Randomized clinical trials (RCTs) and real-world data (RWD) in patients with atrial fibrillation have shown that—compared to vitamin K antagonists (VKAs)—non-VKA oral anticoagulants (NOACs) are at least as effective in the prevention of ischaemic stroke, while decreasing the risk of bleeding.We aim to evaluate the cost-effectiveness of the NOAC apixaban versus other NOACs  and VKA, for stroke prevention in patients with atrial fibrillation by including the available data both from RCT and real-world analyses of all NOACs into one integrative previously published model.The model was updated to the current Dutch healthcare situation. The incremental cost-effectiveness ratio was calculated using either efficacy/effectiveness and safety data derived from a network meta-analysis (NMA) synthesizing NOAC RCTs or RWD. We conducted a systematic literature search to identify eligible publication to best inform the RWD-based analysis. Additional sensitivity and scenario analyses were conducted to test the robustness of the outcomes.In the NMA-based analysis, apixaban appeared to be cost-effective compared to VKA  and dominant (cost-saving and more effective) over dabigatran 110 mg, dabigatran 150 mg, edoxaban and rivaroxaban. In the RWD-based analysis, apixaban was dominant over all other anticoagulants. In the scenario analysis apixaban appeared to be not cost-effective compared to dabigatran 150 mg, when using equal event-unrelated treatment discontinuation rates for each drug. In all other scenarios apixaban is cost-effective or cost-saving compared to VKA and other NOACs.Based on RCTs as well as RWD, we conclude that apixaban is generally cost-effective or even cost-saving (less costly and more effective) compared to VKA and other NOACs in the overall population of patients with atrial fibrillation. Atrial fibrillation (AF) is the most common cardiac arrhythmia and is a major cause of ischemic stroke, heart failure and cardiovascular morbidity . In the AF constitutes a significant public health problem, and estimates suggest that this condition accounts for 1.3% of the national healthcare budget in the Netherlands with a total estimated cost of €583 million in 2009 . In patiIn patients with AF, anticoagulant (AC) therapy with vitamin K antagonists (VKAs) has been the most effective treatment for the prevention of stroke over the last decades. However, on the basis of randomized clinical trial (RCT) results, the European guidelines have adopted the non-vitamin K antagonist oral anticoagulants (NOACs) as the preferred treatment for the prevention of stroke in patients with AF since 2012 . A netwoIn terms of effectiveness, safety and costs it is relevant to investigate whether the results of the cost-effectiveness using RWD support results of analyses based on RCTs . Multiple cost-effectiveness analyses have been published on the different NOACs versus VKAs on the basis of the respective RCTs results, using different models and cost assumptions. Here, we aim to evaluate the cost-effectiveness of the NOAC apixaban versus other NOACs and VKAs for stroke prevention in patients with AF by including the available data both from NMA and real-world analyses of all available NOACs in one integrative previously published model .We aim to evaluate the cost-effectiveness of apixaban compared to VKA, dabigatran, rivaroxaban and edoxaban, by capturing all costs and health outcomes related to the disease, treatment and complications, during the lifetime of a patient with AF. The primary outcome of our analysis is the incremental cost-effectiveness ratio (ICER), calculated by dividing the incremental costs by the incremental health outcomes, presented in life-years LY and quality adjusted life-years (QALY). The ICER is compared to a willingness-to-pay (WTP) threshold of €20,000/QALY. This threshold is based on the disease burden calculation  in the calculation tool as recommended by the Dutch guidelines for pharmacoeconomic research . The ICEWe conducted a systematic literature search to identify eligible publication to best inform the RWD-based analysis. The systematic literature search was conducted with help of the ‘Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement’, to identify RWD studies eligible for the RWD-based analysis . DetailsBoth the NMA-based and the RWD-based analyses were based on a previously published Markov model . The modAll patients entered the model in the ‘AF’ state. Upon the occurrence of a clinical event the patient remained in the corresponding health state for one cycle and then moved to one of the transient or absorbing states. ‘AF’ and ‘AF without AC therapy’ (red squares) reflect transient states, meaning that patients were able to remain in them or move to other states in the model. In absorbing states (e.g. ‘death’ [blue squares]) patients remained until death. Upon the occurrence of ‘event unrelated AC discontinuation’ patients were assumed to discontinue AC therapy permanently.2 score and CHA2DS2-VASc score were 1.7 and 3.1, respectively. In the RWD-based analysis, the patient characteristics were based on the selected real-world study itself. The patients were on average 74.3 years old, 54.1% were male and the average CHA2DS2-VASc score was 3.7.In the NMA-based and RWD-based analyses, all patients entered the model with baseline characteristics as presented in 2 score distribution in Dutch patients  in the ARISTOTLE trial . Hazard patients  with thepatients . Subsequpatients  18]. ‘I. ‘I2 scoThe transition probabilities used in the RWD-based analysis are summarized in In sensitivity analysis, transition probabilities, hazard ratios and distributions were varied with a beta, log normal and Dirichlet distributions, respectively.Mortality rates are summarized in Treatment discontinuation could be related or unrelated to an event. Upon the occurrence of non-fatal other ICH, patients were assumed to discontinue AC therapy for six weeks after which they resumed their original AC . PatientIn case of ‘event unrelated AC discontinuation’, all event risks  were updated to no treatment. These event rates for patients receiving no treatment, summarized in In absence of health-state-specific EQ-5D Dutch utilities, health-state-specific utility scores were obtained from a UK EQ-5D catalogue by Sullivan et al.  Table 1Table 1, Direct and indirect costs in- and outside the healthcare sector were considered, since the ICERs were calculated from a societal perspective, as recommended by the Dutch cost manual for pharmacoeconomic evaluation . All cosEvent costs per health state were divided into acute and long-term maintenance costs. Acute costs were related to hospital and rehabilitation facility costs and maintenance costs reflected medical costs during the patient’s lifetime . Event cIndirect costs included travel expenses and event related informal care costs. Travel costs were included for 45% of the INR monitoring visits (patients who visited thrombosis service themselves) and for each routine care or dyspepsia related GP visits . These cAll costs were inflated to 2018 using the consumer price index from Dutch Statistics (CBS Statline) . Cost ouNext to base case analyses based on the NMA and RWD, sensitivity analyses were conducted to evaluate the influence of uncertainty in input parameters on the ICER. In the probabilistic sensitivity analysis (PSA) costs, utilities, transition probabilities were varied simultaneously over their 95% CIs and ICERs were calculated during 2,000 simulations. Outcomes were presented in cost-effectiveness planes and cost-effectiveness acceptability curves. In the univariate sensitivity analysis input parameters were varied one by one over their 95% CI, to examine the influence on the ICER for each parameter separately. The input parameters with the highest impact on the ICER were presented in a Tornado diagram. Three additional scenario analyses were conducted to capture the effect of healthcare payer’s perspective (scenario 1), equal drug prices for all NOACs (scenario 2) and equal event unrelated AC discontinuation rates for all NOACs (scenario 3).The deterministic results of the NMA-based and RWD-based analyses are summarized in The results of the PSA are plotted in cost-effectiveness planes, shown in Additionally, we conducted a univariate sensitivity analysis. We conducted three additional scenarios to assess the influence of the healthcare payer’s perspective (scenario 1), equal drug costs among NOACs (scenario 2) and equal rates of event unrelated AC discontinuation among NOACs and VKAs (scenario 3). Scenario 1 showed that compared to societal perspective the incremental costs of apixaban versus VKA and edoxaban in the NMA-based analysis increase, resulting in an ICER of €5,787/QALY and €206/QALY.In RWD-based analysis, apixaban is cost-effective compared to VKA (€292/QALY), and cost-saving (dominant) compared to dabigatran and rivaroxaban. In scenario 2, equal drug costs for NOACs resulted in an ICER of €2,884/QALY for apixaban versus edoxaban in the NMA-based analysis. All other ICERs are dominant.In the third scenario of the NMA-based analysis, equal event unrelated AC discontinuation rates for NOACs and VKAs resulted in an ICER of €244,079/QALY for apixaban versus dabigatran 150 mg, which is not considered cost-effective at the WTP threshold of €20,000/QALY. In NMA-based analysis, apixaban appeared to be dominant over dabigatran 110 mg and rivaroxaban and cost-effective compared to VKA  and edoxaban . In RWD-based analysis apixaban was still dominant over VKA, dabigatran and rivaroxaban. Detailed results of the three scenarios are presented in To our knowledge, this is the first cost-effectiveness study comparing different NOACs and VKA with RCT data as well as RWD in patients with AF. The RWD-based analysis was specifically implemented to externally validate conclusions found in the NMA-based analysis based on RCT data. In the NMA-based analysis, apixaban appeared to be cost-effective compared to VKA and cost-saving compared to dabigatran 110 mg, dabigatran 150 mg, rivaroxaban and edoxaban. The RWD-based analysis showed that apixaban is cost-saving compared to all comparators. Apixaban was shown, in both analyses, to be the most cost-effective treatment option at a WTP threshold of €20,000/QALY . In the scenario analysis apixaban appeared to be not cost-effective compared to dabigatran 150 mg, when using equal event-unrelated treatment discontinuation rates for each drug. In all other scenarios apixaban is cost-effective or cost-saving compared to VKA and other NOACs.A previously published cost-effectiveness analysis, which was based on the same model as our analysis, was conducted in the UK setting . They foIn a follow-up publication , it was A Dutch study that compared the cost-effectiveness of apixaban, rivaroxaban and dabigatran to VKA  found thEvent unrelated AC discontinuation rates differ among the anticoagulants . To showNotably, costs and QALYs are highly driven by the patients who discontinue AC therapy. Treatment discontinuation in patients with AF is very undesirable, because from that moment on they are not protected for stroke or other thromboembolic events. In practice, a certain percentage of the patients who discontinue their initial AC therapy, will be encouraged to switch to another AC. However, it is hard to get supportive data on the percentage of patients fully discontinuing AC therapy, as well as reliable data on what that exactly means for stroke and bleeding risks. By emphasizing the importance of AC therapy and discussing the reason for the AC discontinuation the patient can be encouraged to restart or switch AC therapy.The major advantage of this study is that both an NMA and RWD were used for cost-effectiveness. For the RWD-based analysis we used the publication of Lip et al. that best met the inclusion criteria for the systematic literature search underlying the NMA . Differe2/CHA2DS2-VASc scores and age, can obviously differ among populations. Obviously due to the fact that RWD studies include heterogeneous populations from different countries, differences in clinical outcomes can be expected, which potentially also leads to differences in economic outcomes. Therefore, the use of another RWD study might lead to different outcomes. Also, outcomes of NMAs can vary when making use of different combinations of clinical trials, differences in methodology and the differences in definitions of the outcomes [Unfortunately, we were unable to include RWD on Dutch patients. Throughout the next years, a recently launched project “DUTCH-AF registry” aims to include 6,000 Dutch patients with AF to collect and assess data on effectiveness, adherence and bleeding risk of NOACs and VKAs . It is ioutcomes , again pNotably, we included low-dose dabigatran (110 mg) in our analysis. We included this dose since it was officially approved by European Medicines Agency , althougBased on RCT-data as well as RWD, we conclude that generally apixaban is cost-effective or even cost-saving (less costly and more effective) compared to VKA and other NOACs in the overall population of AF-patients. It should be mentioned that, especially compared to other NOACs, only marginal differences in costs and health effects were found and the use of other effectiveness and safety data sources or assumptions, considerations of specific patient populations, costs might lead to different outcomes.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S1 TableAbbreviations: CHADS2 score, congestive heart failure, hypertension, age ≥75 years, diabetes mellitus, prior stroke or transient ischemic attack or thromboembolism; CHA2DS2-VASc score, congestive heart failure, hypertension, age ≥75 years (2), diabetes mellitus, prior stroke or transient ischemic attack or thromboembolism (2) and vascular disease .(DOCX)Click here for additional data file.S2 Tablea The event rates of ischaemic stroke, ICH and other MB were adjusted by CHADS2-scores with the distribution presented in (DOCX)Click here for additional data file.S3 TableaHazard ratio apixaban versus comparator = (1/HR comparator versus apixaban).Abbreviations: CI, confidence interval; HR, hazard ratio; ICH, intracranial haemorrhage; MB, major bleeding, PY, patient-years; SE, systemic embolism; VKA, vitamin K antagonist. (DOCX)Click here for additional data file.S4 Tablea Lambda and Gamma are the natural logarithms of the slope of the survival hazard and age, respectively, which can be used to calculate the predicted survival at any time per age group (0–75 or >75 years) and gender. b Assumed to be equal to mortality risk adjustment factor of AF, since these events we assumed to have no additional effect on mortality risk in the period after the event. Abbreviations: AF, atrial fibrillation; CI, confidence interval; CRNMB, clinically relevant non-major bleeding; HR, hazard ratio; ICH, intracranial haemorrhage; MB, major bleeding; MI, myocardial infarction; SE, systemic embolism.(DOCX)Click here for additional data file.S5 Tablea intracranial haemorrhage including haemorrhagic stroke. Abbreviations: CI, confidence interval; CRNMB, clinically relevant non-major bleeding; CV, cardiovascular; ICH, intracranial haemorrhage; MB, major bleeding; MI, myocardial infarction; PY, patient-years; SE, systemic embolism.(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


Since use of diffusion-weighted imaging (DWI) positivity in the “tissue-based” definition of stroke in patients with a clinical TIA is supported by the high associated 90-day risk of recurrent stroke, we aimed to determine long-term prognostic significance, stratified by etiologic subtype, and whether the same tissue-based distinction is predictive in minor strokes.Consecutive eligible patients with TIA or minor stroke  in the population-based Oxford Vascular Study underwent brain MRI at baseline. Stroke risk on 10-year follow-up was stratified by NIHSS (0/1 vs 2/3) and Trial of Org 10172 in Acute Stroke Treatment classification of the initial event.p = 0.009) or a stroke with NIHSS 0–1 , but not after a stroke with NIHSS 2–3 . Ischemic stroke risk after DWI-positive TIA was at least equivalent to that after DWI-negative stroke . Among all patients, DWI positivity was most predictive of 10-year risk after cryptogenic events .Among 1,033 patients , 248 (24.0%) had acute lesions on DWI . A positive DWI was associated with an increased 10-year risk of recurrent ischemic stroke after an index TIA (hazard ratio [HR] 2.66, 95% confidence interval [CI] 1.28–5.54, DWI positivity is associated with an increased long-term risk of recurrent stroke after TIA and minor stroke, supporting a tissue-based definition of minor stroke as well as TIA. Prognostic value is greatest after cryptogenic events. Second, although DWI-negative minor stroke is widely recognized,17 the prognostic implications remain uncertain.11 Whether patients and physicians should be reassured by normal imaging even in the presence of focal symptoms and signs lasting longer than 24 hours is unclear. Prognostic uncertainty is often greatest after cryptogenic events, but any difference in prognostic value of DWI between etiologic subtypes of TIA and stroke is also uncertain.Two other important issues remain to be clarified: whether acute DWI lesions also predict longer-term outcome and whether they predict risk after minor stroke as well as TIA. First, in terms of long-term prognosis, until recently only a few small studies had evaluated the prognostic implications of DWI positivity beyond 1 year, with conflicting results.We therefore studied the 10-year risk of recurrent ischemic stroke in DWI-positive vs DWI-negative TIA and minor stroke in a large population-based cohort, stratified according to cryptogenic vs noncryptogenic etiology.18 between December 2004 and March 2017. OXVASC is an ongoing population-based study of the incidence and outcome of all acute vascular events in a population of 92,728 individuals, registered with 100 general practitioners in 9 general practices in Oxfordshire, United Kingdom. The multiple overlapping methods used to achieve near complete ascertainment of all individuals with TIA and ischemic stroke have been reported previously.18 Stroke and TIA were defined according to WHO criteria ,19 and for the current analyses, we included all patients with DWI performed as part of the initial assessment of TIA or minor ischemic stroke. The closest event prior to the DWI scan was taken as the index event.We studied consecutive patients referred to the Oxford Vascular Study (OXVASC) with suspected TIA or stroke20Patients were assessed by a neurologist or stroke physician and all presentations and investigations were reviewed by the senior study neurologist (P.M.R.) and TIA or stroke mimics excluded. Demographic data and stroke risk factors were collected from face-to-face interview and cross-referenced with primary care records. Detailed clinical history was recorded in all patients and assessments were made for stroke severity using the NIH Stroke Scale (NIHSS) as recorded on assessment. Minor stroke was defined as NIHSS ≤3. Cause of ischemic events was classified according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria.21 Of note, to exclude hemorrhage and mass lesions close to the area of interest on DWI, the study protocol also included a T2-weighted turbo gradient spin echo sequence and T1-weighted imaging. All brain imaging was reviewed by a senior study neuroradiologist (W.K.), blinded to any information about recurrent events. Restricted diffusion (DWI-positive) was defined as hyperintensity on DWI with decreased signal on apparent diffusion coefficient images. Acute DWI lesions were classified as being positive even if they did not correlate with the presenting symptoms.Patients routinely had brain imaging (CT or MRI), vascular imaging , 12-lead ECG, and standard blood tests. Echocardiography, 24-hour ECG (Holter), and 5-day ambulatory ECG monitoring were done when clinically indicated. During the study period, OXVASC brain imaging protocols changed: from 2002 to 2009, CT brain was the first-line imaging modality with MRI for use in selected cases when clinically indicated; from 2009 to 2012, MRI was used routinely for suspected posterior circulation events; and from 2012 onwards, MRI brain became the first-line imaging modality for all patients with suspected TIA or minor stroke. If a patient was reviewed in the emergency department prior to referral to the TIA clinic, he or she would typically receive a CT brain scan acutely followed by an MRI brain scan in the OXVASC clinic. The parameters for MRI scanners and MRI scanning protocols used in the study have been described elsewhere.Patients were followed up face to face at 1, 6, 12, 24, 60, and 120 months by a study nurse or physician to identify any recurrent stroke, supplemented by review of primary care records. Where patients were seen prior to symptom resolution, review at 1 month confirmed a TIA or minor stroke diagnosis. All recurrent events that occurred during follow-up would also be identified by the ongoing daily case ascertainment. Patients who had moved out of the study area were followed up via telephone at the same time points as face-to-face follow-up. We recorded all deaths during follow-up with the underlying causes by direct follow-up, via primary care records, and by centralized registration with Office for National Statistics.20 (cryptogenic and noncryptogenic). Analyses were censored at the outcomes of interest: death or the end of follow-up . All patients had at least 1 year of follow-up.We used Kaplan-Meier survival analysis to calculate the 10-year risks of recurrent ischemic stroke stratified by baseline DWI positivity and compared risks by Cox regression analysis adjusted for age and sex. Predefined subgroup analysis included index event type (ischemic stroke or TIA), NIHSS stroke category (0–1 and 2–3), and TOAST classificationHeterogeneity in the prognostic values of DWI positivity was assessed for using the Mantel-Haenszel test. A sensitivity analysis was performed to identify any differences relating to the changes in imaging protocol. All statistical analyses were performed with IBM SPSS version 22.0 .Written informed consent or assent from relatives was obtained in all participants. OXVASC was approved by the local research ethics committee (OREC A: 05/Q1604/70).Requests for access to the data reported in this article will be considered by the corresponding author.After exclusion of 745 patients referred in the study period with TIA or stroke mimics, 1,033 patients fulfilled the inclusion criteria. Baseline patient characteristics are shown in The median time (interquartile range) from event to scan and clinic assessment to scan was 3 days (7) and 0 days (1), respectively. Among those cases (n = 632/61.2%) investigated after 2012, when the imaging protocol changed to routine use of MRI in all eligible patients, 537 (90.3%) were imaged within 24 hours of assessment.During 4,778 patient-years of follow-up, there were 70 recurrent ischemic strokes and 141 deaths; 9 and 11, respectively, within 90 days from index event . The overall 10-year risks  of recurrent ischemic stroke and death were 9.5% (7.0–12.1) and 22.5% (18.4–26.6), respectively.p < 0.00001) and of death (p = 0.001). DWI positivity remained predictive for risk of post-90-day recurrent ischemic stroke  and death . Sensitivity analyses stratified by study phase also showed consistently increased risks in DWI-positive patients with recurrent ischemic stroke  and death .Among all patients with TIA or stroke , DWI posp = 0.064), driven by those with minor stroke with NIHSS 0–1 . These risks remained statistically significant following adjustment for age and sex, and for post-90-day risk. However, a positive DWI was not associated with an increased risk of recurrent ischemic stroke in patients with NIHSS 2–3 .When stratified by type and severity of index event , there wp = 0.003) and post 90 days , but not in patients with noncryptogenic event patients (data not shown).A positive DWI was most predictive of recurrent ischemic stroke in patients with index cryptogenic events, both overall , identifying appropriate DWI lesions in 70% of minor strokes and 13% of TIAs.24 Due to the delayed scan time, there is a risk of falsely negative DWI; however, despite this potential bias, we have identified a significantly lower risk in these patients, and therefore any reclassification would strengthen the results.The rate of DWI positivity (13.9% of TIAs and 65.1% of minor strokes with NIHSS 3) in our study is comparable with previous studies that reported a range between 12% and 67%.Redefining TIA to a tissue-based (DWI) definition has the benefits of conveying specific prognostic information, but the same approach has not been applied to minor stroke. Our data show that DWI positivity has similar prognostic importance after minor stroke with NIHSS 0–1, which might also help in managing these patients and is reassuring for those with a negative DWI scan. Furthermore, all acute DWI lesions identified in this study were considered, regardless of whether they correlated with the patient's clinical presentation. Our findings would support standard investigation and treatment of such patients with silent lesions, but further studies regarding the long-term risk of asymptomatic DWI lesions would be required.25In addition to the strong existing evidence that TIA patients with an acute ischemic lesion on DWI have a high 90-day risk of stroke, we showed that the prognostic importance of DWI positivity is maintained on long-term follow-up. The reasons why DWI-positive lesions were associated with long-term risks are less clear, although some investigators have postulated that a positive DWI might suggest increased intrinsic vulnerability of the brain to infarction. We found that patients with DWI-positive lesions were more likely to be male, have a noncryptogenic etiology , and there was a trend to a higher prevalence of diabetes . The preWe showed that DWI positivity is most strongly predictive of recurrent ischemic stroke and death in patients with cryptogenic index events, driven partly by the benign prognosis of patients with DWI-negative cryptogenic events. The lack of a treatable cause of cryptogenic events, such as atrial fibrillation or carotid stenosis, makes secondary prevention in these patients less targeted. The ability to select high-risk cryptogenic TIA/stroke patients on the basis of DWI may allow inclusion into future trials addressing the unmet need for more effective secondary prevention.Strengths of this study include the large population-based cohort, with stroke-specialist confirmed TIA, blinded radiologic review, and high rates of secondary prevention. However, our study did have some limitations. First, our findings were based on a mainly Caucasian population and may not be generalizable to a more ethnically diverse population. Second, some patients with DWI-negative TIA/minor stroke could have had a nonvascular cause for their symptoms, particularly perhaps those with other normal investigations , but we did use standard diagnostic criteria, administered by an experienced vascular neurologist, and so our results are likely to be generalizable to routine clinical practice. Of note, the 745 patients referred in the study period with a clear nonvascular cause for their symptoms were excluded. Furthermore, we cannot be certain that these results can translate to a hyperacute setting, such as in the emergency department, as our study is a predominantly outpatient, more inclusive cohort.DWI positivity conveys useful, long-term prognostic information in patients with TIA and minor stroke, supporting the tissue-based definition of TIA, which could also be extended to include those with minor stroke.


PvNSP2, PvNIN, and PvFLOT2, which are genes controlling the nodulation process were significantly reduced in Pi-deficient common bean seedlings. In addition, whole-genome transcriptional analysis revealed that the expression of hormones-related genes is compromised in Pi-deficient seedlings inoculated with rhizobia. Moreover, we showed that regardless of the presence or absence of rhizobia, the expression of PvRIC1 and PvRIC2, two genes participating in the autoregulation of nodule numbers, was higher in Pi-deficient seedlings compared to control seedlings. The data presented in this study provides a mechanistic model to better understand how Pi deficiency impacts the early steps of the symbiosis between common bean and rhizobia.Phosphate (Pi) deficiency reduces nodule formation and development in different legume species including common bean. Despite significant progress in the understanding of the genetic responses underlying the adaptation of nodules to Pi deficiency, it is still unclear whether this nutritional deficiency interferes with the molecular dialogue between legumes and rhizobia. If so, what part of the molecular dialogue is impaired? In this study, we provide evidence demonstrating that Pi deficiency negatively affects critical early molecular and physiological responses that are required for a successful symbiosis between common bean and rhizobia. We demonstrated that the infection thread formation and the expression of  Phaseolus vulgaris (common bean) represents the principal source of non-animal proteins and minerals for human consumption in the developing world  were high-throughput sequenced in 75 bp single-end at the University of Missouri DNA Core Facility by using an Illumina NextSeq500 sequencer  according to the manufacturer’s instructions. Image analysis and base calling were performed using the Illumina pipeline . The thresholds for Cuffdiff were set at 2-fold change and q-value ≤ 0.05 before calling genes to be differentially expressed, which are usually annotated with their unique identifiers defined by the gene models annotation and gene coordinates files. Data obtained from this differentially expression analysis was independently validated by qRT-PCR.The next step in our pipeline was to quantify the expression level of each transcript/gene by measuring the level of mRNA-seq alignments using the tool Cufflinks . The quaP. vulgaris Version 2.1 gene reference background [p-values for the GO terms were obtained through Fisher’s exact test, and a q-value was computed to produce lists of significant GO terms with an estimated False discovery rate (FDR) of 5%. Enriched GO terms with q < 0.05 were further summarized and visualized on REVIGO. Additionally, MapMan gene functional classification was used [The biological relevance of the differentially regulated genes was assessed by a gene function enrichment analysis using the method Singular Enrichment Analysis (SEA) available in the web-based tool AgriGo ,67, follckground ,60. p-vawas used ,71. For was used , was useAll the statistical analyses were conducted using R software 3.0.1. The specific tests performed are indicated in the legend of the corresponding figure.It has been demonstrated that the reduction in nodule number is one of the negative effects of Pi deficiency in legumes . HoweverR. tropici CIAT899::uidA. Forty-eight hours post inoculation, 95% (n = 190) of the seedlings growing under optimal-Pi conditions showed rhizobia-induced root hair deformation (R. tropici::uidA strain) at the susceptible zone , PvNIN (Phvul009G115800), and PvFLOT2 (Phvul.009G090700) in common bean seedlings growing under optimal- and low-Pi conditions and inoculated with rhizobia. Our analysis revealed that the expression of these three symbiosis-related genes was induced upon one-, 24, and 48 h post-inoculation with R. tropici in common bean seedlings growing under optimal Pi conditions  in Pi-deficient seedlings in response to rhizobia  and PvRIC2 (Phvul011G135900) in common bean seedlings grown under optimal- or low-Pi conditions, and under mock and rhizobia-treated conditions. This analysis revealed that the expression of PvRIC1 is induced as early as one-hour post inoculation with rhizobia in both control and Pi-deficient common bean seedlings and have its maximum induction level at 48 h post-inoculation with rhizobia  Pi-deficient common bean seedlings are preconditioned to reduce the number of rhizobia infections. This hypothesis can be supported by the fact that the overexpression of RIC1 and RIC2 inhibits nodulation in soybean [PvRIC1 and PvRIC2 in response to Pi deficiency might be critical for common bean to properly adapt to Pi deprivation. This hypothesis is supported by the fact that other CLE peptides  [It has been reported that the expression of ectively . Based oectively  and thatrhizobia a. In conculation c. Althoueedlings b,d. Addieedlings b,d. Alto soybean . (2) Hig, CLE14)  play a c, CLE14) ,82.To obtain a better insight into the genetic responses of common bean plants leading to a reduced nodulation under Pi-deficient conditions, we conducted an mRNA-seq analysis. For this genome-wide transcriptional analysis, based on our transcriptional data obtained by qRT-PCR, we selected 48 h post-inoculation to capture more transcriptional responses to rhizobia.P. vulgaris genome reference sequence Version 2.1 [A total of 12 cDNA libraries were generated from control  and Pi-deficient roots (low Pi: LP) inoculated 48 h with mock  or rhizobia . Three independent biological replicates were generated for each condition. These libraries were sequenced using the Illumina NextSeq500 platform. After filtering low-quality reads, a total of 176,365,724 single-end reads (75 bp) were aligned to the sion 2.1 ,60 usingsion 2.1 . Of thession 2.1  under thPvNSP2 (Phvul.009G122700) and PvFLOT2 (Phvul.009G090700) were also classified as differentially regulated by mRNA-seq approach (PvNIN (Phvul009G115800) in our mRNA-seq analysis, which we explain by a difference in the sensitivity of mRNA-seq and qRT-PCR approaches.In order to identify additional genes with a potential role in the regulation of the early stages of the symbiosis between common bean and rhizobia under low-Pi conditions, the mRNA-seq data sets were analyzed using Cuffdiff  with an approach . HoweverTo confirm these mRNA-seq results, the expression of 25 randomly selected genes was analyzed via qRT-PCR and using independent biological material from the one used for mRNA-seq analysis . The expUpon confirmation of the reliability of our mRNA-seq datasets, we performed additional pairwise comparisons to identify genes that might explain the reduction in the nodule number triggered by Pi deficiency. To this end, we compared the differentially regulated genes found in the pairwise comparisons OPR/OPM and LPR/LPM to identify commonly and uniquely regulated genes under these Pi conditions and interacting with rhizobia. This comparative analysis revealed that 234 (180 up-regulated and 54 down-regulated) genes were commonly regulated in both Pi conditions while interacting with rhizobia . In contFurther hierarchical clustering analyses on the 234 common regulated genes revealed a clear variation in the expression levels computed for these genes c,d. For To gain more insights into the molecular functions of the differentially regulated genes identified in this study, we performed a gene-enrichment analysis and a functional classification in MapMan to identify the molecular mechanisms leading to an inefficient symbiosis between common bean and rhizobia under Pi-deficient conditions. This analysis indicated that Regulation of transcription, Hormones, Regulation/Signal transduction, Transport, Biotic interactions and Protein modification/degradation were the most overrepresented functional categories in both control and Pi-deficient common bean seedlings interacting with rhizobia .Phytohormones play a critical role in different stages of the symbiosis between legumes and rhizobia . For insPvNIN and PvFLOT2) and physiological  events observed in Pi-deficient common bean seedlings might be partially explained by defects in the auxin-cytokinin balance.Based on the fact that auxin and cytokinin are crucial regulators of both early and late  stages of the symbiosis between legume and rhizobia , and theIn addition to auxin- and cytokinin-related genes, we also found that genes involved in the biosynthesis of the phytohormones ethylene, abscisic acid (ABA)- and jasmonate, as well as in the signal transduction related to these three phytohormones, were differentially regulated in response to rhizobia inoculation. Although we observed that common bean seedlings growing under optimal-Pi conditions and inoculated with rhizobia showed more differentially regulated genes  related to these three hormones than Pi-deficient seedlings inoculated with rhizobia, the majority of them were down-regulated . In contENOD11), inhibits the initiation and maintenance of the calcium spiking, decreases the number of rhizobia-induced root hair deformation and infection thread, and reduces nodule formation in M. truncatula plants [Ethylene, jasmonate, and ABA are essential to activate a series of molecular and physiological responses allowing plants to cope with Pi deficiency . Howevera plants ,91. Recea plants . Hence, M. truncatula roots, it has been proposed that WAKs might play a critical role in the early steps of this symbiosis, primarily in the cell wall-cytoplasm signaling [The proper recognition of rhizobia by the host legume is crucial for a successful symbiosis. To this end, legumes rely on the LysM-domain receptor kinases NFR5 , NFR1 9 and eNFRignaling . In our The data presented in this study lead us to conclude that Pi deficiency negatively affects early events of the symbiosis between legumes and rhizobia. The negative effects in the symbiosis between the common bean and rhizobia under Pi deficiency might be triggered by an imbalance between auxin and cytokinin, as well as by the activation of the ethylene, jasmonate and ABA-related signaling pathway, which are negative regulators of early molecular events of this symbiosis . Finally


P = 1.38e−03) was determined. Then bioinformatics analysis to recruit putative target genes prediction of hsa-miR-133a-3p and pathway enrichment analysis to reveal what biological processes this miRNA may affect were conducted. It was indicated that pathways were commonly associated with adrenergic signaling in cardiomyocytes, adherens junction, PI3K-Akt signaling pathway and AMPK signaling pathway. Furthermore, STRING and Cytoscape tools were used to visualize the interactions between target genes of hsa-miR-133a-3p. Six genes were detected as hub genes among 576 targets which were CDC42, RHOA, EGFR, VAMP2, PIK3R2 and FN1. After Kyoto Encyclopedia of Genes and Genomes pathway analysis, it was detected that these hub genes were mostly enriched in signaling pathways and cancer. In this meta-analysis, it is stated that circulating hsa-miR-133a-3p may serve as a potential non-invasive biomarker and therapeutic target in PMO.MicroRNAs (miRNAs) have been proven to play a crucial role in postmenopausal osteoporosis (PMO), and studies on their diagnostic value have been increasing. In our study, we aim to identify the key miRNAs in the PMO that might be potential biomarkers. A comprehensive systematic literature search was conducted by searching PubMed, Web of Science, Embase and Cochrane Library databases. In the total of 16 independent miRNA expression studies which contained 327 PMO patients and 328 postmenopausal (PM) healthy control samples, miRNAs were evaluated by using robust rank aggregation (RRA) method. A statistically significant meta-signature of up-regulated hsa-miR-133a-3p ( Osteoporosis is a systemic skeletal disorder, which is common in postmenopausal (PM) women, characterized by an increased risk of bone fragility and a decrease in bone mass . Bone hoMicroRNAs (miRNA) are short noncoding and single-stranded molecules in 18–24 nucleotides length which play key roles in translation and expression of genes via binding to the 3′untranslated region (3′UTR) of target messenger RNAs (mRNA) ,6. miRNAmiRNA profiling datasets were arising rapidly with the improvement of high-throughput technologies. Using of different technological platforms and small sample size in studies results miRNA data sets show inconsistent results between various studies. In our study, we aimed to identify the key circulating miRNAs in the postmenopausal osteoporosis (PMO) that might be non-invasive potential biomarkers. Considering our aim and these problems, we found a substantial way to increase the statistical power of profiling data by combining the results of several studies. We combined these results via conducting a meta-analysis applying the robust rank aggregation (RRA) method , followeWe conducted a systematic literature search to identify miRNA expression profiling studies in PMO published up to 9 October 2018. The databases searched included PubMed, Web of Science, Embase and Cochrane Library through the MESH search headings ‘(miRNA OR microRNA OR miR) AND (osteoporosis)’. The searches were limited to English language studies and only full text published studies were included. We evaluated potentially relevant studies according to their titles and abstracts by using Rayyan which is a free application that dramatically speeds up the process of screening and selecting studies . MoreoveThe inclusion criteria for the selection of eligible studies were defined as: (1) circulating miRNA expression profiling on PMO patients; (2) have to compare PMO samples with PM healthy control samples; (3) miRNA expressions were reported to be profiled by using miRNA microarray, next-generation sequencing (NGS) or qRT-PCR; (4) have to report cut-off criteria of dysregulated miRNAs; (5) they reported fold changes (even if non-explicit); (6) have to report sample sizes. The exclusion criteria were defined as: (1) studies on tissues, cell lines and animals; (2) review literature and case reports.Relevant papers were selected by two authors (E.P. and T. D.) and decided the list of studies be included carefully.miRNAmeConverter available in Bioconductor R package [From the full text and supplementary data of each study, the subsequent eligible information was collected and documented as first author, date of publication, country of study, sample sizes, tissue types, miRNA expression profiling assay type, number of probes, the list of dysregulated miRNAs and their related fold changes if given and cut-off criteria of dysregulated miRNAs. The authors directly contacted the correspondence of the studies to reach the gene lists which were not given in the full text and supplementary information. All miRNA names were standardized according to miRBase v22 by using  package .http://cran.R-project.org/). This approach is utilized for comparison of different ranked gene lists, and is a definite and powerful approach for identification of differentially expressed miRNA integrated signature where a P-value would be assigned for each miRNA in the ranked lists to rerank these miRNAs and determine their significance. This method applies a probabilistic model for aggregation. The RRA method is robust to noise, and it facilitates the computation of significance probabilities to each of the elements in the final ranking [P-values (P<0.05).RRA method was used in our meta-analysis which is a free package in R software  was constructed to predict targets of miRNAs, which cover 14 databases. MultiMiR package was used to predict targets of miRNAs by DIANA-microT, ElMMo, MicroCosm, miRanda, miRDB, PicTar, PITA and TargetScan databases and also, validated targets of miRNAs were acquired from miRecords, miRTarBase and TarBase databases with the criterion of primary score listed in top 35 [In the present study MultiMiR package  is an online program which allows functional annotation of the huge number of genes obtained from several genomic resources . We useded miRNA .http://string-db.org/) is online software that aims to provide a crucial evaluation and integration of protein–protein interaction (PPI), including direct and indirect connections [P-values≤0.05 after Benjamini correction cut-off criterion.The STRING database  were reported in at least two studies. Furthermore four miRNAs  were reported to be dysregulated in both directions .P-value (1.38e−03). Only this miRNA was detected in two datasets of included studies by using RRA.Afterward, using the RRA method, we identified a statistically significant meta-signature of one up-regulated miRNA; hsa-miR-133a-3p considering the permutation in silico predicted and three validated databases was systematically screened by using MultiMiR for miRNA–target interactions. According to our criterion, a total of 3153 target genes were predicted initially, and only 576 genes were essential for further studies.Target genes of hsa-miR-133a-3p were evaluated via bioinformatics analysis. The combination of eight different P<0.05 after Benjamini correction). Also, the KEGG pathway analysis showed that target genes were enriched in adrenergic signaling in cardiomyocytes, adherens junction, PI3K-Akt signaling pathway and AMPK signaling pathway   .According to the World Health Organization, osteoporosis is one of the most common diseases, and 30–50% of all women in the world suffer fractures due to osteoporosis throughout their lives . CirculaDEXA is a gold standard method for the measurement of BMD, used in the diagnosis of osteoporosis. To our knowledge, the present study is the first meta-analysis concerning PMO circulating miRNA expression profiles and identifying specific miRNA as a potential biomarker in PMO. Serum biomarker analyzing is beneficial as dysregulation of specific circulating miRNAs in the serum might present valuable potential diagnostic predictors for PMO.P = 0.00138).Although there are some studies in the literature on PMO and circulating miRNAs, no meta-analysis is available, and consistent and precise data cannot be reached. In the present study, we aimed to use meta-analysis to recognize consistently dysregulated miRNAs, which were mentioned in profiling results to be candidate biomarkers for PMO. To overcome inconsistent results of miRNA dysregulation in PMO patients reported in the literature, RRA method was performed in the present study. Ranked list of miRNAs from each included study was aggregated into a single gene ranking and analyzed using the RRA method, which approved hsa-miR-133a-3p as the only statistically significant miRNA associated with PMO (Dysregulation of miR-133a-3p has been identified in several types of cancers, such as bladder , colorecAlso, there are studies available concerning circulating miRNAs in PMO based on different inclusion patient criteria in the literature. Studies on the circulating miRNAs in PMO patients that were compared with the PM women without osteoporosis were included in this meta-analysis. As noted in MiR-133a was initially examined as a muscle-specific miRNA involved in the regulation of muscle-cell differentiation and pathogenesis of heart disease . Afterwain vitro experiments demonstrated that miR-133a was up-regulated during osteoclastogenesis and overexpression of this miRNA promoted RANKL induced differentiation of RAW264.7 and THP-1 cells into osteoclasts. And also in vivo experiments carried out in the present study showed that osteoclastogenesis related factors levels changed in serum, lumbar spine BMD increased and bone histomorphology changed in miR-133a knockdown ovariectomized rats. These results indicated that miRNA-133a is involved in the regulation of PMO by promoting osteoclast differentiation [In an included study to our meta-analysis, ntiation .Target gene prediction for miR-133a-3p was performed by using MultiMiR. 576 genes were detected as the target of this miRNA and KEGG pathway analysis has shown that these genes were mostly enriched in adrenergic signaling in cardiomyocytes and adherens junction. Some other reports in the literature that show the association of these two pathways with osteoporosis ,55. FurtIn conclusion, hsa-miR-133a-3p seems to be a potential biomarker for PMO, since its levels are up-regulated in patients suffering from PMO. This miRNA would enable the analysis of osteoporosis without requiring DEXA so that radiation exposure will be reduced in these patients. Thus, a deeper examining of the role of miRNAs in osteoporosis can inspire critical implications for the early diagnosis and prevention of osteoporosis. It can also provide unique opportunities to develop novel pharmacologic approaches for osteoporosis.


Four signatures were identified, including an IFN-induced gene signature and a ribosomal protein gene-signature, that indicated pDC activation. Comparison with a dataset of in vitro activated pDCs showed that pSS pDCs have higher expression of many genes also upregulated upon pDC activation. Corroborating this transcriptional profile, pSS pDCs produced higher levels of pro-inflammatory cytokines, including type-I IFN, upon in vitro stimulation with endosomal Toll-like receptor ligands. In this setting, cytokine production was associated with expression of hub-genes from the IFN-induced and ribosomal protein gene-signatures, indicating that the transcriptional profile of pSS pDCs underlies their enhanced cytokine production. In all transcriptional analyses, nSS patients formed an intermediate group in which some patients were molecularly similar to pSS patients. Furthermore, we used the identified transcriptional signatures to develop a discriminative classifier for molecular stratification of patients with sicca. Altogether, our data provide in-depth characterization of the aberrant regulation of pDCs from patients with nSS and pSS and substantiate their perceived role in the immunopathology of pSS, supporting studies that target pDCs, type-I IFNs, or IFN-signaling in pSS.Primary Sjögren's syndrome (pSS) is a systemic auto-immune disease typified by dryness of the mouth and eyes. A majority of patients with pSS have a type-I interferon (IFN)-signature, which is defined as the increased expression of IFN-induced genes in circulating immune cells and is associated with increased disease activity. As plasmacytoid dendritic cells (pDC) are the premier type-I IFN-producing cells and are present at the site of inflammation, they are thought to play a significant role in pSS pathogenesis. Considering the lack of data on pDC regulation and function in pSS patients, we here provided the first in-depth molecular characterization of pSS pDCs. In addition, a group of patients with non-Sjögren's sicca (nSS) was included; these poorly studied patients suffer from complaints similar to pSS patients, but are not diagnosed with Sjögren's syndrome. We isolated circulating pDCs from two independent cohorts of patients and controls (each  Primary Sjögren's syndrome (pSS) is a systemic rheumatic autoimmune disease characterized by mononuclear infiltration of the exocrine glands, associated with dysfunction and destruction of the glands and dryness of primarily mouth and eyes . PatientMX1, IFIT3, IFI44), is present in the majority of pSS patients and is associated with increased systemic disease activity (An interferon (IFN)-signature, defined as the increased expression of a panel of type-I IFN induced genes  are uniquely capable of rapidly producing high levels of type-I IFN upon activation. pDCs constitutively express endosomal Toll-like receptors (TLR) 7 and 9, through which they recognize viral RNA and DNA but can also bind endogenous nucleic acids. Triggering of these TLRs results in production of type-I IFNs or other pro-inflammatory cytokines, depending on the cellular compartment in which the receptors encounter their ligand . ImportaIn patients with pSS, numbers of pDCs in peripheral blood are decreased and associated with increased salivary gland inflammation , 16. Furin vitro experiments, independent donors were included . pDCs were freshly isolated by MACS using BDCA-4+ isolation kit (Miltenyi Biotec). In addition, monocytes were freshly isolated by MACS from a subset of 43 donors  using the CD14+ isolation kit (Miltenyi) after isolation of pDCs to quantify the IFN-score. Cells were lysed in RLTplus buffer  supplemented with beta-mercaptoethanol  for transcriptional analyses. Total RNA was purified using AllPrep Universal Kit (Qiagen), according to the manufacturer's instructions. RNA concentration was assessed with Qubit RNA Kit (Thermo Fisher Scientific) and RNA integrity was measured by capillary electrophoresis using a Bioanalyzer (Agilent) and the RNA 6000 Nano Kit (Agilent Technologies); all samples had RIN-score >7.0.https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). All samples passed the quality check. For each sample, reads were then aligned to the human genome assembly (GRCh38 build 79) , which were used for subsequent analyses.For the discovery cohort samples, RNA sequencing was performed at the Beijing genomics institute on a NextSeq 500 sequencer (Illumina) by applying standard manufacturer's protocols. About 20 million paired-end (91 bp) reads were generated for each sample. For the replication cohort, RNA sequencing was performed at the Beijing genomics institute using an Illumina HiSeq 4000 sequencer (Illumina), by applying standard manufacturer's protocols. About 20 million paired-end (100 bp) reads were generated for each sample. Raw reads obtained from RNAseq were quality checked using the FastQC tool (uild 79)  using STuild 79) . The aliuild 79)  for eachuild 79)  to removuild 79) . We usedin vitro stimulated pDCs using gene set enrichment analysis (GSEA)  . For this (GSEA) . Bioconds (GSEA) . Ten thoTo study the inter-dependence between genes and their interactions, we constructed two gene co-expression networks (one for the discovery cohort and another for the replication cohort) using WGCNA Bioconductor/R package . The genFor each module the gene or the first principal component was calculated and plotted using ggplot2 and ggsci libraries in R. For each gene, the connectivity represents the sum of connection strengths (or the weighted correlations) of each node with its neighbors in a given module. We normalized the connectivities for each module in the range , where 1 represents the maximum and 0 represents the minimum connectivity in the module. We calculated the overlap between the modules constructed from the two independent cohorts and used Fisher's exact test to identify modules with significant overlaps . We perf*105 cells/mL in 96 wells round-bottomed plates (Thermo Fisher Scientific) in the presence of 1 mM of loxoribine  or 1 μM of CPG-C . After 3 h, supernatants were harvested and cells lysed, samples were stored at −80°C. Cytokines were measured in the supernatants using multiplex immunoassay.Isolated pDCs were cultured in RPMI Glutamax (Gibco) supplemented with penicillin, streptomycin (both Gibco), and 10% FBS (Sigma). Cells were cultured at 6GAPDH and GUSB  and the Quantstudio system (Thermo Fisher Scientific). In the cultured pDCs, mRNA expression was measured and normalized to the mean expression of two housekeeping genes: and GUSB . The relIFI44L, IFI44, IFIT3, LY6E, and MX1) was assessed using qPCR. Expression of each sample was normalized to the mean expression of two housekeeping genes: GAPDH and GUSB . The purity of pDCs was (median [IQR]) 92% [87–95%], monocyte purity was 97% [95–98%]; there were no differences in purity between any of the groups. For assessment of CCR5-expression on pDCs, 1 million PBMCs were stained for 30 min at 4°C and the expression of CCR5 on lineage-negative (CD3-CD14-CD16-CD19-CD56-) and HLA-DR+CD123+BDCA-2+ pDCs was determined .k = 1) from a GLM regression of the counts to batch-corrected read counts from discovery and replication cohorts. We used the caret Bioconductor/R package to perform backward feature selection and 10-fold cross validation to get high prediction accuracy. This allowed us to select 467 genes. We further used the SVM algorithm  to develop a classifier using the 467 selected genes to discriminate pSS patients from HC donors.We used the RUVSeq Bioconductor/R package  to consiin vitro experiments, differences between patients and controls were assessed using Mann-Whitney U-test. Changes in expression of ribosomal proteins after stimulation were analyzed using Wilcoxon matched-pairs signed rank test. Correlations were assessed using Spearman's rank correlation coefficient. Two-sided testing was used for all analyses. Differences and correlations were considered to be significant at p < 0.05. As the gene signatures identified were replicated in an independent cohort, no FDR-correction was used for the RNA-sequencing analysis.For RNA-sequencing analysis, we used Wald's test to identify DEGs in each pair-wise comparison performed between the three groups  and used LRT to identify DEGs considering multiple groups. For the n = 31; We recruited two independent cohorts of patients and healthy controls  including RPL23; and the increased expression of C-C chemokine receptor 5 (CCR5), which is an important driver of pDC migration under inflammatory conditions , transcription initiation and regulation (yellow), and IFN-signaling (red) . No pathTo further close in on the most relevant differentially-expressed genes and processes within the signatures, we identified their hub-genes using the normalized connectivity of each gene in both cohorts . ConnectAs the red signature hub-genes contained four of the five genes used to quantify the IFN-score , we expeThe blue signature represents broadly decreased expression of ribosomal proteins. Classical DCs decrease their transcription and translation of RPs upon stimulation with various activating signals, including TLR ligands –38. To iRPL11, RPL27, RPS11). Indeed, all three RPs were downregulated upon stimulation with loxoribine in HC pDCs, while RP expression in pSS patients was typically lower compared to the HC cells consistent with the sequencing data  or TLR9 (CPG-C) and measured the expression of three hub-genes from the blue gene signature  correlated with cytokine production .To assess whether transcriptional data from pDCs could be used for patient stratification, we developed a discriminative classifier using the robust and consistent transcriptional signature of pSS pDCs. Using machine learning, we selected 467 genes from the gene signatures that provided the most discriminative signal between pSS patients and HCs. . This sePatient inclusion was based on the 2002 AECG classification criteria  in additMYD88, TLR7, and TLR9; we did observe clear correlations of TLR7 and MYD88 with the IFN-score in both cohorts, supporting the published data  are top hits in published studies in pSS  with functions in anti-viral immunity and regulation of type-I IFN production  as well as the transcription factors SP1 and TP53, which are central regulators of this process  transcripts  autoimmunity. To examine whether pDC dysregulation may play a role in these patients we included them in this study. Our data indicate that pDCs from nSS patients are similarly but less strongly activated compared to those from pSS patients on a group level. A set of nSS patients, who all did not meet the 2002 classification-criteria  at initiConcluding, we show that pSS pDCs are robustly distinct from HC pDCs, have an activated transcriptional phenotype, and are primed for increased pro-inflammatory cytokine production. nSS patients form an intermediate group in which some patients are molecularly similar to pSS patients in concordance with the most recent classification-criteria. In addition, the expression of hub genes from the two major identified gene signatures correlates with cytokine production, indicating that the transcriptional dysregulation of pSS pDCs underpins their enhanced cytokine production. These data substantiate the notion that pDCs play a role in pSS immunopathology and support studies that target pDCs, type-I IFNs, or IFN-signaling in pSS.https://www.ncbi.nlm.nih.gov/geo/ - GSE135635. The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.The RNA sequencing datasets generated for this study can be found in the GEO database at The medical ethics committee of the UMC Utrecht approved the study , all patients gave their written informed consent.MH, AP, SB, MR, AK, JR, and TR were involved in conception and design of the study. MH, SB, SH, CB, EH, and AK were involved in data acquisition. MH, AP, SB, NS, AK, JR, and TR were involved in data analysis and interpretation. MH and AP drafted the manuscript. All authors revised the manuscript critically for important intellectual content and approved the submitted version.TR is a principal investigator in the immune catalyst program of GlaxoSmithKline, which is an independent research program. He did not receive any financial support other than the research funding for the current project. 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.


Although no consensus exists on whether extracorporeal membrane oxygenation (ECMO) support is an indication for solid-organ transplantation other than heart and lung, this practice continues to be limited. We present a case of a 55-year-old man who was placed on venoarterial ECMO (VA-ECMO) during orthotopic heart transplantation (OHT) because of acute graft failure. Twenty-four hours later, the patient underwent deceased-donor renal transplantation (DDRT) while on VA-ECMO and had no complications. On post-DDRT day 2 (post-OHT day 3), the patient was successfully decannulated from VA-ECMO and ultimately discharged home. This case highlights the potential successful use of ECMO support during solid-organ transplantation other than heart and lung and the importance of trained providers and tailored anticoagulation. To the authors' knowledge, this is the first report of perioperative ECMO use during kidney transplantation after recent heart transplantation. Extracorporeal life support (ECLS) or extracorporeal membrane oxygenation (ECMO) is used to support patients with cardiac or respiratory failure, or both, which is refractory to conventional therapy . IndicatAlthough no consensus exists on whether ECMO support is an indication for solid-organ transplantation other than heart and lung, this practice continues to be limited. To the authors' knowledge, there are no reports of perioperative ECMO support during kidney transplantation. The authors present the case of a 55-year-old man who was placed on VA-ECMO during heart transplantation because of acute graft failure and then underwent single-kidney transplantation while on VA-ECMO, with no complications.A 55-year-old Caucasian male was admitted to our hospital with a history of biventricular systolic heart failure due to ischemic cardiomyopathy and chronic stage IV kidney disease due to diabetic nephropathy; one month earlier, he had been approved for combined heart-kidney transplant. He recently had increased fatigue and dyspnea, weight gain of 8 kg, and serum creatinine that increased from 2.3 mg/dL to 3.7 mg/dL despite outpatient inotropic and diuretic therapy. Other comorbidities included antiphospholipid syndrome under warfarin management, multiple myocardial infarctions, with 2 coronary artery bypass grafting procedures, placement of biventricular automated implantable cardioverter-defibrillator, and placement of left ventricular assist device (HeartWare) 5 months earlier.On hospital day (HD) 14, after optimization of hemodynamics with diuretics and inotropic support, the patient was listed for heart-kidney transplant and transferred to the intensive care unit. On HD 24, the patient was taken to the operating room for combined heart-kidney transplant; however, only orthotopic heart transplantation (OHT) was possible. During the OHT, acute right ventricular graft dysfunction developed, resulting in cardiogenic shock, requiring placement of VA-ECMO support through central cannulation and withholding of the single-kidney transplantation. VA-ECMO was utilized to allow the newly transplanted heart to rest and recover and to optimize hemodynamics and volume status in order for the patient to eventually receive the single-kidney transplantation. Simultaneously, he received support with vasopressin 0.04 U/min, dobutamine 10 mcg/kg/min, epinephrine 0.01 mcg/kg/min, and full-dose anticoagulation with heparin 9.5 U/kg/hour. Tables On post-OHT day 1, after the patient had improved volume and cardiovascular status, adequate urine output , and stable laboratory testing parameters, the heparin infusion was reduced to 5.5 U/kg/hour  and the patient underwent deceased-donor renal transplantation (DDRT). During the DDRT procedure, the patient received 1 unit of packed red blood cells and had no complications. After DDRT, the patient required epinephrine at 0.03 mcg/kg/min, dobutamine at 10 mcg/kg/min, and heparin at 5.5 U/kg/hour.During the first 48 hours after DDRT, the patient's urine output was adequate  and his serum creatinine concentration decreased to 2.0 mg/dL. On post-DDRT day 2 (post-OHT day 3), after repeated transesophageal echocardiography showed improved right ventricular function and optimal volume status, the patient underwent ECMO decannulation, chest washout, and sternal closure without complication. His renal function continued to improve, and his anticoagulation was discontinued. The only inotropic support needed was dobutamine at 7.5 mcg/kg/min. On post-DDRT day 5 (post-OHT day 6), the patient was successfully weaned from mechanical ventilation and on post-DDRT day 18 (post-OHT day 19) was transferred to the posttransplant care unit for further care.The primary indications of ECMO are severe respiratory failure, cardiac failure, or combined cardiopulmonary failure with a high mortality risk, where ECMO provides life-saving cardiopulmonary support for patients who would not otherwise survive with conventional therapy . ECMO isThe perioperative use of ECMO in candidates for heart or lung transplantation  and those who have early severe graft failure has been well described and is considered the standard of care. However, the use of ECMO for patients undergoing solid-organ transplantation other than heart and lung continues to be limited. Transplant surgeons and intensivists have substantial hesitancy, perhaps due to the required anticoagulation state of patients on ECMO; the considerable frequency of adverse events during ECMO for adults, such as cannula site hemorrhage , surgical site hemorrhage , renal failure , and infection ; and the delicate hemodynamic status [Few reports have been published about ECMO use for patients with liver or kidney transplantation and fewer about perioperative ECMO in liver, combined heart-liver, and lung-liver transplantation. To the authors' knowledge, this is the first report of perioperative ECMO use during a single-kidney transplant after heart transplantation.Patients with heart failure have increased prevalence of renal and liver disease, whereby the renal and liver disease worsens heart function even further , 4. AcutFor patients with severe liver or renal failure whose best therapeutic option is transplant receipt, the concomitant need for ECMO support represents a challenge for the treating team, in part due to the substantial debate around anticoagulation management on ECMO (either VA or VV). On one hand, continuous contact between blood and foreign surfaces of the ECMO circuit shifts the hemostatic balance to a hypercoagulable state; on the other hand, the most frequent complications related to ECMO are bleeding events. Additionally, there are no randomized control trials that provide evidence or guidance on this issue. However, use of ECMO with low-dose heparinization may be a good strategy because it has been increasingly reported to reduce perioperative morbidity in lung transplant patients .In the present case, the patient presented with an acute decompensation of his chronic heart failure which had caused acute worsening of his chronic kidney disease, that is, cardiorenal syndrome type 1  . When deA consensus is lacking on whether ECMO support is a contraindication for solid-organ transplantation other than heart and lung. In addition, no evidence shows negative outcomes related to the ECMO system and the anticoagulation status in this patient population. The authors opine that ECMO support is not an absolute contraindication for solid-organ transplantation other than heart and lung. Skilled ECMO management with trained providers, tailored anticoagulation with low-dose heparinization, and health care infrastructure that can help prevent or manage adverse events are measures that ensure successful ECMO use in delicate yet necessary interventions such as solid-organ transplantation other than heart and lung.This case report does not allow for the conclusion that every patient on ECMO who is also a candidate for solid-organ transplantation other than lung or heart should undergo the transplant while on ECMO. Currently, the authors are unable to prove whether this approach can improve outcomes in a large population of patients; more cases are required to arrive at solid conclusions.


Preterm birth is associated with heightened risk for attention-deficit/hyperactivity disorder (ADHD)-like symptoms and neurocognitive impairments, including impairments in performance monitoring. Here, we investigate the cognitive and neurophysiological processes from a performance-monitoring task in preterm-born adolescents and examine whether these processes in preterm-born adolescents reflect identical neurophysiological impairments to those observed in term-born adolescents with ADHD. We compared 186 preterm-born individuals to 69 term-born individuals with ADHD and 135 term-born controls on cognitive-performance measures and event-related potentials (ERPs) of conflict monitoring (N2) and error processing  from a flanker task. Preterm-born adolescents demonstrated reduced N2, ERN and Pe amplitudes, compared to controls, and similar ERN and Pe impairments to term-born adolescents with ADHD. While ADHD symptoms correlated with ERN amplitude at FCz among the preterm-born, ERN amplitude at Fz, N2 and Pe amplitude were not associated with ADHD symptoms. Preterm-born individuals show impairments on neurophysiological indices of conflict monitoring (N2) and error processing (ERN and Pe). Early neurophysiological error processing may be a marker underlying the processes linked to the increased risk for ADHD among preterm-born individuals. Error detection processes are malleable and potential targets for non-pharmacological interventions. Preterm-born individuals are likely to benefit from early interventions. An estimated 15 million babies worldwide are born preterm (before 37 completed weeks of gestation) every year, with a mean preterm birth rate of 8.6% in the developed world , the surPreterm-born individuals are not only at an increased risk for the behavioral symptoms of ADHD, but also for similar cognitive impairments as observed in individuals with ADHD ,4, such Cognitive-performance data, however, only affords indirect insight into covert processing. Electrical potentials generated by the brain following internal or external events (such as stimuli and responses), known as event-related potentials (ERPs) , insteadERP studies in preterm-born individuals in childhood, adolescence and adulthood are scarce. Most ERP research carried out in preterm-born children has focused on auditory ERP measures –15. AudiDirect comparisons between individuals born preterm and individuals with ADHD are rare but can explicate whether the impairments observed in preterm groups are truly identical to those found in individuals with ADHD or part of wider-ranging impairments. This could aid in identifying biomarkers for the underlying processes linked to the increased risk for ADHD among preterm-born individuals, and effectively in planning targeted interventions. Only one small-scale ERP study  investigated attentional processing in preterm-born children of very low birth weight (<34 weeks and <1501g) with and without ADHD, as well as in term-born controls and term-born individuals with ADHD, using a visual oddball paradigm . CompareIn addition to impairments in attention and inhibition, neurocognitive impairments in ADHD include deficits in performance monitoring ,19. The The objective of the current study is to examine the cognitive and neurophysiological processes from a performance-monitoring task in preterm-born adolescents and young adults. Building on the cognitive-performance and ERP measures, which sensitively captured the performance monitoring impairments in adolescents and young adults with ADHD , we furtThe sample comprised 194 adolescents and young adults born preterm (<37 weeks of gestation), 93 ADHD participants and 166 controls. Exclusion criteria for all groups were IQ<70, general learning difficulties, cerebral palsy or any other medical conditions that affects motor co-ordination including epilepsy, as well as brain disorders and any genetic or medical disorder that might mimic ADHD. In addition, preterm birth was an exclusion criterion in the ADHD and control groups, because this study aimed to establish whether the cognitive impairments associated with preterm birth reflect identical neurophysiological impairments in term-born individuals with ADHD.The preterm group was recruited from secondary schools in Southeast England. All preterm participants had one full sibling available for ascertainment, and were born before 37 weeks’ gestation. Siblings of preterm-born individuals were included in the preterm group if they were also born preterm (before 37 weeks’ gestation) to maximize the number of participants in the preterm group. Term-born siblings of preterm-born individuals were not included in this analysis. Seven individuals from the preterm sample were excluded because medical birth records could not corroborate preterm status  < 37 weeks). One individual was excluded because of IQ<70. While preterm-born individuals were unselected for ADHD status, eight preterm-born individuals met diagnostic criteria for a research diagnosis of ADHD. Since here preterm birth is investigated as a potential risk factor for ADHD, preterm-born individuals who demonstrated high levels of ADHD symptoms were not excluded from the analysis   to all pThe task was an adaptation of the Eriksen Flanker paradigm  designedThe EEG was recorded from a 62-channel DC-coupled recording system (extended 10–20 montage), using a 500Hz sampling-rate, impedances under 10kΩ, and FCz as the recording reference. The electro-oculograms (EOGs) were recorded from electrodes above and below the left eye and at the outer canthi. EEG data were analyzed using Brain Vision Analyzer 2.0 . Raw EEG recordings were down-sampled to 256Hz, re-referenced to the average of all electrodes, and filtered using Butterworth band-pass filters . All trials were visually inspected for electrical artifacts or obvious movement, and sections of data containing artifacts were removed manually. Ocular artifacts were identified using the infomax Independent Component Analysis (ICA) algorithm . SectionData were segmented based on 1) stimulus-locked trials where a correct response was made and (2) response-locked (error-related) trials where an incorrect response was made. Individual averages were created based on each condition, requiring ≥20 clean segments for each participant. After averaging, the electrodes and latency windows for ERP analyses were selected based on previous studies ,23,25, t stimulusTwo preterm-born participants (1.1%) were excluded from the ERP analysis of the N2 and 14 preterm-born participants (7.5%) were excluded from the ERP analysis of the ERN/Pe due to having fewer than 20 artifact-free segments available for analysis. Two participants with ADHD (2.9%) were excluded from the ERP analysis of the N2 and seven participants with ADHD (10.1%) were excluded from the ERP analysis of the ERN/Pe due to having fewer than 20 artifact-free segments available for analysis. Fourteen control participants (10.4%) were excluded from the ERP analysis of the ERN/Pe due to having fewer than 20 artifact-free segments available for analysis. These exclusion ratios across groups reflect similar ratios reported in previous studies using this paradigm ,25.Data were analyzed using random intercept models in Stata, to control for non-independence of the data (i.e. data coming from siblings of one family), using the ‘robust cluster’ method to estimate standard errors . We ran The random intercept model yielded a significant main effect of group for MRT , RTV , DSB , N2 , Pe  and ERN  amplitude  and ERN . No significant group-by-congruency interaction emerged for N2 , MRT , RTV  or errors . No significant group-by-congruency-by-electrode site interaction emerged for N2 amplitude .Post-hoc analyses found that the preterm group had significantly reduced N2 and ERN amplitude at FCz, as well as reduced DSB and Pe amplitude compared to the control group, with medium-to-large effect sizes . CompareAmong those born preterm, DIVA ADHD symptoms correlated with ERN amplitude at FCz , but not with ERN amplitude at Fz , Pe amplitude , N2 amplitude at Fz  or N2 amplitude at FCz .Controlling for IQ, group effects on MRT in the congruent condition became significant  and neurophysiological error processing (ERN and Pe). The early (ERN) and late (Pe) error detection impairments observed in the preterm-born individuals were similar to those found in term-born individuals with ADHD. While the impairments in early error processing (ERN) were significantly associated with ADHD symptoms, such that smaller ERN amplitude at FCz was linked to higher ADHD symptom severity, Pe amplitude did not correlate with ADHD symptoms in the preterm group. Early electrophysiological error processing may, therefore, be a putative marker for the underlying processes linked to the increased risk for ADHD among those born preterm.Reduced N2 amplitude at FCz, indexing conflict-monitoring impairments, was observed in the preterm-born individuals. Previous research had found increased NoGo-N2 amplitude in children born very preterm (<32 weeks) using an auditory oddball paradigm . The disPreterm-born individuals further demonstrated impairments in Pe amplitude, compared to term-born controls. While the functional importance of the Pe is still unclear, the Pe has frequently been linked with conscious awareness of having committed an error . In lineWe further identified impaired early error processing, indexed by reduced ERN amplitude at FCz, in the preterm group. This impairment is similar to that in individuals with ADHD who also demonstrated reduced ERN amplitude compared to controls . The sigWe repeated the analyses with IQ as a covariate to examine any potential contribution from IQ to the results. When removing the effects of IQ, differences on MRT and RTV between the groups became non-significant, suggesting that group differences on this measure may reflect impairments related to IQ. These findings may also explain the discrepancy between this study and previous studies ,22, whicOne limitation of this analysis is that we cannot completely rule out the possibility of background factors accounting for our ERP findings, because we were not able to investigate risk factors for preterm birth . Yet, population-based quasi-experimental designs found that the link between psychiatric morbidity and preterm birth is largely independent of shared familial confounds and measured covariates, in line with a causal relationship . NeverthThis study provides evidence for impairments in performance monitoring, including conflict monitoring (N2) and neurophysiological error processing (ERN and Pe) in preterm-born individuals. While the conflict monitoring impairments in the preterm group do not seem to be identical to those in individuals with ADHD, early neurophysiological error processing may be a biomarker linked to the increased risk for ADHD. Since previous studies have demonstrated that error detection processes are malleable  and alsoS1 FileAppendix A–Excluding preterm-born individuals with a research diagnosis of ADHD from the analysis. Appendix B–Analysis controlling for IQ. Appendix C–Analysis of the males only. Appendix D–Analysis of the age-matched subsample.(DOCX)Click here for additional data file.S1 Dataset(CVS)Click here for additional data file.


We present the first-ever autologous stem cell transplantation (ASCT) outcome data from a secondary-care healthcare facility. Albeit exact details of patient and disease characteristics and co-morbidity scores for all patients are not available, the engraftment and survival data is very similar to those published from large tertiary-care cancer centres, both regionally and internationally. Transplant Related Mortality (TRM) of 3.1% is within the expected range and includes a patient who died of acute drug reaction (ADR) during conditioning chemotherapy, prior to the ASCT. Furthermore, cyclophosphamide mobilization chemotherapy is given in the outpatient setting. This study is important in terms of healthcare resource optimization as well as patients’ convenience and highlights that ASCT can be performed in a safe and effective manner with comparable survival rates even at a DGH, provided the centre stays abreast with the recent developments and can offer its patients with standard of care treatment of the era. Autologous stem cell transplantation (ASCT) remains the leading method of bone marrow transplantation (BMT), both in the upfront and in the salvage settings, either with a curative-intent or as a survival-prolonging strategy for multiple myeloma (MM) and lymphoma. Child et al., (2003) demonstrated that high-dose therapy (HDT) followed by ASCT prolongs survival in transplant-eligible MM patients, whereas this strategy holds potential for cure for a fraction of patients with relapsed/refractory Hodgkin’s (HL) and Non-Hodgkin’s Lymphoma (NHL) . Since the stem cells are derived from the same individual, ASCT carries a superior morbidity and mortality profile in comparison with allogeneic transplant. There are fewer acute and chronic transplant-related complications and no graft-vs-host disease (GVHD), albeit at the expense of higher relapse rate . There are also less healthcare resources consumed and, hence, it is being performed both in the inpatient as well as outpatient settings globally . In addition, ASCT may yet rarely be performed at the secondary-level healthcare centres which may house facilities and manpower to perform ASCT and adhere to international standards . Great Western Hospital (GWH) in the southwest city of Swindon, England is one such secondary-level centre which is JACIE-accredited and perform ASCT regularly. It would be enormously convenient for patients to receive this potentially curative, state-of-the-art treatment within their cities/towns or in the vicinity and not have to travel several hundred miles. Though ASCT is being performed in secondary-care centres in Western countries, no long-term ASCT outcomes data is available from them. The purpose of this study was to learn if ASCT outcomes performed at a district general hospital (DGH) in the United Kingdom, equivalent to a secondary-care centre, are any different from those performed at large, tertiary care cancer centres. The outcome variables presented are engraftment, transplant-related mortality (TRM), overall survival (OS) and progression-free survival (PFS). We further present general trends observed during the 18-year study period. 2 whereas those with lymphoma received either ESHAP  or ICE , both with or without Rituximab 375 mg/m2. After the peripheral blood stem cell (PBSC) count of >/= 10 x 106/L was achieved, patients were sent to National Health Services Blood and Transplant (NHSBT) centre at the Oxford University Hospital (OUH), located 30 miles northeast of Swindon, for stem cell apheresis. The harvested cells were then processed and stored at OUH and were requested a week prior to the infusion day. PBSCs were transported to the patient’s room in a dry shipper, in the vapour phase of liquid nitrogen, and the temperature of the water bath was consistently maintained below 40°C. DMSO toxicity could be reduced by limiting infusion to 1ml DMSO/kg of recipient weight/day; the equivalent of no more than 10mls of thawed cells/kg/day (using 10% DMSO). PBSC infusion was then performed after 24 hours of myeloablative chemotherapy.This was a retrospective review of 129 consecutive patients at GWH, between January 1998 and December 2015. All patients underwent ASCT as per the EBMT (European Society for Blood and Marrow Transplantation) criteria. For PBSC mobilisation, MM patients received cyclophosphamide 3,000mg/m6 CD34+ PBSC/kg of body weight (BW) were collected. All patients achieved target stem cell dose. Patients who proceeded to HDT after successful PBSC collection received conditioning with BEAM  or melphalan, either full dose of 200mg/m2 or intermediate dose of < 200mg/m2. PBSC infusion was performed on D0 with a minimum CD34 + PBSC dose of 2 x 106/kg BW. Standard anti-infective prophylaxis and supportive care were rendered in the peri- and post-ASCT period. Anti-infective prophylaxis in the peri-ASCT setting included acyclovir and fluconazole whereby acyclovir was continued until 90 days, post-transplant. In the post-ASCT period, trimethoprim/sulfamethoxazole was continued till 120 days post-transplant and ciprofloxacin until the first 14 days, at a minimum. We retrieved data on patient demographics (age and gender), ASCT trends across 2 decades, haematological disorders during the study period, engraftment: stratified according to conditioning regimen used, TRM: defined by death within 100 days of ASCT, and OS and PFS: stratified according to transplanted disorders. Data was collected via homogenized EBMT registry via Minimal Essential Data-A (MED-A) forms onto spreadsheets. Data was analyzed and survival curves were generated using the Winstat software.Apheresis was continued until at least 6 × 10Over the last decade, the advent of newer chemo/immunotherapeutic agents has changed the entire outlook of haematological malignancies. Novel agents have substantially prolonged survival of MM whereas Rituximab-based combination chemotherapy offered better cure rates for B-cell NHL . GWH has also kept abreast with developments and has rendered its patients with the standard-of-care treatment, either through National Health Service (NHS), industry or via compassionate drug fund (CDF). Since the first ASCT in 1998, GWH has performed more ASCT in males  than in females . The median age was slightly younger in males (53.7 vs 55.3) and the median age at ASCT, regardless of the disorder, was 57.3 (19-71), which increased over the study period. The median number of ASCT performed was 6.5, meeting the JACIE accreditation standard of a minimum of 5 ASCT procedures per annum . This progressively increased over the 18-year period with the least number of transplants performed in 1998 (2) and the most in 2015 (16). MM, Diffuse Large B-Cell Lymphoma (DLBCL) and Hodgkin’s Lymphoma (HL) accounted for the majority of ASCT with other NHL subtypes accounting for the rest. The median ages in different disease subgroups are depicted in BEAM was used in 57.4% of patients conditioning regimen whereas melphalan was used in the remaining 42.5%, either in full or reduced dose. All patients but 2 did not achieve neutrophil engraftment and both had received BEAM conditioning. 5 patients did not accomplish platelet engraftment, 4 from the BEAM arm and 1 from the intermediate-dose melphalan cohort. The engraftment data is shown in Although retrospective, we herein present the first-ever ASCT outcome data from a secondary-level healthcare facility. Albeit exact details of patient, disease characteristics and co-morbidity scores for all patients are not available, the engraftment and survival data are very similar to those published from large tertiary-care cancer centres, both regionally and internationally . TRM of 3.1% is within the expected range and includes a patient who died of acute drug reaction during conditioning chemotherapy, prior to the actual ASCT. Furthermore, cyclophosphamide mobilisation chemotherapy is given in the outpatient setting. In conclusion, this study is important in terms of healthcare resource optimization as well as patients’ convenience and highlights that ASCT can be performed in a safe and effective manner with comparable survival rates even at a DGH, provided the centre stays abreast with the recent developments and can offer its patients with standard of care treatment of the contemporary era.The authors declare no competing financial interests to disclose for this study.


Limited evidence has been obtained on varicella-zoster virus (VZV) infection in patients with breast cancer as a complication related to adjuvant radiotherapy. We conducted a cohort study aimed to assess the risk of VZV infection in this patient setting.We used the National Health Insurance Research Database to identify 65,981 patients with breast cancer in Taiwan who underwent breast surgery between 2000 and 2011. After a 1:1 propensity score match was obtained between patients with and without radiotherapy, a competing risk regression model was constructed to estimate the hazard ratios and the incidence rate difference (IRD) of VZV infection in the patients with breast cancer receiving radiotherapy and those not receiving radiotherapy.After adjusting for covariates, the radiotherapy cohort showed a 1.51-fold higher risk  of subsequent VZV infection than the nonradiotherapy cohort. Furthermore, VZV infection risk was 3.85-fold higher among patients aged >65 years who received radiotherapy than among those of the same age who did not receive radiotherapy . The risk increased with adjusted hazard ratio of 6.6  and 7.08  in follow-up period less than 3 months and 3–5 months respectively.Radiotherapy was associated with an increased risk of VZV infection among patients with breast cancer. The risk was significantly higher in older patient (>65 years old) and/or those who received chemotherapy. Regular clinical follow-up and additional serological testing in the first 5 months after radiotherapy are recommended. Varicella-zoster virus (VZV) infection, which can cause chicken pox and herpes zoster, usually presents as localized skin disease that shows little preponderance to serious symptoms or sequelae such as neuropathy, vasculopathy, or retinal necrosis . VZV infBreast cancer is the most common cancer among women worldwide, with more than one million new diagnoses each year . Many paPatients with breast cancer who underwent breast surgery were identified from the Registry for Catastrophic Illness Patient Database (RCIPD). The RCIPD is a subdatabase of the National Health Insurance Research Database (NHIRD) and contains records of patients with catastrophic illness from 1997 to 2011. The NHIRD contains data from the National Health Insurance program, a single-payer health insurance program mandatory for all citizens (23.74 million residents) with a coverage rate of approximately 99% . The RCIThe NHIRD encrypts patient personal information to protect privacy and provides researchers with anonymous identification numbers associated with relevant claims information, including sex, date of birth, medical services received, and prescriptions. Therefore, patient consent is not required to access the NHIRD. This study was approved to fulfill the condition for exemption by the Institutional Review Board (IRB) of China Medical University (CMUH104-REC2-115-CR3). The IRB also specifically waived the consent requirement.We selected patients with breast cancer (ICD-9-CM: 174) who received and did not receive RT during 2000–2010. The RT date was considered the index date of this study. Demographic data only included patients’ age. Chemotherapy and hormone therapy (tamoxifen and letrozole) records after the index date were the treatment variables. History of hypertension (ICD-9-CM: 401–405), diabetes mellitus (ICD-9-CM: 250), hepatitis B (ICD-9-CM: 070.20–070.33), hepatitis C , HIV (ICD-9-CM: 042), systemic lupus erythematosus , and rheumatoid arthritis (ICD-9-CM: 714) before the index date were considered as comorbidity variables. The main outcome was VZV infection, including chickenpox (ICD-9-CM: 052) and herpes zoster (ICD-9-CM: 053).Patients who had been diagnosed with chickenpox or herpes zoster between entry into the database and the index date were excluded from the study. Propensity score matching was employed to match the RT cohort to the non-RT cohort with a matching ratio of 1:1. The matching criteria were age, index year, diagnosis year of breast cancer, all comorbidities, and all treatments.The age group, comorbidity variables, and treatment variables were defined as categorical variables and tested using Chi-square tests to evaluate the distribution of variables between the RT and non-RT cohorts. The difference in average age between the two cohorts was tested using the two-sample Student’s t-test. We calculated the incidence rate (IR) and incidence rate difference (IRD) to assess the absolute risk of VZV infection. Given the mortality of breast cancer, the competing risk regression model was applied to calculate the subhazard ratios (SHRs) of outcome with 95% confidence intervals (95% CIs). Adjusting for age group, comorbidities, and treatments, the multivariable competing risk regression model was used. Stratified analyses of follow-up period were conducted to assess the risk at different time intervals. Statistical analysis was performed using SAS version 9.4 . Statistical significance was accepted at p value < 0.05.In total, 65,981 patients with breast cancer who underwent breast surgery during 2000–2010 were identified in the RCIPD . After eMost patients who underwent RT were in the age range 41 to 65 years (73.7%) , as wereUsing the Kaplan–Meier method, the cumulative incidence of VZV infection in patients with breast cancer was discovered to be significantly higher in the RT cohort than in the non-RT cohort (p = 0.007) . In totaZoster incidences were 7.47 and 12.45 per 10000 person-years in the non-RT and RT cohorts, respectively . RT patiThe analysis of follow-up period stratification indicated that VZV infection risk was significantly high within 5 month post receiving RT . The adjIn this population-based study, we determined that the risk of VZV infection among patients with breast cancer was significantly higher in those who received RT than in those who did not . In the subgroup of patients who received chemotherapy, the patients with RT treatment showed a higher risk of VZV infection than those without RT . Among the patients over 65 years old, the highest risk was posed by RT . The VZV infection risk causing by RT was only significant within the first 5 months . Based on our findings, we conclude that RT contributes to the risk of VZV infection in patients with breast cancer. The RT-induced VZV infection is likely to be seen within the first 5 months, and later occurrences are less clearly related to the therapy.The activation of VZV is associated with compromised immunity due to conditions such as HIV infection , diabeteThe possible mechanism of RT induced VZV infection has been well discussed in the review article written by Ramirez-Fort et al . They poHowever, this study was subject to limitations. First, the NHIRD does not provide detailed information on potential confounding factors such as body mass index, smoking habits, alcoholic consumption, family history, or cancer stage. Second, we were unable to eliminate the impact of unobserved confounders because a retrospective study was conducted. Third, clinical data prior to 1995 was not available because the NHI of Taiwan was not implemented before this time. Therefore, we were unable to separately analyze the subgroup of patients who had ever (lifetime) received the related vaccination or experienced previous episodes of VZV infection. Moreover, the NHIRD provides no information on RT dose level, energy level, technique, target volume, or distribution. This precluded us from performing any analysis involving these variables.In conclusion, our study was the first to provide evidence using a large-scale nationwide cohort study to determine the role of RT in VZV infection among patients with breast cancer. Our finding demonstrated that the risk of VZV infection is higher in RT cohort than in non-RT cohort. Based on our study, we suggest additional attention to older breast cancer patients (>65 years old) and/or those who receive chemotherapy. Regular clinical follow-up and additional serological testing in the first 5 months after RT are recommended to ensure early diagnosis and appropriate treatment and thus prevent serious sequelae.