. The manufacturer established these proprietary cut-offs, so we do not have specific details on how they are calculated. As we did not have DPA1 types on most subjects, antibody specificities against potential DP autoantigens were not included in this analysis.Subject HLA types were imputed at two-field resolution as previously described.2 test. Comparisons of mean values between two groups were done by unpaired t-test. Differences were reported as significant if p<0.05.Prism  was used for all statistical analysis. Comparisons of antibody levels between multiple groups were made with one-way analysis of variance (ANOVA) using Dunnett’s post-test to compare each case group to healthy controls. Comparisons of frequencies were made by χAnti-HLA antibodies can be found in many healthy females as the result of previous pregnancies, but only rarely in males or in nulliparous females.10.1136/lupus-2018-000278.supp1Supplementary dataThe levels  and the We next evaluated the specificities of these anti-HLA antibodies to determine if any of them were specific for autoantigens. For the parous females, all healthy subjects positive for antibodies were evaluated, while a subset of 25 class I positive and 25 class II positive subjects were selected from among the SLE subjects and from among the RA subjects. Levels of anti-HLA antibodies in selected samples were compared against those from the full pool of positive samples to confirm a representative distribution of antibody levels . Three d10.1136/lupus-2018-000278.supp2Supplementary dataFinally, we looked to see if the presence of these anti-HLA antibodies was linked with any clinical outcomes. For the SLE cases, we looked at dsDNA antibodies, abnormal  immunoglobulin (Ig)G levels, abnormal IgM levels, age at diagnosis and lupus nephritis. For the RA subjects, we evaluated age at diagnosis, cyclic citrullinated peptide antibodies, extra-articular manifestations, rheumatoid factor and erosions.For the parous female SLE cases, the number of class II antibody epitopes was significantly higher among those with dsDNA antibodies, and the level of class I antibodies was associated with abnormal IgG levels. These differences were modest and did not appear in the nulliparous group, but they were consistent with the hypothesis that anti-HLA antibodies are more likely to occur alongside other abnormal antibody responses (data not shown).For the nulliparous SLE cases, higher levels of both class I and class II HLA antibodies were associated with lupus nephritis . This waEarlier age of diagnosis was associated with the presence of anti-class I HLA antibodies in both the nulliparous SLE  and nullIn this study, we have demonstrated a doubling of the frequency of anti-HLA antibodies among parous women with SLE or RA compared with healthy controls with similar numbers of pregnancies. In addition, anti-HLA antibodies were found in many SLE and some RA subjects without any known alloexposures. While most antibody specificities targeted allogeneic HLA, a significant portion of the SLE and RA subjects carried one or more antibodies against autologous HLA. Increased levels of anti-HLA antibodies were associated with nephritis among nulliparous SLE subjects, and anti-class I HLA antibodies were associated with earlier age of diagnosis for nulliparous SLE and RA subjects.The higher levels and frequency of antibodies found among parous SLE and RA subjects compared with the healthy parous controls is consistent with the overly sensitive immune system associated with these diseases. This is also reflected in the diversity of antibody specificities, particularly for anti-class II antibody responses, which were far more diverse in the RA and SLE cases compared with controls . In addiWhile the majority of anti-HLA antibodies were targeting allogeneic HLA antigens, many of the SLE subjects and some of the RA subjects carried one or more antibodies targeting autologous HLA . The speWhile both the SLE and RA cases responded similarly to alloexposure via pregnancy, they differed significantly in the absence of allogeneic exposure. Some anti-HLA antibodies were detected in the nulliparous female and male RA cases, but levels were only significantly higher than controls among nulliparous females, and only for anti-class I antibodies. It is possible that these higher levels of HLA antibodies in nulliparous RA females compared with RA males could be the result of undocumented early miscarriage, but may also represent some other sex effect. In contrast, the nulliparous female and male SLE groups had significantly higher antibodies detected as compared with controls for both class I and class II, and for class II, the frequencies were not significantly different from the alloexposed (parous) SLE group . FurtherThe anti-class I and anti-class II antibody responses also differed significantly, particularly among the SLE cases. There were far more antibodies generated against class II antigens for SLE, even in the absence of alloexposure. Furthermore, the frequency of autoreactive antibodies against class II was much higher than those targeting class I, and this is in spite of our inability to include the DP loci in this analysis due to incomplete typing. One possibility is that tolerance mechanisms protecting against anti-class I responses may be more robust than those for class II. This does not seem unreasonable given the ubiquitous expression pattern of class I HLA compared with the more restricted expression of class II. Interestingly, the link seen between age of diagnosis and anti-HLA antibodies was only seen for class I antibodies, even though this association was observed in both the SLE and RA nulliparous groups. This may suggest that the appearance of anti-class I HLA antibodies in the absence of alloexposure may be linked with an increased genetic burden, as this is generally associated with an earlier age of diagnosis.It is difficult to assess if these anti-HLA antibodies are contributing to disease or simply an indicator of an imbalanced immune system. We did see a correlation between nephritis and both anti-class I and anti-class II antibodies for the nulliparous SLE cases but were unable to establish a causal relationship. As with age of diagnosis, differences were not observed in the parous female group, again, presumably due to the higher levels of antibodies seen following pregnancy. In addition, both SLE and anti-HLA antibodies have been linked to increased risk of pregnancy loss or preterm labour.In summary, we believe that this study is the first to show an increase in antibodies against HLA associated with SLE and RA. We have found that these antibodies occur more frequently and at higher levels in parous SLE and RA women compared with parous controls with similar exposures. Furthermore, these antibodies occur in the absence of allogeneic exposure, particularly in SLE. While the majority of the antibodies generated are against non-self antigens, we found many of the SLE and some of the RA subjects tested positive for autoreactive anti-HLA antibodies. Further work will be required to determine what role these antibodies play in disease pathogenesis.