A propensity score-integrated approach for leveraging external data in a randomized controlled trial with time-to-event endpoints.

In a randomized controlled trial with time-to-event endpoint, some commonly used statistical tests to test for various aspects of survival differences, such as survival probability at a fixed time point, survival function up to a specific time point, and restricted mean survival time, may not be directly applicable when external data are leveraged to augment an arm (or both arms) of an RCT. In this paper, we propose a propensity score-integrated approach to extend such tests when external data are leveraged. Simulation studies are conducted to evaluate the operating characteristics of three propensity score-integrated statistical tests, and an illustrative example is given to demonstrate how these proposed procedures can be implemented.

lncRNA RMST suppresses the progression of colorectal cancer by competitively binding to miR-27a-3p/RXRα axis and inactivating Wnt signaling pathway.

Colorectal cancer (CRC) ranks the 3rd in cancer types globally. Long noncoding RNAs (lncRNAs) are related to the initiation and progression of CRC. The current study plans to reveal the action of rhabdomyosarcoma 2-associated transcript (RMST) in CRC. The results show that RMST is downregulated in CRC specimens and cell lines relative to normal specimens and a fetal normal colon cell line (FHC), respectively. Elevation of RMST represses cell proliferation and colony formation and induces cell apoptosis in CRC cells. Bioinformatic analysis reveals a binding site in RMST for miR-27a-3p. The direct association between RMST and miR-27a-3p is confirmed by dual luciferase reporter assay, RNA pull-down assay, and real time-quantitative polymerase chain reaction (RT-qPCR). miR-27a-3p is upregulated in CRC tumor specimens relative to normal specimens, and there is a negative correlation between RMST and miR-27a-3p in CRC tumor specimens. In addition, the effects of RMST overexpression are weakened by the elevation of miR-27a-3p. RMST and retinoid X receptor (RXRα) share the same complementary site with miR-27a-3p. The direct association between RXRα and miR-27a-3p is confirmed by RNA pull-down assay, RT-qPCR and western blot analysis. Overexpression of RMST induces RXRα expression and inactivates the Wnt signaling pathway by decreasing ß-catenin levels in CRC cells. Collectively, our findings reveal a pivotal role of RMST in regulating miR-27a-3p/RXRα axis and counteracting Wnt signaling pathway during the progression of CRC.

Long non-coding RNA RMST serves as a diagnostic biomarker in patients with carotid artery stenosis and predicts the occurrence of cerebral ischemic event: A retrospective study.

OBJECTIVES: The purpose of this retrospective study is to explore the diagnostic and prognostic roles of serum RMST in carotid artery stenosis (CAS). METHODS: Serum levels of RMST were detected in CAS patients, and the relationship between degree of carotid stenosis and RMST levels was analyzed. The ROC curve was drawn to evaluate RMST value in predicting the risk of CAS. Then, all CAS patients received a 5-year follow-up. K-M curve was used to analyze the significance of RMST on prognosis of CAS patients. Multi-factor cox logistic regression analysis was conducted to evaluate independent factors for outcome of CAS patients. RESULTS: An increased RMST expression was certified in CAS patients when compared with healthy controls. The increase of serum RMST expression was related to high degree of carotid stenosis. In addition, serum RMST was a possible diagnosis and an independent influencing factor of prognosis in patients with CAS. CONCLUSIONS: Raised serum RMST level was found in patients with CAS. Detecting RMST expression levels was of high value for predicting the occurrence and outcomes in CAS.

Analyzing differences between restricted mean survival time curves using pseudo-values.

Hazard ratios are ubiquitously used in time to event analysis to quantify treatment effects. Although hazard ratios are invaluable for hypothesis testing, other measures of association, both relative and absolute, may be used to fully elucidate study results. Restricted mean survival time (RMST) differences between groups have been advocated as useful measures of association. Recent work focused on model-free estimates of the difference in restricted mean survival through follow-up times, instead of focusing on a single time horizon. The resulting curve can be used to quantify the association in time units with a simultaneous confidence band. In this work a model-based estimate of the curve is proposed using pseudo-values allowing for possible covariate adjustment. The method is easily implementable with available software and makes possible to compute a simultaneous confidence region for the curve. The pseudo-values regression using multiple restriction times is in good agreement with the estimates obtained by standard direct regression models fixing a single restriction time. Moreover, the proposed method is flexible enough to reproduce the results of the non-parametric approach when no covariates are considered. Examples where it is important to adjust for baseline covariates will be used to illustrate the different methods together with some simulations.

Mortality of inactive hepatitis B virus carriers in Japan is similar to that of the general population.

AIMS: Data on the long-term outcomes of individuals with hepatitis B virus (HBV) infection who are hepatitis envelope antigen (HBeAg)-negative inactive carriers (ICs) are limited due to small numbers. We compared the long-term prognosis of well-defined ICs with that of age- and gender-matched general population controls. METHODS: A total of 526 HBeAg-negative patients who demonstrated alanine aminotransferase (ALT) level ≤40 U/L and HBV DNA level ≤4.3 log IU/ml at least three times within 1 year after the start of follow-up were enrolled as ICs. Inactive carriers were divided into two groups: Group A (n = 332), whose ALT level was ≤30 U/L and HBV DNA level was ≤3.3 log IU/ml, and Group B (remaining patients, n = 194). We determined the long-term prognosis of ICs and compared it with that of general population controls. We also analyzed factors associated with hepatitis B surface antigen (HBsAg) clearance and phase transition in ICs. RESULTS: There were no significant differences in hepatocellular carcinoma development or all-cause, liver-related, or non-liver-related mortality between Groups A and B. There was no significant difference in all-cause mortality between ICs and the general population. Low HBsAg level (≤3.0 log IU/ml) and the presence of fatty liver were associated with HBsAg clearance and high alpha-fetoprotein level was associated with phase transition. CONCLUSIONS: The long-term prognosis of well-defined ICs was similar to that of general population controls. In addition, the ICs had a high HBsAg clearance rate and low phase transition rate.

Significant reduction of long non-coding RNAs expression in bipolar disorder.

Long non-coding RNAs (lncRNAs) have been recently emerged as critical modulators of oxidative stress pathway. Likewise, rising evidence currently highlights dysfunction of oxidative stress pathways in bipolar disorder (BD) patients.In the current study, we evaluated the expression levels of H19, SCAL1 (LUCAT1), RMST, MEG3 and MT1DP lncRNAs in the PBMC from 50 patients with BD and 50 control subjects (male/female ratio in each group: 70%/30%). Expression levels of SCAL1, RMST and MEG3 but not H19 and MT1DP were considerably decreased in BD patients compared with healthy individuals. Such significant decrease in the expression of MEG3, RMST and SCAL1 was only reported in male BD patients compared with male controls. Substantial pairwise correlations were observed between expression levels of these lncRNAs in BD subjects. The area under curve values for RMST, MEG3 and SCAL1 were 0.70, 0.63 and 0.61 respectively. On the basis of this finding, RMST had the best efficiency in the discrimination of disease status between BD patients and controls. Taken together, the current results suggest a role for MEG3, RMST and SCAL1 lncRNAs in the pathogenesis of BD. In addition, peripheral expression levels of these lncRNAs might serve as potential peripheral markers for BD.

Knockdown of RMST Impedes Neuronal Apoptosis and Oxidative Stress in OGD/R-Induced Ischemic Stroke Via Depending on the miR-377/SEMA3A Signal Network.

Long non-coding RNAs (lncRNAs) have pivotal roles in regulating ischemic stroke (IS), including lncRNA rhabdomyosarcoma 2-associated transcript (RMST). The purpose of this report is to discover the functional mechanism of RMST. The expression detection of RMST, microRNA-377 (miR-377) and Semaphorin 3A (SEMA3A) was performed by quantitative real-time polymerase chain reaction (qRT-PCR). Oxygen and glucose deprivation/reperfusion (OGD/R) in N2a cells was used to mimic IS environment in vitro. Cell Counting Kit-8 (CCK-8) and flow cytometry were implemented to assess cell viability and apoptosis. Oxidative stress was analyzed via assaying the associated indicators. Dual-luciferase reporter, RNA pull-down and RNA immunoprecipitation (RIP) assays were jointly administrated for binding analysis between targets. SEMA3A protein level was measured using western blot. We found in IS serum samples, RMST was upregulated while miR-377 was downregulated. After the establishment of OGD/R-induced IS model, we found that the decreased RMST abrogated the OGD/R-triggered apoptosis and oxidative stress. Through the target analysis, miR-377 was shown to be sponged by RMST and the effects of RMST knockdown on OGD/R-induced cell injuries were related to miR-377 upregulation. Besides, SEMA3A served as a target gene of miR-377 and the mitigation of miR-377 for ischemic brain damages was achieved by downregulating SEMA3A. What's more, RMST could regulate SEMA3A by playing the sponge action on miR-377. Collectively, all these findings clarified that RMST repression retarded IS progression in vitro via SEMA3A downregulation by targeting miR-377, which represented a different perspective in the pathological development of IS.

RETRACTED: lncRNA RMST Enhances DNMT3 Expression through Interaction with HuR

Large bodies of studies have shown that the CRISPR/Cas9-based library screening is a very powerful tool for the identification of gene functions. However, most of these studies have focused on protein-coding genes, and, furthermore, very few studies have used gene reporters for screening. In the present study, we generated DNA methyltransferase 3B (DNMT3B) reporter and screened a CRISPR/Cas9 synergistic activation mediator (SAM) library against a focused group of lncRNAs. With this screening approach, we identified Rhabdomyosarcoma 2-Associated Transcript (RMST) as a positive regulator for DNMT3B. This was confirmed by activation of the endogenous RMST by SAM or ectopic expression of RMST. Moreover, RMST knockout (KO) suppresses DNMT3, while rescue with RMST in the KO cells restores the DNMT3 level. Finally, RMST KO suppresses global DNA methylation, leading to the upregulation of methylation-regulated genes. Mechanistically, RMST promotes the interaction between the RNA-binding protein HuR and DNMT3B 3' UTR, increasing the DNMT3B stability. Together, these results not only provide the feasibility of a reporter system for CRISPR library screening but also demonstrate the previously uncharacterized factor RMST as an important player in the modulation of DNA methylation.

EGb761 Protects Brain Microvascular Endothelial Cells Against Oxygen-Glucose Deprivation-Induced Injury Through lncRNA Rmst/miR-150 Axis.

In the present study, we aimed to illustrate the roles and working mechanisms of long non-coding RNA (lncRNA) rhabdomyosarcoma 2-associated transcript (Rmst) and EGb761 in oxygen-glucose deprivation (OGD)-induced brain microvascular endothelial cells (BMECs). OGD exposure augmented the level of Rmst while reduced the expression of miR-150 in bEnd.3 cells. MiR-150 could directly bind to Rmst in bEnd.3 cells. Rmst silencing abrogated the inhibitory influences on the proliferation and migration and the promoting impact on the apoptosis of bEnd.3 cells caused by OGD exposure. Rmst overexpression intensified OGD-induced injury in bEnd.3 cells. OGD induced the injury of bEnd.3 cells through Rmst/miR-150 axis. EGb761 attenuated the damage in bEnd.3 cells induced by OGD through targeting Rmst/miR-150 axis. EGb761 might be an effective therapeutic agent to protect brain microvascular endothelial cells from hypoxia-ischemia induced injury.

On the empirical choice of the time window for restricted mean survival time.

The t-year mean survival or restricted mean survival time (RMST) has been used as an appealing summary of the survival distribution within a time window [0, t]. RMST is the patient's life expectancy until time t and can be estimated nonparametrically by the area under the Kaplan-Meier curve up to t. In a comparative study, the difference or ratio of two RMSTs has been utilized to quantify the between-group-difference as a clinically interpretable alternative summary to the hazard ratio. The choice of the time window [0, t] may be prespecified at the design stage of the study based on clinical considerations. On the other hand, after the survival data have been collected, the choice of time point t could be data-dependent. The standard inferential procedures for the corresponding RMST, which is also data-dependent, ignore this subtle yet important issue. In this paper, we clarify how to make inference about a random "parameter." Moreover, we demonstrate that under a rather mild condition on the censoring distribution, one can make inference about the RMST up to t, where t is less than or even equal to the largest follow-up time (either observed or censored) in the study. This finding reduces the subjectivity of the choice of t empirically. The proposal is illustrated with the survival data from a primary biliary cirrhosis study, and its finite sample properties are investigated via an extensive simulation study.

Dynamic RMST curves for survival analysis in clinical trials.

BACKGROUND: The data from immuno-oncology (IO) therapy trials often show delayed effects, cure rate, crossing hazards, or some mixture of these phenomena. Thus, the proportional hazards (PH) assumption is often violated such that the commonly used log-rank test can be very underpowered. In these trials, the conventional hazard ratio for describing the treatment effect may not be a good estimand due to the lack of an easily understandable interpretation. To overcome this challenge, restricted mean survival time (RMST) has been strongly recommended for survival analysis in clinical literature due to its independence of the PH assumption as well as a more clinically meaningful interpretation. The RMST also aligns well with the estimand associated with the analysis from the recommendation in ICH E-9 (R1), and the test/estimation coherency. Currently, the Kaplan Meier (KM) curve is commonly applied to RMST related analyses. Due to some drawbacks of the KM approach such as the limitation in extrapolating to time points beyond the follow-up time, and the large variance at time points with small numbers of events, the RMST may be hindered. METHODS: The dynamic RMST curve using a mixture model is proposed in this paper to fully enhance the RMST method for survival analysis in clinical trials. It is constructed that the RMST difference or ratio is computed over a range of values to the restriction time τ which traces out an evolving treatment effect profile over time. RESULTS: This new dynamic RMST curve overcomes the drawbacks from the KM approach. The good performance of this proposal is illustrated through three real examples. CONCLUSIONS: The RMST provides a clinically meaningful and easily interpretable measure for survival clinical trials. The proposed dynamic RMST approach provides a useful tool for assessing treatment effect over different time frames for survival clinical trials. This dynamic RMST curve also allows ones for checking whether the follow-up time for a study is long enough to demonstrate a treatment difference. The prediction feature of the dynamic RMST analysis may be used for determining an appropriate time point for an interim analysis, and the data monitoring committee (DMC) can use this evaluation tool for study recommendation.

LncRNA RMST activates TAK1-mediated NF-κB signaling and promotes activation of microglial cells via competitively binding with hnRNPK.

This study aimed to explore the biological role and molecular mechanism of long noncoding RNA (lncRNA) rhabdomyosarcoma 2-associated transcript (RMST) in regulating microglial activation. Mouse microglial BV2 cells were cultured to establish the cell model of cerebral ischemic stroke by oxygen-glucose deprivation (OGD). We observed highly expressed RMST, increased expression of M1, and decreased expression of M2 markers in BV2 microglial cells stimulated with OGD. These alterations were reversed by RMST knockdown. Activation of transforming growth factor-beta-activated kinase 1 (TAK1)-mediated nuclear factor-κB (NF-κB) pathway was observed upon OGD stimulation, which was promoted by RMST through competitively binding with heterogeneous nuclear ribonucleoprotein K (hnRNPK), confirmed by RNA pull down and RNA immunoprecipitation (RIP) assays. Furthermore, RMST overexpressing-BV2 cells effectively enhanced neuronal apoptosis. In conclusion, RMST promoted OGD-induced microglial M1 polarization by competitively interacting with hnRNPK via TAK1-mediated NF-κB pathway, which will provide a basis for understanding the pathogenesis of cerebrovascular diseases.

Measuring Survival Benefit in Health Technology Assessment in the Presence of Nonproportional Hazards.

BACKGROUND: Proportional hazards (PH) is an assumption often made by researchers, despite evidence of nonproportionality in a significant proportion of clinical trials. In the presence of non-PH, the interpretation of hazard ratios, medians, and landmark survival as summary measures of treatment effect can become problematic. Several recent studies have recommended restricted mean survival time (RMST) as an alternative metric for survival analysis, particularly where non-PH may apply. OBJECTIVES: To determine the current approaches of health technology assessment (HTA) agencies to value assessment in the presence of non-PH, and the extent to which RMST is accepted as an alternative measure of treatment benefit. METHODS: Methodological guidelines published by 10 HTA agencies were reviewed to establish recommended approaches for presenting survival benefit from clinical trials. Published HTA reports for 23 oncology agents approved by the US Food and Drug Administration and the European Medicines Agency since 2014 were reviewed to determine how guidelines are implemented in practice and identify instances where the PH assumption was tested and RMST analyses reported. RESULTS: Testing for non-PH is not widely incorporated into HTA except by the UK National Institute for Health and Care Excellence. RMST is used infrequently but has been used in a number of countries, particularly by agencies that focus on cost effectiveness. CONCLUSIONS: HTA agencies vary in their approaches to non-PH. Most do not routinely check the PH assumption. RMST has played a role in assessing clinical benefit within HTA, although not consistently within countries (across drugs) or across countries (for the same drug).

Long non-coding RNA (LncRNA) RMST in triple-negative breast cancer (TNBC): Expression analysis and biological roles research.

Microarray showed that lncRNA RMST was differentially expressed in cervical cancer. Further experiments were conducted to detect the expression and biological function of RMST in triple-negative breast cancer (TNBC). Microarray was used to screen the differentially expressed lncRNAs in TNBC. QRT-PCR was applied to uncover the expression of RMST in TNBC tissues. The cell viability of RMST-transfected TNBC cells were probed by CKK-8 assay and colony formation assay. TUNEL assay was conducted to test the cell apoptosis and FCM assay was exerted to detect the cell cycle. The invasion and migration ability of transfected cells were examined by transwell assay. RMST played its biological function through regulating the mRNA or protein expression in cytoplasm. CCK-8 and colony formation assay unveiled that RMST could slow down the proliferation of TNBC cells to influence the tumor progression. TUNEL results revealed that RMST could enhance cell apoptosis in TNBC. The cell cycle detected by FCM assay indicated that RMST might induce the block of G0/G1 phase thus inhibiting TNBC cell proliferation. RMST overexpression could also restrain the invasion and migration abilities of TNBC cells. RMST played a role of tumor suppressor in TNBC through inhibiting cell proliferation, invasion and migration, enhancing cell apoptosis, and regulating cell cycle.

The oxidative stress-associated long non-coding RNAs in pancreatic cancer.

PURPOSE: A lot of people are dying from pancreatic cancer (PC) annually. The early detection of this cancer is particularly challenging due to the fact that symptoms tend to appear in advanced stages. It has been suggested that oxidative stress may play a role in the development of PC. Several genes regulate this process, including long noncoding RNAs (lncRNAs). There is no comprehensive study on the expression pattern of lncRNAs related to oxidative stress in PC patients. In the present case-control study, we quantified levels of oxidative stress-associated lncRNAs in PC patients versus healthy controls. PATIENTS AND METHODS: In the present study, we investigated the expression levels of lincRNA-p21, LUCAT, RMST, FOXD3-AS1, and MT1DP lncRNAs in the peripheral blood mononuclear cells (PBMCs) of 53 â€‹PC patients and 50 healthy controls. The association between lncRNA expression and clinical and pathological characteristics was also evaluated. RESULTS: The expression of lincRNA-P21 and rhabdomyosarcoma 2-associated transcript (RMST) lncRNAs in PC patients has significantly decreased. Expression of lncRNA RMST was significantly higher in TNM stage III-IV patients in comparison to TNM stage I-II patients. In addition, a significant positive association was recognized between candidate lncRNA expression, and finally, the AUC values of the expression levels of lincRNA-p21 and RMST were 0.60 and 0.61, respectively. CONCLUSIONS: Altogether, our study suggests a possible role of lincRNA-p21 and RMST lncRNAs in the etiology of PC pathobiology, and their biomarker role may be understood in future studies.

Sample size calculation for multi-arm parallel design with restricted mean survival time.

With the recent advances in oncology treatment, restricted mean survival time (RMST) is increasingly being used to replace the routine approach based on hazard ratios in randomized controlled trials for time-to-event outcomes. While RMST has been widely applied in single-arm and two-arm designs, challenges still exist in comparing RMST in multi-arm trials with three or more groups. In particular, it is unclear in the literature how to compare more than one intervention simultaneously or perform multiple testing based on RMST, and sample size determination is a major obstacle to its penetration to practice. In this paper, we propose a novel method of designing multi-arm clinical trials with right-censored survival endpoint based on RMST that can be applied in both phase II/III settings using a global χ2 test as well as a modeling-based multiple comparison procedure. The framework provides a closed-form sample size formula built upon a multi-arm global test and a sample size determination procedure based on multiple-comparison in the phase II dose-finding study. The proposed method enjoys strong robustness and flexibility as it requires less a priori set-up than conventional work, and obtains a smaller sample size while achieving the target power. In the assessment of sample size, we also incorporate practical considerations, including the presence of non-proportional hazards and staggered patient entry. We evaluate the validity of our method through simulation studies under various scenarios. Finally, we demonstrate the accuracy and stability of our method by implementing it in the design of two real clinical trial examples.

Inference for restricted mean survival time as a function of restriction time under length-biased sampling.

The restricted mean survival time (RMST) is often of direct interest in clinical studies involving censored survival outcomes. It describes the area under the survival curve from time zero to a specified time point. When data are subject to length-biased sampling, as is frequently encountered in observational cohort studies, existing methods cannot estimate the RMST for various restriction times through a single model. In this article, we model the RMST as a continuous function of the restriction time under the setting of length-biased sampling. Two approaches based on estimating equations are proposed to estimate the time-varying effects of covariates. Finally, we establish the asymptotic properties for the proposed estimators. Simulation studies are performed to demonstrate the finite sample performance. Two real-data examples are analyzed by our procedures.

lncRNA RMST is associated with the progression and prognosis of gastric cancer via miR-204-5p.

BACKGROUND: Exploring novel biomarkers for gastric cancer holds promise for enhancing patients' therapy and survival rates. lncRNAs and miRNAs have emerged as important biomarkers for various human cancers. However, the role of lncRNA RMST (RMST) in gastric cancer development and the mechanism underlying its function remains unclear. RESULTS: Significant upregulation of RMST was observed in gastric cancer tumor tissues. RMST levels showed strong correlation with patients' lymph node metastasis and TNM stage and serving as a predictor of adverse prognosis RMST negatively regulated miR-204-5p, which in turn mediated the inhibitory effects of RMST knockdown on gastric cancer cell growth and metastasis. CONCLUSION: RMST served as both a prognostic biomarker and tumor promoter by modulating miR-204-5p. Inhibiting RMST could represent a novel and potential therapeutic strategy for gastric cancer.

Therapeutic silencing of lncRNA RMST alleviates cardiac fibrosis and improves heart function after myocardial infarction in mice and swine.

Rationale: Cardiac fibrosis is an adverse consequence of aberrant fibroblast activation and extracellular matrix (ECM) deposition following myocardial infarction (MI). Recently, long noncoding RNAs (lncRNAs) have been reported to participate in multiple cardiac diseases. However, the biological functions of lncRNA rhabdomyosarcoma 2-associated transcript (RMST) in cardiac fibrosis remain largely unknown. Methods: The role of RMST in regulating cardiac fibroblast (CF) proliferation, fibroblast-to-myofibroblast transition (FMT), and ECM production, which were induced by transforming growth factor-ß1, was evaluated through immunofluorescence staining, cell contraction assay, cell migration assay, qRT-PCR, and western blot. The therapeutic effect of RMST silencing was assessed in murine and porcine MI models. Results: The present study showed that RMST expression was upregulated and associated with cardiac fibrosis in murine and porcine MI models. Further loss-of-function studies demonstrated that RMST silencing in vitro significantly inhibited CF proliferation, FMT, and ECM production. Accordingly, RMST knockdown in vivo alleviated cardiac fibrosis and improved cardiac contractile function in MI mice. Moreover, RMST acted as a competitive endogenous RNA of miR-24-3p. miR-24-3p inhibition abolished, while miR-24-3p agomir reproduced, the RMST knockdown-mediated effects on CF fibrosis by regulating the lysyl oxidase signaling pathway. Finally, the therapeutic potential of RMST knockdown was evaluated in a porcine MI model, and local RMST knockdown significantly inhibited cardiac fibrosis and improved myocardial contractile function in pigs after MI. Conclusion: Our findings identified RMST as a crucial regulator of cardiac fibrosis, and targeting RMST may develop a novel and efficient therapeutic strategy for treating fibrosis-related cardiac diseases.