Barrett's oesophagus with indefinite for dysplasia (BE‐IND) is a subjective diagnosis with a low interobserver agreement (IOA) among pathologists and uncertain clinical implications. This study aimed to assess the utility of p53 immunohistochemistry (p53‐IHC) in assessing BE‐IND specimens.P < 0.001) and increased IOA for all BE grades . An aberrant p53‐IHC pattern significantly increased the likelihood of reclassifying BE‐IND to definite dysplasia .Archive endoscopic biopsies with a BE‐IND diagnosis from two academic centres were analysed. First, haematoxylin and eosin‐stained slides (H&E) were reviewed by four expert gastrointestinal (GI) pathologists allocated into two groups (A and B). After a washout period of at least 8 weeks, H&E slides were reassessed side‐to‐side with p53‐IHC available. We compared the rate of changed diagnosis and the IOA for all BE grades before and after p53‐IHC. We included 216 BE‐IND specimens from 185 patients, 44.0 and 32.9% of which were confirmed after H&E slide revision by groups A and B, respectively. More than half the cases were reclassified to a non‐dysplastic BE (NDBE), while 5.6% of cases in group A and 7.4% in group B were reclassified to definite dysplasia. The IOA for NDBE, BE‐IND, low‐grade dysplasia (LGD) and high‐grade dysplasia (HGD)/intramucosal cancer (IMC) was 0.31, 0.21, −0.03 and −0.02, respectively. Use of p53‐IHC led to a >40% reduction in BE‐IND diagnoses (P53‐IHC reduces the rate of BE‐IND diagnoses and improves the IOA among pathologists when reporting BE with equivocal epithelial changes.  Barrett's oesophagus (BE) is an acquired metaplastic condition of the oesophagus, which develops in the context of chronic gastro‐oesophageal acid reflux.Indefinite for dysplasia in BE (BE‐IND) is diagnosed when the degree of cellular atypia is suggestive but not definite for dysplasia. The risk for BE‐IND progression is higher than non‐dysplastic BE (NDBE) and quoted in approximately 1.5 cases per 100 person‐years.et al. demonstrated that p53‐IHC improved IOA for dysplasia among 10 pathologists with no previous experience in p53‐IHC.Immunohistochemical staining for the tumour suppressor protein p53 (p53‐IHC) is a clinically applicable candidate marker. Evidence shows that aberrant p53 expression correlates with an increased risk of both incident and prevalent dysplasia.With this background, we set out to investigate the utility of p53‐IHC in the assessment of BE‐IND cases. We established a large multicentre cohort of samples with a previous BE‐IND diagnosis and evaluated the IOA and the rate of confirmed BE‐IND diagnosis by multiple pathologists.In this multicentre retrospective cohort study, we have analysed endoscopic tissue samples originating from patients with BE‐IND diagnosis from two academic referral centres in the United Kingdom  and the United States . Patients were identified within prospective research databases at each institution. At the time of screening, the Cambridge and Mayo Clinic databases included patients with BE diagnosis from 1999 to 2018 and 1997 to 2017, respectively. Inclusion criteria were: (i) age 18 years or older, (ii) endoscopic evidence of BE ≥1 cm in length; (iii) the presence of intestinal metaplasia (IM) on biopsies; and (iv) at least one biopsy from the study endoscopy was reported as BE‐IND. We excluded patients with a diagnosis of definite dysplasia of any grade either at a previous endoscopy or at the study endoscopy showing concomitant BE‐IND. All patients provided written, informed consent to be included in the research databases, and an ethics committee approval for this study was granted at both institutions . All authors had access to the study data and reviewed and approved the final manuscript.‘no dysplasia’, when study pathologist‐diagnosed gastric or intestinal metaplasia without dysplasia, or‘dysplasia’, when study pathologist diagnosed any degree of definite dysplasia .The slides from eligible patients were reviewed by four expert GI pathologists. All pathologists involved in the study are experienced in assessing BE samples, each having a regular case volume of ~10–20 cases per week for 8–20 years. Pathologists were divided into two groups (groups A and B) to ensure that each sample was assessed independently by one pathologist from group A and one pathologist from group B. To avoid clustering in the assessment, pathologists from the two groups were paired randomly so that one pathologist could be compared with any of the two pathologists from the other group. In the first stage, only H&E slides were evaluated. The pathologists did not take part in the selection of study cases. Although some of the Cambridge study pathologists might have reported the original BE‐IND diagnosis, we did not look at the intraobserver diagnosis, as this was outside the scope of the study. The histological assessment was made using the Vienna classification.The interobserver agreement (IOA) between pathologists in group A versus group B for each BE grade  was calculated. All authors had access to the study data and reviewed and approved the final version of the manuscript.Sections from historical blocks corresponding to slides with the original diagnosis of BE‐IND were cut. Immunostaining was carried out on the Leica BOND‐MAX™ system using Bond polymer refined detection reagents  and a monoclonal antibody for wild‐type and mutant forms of p53, NCL‐L‐p53‐D07 (Leica Microsystems UK Ltd). The antibody was diluted at 1:50. Following staining, slides were counterstained using the Leica Autostainer XL and then cover‐slipped using the Leica fully automated glass coverslip.Following a washout period of at least 8 weeks, each H&E slide was reviewed with matched p53‐IHC available by the same pathologists in groups A and B who assessed the samples in the first stage. The pathologists were blinded to their initial reading from the first stage of assessment. Aberrant p53‐IHC was regarded as either strong p53 staining compared to background level (overexpression) or focal absence of staining compared to background level (absent pattern), as illustrated in Figure et al. 2019Quantitative variables were described as means, medians with standard deviation (SD) and interquartile ranges (IQRs), where appropriate. Categorical variables were presented as counts and percentages of the cohort. The rates of changed diagnosis in the first and second histological assessment were compared using McNemar's test. The IOA for BE grades were generated using Cohen's kappa correlation coefficients (κ), with values ≤0 indicating no agreement, 0.01–0.20 no to poor agreement, 0.21–0.40 fair agreement, 0.41–0.60 moderate agreement, 0.61–0.80 substantial agreement and 0.81–1.00 almost perfect agreement. The κ values were generated with the ‘irr’ package  statement for cohort studies.The impact of p53‐IHC patterns on reclassifying the initial diagnosis (both to NDBE and dysplasia) was reported using odds ratios (ORs) calculated by median‐unbiased estimation (mid‐p) with 95% confidence intervals (CI). For all analyses, a No funders had any role in study design, data collection, data analyses, interpretation or writing of the report.n = 65, 35.1%; Mayo Clinic n = 120, 64.9%) were included in the study . The median BE length was 4 cm (IQR = 1–7 cm). Forty‐two patients had evidence of multifocal IND (22.7%) and 63 patients had persistent IND during the follow‐up . The median FU time was 5.3 years (IQR = 1.3–9.2 years).A total of 216 BE‐IND samples from 185 patients  and cellular atypia of unknown significance , and less often due to technical artefact . Cohort characteristics are presented in Table At the time of the BE‐IND diagnosis, 75 samples (34.7%) had no features of background inflammation and 132 samples (61.1%) had evidence of inflammation (98 samples with mild and 34 with moderate inflammation). In most cases, the diagnosis of BE‐IND was made due to background inflammation  and 129 cases in group B (59.7%) Figure . The BE‐The overall agreement among pathologists was only fair, with a κ = 0.23 (95% CI = 0.12–0.34) and a 57.4% agreement. On a BE grade level, the IOAs for NDBE, BE‐IND, LGD and HGD/IMC were 0.31 (95% CI = 0.19–0.44), 0.21 (95% CI = 0.07–0.34), −0.03 (95% CI = −0.50 to 0.43) and −0.02 (95% CI = −0.64 to 0.60), respectively. For all dysplastic cases, the IOA was 0.17 (95% CI = −0.17 to 0.51).Following at least 8 weeks' washout period, individual slides were reviewed by the same pathologists with p53‐IHC available Figures  and 2. TIn group A, 25 samples were scored as an aberrant p53‐IHC pattern , 19 as equivocal (7.7%) and 141 as a normal pattern (65.3%). In group B, 16 samples were reported as an aberrant pattern , eight as equivocal (3.7%) and 168 as a normal pattern (77.8%). There was a substantial agreement between pathologists recognizing the p53‐IHC aberrant pattern with a κ value of 0.64 (95% CI = 0.47–0.82) and a percentage agreement of 92.9%.n = 137) in group A and 71.8% (n = 155) in group B . Conversely, the rate of confirmed BE‐IND cases decreased significantly to 48 cases (22.2%) and 47 cases (21.8%) in groups A and B, respectively . Finally, a reclassification to definite dysplasia was made in 31 cases  in group A and 14 cases  in group B. The total number of cases with dysplasia also increased significantly , whereas the number of HGD/IMC remained similar, with 10 cases in stage I and 11 in stage II (P = 1.00). Figure The fraction of cases reclassified to NDBE in stage II assessment increased to 63.4%  increased from κ = 0.23 to κ = 0.39 (95% CI = 0.30–0.50), with a percentage agreement of 69.9%. The IOA for NDBE, BE‐IND, LGD and HGD/IMC increased to 0.46 (95% CI = 0.32–0.58), 0.26 (95% CI = 0.09–0.43), 0.49 (95% CI = 0.26–0.73) and 0.35 (95% CI = −0.13 to 0.83), respectively. Finally, for all dysplastic cases, the agreement increased to 0.44 (95% CI = 0.22–0.66). The changed IOA values are summarized in Table We then looked at how the p53‐IHC staining pattern impacted upon the subsequent grading of the initial BE‐IND diagnosis. We found a striking increase in the likelihood of a pathologist diagnosing definite dysplasia in the presence of an aberrant p53‐IHC pattern with an OR of 44.3 (95% CI = 18.8–113.0). Conversely, a non‐aberrant p53‐IHC pattern  was associated with an increased likelihood of NDBE diagnosis .n = 19), most of the samples were reclassified to dysplasia both within groups A and B pathologists . On the contrary, within the cases with non‐aberrant p53 pattern (n = 163), most of the samples were reclassified to NDBE  reviewed smaller cohorts of cases (n = 6–70).This study has several strengths. We have studied a large cohort of patients with BE‐IND from two high‐volume centres with geographical diversity, which helps to draw a clear conclusion on the role of p53‐IHC in this subgroup of patients. Although previous studies had demonstrated the added benefit of p53‐IHC in BE, BE‐IND cases were either absent or sparsely represented in these cohorts. We have used a robust methodology with four expert GI pathologists randomly paired, which allowed us to calculate the IOA of different pairs of observers. We chose this design as the number of cases was high, and each slide's assessment by all four pathologists would be unfeasible. In other studies, a larger group of pathologists (This study has a few limitations. The four expert pathologists involved in this study work in the same institution; therefore, the results might not be generalized to a broader community of pathologists with different degrees of experience. However, clinical guidelines recommend expert pathologist review in case of suspected dysplasia; therefore, BE‐IND should be reviewed by a pathologist with interest and experience in BE diagnosis. When the study was conceived digital pathology was not widely available, and a decision to have pathologists from a single institution had to be taken. In addition, criteria for BE‐IND diagnosis might have changed over time, although this is difficult to characterize. For this reason, some of the historical cases included in this study might not fully represent biopsies currently reported as BE‐IND; however, we felt inclusion for historical cases was required to build a sufficiently large cohort to address the study objective with statistical power.In summary, we provide evidence that p53‐IHC has a robust clinical utility to help the pathologist report cases with atypical cytological changes, with and without definite dysplasia features, and should be routinely used, particularly when BE‐IND diagnosis is considered.All the authors disclose no conflicts of interest.W Januszewicz: concept and design of the study, data analysis and interpretation, drafting and revising the manuscript; N D Pilonis: acquisition of data, drafting and revising the manuscript. T Sawas: acquisition of data, drafting and revising the manuscript; R Phillips: acquisition of data, drafting and revising the manuscript; M O'Donovan: histopathological assessment of the samples, analysis and interpretation of data, drafting and revising the manuscript; A Miremadi: histopathological assessment of the samples, analysis and interpretation of data, drafting and revising the manuscript; S Malhotra: histopathological assessment of the samples, analysis and interpretation of data, drafting and revising the manuscript; M Tripathi: histopathological assessment of the samples, analysis and interpretation of data, drafting and revising the manuscript; A Blasko: p53‐IHC staining and interpretation, acquisition of data, analysis and interpretation of data, drafting and revising the manuscript; D A Katzka: concept and design of the study, drafting and revising the manuscript; R C Fitzgerald: concept and design of the study, data analysis and interpretation, drafting and revising the manuscript; M di Pietro: concept and design of the study, data analysis and interpretation, drafting and revising the manuscript; all authors revised and accepted the final version of the manuscript.Figure S1. Sankey plot representing the impact of p53 immunostaining patterns in reclassifying the original BE‐IND diagnosis separately in Group A and Group B pathologists. BE‐IND; Barrett's oesophagus indefinite for dysplasia, NDBE; non‐dysplastic Barrett's oesophagus.Click here for additional data file.


TP53 mutations is associated with an unfavorable outcome in patients allografted for acute myeloid leukemia (AML), leading some to question the benefit of an allogeneic stem cell transplantation  for this patient group, although this has not been studied in a large cohort.The presence of TP53 mutation status was reported, were included in this study from the European Society for Blood and Marrow Transplantation.A total of 780 patients with AML in first complete remission, with either intermediate‐ or adverse‐risk cytogenetics, whose TP53 mutation at diagnosis  as compared to the cohort without (n = 601) . In patients with mutant TP53 AML with no evidence of either chromosome 17p loss (17p–) and/or complex karyotype (CK) , 2‐year OS was 65.2% . This was not significantly different to patients without TP53 mutations. In patients with mutant TP53 AML with either 17p– and/or CK , the OS was lower .Two‐year overall survival (OS) was impaired in patients (n = 179) with evidence of a TP53 mutations in AML following an allo‐SCT is not evident in patients with neither co‐occurring 17p– and/or CK, and these data inform decisions regarding allo‐SCT in patients with TP53 mutant AML.In summary, the adverse prognostic effect of  Allogeneic stem cell transplantation  provides a potentially curative treatment for patients with acute myeloid leukemia (AML) due to an ability to deliver both intensified chemotherapy and a potent graft‐versus‐leukemia effect.TP53 mutations account for approximately 10% of AML casesTP53 mutant AML post allo‐SCT.TP53 mutant myelodysplastic syndrome (MDS) after allo‐SCTTP53 myeloid malignancies. It is also unknown whether other cytogenetic and genetic features may affect the outcome of patients with TP53 mutant AML after an allo‐SCT, but is established in the nontransplant setting.TP53 mutant MDS present conflicting results as to the significance of coexisting cytogenetic abnormalities.TP53 mutations and coexisting complex karyotype in patients with MDS may lead to reduced overall survival (OS) due to increased relapse rates,TP53 with these other genetic features associated with increased risk of relapse post allo‐SCT,TP53 allele.TP53 mutant AML.TP53 mutations in patients with AML in first complete remission (CR1) that undergo an allo‐SCT and to identify any further determinants of transplant outcomes in this subgroup of patients.The aim of this Acute Leukemia Working Party (ALWP) study from the European Society for Blood and Marrow Transplantation (EBMT), was to elucidate the prognostic value of TP53 mutation status. Patients were only included in this analysis if their TP53 mutation status was reported to the EBMT registry. There was no restriction to inclusion with regards to age, conditioning intensity, or donor source. Monosomal karyotype was defined as per previous studies,We analyzed the outcomes of patients with AML who had intermediate or adverse risk cytogenetic abnormalities, who received an allo‐SCT in CR1 between 2015 and 2019, according to their The review committee of the ALWP of the EBMT registry approved and provided the data for this study. The EBMT is a voluntary group of more than 600 transplant centers that are required to report consecutive stem cell transplantations and follow‐ups once a year . PatientTP53 mutation, present vs absent) were compared by using χ2 statistics for categorical variables and the Mann–Whitney test for continuous variables. Acute and chronic GVHD was graded as per previous definitions.TP53 mutant AML to identify factors which might influence posttransplant outcomes in this group. In the Cox model, we included all major variables  and performed a stepwise selection of cytogenetics abnormalities . Results were expressed as the hazard ratio (HR) with a 95% confidence interval (95% CI). Two‐sided tests were used. To determine factors associated with time‐to‐event outcomes, a type 1 error rate was fixed at 0.05. Analyses were performed using R 4.1.1.Patient‐, disease‐, and transplant‐related characteristics for the 2 cohorts , which is consistent with previous observations.TP53 mutant subgroup is lower than in previous reports of newly diagnosed patients with TP53 mutant AMLIn patients with TP53 mutation, only 16% harbored a normal karyotype. In contrast, there was a highly statistically significant enrichment of chromosomal abnormalities in chromosomes 17p (31% vs 3%), 5q (49% vs 5%), and 7q (34% vs 8%) as well as complex (65% vs 12%) and monosomal karyotypes (41% vs 8%) in the cohort of patients with mutated TP53 mutation on transplant outcome was first examined by performing a univariate analysis, results of which are shown in Supporting Table TP53 mutation had an increased CIR of 55%  at 2 years, in comparison to those without, who had a CIR of 25.2%   in those without a mutation in TP53. TP53 mutations had no impact on rates of NRM but LFS at 2 years was also significantly reduced in the presence of a TP53 mutation . Other adverse‐risk cytogenetic subgroups such as complex and monosomal karyotypes, loss of 17p (−17 or del(17p)), del(7q), and del (5q) increased risk of relapse after allo‐SCT in this cohort and similarly resulted in a statistically significantly reduced OS  and OS and LFS remained impaired at 2 years .Given the increased co‐occurrence of CT Table . When adP = .017) and the presence of a complex karyotype . Moreover, increasing age was associated with a higher NRM risk  and shorter OS .In this multivariate analysis, the other factors that increased relapse risk and reduced OS in this cohort of AML patients with intermediate and adverse risk cytogenetics who received an allo‐SCT in CR1 were the presence of either deletion or loss of chromosome 5  and OS of 16.3% . In contrast, patients without losses at chromosome 17p had a reduced CIR at 2 years of 47.4%  and improved OS of 44.7% . Similarly, patients with a complex karyotype had a CIR at 2 years of 66.4%  and an OS of 23.1% . In contrast, patients without a complex karyotype had an improved CIR of 30.8%  and OS of 61.9% . Neither transplant intensity nor use of in vivo T‐cell depletion impacted relapse risk. The presence of pretransplant MRD was associated with an inferior overall survival  and increased the risk of relapse . Similarly, chromosome 17p losses was associated with a reduced OS .We used a Cox proportional hazards regression model to account for other prognostic factors related to patient, donor, and transplant characteristics and co‐occurrence of more than 1 poor prognostic variable (Supporting Table TP53 mutations who had either a loss of 17p and/or complex karyotype (n = 126) or neither of these karyotypic abnormalities (n = 53). Patients with TP53 mutant AML with neither 17p losses and/or complex karyotype had a favorable OS at 2 years of 65.2% , with a CIR at 2 years of 27.5% . In contrast, in the presence of either or both loss of 17p and/or complex karyotype, the OS at 2 years was only 24.6%  with a higher CIR of 65.4%   for OS. In contrast, patients with mutated TP53 and either 17p loss and/or complex karyotype had an inferior OS with an adjusted HR of 2.9   Table .TP53‐mutated AML who have undergone an allo‐SCT in CR1 had an inferior OS to those without a mutation in TP53, regardless of the presence of concomitant high risk cytogenetic features, such as loss of chromosome 5 or 7. However, the poor prognosis of patients with mutations at TP53 seems to be restricted to those patients with concomitant loss of 17p and/or complex karyotypes. Even in this latter subgroup with additional high‐risk features, 2‐year OS was possible in a sizeable cohort of patients, suggesting that allo‐SCT remains the only curative strategy for this group. This is of notable clinical significance due to the ongoing debate surrounding the issue of whether allo‐SCT can provide long‐term survival in patients with TP53 mutant AML.In this study from the ALWP of the EBMT, we demonstrate that patients with TP53 mutant AML posttransplant. Previous large studies have shown varying results for whether a complex karyotype may additionally provide prognostic information on TP53 mutant MDS following an allograft,Our data clearly show that a complex karyotype significantly increases the risk of relapse in patients with TP53 activity in AML following an allograft, as seen in the additional poor prognostic implications of concomitant 17p abnormalities in patients with TP53 mutant AML. Recently, in a largely nontransplant setting, biallelic loss of TP53 activity has been shown to be important in MDS,TP53 activity was inferred through several routes, including variant allelic frequency (VAF). In 2 recent studies, VAF was an independent prognostic factor in predicting response to hypomethylating agents in patients with TP53 mutant MDS,TP53 VAF may be dependent on treatment intensity.This study supports the importance of biallelic loss of TP53 mutation, lower intensity regimens such as venetoclax‐based regimens may have at least comparable responses to patients treated with intensive treatment, and they may proceed to an allograft in a fitter state.In patients with a TP53 mutant AML. Recent findings suggest that TP53‐mutated AML is associated with an immunosuppressive microenvironment.TP53 mutant AML. Pretransplant MRD is of clear significance in predicting posttransplant outcomes through a range of different methodologies.TP53 mutant AML.TP53 mutant AML. Future studies should address this issue comprehensively. Given the recent data on the impact of conditioning intensity on outcomes of patients with pretransplant MRD,conditioning) had no impact on outcomes of TP53‐mutant patients undergoing an allo‐SCT , allo‐SCT still remains an important treatment strategy. These novel data are not only of immediate clinical relevance but also have importance for basic scientific studies focused on understanding the mechanism of TP53 mutations.In summary, this report provides the first comprehensive analysis of post‐transplant outcomes of patients with This work was supported by research support and clinical trials funding from Cancer Research UK, Cure Leukemia, and Bloodwise.Gesine Bug reports a grant (paid to her institution) from Novartis; honoraria from Jazz Pharmaceuticals (paid to her institution), Celgene, and Gilead; support for attending meetings and/or travel from Jazz Pharmaceuticals, Gilead, and Neovii; and payment for participation on an advisory board from Novartis, Pfizer, Eurocept, Gilead, and Celgene. Charles Craddock reports grants from Celgene, Bloodwise, Cancer Research UK, and Cure Leukemia; and honoraria from Celgene, Daichi‐Sankyo, Novartis, and Pfizer. Justin Loke reports payment or honoraria from Pfizer, Janssen and Amgen; and travel support from Novartis and Daichi‐Sankyo. The other authors made no disclosures.Justin Loke: Study design, analysis, and writing. Myriam Labopin: Study design, analysis, and writing. Charles Craddock: Study design, analysis, and writing. Arnon Nagler: Study design, analysis, and writing. Jordi Esteve: Study design, analysis, and writing. Mohamad Mohty: Study design, analysis, and writing. Jan J. Cornelissen: Patient recruitment, study analysis, and writing. Hélène Labussière‐Wallet: Patient recruitment, study analysis, and writing. Eva Maria Wagner‐Drouet: Patient recruitment, study analysis, and writing. Gwendolyn Van Gorkom: Patient recruitment, study analysis, and writing. Nicolaas P.M. Schaap: Patient recruitment, study analysis, and writing. Nicolaus M. Kröger: Patient recruitment, study analysis, and writing. Joan Hendrik Veelken: Patient recruitment, study analysis, and writing. Montserrat Rovira: Patient recruitment, study analysis, and writing. Anne Lise Menard: Patient recruitment, study analysis, and writing. Gesine Bug: Patient recruitment, study analysis, and writing. Ali Bazarbachi: Patient recruitment, study analysis, and writing. Sebastian Giebel: Patient recruitment, study analysis, and writing. Eolia Brissot: Patient recruitment, study analysis, and writing.Supporting information S1Figure S1Click here for additional data file.Supporting information S2Table S1sClick here for additional data file.Supporting information S3Table S2Click here for additional data file.


Atrial fibrillation (AF) is common arrhythmia in valvular heart disease (VHD) and is associated with adverse outcomes.εs/εe/εa) and their corresponding strain rate (SRs/SRe/SRa).To evaluate the left atrial (LA) function in patients with AF‐VHD by cardiovascular magnetic resonance imaging feature tracking (CMR‐FT) using LA strain  underwent MRI scans performed with drug‐controlled heart rate before correcting the rhythm and valves through maze procedure. Fifteen participants with normal cardiac MRI were included as healthy control. εs/SRs, εe/SRe, and εa/SRa, corresponding to LA reservoir, conduit, and booster‐pump function, were assessed using Feature Tracking software (CVI42 v5.12.1).This was a retrospective cross‐sectional inter‐reader and intra‐reader reproducibility conducted from July 1, 2020, to January 31, 2021. A total of 39 patients with AF‐VHD  were significantly decreased , while LA size and volume were increased in AF‐VHD group . In the subgroup, RHVD group showed lower LA total ejection fraction (LATEF) and strain data than DHVD group . Decreased LATEF was significantly related to altered LA strain and strain rate, especially in εs, εe, and SRs . Interstudy and intrastudy reproducibility were consistent for LA volumetry and strain parameters (intraclass correlation coefficient: 0.88–0.99).Compared with healthy controls, LA global strain parameters (CMR‐FT can be used to assess the LA strain parameters, and identify LA dysfunction and deformation noninvasively, which could be a helpful functional imaging biomarker in the clinical treatment of AF‐VHD. The LA normal function includes reservoir (collection of pulmonary venous blood during left ventricular [LV] systole), conduit (passage of pulmonary venous blood flow to the LV during LV early‐diastole), and booster pump function .LA strain assessed by cardiovascular magnetic resonance imaging feature tracking (CMR‐FT) has been used in many cardiovascular diseases and enhanced the diagnostic value,CMR‐FT is a new method to evaluate myocardial strain and strain rate; it can be applied to routine cine images and has the advantages of high spatial resolution, large field of view, good reproducibility, and it can more sensitively reflect the functional characteristics of myocardial tissue.2This study was approved by the local ethics committee. All patients or their families provided written informed consent for the examination.2.1From July 1, 2020, to January 31, 2021, 39 consecutive patients with AF‐VHD who were admitted to the department of cardiac surgery in our hospital, and performed MRI to assess the left atrium function before valve replacement surgery only or valve replacement surgery with Maze procedure to correct valves and rhythm were included in this study. All patients were with persistent AF and mitral stenosis. The clinical characteristics, echocardiography findings on admission, and cardiac MRI data were retrospectively collected.The inclusion criteriaAt the same time, 15 healthy participants with normal cardiac MRI in our hospital were included as the healthy control group. The healthy control group excluded claustrophobia, coronary/congenital/valvular heart disease, hypertension, diabetes, severe arrhythmia , chronic kidney disease, myocarditis, and cardiomyopathy.2.2CMR studies were performed using a 3.0T MR scanner  and a 32‐channel phased‐array body coil. All images were ECG gated; patients were placed in the supine position and required to hold their breath during image capture. Cine images were acquired in the short‐axis views and longitudinal two‐, three‐ and four‐chamber views using True Fast imaging with Steady‐State Precession (TrueFISP) imaging sequences covering the entire LV and LA . In addition, we used the cardiac shim model of SIEMENS to adapt adjustment volume to reduce dark band artifacts.2.3(1)LV volumetric parameters: LV end‐diastolic volume , end‐systolic volume , ejection fraction (LVEF), cardiac output (LVCO), cardiac index (LVCI) and LV mass (LVM) were measured. LV endocardial and epicardial contours were drawn on LV short‐axis cine images, excluding the papillary muscles. All parameters of LV and LA were corrected according to body surface area (BSA), for example, LVEDV index (LVEDVi) = LVEDV/BSA × 100%, and so forth.(2)εs), active strain (εa), passive strain , peak first positive strain rate (SRs), peak early (first) negative strain rate (SRe), and peak late (second) negative LA strain rate(SRa). εs and SRs, corresponding to LA reservoir function; εe and SRe, corresponding to LA conduit function; εa and SRa, corresponding to LA booster pump functionLA strain and strain rate: the LA myocardial deformation was quantified using CVI 42 Tissue Tracking software.(3)max, before the end‐systolic mitral valve was opened) and the minimum volume . LA total ejection fraction (LATEF) = (LAVmax − LAVmin)/LAVmax × 100%.LA volume (LAV): The LA endocardium was manually labeled by the end‐diastolic atrial minimum volume at the four‐chamber and two‐chamber levels using CVI 42. The pulmonary vein and LA appendage were not included. The parameters of the LA volume were obtained using Simpson's method. The parameters of LAV included the maximum volume (LAV(4)The intraobserver and interobserver variability for the LA parameters, measurements were assessed by the intraclass correlation coefficient (ICC).Images were analyzed using CVI42 .2.4t‐test or Mann–Whitney U test. χ2 or Fisher exact test was used to assess categorical data. Pearson or Spearman correlation was performed to investigate the potential relationship between LA strain parameters and LA function. Moreover, we assessed the ICC to evaluate the accuracy and the precision of the method to measure each LA parameters. ICC was scored as follows: poor reliability, ICC < 0.50; moderate reliability, ICC: 0.50–0.75; good reliability, 0.75–0.9; excellent reliability, ICC > 0.9.p‐value < 0.05 was considered statistically significant.Data were analyzed using SPSS, version 20.0 . Mean ± SD or median (quartiles) was used to express measurement data in accordance with normal distribution, and the continuous variables were analyzed by independent sample 33.1p < 0.001), especially in the RHVD group. RHVD group showed lower body mass index  and BSA  than DHVD group. RHVD group had more diagnoses of mitral stenosis , and all patients in the AF‐VHD group had mitral regurgitation; there was no statistical significance for tricuspid regurgitation . There was no statistical significance for severity of valvular heart disease and proportion of presurgery medications  between the two groups .A total of 39 patients (22 DHVD and 17 RHVD) and 15 control participants were included in the study. Baseline characteristics and volumetric chamber indices for all control participants are summarized in Table 3.2p < 0.001, Table p = 0.877).Compared with the control group, the AF‐VHD group showed higher LVEDD, LVEDV(i), LVESV(i), and lower LVEF  and LATEF than did control participants  and lower LATEF .Patients with AF‐VHD had higher LA size (LAAD and LAV), lower strain values  and negatively correlated with SRe, SRa . For interobserver reproducibility, the LAVmax and LAVmin had the highest reproducibility . The least reproducible segmental measurement for interobserver reproducibility was the SRe .Intraobserver and interobserver reproducibility of global LA strain, strain rate, and volumetric parameters using CMR are shown in Table 4Our study evaluated the LA function in patients with AF‐VHD using CMR‐FT. We found that LA function in AF ‐VHD was lower than the Control healthy participants. AF‐VHD patients with reduced reservoir and conduit function, reduced or absent booster pump function, which showed in strain parameters. LATEF has a linear correlation with LA strain parameters. LA enlargement was also observed in AF‐VHD, which means LA remodeling. In the subgroup, DHVD had higher LV size, volume, mass, and stain parameters than those in RHVD. And LA strain and volumetric parameters showed good reproducibility.LA remodeling consists of both structural and functional changes; enlargement of the LA and fibrosis of the atrial muscle promotes the persistence of AF,2 and are treated medically have increased mortality and more cardiac events (AF and heart failure). After mitral valve surgery, the cutoff of LAVi ≥ 60 ml/m2 lost its prognostic value. Caso P et al.εs in patients with asymptomatic rheumatic mitral stenosis followed for 3–4 years. These markers of risk are particularly important because in clinical practice, risk stratification for mitral surgery helps with clinical decision‐making in these patients.LA size by volumetric index is widely accepted as a significant prognostic marker of mortality and outcomes in many cardiovascular diseases.εa and SRa) is very low or absent in patients with AF‐VHD,a and SRa show good reproducibility.Truong VT et al.Previous size (volume) parameters can only reflect the changes in LA structural remodeling, whereas strain parameters can better reflect the early changes in LA in functional parameters. The comprehensive assessment continues to provide more insight into LA function, and therefore more information is available to guide clinical treatment. Future large‐scale studies are warranted to assess whether individual strain parameters using CMR‐FT may provide additional clinical value in stroke risk assessment and guidance of anticoagulation therapy.5This was a single‐center study with a relatively modest sample size. We only assessed global longitudinal strain and did not assess radial strain. Radial strain has been noted as a parameter that is difficult to obtain and reproduce consistently.6CMR‐FT is a reliable tool with good clinical feasibility and repeatability for noninvasive quantitative assessment of LA strain and strain rate (LA function) without using a contrast agent and may provide insight into assessment of the LA performance over time in patients with AF‐VHD, which has potential clinical value in guiding the treatment, prognosis, evaluation and risk stratification.The authors declare no conflicts of interest.Huishan Wang and Benqiang Yang contributed to the conception and design of the study; Jie Hou, Yu Sun, and Wei Wang contributed significantly to manuscript preparation; Jie Hou and Yu Sun analyzed the data; Jie Hou wrote the manuscript; Libo Zhang, Hongrui You and Rongrong Zhang helped perform the analysis with constructive discussions and revised the manuscript. All authors received the final approval of the submission.Supporting information.Click here for additional data file.


Although multilineage cells derived from oral tissues, especially the dental pulp, apical papilla, periodontal ligament, and oral mucosa, have neural crest‐derived stem cell (NCSC)‐like properties, the differences in the characteristics of these progenitor cell compartments remain unknown. The current study aimed to elucidate these differences.Sphere‐forming apical papilla‐derived cells (APDCs), periodontal ligament‐derived cells (PDLDCs), and oral mucosa stroma‐derived cells (OMSDCs) from the same individuals were isolated from impacted developing teeth. All sphere‐forming cells were characterized through biological analyses of stem cells.in vitro. In immunocompromised mice, APDCs formed hard tissues better than PDLDCs and OMSDCs.All sphere‐forming cells expressed neural crest‐related markers. The expression of certain tissue‐specific markers such as CD24 and CD56 (NCAM1) differed among tissue‐derived cells. Surprisingly, the expression of only CD24 and CD56 could be discriminated in human tissues. Although APDCs and PDLDCs exhibited greater mineralized cell differentiation than OMSDCs, they exhibited poorer differentiation into adipocytes Although cells with NCSC‐like properties present the same phenotype, they differ in the expression of certain markers and differentiation abilities. This study is the first to demonstrate the differences in the differentiation ability and molecular markers among multilineage human APDCs, PDLDCs, and OMSDCs obtained from the same patients, and to identify tissue‐specific markers that distinguish tissues in the developing stage of the human tooth with immature apex. This study illustrates that neural crest‐derived cells from distinct oral tissues, namely the apical papilla, periodontal ligament, and oral mucosa, have varying differentiation potential, and tissue‐derived cell‐specific molecular markers have been identified. CD24 and NCAM1/CD56 expression were found to differ among multilineage oral tissue‐derived cells, similar to our observation in human tissue. A sufficient number of stem cells can be obtained from the third molar tissue without causing aesthetic issues or collecting surplus tissue. Dental pulp stem cells (DPSCs),et al. reported that stem cells from the apical papilla have higher proliferative potential, higher telomerase activity, and a greater capacity for hard/mineralized‐tissue formation than stem cells from DPSCs.Although DPSCs are present in the dental pulp tissue of human teeth with completely formed roots, the apical papilla from human teeth with immature root apices is a developing tissue, and the stem cells present play a crucial role in complete root formation. Sonoyama Another source of stem cells, the oral mucosa with a very high regenerative ability following injury, and can be obtained even from patients without teeth.The apical papilla, periodontal ligament, and oral mucosa can be obtained concomitantly during the extraction of an impacted third molar with an immature apex. To the best of our knowledge, differences in the differentiation ability and expression of molecular markers among human apical papilla‐derived cells (APDCs), periodontal ligament‐derived cells (PDLDCs), and oral mucosa stroma‐derived cells (OMSDCs) obtained from individuals simultaneously and from the same sites have not been reported previously. This study was performed to characterize human NCSC‐like cells from tissues obtained through the extraction of impacted developing third molars and to confirm the differences in cellular characteristics and molecular markers of individual tissue‐derived stem cells, which have a great clinical scope and are potentially valuable in dental regenerative medicine.2Detailed materials and methods are described in the supplementary material .2.1Human apical papilla, periodontal ligaments, and oral mucosa tissues were obtained for the extraction of impacted developing third molars. Written informed consent was obtained from all donors.2.2Prepared sections were used for hematoxylin–eosin (H&E) and immunohistochemical staining for nestin, CD44, CD24, and CD56 (NCAM1).2.3APDCs, PDLDCs, and OMSDCs were cultured in Isocove's modified Dulbecco's medium  supplemented with 10% fetal bovine serum (FBS) . APDCs, PDLDCs, and OMSDCs were passaged and cryopreserved.Oral mucosal epithelial cells were cultured in keratinocyte SFM (Gibco Life Technologies). The human tongue squamous cell carcinoma cell line (SCCKN) was cultured in 45% Dulbecco's modified Eagle medium (DMEM) (Gibco Life Technologies), 45% RPMI 1640 medium (Gibco Life Technologies), and 10% FBS.2.4The growth assay was performed as previously described.2.5APDCs, PDLDCs, and OMSDCs from five patients were seeded at a density of 500 cells/well in 6‐well plates. After 14 days of culture, the colonies were stained and counted as previously described.2.62 supplements (Gibco Life Technologies), 20 ng/ml basic fibroblast growth factor , and 20 ng/ml epidermal growth factor (PeproTech) for seven days.APDCs, PDLDCs, and OMSDCs were cultured in 24‐well super‐hydrophilic plates  in serum‐free DMEM/F12 (1:1) containing N2.7Immunohistochemistry was performed as previously described.2.8Sphere‐forming APDCs, PDLDCs, and OMSDCs were enzymatically dissociated and washed with PBS. The cells were incubated at room temperature for 1 h with primary antibodies . After incubation, the cells were washed with PBS, then incubated with anti‐mouse or anti‐rat IgG conjugated with Alexa 488 or 594  at room temperature for 30 min. Subsequently, the cells were analyzed with a FACSCant™ .2.9Total RNA was extracted from the cells using the RNeasy Mini Kit , according to manufacturer instructions.2.10The total RNA of sphere‐forming APDCs, PDLCs, and OMSCs (100 ng) was reverse‐transcribed into cDNA using the Low Input Amp Labeling Kit . Afterward, cDNA was mixed with a hybridization buffer and hybridized to a SurePrint G3 Human GE microarray 8X60k v3 (Agilent Technologies) for 17 h by following manufacturer instructions. The chips were washed and subsequently scanned using the Agilent microarray scanner (Agilent Technologies). Raw data were analyzed using the feature extraction software , and microarray data were mined and analyzed using GeneSpring (Agilent Technologies). The signal intensity was normalized by adjusting the data to a 75th percentile baseline (GEO accession number for the microarray gene expression data: GSE164327).2.11Semi‐quantitative RT‐PCR was performed using the PrimeScript One‐Step RT‐PCR Kit Ver.2 . Quantitative RT‐PCR was performed using the One‐Step PrimeScript RT‐PCR Kit . The primer sequences used for RT‐PCR are listed in Table 2.125 cells/tube) were maintained in MSC chondrogenic differentiation medium  using a pellet culture.For mineralized cell differentiation, the cells were cultured in mesenchymal stem cell (MSC) osteogenic differentiation medium  supplemented with 100 ng/ml bone morphogenic protein 2 . For adipogenic differentiation, the cells were cultured in adipogenic differentiation medium, . For chondrogenic differentiation, enzymatically dissociated cells  scaffolds . These scaffolds were implanted into subcutaneous pouches in the dorsum of five‐week‐old male BALB/cAJcl nude mice  after mineralization‐induced cell differentiation as described above.2.14SEM analysis was performed as previously described.2.15p < 0.05 was considered significant. All statistical analyses were performed using EZR , a graphical user interface for R .Mean values were compared using one‐way analysis of variance with post hoc Tukey's multiple comparison test. 33.1The developing impacted third molar had an immature apex Figure . The api3.2in vitro monolayer culture ; CD56 (NCAM1), LRRC17, KCNK12, and HAPLN1 (group 2); and SUSD2, PTPRE, FBLN2, and DLL1 (group 3) were identified. These genes were selected from the top 10 common genes with significantly different expression levels according to the data when compared between groups  were also identified in these cells and clustered into three groups Figure . Group 1s Figure . The quas Figure . Only SUy Figure . CD24 exy Figure . CD56  was confirmed in APDCs, PDLDCs, and OMSDCs  was significantly upregulated in OMSDCs compared with that in APDCs and PDLDCs  and α‐SMA (protein) expression were confirmed in all cells  and β3‐tubulin (protein) expression were confirmed in all cells  imaging. We found that all kinds of cell cultures after differentiation contain cells showing a marked increase in Ca2+ concentration in response to extracellular administration of glutamate, a neurotransmitter, whereas there were no glutamate‐induced Ca2+ responses in undifferentiated control cells   and OCN expression  than PDLDCs and OMSDCs , they may have been derived from NCs.To date, specific markers for the localization of NCSCs in human tissues have not been identified. We previously reported that nestinNES, CD44, SNAI1, SNAI2, MSX1, and HES1 which were NC‐associated genes.The sphere culture system has been widely used for the enrichment of NSCs and other types of stem cells.et al. reported that CD24 is a specific marker of the apical papilla, whereas it is not detected in DPSCs and bone marrow MSCs, and that mineralized cells differentiated from APDCs do not express CD24.et al. recently reported that CD24 expression increases by 72.9% during sphere formation in mouse APDCs.et al. reported an average of 7.56% expression in CD24‐positive cells.et al. detected adipocyte differentiation in colonies derived from MSCA‐1+/CD56− MSCs but not in those derived from MSCA‐1+/CD56+ MSCs.− OMSDCs exhibited significantly higher adipocyte differentiation than CD56+ APDCs and PDLDCs.We detected genes with different expression levels among distinct tissue‐derived cells. CD24 has been reported as a marker of several normal tissue‐derived stem/progenitor cells and cancer stem cells.et al. reported that HAPLN1 is expressed de novo in EPCAM1−/CD56 (NCAM1)+ mesoderm‐committed progenitor cells and fibroblastic hepatocellular carcinoma cells, and during the dedifferentiation of hepatocyte‐like cells to liver progenitors. Silencing of HAPLN1 downregulates the markers of EMT.et al. reported that LRRC17, a member of the LRR superfamily, acts as a negative regulator of RANKL‐induced osteoclast differentiation and is highly expressed in osteoblasts.in vitro than did OMSDCs , and metastatic melanomas.s Figure . Althougin vitro.in vitro differentiation abilities of APDCs and PDLDCs were similar, and that the mineralized cell differentiation abilities of these cells were high, although their adipogenic ability was low (Figure et al. reported that GMSCs have a higher adipogenic differentiation ability than DPSCs, and that DPSCs exhibit a higher osteogenic potential than GMSCs.et al. However, we did not observe any difference in chondrocyte differentiation between tissues. Previous studies isolated these stem/progenitor cells through monolayer culture, whereas we used enriched stem/progenitor cells isolated using the sphere culture technique in this study. We believe that differentiation abilities may differ with the stem/progenitor cell isolation technique.Human stem/progenitor cells derived from APDCs, PDLDCs, and OMSDCs can differentiate into NC lineage cells w Figure . OMSDCs w Figure . Monteruin vivo hard tissue regenerative capacity of APDCs, PDLDCs, and OMSDCs has been reported previously.in vitro and in vivo transplantation experiments. Grzesik et al. reported that hard tissue is not formed upon transplantation of PDLDCs, although these cells form calcified nodules and express mineralized‐cell differentiation markers when cultured in mineralized‐cell differentiation medium in vitro.et al. reported that, unlike DPSCs, PDLSCs have the potential to generate cementum/PDL‐like tissue, in which hard tissue is thinly formed around HA in vivo.et al. suggested that differentiated mineralized cells are lost in the early stage of differentiation and that the remaining cells do not form hard tissue in vivo; alternatively, the differentiated mineralized cells may be negatively regulated by other fibroblastic populations that exert a suppressive influence in vivo.in vivo is closely associated with biological roles. For example, APDCs have a high ability to form hard tissues, such as tooth roots. In contrast, in the periodontal ligament, the ability to form cementum remains unaltered under application of occlusal force. In addition, ectopic bone formation in the oral mucosa is extremely rare.The in vitro multipotential phenotype. Recently, Chan et al. identified the skeletal stem cells from human adult bone that also have a hierarchy and have the highest self‐renewal and differentiation potential, even in cells isolated according to the conventional MSCs definition.Previous reports have suggested that human “NCSC‐like cells” are defined based on their sphere‐forming capacity, expression of NCSC‐related markers, and This study, however, showed that the differentiation potential and expression of markers differed depending on the tissue from which the cells were obtained, suggesting that these tissue‐specific characteristics should be considered during application in regenerative medicine.5We demonstrated that multilineage sphere‐forming APDCs, PDLDCs, and OMSDCs share the same phenotypes as other stem/progenitor cells, although expression of certain tissue‐specific markers and differentiating abilities vary based on tissue source. We showed for the first time that APDCs, PDLDCs, and OMSDCs obtained from the same patients and, concomitantly, the same sites, can be used individually in regenerative medicine‐based therapy. These differences in the differentiation capacity between each type of tissue‐derived cells should be taken into consideration when administering stem cell‐based therapy in clinical settings. In addition, we identified human tissue‐specific markers in the currently unknown human developing tooth with immature apex. Our study identified important tissue‐specific markers that distinguish between apical papilla, periodontal ligament, and oral mucosa in developing stage of human third molar, as well as serves as a basis for future regenerative medicine research.SA contributed to study conception and design, methodology, investigation, data analysis and interpretation, and manuscript writing. AK, KK, and KN contributed to study conception and design, methodology, investigation, and data analysis and interpretation. NY and YK contributed to study conception and design, methodology, and investigation. MM, TM, CH, HK, and TY contributed to study conception and design and methodology. All authors read and approved the final manuscript.The authors declare that they have no competing interests.Appendix S1Click here for additional data file.Table S1Click here for additional data file.


Gamma‐glutamyl transferase (GGT) is considered a marker of oxidative stress in vivo. In this study, we aimed to examine the association of serum GGT levels with 3‐month and 1‐year stroke recurrence in patients with acute ischemic stroke or transient ischemic attack (TIA).We conducted a large and multicenter cohort study. Participants with ischemic stroke or TIA who had a baseline GGT measurement were enrolled in the China National Stroke Registry‐3 study from August 2015 to March 2018. They were divided into four groups according to sex‐specific quartiles of GGT levels. The effect of GGT on stroke recurrence and other vascular events was examined during the 1‐year follow‐up period. Multivariate Cox regression models were performed to evaluate the association. Discrimination tests were used to examine the degree to which incorporating GGT into the conventional model predicted stroke adverse outcomes.A total of 12,504 patients were enrolled. At both the 3‐month and 1‐year follow‐ups, patients in the highest quartile group of GGT levels exhibited a higher risk of stroke recurrence , ischemic stroke , and combined vascular events  than those in the lowest quartile group. Moreover, the Kaplan–Meier curves revealed that the incidence rates of stroke adverse outcomes were quite different in the four groups. The highest quartile group showed the highest cumulative incidence, while the lowest quartile group showed the lowest cumulative incidence. After applying discrimination tests, adding GGT into the conventional model resulted in slight improvements in predicting stroke adverse outcomes (NRI: 10%–14%).This study demonstrated that elevated GGT levels were positively associated with an increased risk of stroke adverse outcomes, namely, recurrence, ischemic stroke, and combined vascular events. This large, multicenter cohort study reveals elevated GGT levels are positively associated with the increased risk of stroke adverse outcomes, namely, recurrence, ischemic stroke, and combined vascular events. There is a slight improvement in predicting the risk of stroke adverse outcomes when adding GGT into the conventional model after applying discrimination tests. KY2015‐001‐01). All participants signed a written informed consent form prior to their inclusion in the study.2.2The baseline data of the enrolled participants were collected following the standard data collection protocol developed by the steering committee. The protocol and the statistical analysis plan have been published in a previous study.The information from all participants was collected by comprehensive assessments on admission and detailed medical records, including baseline characteristics , medical histories  and physical examination results  score, the National Institutes of Health Stroke Scale (NIHSS) score). All imaging data were collected in DICOM format on disks and analyzed by two professional neurologists. Moreover, exposure to various medications during hospitalization  was assessed. In addition, serum GGT, fasting blood glucose (FBG), total cholesterol (TC), triglyceride (TG), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels from fasting blood samples were collected carefully and with high quality within 24 h of admission to address potential sources of bias. All blood samples were stored in EDTA anticoagulant collection tubes and serum separation tubes in a −80°C refrigerator.2.3The primary clinical outcome was stroke recurrence . The secondary clinical outcomes were ischemic stroke and combined vascular events . The outcomes mentioned above were observed at 3 months and 1 year after stroke onset. According to the World Health Organization criteria, the diagnostic criteria for stroke rely on symptoms, physical signs, scale evaluations, and neuroimaging (magnetic resonance or brain computed tomography).2.42‐test or Fisher's exact test.The data were tested for normal distribution using the Kolmogorov–Smirnov test. Continuous variables conforming to a normal distribution are presented as the means ± standard deviations (SDs) and were compared using ANOVA. Continuous variables that did not exhibit a normal distribution are presented as medians with interquartile ranges (IQRs) and were compared using the Kruskal–Wallis U test. Categorical variables are expressed as numbers (proportions) and were compared using the χp‐values < 0.05 were considered to be statistically significant and were two‐sided.First, patients participating in this study were divided into four groups according to sex‐specific quartiles of GGT, which was assessed on admission. Then, the association of GGT levels with stroke recurrence, ischemic stroke, and combined vascular events was estimated by using a multivariate Cox regression model. In addition, hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated after adjusting for potential confounding factors. The first model was adjusted for age and sex. In the second model, body mass index (BMI), smoking status, alcohol consumption and medical histories were added. In the third model, systolic blood pressure (SBP), diastolic blood pressure (DBP), the NIHSS score at admission, the mRS score, the TOAST classification ,All statistical analyses were conducted using SAS version 9.4 (SAS Institute Inc.).33.1As shown in Figure 3.2During the 1‐year follow‐up period, among the eligible participants, 787 6.29%) patients had stroke recurrence, 736 (5.89%) had ischemic stroke, and 815 (6.52%) had combined vascular events within 3 months. At the 1‐year follow‐up, 1235 (9.88%), 1134 (9.07%), and 1307 (10.45%) patients had recurrent stroke, ischemic stroke, and combined vascular events, respectively. The associations of GGT levels with stroke adverse outcomes are presented in Table 9% patienIn addition, according to serum GGT levels, the Kaplan–Meier curves showed the time to event for stroke recurrence, ischemic stroke, and combined vascular events at the 3‐month and 1‐year follow‐ups , NRI 14.00% (p = 0.0003), NRI 14.00% (p < 0.0001); 1 year: NRI 11.00% (p = 0.0005), NRI 11.00% (p = 0.0003), NRI 10.00% (p = 0.0012), respectively]  [3 months: NRI 14.00% , medium‐GGT (m‐GGT), small‐GGT (s‐GGT), and free‐GGT (f‐GGT).3+, thus oxidizing low‐density lipoprotein (LDL), which results in the formation of oxidized LDL. In addition, serum GGT partially adsorbs onto circulating LDL, localizing with oxidized LDL and CD68+ foam cells, and has been found within cerebral, carotid, and coronary atherosclerotic plaques.Moreover, oxidative stress events are related to the pathogenesis of atherosclerosis and a series of cardiovascular‐cerebrovascular diseases (CCDs). During cerebrovascular events, the interruption of blood flow leads to cerebral oxygen deprivation, and the energy failure caused by hypoxia destroys the oxidant‐antioxidant balance, leaving the body in a state of oxidative stress.This study also had some limitations. First, since only patients with minor stroke (a low NIHSS score) and TIA were included in this study, these findings need validation in other cohorts. Second, for the interesting results of this study to be of greater breadth and relevance, further studies with longer follow‐ups should be considered. Third, because of missing data for some variables of interest, the potential impact of residual confounders, such as the location and size of stroke lesions, partial drug use and drug dosage, on the results were not completely excluded. Furthermore, this study collected only baseline GGT measurements; nevertheless, as a biomarker that reflects metabolism, GGT levels could present a dynamic change characteristic. Thus, we cannot rule out the possibility that this change may influence the role of GGT in stroke recurrence to some extent. The effect of dynamic changes in GGT on stroke recurrence needs to be further verified.Biomarkers for the diagnosis and prognosis of stroke have always been a focus in this field. Recent research suggests that gut microbiomes related to inflammation may serve as biomarkers and therapeutic targets for stroke.5In conclusion, as a low‐cost, measurable and accurate biomarker, GGT activity was positively associated with the risk of stroke adverse outcomes, namely, recurrence, ischemic stroke, and combined vascular events. This suggests that for stroke patients with high‐GGT levels, more effective medical and lifestyle interventions should be performed as early as possible. More studies are still needed to further investigate the pathophysiological mechanism of GGT activity in the stroke population.SQL and AXW designed the study and drafted the manuscript. XT and YTZ analyzed the data. AXW and XM contributed to reviewing the statistical problems. SQL and YMZ interpreted the data. YMZ reviewed the manuscript. All authors approved the final version of the manuscript.All authors have nothing to disclose.TABLE S1–S5Click here for additional data file.Appendix S1 Supporting informationClick here for additional data file.


There is substantial variability in prostate cancer (PCa) mortality rates across Caucasian American (CA), African American (AA), Asian, and Hispanic men; however, these estimates are unable to disentangle race or ethnicity from confounding factors. The current study explores survival differences in long‐term PCa outcomes between self‐reported AA and CA men, and examines clinicopathologic features across self‐reported CA, AA, Asian, and Hispanic men.This retrospective cohort study utilized the Center for Prostate Disease Research (CPDR) Multi‐center National Database from 1990 to 2017. Subjects were consented at military treatment facilities nationwide. AA, CA, Asian, or Hispanic men who underwent radical prostatectomy (RP) for localized PCa within the first year of diagnosis were included in the analyses. Time from RP to biochemical recurrence (BCR), BCR to metastasis, and metastasis to overall death were evaluated using Kaplan–Meier unadjusted estimation curves and adjusted Cox proportional hazards regression.p = 0.01); however, no difference was observed between AA and CA men for a time from BCR to metastasis  and time from metastasis to overall death .This study included 7067 men, of whom 5155 (73%) were CA, 1468 (21%) were AA, 237 (3%) were Asian, and 207 (3%) were Hispanic. AA men had a significantly decreased time from RP to BCR compared to CA men (HR = 1.25, 95% CI = 1.06–1.48, In an equal access health care setting, AA men had a shorter survival time from RP to BCR, but comparable survival time from BCR to metastasis and metastasis to overall death. Previous research has demonstrated significant differences in long‐term prostate cancer outcomes in Caucasian American compared to African American men. This study explored whether racial differences in long‐term oncological outcomes exist among men diagnosed with prostate cancer and treated surgically, in equal access health care settings. While African American men had slightly poorer biochemical recurrence‐free survival compared to Caucasian American men, there were no racial differences observed for distant metastasis‐free survival or overall survival, controlling for detailed clinical factors. In a large racially heterogeneous cohort of men within an equal access health care setting, long‐term prostate cancer outcomes were comparable across race. The primary aim of this study was to examine the relationship between CA and AA race and survival time from radical prostatectomy (RP) to BCR, BCR to metastasis, and metastasis to overall survival, controlled for a wide array of clinicopathologic relevant variables. A secondary study aim was to compare clinicopathologic variables across CA, AA, Hispanic, and Asian men.22.1A retrospective cohort study was conducted on patients enrolled in the Center for Prostate Disease Research (CPDR) Multi‐Center National Database in men with biopsy‐confirmed PCa who underwent RP treatment within 12 months of diagnosis between January 1, 1990 and December 31, 2017. Enrollees in the CPDR database are military health care beneficiaries who are eligible for TRICARE‐for‐life health care coverage. Demographic, clinical, treatment, and outcomes data were collected as part of routine patient follow‐up on all CPDR enrollees. Informed consent was obtained at the time of transrectal ultrasound‐guided biopsy (TRUS) for suspicion of PCa, as described previously.2.2Patient characteristics of interest in this study included: Age at diagnosis (years), time from RP to last follow up (years), time from diagnosis to RP (months), self‐reported race or ethnicity , obesity , number of medical comorbidities , PSA (ng/ml) at time of diagnosis, pathologic stage , 2014 ISUP Gleason score , surgical margin status , primary treatment year , post‐BCR PSA doubling time (PSADT) , any secondary treatment, BCR, metastasis, metastasis after BCR, death from any cause, and death from any cause after metastasis.2.3BCR was defined as a PSA value ≥0.2 ng/ml observed at ≥8 weeks post‐operatively, followed by a subsequent confirmatory PSA level ≥0.2 ng/ml or the initiation of salvage therapy.BCR, distant metastasis, and overall death were modeled as time‐dependent study endpoints with three possible results: Achieved endpoint, censored, or achieved end of study with no event. Men with <6 months of follow‐up time were excluded from each model. As an endpoint, distant metastasis must have occurred more than 1 year after PCa diagnosis. For the BCR endpoint, men were censored at treatment date between RP and BCR, date of last known medical visit, or date of death. For the distant metastasis and death endpoint, men were censored at the date of last known medical visit or date of death. Exclusions were also made for men who underwent adjuvant treatments between RP and BCR, between BCR and metastasis, or between metastasis and death, due to the variability and strong correlation of receipt of treatment with PSADT.2.4U test was used to compare categorical and continuous variables, respectively.All statistical analyses were performed using SAS version 9.4 . Frequencies and distributions of patient features were calculated for the study cohort, and stratified by race/ethnicity . The chi‐square test and Mann–Whitney P‐values were computed using two‐sided statistical tests (α = 0.05). PH assumptions were checked using the ASSESS PH statement in SAS, and each variable met the PH assumption.A Kaplan Meier curve analysis was used to produce 5‐, 10‐, and 15‐year probability estimates for survival from RP to BCR, BCR to metastasis, and metastasis to overall death as a function of race and race by obesity, restricted to AA and CA men. Cox proportional hazards (PH) analysis was used to model RP to BCR, BCR to distant metastasis, and distant metastasis to overall death as a time‐dependent outcome, as a function of race (CA versus AA). The Cox PH model from RP to BCR was controlled for age at RP, time from diagnosis to RP, primary treatment year, diagnostic PSA, obesity, pathologic stage, Gleason score, surgical margin status, and the number of major comorbidities. The BCR to metastasis Cox PH model was controlled for age at BCR, time from RP to BCR, obesity, pathologic stage, Gleason score, surgical margin status, and PSADT. The metastasis to overall death model was adjusted for age at metastasis, time from RP to metastasis, obesity, and the number of major comorbidities. Hazards ratios (HR) and 95% confidence intervals (CI) were reported for Cox PH models. 3p < 0.001), have a longer time from RP to last follow‐up , have PCa treatment in earlier years (p < 0.001), and have a longer post‐BCR PSADT (p = 0.11). AA men were less likely to have major comorbidity . Hispanic men were more likely to have a lower diagnostic PSA level . Asian men were less likely to be obese , and more likely to have a shorter time from diagnosis to RP , be diagnosed with the lowest pathologic prostate tumor stage and grade  and to receive another treatment after RP  , 1468 21%), 237 (3%), and 207 (3%) self‐reported as CA, AA, Asian, and Hispanic, respectively Figure . Median 1%, 237 (p = 0.005) .  men who had BCR in the overall cohort: 821 (20%) CA men and 256 (23%) AA men (Table ) Figure . Additio. Figure .p = 0.154)   men who had distant PCa metastasis following BCR in the overall cohort: 71 (80%) CA men and 16 (18%) AA men. The Kaplan–Meier analysis demonstrated that there was no difference in survival from BCR to metastasis among CA men compared to AA men (log‐rank ) Figure . There w) Figure .p = 0.035)   men who died following their metastatic PCa in the overall cohort: 82 (84%) CA men and 14 (14%) AA men (Table ) Figure . When ev) Figure .Survival time from RP to BCR was shorter for AA men compared to CA men , controlled for a wide array of demographic, clinical, and pathological relevant variables was examined as the primary study aim. Comparison of clinicopathologic variables across CA, AA Hispanic, and Asian race or ethnicity was assessed as a secondary study aim. Both analyses were conducted in a large retrospective cohort of racially diverse PCa patients treated with RP and enrolled in an equal access military health care system over a 28‐year period. This setting reduces the disparity in health care access, treatment, and education, which may influence PCa progression and provides a deeper analysis adjusted for potential confounders. This study supports that there is a positive association between AA race and BCR‐free survival when compared to CA race, but no difference between CA and AA race and survival time between BCR and metastasis, and metastasis and overall death. Furthermore, this analysis revealed that Asian and Hispanic American men have similar or better clinical and pathologic PCa features when compared to CA men. To our knowledge, this is the first study to look at long‐term PCa outcomes on a continuum: From RP to BCR, BCR to metastasis, and metastasis to overall survival, one of the largest RP cohorts comparing long‐term PCa outcomes for CA and AA men, and one of the largest known post‐RP cohorts of Asian and Hispanic American men.p = 0.18). However, despite a shorter BCR‐free survival time for AA men with a near equal proportion of men receiving treatment after BCR, there was no difference in survival time between BCR and metastasis and metastasis and overall death when comparing AA men to CA men. These data are confirmed by a recent analysis on patients who received HT after BCR in an equal access system, showing that race was not a predictor of DM or other adverse outcomes. Additionally, a recent analysis with the entire Department of Defense database revealed no difference in overall survival between AA and CA men diagnosed with PCa.Similar to other studies that have examined race and BCR‐free survival, our analysis also revealed a moderate significant decrease in BCR‐free survival time when comparing AA men to CA men, adjusted for confounding variables. When BCR occurs, the treatment team often recommends either androgen‐deprivation (HT) and/or salvage external beam radiation treatment (EBRT) usually happens. In our cohort, there was no significant difference between race and receipt of treatment after BCR  and do not necessarily reflect the views, opinions or policies of Uniformed Services University of the Health Sciences (USUHS), The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., the Department of Defense (DoD), the Departments of the Army, Navy, or Air Force. Mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. Government.Among men undergoing radical prostatectomy in an equal access health care setting, we identified and described a cohort of African American, Caucasian, Asian, and Hispanic men. While African American men had an increased risk of biochemical recurrence compared to Caucasian American men after controlling for prostate cancer prognostic factors, African American men experienced a similar risk of metastasis and overall death compared to Caucasian American men.Figure S1Click here for additional data file.


BUB1 and EXO1) and one (RRM2) survival‐significant ceRNA(s) shared across breast‐colorectal‐endometrial and prostate–colorectal–endometrial combinations, respectively. Both BUB1 and BUB1B genes were identified as shared ceRNAs across more than two HD cancers of interest. These genes play a critical role in cell division, spindle‐assembly checkpoint signalling, and correct chromosome alignment. Furthermore, shared ceRNAs across multiple HD cancers have been involved in essential cancer pathways such as cell cycle, p53 signalling, and chromosome segregation. Identifying ceRNAs' roles across multiple related cancers will improve our understanding of their shared disease biology. Moreover, it contributes to the knowledge of RNA‐mediated cancer pathogenesis.Competing endogenous RNAs (ceRNAs) have become an emerging topic in cancer research due to their role in gene regulatory networks. To date, traditional ceRNA bioinformatic studies have investigated microRNAs as the only factor regulating gene expression. Growing evidence suggests that genomic , transcriptomic , and epigenomic  factors can influence ceRNA regulatory networks. Herein, we used the Least absolute shrinkage and selection operator regression, a machine learning approach, to integrate DM, CNA, and TFs data with RNA expression to infer ceRNA networks in cancer risk. The gene‐regulating factors‐mediated ceRNA networks were identified in four hormone‐dependent (HD) cancer types: prostate, breast, colorectal, and endometrial. The shared ceRNAs across HD cancer types were further investigated using survival analysis, functional enrichment analysis, and protein–protein interaction network analysis. We found two ( However, in recent years, the investigation of noncoding RNAs (ncRNAs) has increased attention. For example, long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) are two types of ncRNAs that play crucial roles in transcriptional and posttranscriptional gene regulation.Existing ceRNA network analyses have described many risk‐associated ceRNAs in common cancers.Herein, we used a supervised machine learning approach, ‘Cancerin (Cancer‐associated ceRNA interaction networks)’,22.1https://gdc.cancer.gov/access-data/gdc-data-portal).https://starbase.sysu.edu.cn/) and miRcode (http://www.mircode.org/) databases.The genomic and clinical data of five HD cancer types  were excluded to increase the sensitivity and precision of the differential expression analysis. After excluding low‐expressed genes, RNA and miRNA raw counts data were re‐normalized using the trimmed mean of M values method implemented in the edgeR R package.2.3β values) using the Linked Omics database (http://www.linkedomics.org/login.php),β value was estimated as the ratio of the methylated probe intensity to the overall intensity (sum of methylated and unmethylated intensities). Therefore, the β value ranges between 0 and 1, with 0 being hypomethylated and 1 being hypermethylated. Linked Omicsβ values. Therefore, we integrated COAD and READ RNA and miRNA expression samples into a single analysis of colorectal cancer (COLCA). We use the ‘COLCA’ term to explain results in the combined analysis of COAD and READ data.We obtained gene‐level copy number values and gene‐level DM values on CpG sites  were retrieved from the starBase2.Selecting miRNAs that contribute to the RNA expression level.We used a regularized regression method, the least absolute shrinkage and selection operator (LASSO), to evaluate the effect of miRNA regulators on RNA expression levels.λ denotes the amount of shrinkage and the green‐coloured section defines the sum of the absolute value of the magnitude of coefficients. The LASSO models are equivalent to the linear regression models when λ = 0. Increasing/decreasing λ value cause increased biasedness/increased variance, respectively. We have used the 10‐fold cross‐validation approach to find the optimal λ value for each LASSO model.The blue‐coloured part implies the residual sum of squares calculation, The LASSO models are constructed considering transcription‐mediating factors such as DM, TF and CNA. Two model types are fitted for DE mRNAs  Pearson correlation analysis to filter strongly positive correlated ceRNA candidate pairs; (ii) hypergeometric test to identify lncRNA–mRNA sharing the significant number of miRNAs; (iii) using the bnlearn R packageIdentifying ceRNAs common among multiple HD cancer types may assist in understanding their shared molecular pathogenesis and drug repositioning. Therefore, we evaluated shared ceRNA pairs (lncRNA–mRNA or mRNA–mRNA) among HD cancer types. The statistically significant ceRNA pairs (RNA A–RNA B) in each HD cancer were compiled into a single table. Then, we coded a new variable combining ceRNA candidate pairs, such as ‘RNA A ensemble ID_RNA B ensemble ID.’ Then, the one‐way table was created to identify shared values of the derived variable across all four HD cancer types. Suppose one‐way frequency equals 4 for a given RNA A–RNA B combination. In that case, a ceRNA association is classified into ‘the shared ceRNA network of HD cancers.’ Similarly, if frequency equals 2 or 3, these ceRNAs have been associated with two or three cancer types, respectively.Shared ceRNAs across multiple HD cancer types were applied in survival analysis, functional enrichment analysis, and PPI network analysis to evaluate their predictive ability and biological functions in cancer. We used survival3After the quality‐control process, we retrieved 495/52 (cases/controls), 1091/113, 456/41, 166/10, and 543/35 samples from PRAD, BRCA, COAD, READ, and UCEC. In the differential expression analysis, we used 15,509, 15,244, 14,771, 14,866, and 15,197 genes from PRAD, BRCA, COAD, READ, and UCEC after removing those that were low‐expressed.3.1The number of DE RNAs, DE miRNAs, DM, CNA, TF–target interactions and miRNA–lncRNA/mRNA interactions used in each HD cancer‐specific ceRNA analysis are given in Table In the next analytical steps (machine‐learning approach and inferring ceRNAs), DE mRNA/miRNA expression data of COAD and READ combined as colorectal cancer (COLCA) analysis.3.2β estimates, CNA values, miRNA–lncRNA/mRNA and TF–target interactions. The statistically significant LASSO predictors (estimated by the bootstrap procedure) were used to identify potential ceRNA pairs. After that, we applied three user‐defined thresholds  has occurred. The hazard ratio compares two groups, here low and high expressed. The hazard ratios and p values in blue/red fonts indicate that the gene's low/high expressed level is associated with the survival of cancer in interest. In Table The ceRNAs common among HD cancer combinations were checked for the prognosis in each cancer (included in the combination). Table BUB1 and EXO1) from the BRCA‐COAD‐UCEC and the RRM2 from PRAD‐COAD‐UCEC were prognostic in all three cancer types included in the combination of interest.As described in Table BUB1 , EXO1 , and RRM2  are illustrated in Figure The K‐M survival curves for BUB1 and EXO1 show better survival. Nevertheless, high‐expressed BUB1 and EXO1 are associated with a higher survival probability in COAD (B). The low‐expressed RRM2 gene has shown the highest survival probability (hazard ratio = 9.13) in PRAD (A) compared with other cancers, COAD (B) and UCEC (C).In BRCA (A) and UCEC (C), low‐expressed 3.4We conducted the functional enrichment analysis for shared ceRNAs in each HD cancer combination. The genes shared across two/three HD cancers (listed in Supporting Information) was used for the functional enrichment analysis. Table Enrichment analyses result of ceRNAs shared among three‐HD cancers are illustrated in Figure 3.5PPI network analyses for ceRNAs shared among HD cancer combinations were conducted using the STRING online platform.4The aetiology of complex diseases such as cancer is often related to aberrant gene expression at the transcriptional and posttranscriptional levels. Over the last decade, ceRNAs have emerged as an important class of posttranscriptional regulators that alter gene expression through a miRNA‐mediated mechanism. This study has extended the knowledge of ceRNA networks in HD cancers  by integrating other molecular effects such as DM, TF and CNA, which influence the ceRNA mechanism. Moreover, it expands the knowledge of HD cancers shared biology by investigating and comparing gene regulatory networks of more than two HD cancer types.First, we investigated regulating factors‐mediated ceRNAs in individual HD cancers. After that, we identified ceRNAs shared across two/three/four HD cancers. However, none of the ceRNAs was shared among the four cancers of interest. The shared ceRNAs across two/three HD cancers were involved in survival analysis and two downstream analyses, functional enrichment analysis and PPI network analysis.BUB1 and EXO1 were associated with patient survival of all three cancers of interest. BUB1 gene encodes a serine/threonine‐protein kinase that plays a central role in mitosis. It also involves DNA damage response and cell cycle regulation.BUB1 gene has been previously reported in BRCA, COLCA, and multiple digestive tract cancers but not in UCEC.BUB1 appears for the first time in UCEC risk. The BUB1 β (BUB1B) was recognized as a shared ceRNA among PRAD, BRCA and UCEC. These BUB1 and BUB1B genes are central components of the mitotic checkpoint for spindle assembly (SAC).BUB1 and BUB1B genes in HD cancers should be further examined. Several experimental and bioinformatic studies have shown the importance of EXO1 in replication, DNA repair pathways, cell cycle checkpoints, and its association to cancer.EXO1 gene as risk alleles for different types of cancers.We found 16 ceRNAs shared across BRCA, COLCA, and UCEC. Among them, DEPDC1B, BUB1 and RRM2, were identified as shared ceRNAs among PRAD, COLCA and UCEC. DEPDC1B has been identified as a prostate cancer metastasis oncogene. It was positively correlated with metastasis status, high Gleason score, advanced tumour stage and poor prognosis. Moreover, mechanistic investigations found that DEPDC1B induced epithelial–mesenchymal transition (EMT) and enhanced proliferation by binding to Rac1 and enhancing the Rac1‐PAK1 pathway.DEPDC1B in COLCA and UCEC has not been described in previous bioinformatics or functional studies. According to the survival analysis result, the RRM2 gene was a prognostic biomarker in PRAD, COLCA and UCEC. The well‐established role of RRM2 is to maintain the balance of deoxynucleoside triphosphate pools for DNA synthesis and DNA repair.RRM2 is associated with inducing activities of various oncogenes, including those encoding nuclear factor‐κB, Myc proto‐oncogene protein, tyrosine‐protein kinase transforming protein Fes and ornithine decarboxylase.RRM2 correlates with cellular invasiveness, metastasis and tumourigenesis of cervicalRRM2 expression has also been described in ovarianRRM2 serves as a prognostic biomarker in colorectal cancer. None of the ceRNAs was shared among PRAD, BRCA and COLCA, and hence common ceRNAs were not found across four HD cancer types  included in this study.Three genes, KIF4A, KPNA2, TPX2, TUBA1C, RAD54L, MTFR2, ANLN, RACGAP1, FAM83D, KNSTRN, CCNE1 and DSCC1) out 88 and 6  out of 77 ceRNAs shared among BRCA‐UCEC and COLCA‐UCEC pairs, respectively, have exhibited the survival significance in both cancers included in the combinations. These 18 genes have been previously described in HD studies due to their tumour‐suppressive/oncogenic/cancer‐driven nature.The survival analyses of shared ceRNAs among two HD cancer combinations have shown that 12  and chromosome related GO pathways , which play an important role in cancer. The shared ceRNAs across PRAD, COLCA and UCEC were involved in five unique KEGG pathways, drug metabolism‐other enzymes, pyrimidine metabolism, glutathione metabolism, oxidoreductase activity, acting on CH or CH2 groups, and ferric iron‐binding. Experimental studies might be required to investigate these KEGG pathways' involvement in the shared biology of PRAD, COLCA and UCEC.p was <1.0E − 16. Four and 16 ceRNAs shared among PRAD‐BRCA‐UCEC (p = 1.12E − 09) and BRCA‐COLCA‐UCEC (p = 2.04E − 09) exhibited strong PPI network connecting all the ceRNA nodes. Therefore, these gene sets should be further studied to evaluate their combined effect on HD cancers.The PPI network describes the physical interaction between identified shared ceRNAs in this study, mediating the assembly of proteins into protein complexes, for example, mediating signalling/regulation and transport events in the cell. According to PPI network analysis results, ceRNAs lists of each two/three HD cancer combinations have interacted with stronger statistical evidence. Five out of nine HD cancer combinations' PPI network enrichment The major limitation of this study is restricting miRNA–mRNA and miRNA–lncRNA selection (in Step 1). We have chosen both predicted and experimentally validated miRNA–target interaction only from two miRNA–mRNA/lncRNA databases, starBase and miRcode. We followed this limitation to get a substantial set of miRNA–mRNA/lncRNA interactions. Consideration of more possible ceRNA associations will improve the machine‐learning outcome by increasing the number of predictors. Moreover, we have not included circRNAs and pseudogenes in the ceRNA network analyses. This study retrieved only a limited number of statistically significant miRNAs from the differential expression analysis of miRNA‐seq data in TCGA‐PRAD. Therefore, the number of miRNAs (independent variables) involved in PRAD‐LASSO models was comparatively lower. Nevertheless, selections of TFs‐target interactions and CNAs were independent of PRAD differential expression analyses.Our previous bioinformatic study identified shared ceRNAs (both lncRNAs and mRNAs) across PRAD, BRCA, COLCA and UCEC, considering miRNAs as the only gene regulator in the ceRNA network.Our machine learning‐based study of omics data provides a set of pivotal ceRNAs associated with two or three HD cancers of interest. According to functional enrichment analyses, shared ceRNAs across two/three HD cancers are involved in hallmarks of cancer, such as cell cycle, DNA replication/repair, chromosomal segregation, mitotic checkpoint, nuclear division, p53 signalling, glutathione metabolism, pyrimidine metabolism and ferric iron binding. Most of these pathways are directly/indirectly associated with RNA‐processing steps that influence RNA regulatory networks, including ceRNAs. A recent study found the complex biology of glutamine (precursor of glutathione) metabolism in driving BRCA growth.5It is noteworthy that, to our knowledge, this is the first study that integrates genomic (CNA), transcriptomic  and epigenetic (DM) regulatory factors to infer genome‐wide ceRNA interactions shared across a group of cancers. These cross‐HD cancer ceRNAs have shown tumour‐suppressive/tumour‐promoting and survival‐prognosis properties in previous HD cancer studies. The main bottleneck of this machine learning approach is the lack of RNA, DM and CNA data from a single study, especially for other diseases, except for cancers. Besides these limitations, cross‐cancer shared ceRNAs may help identify potential unexpected targets applied for a subset of cancers . Furthermore, it will help to understand the shared disease biology of HD cancers.Dulari K. Jayarathna, Neha S. Gandhi, Miguel E. Rentería and Jyotsna Batra designed the study. Dulari K. Jayarathna performed data acquisition, data analysis and data interpretation. Dulari K. Jayarathna wrote the original manuscript. Neha S. Gandhi, Miguel E. Rentería and Jyotsna Batra revised the manuscript critically. All authors read and approved the final manuscript.The authors declare no conflict of interest.This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committees of Queensland University of Technology (19 December 2019/1900001147) and QIMR Berghofer Medical Research Institute (23 August 2019/P1051).Supplementary information.Click here for additional data file.