ENCORE, an observational, prospective, open‐label study, investigated real‐world treatment practices and outcomes with cetuximab plus platinum‐based therapy (PBT) in first‐line (1L) recurrent and/or metastatic squamous cell carcinoma of the head and neck (R/M SCCHN).This multinational study aimed to investigate the long‐term use of cetuximab plus PBT for 1L R/M SCCHN in a clinical setting. In particular, this study aimed to explore clinical considerations such as the decision to prescribe cetuximab plus PBT in R/M SCCHN, the mode and duration of treatment, and patient outcomes.Previously untreated patients with R/M SCCHN whose planned treatment was cetuximab plus PBT were enrolled from 6 countries. Among 221 evaluable patients, planned treatments included cetuximab plus carboplatin (31.2%), cisplatin plus 5‐fluorouracil (31.7%), or carboplatin plus 5‐fluorouracil (23.1%); 3.2% included a taxane, and 45.2% did not include 5‐fluorouracil. Cetuximab treatment was planned for a fixed duration (≤24 weeks) in 15 patients (6.8%) and until disease progression in 206 (93.2%). Median progression‐free survival and overall survival were 6.5 and 10.8 months, respectively. Grade ≥3 adverse events occurred in 39.8% of patients. Serious adverse events occurred in 25.8% of patients; 5.4% were cetuximab‐related.In patients with R/M SCCHN, first‐line cetuximab plus PBT was feasible and modifiable in a real‐world setting with similar toxicity and efficacy as in the pivotal phase III EXTREME trial. Clinical trial registration number: EMR 062202‐566. Observational studies are therefore necessary to ensure that benefits observed in controlled clinical trials translate accordingly to patients in the real‐world setting. ENCORE  was a multinational , observational, prospective, open‐label study that investigated the real‐world treatment practices, efficacy, and safety of cetuximab plus PBT in 1L R/M SCCHN. The ENCORE study aimed to investigate the widespread use of cetuximab plus PBT for R/M SCCHN in a clinical setting over many years.In particular, the ENCORE study aimed to explore clinical considerations regarding cetuximab plus PBT usage in R/M SCCHN, such as the decision to prescribe, the mode and duration of treatment, and patient outcomes. This study aimed to create one centralized database with the intention of evaluating results from different regions and countries, thereby permitting a descriptive comparison across the selected countries.22.1ENCORE was a post authorization, non‐interventional, prospective, non‐comparative and open‐label phase IV study that investigated the use of cetuximab plus PBT in 1L R/M SCCHN. Patients were eligible if they were ≥18 years of age; had confirmed SCCHN of the oral cavity, oropharynx, hypopharynx, or larynx with recurrence not amenable to curative locoregional therapy and/or distant metastases; were suitable for 1L combination treatment with cetuximab plus PBT with or without local palliative treatments; and had a planned treatment regimen that included cetuximab. Patients were excluded from the study if they had previously received chemotherapy for R/M SCCHN , or if they were not considered suitable for the treatment and the study, as per normal clinical practice for 1L treatment of R/M SCCHN at the investigator's site in the participating countries. The study planned to enroll up to 300 patients, or the maximum number of eligible patients in the participating countries by December 2016.Treatment involved cetuximab in combination with PBT for the 1L treatment of R/M SCCHN. All agents were given according to their locally approved labels, and planned treatment duration was according to normal clinical practice for each treating physician. The clinical decision to use cetuximab with PBT and the recording of patient baseline characteristics, planned treatment regimens, administration schedule, and efficacy/safety outcomes were at the sole discretion of the investigator. Patient follow‐up was for 1 year after the first dose of cetuximab, or until death, whichever occurred first . Patients were considered as receiving cetuximab maintenance therapy if any administration of cetuximab was given >7 days after the last administration of chemotherapy.The primary objective was to assess treatment patterns for cetuximab, especially duration of use, for the treatment of 1L R/M SCCHN in combination with platinum‐based chemotherapy across different regions worldwide. Key secondary objectives were to assess treatment outcomes, including the endpoints of best overall response (BOR), disease control rate (DCR), ORR, PFS and OS, and adverse events (AEs) related to cetuximab. Only serious adverse events (SAEs), non‐serious AEs related to study treatment, and AEs leading to a delay or the permanent discontinuation of study treatment were to be recorded until the 30‐day posttreatment safety follow‐up assessment; AEs were graded according to the National Cancer Institute Common Terminology for Adverse Events v4.03.Because this was a prospective observational study, several patients were enrolled who had started cetuximab treatment prior to enrollment; these patients were included in the analysis providing that consent had been given and that retrospective data were available. The full analysis set (FAS) therefore included all enrolled patients who received cetuximab; primary endpoints and safety were analyzed in the FAS. As some patients began cetuximab treatment prior to enrollment, the target analysis set (TAS) was created to allow for output generation of patients in the FAS. The TAS included all patients who received their first dose of cetuximab on or after the date of enrollment. Secondary efficacy endpoints were analyzed in both the FAS and TAS.2.2Descriptive and exploratory statistical methods were used to analyze the study data; all statistical methods were of exploratory nature only.BOR, ORR and DCR: these qualitative variables were summarized as a number and percentage for the FAS and TAS. The Cochran–Mantel–Haenszel test was used to test for differences in ORR and DCR between patients with planned cetuximab treatment until PD versus those with a planned fixed duration of cetuximab treatment. The corresponding odds ratios and 95% confidence intervals (CIs) for the odds ratios were determined. The association between ORR and planned duration of cetuximab treatment, respectively, and possible explanatory variables were analyzed using logistic regression methods.PFS and OS: The Kaplan–Meier method was used to estimate median PFS and OS and the corresponding 95% CIs. A log‐rank test was used to compare PFS and OS between patients who received cetuximab until PD and those who received cetuximab for a fixed duration. The hazard ratios for PFS and OS (including the 95% CIs) were determined to assess the effect of planned cetuximab treatment duration (not until PD vs. until PD). Cox proportional hazard methods were used to investigate the association between PFS and OS and possible explanatory variables.Due to a protocol amendment, the planned follow‐up time was reduced from 2 years to 1 year to allow for an earlier study end. However, the follow‐up date for patients enrolled under the original plan of 2 years of follow‐up was not censored; all evaluable data were used.33.1n = 1 [0.5%]), France (n = 46 [20.8%]), Italy (n = 144 [65.2%]), Portugal (n = 8 [3.6%]), Russia (n = 21 [9.5%]), and South Africa (n = 1 [0.5%]). The enrollment period was approximately 1.5 years, with the first patient enrolled in May 2015 and the last patient enrolled in December 2016. A total of 159 patients in the FAS (71.9%) with prospective data collection were included in the TAS. Most patients had a follow‐up of 1 year; however, patients who began cetuximab treatment before enrollment, or were enrolled >1 year before the protocol amendment, had longer follow‐ups. Baseline characteristics are presented in Table A total of 225 patients were enrolled; 221 patients received ≥1 dose of cetuximab and were included in the FAS and the safety population , carboplatin plus 5‐FU (n = 51 [23.1%]), or carboplatin alone (n = 69 [31.2%]). A taxane was included in 3.2% of planned treatments. In total, 45.4% of planned treatments did not include 5‐FU. In the FAS, cetuximab treatment was planned until PD for most patients (n = 206 [93.2%]), of which chemotherapy was planned until PD for 111 (53.9%), and for a fixed duration (3–30 weeks) for 95 (46.1%); both cetuximab and chemotherapy were planned for a fixed duration of ≤24 weeks for 15 patients (6.8%) (Supplementary Table The most commonly planned treatments for the ENCORE patient population included chemotherapy backbones consisting of cisplatin plus 5‐FU (n = 70 3.7%), car3.2n = 72 [32.6%]), occurrence of SAE (n = 12 [5.4%]), investigator decision (n = 25 [11.3%]), treated according to a planned fixed duration (n = 13 [5.9%]), and death (n = 11 [5.0%])  are outlined in Table n = 72 2.6%), oc3.3n = 206) or for a fixed duration (n = 15), a BOR of complete response was achieved by 12 patients (5.8%) and 0 patients, and a BOR of partial response was achieved by 43 patients (20.9%) and 6 patients (40.0%), respectively. Among patients in the TAS who received cetuximab until PD (n = 144) or for a fixed duration (n = 15), a BOR of complete response was achieved by 6 patients (4.2%) and 0 patients, and a BOR of partial response was achieved by 28 patients (19.4%) and 6 patients (40.0%), respectively. ORR and disease control rate were 27.6%  and 65.6% , respectively, in the FAS and 25.2%  and 57.9% , respectively, in the TAS. There were 26 (11.8%) patients who discontinued PBT <6 months and may be considered platinum‐refractory and 195 (88.2%) patients who received no platinum or discontinued ≥6 months. The ORR  was comparable between both subgroups: 23.1% in patients who had discontinued platinum‐based chemotherapy <6 months versus 28.2% in those who had not received or had discontinued platinum‐based chemotherapy ≥6 months .Efficacy outcomes for the FAS and TAS are presented in Table Median PFS was 6.5 months overall in the FAS . Furthermore, previous cetuximab therapy, chemotherapy regimen, or disease status did not impact efficacy results. In the multivariable Cox proportional hazard analysis for PFS, total number of sites with distant metastases (2 or ≥3 vs. 0–1), a higher baseline ECOG PS score (≥2 vs. 0–1) and the presence of nervous system disease were associated with a shorter duration of PFS (Supplementary Table p = .0129)  and Italy (17.9%) compared with the other countries combined , AEs  occurred in 170 patients (76.9%) and were related to cetuximab in 104 patients (47.1%). The most frequent AE of any grade related to cetuximab was rash in 41 patients (18.6%), followed by asthenia in 15 patients (6.8%), skin reactions in 14 patients (6.3%), and dermatitis acneiform in 12 patients (5.4%)  and were related to cetuximab in 102 patients (46.2%). Grade ≥3 AEs occurred in 88 patients (39.8%), and 24 patients (10.9%) had an AE leading to death. Fatal anaphylactic shock attributable to cetuximab occurred in one patient (0.5%) on the first day of treatment; all other AEs leading to death were not considered to be related to cetuximab.4The ENCORE study suggests that cetuximab plus PBT is an effective and widely used treatment in real‐world clinical settings across several countries according to current guidelines and continues to be a recommended 1L treatment for patients with R/M SCCHN. Of note, 45.2% of planned treatments did not include 5‐FU, and 3.2% included a taxane. Additionally, the median OS and PFS times in this study with 1L cetuximab plus platinum with/without 5‐FU were comparable to results with the EXTREME regimen (cetuximab plus platinum plus 5‐FU) in the randomized phase III EXTREME trial.The baseline characteristics of patients in the ENCORE study and the EXTREME trial were similar, although the median age was lower in the EXTREME trial (56 years) compared with the ENCORE study (64 years).An important finding of the ENCORE study was that the planned cetuximab treatment duration for most patients with R/M SCCHN treated with cetuximab plus PBT was until PD (206 of 221 [93.2%]). In contrast, a very small proportion of patients (15 of 221 [6.8%]) were treated with a fixed duration of cetuximab. This indicates that treating clinicians would rather rely on treatment until PD, which was shown to be efficacious in the pivotal EXTREME trial.The EXTREME regimen was recently evaluated in patients with R/M SCCHN who were ≥70 years old in the ELAN (ELderly head And Neck cancer) trials.There were limitations associated with the ENCORE study, which are typical of the observational study design. Some patients began cetuximab treatment before the date of enrollment, potentially leading to selection bias and study outcomes bias. Their outcomes may have a positive bias, particularly for time‐to‐event endpoints, because patients who were already on treatment without progression upon enrollment were more likely to be responders and have longer survival times than patients starting treatment at enrollment. The TAS was introduced for those patients in the FAS who had only started cetuximab after their enrollment. The difference between the results in the FAS and TAS allows an assessment of the effect of the selection bias. Efficacy outcomes from the TAS may therefore be considered more reliable than those of the FAS. Furthermore, conducting the safety analysis on the TAS may be more representative of real‐world clinical practice as any AEs occurring before study enrollment may not be captured in the FAS, potentially underestimating treatment toxicity. Another limitation was that patients were enrolled from only 6 countries ; therefore, a true global perspective of real‐world practices for 1L R/M SCCHN treatment may not have been gained. The low number of patients whose treatment was planned for a fixed duration does not allow a valid conclusion regarding whether the planned treatment duration (until PD or for a fixed duration) had an impact on the treatment outcome; however, this analysis does show that treatment until PD is a well‐accepted regimen, consistent with results from the EXTREME study.In summary, ENCORE provides insight into real‐world clinical practices for 1L R/M SCCHN treatment and further contributes to the growing body of evidence suggesting that cetuximab plus PBT is a safe and effective treatment across a broad patient population. In an international real‐world setting, median OS and PFS times with 1L cetuximab plus platinum with/without 5‐FU were comparable to the results observed in the randomized pivotal phase III EXTREME trial.Christophe Le Tourneau: Conceptualization ; data curation ; formal analysis ; investigation ; resources ; writing – original draft . Massimo Ghiani: Conceptualization ; data curation ; formal analysis ; investigation ; resources ; writing – original draft . Maria Chiara Cau: Conceptualization ; data curation ; formal analysis ; investigation ; resources ; writing – original draft . Roberta Depenni: Conceptualization ; data curation ; formal analysis ; investigation ; resources ; writing – original draft . Graziana Ronzino: Conceptualization ; data curation ; formal analysis ; investigation ; resources ; writing – original draft . Pierluigi Bonomo: Conceptualization ; data curation ; formal analysis ; investigation ; resources ; writing – original draft . Vincenzo Montesarchio: Conceptualization ; data curation ; formal analysis ; investigation ; resources ; writing – original draft . Luigi Leo: Conceptualization ; data curation ; formal analysis ; investigation ; resources ; writing – original draft . Jeltje Schulten: Conceptualization ; formal analysis ; resources ; writing – original draft . Satu Salmio: Conceptualization ; formal analysis ; resources ; writing – original draft . Diethelm Messinger: Conceptualization ; formal analysis ; writing – original draft . Andrea Sbrana: Conceptualization ; data curation ; formal analysis ; investigation ; resources ; writing – original draft . Edith Borcoman: Conceptualization ; data curation ; formal analysis ; investigation ; resources ; writing – original draft . Maria Grazia Ghi: Conceptualization ; data curation ; formal analysis ; investigation ; resources ; writing – original draft .CLT reports receiving honoraria and travel expenses and providing consulting or advisory services for AstraZeneca, Bristol Myers Squibb, Celgene, EMD Serono, GlaxoSmithKline, Merck & Co., Nanobiotix, Rakuten Medical, Roche, and Seattle Genetics. PB reports providing consulting or advisory services for Angelini Pharma and EMD Serono. VM reports receiving honoraria from Bristol‐Myers Squibb and Italfarmaco. JS and SS are employees of the healthcare business of Merck KGaA, Darmstadt, Germany. DM was an employee of Prometris GmbH. All other authors report no conflicts of interest.All patients provided written informed consent prior to enrollment, and the protocol was approved by the local regulatory and ethics committee at each participating center. The study was conducted in accordance with the Declaration of Helsinki and the International Council on Harmonization Guidelines on Good Clinical Practice.Supplementary Table 1: Planned duration of chemotherapy in the FAS.Supplementary Table 2: Cetuximab treatment exposure in the FAS.Supplementary Table 3: Cetuximab cumulative dose and relative dose intensity in the FAS†, ‡.Supplementary Table 4: Multivariable Cox proportional hazards model to identify prognostic factors for PFS in the TAS.Supplementary Table 5: Multivariable Cox proportional hazards model to identify prognostic factors for OS in the TAS.Supplementary Table 6: Reasons (ratings) for planned treatment (FAS).Supplementary Table 7: Association between baseline and disease characteristics and planned cetuximab treatment duration – multiple logistic regression analysis (TAS).Click here for additional data file.


Lung cancer patients face a high risk of thromboembolism (TE), which is considered to be a poor prognostic factor. However, the impact of symptomatic cerebral infarction (CI) and pulmonary embolism (PE) on the prognosis of advanced non‐small cell lung cancer (NSCLC) patients is not fully understood.We retrospectively identified 46 patients with advanced NSCLC who developed symptomatic CI or PE at five hospitals in Japan between January 2010 and December 2019. Prognosis and biomarker levels after incident CI and PE were investigated.p = 0.16), the overall survival (OS) after incident TE tended to be worse in patients with CI than in those with PE . Multivariate analysis showed that OS after CI was worse in patients with high D‐dimer (DD) levels than in those with low DD levels at the time of incident CI .Of the 46 patients, 36 developed symptomatic CI, and 10 developed symptomatic PE. The median follow‐up duration after incident CI and PE was 18.2 months. Although the proportion of Common Terminology Criteria for Adverse Events grade 4 tended to be higher in patients with PE than in those with CI (30% vs. 11%, This retrospective study demonstrated that the prognosis of patients tended to be poorer after CI than after PE. The DD levels at the time of incident CI might be a promising predictor of clinical outcomes in advanced NSCLC patients who develop CI. We retrospectively analyzed the prognosis and predictors of survival in patients with advanced non‐small cell lung cancer who developed symptomatic thromboembolism. In this study, the prognosis of patients with cerebral infarction tended to be poorer than that of patients with pulmonary embolism. In addition, we showed that D‐dimer levels were a promising prognostic predictor in patients who developed cerebral infarction. However, few studies have focused on advanced NSCLC patients with TE, especially those with CI.Therefore, this study aimed to determine the prognosis and identify potential predictors of survival in advanced NSCLC patients who developed symptomatic CI and PE in a real‐world setting.22.1In this study, we retrospectively enrolled patients with advanced NSCLC who developed Common Terminology Criteria for Adverse Events (CTCAE) grade 2  and more severe CI or CTCAE grade 3  and more severe PE at five hospitals in Japan between January 2010 and December 2019 by reviewing medical records. We gathered the following data: sex, age at incident CI and PE, smoking history, history of stroke, history of antithrombotic drugs usage, body mass index at incident CI and PE, laboratory results at incident CI and PE, Eastern Cooperative Oncology Group Performance Status (ECOG‐PS) before incident CI and PE, TNM stage based on classification system version 8, presence of brain metastasis, liver metastasis, pleural effusion, and double cancer; histological subtype, presence of epidermal growth factor receptor (EGFR) mutation; programmed death‐ligand 1 expression level in tumors, radiation history, treatment line, treatment for cancer; TE type, severity classified using the CTCAE version 5.0, treatment for CI and PE; and OS after incident CI and PE. The Ethics Committees of the Kyoto Prefectural University of Medicine , and each participating hospital approved the study protocol.2.2p < 0.05. All statistical tests were two‐tailed.Data were collected retrospectively in June 2020. The median follow‐up duration after incident CI and PE was 18.2 months. During the follow‐up period, we identified 38 deaths out of 46 patients. The Mann–Whitney U test was used to compare the means of the continuous variables. The Fisher's exact test was used to compare the proportions of categorical variables. The Kaplan–Meier method was used to calculate survival curves, and the log‐rank test was used to compare the differences. When comparing OS after incident CI and PE in patients with CI and PE, we performed propensity‐score matching using the following variables: sex, age, ECOG‐PS, and presence of treatment for cancer. Nearest neighbor matching was performed at a ratio of 1:1. Caliper was set at 0.2. Hazard ratios and 95% confidence intervals were calculated using Cox proportional hazards models. Patients who were alive were censored on the date of the last follow‐up. The cutoff values of platelet count, lactate dehydrogenase, albumin, C‐reactive protein, and D‐dimer (DD) levels were set at the respective median value. Because the cutoff value of DD levels as a prognostic factor remained unclear, we performed receiver operating characteristic (ROC) curve analysis to identify the optimal cutoff value for DD. In the ROC analysis, the sum of specificity and sensitivity for 30‐days mortality was maximized at 16.1 μg/mL. Therefore, we considered that it is suitable for the median value of 15.6 μg/mL as a reasonable cutoff value for DD Figure . Patient33.1We identified 46 patients with advanced NSCLC who developed symptomatic CI or PE. Of these, 36 and 10 patients developed CI and PE, respectively.p = 0.033). The proportion of patients with pleural effusion at incident CI and PE was higher in the PE group than in the CI group .The characteristics of the patients classified according to the TE type are shown in Table p = 0.030). The median DD level was high in patients with both CI and PE .The laboratory data classified by TE type are presented in Table 3.2The CTCAE grades in patients with CI and PE are shown in Figure p = 0.17). To control for confounding variables, we performed the propensity‐score analysis, which showed no significant difference in OS after incident CI and PE between patients with CI and those with PE   Figure .3.3p < 0.001).The results of univariate analyses for OS after incident CI and PE are shown in Table p < 0.001) ; formal analysis (lead); methodology ; writing – original draft . Tadaaki Yamada: Conceptualization (lead); methodology ; project administration (lead); writing – original draft . Satomi Tanaka: Conceptualization (supporting); resources . Aosa Sasada: Resources . Shinsuke Shiotsu: Resources . Nozomi Tani: Resources . Takayuki Takeda: Resources . Yusuke Chihara: Resources . Soichi Hirai: Resources . Yoshizumi Takemura: Resources . Akihiro Yoshimura: Writing – review and editing . Kenji Morimoto: Writing – review and editing . Masahiro Iwasaku: Writing – review and editing . Shinsaku Tokuda: Writing – review and editing . Young Hak Kim: Writing – review and editing . Koichi Takayama: Supervision (lead).TY received commercial research grants from Pfizer, Ono Pharmaceutical, Janssen Pharmaceutical K.K., AstraZeneca, and Takeda Pharmaceutical Company Limited and personal fees from Eli Lilly. KT received research grants from Chugai‐Roche and Ono Pharmaceutical and personal fees from AstraZeneca, Chugai‐Roche, MSD‐Merck, Eli Lilly, Boehringer‐Ingelheim, and Daiichi‐Sankyo. The other authors have no conflicts of interest to declare.The Ethics Committee of the Kyoto Prefectural University of Medicine authorized the study protocol (ERB‐C‐1733‐4), and the study was carried out in compliance with the Declaration of Helsinki on the handling of patients' personal data.Owing to the retrospective nature of the study, the requirement for informed consent was waived, and the Ethics Committee of each individual hospital accepted the use of the official website to offer an opt‐out option.Not applicable.Figure S1.Click here for additional data file.Figure S2.Click here for additional data file.Table S1.Click here for additional data file.


Many children experience adversity, yet few receive needed psychiatric services. Pediatric primary care providers (PCPs) are uniquely positioned to intervene but often lack training and resources to provide patients with adverse childhood experiences (ACEs) the psychiatric support they need. The current study examines characteristics of youth with and without ACEs who were the focus of PCP contacts with a statewide child psychiatry access program (CPAP). Compared to those without ACEs, patients with ACEs were more often receiving medication treatment at time of CPAP contact, prescribed two or more psychotropic medications, and diagnosed with two or more mental health disorders. Study findings indicate that patients with ACEs for whom PCPs sought CPAP support were experiencing more clinically severe and complex mental health concerns. These findings underscore the important role of CPAPs in supporting PCPs with pediatric patients who have ACEs and will inform training provided by CPAPs to PCPs. However, many pediatric PCPs are infrequently inquiring about ACEs and may not be familiar with the long-term implications.14 timely referral from pediatric PCPs to specialized care is often not possible. Child psychiatry access programs (CPAPs) are one way to bridge this gap and support PCPs as they treat pediatric patients exposed to ACEs. CPAPs support PCPs in managing the mental health concerns of their pediatric patients through telephone consultation with child and adolescent psychiatrists, resource/referral networking, and continuing education, with some programs also offering direct-to-patient services  through co-located behavioral health or telehealth models.15 CPAPs can play a specific role in supporting pediatric PCPs in identifying and addressing ACEs, through training and consultations on screening and effective treatments as well as referrals for follow-up mental health care.16 Barclay and colleagues demonstrated that CPAPs receive a significant number of consultations about patients with trauma-related concerns, and this population presents with a high level of clinical severity.17 This previous research inspired the current study which expands to focus on adverse childhood experiences and analysis of consultation as well as co-located behavioral health service data.Due to the national shortage in child mental health specialists,18 these findings are even more critical in guiding future CPAP training and service offerings and in identifying treatment gaps for ACE exposed youth.The purpose of the current study is to understand the types and rates of ACEs reported by pediatric patients who were the focus of contacts with one statewide CPAP—Maryland Behavioral Health Integration in Pediatric Primary Care (BHIPP). This study explores the characteristics and service use of these youth with and without ACEs, including (1) demographics, clinical severity, and presenting concerns, (2) rates and types of treatment received prior to BHIPP contact, and (3) types of psychiatric medications employed. In light of the fact that the US Department of Health and Human Services has expanded funding to CPAPs nationwide,BHIPP provides an array of services including consultation and co-located social work services. BHIPP’s warm line is staffed by licensed master's-level behavioral health consultants, from whom PCPs can request general information, resource/referral networking tailored to patients, or consultation with a child and adolescent psychiatrist. BHIPP also provides embedded social work services in selected primary care practices where master’s level social work interns conduct screening and brief behavioral health interventions . Pediatric patients are connected with this service through PCP or self/family referral. This study is a secondary analysis of programmatic data collected during the provision of normal BHIPP services.N = 24) or repeat contacts concerning the same patient  were excluded from analyses. This study includes the remaining 4993 unique patient-specific BHIPP contacts for psychiatric consultation  and co-located social work services . IRB approval was obtained from Johns Hopkins University, University of Maryland, and Maryland’s Department of Health.From October 2012 through September 2020, 8228 patient-specific BHIPP contacts were completed through telephone consultation with a BHIPP child and adolescent psychiatrist and in-person visits with an embedded BHIPP social work intern. These contacts pertained to pediatric patients seen in a primary care setting in the state of Maryland. As of September 2020, there were 1127 pediatric providers across the state who had engaged with BHIPP through enrolling for services, calling the warm line, or both. Co-located social work services were provided in selected primary care practices in one rural region of Maryland by an average of eight BHIPP interns per academic year. Data on patients above 24 years old  tools hosted at Johns Hopkins University.21During all patient-specific contacts, BHIPP staff collected de-identified data on patient demographics , insurance type , presenting concerns , and prior history of behavioral health treatment. Providers or families  were asked if the patient had experienced any adverse childhood experiences or trauma. BHIPP staff selected the reported ACE(s) or trauma from a list of 23 options such as child maltreatment, separation from caregiver, and loss of a loved one  = 99.89, p < 0.001 and between the ages of 13 and 18 χ2(8) = 202.11, p < 0.001, than those with information on ACE history. In terms of clinical characteristics, those with unknown ACE history more often reported attention/concentration problems χ2(2) = 26.00, p < 0.001; were more often diagnosed with attention deficit/hyperactivity disorder (ADHD) χ2(2) = 156.03, p < 0.001; had more comorbid mental health diagnoses χ2(2) = 414.72, p < 0.001; and were more often rated as clinically severe (27.8% CGI-S > 4) χ2(4) = 264.21, p < 0.001. Furthermore, those with unknown ACE history were more often receiving medication treatment χ2(2) = 702.46, p < 0.001 and outpatient psychotherapy χ2(2) = 187.58, p < 0.001; had higher rates of polypharmacy χ2(12) = 507.30, p < 0.001; and were more often recommended by BHIPP staff for a medication evaluation/change χ2(2) = 1209.31, p < 0.001 than those included in the study. Thus, the final analytic sample includes 4113 patient-specific contacts with information on ACEs.A subset of patient-specific contacts  focused on children with a history of ACEs or a diagnosis of a trauma and stressor-related disorder , hereafter referred to as the “ACE group.” The remaining patients (N = 3306) who did not report a history of ACEs or trauma are referred to as the “no ACE group” , loss of a loved one , and impaired caregiver . Among those reporting ACEs (N = 682), 67.2% reported one ACE, 20.1% reported two ACEs, and 12.8% reported three or more ACEs.Among the 4113 unique patient-specific contacts included in this study, 19.6% (χ2(3) = 96.38, p < 0.001 and aggression (32.8%) χ2(3) = 54.22, p < 0.001. For patients aged 6–12, behavior problems at school (27.5%) χ2(3) = 40.87, p < 0.001 and attention/concentration problems (22.2%) χ2(3) = 27.28, p < 0.001 were the most frequently reported. Patients between 13 and 18 years old more frequently presented with depressed mood (52.7%) χ2(3) = 172.83, p < 0.001 and suicidal thoughts or gestures (16.5%) χ2(3) = 38.91, p < 0.001, compared to those in other age groups. Finally, young adults (age 19 and up) more frequently presented with anxiety (70.6%) χ2(3) = 64.79, p < 0.001, compared to younger age groups.Overall, the most frequently endorsed presenting problems for those in the ACE group were anxiety (39.4%), behavior problems at home (30.1%), and depressed mood 26.8%), see Table 6.8%, seeχ2(2) = 65.57, p < 0.001. Numerous significant differences in the presenting problems were identified between those in the ACE and no ACE groups, for which the ACE group more frequently endorsed problems (Table χ2(1) = 31.07, p < 0.001; sleep problems (12.8%), χ2(1) = 42.33, p < 0.001; and impulsivity (11.0%), χ2(1) = 43.10, p < 0.001. There were also significant differences in diagnostic impressions across the ACE and no ACE groups , ages 6–12 (44.1%), and Caucasian (55.5%) as compared to those in the no ACE group Table . Those iχ2(2) = 142.54, p < 0.001, than those in the no ACE group, with 24.4% of patients in the ACE group receiving a CGI-S > 4. Patients in the ACE group were more often identified as having comorbid diagnoses , (50.1%) χ2(1) = 229.83, p < 0.001, and were more often already receiving behavioral health treatment at the time of BHIPP contact, (48.7%) χ2(2) = 51.61, p < 0.001, than those in the no ACE group. Among those already receiving treatment in the ACE group, the most frequent treatments were medication (27.0%) and outpatient psychotherapy  = 68.92, p < 0.001, with the most frequent medications prescribed being stimulants (15.1%) and SSRIs (13.4%). BHIPP recommendations made at the time of contact also varied by group with those in the ACE group more often recommended for referral to mental health or community resources (49.8%), χ2(1) = 141.96, p < 0.001 and medication management (36.1%), χ2(1) = 143.94, p < 0.001, whereas in-office behavioral interventions were more commonly recommended for those in the no ACE group (40.7%) χ2(1) = 20.39, p < 0.001.Those in the ACE group were rated more clinically severe by BHIPP staff χ2(3) = 425.30, p < 0.001. In nearly all cases, a higher proportion of patients who were the subject of consultation services reported each type of ACE including having an impaired caregiver χ2(1) = 4.18, p = 0.041; experiencing physical abuse χ2(1) = 5.13, p = 0.024; and hospitalization χ2(1) = 6.97, p = 0.008, relative to co-located social work services (Table χ2(1) = 5.28, p = 0.022 and being bullied χ2(1) = 4.27, p = 0.039. Those with ACEs receiving services through consultation were also rated as more clinically severe (37.3% CGI-S > 4) χ2(1) = 78.32, p < 0.001 and identified as having more comorbid diagnoses (33.5% with 3 or more diagnoses) χ2(1) 57.86, p < 0.001 (Table χ2(2) = 518.90, p < 0.001, and more often prescribed multiple psychiatric medications, compared to those receiving co-located social work services.Characteristics of patients in the ACE group were also examined across BHIPP service types to identify any differences in patient characteristics between those who were the subject of telephone consultation with a child and adolescent psychiatrist and co-located social work services. Those with ACEs receiving co-located social work services were more often female 59.6%) and under the age of 6 22.1%) as compared to those who were the subject of consultation. However, the majority of patients across both services were 6–12 years old . Numerous significant differences were found between the frequencies of ACEs reported across service types. Of those receiving co-located social work services, 9.4% reported at least one ACE, whereas 36.4% of those receiving services through consultation reported at least one ACE % as comp9.6% and The study findings indicate that pediatric patients who are the subject of CPAP contact and report a history of ACEs present to primary care offices with higher clinical severity and complexity than those who do not have a history of ACEs. Specifically, the patients with ACEs who are the focus of CPAP contacts have higher rates of mental health diagnoses, are more frequently diagnosed with multiple comorbid mental health conditions, are more often prescribed psychiatric medications, and are more often taking multiple psychiatric medications. Nearly 20% of the current sample reported at least one ACE with the most common being separation from primary caregiver, loss of a loved one, and impaired caregiver. In addition, relative to co-located social work services, patients who were the focus of consultations reported nearly four times the number of ACEs and demonstrated greater clinical severity and complexity of mental health concerns.11 Lack of routine screening is likely one of the primary reasons for which the current study had a portion of patients with missing data on ACEs. We posit two potential explanations for the differences between those included in this study and this missing data—for which even higher clinical severity was found. First, it is possible that these patients did not disclose a history of ACEs to PCPs as they were already receiving behavioral health treatments and may not have felt that the disclosure of ACE history was necessary. Second, the most frequent diagnosis among the unknown group was ADHD. While many of these cases are likely to have an accurate ADHD diagnosis, it is also possible that some of these patients are presenting with posttraumatic stress symptoms that may be misdiagnosed as ADHD . Whereas some research suggests that trauma is a risk factor for ADHD, others have found that trauma is frequently misdiagnosed as ADHD,22 thus underscoring the importance of thorough screening for ACEs and a more in-depth assessment of mental health concerns to support appropriate diagnosis and treatment.However, research has shown that routine screening for ACEs in pediatric primary care is not a common practice.23 For example, in 2018–2019, the rate of ACEs among children in Maryland was 38.4%, considerably higher than the 19.6% reported in the current sample.23 The majority of patients in the current study only reported experiencing one ACE which may be accurate or may be another sign of underreporting. Regardless, prior work suggests that experiencing even one ACE is enough to contribute to negative mental and physical health outcomes, though a dose–response relationship is observed in that those with multiple ACEs are at an increased risk for more negative outcomes.24 It is also important to note that ACEs have differential impacts. Recent research suggests that household dysfunction is more highly associated with health risks, while child maltreatment is more highly associated with psychological difficulties.25 In addition, the pediatric patients included in this study have been identified as having some level of mental health concerns necessitating a BHIPP consultation call or referral to BHIPP co-located social work services—making this sample not generalizable to the population of youth presenting to primary care.There are some limitations related to the method for collecting data on ACE exposure. Providers and patients/families were not necessarily presented with a list of ACEs but were rather asked a general question about ACE exposure. The knowledge of what experiences constitute ACEs may vary by the provider, and patients/families may feel uncomfortable disclosing this information. Likewise, pediatric PCPs may feel uncomfortable inquiring about ACEs or may not have time to address ACE-related concerns. Additionally, data from consultations are PCP-reported and not directly collected from the patient/family—creating a filter in which information may be lost. It seems likely that the current sample of pediatric patients had a higher number of ACEs than were actually reported to PCPs or BHIPP staff, as the rate of ACEs in the current study is much lower than other community samples.26 Future work should examine methods for ACE screening across settings and with diverse patient populations. Results from the current study show that Caucasian female pediatric patients most often reported ACEs; however, research has demonstrated a disproportionate trend in ACE exposure for systematically marginalized populations with Black and Latinx youth experiencing more cumulative ACEs than their White peers.27 Cultural implications for disclosure of ACEs in the pediatric primary care setting should be studied to inform culturally responsive care, to reduce ethnic and racial disparities in ACE exposure, and increase access to effective treatments. One potential way to increase culturally responsive care may be to employ diverse, bilingual support staff that may assist with screening and referral services.28 Future work should explore the impacts of such practices on disclosure rates of ACEs within the pediatric primary care setting. Finally, future research should explore the impact of receipt of CPAP services on changes to the care PCPs provide to their patients who have a history of ACEs.Future research should address best practices for inquiring about ACEs in pediatric primary care including how to help families feel comfortable disclosing ACEs and strategies for helping pediatric PCPs incorporate ACE screening into routine visits. In a recent study by Thakur and colleagues, surveying patients about ACEs in aggregate  instead of by individual ACE resulted in more ACEs being reported.15 These findings about Maryland’s CPAP are relevant to nationwide efforts by CPAPs to bridge the gap between the need for and access to mental health treatment for ACE-exposed youth.The current study’s findings will help inform training provided by CPAPs to pediatric PCPs across the country. Specifically, findings indicate that additional training is needed to increase pediatric PCP knowledge of the types of ACEs, which are most common, and the psychosocial, emotional, and physical consequences of ACE exposure for child health and development. Training could increase provider comfort with asking about ACEs by supporting pediatric PCPs in identifying what screening tools to use, how to respond to patients’ report of ACEs, and best practices for follow-up. Providers also need support in the management of mental health concerns related to ACE exposure and identification of appropriate community resources—child psychiatry access programs like BHIPP reflect one model for providing this support.10 Routine screening is imperative to identify youth at risk of developing significant mental and physical health concerns. As of January 2020, California launched universal ACE screening in the context of primary care, and providers are now able to receive reimbursement for using the Pediatric ACEs and Related Life Events Screener.29 In order to successfully implement routine ACE screening, pediatric providers nationwide will need access to reimbursement and support in appropriately responding to and providing care for youth with ACE exposure. CPAPs can provide training and technical assistance to pediatric PCPs in screening for ACEs to help promote PCPs’ use of validated ACE screening tools with their patients to inform the consultation process and ensure that appropriate treatment options are provided.Mental health promotion for pediatric patients with a history of ACEs is in critical need.30 Pediatric PCPs can also be trained to provide brief in-office interventions that include psychoeducation on how ACEs impact health and anticipatory guidance for caregivers regarding developmental periods that are typically challenging.31 CPAPs could provide such training and support to pediatric PCPs in implementing these interventions and identifying available mental health and community resources. Finally, collaborative care practices between behavioral health providers and pediatric PCPs regarding treatment planning can improve the continuity of care for youth exposed to ACEs.Avenues for supporting youth with ACE exposure in the context of primary care include continuous monitoring of youth behavioral and physical health and referral to specialty mental health care  and community supports .


Subgroup analysis revealed a statistically significant association between the anterior–lateral electrode and increased cardioversion rate of AF in subgroups of less than five shocks, patients with 60 years old or more and patients with left atrial (LA) diameter >45 mm , , and , respectively. Anteriolateral electrode is more effective than anterior–posterior electrode in external cardioversion of AF, particularly in patients who have received less than 5 shocks, are 60 years old or older and have a LA diameter greater than 45 mm.The efficacy of anteriolateral versus anterior–posterior electrode positions in the success of atrial fibrillation's (AF) electrical cardioversion is unclear. Our aim is to perform a meta‐analysis to compare the success rate of both electrode positions. PUBMED, WOS, OVID, and SCOPUS were searched. Inclusion criteria were clinical trials that compared anterior–lateral with anterior–posterior electrodes in external cardioversion of AF. After the full‐text screening, 11 trials were included in the analysis. The total number of patients included in the study is 1845. The pooled analysis showed a statistically significant association between anterior–lateral electrode and increased cardioversion rate of AF (odds ratio [OR] = 1.40, 95% confidence interval [CI] = 1.02–1.92,  Anterior–lateral versus anterior–posterior electrodes in external cardioversion of atrial fibrillation. A meta‐analysis The global burden of AF has increased significantly due to the aging population. By 2030, the United States may have over 12 million people with AF.Electrical cardioversion is the most popular method of rhythm control in cardiology practice. It effectively terminates the AF in more than 90% of the cases.2Ethical approval is not required as this study is a meta‐analysis of published clinical trials. The present meta‐analysis was performed in accordance with Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA) guidelines and the Cochrane handbook.We systematically searched PUBMED, Web of Science, OVID, and SCOPUS from inception to January 30, 2022. The search included the following key terms: “Anterior lateral” OR “anterolateral” AND “Electrode” AND “Anterior posterior” OR “Anteroposterior” AND “atrial fibrillation” OR “AF”. We further reviewed the reference list of articles included in this review to include other relevant studies. Primary source studies published in peer‐reviewed journals were eligible for inclusion if they met the below criteria.2.1Clinical trials that compared anterior–lateral electrode with anterior–posterior electrode in external cardioversion of AF. We excluded cohort studies, case reports, editorials, conference abstracts, and animal studies. And thus, the PICO criteria for our meta‐analysis will be:Population: Patients with AF.Intervention: Anterior–lateral electrode.Comparison: Anterior–posterior electrode.Outcome: Cardioversion success rate between anterior–lateral electrodeposition group and the anterior–posterior group.2.2Initial title and abstract screening were conducted by two reviewers (S. S. and H. F.) and all disagreements were discussed to reach a consensus, otherwise, a third opinion from (K. R. M.) was obtained.n), other baseline diseases, duration of AF, and baseline treatment.Potentially eligible articles were imported for full‐text review and assessed for inclusion. We extracted data using an Excel sheet. Examples of data collected are study arms, number of patients in each group, age, sex (We used the Cochrane tool (Risk of Bias 2) to assess the risk of bias in randomized trials. The following items were assessed .2.3χ2 p < .05), a random effect model was used otherwise, a fixed‐effect model was employed, in general; the results were considered significant if the p value was less than .05.We used the Review Manager Software version 5.4 to perform the meta‐analysis; the dichotomous outcomes were presented as odds ratios (OR) with a 95% confidence interval. In case of heterogeneity  diameter. The number of shocks subgroup was divided into two subgroups: five shocks or less than five shocks. The age subgroup was divided into two subgroups: 60 years old and more or less than 60 years old. The LA diameter subgroup was divided into two subgroups: more than 45 mm or 45 mm and less than 45 mm. The overall risk of bias was high in most of the studies, as we found significant bias in deviation from the intended interventions domain in six studies, missing outcome data domain in one study, and measurement of the outcome domain in three studies; however, we found no significant bias in randomization process domain in the included studies as shown in Figure The total number of patients included in the study is 1845 patients, 931 patients in the anterior–lateral electrode group, and 941 patients in the anterior–posterior electrode group, other baseline data are shown in Table 3.23.2.1p = .04), indicating that anterior–lateral electrodeposition is better than anterior–posterior electrodeposition in the external cardioversion of AF. We observed no heterogeneity among studies , Figure The pooled analysis showed a statistically significant association between the anterior–lateral electrode and increased cardioversion rate of AF compared with anterior–posterior electrode . We observed no significant heterogeneity among studies in this subgroup , Figure p = .006). We observed no significant heterogeneity among studies in this subgroup , Figure The pooled analysis showed a statistically significant association between anterior–lateral electrode and increased cardioversion rate of AF compared with anterior–posterior electrode in a subgroup of less than 5 shocks . We observed no significant heterogeneity among studies in this subgroup , Supporting Information: Figure p = .57). We observed a significant heterogeneity among studies in this subgroup , Supporting Information: Figure p = .48, I2 = 0%), and the pooled analysis showed no difference between the two groups .The pooled analysis showed no statistically significant difference between anterior–lateral electrode and anterior–posterior electrode in a subgroup of less than 60 years old . We observed a significant heterogeneity among studies in this subgroup , Supporting Information: Figure p = .59, I2 = 0%), and the pooled analysis showed a statistically significant association between the anterior–lateral electrode and increased cardioversion rate of AF compared with anterior–posterior electrode in a subgroup of more than 45 mm .p = .83). We observed no significant heterogeneity among studies in this subgroup , Supporting Information: Figure The pooled analysis showed no statistically significant difference between anterior–lateral electrode and anterior–posterior electrode in a subgroup of 45 mm and less than 45 mm  and asymmetry index, ASI , as markers for LA remodeling. They found that paroxysmal AF is more likely to have greater LAV and relatively higher ASI than the average population. Progression to persistent A.fib is more likely to have greater LAV with stable ASI. Eventually, long‐term persistent A.fib was found to have similar LAV to persistent A.fib with larger ASI.Moreover, the progression of A.fib is associated with poor outcomes after transcutaneous ablation.Second, a divergent patient population seems to be a valid prognostication factor in determining the safety and efficacy of various approaches. Our pooled analysis revealed that anterior–lateral position is more likely to attain higher success chances in patients aged 60 years old or more.Third, it is essential to consider that some medical practices have been recently obsolete and are no longer clinical guidelines. One is the use of monophasic shocks in cardioversion which have been widely replaced with biphasic shocks that have shown optimal efficacy in managing different types of arrhythmias.Given the current data, it is reasonable to consider the anterior–lateral position as a first‐line modality in AF cardioversion, given the relatively higher success rates, especially in elderly patients and those with enlarged left atrium indicating LA remodeling. Additionally, the anterior–lateral position has anatomical convenience and easier accessibility, particularly in critically ill patients.Nevertheless, our study has some limitations. Variable protocols for cardioversion have been applied. While some papers followed the step‐up energy levels approach, others used initial high‐energy levels of 200 J and higher on failed attempts. We found some bias in deviation from the intended interventions, missing outcome data, and measurement of the outcome domains in some studies; however, we found no significant bias in the randomization process domain in the included studies. Lastly, our included articles had variable proportions of persistent and permanent AF, which are hard to treat and might alter the outcomes.5Our meta‐analysis revealed that anterior–lateral electrodeposition is more effective and better than anterior–posterior electrodeposition in external cardioversion of AF, particularly in patients who have received less than five shocks, are 60 years old or older, and have a LA diameter greater than 45 mm. More multicenter randomized clinical trials are warranted to support our findings.Conceptualization: Karam R. Motawea. Research design, data collection, literature search, statistical analysis, and manuscript preparation: Karam R. Motawea, Mostafa R. Mostafa, Mohamed Magdi, Mohamed Magdi, Hager Fathy, Sarya Swed, Mohamed M. Belal, Dina M. Awad, Rowan H. Elhalag, Nesreen E. Talat, Samah S. Rozan, Abdulqadir J. Nashwan, Naim Battikh, Bisher Sawaf, Mhd Kutaiba Albuni, Elias Battikh, Gihan M. Mohamed, Amr Farwati, and Hani Aiash: All authors read and approved the final manuscript. All authors take responsibility for all aspects of the reliability and freedom from bias of the data presented and their discussed interpretationThe authors declare no conflict of interest.Supplementary information.Click here for additional data file.Supplementary information.Click here for additional data file.


Transplant recipients have a 2‐ to 4‐fold increased risk of developing malignancies over the general population. Cancer is the second most common cause of death for recipients. The magnitude of the risk depends on the cancer type and increases in viral‐related malignancies. Skin cancer is the most common. However, data in most cancer registries is limited to cutaneous melanomas, thereby limiting the epidemiologic examination of cancer risk in non‐melanoma skin cancer. Our goal was to evaluate post‐kidney transplant cancer cases and sites in our population to guide screening recommendations.Between 2009 and 2015, a retrospective study of adult kidney recipients transplanted at East Carolina University was conducted. The first cancer diagnosis after transplant through February 18, 2020, was captured and analyzed. Patient demographics, cancer sites, and histological diagnoses were analyzed and compared. p16 immunohistochemistry was used as a surrogate marker for high‐risk human papillomavirus (HPV) infection.N = 439), the majority were non‐Hispanic Black (NHB) individuals, 312 (71.1%), and 127 (28.9%) were non‐Hispanic White (NHW) individuals. Of these, 59 (13.4%) developed a posttransplant malignancy, with the majority on sun‐exposed skin found in NHW. NHB had all anogenital/mucosa skin cancers on non‐sun‐exposed skin. Of these detected in NHB, all were squamous cell carcinomas, with five out of six (83.3%) being positive for p16.Retrospectively, kidney transplant recipients were analyzed (Posttransplant malignancy differed significantly by race, site, and potential source of etiology. The majority of malignancies are likely explained by acceleration of precursor lesions from prior exposure to ultraviolet rays or HPV. This study demonstrated that posttransplant skin malignancies differed significantly between non‐Hispanic White and non‐Hispanic Black kidney recipients by cancer site and patient skin tone. Comprehensive full body dermatologic inspection following kidney transplantation is critical and should include close inspection of all skin, including the anal and genital regions. We identified 538 adult kidney transplant recipients during these years. Any cancer diagnosis after transplantation surgery through February 18, 2020, was captured through detailed medical record review. We did not have adequate sample sizes to produce reliable estimates to investigate the intersections of all races and ethnicities. Therefore, only data from NHW and NHB were compared.Of the 538 patients, 16 were excluded; of these, 11 were of Hispanic ethnicity and 5 were of Asian race. Of these individuals, only two, who were Hispanic developed a post‐kidney transplant cancer. An additional 81 patients were excluded because of a history of pretransplant malignancy. Two patients were excluded as clinical information was not available entirely. Thus, 439 recipients were included.The study evaluated demography, time interval from transplant to the first cancer diagnosis, and histologic and immunohistochemical features. Race and ethnicity are based on the information reported in the electronic medical record (EMR).The regarded cancer diagnoses were solely based on histopathological examination by an average of two pathologists, each in the Department of Pathology at ECU. If patients developed multiple cancers over time, only the first cancer diagnosis was included in the investigation.Cancer‐containing tissues including skin specimens were formalin‐fixed, paraffin‐embedded, and sectioned 5 μm in thickness. Hematoxylin–eosin staining was performed in a standardized method. By immunohistochemistry, we examined rates of p16 positivity in the squamous cell carcinomas. P16 positivity is often considered a surrogate marker of integration of high‐risk human papillomavirus (HPV) DNA into the host genome.Stata Statistical Software: Release 17: StataCorp LLC. This retrospective study was approved by ECU and Medical Center Institutional Review Board (IRB‐99‐0282) and authorization for waiver of consent was obtained.Descriptive statistics, chi square tests and Fisher's Exact analyses were conducted in STATA StataCorp. 2021. 33.1p < 0.001. At the time of transplant, no significant gender differences were seen, we had 239 males (54.4%) versus 200 females (45.6%), p = 0.17. The median age at the kidney transplant was 58.50  years of age with an age range of 17–82 years (Table The baseline characteristics of 439 recipients are summarized in Table 3.2N = 439), 59 (13.4%) patients developed a post‐kidney transplant malignancy. Although our recipients were significantly more likely to be NHB, the overall number of new cancer cases in NHW was higher than that in NHB; 33 cancers among 127 NHW (55.9%) and 26 cancers among 312 NHB (44.1%). Of these, males were more affected, 37 (62.7%), than 22 (37.3%) females. The group aged 50 years and older had significantly more cancers than those under age 50; 46 (82.1%) versus 10 (17.9%), p = 0.003.Of the overall sample , followed by basal cell carcinoma (3 cases). Basal cell carcinoma was only seen on sun‐exposed skin in NHW. Malignant melanoma, Merkel cell carcinoma or Kaposi sarcoma were not observed in this study.3.4We found striking racial and ethnic differences in sun‐exposed skin versus non‐sun‐exposed skin cancer Figure . Among 23.5N = 439) in our study, 94 (21.4%) died between date of transplantation and February 18, 2020. (None of the deaths were attributed to COVID 19 on the death certificate.) Of note, one patient, a 57‐year‐old NHB female, developed and died from a previously non‐diagnosed, anal squamous cell carcinoma.Of the total transplant population  is an important risk factor for lighter‐skinned NHW as NHB are more protected from sun damage due to more deeply pigmented skin and higher melanosomes levels.4.1A well‐recognized fact is that immunological control of oncogenic viral infections is impaired, resulting in more common posttransplant cancers like Kaposi sarcoma and HPV‐related cancers. Squamous cell carcinoma arising in a genital area is frequently associated with preceding high‐risk HPV infection.Although the social determinants of health are well‐known factors that increase cancer risk, some studies show that some differences might be related to infection of different HPV genotypes among races and ethnicities.In contrast to the direct relationship between HPV and p16 positivity in genital squamous cell carcinomas, the significance of p16 positivity in posttransplant squamous cell carcinoma on sun‐exposed skin should be interpreted cautiously. We found that p16 was positive in 57.1% of cases; however, this may not be related to infection by high‐risk HPV which is rare in cutaneous cancers in general. It is recognized that p16 positivity in some squamous cell carcinomas, that occur in the head and neck, esophagus, and lung, are unlikely due to high‐risk HPV infection.Rather, posttransplant squamous cell carcinomas on the sun‐exposed skin is at least partially caused by a β‐HPV infection as in epidermodysplasia verruciformis type or cutaneous type (HPV 5 and 8) which are different from high‐risk HPV such as HPV16 and 18.4.2Epidemiologic evidence supports the association of HPV in the cervical vaginal microbiome with an increased risk of inflammation and HPV progression to cervical cancer.One limitation in this study is that racial and ethnic reporting is limited to that reported in the EMR, and we suggest it as a proxy for lighter and darker skin tones. We also recognize that race is a co‐created social construct, not a biological variable.In conclusion, transplant medicine has been growing continuously and prevention of posttransplant malignancy with comprehensive skin inspection, including anogenital areas, is crucial. Skin cancer was the most common malignancy, and squamous cell carcinoma is the most common histologic type. In addition, 95% of known skin cancer sites in NHW developed on sun‐exposed areas; in contrast, 100% developed on non‐sun‐exposed, anogenital areas in NHB individuals. Different oncogenic mechanisms involving ultraviolet rays, possible different HPV type infections, the skin microbiome, potential genetic or biological differences among races, and the types of immunosuppression used all may play a role. This study indicates that screening with attention to risk stratification for kidney transplant recipients based on cancer site, cancer type, and potentially race and ethnicity may aid in the early detection of malignancies.Kotaro Takeda: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; writing – original draft (lead); writing – review and editing (lead). Carolann Risley: Formal analysis ; funding acquisition ; investigation ; methodology ; writing – original draft (lead); writing – review and editing (lead). Aisha Kousar: Data curation ; investigation . Kimberly P. Briley: Data curation . Karyn Prenshaw: Investigation . Rajesh Talluri: Formal analysis ; writing – review and editing (supporting). Kim R. Geisinger: Conceptualization ; investigation ; methodology ; supervision ; validation ; writing – original draft (lead); writing – review and editing . Lorita M. Rebellato: Conceptualization ; data curation ; investigation ; project administration ; supervision ; writing – original draft ; writing – review and editing .This work was supported by the Mississippi Center for Clinical and Translational Research Center – National Institute of General Medical Sciences of the National Institutes of Health under Award Number 1U54GM115428.The authors do not have any conflict of interests to disclose.The views expressed in this manuscript are those of the authors and do not reflect the official policy of the Department of Defense or the US government.The use of patient data was approved by the East Carolina University Brody School of Medicine Institutional Review Board for human studies and authorization for waiver of consent was obtained. All clinical and research activities are consistent with the Principles of the Declaration of Istanbul.


Prostate cancer (PCa) is a major cause of cancer morbidity and mortality for men globally, and androgen signaling clearly drives its onset and progression. Androgen receptor (AR) regulation is complex and remains elusive, despite several studies tackling these issues. Therefore, elucidating the mechanism(s) underlying AR regulation is a potentially promising approach to suppressing PCa.We report that Par14, one isoform of the prolyl isomerases homologous to Pin1, is a critical regulator of AR transcriptional activity and is essential for PCa cell growth.Par14 was shown to be overexpressed in PCa, based on analyses of deposited data. Importantly, overexpression of Par14 significantly enhanced androgen‐sensitive LNCap cell growth. In contrast, silencing of Par14 dramatically decreased cell growth in LNCap cells by causing cell cycle arrest. Mechanistically, silencing of the Par14 gene dramatically induced cyclin‐dependent kinase inhibitor p21 at both the mRNA and the protein level through modulating the localization of p53. In addition, suppression of Par14 in LNCap cells was shown to downregulate the expressions of androgen response genes, at both the mRNA and the protein level, induced by dihydrotestosterone. Par14 was shown to directly associate with AR in nuclei via its DNA‐binding domain and augment AR transcriptional activity.Thus, Par14 plays a critical role in PCa progression, and its enhancing effects on AR signaling are likely to be involved in the underlying molecular mechanisms. These findings suggest Par14 to be a promising therapeutic target for PCa. Prolyl isomerase Par14 directly associates with the androgen receptor. Par14 is mandatory for cell proliferation and its transcriptional activity. Moreover, Par14 interacts with AR and enhances its transcriptional activity. These findings suggest Par14 to be an attractive target for the discovery of drugs targeting PCa.22.13 and antibiotics. They were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37°C. For DHT stimulation, LNCap cells were incubated in the medium supplemented with 10% charcoal‐stripped serum (CSS) overnight, and were then exposed to 10 nM DHT for the indicated times. DHT was purchased from Selleck Chemicals.LNCaP cells were purchased from RIKEN. All cells were routinely cultured in RPMI1640 (Life Technologies) containing 10% fetal calf serum, glutamine, NaHCO2.2https://tnmplot.com/analysis/). This platform includes a dataset from GTex, TCGA and TARGET.http://gepia.cancer‐pku.cn/).The expressions were compared between normal and tumor tissues based on TNM plot data (2.3Plasmid transfection was performed by using X‐treme reagent (Roche). The plasmid and X‐treme were both placed in Opti‐MEM to allow formation of the complex for 15 min. Subsequently, these complexes were added to the cells.For Par14 knockdown, siRNA treatment was conducted by the reverse‐transfection method. Briefly, all stars Negative or Par14 siRNA was mixed with RNAiMAX in Opti‐MEM for 15 min. The mixture was applied to each well, and the cells were then seeded. The following siRNA sequences were used.Par14 si‐1: CCGCACAGTATAGTGAAGATAPar14 si‐2: GGCCGCACAGTATAGTGAAGATAAA2.4Cell proliferations were examined employing WST‐1 reagent (Takara Bio). Each cell was treated with Par14 plasmid or siRNAs. After 5 days, WST‐1 reagent was added to fresh medium and the cells were incubated for 3 h at 37°C. The absorbance of the supernatant was measured at 450 nm employing a Varioscan (Thermo Fisher Scientific).2.5http://n2t.net/addgene:171240; RRID: Addgene_171240). AR deletion mutants were inserted into a myc‐TEV‐Flag‐pcDNA vector. PSA5.8k‐Luc reporter vector, which includes the 5.8 kb PSA enhancer/promoter region, was kindly provided by Dr. A. Mizokami (Kanazawa University).The human Par14 gene was inserted into pcDNA3.1 (‐) with a Flag tag sequence in the N‐terminal. Flag‐M4‐AR was a generous gift from Steven Balk  was conducted, as described in a previous report.2.7LNCap cells were treated with siRNAs. After 3 days, the cells were collected. Subsequently, they were fixed with ice‐cold ethanol for 2 h at 4°C. After washing with PBS, the cells were mixed with propidium iodide/RNase staining buffer (BD Biosciences) for 15 min. Samples were washed again and analysis of the cell cycle was carried out employing the LSR Fortessa X‐20 (BD Biosciences).2.8Western blotting was performed, as described in previous reports.Primary antibodies were diluted as follows: AR , GAPDH , PSA , NKX3.1 , KLK2 , p21 , Par14 , and Flag antibody . Par14 antibody was also raised by the injection of GST‐Par14 into rabbits. Primary and secondary antibodies were diluted in 3% skim milk or Can Get Signal (Toyobo).2.9http://www.heatmapper.ca/). The primers used were as follows.Total RNA was extracted using a NucleoSpin RNA kit, according to the manufacturer's protocol. After RNA concentrations had been measured using Nanodrop, the same amount of cDNA was then created using reverse‐transcriptase with a verso cDNA kit. Then, real time PCR was conducted employing SYBR Green and each specific primer. Heatmap was created by Heatmapper (hPSA (KLK3) F: atgacgtgtgtgcgcaagtt R: atggttcactgccccatgachKLK2 F: aggcacacacacagcaagga R: gggggacctgaacaaacctchNKX3.1 F: cttccaaggcttccccaaac R: agctccgaggagagctgctthPUMA F: tggactcagcatcggaaggt R: tgaaggagcaccgagaggaghBAX F: gcgtccaccaagaagctgag R: gccttgagcaccagtttgcthGADD45 F: atggataaggtgggggatgc R: gcctggatcagggtgaagtgh p21 F: tcaaatcgtccagcgacctt R: tggaaggtgtttggggtcagh p27 F: gggcaagtacgagtggcaag R: gtccaccaaatgcgtgtccthPar14 F: aaggggcttgtacggcaact R: actgcattgccaccaccttthGAPDH F: ggtgaaggtcggagtcaacg R: agggatctcgctcctggaag2.10LNCaP cells were cultivated on a 48‐well plate overnight, and then transfected with both PSA‐Luc and TK‐rluc vector, using X‐treme reagent. One day thereafter, the cell medium was switched to medium containing CSS. The next day, the cells were stimulated with DHT for 24 h, and the luciferase assay was performed using a Dual‐Glo Luciferase assay kit (Promega), according to the manufacturer's protocol.2.11The indicated plasmids were transfected into LNCap cells. After 2 days, the cells were solubilized by lysis buffer containing Tris–HCl, NaCl, ethylenediaminetetraacetic acid, 0.1% Triton, phenylmethylsulfonyl fluoride, NaF and orthovanadate. After centrifugation, the cell lysates were transferred into new tubes and flag beads were added. Samples were rotated for 2 h at 4°C, and the beads were then washed with lysis buffer four times. Finally, 2DB was added to each tube and the samples were heated at 95°C for 10 min.2.12Cells were cultivated in an 8‐well chamber. They were fixed with 10% formalin for 10 min, then for 5 min in 0.1% Triton. For blocking, 3% goat serum was added to each well for 30 min. Thereafter, cells were reacted with primary antibody (1:300) at 4°C overnight. Subsequently, the cells were mixed with Alexa488 (1:400) at room temperature for 1 h. Finally, cells on the slide were mounted with 4′,6‐diamidino‐2‐phenylindole and observed by BZ8000. After each process, the chamber slide was washed with PBS three times.2.13All data are presented as means + standard error of mean. Statistical significance was calculated employing the BellCurve for Excel.t test was used for the analysis of WST‐1, flow cytometry, real time PCR  which pools the previously deposited RNA‐seq data.Although both Pin1 and Par14 belong to the Parvulin family, the WW domain in the N‐terminal of Pin1 is replaced with a Basic domain in Par14, which accounts for the difference in their binding motifs Figure . As Pin1http://gepia.cancer‐pku.cn/). The patients with PCa expressing high levels of Par14 had shorter overall survival (OS) periods as well as disease‐free survival (DFS) periods than those expressing low levels of Par14  Figure  accountsIn this study, we began by demonstrating Par14 to be overexpressed in PCa as compared with normal tissues, based on analyses of deposited data, and this overexpression was more evident than that of Pin1 Figure . ImportaA direct association between Par14 and AR was demonstrated. As to PCa, there are studies indicating Pin1 to be a critical regulator for both PCa proliferation and AR signaling.However, as is well known, ADT eventually leads to ligand‐independent AR activation, so‐called CRPC. This phenomenon is partially explained by the manifestation of AR‐V7 which is LBD lacking a receptor.https://thebiogrid.org/111320/table/homo‐sapiens/pin4.html), which is a database of protein interactions, showed the presence of numerous Par14 binding partners. These findings raise the possibility that Par14 has divergent roles in cell growth and other physiological functions.We cannot exclude the possibility that Par14 promotes growth through an AR‐independent pathway, in addition to AR‐dependent signaling. Par14 is known to be expressed in both the nucleus and the cytosol according to the Human Protein Atlas database. We assume that Par14, like Pin1, interacts with a number of substrates to modulate cellular functions. In fact, both Pin1 and Par14 associate with insulin receptor substrate‐1 (IRS‐1), enhancing its insulin‐induced phosphorylation.As to the limitations of this study, first, we used only single AR‐positive cells, because we cannot obtain others from cell banks in Japan. As is well known, androgen ablation therapy causes the development of CRPC which was, at least partially, triggered by the expression of AR‐V7. Cancer tissues are of course heterogonous, and Par14 might have distinct roles, depending on cell types. Accordingly, future studies are required to fully clarify the roles of Par14 in the functions of AR and its splicing variants, such as AR‐V7. In addition, unfortunately, no Par14 inhibitors have as yet been developed and we thus cannot examine their effects on the in vivo growth of PCa. Creating Par14 inhibitors and conducting in vivo trials might facilitate understanding the effects of Par14 on the survival rate and/or proliferation under physiological conditions.In conclusion, the findings of our current study reveal that Par14 plays a critical role in PCa progression, and its enhancing effect on AR signaling may well contribute to the underlying molecular mechanisms. Accordingly, Par14 is a key molecule for suppressing PCa and its inhibitors are promising targets for novel therapies.Miki Naito: Data curation (lead); investigation (lead). Kenichiro Ikeda: Data curation (supporting); investigation (supporting). Shunya Aoyama: Investigation (supporting). Mayu Kanamoto: Investigation (supporting). Yasuyuki Akasaka: Investigation (supporting). Yuri Kido: Investigation (supporting). Mikako Nakanishi: Investigation (supporting). Machi Kanna: Supervision (supporting). Takeshi Yamamotoya: Supervision (supporting). Akio Matsubara: Project administration (supporting); supervision . Nobuyuki Hinata: Project administration (supporting); supervision (supporting); writing – original draft (supporting). Tomoichiro Asano: Conceptualization (supporting); funding acquisition (lead); project administration ; supervision ; validation ; writing – original draft ; writing – review and editing . Yusuke Nakatsu: Conceptualization (lead); data curation (lead); funding acquisition ; investigation (lead); project administration ; supervision ; validation ; visualization ; writing – original draft ; writing – review and editing .This work was partially supported by Grants‐in‐Aid for Scientific Research (C)  and (B) (To T.A. 20H03732). This research was also supported by the Nozomi H Foundation, Nagao Takeshi Orphan Disease Research Foundation and the Institute for Adult Diseases Asahi Life Foundation.The authors have no conflicts of interest.N/A.Figure S1.Click here for additional data file.


Health literacy and e‐health literacy are important factors helping people shape awareness of health behaviours in different aspects, including sleep hygiene behaviours. Good sleep hygiene behaviours promote sleep quality and are beneficial to overall mental wellbeing.We aimed to examine if sleep hygiene behaviours may mediate the association between health literacy/e‐health literacy and mental wellbeing.n = 9775; mean [SD] age = 36.44 [11.97] years; 67.3% females) completed the Health Literacy Instrument for Adults, eHealth Literacy Scale, three items on sleep hygiene behaviour that have been used in prior research and the Short Warwick Edinburgh Mental Wellbeing Scale. Data were then subjected to structural equation modelling (SEM) including 500 bootstrapping resampling to examine whether sleep hygiene is a mediator in the relationship between health literacy/e‐health literacy and mental wellbeing.Adult Iranian subjects  and sleep hygiene behaviours . Sleep hygiene behaviours were significantly associated with mental wellbeing . Moreover, SEM that incorporated bootstrapping approaches indicated that sleep hygiene behaviours were significant mediators in the association between health literacy/e‐health literacy and mental wellbeing.Both health literacy and e‐health literacy were significantly associated with mental wellbeing (We conclude that health literacy and e‐health literacy are associated with mental health wellbeing in the Iranian population. Additionally, the association could be mediated via sleep hygiene behaviours.The study was co‐designed with healthcare providers from the vice‐Chancellor's Office for Health Affairs of Qazvin University of Medical Sciences as equal partners. Moreover, the women's health volunteers were involved in the design of the study. Moreover, we hypothesized that (i) sleep hygiene behaviours are significant mediators in the association between traditional health literacy and mental wellbeing and (ii) sleep hygiene behaviours are significant mediators in the association between e‐health literacy and mental wellbeing.22.1This study is part of a broader study focused on health literacy and mental wellbeing amongst Iranian citizens residing in the city of Qazvin.2.22.2.1The HELIA is a 33‐item questionnaire administered via self‐report. Amongst the 33 items, five domains are defined: reading (4 items), access to information (6 items), understanding (7 items), appraisal (4 items) and decision‐making/behavioural intention (12 items).2.2.2The eHEALS is an eight‐item questionnaire administered via self‐report. Amongst the eight items, one domain of e‐health literacy has been validated.2.2.3Three items were used to assess sleep hygiene behaviours: (i) How many times did you make your bedroom a restful place over the past week? (ii) How many times did you avoid going to bed feeling hungry or thirsty? (iii) How many times did you avoid anxiety and stress‐provoking activities before going to bed? All items are rated on a 5‐point Likert scale ranging from 1 (never) to 5 . Then, the three items were averaged to indicate sleep hygiene behaviours. A higher score in the sleep hygiene behaviours measure indicates better sleep hygiene. Moreover, these three sleep hygiene behavioural items in the Persian language showed satisfactory properties in their internal consistency and criterion‐related validity with the Pittsburgh Sleep Quality Index.2.2.4The SWEMWS is a seven‐item questionnaire administered via self‐report. Amongst the seven items, one domain of mental wellbeing has been validated.2.2.5All participants provided information on their age, gender, educational status, residence and marital status.2.3Demographics were initially presented using descriptive statistics . Pearson correlations and descriptive statistics were then used to examine the associations and central tendencies of the studied variables . Finally, three models were constructed via structural equation modelling (SEM) to examine the mediation effects of sleep hygiene behaviours in the association between health/e‐health literacy and mental wellbeing.In the first model, mediation effects of sleep hygiene behaviours were tested in the association between health literacy (assessed via HELIA with separated domains) and wellbeing Figure . In the For all the SEM models, age, gender, residence and educational status were controlled. Moreover, the following fit indices were used to examine if the first and the second models are supported: comparative fit index (CFI) > 0.9, Tucker–Lewis index (TLI) > 0.9, root mean square error of approximation (RMSEA) < 0.08 and standardized root‐mean‐square residual (SRMR) < 0.08.3n = 6576) with 1.3% not indicating gender. Nearly half of the participants were residing in Qazvin  and 74.6% lived in a city (n = 7287). Most of the participants had completed higher education (32.9% had high school diplomas and 39.4% had university degrees) and 71.5% were currently married ; and significantly associated with e‐health literacy in a moderate magnitude positively . Similarly, mental wellbeing was significantly associated with all the HELIA domains in a moderate to strong magnitude positively  and significantly associated with e‐health literacy in a moderate magnitude positively . Moreover, sleep hygiene behaviours were significantly associated with mental wellbeing positively .Table χ2 (df) = 22593.172 (973), p < .001, SRMR = 0.04, RMSEA (90% CI) = 0.05 (0.05–0.05), TLI = 0.92 and CFI = 0.93; for eHEALS  = 7066.205 (192), p < .001, SRMR = 0.04, RMSEA (90% CI) = 0.06 (0.06–0.06), TLI = 0.93 and CFI = 0.94. Furthermore, sleep hygiene behaviours were found to be a positive and significant mediator in the following paths: (i) from HELIA reading domain to mental wellbeing , (ii) from HELIA access to information domain to mental wellbeing , (iii) from HELIA appraisal domain to mental wellbeing , (iv) from HELIA decision making/behavioural intention domain to mental wellbeing  and (v) from eHEALS to mental wellbeing . Also, health literacy domains , except for access to information (p = .15) and e‐health literacy (β [p] = .24 [<.001]) had significantly direct effects on mental wellbeing (Table Two proposed mediation models using latent constructs (one for separated HELIA domains and another for eHEALS) had satisfactory fit statistics. For HELIA Figure : χ2 (dfχ2 (df) = 104,420.76 (190), p < .001, RMSEA (90% CI) = 0.237 (0.236–0.238), TLI = 0 and CFI = 0]  and e‐health literacy  with mental wellbeing  but not e‐health literacy  had significantly direct effects on mental wellbeing  could not be identified [4The present study used a large Iranian sample to examine if sleep hygiene behaviours mediate the association between health literacy/e‐health literacy and mental wellbeing. Our findings suggest that all domains of health literacy, except for the understanding domain, and e‐health literacy were positively and significantly associated with mental wellbeing via sleep hygiene behaviours. Moreover, all health literacy domains and e‐health literacy were positively and significantly associated with sleep hygiene behaviours and with mental wellbeing. In addition, sleep hygiene behaviours were also positively and significantly associated with mental wellbeing and emerged as a mediator in the relationship between mental well‐being and health literacy. Thus, it is reasonable to assume that any improvements in health literacy may promote people's sleep hygiene behaviours, and subsequently enhance their mental wellbeing.The associations found between health literacy and sleep hygiene behaviours in the present study concur with prior findings. Indeed, improved health literacy may reduce the prevalence and severity of sleep disorders,The associations found between sleep hygiene behaviours and mental wellbeing in the present study may be ascribable to improved sleep as facilitated by sleep hygiene. Specifically, prior evidence shows that enhanced sleep hygiene behaviours are associated with quality sleep,In addition, as shown herein, both health literacy and e‐health literacy may improve mental wellbeing as a result of better sleep hygiene behaviours.There are some limitations in the present study that deserve mention. First, the present study collected data using self‐reports. Therefore, the present findings are subject to several biases related to this type of data collection method, namely single rater bias, recall bias and social desirability bias. Second, given that health literacy could be different between Western and Eastern countries and the present study only recruited Iranians, the present findings may not be generalizable. Future studies are thus needed to examine if the present findings can be replicated in other countries. Third, the measure of sleep hygiene behaviours was not strong. Specifically, we only used three items to assess sleep hygiene behaviours; thus, they might not capture the whole construct of sleep hygiene behaviours. Future studies are thus suggested using other standardized and validated instruments on sleep hygiene behaviours .5In summary, sleep hygiene behaviours emerged as a significant mediator in the association between health/e‐health literacy and mental wellbeing in a large sample of Iranians. These findings indicate the importance of improving health literacy and e‐health literacy in the population, whereby improved health literacy correspondingly ameliorates sleep hygiene behaviours leading to better preservation of mental wellbeing. However, future experimental studies are needed to examine if improving health literacy and e‐health literacy could increase sleep hygiene behaviours, and promote mental wellbeing.Mehran Alijanzadeh, Rafat Yahaghi and Amir H. Pakpour: Conceptualization. Mehran Alijanzadeh, Rafat Yahaghi and Amir H. Pakpour: Methodology. Jalal Rahmani, Nahid Yazdi, Elahe Jafari, Najmeh Shahmahdi and Hashem Alijani: Software. Mehran Alijanzadeh, Rafat Yahaghi and Amir H. Pakpour: Validation. Amir H. Pakpour: Formal analysis Narges Zamani, Rafat Yahaghi, Razie Fotuhi, Masoume Zarenejad, Elham Taherkhani, Zeinab Buchali, Narges Mahmoudi, Leila Poorzolfaghar, Safie Ahmadizade, Azam Shahbazkhania and Mehran Alijanzadeh: Investigation. Amir H. Pakpour: Resources. Chung‐Ying Lin and Amir H. Pakpour: Data curation. Chung‐Ying Lin and Amir H. Pakpour: Writing—original draft preparation. Amir H. Pakpour, David Gozal: Writing—review and editing. Chung‐Ying Lin: Visualization. Amir H. Pakpour: Supervision Rafat Yahaghi, Mehran Alijanzadeh, Zeinab Buchali, Jalal Rahmani, Hashem Alijani and Amir H. Pakpour: Project administration. Amir H. Pakpour: Funding acquisition. All authors read and agreed to the published version of the manuscript.The authors declare no conflict of interest.This research was reviewed and approved by the Qazvin University of Medical Sciences (IR.QUMS.REC.1400.225).


Furthermore, we used the Human Protein Atlas database and our single‐cell RNA‐seq datasets of BLCA patients to verify the expression of 13 EMRGs at the protein and single‐cell levels. We constructed a risk score model; the area under the curve of the model at 5 years was 0.792. The risk score was significantly correlated with the immune markers M0 macrophages, M2 macrophages, CD8 T cells, follicular helper T cells, regulatory T cells, and dendritic activating cells. Furthermore, eight immune checkpoint genes were significantly upregulated in the high‐risk group. The risk score model can accurately predict the prognosis of BLCA patients and has clinical application value. In addition, according to the differences in immune infiltration and checkpoints, BLCA patients with the most significant benefit can be selected for immune checkpoint inhibitor therapy.Bladder urothelial carcinoma (BLCA) is a common malignant tumor of the human urinary system, and a large proportion of BLCA patients have a poor prognosis. Therefore, there is an urgent need to find more efficient and sensitive biomarkers for the prognosis of BLCA patients in clinical practice. RNA sequencing (RNA‐seq) data and clinical information were obtained from The Cancer Genome Atlas, and 584 energy metabolism‐related genes (EMRGs) were obtained from the Reactome pathway database. Cox regression analysis and least absolute shrinkage and selection operator analysis were applied to assess prognostic genes and build a risk score model. The  Reprogramming of energy metabolism is closely related to cancer. In this study, we constructed a prognostic model of bladder urothelial carcinoma based on energy metabolism‐related genes and explored the differences in the immune microenvironment and immune checkpoints between high‐risk and low‐risk groups, which will help to accurately select patients who are more likely to benefit from immunotherapy. AUCarea under the curveBLCAbladder urothelial carcinomaCIconfidence intervalC‐indexindex of concordanceDCAdecision curve analysisEMRGsenergy metabolism‐related genesFCfold‐changeFDRfalse discovery rateGOGene OntologyHPAHuman Protein AtlasHRhazard ratioKEGGKyoto Encyclopedia of Genes and GenomesLASSOleast absolute shrinkage and selection operatorOSoverall survivalRNA‐seqRNA sequencingROCtime‐dependent receiver operating characteristicTCGAThe Cancer Genome AtlasBladder urothelial carcinoma (BLCA) is a frequent malignant tumor of the human genitourinary system and is estimated to be the tenth most common cancer worldwide by 2020, with approximately 573 000 new instances and 213 000 deaths , there iet al. | > 1, false discovery rate (FDR) < 0.05 and P < 0.05 were the criteria for determining differentially expressed EMRGs. Then, Gene Ontology (GO) [clusterprofiler’ r package. In addition, gsea v4.1.0 software was run to reveal the potential biological pathways and mechanisms of the groups. Gene sets with P < 0.05 and FDR < 0.25 after 1000 permutations were considered to be significantly enriched [The mechanism of EMRGs in the progression of BLCA was investigated using functional analysis. First, differentially expressed EMRGs between the high‐risk group and low‐risk group were screened by the ‘ogy (GO)  functionenriched .http://www.proteinatlas.org) [cell ranger software  was used to preprocess the sequencing data. A heatmap of the expression levels of the EMRGS was drawn using the ‘complexheatmap’ and ‘pheatmap’ packages.Data from the HPA database (las.org)  were chor software (version 4.0.4). Statistical tests were two‐sided, and P < 0.05 was regarded as statistically significant.All data analyses were carried out in ACSM2A, ESD, GALK1, GLCE, HSPG2, HYAL3, IDUA, MAT2B, MECR, NR1H4, NUP188, PPP2CB, and TPST1) were determined using LASSO regression analysis . We ranked the patients' risk scores from lowest to highest  was an independent and important prognostic factor  and M2 macrophages (P = 0.002) in the high‐risk group were higher. Compared with the high‐risk group, the infiltration levels of plasma cells (P = 0.004), CD8 T cells (P = 0.001), follicular helper T cells (P = 0.011), regulatory T cells (Tregs) (P < 0.001), and dendritic activating cells (P = 0.005) were higher in the low‐risk group. In addition, the distribution of 22 immune cells was different in each BLCA patient , PDCD1 (PD‐1), HAVCR2, IDO1, PDCD1LG2 (PD‐L2), CTLA4, and TIGIT were greater in the high‐risk group , pathways in cancer , amino sugar and nucleotide sugar metabolism , cell cycle , and purine metabolism  , oxidative phosphorylation , fatty acid metabolism , glycerophospholipid metabolism  and tyrosine metabolism , were enriched in the low‐risk group  Fig. . In addioup Fig. .ACSM2A, ESD, HSPG2, HYAL3, IDUA, NUP188, and PPP2CB in BLCA tissues were higher than those in normal bladder tissues.To verify the expression levels of these 13 EMRGs (note that NR1H4 was absent), we detected the protein levels of normal samples and BLCA tissues on the HPA website Fig. . In the TPST1 and IDUA were mainly expressed in fibroblasts. ACSM2A and HSPG2 were highly expressed in the endothelium. Genes such as HYAL3 and MAT2B were expressed at a low level in particular cell types.To further validate the expression levels of the 13 EMRGs in single cells, our BLCA scRNA‐seq dataset was analyzed Fig. . The resBladder urothelial carcinoma is a kind of molecular heterogeneous malignant tumor, and its molecular characteristics are closely related to the prognosis of BLCA . In receThrough univariate and multivariate Cox regression analysis, EMRGs related to BLCA prognosis were screened and the model was optimized. Finally, a risk score model was developed based on 13 EMRGs. Survival analysis showed that patients in the high‐risk group had shorter OS and worse prognosis. TNM staging is helpful to predict the prognosis of cancer patients. Multivariate Cox regression analysis showed that the risk score model, age, and N stage were independent prognostic factors for BLCA patients. ROC curve analysis showed that the AUC value of the risk score model was higher than that of the traditional clinical parameters , suggesting that the predictive efficacy of the risk score model was better than that of the commonly used clinical prognostic indicators.et al. [et al. [Previous researchers have constructed a prediction model for BLCA prognosis based on the TCGA‐BLCA dataset. For example, Peng et al.  develope [et al.  construc [et al. . In summLAG3, PD‐L1, PD‐1, HAVCR2, IDO1, PD‐L2, CTLA4, and TIGIT) were significantly upregulated in the high‐risk groups. Moreover, 13 EMRGs were mainly enriched in tumor‐related pathways and metabolism‐related pathways. Finally, the expression levels of 13 EMRGs were proven in the HPA database and our single‐cell sequencing dataset.Combined with age, N stages, and risk score, a nomogram prediction model was established. The nomogram prediction model has a good clinical application value, and its prediction efficiency (AUC = 0.817) is better than that of a single predictor. Our results also showed that the immune microenvironment differed notably among different risk groups, and eight checkpoint genes , and mutations in GALK1 can lead to GALK deficiency or type 2 galactosemia [GALK‐deficient individuals are unable to phosphorylate galactose and thus accumulate galactose and galactitol [GALK1 and BLCA, but our results provide new evidence that it may serve as a meaningful marker and immunotherapeutic target for BLCA patients. PPP2CB was confirmed to be a risk factor for the activity, proliferation, and migration of BLCA cells [TPST1 upregulated expression levels in BLCA, breast cancer, tongue squamous cell carcinoma, and nasopharyngeal carcinoma [et al. [TPST1 expression levels in BLCA were closely linked with a low survival rate and high tumor pathological stage; therefore, TPST1 could be used as a prognostic indicator. It has been reported that IDUA is involved in chondroitin sulfate/dermatan sulfate metabolism and glycosaminoglycan metabolism, and IDUA_GNPDA1 could be used as a beneficial prognostic factor in hepatocellular carcinoma [IDUA in breast cancer patients was decreased compared with that in normal controls, and IDUA was confirmed to be a potential target for visceral metastasis in breast cancer patients [The majority of the genes in our risk score model have previously been linked to cancer. However, the role of several genes in BLCA remains unknown. ctosemia . In addilactitol . Becauselactitol . In addiCA cells , which carcinoma , 43, 44 arcinoma , 46. Che [et al.  confirmearcinoma . The exppatients .The tumor microenvironment and immune cells affect the treatment and prognosis of BLCA patients. Immune cell infiltration in tumors has been shown to correlate with patient prognosis , 50, 51.Notably, we observed that tumor‐related pathways were significantly enriched in high‐risk patients, while metabolism‐related pathways, such as fatty acid metabolism, glycerophospholipid metabolism, linoleic acid metabolism, and tyrosine metabolism pathways, were significantly enriched in low‐risk patients. Our results provide some new perspectives for the personalized treatment of BLCA. For example, in the future, low‐risk patients might benefit more from the targeted treatment of fatty acid metabolism.Our research is the first to validate the expression levels of EMRGs of the risk score model using single‐cell sequencing data of BLCA, which is innovative, but there are also some deficiencies that need to be improved. First, because the research subjects came from Europe, we need further validation in a large global multicenter cohort. Second, other traditional clinical parameters, such as smoking history, family history, and treatment method, were not included in the analysis. At present, we cannot determine whether these factors affect the predictive effect of the risk score model. Finally, genes have a variety of biologically and pathologically relevant functions in most cancer cells and are closely related to the internal features of cancers, thus serving as precise predictors of cancer development and prognosis . TherefoLAG3, PD‐L1, PD‐1, HAVCR2, IDO1, PD‐L2, CTLA4, and TIGIT) were significantly upregulated in the high‐risk groups. This could facilitate the precise selection of patients who would benefit more from immunotherapy.In summary, we developed a risk score model based on 13 EMRGs with better predictive efficacy than traditional clinical parameters. The immune microenvironment differed significantly among different risk groups, and eight checkpoint genes (CH, YL, QL, CW, BF, XM, and RY collected the data, and performed data cleaning and statistical analysis. CH was accountable for writing the manuscript and preparing diagrams. YL, QL, and LZ revised the article. SH, NL, JC, and FW were accountable for the research content framework building. LL, ZM, and LM guided research design and promoted project progress. All authors approved the submitted manuscript.The authors declare no conflict of interest.Table S1. EMRGs associated with BLCA patients' OS in TCGA‐BLCA data.Click here for additional data file.Table S2. Differential metabolism‐related genes between the high‐ and low‐risk groups in TCGA‐BLCA data.Click here for additional data file.