ClinicalTrials.gov identifier, NCT03823287) and LUCERNE  trials that aimed to assess efficacy, safety, and durability of faricimab, the first bispecific antibody for intraocular use, which independently binds and neutralizes both angiopoietin-2 and vascular endothelial growth factor-A (VEGF-A) versus aflibercept in patients with neovascular age-related macular degeneration (nAMD).To describe the design and rationale of the phase 3 TENAYA , and investigator assessment. The primary efficacy end point was BCVA change from baseline averaged over weeks 40, 44, and 48. Secondary end points included the proportion of patients receiving every-8-week, every-12-week, and every-16-week faricimab and anatomic outcomes. Safety outcomes included incidence and severity of ocular and nonocular adverse events. From week 60, faricimab-treated patients followed a personalized treatment interval (PTI), a novel protocol-driven treat-and-extend regimen with interval adjustment from every 8 weeks to every 16 weeks based on individualized treatment response measured by anatomic criteria, functional criteria, and investigator assessment of patients’ disease activity.Rationale for trial design and PTI approach.The TENAYA and LUCERNE trials were the first registrational trials in nAMD to test fixed dosing regimens up to every 16 weeks based on patients' disease activity in year 1 and incorporate a PTI paradigm during year 2. The PTI approach was designed to tailor treatment intervals to individual patient needs, to reflect clinical practice treatment practice, and to reduce treatment burden.The innovative trial design rationale for the TENAYA and LUCERNE trials included maximizing the benefits of angiopoietin-2 blockade through dosing up to every 16 weeks and PTI regimens based on patients' disease activity while fulfilling health authority requirements for potential registrational efforts. Patients were randomized in a 1:1 fashion to 2 treatment arms. Randomization was stratified by baseline BCVA Early Treatment Diabetic Retinopathy Study letter score as assessed on day 1 , low-luminance deficit on day 1 (< 33 letters or ≥ 33 letters), and region . Low-luminance BCVA was measured by placing a 2.0-log unit neutral density filter (Kodak Wratten 2.0) over the best correction for that eye and having the participant read the normally illuminated Early Treatment Diabetic Retinopathy Study chart.Patients randomized to the aflibercept arm received intravitreal aflibercept 2.0 mg every 4 weeks for 3 monthly initial doses and then continued on an every-8-week regimen up to week 108 according to the approved label of aflibercept. Patients randomized to the faricimab arm initially received intravitreal faricimab 6.0 mg every 4 weeks up to and at week 12 (4 injections) and then received every-16-week, every-12-week, or every-8-week dosing up to week 60 based on disease activity assessments at weeks 20 and 24. Disease activity assessments were guided by central subfield thickness (CST) and BCVA criteria and by investigator assessment. Patients with anatomic or functional signs of disease activity at weeks 20 or 24 were treated with every-8-week or every-12-week faricimab, respectively . From weFrom weeks 60 to 108, patients randomized to faricimab received treatment according to a personalized treatment interval (PTI), further described below . A study duration of 112 weeks allowed for evaluation of the role of faricimab in the treatment of patients with nAMD under a flexible, individualized regimen approach during the second year of the study.These were double-masked studies, with the assessor physician, BCVA examiner, and patients masked to treatment assignment. All patients attended all visits, with a sham procedure administered when no study treatment was administered. No rescue treatments were permitted within the study, given that the purpose was to evaluate extended dosing with faricimab. However, patients could be withdrawn from the study if they were deemed to require rescue therapy.To participate in the trials, patients were required to have active treatment-naïve macular neovascularization  or OCT. Patients were included if they had a CNV lesion size of up to 9 disc areas on FFA with a CNV component area of more than 50% of the total lesion size on FFA.Only 1 eye was assigned as the study eye. If both eyes were considered eligible (per the inclusion and exclusion criteria), the eye with the worse BCVA, as assessed at screening, was to be selected as the study eye.Patients were excluded if, among other factors, they had CNV resulting from causes other than nAMD or had undergone cataract surgery or treatment for complications of cataract surgery with steroids or yttrium–aluminum–garnet laser capsulotomy within 3 months before day 1. A complete list of inclusion and exclusion criteria is provided in Central reading centers (CRCs) conducted a masked assessment of patient eligibility during screening based on a review of color fundus photographs, OCT images, and FFA images to ensure that CNV secondary to AMD met the ocular inclusion criteria for the study. During the study treatment period, the CRCs provided a masked evaluation of all ocular images, including color fundus photographs, FFA images, optional indocyanine green angiography images, OCT images, and optional OCT angiography images to allow for accurate and unbiased assessment of the disease at study visits, including at weeks 20 and 24.Because of the global nature of the trials executed across 271 sites, 2 CRCs, the Duke Reading Center and the Vienna Reading Center, needed to be included to manage the images and methods across a wide span of time zones and to provide site support and training, if needed. This highlighted the need to ensure that assessments could be reproduced and replicated by both CRCs. A harmonization process was undertaken that yielded excellent concordance in grading of images between the CRCs, and a manuscript on this exercise is in progress. For the trial, dual reads for CST were performed by 2 independent readers. If discrepancies were noted, these were adjudicated by a third reader. In the TENAYA and LUCERNE trials, spectral-domain OCT images were obtained with the Spectralis , Cirrus (Carl Zeiss Meditec), and Topcon  devices.Patients randomized to aflibercept were treated at every-8-week intervals starting at week 16 after the 3 every-4-week initiation doses  for the entire duration of the study. Although every-4-week ranibizumab was used as a comparator in the phase 2 program, aflibercept was chosen as a comparator in the faricimab phase 3 programs for both diabetic macular edema and nAMD because it has a single approved labeled dose (2.0 mg) and treatment regimen worldwide, which provides the same comparator in all countries for the phase 3 faricimab development program, satisfying regulatory requirements globally. Furthermore, aflibercept was dosed at fixed every-8-week intervals, providing a suitable comparator against which to evaluate the potential for extended dosing with faricimab dosed up to every 16 weeks. The decision to use aflibercept as a comparator aligned well with the findings from the 2020 American Society of Retina Specialists Preferences and Trends survey, which reported that aflibercept was the agent that retina specialists most commonly use as first-line therapy.After the 4 monthly initiation doses of faricimab, patients subsequently were treated at fixed intervals ranging from every 8 weeks to every 16 weeks based on disease activity assessments at weeks 20 and 24. Disease activity criteria were prespecified and are noted in At week 24, 12 weeks after the last initiation dose, patients underwent the second disease activity assessment, and if any criteria were met, they were treated and kept on a fixed every-12-week dosing regimen until week 60. Patients already receiving every-8-week treatment were assessed for disease activity at week 24, but were not treated because they already were receiving every-8-week treatment. Finally, the remaining patients who did not meet disease activity criteria at weeks 20 and 24 were treated at week 28 after the last initiation dose and continued on a fixed every-16-week regimen until week 60 .Figure 1The treatment intervals in the phase 3 trials were based on the phase 2 STAIRWAY and AVENUE trial designs. The extended dosing was based on the results of the STAIRWAY trial, which showed that nearly two-thirds of all faricimab-treated patients did not show disease activity 12 weeks after the last initiation dose and were eligible to try every-16-week dosing. In the STAIRWAY trial, a single disease activity assessment (based on OCT and BCVA criteria and investigator assessment) at week 24 enabled patients in the every-16-week arm with active disease to be dosed at an every-12-week interval.,The AVENUE trial demonstrated that faricimab 6.0 mg administered at every-8-week or every-4-week intervals was well tolerated and resulted in comparable vision and anatomic gains as those achieved with every-4-week ranibizumab. In a pharmacokinetic analysis of samples from the phase 2 trials, faricimab maintained high concentrations in the vitreous and demonstrated durable intraocular VEGF suppression in the aqueous humor for at least 8 weeks compared with 4 weeks with ranibizumab. In addition, sustained suppression of intraocular angiopoietin-2 was observed with faricimab. Although faricimab 6.0 mg every 4 weeks was shown to be well tolerated in the AVENUE trial, no efficacy advantage over every-8-week dosing was shown. For this reason, every-8-week dosing was selected as the minimum treatment interval in the TENAYA and LUCERNE trials based on disease activity status at week 20, and every 16 weeks was selected as the maximum treatment interval based on data from the STAIRWAY trial and pharmacokinetic and pharmacodynamic analyses from the phase 2 trials.At week 60, all patients in the faricimab arm were scheduled to receive an active dose of faricimab and the second phase of the study began, in which faricimab-treated patients were dosed based on a PTI regimen. The PTI was a protocol-driven modified treat-and-extend (T&E) regimen with interval adjustment based on individualized treatment response as measured by CST and BCVA criteria. Treatment intervals could be extended in 4-week increments or reduced in 4- or 8-week increments to a minimum of every 8 weeks or a maximum of every 16 weeks or could be maintained according to calculations based on functional and anatomic criteria and clinical assessment by the investigator ; Fig S1 For example, for a patient with an at least 10-letter decrease in BCVA, compared with the highest BCVA during a study drug dosing visit that was attributable to nAMD activity, the treatment interval was reduced by 4 weeks. In contrast, for a patient who showed a similar worsening in BCVA but also showed an at least 50-μm increase in CST compared with the average from the last 2 study drug dosing visits, the treatment interval was reduced by 8 weeks. Some scenario examples are illustrated in The primary efficacy end point was the change from baseline in BCVA averaged over weeks 40, 44, and 48. The BCVA outcome measure is based on the Early Treatment Diabetic Retinopathy Study visual acuity chart assessed at a starting distance of 4 m. Averaging BCVA over 3 time points reduced the impact of measurement variability between visits, as well as intraobserver and interobserver variability. Thus, it may be a more robust measure of the true treatment effect on BCVA than measurement at a single time point, potentially providing a more precise measurement of treatment effect. Additionally, given the design of the TENAYA and LUCERNE trials, and therefore the variation in treatment schedules, averaging also minimized any potential impact of time since last active treatment on outcomes, and therefore allowed for a fairer comparison across treatment arms.,Secondary efficacy objectives included the proportion of patients receiving faricimab every 16 weeks, every 12 weeks, and every 8 weeks; evaluation of efficacy of faricimab on additional BCVA outcomes; and anatomic outcomes, such as change from baseline in CST over time, proportions of patients with absence of intraretinal fluid, and proportions of patients with absence of subretinal fluid over time. Other secondary objectives included the number of study drug injections received through weeks 48, 60, and 112. Preclinical evidence on dual angiopoietin-2 and VEGF-A inhibition suggests that the vessel-stabilizing effects of angiopoietin-2 impact vascular leakage and inflammation,One of the key objectives of the TENAYA and LUCERNE trials was to investigate the safety of faricimab. To ensure the safety of all patients during the conduct of the study, several safety assessments were included in the study design for year 1 and continue to be included through year 2. These include regular ophthalmologic assessment, adverse event monitoring, and protocol-specified laboratory safety tests. Additional safety imaging assessments were permitted at the principal investigator’s discretion, as required. An independent data monitoring committee monitored safety and study conduct on an ongoing basis.ocular vasculitis and retinal vasculitis. Cross-checks between the electronic case report form recorded clinical data and reports of adverse events were performed on an ongoing basis by the sponsor as part of the medical data review plan. Safety was assessed through descriptive summary of ocular and nonocular adverse events, deaths, and ocular assessments. Clinically significant laboratory abnormalities and clinically significant vital sign abnormalities were reported as adverse events and were evaluated as part of the adverse event assessments. Participant description of treatment-emergent adverse events was matched with Medical Dictionary for Regulatory Activities thesaurus terms, and the incidence and severity were summarized by treatment arm.All adverse events, including serious adverse events and adverse events of special interest, were required to be recorded on the adverse event electronic case report form and reported to the sponsor. Intraocular inflammation; infectious endophthalmitis associated with intravitreal injection; retinal detachment, tear, or both; iatrogenic traumatic cataracts; and increased intraocular pressure, as well as the potential nonocular risk of arterial thromboembolic events were adverse events of special interest based on experience with other intravitreal anti-VEGF monotherapies. Individual occurrences of these events were evaluated and documented by the study sites. The protocol defined the verbatim terms to be used when recording intraocular inflammation adverse events in the electronic case report form, which corresponded to Medical Dictionary for Regulatory Activities Preferred Terms. The Medical Dictionary for Regulatory Activities version 23.1 dictionary used in the study included the terms t test; 2.5% 1-sided type I error rate; and a 10% dropout rate. A nominal type I error penalty of 0.0001 was taken for each time the independent data monitoring committee reviews unmasked data before the formal analysis of the primary efficacy end point. The intention-to-treat population consisted of 1329 patients  randomized in the study.The study aimed to enroll a total sample size of approximately 320 patients per arm, which would provide more than 90% power to show noninferiority of faricimab compared with aflibercept in the intention-to-treat population, using a noninferiority margin of 4 letters in BCVA and under the following assumptions: no difference in the mean change from baseline in BCVA between the 2 treatment arms; standard deviation of 14 letters for the change from baseline in BCVA averaged over weeks 40, 44, and 48; 2-sample The per-protocol population consisted of all patients randomized who received at least 1 dose of study treatment and who did not have a major protocol violation that impacted the efficacy evaluation or treatment interval determination. The safety-evaluable population consisted of all patients who received at least 1 injection of active study drug (faricimab or aflibercept). Efficacy end points were analyzed using the intention-to-treat population, and safety outcomes were summarized using the safety-evaluable population. Additional efficacy analyses based on the per-protocol population also have been conducted.Changes in BCVA from baseline (primary outcome) were compared using a mixed model for repeated measures, which assumed an unstructured covariance structure. The model included the change from baseline at weeks 4 to 48 as the response variable, the categorical covariates of treatment group, visit, visit by treatment group interaction, and baseline BCVA (continuous), as well as randomization stratification factors as fixed effects. Comparisons between the 2 treatment arms were made using a composite contrast over weeks 40, 44, and 48. Missing data were imputed implicitly by the mixed model for repeated measures. Although continuous secondary outcomes were analyzed using a mixed model for repeated measures, binary secondary end points were analyzed using stratified estimation for binomial proportions. The proportion of patients in each treatment group and the overall difference in proportions between treatment groups were estimated using the weighted average of the observed proportions and the differences in observed proportions over the strata defined by randomization stratification factor of baseline BCVA score, low-luminance deficit, and region. The estimates and confidence intervals were provided for the adjusted mean (for continuous variables) or weighted proportion (for binary variables) for each treatment group and for the difference between the 2 treatment groups. The efficacy analyses were tested at a significance level of 0.0497, and all confidence intervals were 2-sided and at the 95.03% level.The TENAYA trial commenced recruitment in February 2019, and the LUCERNE trial commenced recruitment in March 2019. Primary end point analysis was completed for both trials in January 2021, and both studies are ongoing as of the date of this publication.The TENAYA and LUCERNE trials are global phase 3 studies that enrolled more than 1300 patients with treatment-naïve nAMD to evaluate extended fixed treatment regimens of every-8-week to every-16-week faricimab immediately after the initial doses in the first year of the study based on disease activity assessment criteria. This description of the study design provides an understanding of the unique features of the TENAYA and LUCRENE trials, which were informed by the early clinical results for faricimab.,Although intravitreal anti-VEGF therapy targets the abnormal blood vessels and decreases permeability, potentially leading to regression of pathologic vasculature, it does not address inflammation and possibly fibrosis mediated by angiopoietin-2. Given the effects of angiopoietin-2 in vascular destabilization, neutralization of angiopoietin-2 combined with VEGF-A may have the potential to restore vascular stability and to induce maturation of vessels through restoring angiopoietin-1 and tyrosine kinase with immunoglobulin-like domains signaling, which in turn reduces vascular leakage, neovascularization, and inflammation, as well as vascular responsiveness to the effects of VEGF-A. Together, these effects contribute to a multitargeted approach to achieving vascular maturity compared with intravitreal anti-VEGF monotherapy.,In the phase 2 STAIRWAY trial, faricimab demonstrated sustained efficacy through extended durability on fixed every-12-week and every-16-week dosing, with comparable vision and anatomic gains versus every-4-week ranibizumab.4,,,,,Alternate treatment regimens to fixed monthly dosing have been investigated in patients with nAMD, with a view to minimizing treatment burden and using a more individualized treatment approach.,In the TENAYA and LUCERNE trials, faricimab-treated patients follow a PTI approach from week 60. Extended treatment intervals adjusted based on patients’ individual responses to treatment allow personalized faricimab dosing, which may reduce the number of treatment visits, thereby reducing the treatment burden while still maximizing visual gains. This reflects a significant unmet need in patients with nAMD.In conclusion, the phase 3 TENAYA and LUCERNE trials are evaluating the potential of faricimab, the first bispecific antibody that independently binds and neutralizes both VEGF-A and angiopoietin-2, to address an unmet clinical need for more durable therapies in nAMD that sustain BCVA gains over time.


The proposed multi-channel system is validated by measuring the ballistocardiogram (BCG) and pulse wave, which paves the way for future portable remote human physiological signals monitoring devices.Flexible sensor arrays are widely used for wearable physiological signal recording applications. A high density sensor array requires the signal readout to be compatible with multiple channels. This paper presents a highly-integrated remote health monitoring system integrating a flexible pressure sensor array with a multi-channel wireless readout chip. The custom-designed chip features 64 voltage readout channels, a power management unit, and a wireless transceiver. The whole chip fabricated in a 65 nm complementary metal-oxide-semiconductor (CMOS) process occupies 3.7 × 3.7 mm Flexible electronic devices are widely used in detecting human physiological signals for their advantages of being attachable and wearable ,2,3,4,5.Among the sensors, the flexible pressure sensors, which imitate the human sensory system, are widely applicated in wearable devices ,9,10. FlThe combination of sensors and near-sensor chips reduces communication power consumption . The speThis paper proposes a remote health monitoring system to acquire ballistocardiogram (BCG) and pulse waves, which integrates a flexible piezoresistive pressure sensor array and a wireless multi-channel voltage readout chip. We also proposed a nonlinear resistance readout circuit to calibrate the inversely proportional resistive response of the pressure sensor.R2 = 0.99. The pressure sensor performance is studied. The electronic signal from the pressure sensors is collected and processed. In our sensor system, the relationship between resistance and voltage is nonlinear. A specific chip is needed to process such signal. The inversely proportional resistance-to-voltage converter could be used for nonlinearity correction. The sensitivity of the whole signal path, including the sensor and readout circuit, is obtained by taking Equation (1) into Equation (4). Assuming that the pressure measurement range is from 50 kPa to 1000 kPa, the sensitivity curves of the signal path of the linear and inversely proportional readout are shown in The minimum identifiable input change ocessing ,21.2. To further reduce the size and power of the proposed system, we utilize a self-designed 64-channel signal readout system on chip (SoC) to process the voltage-to-digital conversion and data transmission. As shown in This chip supports dual-mode communication. In the wireless mode, the chip core consumes 2.28 mW from a 1 V supply, and the power distribution is shown in We set up a high-density sensor array which is compatible with multi-channel signal readout. Here we show a case study to collect and process human physiological signals. BCG is an indicator of human heart activity. As shown in The sensor signal is amplified and recorded by the signal processing circuit described in The pressure sensor array can also be used to monitor the pulse wave during sleep. As shown in The flexible pressure sensor array, together with the custom-designed multi-channel signal readout chip provides a low-power solution to wearable health monitoring devices. The nonlinear resistance readout circuit calibrates the inversely proportional characteristic of the pressure sensor. The high linearity between output voltage and pressure simplifies the reconstruction process compared to traditional designs. This method is suitable for applications that need to directly record the pressure change. The nonlinear resistance-to-voltage conversion is easy to implement by simply decreasing the reference resistance in the resistor divider. The readout circuit becomes more sensitive at high-pressure conditions, common in BCG applications using bodyweight to provide pressure.The robustness of physiological signal recording in a moving human body is important in wearable devices. As the size of each sensor used in this paper is small compared to the curvature radius of the wrist and heel, the bending of the foot and the wrist causes little sensor deformation. Meanwhile, the good bending reliability of the flexible sensor leads to little signal distortion. The proposed non-linear readout scheme is not limited to the specific material used in the paper, but can also be applied to other resistive sensors with similar inversely proportional responses.Recent studies exhibit the feasibility of the cardiovascular (CV) parameters monitoring based on BCG. The amplitude between the second major wave and the third major wave is called J-K amplitude, which is an indicator of diastolic and systolic pressure . MultivaThis paper reports a remote health monitoring system based on a pressure sensor array with a wireless multiple channels sensor signal processing chip. The system includes a flexible resistive pressure sensor array, a data transmission module, and a display module. The system can detect BCG signals and multi-channel pulse waves in sitting, sleeping, and other life scenes and exhibits great potential in human health monitoring and medical diagnostics. The proposed system has the potential to record from much more sensor sites, leading to a more precise measurement.


Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer. Developing targeted therapies for MCC requires increased understanding of the mechanisms driving tumor progression. In this study, we aimed to identify genes, signaling pathways, and processes that play crucial roles in determining disease-specific survival in MCC. We analyzed the gene expression of 102 MCC tumors and identified genes that were upregulated among survivors and in patients who died from MCC. We cross-referenced these genes with online databases to identify the pathways and processes in which they function. Genes upregulated among survivors were mostly immune response related and genes upregulated among patients who died from MCC function in various pathways that promote cancer progression. These results could guide future studies investigating whether these genes and pathways could be used as prognostic markers, as markers to guide therapy selection, or as targets of precision therapy in MCC.Background: Merkel cell carcinoma (MCC) is a rare but highly aggressive neuroendocrine carcinoma of the skin with a poor prognosis. Improving the prognosis of MCC by means of targeted therapies requires further understanding of the mechanisms that drive tumor progression. In this study, we aimed to identify the genes, processes, and pathways that play the most crucial roles in determining MCC outcomes. Methods: We investigated transcriptomes generated by RNA sequencing of formalin-fixed paraffin-embedded tissue samples of 102 MCC patients and identified the genes that were upregulated among survivors and in patients who died from MCC. We subsequently cross-referenced these genes with online databases to investigate the functions and pathways they represent. We further investigated differential gene expression based on viral status in patients who died from MCC. Results: We found several novel genes associated with MCC-specific survival. Genes upregulated in patients who died from MCC were most notably associated with angiogenesis and the PI3K-Akt and MAPK pathways; their expression predominantly had no association with viral status in patients who died from MCC. Genes upregulated among survivors were largely associated with antigen presentation and immune response. Conclusion: This outcome-based discrepancy in gene expression suggests that these pathways and processes likely play crucial roles in determining MCC outcomes. Merkel cell carcinoma (MCC) is a neuroendocrine carcinoma of the skin with a poor prognosis. The survival rate of MCC varies significantly; population-based studies from New Zealand, Finland, and the United States revealed 5-year disease-specific survival rates of 45%, 59%, and 73%, respectively ,2,3. In In a recent study by Harms et al., targeted DNA and transcriptional profiling of 63 and 26 pre-defined, cancer-relevant genes, respectively, was performed. The study revealed that oncogene-activating mutations in PIK3CA, IDH2, and JAK2 were associated with poorer disease-specific survival in MCPyV-positive cases. Improved disease-specific survival was associated with higher expression of the pro-inflammatory transcripts IDO1, IFNG, and GZMA in MCPyV-positive cases, whereas high expression of the oncogene transcripts BRAF, RET, and UBE2C was associated with poorer survival in MCPyV-negative cases . A previIn this study, we aimed to identify genes, processes, and pathways that play crucial roles in causing MCC-specific death and MCC-specific survival that may be targeted in the treatment of MCC or may be used in guiding therapy selection or as prognostic markers. We investigated transcriptomes from the primary MCC tumors of 102 patients to identify the genes that were most significantly upregulated among survivors and among patients who died from MCC. We subsequently cross-referenced these genes with gene ontology and pathway databases to identify the biological processes and signaling pathways associated with these differentially expressed genes.Data on patients diagnosed with MCC in Finland from 1983 to 2013 were obtained from the Finnish Cancer Registry and Helsinki University Hospital files. Clinical details were extracted from hospital records. Formalin-fixed, paraffin-embedded (FFPE) tissue blocks from primary tumors were retrieved from the pathology archives. MCC diagnoses were confirmed in a blinded fashion from our earlier studies according to well-established criteria by two researchers with special expertise in MCC pathology . The stuThe patients were subdivided into the following groups: the poor prognosis group (patients who died from MCC) and the good prognosis group . The genes that were upregulated in the tumors of patients in the poor prognosis group when compared with the good prognosis group will be referred to as death-associated genes (DAGs); genes that were upregulated in tumors of patients in the good prognosis group when compared with the poor prognosis group will be referred to as survival-associated genes (SAGs).MCPyV detection from paraffinized tumor blocks was performed previously and is described in detail elsewhere . BrieflyRNA extraction and sequencing were performed on FFPE primary tumor samples from 120 patients from whom sufficient representative tumor tissue was available. Fourteen patients were excluded from the study as the corresponding samples did not pass the quality control during the processing of sequencing data. A further four patients were excluded because their tumor MCPyV status was not known. The final sample size was 102 patients.Two 10 µM sections from FFPE MCC samples of a cancer-representative area were used as starting material for the RNA extractions. RNA extraction was performed with a QIASymphony SP instrument  and QIAsymphony RNA extraction kit , following the manufacturer’s protocol. The quality and quantity of the extracted RNA were measured using a 2100 Bioanalyzer . The average RNA integrity number (RIN) was 2.1.RNA sequencing was performed by the sequencing unit of the Institute for Molecular Medicine Finland (FIMM) Technology Centre, University of Helsinki. Prior to library preparation, 1 µL of 1:1000 ERCC RNA spike-in control was added to each sample. A QuantSeq 3′ mRNA-Seq Library kit  was used to prepare the RNA sequencing library in 48 sample batches. The library preparation was performed according to the manufacturer’s instructions and is described in detail elsewhere [Htseq-count files from Bluebee sequencing process were read into R   . Data frp-value < 0.05; these were genes found to be upregulated in samples of the poor prognosis group. SAGs had a negative logFC with BH corrected p-value < 0.05; these were genes found to be upregulated in samples of the good prognosis group. Altogether, we identified 79 genes differentially expressed between patients in these two groups.A differential expression test of genes between the good and poor prognosis groups was performed using estimateDisp and exactTest function from edgeR with default parameters. DAGs were defined as those with a positive logFC with Benjamini–Hochberg (BH) corrected x-axis and the 79 genes on the y-axis. Both axes were clustered using 1-Pearson correlation coefficient as the distance measure and the linkage method Ward.D2. The rows of the matrix were scaled to have a mean of 0 and a standard deviation of 1.To visualize the gene expression patterns of these 79 identified genes, we drew a conventional heatmap with 102 patients on the http://amp.pharm.mssm.edu/Enrichr, accessed on 31 December 2021) [p-values.The gene ontology and signaling pathway analyses of DAGs and SAGs were performed by separately cross-referencing the lists of DAGs and SAGs with the Gene Ontology Biological Process (GO BP), the Gene Ontology Molecular Function (GO MF), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway databases using Enrichr ,20. EnriStatistical analysis was performed with SPSS statistics 26.0 software . The MCC-specific prognostic effects of tumor MCPyV status, sex, age at the time of diagnosis, tumor location, and the presence of lymph node or systemic metastasis at diagnosis were assessed by Kaplan–Meier analysis and logrank test. The factors that were significantly associated with MCC-specific survival in univariate analyses were included in a Cox regression multivariate analysis. In both uni- and multivariate survival analyses, MCC-specific survival was calculated from the date of diagnosis to death considered to be due to MCC, censoring subjects alive on their last follow-up date, and subjects who died from a cause unrelated to MCC. The correlation between the abundance of tumor-infiltrating, CD3-positive lymphocytes and the expression levels of the genes CD74, TAP2, PSME1, HLA-DRA, and HLA-E was investigated by calculating Pearson’s product–moment correlation in each case. The abundance of tumor-infiltrating lymphocytes was investigated previously and is described in detail elsewhere; data were available in 80/102 cases [In order to evaluate the consistency of our findings, we repeated the differential expression test of genes between the good and poor prognosis group, this time only including the 40 patients who survived at least 3 years after initial diagnosis in the good prognosis group. We subsequently cross-referenced the differentially expressed genes yielded by this control analysis with the GO and KEGG databases in the manner described in n = 15) and MCPyV-negative (n = 13) tumors of patients who died from MCC.In order to investigate potential relationships between DAG-expression and negative MCPyV status, we repeated the differential expression test of genes between MCPyV-positive (p < 0.001). Detailed patient clinical data are shown in p-value < 0.05. A summary of the upregulated genes, along with their corresponding logFC (fold change) values, false detection rates (FDR), and p-values, can be found in The differential expression test of genes revealed a total of 50 DAGs and 29 SAGs with a BH corrected p-values in p-value calculated using the BH method for correction for multiple hypotheses testing. The top 10 KEGG pathway terms most significantly enriched by DAGs and SAGs can be found in the same format in The top 10 GO BP and GO MF terms most significantly enriched by the DAGs and SAGs, respectively, can be found arranged according to their Cancer-relevant processes, functions, and pathways enriched by the DAGs included the PI3K-Akt signaling pathway, the MAPK signaling pathway, and angiogenic processes. Considering angiogenesis, the most significantly enriched GO terms included regulation of vascular associated smooth muscle cell migration, positive regulation of vascular endothelial cell proliferation, vascular endothelial growth factor receptor 2 binding, and vascular endothelial growth factor receptor binding. Among the most significantly enriched KEGG pathway terms was VEGF signaling pathway and among the DAGs was vascular endothelial growth factor A (VEGFA). Five of the DAGs were associated with the KEGG pathway term PI3K-Akt signaling pathway  and four of the DAGs were associated with the KEGG pathway term MAPK signaling pathway .The DAGs also exhibited enrichment of developmental pathways and processes, such as the GO PB terms chordate embryonic development , in utero embryonic development , and skeletal system development .The processes, functions, and pathways most significantly enriched by the SAGs were mostly related to immune response, particularly to antigen processing and MHC-dependent antigen presentation, and to T-cell mediated immune response. Examples include the KEGG pathway term antigen processing and presentation  and the GO BP terms T cell receptor signaling pathway  and positive regulation of lymphocyte proliferation .p-values of <0.05, enriched by the DAGs and SAGs, are presented in Supplementary Tables S1 and S2.Complete lists of all the GO BP, GO MF, and KEGG pathway terms that had p-values were no longer significant owing to the increased number of differentially expressed genes. The DAGs in this control analysis also exhibited enrichment of several terms related to mitosis as well as chromatin binding, the latter in part caused by the inclusion of several genes encoding histone proteins; the main finding that was lost in this control analysis was the DAG VEGFA, and thereby several GO and KEGG pathway terms related to angiogenesis. Complete lists of the differentially expressed genes yielded by this control analysis, as well as the GO and KEGG pathway terms significantly enriched by them, are provided in Supplementary Tables S3–S5.Including only patients who were still alive 3 years after diagnosis in the good prognosis group resulted in an increased amount of differentially expressed genes. The number of genes upregulated in the good prognosis group increased to 82, including 22/29 of the original SAGs; the number of genes upregulated in the poor prognosis group increased to 118, including 40/50 of the original DAGs. Considering the SAGs of this control analysis, the vast majority of the most significantly enriched GO and KEGG pathway terms were still immune response-related, especially related to antigen processing and MHC-dependent antigen presentation. The GO and KEGG pathway terms most significantly enriched by the DAGs in this control analysis still included embryonic developmental processes. The KEGG pathways PI3K-Akt and MAPK signaling pathway were still enriched by the same DAGs, with the exception of FGFR2 instead of VEGFA in both cases; however, the The differential expression test of genes between MCPyV-positive and MCPyV-negative tumors of patients who died from MCC yielded 100 genes significantly upregulated in MCPyV-negative tumors. Of these genes, eight  were among the DAGs. A complete list of these 100 genes is provided in Supplementary Table S6.The correlation analysis between an abundance of CD3-positive, tumor-infiltrating lymphocytes and the expression levels of the five SAGs causing the enrichment of the KEGG pathway antigen processing and presentation  revealed a significant and positive Pearson’s product–moment correlation in each case. Detailed results are presented in p < 0.001). A list of these 136 genes, together with their logFC-values in GSE39612 and our data, as well as a scatter plot of their expression, is provided in Out of the 250 genes most significantly differentially expressed between MCPyV-positive and MCPyV-negative tumors in GSE39612, 136 genes were present in our FFPE data. The correlation analysis between the logFC-values of these 136 genes based of MCPyV status in GSE39612 and our data yielded a Pearson correlation coefficient of 0.82 , a protumorigenic growth factor, and IGFBP5, which can either inhibit or potentiate insulin-like growth factor-signaling depending on the context . InsulinConsidering clinical implications, the aforementioned pathways and processes include several potentially viable targets for pharmacological intervention. Kervarrec et al. suggested that VEGFA may be a potential therapeutic target in MCC following promising results from drug trials in mouse models using the monoclonal antibody bevacizumab . The PI3Other notable DAGs included MYB, DST, and KIF23. MYB (c-MYB) encodes an oncogenic transcription factor that regulates processes such as cell proliferation and apoptosis in several other malignancies. Small-molecule inhibitors of c-MYB, such as celastrol and blumbagin, have shown promising results in cell cultures and mouse models . DST encAnother curious finding considering DAGs was enrichment of developmental pathways and processes. This may suggest that the cancer cells of more aggressive MCC revert to a more primitive, stem-cell-like state. An embryonic stem-cell-like gene expression pattern has been found to correlate with poor tumor differentiation and poor prognosis in other malignancies . So-callFor SAGs, we found a clear association with pathways and processes related to immune response. Almost all of the most significantly enriched GO BP, GO MF, and KEGG pathway terms were immune-response-related and were especially related to antigen processing, MHC-dependent antigen presentation, and T-cell mediated immune response. These findings underline the importance of a functional immune system, capable of creating a hostile tumor microenvironment, for MCC-specific survival. These findings are also consistent with previous findings on the abundance of tumor-infiltrating lymphocytes, notably CD8-positive T-cells, as a strong predictor of good survival in MCC ,8,9,11.The positive correlation between the abundance of CD3-positive, tumor-infiltrating lymphocytes and the expression levels of the SAGs causing the enrichment of the KEGG pathway antigen processing and presentation suggests that the abundance of CD3-positive lymphocytes functions as a surrogate marker for an immunogenic gene expression signature.Notable SAGs involved in processes not related to immune response included DUSP2, LLGL1, STAT1, and OGFR. DUSP2 encodes a phosphatase that deactivates protumorigenic MAP-kinases, the loss of which predicts a poor prognosis in bladder cancer . LLGL1 eOwing to the strong correlation between MCPyV-negativity and MCC-specific death, we repeated the differential expression test of genes based on MCPyV status within the poor prognosis group. Of the 50 DAGs, eight were significantly upregulated in MCPyV-negative tumors, suggesting that their prognostic relevance resulted at least in part from an association with MCPyV-negativity. The remaining 42 DAGs were not significantly differentially expressed based on MCPyV status within the poor prognosis group, suggesting a prognostic relevance regardless of viral status.It should be noted that the average quality of the extracted RNA was fairly poor (average RIN of 2.1), as is often the case with FFPE samples. However, it has been shown that 3′ tag counting, such as that used in this study, markedly decreases the amount of false positives when studying differential expression of genes in samples of varying RIN at the expense of decreased sensitivity . This stIn summary, we found several novel genes associated with disease-specific survival in MCC. The DAGs were most notably associated with angiogenesis, the PI3K-Akt signaling pathway, the MAPK signaling pathway, and embryonic developmental processes. The SAGs were most notably associated with antigen presentation and immune response. Further studies are required to determine if some of these genes could be used clinically as prognostic markers, as markers to guide therapy selection, or as targets of precision therapy in MCC.


The incidence of renal cell carcinoma (RCC) has doubled in the developed world within the last fifty years, and now it is responsible for 2–3% of diagnosed cancers. The delay in diagnosis and the not fully understood pathogenesis are the main challenges that have to be overcome. It seems that extracellular vesicles (EVs) are one of the key players in tumor development since they ensure a proper microenvironment for the tumor cells. The stimulation of angiogenesis and immunosuppression is mediated by molecules contained in EVs. It was shown that EVs derived from cancer cells can inhibit T cell proliferation, natural killer lymphocyte activation, and dendritic cell maturation by this mechanism. Moreover, EVs may be a biomarker for the response to anti-cancer treatment. In this review, we sum up the knowledge about the role of EVs in RCC pathogenesis and show their future perspectives in this field. Renal cell carcinoma (RCC) accounts for 2–3% of global cancer diagnoses . RCC is RCC is divided into three major histological subtypes: clear cell RCC (ccRCC), papillary RCC (pRCC), and chromophobe RCC (chRCC). Clear cell RCC is the most frequent type of RCC (responsible for 75% of cases). The second most common type of RCC is p-RCC (15%), followed by ch-RCC (5%) . Other tVHL). Its product—the VHL protein, —promotes the proteolysis of the hypoxia-inducible factors 1a (HIF-1a) and 2a (HIF-2a). These proteins are the key regulators of the hypoxic response in cells. The loss of VHL function results in increased expression and stabilization of HIF, which is necessary for tumor growth in cancer cells [The most common RCC subtype is associated with a defect in the von Hippel-Lindau gene .Yang et al. (2013) provided the first study about the potential role of RCC-derived EVs in the induction of immunosuppression. They found that EVs isolated from human kidney adenocarcinoma (ACHN) cells can inhibit T-cell proliferation by delivering Fas-ligand (FasL) on their surface . In their study, exosomes isolated from Rencacell culture induced the suppressive activity of MDSCs by Hsp70-related p-Stat3 activation . Stat3 iNext, it has been revealed that EVs can trigger dysfunction of NK cells in ccRCC patients. Xia et al. (2017) compared the NK cell function of ccRCC patients with benign tumor patients and healthy donors. Tumor-infiltrating NK cells of ccRCC patients presented an inhibitory phenotype and impaired function, which were associated with tumor progression. Exosomes from primary tumor cells of these patients were enriched in TGF-β1 (Transforming Growth Factor-β), which induces the TGF-β/SMAD pathway within NK cells. The SMAD (Suppressor of Mothers against Decapentaplegic) transcription factor regulates the proper activity of NK cells and its overexpression leads to their dysfunction , CD70, and CD147 are upregulated during RCC development and progression. A recent study by Himbert et al. (2020) showed increased expression of CA9, CD70, and CD147 in cells and EVs compared to normal tissues and indicated these proteins as specific markers of EVs in ccRCC .  and JNK  pathways. However, continuous stimulation caused by the strong expression of CD70 may disturb the proper regulation of immune response and induce an exhaustion effect of T-cells or even their apoptosis ,51. In nCD70 mediates apoptosis of lymphocytes in RCC culture, which can be partially blocked with anti-CD27 and anti-CD70 antibodies . Interesp < 0.0001, HR 1.95, 95% CI: 1.3–2.7) [CA9 is an enzyme that catalyzes the hydration of carbon dioxide to carbonic acid, expelling protons to the extracellular matrix. Therefore, it is in charge of intracellular pH regulation of tumor cells  increasing at the same time extracellular acidity, which has distinct effects on the immune response and plays an important role in the suppression of anticancer immunity within the TME ,60. For 1.3–2.7) . Circula1.3–2.7) . TherefoThe metabolic interplay between tumor cells and tumor-infiltrating lymphocytes (TILs) can be also altered by CD147, which contributes to cancer immune escape by modulation of tumor glycolysis and the transport of monohydrates . CD147, Additionally, it was shown that EVs can play a role in immune escape by transferring PD-L1 (Programmed cell death protein ligand 1). It is a transmembrane protein responsible for the modulation of the immune response. By binding to its receptor PD-1 on the T-cell surface it can inhibit their cytotoxic activity and keep the immune response in check. In the case of cancer, tumoral PD-L1 expression is an important mechanism of immune escape. PD-L1-transfer via EVs derived from T-cells inhibits their function and plays a key role in evading the immune response. Poggio et al. (2019) showed that this effect is not restricted to the local tumor site but may also occur, due to EV-mediated transport, in the lymph nodes in a syngeneic mouse model of prostate cancer . Wang etBesides proteins, EVs may improve the ability of tumors to avoid immunosurveillance by delivering also specific miRNA cargo. A miRNA is a short non-coding RNA of about 22 nucleotides that mediates gene silencing by guiding Argonaute proteins to target sites in the 3′untranslated region of target mRNAs . RCC tisLate diagnosis is one of the main reasons for the high mortality rate among patients with RCC. Metastasis is often found at initial diagnosis or even after curative treatment . For thiIn some cases, the authors have revealed that EVs differ between patients and healthy controls . Moreovep = 0.037). They also noted that this activity is positively correlated with microvascular invasion of RCC. Interestingly, these results were not observed in serum GGT  [As it was mentioned before, EVs can be useful not only for diagnosis but also for the staging of RCC. Horie et al. observed in cell culture experiments an increased activity of exosomal γ-glutamyl transferase (GGT) in the cell culture supernatant from neoplastic cells in comparison to the supernatant of normal ones. Additionally, they observed higher activity of the exosomal fraction of this enzyme in patients with advanced tumor stages  . More exRCC is a chemotherapy and radiotherapy-resistant tumor. Immunotherapy and target treatment  are the basis of available treatment protocols. However, some patients do not respond to the applied therapy, so new therapeutic options or methods which can increase the vulnerability of the tumor to used treatments are needed. The role of EVs in oncological therapy is still not well investigated. However, recently an increasing number of researchers have been conducting studies investigating the hypothesis that EVs may be crucial in tumor homeostasis.AXL and c-MET). These genes can be essential in sunitinib resistance development. Indeed, it was confirmed that an increased level of lnc-ARSR was negatively correlated with sunitinib response. Additionally, the authors have proven that the sEVs are the main way of intracellular transfer of lnc-ARSR between resistant and sensitive cells. Moreover, it was shown that the inhibition of sEVs formation or the packing of lnc-ARSR into them can be a method to restore sunitinib response [It was observed that sEVs can be a mediator of drug resistance in renal cell cancer. Qu et al. showed that the long non-coding RNA lnc-ARSR can compete with endogenous miR-34 and miR-449, and therefore increases the expression of their target genes  when T lymphocytes were stimulated by sEVs/IL-12 in comparison to PBS and non-modified EVs. Additionally, the usage of sEVs/IL-12 in research trials resulted in the highest killing rate and antigen-specific cytotoxic effect against cancer cells [The EVs may be also used in anti-tumor therapy. Zhang et al. showed that EVs derived from genetically modified RCC cells may increase the activation of T cytotoxic lymphocytes and the production of IFN-gamma . The moder cells . The poter cells , however+ and CD8+ T lymphocytes. The authors also conducted an experiment showing that intravenous injection of these EVs does not induce tumor immune tolerance in animals [Allium sativum)-derived sEVs can inhibit tumor cell proliferation and induce their apoptosis [Another research group noted that tumor-derived EVs effectively stimulate dendritic cell (DC) activation and upregulate CD11c, MHCII, and IL12 expression in DC. The use of DC loaded with tumor-derived (from RENCA cell line) EVs in a mice model of RCC significantly prolonged the survival time of the animals. Additionally, these EVs strongly promoted the recruitment of CD4 animals . Recentl animals . Furtherpoptosis . Additiopoptosis . Anotherpoptosis .Furthermore, it was noted that EVs can take part in the progression of metastatic disease. It seems that they can be a target in the treatment of advanced RCC. Jin et al. observed that EVs derived from RCC cells (cell line 786-O) can promote lung metastasis formation and tumor growth. They postulate that these effects are induced by MALAT-1. Potentially, the suppression of this transcript factor can reduce cells’ malignant behavior . All thiThe results of both experimental and clinical studies indicate that EVs are one of the key players in the immune response against RCC. The presence and content of EVs can induce immunosuppression that can be crucial in cancer development. Additionally, the molecular cargo of EVs may have a negative impact on intercellular signaling in the surrounding tissue and may reshape the TME towards facilitating tumor growth. Based on this information, it can be concluded that EVs are involved in resistance to treatment of RCC. They can decrease the sensitiveness for immune-check-point inhibitors in patients and block the stimulation of lymphocytes. Moreover, EVs may be a new therapeutic option in the future, due to positive results of in-vitro and in-vivo preclinical studies. Perhaps the detection of circulating EVs for the prediction and prognosis of oncological patient outcomes could have greater clinical implications in the future. However, randomized controlled trials are needed to verify these hypotheses.


Subgroup analysis showed that the association was significant in organ-specific diseases but not in systemic diseases. Risk magnitudes were generally higher among men compared to women. In conclusion, our findings provide evidence for an increased risk of autoimmune diseases in patients with TRD. Controlling chronic inflammation in hard-to-treat depression might play a role in preventing subsequent autoimmunity.Recent literature indicates that patients with depression had increased immune activation. We hypothesised that treatment-resistant depression (TRD), an indicator of non-responsive depression with long-term dysregulated inflammation, could be an independent risk factor for subsequent autoimmune diseases. We performed a cohort study and a nested case-control study to examine the association between TRD and risk of autoimmune diseases, and to explore potential sex-specific difference. Using electronic medical records in Hong Kong, we identified 24,576 patients with incident depression between 2014 and 2016 without autoimmune history and followed up from diagnosis to death or December 2020 to identify TRD status and autoimmune incidence. TRD was defined as having at least two antidepressant regimens and the third regimen to confirm previous treatment failures. Based on age, sex and year of depression, we matched TRD patients 1:4 to the non-TRD in the cohort analysis using nearest-neighbour matching, and matched cases and controls 1:10 using incidence density sampling in the nested case-control analysis. We conducted survival analyses and conditional logistic regression respectively for risk estimation, adjusting for medical history. Across the study period, 4349 patients without autoimmune history (17.7%) developed TRD. With 71,163 person-years of follow-up, the cumulative incidence of 22 types of autoimmune diseases among the TRD patients was generally higher than the non-TRD . Cox model suggested a non-significant association (HR:1.48, 95% CI: 0.99–2.24,  Approximately 280 million people worldwide live with depression, which is ranked a leading cause of disability and a major contributor to the global disease burden , 2. AntiThe multifactorial nature of depression also stimulates a growing interest in its impact on physical health. Due to the bidirectional crosstalk between the endocrine and immune systems, depression and inflammation often influence each other via the balance of cytokine messengers , 7. ReceEvidence on the association between TRD and autoimmune diseases was scarce. To date, a population-based study from Israel reported significantly higher rates of allergic diseases and positive antinuclear antibodies among patients with TRD than responders, whilst systemic autoimmune diseases had significantly increased likelihood of TRD . The croWe used the territory-wide routine EMR database  between 1993 and 2020 managed by the Hospital Authority, a statutory body which manages all public hospitals and clinics in Hong Kong and provides publicly funded healthcare services to all eligible residents (>7 million). Real-time updated patient data including demographics, dates of birth and death, dates of service attendances, all-cause diagnoses coded based on the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) and prescriptions were comprehensively recorded across the outpatient, inpatient and emergency settings for research and auditing purposes. The database has been used and described in previous epidemiological studies on depression, suicidal attempts and autoimmune diseases in Hong Kong , 20–25.We conducted a population-based study using both cohort design and nested case-control design in parallel, with the intention to preserve the advantages and complement the limitations of the other as we consider that cohort studies conventionally have a higher level of evidence, whereas case-control analysis was more appropriate for evaluating rare outcomes. The study period started in January 2014 and ended in December 2020. The cohort consisted of all incident patients aged above 10 years with diagnosis codes for depression  between January 2014 and December 2016 without history of diagnosis for depression since 1993, when the database first became available. Patients were excluded if they had history of studied autoimmune diseases before onset of depression, or if they died immediately after cohort entry.treatment-resistant (exposed) if they had taken at least two antidepressant regimens of adequate duration  and hadsiveness , 27.We analysed the prescription trajectory of all cohort participants from the date of depression onset until December 2020 to ascertain their TRD status, and matched patients with TRD 1:4 to the non-TRD based on their year of first depression diagnosis, and then age and sex using nearest-neighbour matching. The follow-up started from the prescription date of the third regimen (index date for patients with TRD) until any censoring events. The same index date was applied to the four non-TRD matches. Patients whose deaths or onset of autoimmune diseases occurred earlier than the assigned index dates were further excluded  for cases was assigned the same for their matched controls, who were alive and had no history of autoimmune diseases on the index date. We then determined the TRD status of all patients any time before index date and fitted a conditional logistic regression to estimate odds ratios (ORs) of onset autoimmune diseases associated with TRD. The model was adjusted for the same type of covariates as the cohort study.We replicated the analysis by changing the definition of autoimmune diseases to only organ-specific diseases or systemic diseases as outcome (cohort design) or case definition (nested case-control design) and conducted subgroup analysis by sex. Additionally, to address potential competing risk due to premature death, we applied Fine and Grey’s model to assess the subdistribution hazard ratio of autoimmune diseases in the cohort study as a sensitivity analysis. All analyses were performed using R 4.0.3 and cross-checked by two investigators.Figure p = 0.003). The mean time for patients to develop treatment resistance was 2.0 years (±1.6 years). After the index date, the mean follow-up periods were similar between groups .Across the follow-up in the cohort study, 4448 patients 17.7%) developed TRD. After matching and excluding patients who died or had history of autoimmune diseases by index date, 4349 patients with TRD and 16,253 patients with non-TRD had similar baseline age, sex distribution and overall history of psychiatric conditions on index date (Table 7.7% devep = 0.029).In the overall cohort of the nested case-control study, we excluded 614 patients 2.4%) who had a history of autoimmune diseases prior to depression. Among the remaining 24,576 patients, 226 patients (0.9%) had developed autoimmune diseases by December 2020 (cases). Of which, 225 had eligible matches of 2250 controls. There were no marked differences between the cases and controls in baseline age, sex distribution, history of physical and psychiatric conditions and depression duration on index date  per 10,000 person-years], including 77 diagnosed with organ-specific diseases and 36 with systemic diseases. In the cohort analysis, the most frequently developed types of autoimmune diseases were Graves’ disease , rheumatoid arthritis  and psoriasis . A similar pattern of autoimmune incidence was found in the nested case-control analysis , whilst the conditional logistic model in the nested case-control study showed a significant association between TRD and risk of autoimmune diseases with an adjusted OR of 1.67 . Similar results were obtained when the study outcome was stratified into organ-specific autoimmune diseases only, with an adjusted HR of 1.59  and an adjusted OR of 1.89 . The association between TRD status and systemic autoimmune diseases was not significant in the analyses using both study designs.Figures p = 0.032) in the nested case-control study. Despite non-significant model results, the effect magnitudes were generally higher among men in the association between TRD status and risk of overall autoimmune diseases in the cohort study (adjusted HR: 1.88 vs. 1.40), and in the association between TRD status and risk of organ-specific diseases in both study designs , compared with women.In the subgroup analysis, the association between TRD status and risk of autoimmune diseases was significant only in women with an adjusted OR of 1.67 , the magnitude and direction of which were consistent with that in the main analysis, after adjusting for competing risk due to premature death.In the sensitivity analysis using the Fine and Grey model in the cohort study, the HR of TRD status towards risk of overall autoimmune diseases was 1.47  at marginal significance in the cohort study and a significant adjusted OR of 1.67 (95% CI: 1.10–2.53) in the nested case-control study. We also identified that the increased risk more likely stemmed from organ-specific autoimmune diseases than systemic autoimmune diseases. The magnitude of risk among men appeared to be generally higher than in women in the studied associations from both study designs and consistent across subtypes of autoimmune diseases.Triangulation is the practice of increasing reliability of answers to epidemiological questions via integrating results from different study approaches, in which the approaches possess distinct and unrelated assumptions and potential bias . In our Our main findings were largely consistent with current observational studies. Prior to this work, a Hungarian retrospective cohort study analysed six types of somatic comorbidities and reported 22% increase in odds of autoimmune diseases among patients with TRD, despite the inability to determine whether TRD development preceded onset of comorbidities . A crossAs the patients with depression and autoimmune diseases in our study are predominantly women, it is reasonable that a significant association was observed only among women. However, the risk magnitudes of association appeared to be higher among men instead across study designs and outcome types. Despite non-significance, the observed trend was consistent with previous studies which found that the increased CRP was stronger in male patients with depression compared with female patients , 40. In Our findings reported herein underpin several implications with regards to understanding and managing hard-to-treat depression. Alongside existing etiological evidence, our study findings adds to support of the key role that inflammation plays in the pathogenesis of depression and treatment resistance, which renders ongoing exploration of anti-inflammatory agents and cytokine inhibitors as antidepressants valuable for patients without satisfactory treatment responses . In the There were also several limitations to our study. First, similar with other studies utilising nation-wide EMR as the data source, we lacked detailed psychometric measurements and clinical information, such as symptom severity or reasons for switching of drug regimens and dosage adjustment; it was thus challenging to determine whether patients stopped or switched medications due to inadequate initial dosing, atypical pharmacokinetics, adverse events, medication compliance or genuine lack of treatment responses. Given the absence of a universal definition of TRD, pseudo-resistance could not be entirely ruled out. Second, despite the higher evidence level of cohort study, the index dates manually assigned to the non-TRD patients may have occurred late if their matched TRD patients received the third regimen near the later stage of follow-up, therefore yielding a reduced number of events following a shorter observation period. The risk comparison using the cohort study design therefore presents a more conservative estimate. To address this, we performed the nested case-control analysis in parallel to maximise the number of observable events since the index dates were defined differently, with the study design less prone to selection bias compared with a conventional case-control study . Third, We found increased risk of autoimmune diseases in patients with TRD compared with patients who responded to antidepressants. Inflammation control in hard-to-treat depression might play an important role in the prevention of subsequent autoimmune conditions. Association of sex-specific disparities deserves further mechanistic investigation.Supplementary legendSupplementary materials


P = 0.026). Early mortality after surgical treatment was seemingly higher in the pre-Covid-19 era but the difference did not reach statistical significance . Mortality in patients treated conservatively, or turned down for surgery, was lower during the Covid-19 pandemic . The crude mean prevalence of the use rate of conservative or surgical treatment across the studies during Covid-19 and in the pre-Covid-19 era was comparable. The current increased incidence of MI mechanical complications might be a consequence of delayed presentation or restricted access to hospital facilities. Despite the general negative impact of Covid-19 on cardiac surgery volumes and outcomes and the apparent increase of the incidence of MI complications, the outcomes of their surgical and clinical treatment seem not to have been affected during the pandemic.This study aims to compare treatments and outcomes of mechanical complications of acute myocardial infarction (MI) during the Covid-19 and in the pre-Covid-19 era. Electronic databases have been searched for MI mechanical complications during the Covid-19 era and in the previous period from January 1998 to January 2020 (pre-Covid-19 era), until October 2021. To perform a quantitative analysis of non-comparative series, a meta-analysis of proportion has been conducted. Early mortality after surgical treatment was 15.0% while it was significantly higher after conservative treatment (62.4%) ( An 2Study design is available online (PROSPERO registration CRD42021276091), and database search is updated to the end of October 2021. Full details are included in Supplemental Material. This study complies with the Declaration of Helsinki. Local ethics committee approval was not required considering the study design .2.1MEDLINE and the Cochrane Library databases have been systematically. Search strategies including exploded MeSH terms have been used. Search strings have been reported as Supplementary Material. English language restriction was imposed. Additional articles by manually searching the reference lists from recent reviews and the extracted papers have been looked for. Attempts have been made at collecting unpublished data from the authors of potentially pertinent papers.2.2Letters, editorial, reviews, animal studies, and reports with duplication data have been excluded. PICOS study design was used for inclusion/exclusion criteria. To identify eligible studies, a two-step selection process has been applied. Three reviewers  checked the eligibility criteria and selected the studies for inclusion in the present systematic review. Three researchers  independently screened records for inclusion. They were blinded to each other’s decisions. Disagreements between individual judgements have been resolved by consensus. Studies were excluded if they did not meet the criteria.2.3Three investigators  independently extracted data from all eligible studies using a standardized Excel file, focusing on study design, study size, type of intervention, and outcomes. Any disagreement was solved by consensus. We assessed the study quality with the Newcastle–Ottawa quality assessment scale.2.42 = 0 indicates no between-study heterogeneity. Double arcsine transformations have been applied to the observed proportions identified across a collection of studies to make the transformed proportions follow a normal distribution to accurately estimate the summary proportion and increase the validity of the associated statistical analyses. Multiple meta-regression with the Knapp–Hartung adjustment has been used to test the influence of the publication date and Covid-19 on early mortality. A P value of 0.05 was considered statistically significant. Statistical analyses have been done using the packages “meta” and “metafor” of R software, version 4.0.5.To perform a quantitative analysis of non-comparative series, a meta-analysis of proportion has been conducted. To draw statistical inferences from heterogeneous studies, we employed non-iterative estimate of the inter-study variance component based on a random effects model (s2), taking into account that statistical heterogeneity is believed to be due to clinical diversity. TauEthic statement: This study complies with the Declaration of Helsinki. Reviews, meta-analyses, or descriptions of educational materials do not involve human subjects and do not require IRB review : 5–6. doi:10.4300/JGME-D-11-00005.1). This study does not directly involve human participants.Patient and public involvement: Patients or the public were not involved in the design, or conduct, or reporting, or dissemination plans of our research3P = 0.24). Early mortality after surgical treatment was seemingly higher in the pre-Covid-19 era, but the difference did not reach statistical significance (P = 0.135). Conversely, mortality in patients treated conservatively, or turned down for surgery, was lower during the Covid-19 pandemic (P = 0.001). The crude mean prevalence of the use-rate of conservative or surgical treatment across the studies during Covid-19 and in the pre-Covid-19 era was comparable, when considering only the studies reporting both the approaches . After adjusting for the publication date, the period relative to Covid-19 pandemic seemed to influence the effect size for early mortality after conservative treatment but did not have impact in the surgical group. Results are summarized in The pooled prevalence of early mortality independently on the treatment received during the Covid-19 pandemic was 31.1% . When compared to patients treated before the Covid-19 pandemic , there was no difference in the overall early mortality (independently on the treatment received) (46.4% vs 31.1%; 4Mortality for MI mechanical complications during Covid-19 pandemic was not dissimilar from the pre-Covid-19 era. However, underreporting of submersed populations of patients not reaching medical attention because of the gravity of the disease and/or the restricted access to specialized hospital facilities could have clearly affected our results. In support of this hypothesis, an increase in the incidence of out-of-hospital cardiac arrests and in the number of patients declared deceased on scene has been demonstrated in areas at high Covid prevalence ,11.Despite many reports advocated a worrisome disruption of the cardiac surgical and interventional activities due to the unprecedented overload of the healthcare facilities ,3,6,8,12Reasons underlying the results of surgery for MI complications and the apparent improved outcomes of conservative management during Covid-19 pandemic cannot be inferred with the present analysis. The modern improvements of intensive care and medical management might have played a role in the outcomes of the conservative subgroup, as suggested by the meta-regression analysis. However, significant selection and publication biases, implying a large population of critically ill patients not reaching medical attention, or a relatively more stable subset of patients being treated and reported during the pandemic, impedes to establish any explanatory or causative link in this context. However, it is intriguing to note that in the general impetus for publication during the Covid-19 era, there is a seemingly more frequent report of conservative treatments in the surgical literature. Whether this finding suggests that surgery is less offered in the recent era is difficult to ascertain, but it could at least partially balance the publication selection bias typical of the pre-Covid-19 periods. However, as this analysis is comparing consecutive rather than contemporary cohorts, the impact of the progressive advancements in the practice across the years in all the fields of medicine should also be considered. As additional limitations of this study, it remains difficult to evaluate if emergency cardiac surgical services have returned to normal in each country, and therefore, the “Covid era” has a different time span in each country. Moreover, there are no data about the percentage of patients with mechanical complications being offered or receiving surgery to be compared with the percentage of patients declined or turned down for surgery, in both eras; these data were not derivable from published studies and a possible “cherry picking” of low-risk cases by surgeons cannot be disproved.5Besides the inherent limitations of this study, this meta-analysis first and comprehensively described the current evidence on the management of MI complications during the pandemic. Despite the general negative impact of Covid-19 on cardiac surgery volumes and outcomes and the apparent increase of the incidence of MI complications, the outcomes of their surgical and clinical treatment seem not to have been affected during the pandemic.