Similarly, a low intra-sensor variability has been observed, with a RSD of 6.6% (n = 3). Moreover, the sensor shows a long-term stability with reproducible performances for at least 2 months (RSD: 7.8%). Tests in saliva samples show the applicability of Pt nanozymes to commercial systems for non-invasive monitoring of hyperglycemia in saliva, with recoveries ranging from 92 to 98%.The ability of shape-controlled octahedral Pt nanoparticles to act as nanozyme mimicking glucose oxidase enzyme is reported. Extended {111} particle surface facets coupled with a size comparable to natural enzymes and easy-to-remove citrate coating give high affinity for glucose, comparable to the enzyme as proven by the steady-state kinetics of glucose electrooxidation. The easy and thorough removal of the citrate coating, demonstrated by X-ray photoelectron spectroscopy analysis, allows a highly stable deposition of the nanozymes on the electrode. The glucose electrochemical detection (at −0.2 V vs SCE) shows a linear response between 0.36 and 17 mM with a limit of detection of 110 μM. A good reproducibility has been achieved, with an average relative standard deviation (RSD) value of 9.1% (The online version contains supplementary material available at 10.1007/s00604-023-05992-9. Nanozymes are nanomaterials with intrinsic enzyme-like features –3. Their2O2 generated by GOx-mediated glucose oxidation. Such systems do not overcome the above-mentioned issues related to protein fragility as they still require the presence of a natural enzyme. Moreover, the need of additional chromogenic substances for glucose optical detection creates further problems to the system. When used in electrocatalytic applications, severe barriers to establish electrochemical communication between the electrode and the nanozyme are often encountered possibly due to electron transfer resistance at the electrode interface introduced by nanozyme coatings or binding matrices used for nanozyme linkage to the electrode. For overcoming these difficulties, Pt nanomaterials are often integrated with other components, as metal-organic-frameworks, redox mediators, and conductive polymer matrices 40]. ResuOne major feature of the proposed system is the demonstrated absence of polymers and surfactants which provide clean nanostructured surfaces. For demonstrating the enhanced catalytic properties ascribable to the coating-free property of Pt NPs, their electrocatalytic activity has been compared with that of similarly sized octahedral Pt NPs coated with a PVP shell. From data reported in Figure The ability of octahedral Pt nanoparticles to act as GOx-mimicking nanozymes has been proved by testing their analytical performances in glucose electrooxidation with the aim to make a quantitative evaluation of parameters that can effectively foster the natural enzyme replacement in glucose monitoring practices.The limit of detection , 56 (LODn = 3). Similarly, a low intra-sensor variability has been observed, with a percentage RSD value of 6.6% (n = 3). Moreover, the sensor is long-lasting and can be used for at least 60 days, with only 7.8% response variability. This is clearly proven by comparing the freshly prepared sensor response with that observed after 10, 15, 30, 45, and 60 days  value of 9.1% n = . SimilarTo establish the selectivity of Pt nanozymes sensor response to glucose, MPAD measurements have been performed in the presence of interfering organic molecules, namely fructose, sorbitol, ascorbic acid, and lactic acid, each at 1 mM. Results are shown in Fig. Glucose detection by the nanozyme-based sensor was performed in spiked saliva samples. As shown in Table imax and Km, have been estimated by using the Lineweaver-Burk linearization of Michaelis-Menten equation as follows  is a key parameter that indicates the enzyme affinity towards its substrate, with a lower Km value typically referring to a stronger affinity. The value of Km also represents the substrate concentration at which the enzyme operates at one-half of its vmax. If an enzyme has a low Km value, it achieves a maximal catalytic efficiency at a low substrate concentration [Km is significantly comparable to the reported Km value reported for glucose-oxidase (14.2 mM) [From the data fitting, ntration . In our 14.2 mM) . This reSuch results, together with glucose sensing performances, reveal the strength of the proposed sensor representing an artificial system mimicking a natural enzyme with the advantage of reduced costs and higher stability. The observed limit of detection, possibly representing a limitation in comparison with the natural enzyme as it restricts the application in physiologic conditions, makes the system indeed suitable for monitoring hyperglycemia in saliva. To the best of our knowledge, this is the first report that proves that well-defined and extended {111} surface facets on octahedral nanoparticles closely mimic and replace GOx enzyme Table .Km value. Glucose detection shows a wide linear response, interesting limit of detection and excellent reproducibility coupled with long-term stability. The performance of the device in saliva samples reveals the applicability of Pt nanozymes in non-invasive detection of hyperglycemia thus leveraging the unique peculiarity of saliva of being largely available and easily accessible in a non-invasive and pain-free way.Nanozymes represent a great opportunity to overcome the limitations of natural enzymes, providing a reliable, low cost, and highly durable alternative for biomedical and diagnostic applications. Here we describe nanozymes based on shape controlled single-crystal octahedral nanoparticles characterized by well-defined extended {111} surface facets, low polydispersity in size, and an easy-to-remove coating, able to mimic glucose oxidase enzyme, with similar ESM 1(DOCX 6.00 MB)


The value of SARS-CoV-2 lateral flow immunoassay (LFIA) tests for estimating individual disease risk is unclear. The REACT-2 study in England, UK, obtained self-administered SARS-CoV-2 LFIA test results from 361,801 adults in January-May 2021. Here, we link to routine data on subsequent hospitalisation (to September 2021), and death (to December 2021). Among those who had received one or more vaccines, a negative LFIA is associated with increased risk of hospitalisation with COVID-19 , death  , and death with COVID-19 as underlying cause . For people designated at high risk from COVID-19, who had received one or more vaccines, there is an additional risk of all-cause mortality of 1.9 per 1000 for those testing antibody negative compared to positive. However, the LFIA does not provide substantial predictive information over and above that which is available from detailed sociodemographic and health-related variables. Nonetheless, this simple test provides a marker which could be a valuable addition to understanding population and individual-level risk. The value of SARS-CoV-2 lateral flow immunoassay tests for estimating individual disease risk is unclear. Here, the authors link testing data from the REACT-2 study in England to hospital and death records and show that vaccinated individuals with a negative LFIA test were at a higher risk of hospitalisation and death. The clinical value of these simple rapid tests at an individual level is less clear, although they have reasonable sensitivity and specificity in detecting SARS-CoV-2 antibodies at a threshold relevant to viral neutralisation3. Clinical use of LFIAs has been suggested for tracking seroconversion post-infection4 and post-vaccination5, and monitoring waning antibody levels in the months following vaccination6, for example in those with immunosuppression7. Further, IgG antibody positivity using self-administered LFIAs has been shown to predict COVID-19 infection risk over a 200-day follow-up period in an unvaccinated population8. Nonetheless, to date there has been limited uptake of antibody LFIAs at an individual level to guide understanding of personal risk from COVID-19 and inform decision-making about behaviour and need for boosters or other protections.Lateral flow immunoassay tests (LFIAs) have been used at a population level to track antibody prevalence9. Here we follow up data from adults who took part in the final two rounds of REACT-2, conducted in January–May 2021, after the start of the national vaccination campaign, using their linked National Health Service (NHS) administrative health records. We use these data to assess whether LFIA tests have predictive value for adverse health outcomes in the general population, and whether they might be useful in a clinical setting. Specifically, we explore whether LFIA results are associated with future hospitalisation  and mortality .The REal-time Assessment of Community Transmission-2 (REACT-2) study was a nationwide study of SARS-CoV-2 antibody prevalence in adults in England in which randomly selected adults were invited to complete an LFIA test at home and to complete a short online or telephone surveyThe study population included 361,801 participants, of whom 143,774 (39.7%) tested positive on self-administered LFIAs in rounds 5 and 6  of the REACT-2 study. Table Among the 192,637 people who had received one or more vaccinations at least 14 days prior to the LFIA test, 128,282 (66.6%) were LFIA positive; 88,026 people had received two vaccinations, of whom 74,534 (84.7%) tested positive. Of the 14,271 with prior COVID-19 confirmed with a test, 12,065 (84.5%) were LFIA positive; of the 57,317 who reported confirmed or suspected prior COVID-19, 29,700 (51.8%) were LFIA positive.We obtained follow-up data on hospitalisations to September 30, 2021, with mean follow-up time of 184 days and a total of 182,659 person years of follow-up. For mortality we obtained data to December 1, 2021, with a mean follow-up time of 245 days and a total of 243,125 person years of follow-up.For hospitalisations, 16,802 (4.6%) people were admitted for any cause , 5330 of whom were unplanned (emergency) admissions, 91 had COVID-19 as the primary diagnosis and 151 had COVID-19 listed anywhere on their hospital record. There were 389 deaths, including 19 with COVID-19 as the underlying condition and 21 with COVID-19 mentioned anywhere on the death certificate . Those testing negative on the LFIA antibody test had higher cumulative hazards for all outcomes at almost all time points during follow-up. Findings were similar when stratified by risk group, by age and by vaccination status , as well as increased risk of hospitalisation with COVID-19 as the primary diagnosis .After two or more vaccinations, those with a negative LFIA had increased all-cause mortality , mortality with COVID-19 as underlying cause , hospitalisation with COVID-19 as the primary diagnosis  and hospitalisation with COVID-19 mentioned  compared to people who tested positive (Table .13), morWe used 100x subsampled Least Absolute Shrinkage and Selection Operator (LASSO)-penalised Cox models with demographic, biological and health variables together with LFIA result to identify which were preferentially selected as predictive of outcomes (see “Methods”). In these multivariable models, LFIA result was not selected as a predictor of subsequent hospitalisation, emergency hospitalisation, hospitalisation with COVID-19 or death in the population with one or more vaccines, nor when stratified by risk group Fig. . The maiWe used boosted tree models trained on different subsets of predictors, with and without LFIA, cross-validated on unseen data, to assess the predictive value of LFIA result over and above the other variables  in the population who had received one or more vaccines. Adding LFIA result to the boosted tree model did not substantially improve the prediction of hospitalisation or death Fig. ; howeverIn this large, representative study of the population of England, we found that a negative LFIA antibody test result from early 2021, after receiving one or more vaccines at least 14 days previously, was associated with increased risk of hospitalisation from COVID-19 and of death from all causes and those attributed to COVID-19 over the subsequent 6–9 months. These associations persisted after adjustment for multiple factors including age. However, a combination of variable selection approaches and tree-based predictive modelling indicated that the information contained in the LFIA result could be adequately captured by the responses to a set of survey questions on basic demographics and health status.10 and negativity following vaccination or infection may be a biomarker of a limited specific immune response11 and of frailty12 and therefore may be linked to hospitalisation and all-cause mortality. People with immunosuppression have lower antibody titres post-vaccination, and approximately one in five people with solid organ transplants, rare autoimmune rheumatic diseases and lymphoid malignancies are LFIA negative after three vaccine doses13.Nonetheless, the LFIA test might provide a simple and rapid means to identify those at increased risk who might benefit from additional booster doses or rapid access to antiviral medication. The LFIA is a threshold test which correlates with SARS-CoV-2 antibody titres and live virus neutralisation14.In addition, the LFIA provides information, which can be obtained rapidly and at scale, on population antibody levels which may inform the timing and prioritisation of vaccination booster campaigns10; therefore, our LFIA test results will likely include some false negatives which may have reduced (biased) our risk estimates for hospitalisations and mortality following a negative test post-vaccination. Furthermore, the sensitivity of LFIAs may be lower in the weeks following vaccination; although we considered here a vaccine dose to be effective 14 days after injection, we cannot rule out the possibility of bias from time-since-vaccination, which is related to age and comorbidities15. In addition, self-administered LFIAs depend on user interpretation of the finding; nonetheless, validation analysis of the user-interpreted results from REACT-2 showed substantial concordance between user submitted results and those generated both by experts (Cohen’s kappa: 0.89) and by a computer vision machine learning algorithm (Cohen’s kappa: 0.80)18. There are also limitations to the routine data on outcomes including the duration of follow-up which was limited so far to between 6 and 9 months; in addition hospital and mortality data are from the monthly release of provisional data, while full quality assurance is only provided for annual extracts. It is therefore possible that some misclassification may have occurred. A further limitation is possible recruitment bias. Although the REACT-2 study included a large random population sample2, there may have been differential participation linked to risk of severe outcomes.Our study has limitations. LFIAs have imperfect accuracy: the tests used in this study were reported to have sensitivity of 78.7% In conclusion, a negative LFIA result following vaccination is a marker of elevated risk of hospitalisation and death over a 6–9-month time horizon. For people designated at high risk from COVID-19, there was an additional risk of all-cause mortality of 1.9 per 1000 for those testing antibody negative compared to positive. However, the LFIA does not provide substantial predictive information over and above that which is available from detailed sociodemographic and health-related variables. Nonetheless, this simple test provides a marker which could be a valuable addition to understanding population and individual level risk.9 and summary reports for each round21 have been published. Briefly: every 6–8 weeks, personalised invitations were sent to 315,000 adults aged 18 years and above on the NHS register, to achieve approximately equal numbers of responses from each of the 315 lower-tier local authority areas in England. Registration was done by telephone or online, and recruitment ceased when ~120,000 people had signed up. In the sixth and final round (May 2021), a boosted sample of adults aged 50 and above was obtained, with 749,225 invitations sent out resulting in 255,750 (34.1%) registrations and 209,482 (30.0%) completed tests.The REACT-2 programme monitored the community prevalence of SARS-CoV-2 antibodies among adults in England, through a series of six large representative random samples of the population between July 2020 and May 2021. The study protocolhttps://www.imperial.ac.uk/medicine/research-and-impact/groups/react-study/for-researchers/react-2-study-materials/).Respondents were sent a LFIA  test kit for SARS-CoV-2 antibody self-testing and asked to report their test result and upload a photo of the completed test. Respondents also filled out a questionnaire including details of COVID-19 vaccination history, self-reported comorbidities and any history of suspected or confirmed COVID-19  and 6 (May 2021). We obtained Hospital Episode Statistics Admitted Patient Care data  and Civil Registration Deaths data  from NHS Digital for all participants with a valid LFIA result (positive or negative) who consented to this data linkage. We assessed the following outcomes from the date of completion of the LFIA to end of follow-up  or date of death: hospital admission for any reason , unplanned/emergency admission for any reason (with the same exclusions), admission with COVID-19 as the primary diagnosis, admission where COVID-19 was listed anywhere on the record, all-cause mortality, deaths where COVID-19 was reported as the underlying condition, and deaths with COVID-19 mentioned anywhere on the death certificate ; male or female; previous history of COVID-19 ; and self-reported vaccination status 0, 1, 2) with people considered vaccinated 14 days after the reported date of vaccination. Participants who had received 3 or more vaccines at the time of the fieldwork were excluded from analysis. Participants were categorised as “high” or “low” risk based on the UK Joint Committee on Vaccination and Immunisation (JCVI) definition of being clinically extremely vulnerable for COVID-19 with peoN = 192,604) and for all participants, for each of the outcomes of interest. Plots were stratified by risk group, and then further stratified by age group and, for the full population, by vaccination status. We used multivariable Cox regression models to test the association between LFIA result and each outcome stratified by vaccination status in the full cohort. For each outcome, we constructed a series of nested models with increasing adjustments by age group, sex, (vaccination history), prior infection status and risk group. Hazard ratios (HRs) and 95% confidence intervals are reported.Using Kaplan–Meier plots we calculated the rate of each outcome by LFIA result and compared the cumulative hazard functions for those testing negative for the vaccinated group , we used two multivariable approaches and included a range of potentially relevant covariates alongside the LFIA result. First, we used LASSO-penalised Cox regression models with stability analysis as implemented in the SHARP R package25 to predict each of the binary outcomes in unseen data using a five-fold cross validation procedure. We evaluated various combinations of predictors  and evaluated model performance on unseen data using the area under the Receiver Operating Characteristic (ROC) curve (which provides the predictive accuracy of a binary classifier at different discrimination thresholds). Again, we conducted this analysis among individuals who had received one or more vaccinations and in the full cohort.Second, we used extreme gradient boosted tree models as implemented in CatboostAll data collection for the REACT2 study was captured with Questback. All data were analysed using R v4.0.5 (2021).The REACT-2 study holds ethical approval from South Central—Berkshire B Research Ethics Committee . Participants provided informed consent for their data to be used and, separately, indicated whether they were willing for their data to be linked to their NHS records.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Is a brief automated bilingual computerized alcohol screening and intervention (AB-CASI) health information technology tool superior to standard care in reducing alcohol consumption and alcohol-related adverse health behaviors and consequences among US Latino emergency department (ED) patients?In this randomized clinical trial of 840 self-identified adult Latino ED patients with unhealthy drinking, the number of binge drinking episodes within the last 28 days was significantly lower in the AB-CASI group compared with the standard care group  at 12 months after randomization.These findings suggest that AB-CASI is a viable approach to addressing alcohol-related health disparities in the ED, a setting that serves as a national health care front line and safety net. Alcohol use disorders have a high disease burden among US Latino groups. In this population, health disparities persist, and high-risk drinking has been increasing. Effective bilingual and culturally adapted brief interventions are needed to identify and reduce disease burden.To compare the effectiveness of an automated bilingual computerized alcohol screening and intervention (AB-CASI) digital health tool with standard care for the reduction of alcohol consumption among US adult Latino emergency department (ED) patients with unhealthy drinking.This bilingual unblinded parallel-group randomized clinical trial evaluated the effectiveness of AB-CASI vs standard care among 840 self-identified adult Latino ED patients with unhealthy drinking . The study was conducted from October 29, 2014, to May 1, 2020, at the ED of a large urban community tertiary care center in the northeastern US that was verified as a level II trauma center by the American College of Surgeons. Data were analyzed from May 14, 2020, to November 24, 2020.Patients randomized to the intervention group received AB-CASI, which included alcohol screening and a structured interactive brief negotiated interview in their preferred language (English or Spanish) while in the ED. Patients randomized to the standard care group received standard emergency medical care, including an informational sheet with recommended primary care follow-up.The primary outcome was the self-reported number of binge drinking episodes within the last 28 days, assessed by the timeline followback method at 12 months after randomization.P = .01 for interaction).Among 840 self-identified adult Latino ED patients , 418 were randomized to the AB-CASI group and 422 to the standard care group. A total of 443 patients (52.7%) chose Spanish as their preferred language at enrollment. At 12 months, the number of binge drinking episodes within the last 28 days was significantly lower in those receiving AB-CASI  vs standard care . Alcohol-related adverse health behaviors and consequences were similar between groups. The effect of AB-CASI was modified by age; at 12 months, the relative reduction in the number of binge drinking episodes within the last 28 days in the AB-CASI vs standard care group was 30% in participants older than 25 years  compared with an increase of 40% in participants 25 years or younger (RD, 1.40; 95% CI, 0.85-2.31; In this study, US adult Latino ED patients who received AB-CASI had a significant reduction in the number of binge drinking episodes within the last 28 days at 12 months after randomization. These findings suggest that AB-CASI is a viable brief intervention that overcomes known procedural barriers to ED screening, brief intervention, and referral to treatment and directly addresses alcohol-related health disparities.NCT02247388ClinicalTrials.gov Identifier:  This randomized clinical trial compares the effectiveness of an automated bilingual computerized alcohol screening and intervention with standard care for the reduction of alcohol use among US adult Latino emergency department patients with unhealthy drinking. Health disparities related to alcohol are also frequently observed in the ED. Data from the National Hospital Ambulatory Medical Care Survey3 reveal that in 2019, US EDs had more than 150 million visits. This number included more than 26 million ED visits by Latino patients; in the chronic condition at ED visit category, more than 5.1 million visits were categorized as alcohol misuse, abuse, or dependence ED visits.3 From 2006 to 2014, alcohol-related visits to EDs increased by 61.6%, including increases of more than 51% for acute alcohol-related visits and more than 75% for chronic alcohol-related visits.4 Furthermore, from 1999 to 2017, among those aged 16 years or older, national annual alcohol-related deaths doubled (from 35 914 to 72 558), and from 2015 to 2019, 20% of all alcohol-attributable deaths were among individuals aged 20 to 49 years.6Alcohol use disorders (AUDs) are major contributors to US disease burden .7 Over time, ED-SBIRT has been evaluated and found to be associated with reductions in alcohol consumption as well as decreases in adverse physical and social consequences.11Conceptualization and testing of ED screening, brief intervention, and referral to treatment (ED-SBIRT) for alcohol use, which began more than 20 years ago, has provided an opportunity to identify and address patients with AUDs in the ED.13 have provided evidence to support screening and behavioral counseling interventions for unhealthy alcohol use, and the US Preventive Services Task Force has included this approach in its recommendations.14 A recent systematic review and meta-analysis13 reported that ED-SBIRT was associated with a small reduction in drinking, and its authors along with other experts16 made specific note of the potential population benefits of cumulative small individual reductions in alcohol use as a result of SBIRT efforts.High-quality systematic reviews19 Perceived barriers to its regular implementation have included practitioner time burden, personnel cost, and maintenance of intervention fidelity. Moreover, the inability to deliver the intervention in the patient’s preferred language  is a fundamental rate-limiting step. Without language concordance, meaningful patient communication about unhealthy alcohol use and disease prevention is a failed endeavor. The inability to provide ED-SBIRT easily and readily in a language other than English limits the reach of prevention efforts. Furthermore, for US Latino patients, who represent the largest ethnic minority group , this language barrier limits the ability of ED-SBIRT to address alcohol-related health disparities.20In the ED, routine implementation of SBIRT lags behind national guidelines.22 with few exceptions, most US ED-SBIRT clinical trials have excluded enrollment of predominantly Spanish-speaking patients. Research has revealed that daily heavy drinking and high rates of chronic liver disease and cirrhosis-related death are prevalent among Latino men who drink.31 While non-Latino White individuals are more likely to become dependent, once alcohol dependent, Latino individuals have a higher prevalence of recurrent or persistent dependence.34 Latino individuals also have higher rates of alcohol-related adverse consequences .38 Because high-risk drinking among US Latino individuals has increased39 and related health disparities have persisted, and given the continued growth of the US Latino population , a national research priority plan has been developed and disseminated.40 This plan was formulated from the existing literature to systematically address and more rapidly advance research in alcohol-related health disparities. The design and outcomes of the present randomized clinical trial (RCT) are highly relevant to this important research priority plan because they address screening and brief intervention methods for ethnic minority individuals and evaluate the effectiveness of a new tailored intervention among a large ethnic minority group and some of its subgroups. The objective of this RCT was to compare the effectiveness of our automated bilingual computerized alcohol screening and intervention (AB-CASI) digital health tool with standard care in reducing alcohol use among US adult Latino ED patients with unhealthy drinking.Despite studies reporting that US Latinos are less likely to receive alcohol intervention,CONSORT) reporting guideline for RCTs.This study was approved by the Human Investigation Committee of Yale School of Medicine . All parDiagnostic and Statistical Manual of Mental Disorders  was widely in use.41 The trial included the whole spectrum of unhealthy drinking, including individuals with drinking levels higher than the low-risk limits  through individuals with alcohol dependence.41 The design of this RCT has been previously published in full detail.42We conducted a bilingual unblinded parallel-group RCT evaluating the effectiveness of AB-CASI in reducing alcohol use when compared with standard care among adult Latino ED patients. The trial was conducted from October 29, 2014, to May 1, 2020, and ended when recruitment goals were met. Data were analyzed from May 14, 2020, to November 24, 2020. During development of this trial, the Enrolled study participants were self-identified adult Latino ED patients (English or Spanish speaking) found to have unhealthy drinking. Exclusion criteria included current enrollment in a treatment program, pregnancy at enrollment, and the presence of conditions precluding the use of interviews or the AB-CASI tool . The trial setting was the ED of a large urban community tertiary care center in the northeastern US that was verified as a level II trauma center by the American College of Surgeons.44 Using computer tablets , AB-CASI was administered at the patient’s bedside by a trained bilingual research assistant. Participants selected their AB-CASI interface language, and questions and messages were displayed and spoken through headphones for privacy. The AB-CASI tool automatically collected demographic characteristics, administered the AUDIT, delivered a BNI   digital health tool developed for automated ED-SBIRT encompassing the Alcohol Use Disorders Identification Test (AUDIT) and brief negotiation interview (BNI) with 4 core components: (1) raise subject, (2) provide feedback, (3) enhance motivation , and (4) negotiate advice.Participants not randomized to the AB-CASI group received standard emergency medical care, including an informational sheet with recommended primary care follow-up. Screening and referral requirements were performed according to American College of Surgeons level II trauma center designation guidelines. Social worker consultation was at the full discretion of the treating clinician.Prospective participants were approached in the ED by trained bilingual and bicultural research assistants. Patients who self-identified as having Latino ethnicity received a health quiz . Three standard questions about the quantity and frequency of alcohol use were asked: (1) on average, how many days per week do you drink alcohol? (2) on a typical day when you drink, how many drinks do you have? and (3) how many times in the past month have you had X [4 for women or 5 for men] or more drinks on any occasion? Patients reporting a consistently unhealthy pattern of drinking were approached for written informed consent in their language choice of English or Spanish.45 to measure alcohol use severity (administered at baseline); the timeline followback (TLFB) method to measure the number of binge drinking episodes and the number of weekly standard drinks within the last 28 days48 ; the Revised Injury Behavior Checklist49 to measure any physical injuries (administered at baseline and 12 months); the Short Inventory of Problems51  and brief event data53 report to measure alcohol-related adverse behaviors and consequences, such as driving after binge drinking, missing days of work, and getting arrested for any reason ; and the Treatment Services Review54 to measure receipt of specialized treatment services . Bilingual supervised trained research assistants, blinded to group assignment, completed scripted follow-up assessments by telephone. Follow-up assessments lasted 15 to 20 minutes. Participants received gift cards .Baseline and follow-up assessments  included the AUDITThe primary outcome measure was the self-reported number of binge drinking episodes  over the previous 28 days, assessed using 28-day TLFB at 12 months after randomization. We hypothesized that at 12 months, AB-CASI would be superior to standard care in reducing the number of binge drinking episodes and the mean number of weekly standard drinks over the last 28 days. Secondary outcomes included the self-reported mean number of weekly standard drinks measured by 28-day TLFB at 12 months after randomization and alcohol-related adverse health behaviors and consequences over the 12-month period. We hypothesized that at 12 months, AB-CASI would be superior to standard care in reducing alcohol-related adverse health behaviors and consequences.55 demonstrated that the number of binge drinking episodes in the past 30 days was reduced by 1.14 in the intervention compared with the control condition. D’Onofrio et al56 reported similar findings in an RCT conducted among individuals with hazardous and harmful drinking. Given a power of 80%, a significance level of 2-sided α = .05, an SD of 5.2 for the number of binge drinking episodes in the past 28 days, and 1:1 intervention allocation, a sample of 327 participants per group was required to detect a 1.14-episode difference between AB-CASI and standard care. A total of 840 patients with unhealthy drinking were enrolled and randomized to accommodate a study withdrawal rate of up to 20%.Sample size estimation was based on randomizing and following up a sufficient number of patients with unhealthy drinking to evaluate the primary hypothesis that AB-CASI would result in greater 12-month reductions in the primary outcome compared with standard care. An RCT by Fleming et al45 and preferred language. The statistician (Dr Dziura) used a computer algorithm to generate a permuted block randomization sequence. The sequence was concealed in the OnCore system and allocated after enrollment of a participant by a trained bilingual research assistant.Participants were randomized 1:1 using a stratified randomization procedure implemented by the OnCore Clinical Trials Management System (Forte Research). Stratification factors were alcohol use severity measured by AUDIT score 57 The model included fixed effects for intervention, time, and the interaction of intervention with time. Additional fixed effects were included for baseline covariates . Analysis permitted the inclusion of all randomized participants and assumed that missing data occurred at random. Inclusion of baseline, 1-month, 6-month, and 12-month outcome data in the model assisted in meeting this assumption. Linear contrasts with a significance level of 2-sided P = .05 were used to estimate intervention group differences and 95% CIs at 1 month, 6 months, and 12 months. Because the negative binomial model was multiplicative, the relative difference (RD) between groups  with 95% CIs was reported.Analyses were conducted according to the intention-to-treat principle and performed using SAS software, version 9.4 (SAS Institute Inc) . A repeaP = .01 to control inflated type 1 errors from multiple significance testing.Similar repeated-measures mixed-model analyses were implemented for each secondary outcome. Comparison of all secondary outcomes between groups was evaluated at a significance level of 2-sided 58; biculturalism, Hispanicism, and Americanism are categories approximating acculturation referring to respective levels of comfort and involvement in activities that are classified as more American or more Hispanic or Latino. These exploratory subgroup analyses were conducted within the GLMM framework by including the interactions of treatment, time, and subgroup factors. Differences between AB-CASI and standard care were calculated for each level of the subgroup and were compared using linear contrasts.Along with stratification factors, the heterogeneity of treatment effects on the primary outcome was assessed for subgroups based on prespecified factors assessed at baseline . The biculturalism score (which includes Hispanicism and Americanism scores) was measured by the Bicultural Involvement Questionnaire–Short VersionOf 1799 Latino ED patients who screened positive for unhealthy drinking, 1357 met eligibility criteria. A total of 840 patients (61.9%) were enrolled and randomized; of those, 418 patients received AB-CASI and 422 received standard care . Four paIn the AB-CASI group, the GLMM analysis revealed that the mean number of binge drinking episodes within the last 28 days was 7.7  at baseline and decreased to 3.5  at 1 month, 3.4  at 6 months, and 3.2  at 12 months . In the P = .01 for secondary outcomes. Alcohol-related adverse health behaviors and consequences over 12 months were similar between groups  at baseline to 12.4  at 1 month, 11.6  at 6 months, and 10.0  at 12 months . The stan groups .P = .01 for interaction) in the AB-CASI group vs the standard care group, although 95% CIs were wide due to the small size of the subgroup 25 years or younger (n = 176) (P = .04 for interaction). No intervention-related serious adverse outcomes were encountered.At 12 months, the effect of AB-CASI on the number of binge drinking episodes within the last 28 days was modified by age and primary reason for ED visit. In those older than 25 years, binge drinking episodes were 30% lower  vs those 25 years or younger, for whom the point estimate for binge drinking episodes was 40% higher  . Further60 and effectively identified patients with unhealthy drinking while providing tailored bilingual ED-SBIRT. The AB-CASI tool outperformed standard care specifically for reduction in the mean number of binge drinking episodes within 28 days by 21%. Although the mean number of weekly standard drinks was 19% lower in the AB-CASI group at 12 months, the difference did not meet the prespecified threshold for significance. However, while both groups had reductions in alcohol use at 1 month, which could suggest an effect because of the need to initially seek emergency care in itself, the reduction observed in the AB-CASI group endured at 12 months. Identifying that the AB-CASI group had nearly 1 less binge drinking episode than the standard care group is clinically meaningful and worthy of advocating for routine ED screening. This reduction is of particular note when considering that 1 binge drinking episode entails consuming several drinks over a short time, and binge drinking has a well-known association with other health-risking behaviors and a higher risk of acute toxic exposure to end organs. More than one-half (52.6%) of the participants chose to receive AB-CASI in Spanish. In the current state of national ED-SBIRT implementation, it is plausible that this specific vulnerable group would not have received intervention due to the limitations of ED-SBIRT delivery in Spanish and intervention knowledge.To our knowledge, this study is the first large US RCT of ED-SBIRT using a bilingual automated digital health tool to show effectiveness in reducing unhealthy alcohol use. The AB-CASI tool required little time to administer 20 While studies have reported increases in alcohol use and misuse among US Latino individuals,39 they have also highlighted continued challenges in receiving intervention and treatment, which are magnified for monolingual US Latino immigrants.62 Given our findings, it is important to keep in mind the heterogeneity of the US Latino population. Moreover, it has been well established that drinking patterns in this population are also heterogeneous, having implications for the treatment and recovery of those with AUDs.64 Previous studies64 have found that Puerto Rican women who drink have the highest binge drinking rates among all US Latina women. We found no differential intervention effect among men and women, showing that AB-CASI was equally effective. This finding indicates relevant benefits at a population level when considering sex differences in AUDs, particularly because the alcohol use and misuse gap between men and women continues to narrow, with detriment to women.68 While exploratory, we observed a significantly greater reduction in the number of binge drinking episodes within the last 28 days among those in the AB-CASI group who were older than 25 years compared with those who were 25 years or younger. Given the smaller subgroup of participants 25 years or younger (n = 176), further investigation of the impact of the intervention in younger individuals with unhealthy drinking is warranted. We also observed greater reductions for those with a primary alcohol-related reason for visiting the ED. Taken together, these findings can inform ED-SBIRT programs and research addressing alcohol-related health disparities among Latino ED patients with unhealthy drinking.20The AB-CASI tool can provide an effective and cost-feasible strategy to address alcohol-related health disparities.70 bolster evidence for cost savings and improved health. A study by Barbosa et al69 evaluated the cost-effectiveness of delivering ED-SBIRT and compared it with SBIRT delivery in other outpatient medical settings. The ED was found to be the most cost-effective delivery site based on dollars paid per quality-adjusted life years gained . Furthermore, ED-SBIRT provided superior effectiveness at lower cost, with larger reductions in social cost. A recent study70 found that ED-SBIRT can be an effective cost-reducing strategy to address unhealthy alcohol use, and the authors advocated for policy makers and payers to prioritize these types of interventions.Previous ED-SBIRT studiesDSM-IV criteria of AUDs. In mid-2013, the Diagnostic and Statistical Manual of Mental Disorders 71 was released with different AUD terms and threshold criteria. While extensive overlap exists between the DSM-IV and DSM-5, being informed of the criteria differences would be prudent when interpreting trial findings.This study has limitations. First, trial enrollment occurred only at 1 ED in a large urban community tertiary care center in the northeastern US. Second, enrolled participants were predominantly of Puerto Rican descent. While not surprising given the geographic location of the enrollment site and the historic predominance of the Puerto Rican population compared with other Latino groups in the northeastern US, consideration of variability in cultural stressors within different US Latino subgroups could be relevant to interpretation of the findings. Third, while our participant retention was successful, we are not able to comprehensively account for all outcomes among those unavailable for follow-up at some point after the initial baseline assessment. Given the short engagement with the interventions, it is unlikely that mechanisms leading to study withdrawal and missing data months after the intervention were differential between the 2 groups. Rates and reasons for missing outcomes were similar across intervention groups at all time points. Fourth, this trial was originally designed before 2013 and conducted using long-standing In this RCT, the use of AB-CASI in the ED was effective in reducing alcohol use among US English- and Spanish-speaking Latino patients who were identified along the spectrum of unhealthy alcohol use. Highly acceptable to patients and brief in delivery, the AB-CASI tool overcame several long-reported barriers to ED-SBIRT, successfully addressing alcohol-related health disparities among the growing US Latino population.


Background: Chromothripsis caused massive, clustered genomic rearrangements is prevalent in cancer and is considered a new paradigm for tumorigenesis and progression. In this study, we investigated the association among chromothripsis, anti-tumor immune responses, and responsiveness to immune checkpoint blockade (ICB).Methods: Quantification of immune cell infiltration and functional enrichment of immune-related signaling pathways were performed in the discovery set (n = 9403) and the validation set (n = 1140). we investigated the association between chromothripsis and anti-tumor immune responses. In the immunotherapy cohort, copy number alteration-based chromothripsis scores (CPSs) were introduced to assess the extent of chromothripsis to evaluate its association with responsiveness to ICB.Results: In the discovery set and the validation set, the ratios of CD8+ T cells to Tregs, TAMs, and MDSCs were significantly lower in tumors with chromothripsis . The relevant pathways underlying the antitumor immune effect were significantly enriched in tumors without chromothripsis. Chromothripsis can be used as an independent predictor, and patients with low-CPSs experienced longer overall survival (OS) after immunotherapy .Conclusions: Our findings highlight the reduced cytotoxic immune infiltration in tumors with chromothripsis and enhanced immunosuppression in the tumor microenvironment. Chromothripsis can thus be used as a potential indicator to help identify patients who will respond to ICB, which could complement established biomarkers. Chromothripsis is typically associated with massive genomic rearrangements accompanied by copy number alterations in a small region of one or several chromosomes Chromosomal instability (CIN), a hallmark of chromothripsis, has been extensively studied in terms of its association with immunity Here, our objective was to investigate the relationship among chromothripsis, anti-tumor immune responses, and responsiveness to immune checkpoint blockade (ICB) immunotherapy. In both a discovery and validation dataset, we identified consistently reduced immune cell infiltration in tumors with chromothripsis, along with impaired cytolytic activity. We also explored the association between tumor chromothripsis and broad manifestations in the immune microenvironment. In addition, we constructed chromothripsis prediction models from copy number alteration (CNA) signatures and obtained chromothripsis scores (CPSs) from them to elucidate the relationship between chromothripsis and therapeutic outcomes in patients receiving ICB immunotherapies.To investigate the effect of chromothripsis on the tumor microenvironment, we examined multi-omics data from 24 cancer types (solid tumors only) from TCGA. For copy number profiles derived from the SNP6 microarray, we used CTLPScanner to detect and annotate chromothripsis in patients -1 vs 1.05 × 10-1, P < 2.2 × 10-16, Figure ). The enrichment of 28 tumor-infiltrating immune cells in the tumor  cells), which are considered the primary executor of antitumor immunity Patients were divided into two groups based on the occurrence of chromothripsis, where the total immune cell infiltration score was significantly lower in the chromothripsis group than in the non-chromothripsis group , suggesting an immunosuppressive microenvironment in these tumors. In addition, the expression ratio of proinflammatory cytokines  to immunosuppressive molecules  was significantly reduced in tumors with chromothripsis  In accordance with the decrease in tumor-infiltrating cytotoxic and immunosuppressive cells in tumors with chromothripsis, we then investigated the pro-tumorigenic versus anti-tumorigenic properties of the immune microenvironment, as previously reported t = 9.61, P = 6.4 × 10-21, antigen processing and presentation), antigen recognition , effector cell activation , and immune-mediated cytotoxicity  were significantly enriched in tumors without chromothripsis . The analysis of individual tumor types also resulted in generally consistent results .Further, Gene Set Variation Analysis (GSVA) revealed partial signaling pathways involved in anti-tumor immune effects, including antigen presentation  data, based on which we can call CNA and Structure Variantion (SV). Using SV information, ShatterSeek can be used to detect and annotate tumor chromothripsis -1 vs 3.09 × 10-1, P = 9.2 × 10-6, Figure ). In addition, the distribution of tumor-infiltrating immune cell enrichment results was almost unchanged . The ratios of CD8+ T cells to Tregs, TAMs, and MDSCs, including the expression ratio of pro-inflammatory cytokines to immunosuppressive molecules, were significantly lower in the chromothripsis group . The relevant pathways underlying the antitumor immune effect were significantly enriched in tumors without chromothripsis , and the results of GSEA were consistent with this . The results of the enrichment analysis for individual cancer types were also largely consistent with the previous data . The tumor microenvironment of the chromothripsis group was significantly less sensitive to ICB treatment than that of the non-chromothripsis group , which is consistent with the results obtained from TCGA dataset. The results from the discovery and validation datasets simultaneously show that chromothripsis is associated with a reduction in cytotoxic immune cell infiltration in the tumor microenvironment and that patients with chromothripsis have reduced sensitivity to immunotherapy.Consistent with the previous results, the total immune cell infiltration score was significantly lower in the chromothripsis group than in the non-chromothripsis group .In addition to transcriptomic expression profiling, we next focused on genetic features of cancer, particularly immune-related predictors. From patient samples, we obtained the mutation frequencies in ICB-responsiveness-related genes , suggesting that chromothripsis can lead to an increase in somatic mutations and represents a possible increase in tumor neoantigens. Expectedly, the SCNA levels, represented by wGII, were also significantly higher in tumors with chromothripsis , which resulted from the massive, clustered genomic rearrangements mediated by chromothripsis. Chromothripsis could thus lead to increased wGII.The TMB was modest and significantly higher in tumors with chromothripsis . TP53 malfunction could be a predisposing factor for chromothripsisTP53 mutations in tumors with chromothripsis in the discovery and validation set, >60%  of the tumors with chromothripsis never showed TP53 mutations or amplifications. This suggested that TP53 (and other representative genes) might not be a significant factor in suppressing the anti-tumor immune response in tumors with chromothripsis.Similarly, we found that the -1 vs 1.70 × 10-2, P < 2.2 × 10-16; median 1.86 × 10-1 vs 4.09 × 10-1, P < 2.2 × 10-16, Figure , Figure , respectively). A high TMB was associated with favorable survival outcomes in patients receiving ICB immunotherapy Both TMB and wGII are also significantly increased in the chromothripsis group based on the validation PCAWG dataset . In the validation PCAWG dataset, the CNA burden was significantly lower in tumors with chromothripsis . There were many possibilities for this difference, and this difference suggested a possible stochastic association between the CNV burden and chromothripsis. The CNV burden cannot be a major factor in the reduced antitumor immune response in tumors with chromothripsis. Collectively, we believed that chromothripsis can be used as an independent predictor.In addition, we observed inconsistent results. In the discovery TCGA dataset, the burden of copy number alterations  was significantly higher in the chromothripsis group . We obtained CPSs based on a CN signature prediction model. Notably, we integrated all three immunotherapy cohorts to expand the sample size and ensure the reliability of results from the prediction model. In addition, we accounted for the TMB, wGII, CNA burden, PD-L1 expression level, and CD8A expression level . The time-dependent ROC curve for CPS was continuously superior . According to the survROC curves for 1-, 2-, and 3-year OS for CPS, the ROC curve was found to be greater for 1- and 3-year OS . The univariate Cox regression analysis showed that CPS was significantly associated with survival in patients treated with ICB immunotherapy . The remaining predictors did not show significance, which is consistent with previous results.We compared the sequential trends associated with these predictors in terms of overall survival (OS) by generating time-dependent receiver operating characteristic (ROC) curves . Grouping patients again using the median as the threshold, it was found that other biomarkers could not be adequately used to classify patient responses in these cohorts . Furthermore, we observed a numerical trend towards lower response rates and objective response rates in the CPShigh group compared to the CPSlow group . These results support the potential of CPS to complement established biomarkers in identifying patients who are likely to respond favorably to immunotherapy.We then defined patients with CPSs greater than the median as the CPSThe role of chromothripsis in anti-tumor immunity is unclear, even though it is prevalent in tumors and plays an important role in tumor evolution. Here, we report that chromothripsis is associated with reduced cytotoxic immune cell infiltration and that its copy number signature-based score can be used to reliably predict survival outcomes for patients receiving ICB treatment. In both discovery and validation datasets, we found that immune infiltration in tumors with chromothripsis was reduced and that immune suppression in the tumor microenvironment was enhanced. These results all suggested an unfavorable survival outcome for patients harboring tumors with chromothripsis receiving immunotherapy.Chromothripsis is involved in the cGAS-STING signaling pathway through micronucleus formation. Activation of the cGAS-STING signaling pathway in innate immune cells induces the production of type I interferon, which initiates an antigen-specific immune response leading to tumor killing Kras, etc.) and the CNV burden were not consistent in both the discovery and validation datasets. One possible explanation for this is that chromothripsis is heterogeneous. Chromothripsis could exhibit certain chromosomal preferences; for example, chromothripsis is enriched in chromosome 12 in liposarcomas and chromosomes 3 and 5 in kidney renal cell carcinomas The frequencies of mutations in ICB-responsiveness-related genes  that were derived from MSigDB gene sets and involved anti-tumor immune effects. GSEA, which is also a gene set enrichment method, was used to validate the GSVA results VCF or MAF files containing somatic mutations and copy number alteration profiles were downloaded from TCGA and PCAWG. Using the R package Maftools (version 2.8.05), we converted VCF files to MAF files for the subsequent statistical analysis TP53, KRAS, PTEN, JAK1/2, and B2M in different groups. The burden of copy number alterations indicates the total number of genes with copy number gains or losses. Bedtools (version 2.30.0) Patients were divided into two groups, namely the chromothripsis and non-chromothripsis groups. Then, we determined the frequency of mutations in Tumor ploidy is expressed as a weighted median integer copy number (the weight is the length of the copy number segment). With tumor ploidy, we obtained the percentage of genomic material gained and lost per chromosome. Ultimately, the average of this percentage for all autosomes is the wGII of a sample Raw sequencing data originated from the immunotherapy cohorts. Reads aquired by paired-end sequencing were alignd to the human reference genome (HRCh38) to obtain BAM files  Processed paired BAM files  were used as input to MuTect2  to identify somatic single nucleotide variants and small insertions or deletions. We further filtered the acquired mutations based on three rules. First, we filtered for high-confidence variants . Second, non-silent variants  were selected. Third, only rare variants  were selected. loss-of-heterozygosity events and CNAs were acquired by Fraction and Allele-specific Copy number Estimate from Tumor/normal Sequencing (FACETS) Based on the processing protocol in the publication For whole exome sequencing data, the obtained copy number variation profiles were used for CNV signature analysis. First, we filtered the CNV profiles and removed the corresponding regions on X chromosome. The optimal number of categories was obtained by clustering the six CN basic features in the CNV profiles. Then we extracted the CN features from scratch by the hierarchical Dirichlet process (hdp), and these features were input to the established CN signatures prediction model  as basic elements to obtain the prediction metrics of chromothripsis, which are CPSs.To ensure comparability among individual indicators, CPSs obtained from the CN feature prediction model were grouped in the same way as other indicators . The median of all indicators was used as a threshold to group patients, and the one higher than the median of this indicator was the high group. Univariate Cox regression analysis was applied to determine HRs. Kaplan-Meier analysis was employed to estimate survival and log-rank test was used to determine the p-values.Our analysis was performed with R version 4.0.5. The Fisher's exact test was used for 2 × 2 tables of categorical variables and Wilcoxon rank-sum test was applied for differences in continuous variables unless otherwise specified. The noted software tools  that we used throughout the analysis are publicly available.https://tcga-data.nci.nih.gov). The datasets for clinical parameters, SCNAs (the next-generation sequencing-based data), mutations, and RNA-seq came from the PCAWG database (https://dcc.icgc.org/pcawg/). To ensure the reliability of the hierarchical Dirichlet process, we integrated all immunotherapy cohorts to expand the sample size. For immunotherapy cohorts, we utilized raw whole exome sequencing data and RNA-seq clinical parameters from the following studies: 1) Hugo et al., an advanced melanoma anti-PD-1 treated cohort; 2) Riaz et al., an advanced melanoma anti-PD-1 treated cohort; 3) Zhao et al., an advanced glioblastoma anti-PD-1 treated cohort. RECIST 1.1-based quantifications of responses were then used to designate patients as responders (stable disease (SD) for ≥ 6 months, partial response (PR), or complete response (CR)) or non-responders (SD with <6-month duration or progressive disease (PD)). Similarly, patients could also be distinguished as objective (CR or PR) or non-objective (PD or SD) responders. In TCGA and PCAWG databases, the types of tumor samples include fresh tissue, liquid nitrogen cryopreserved tissue, dry ice cryopreserved tissue, or paraffin tumor tissue. In the immunotherapy cohort, Zhao cohort did not specify the type of tumor sample, and both Riaz and Hugo cohorts included fresh tissue samples.All data are available in the main text or the Supplementary figures and tables.Click here for additional data file.


How do treatment-free survival outcomes differ between various first-line immune checkpoint inhibitor therapy regimens for patients with advanced melanoma?In a muticenter cohort study of 316 patients with advanced melanoma treated with first-line immune checkpoint inhibitor therapy, patients treated with nivolumab-ipilimumab experienced a mean treatment-free survival of 12.4 months compared with a mean of 8.9 months for single-agent nivolumab and 11.1 months for single-agent pembrolizumab.This study suggests that patients with advanced melanoma treated with first-line nivolumab-ipilimumab experienced numerically longer treatment-free survival compared with single-agent nivolumab or pembrolizumab. Treatment-free survival (TFS) represents an alternative time-to-event end point, accurately characterizing time spent free of systemic therapy, providing a more patient-centric view of immune checkpoint inhibitor (ICI) therapy regimens. There remains a lack of studies evaluating TFS outcomes among patients with advanced melanoma who are receiving immunotherapy, especially outside of the clinical trial setting.To evaluate TFS outcomes for patients with advanced melanoma receiving first-line ICI therapy outside of a clinical trial setting.This multicenter cohort study of patients with advanced melanoma receiving first-line ICI therapy between August 1, 2013, and May 31, 2020, was conducted in Alberta, Canada. Data analysis was performed in August 2022.Patients received standard-of-care, first-line ICI therapy treatment regimens including single-agent nivolumab, single-agent pembrolizumab, or ipilimumab-nivolumab.Treatment-free survival was defined as the difference in the 36-month restricted mean survival time between 2 conventional survival end points: (1) time from treatment initiation to ICI cessation, death, or censoring at last follow-up and (2) time from treatment initiation to subsequent systemic anticancer therapy, death, or censoring at last follow-up.BRAF V600E positive than those receiving anti–programmed cell death protein 1 (anti–PD-1) monotherapy. The restricted mean TFS was longer for nivolumab-ipilimumab  compared with nivolumab  and pembrolizumab . During the 36-month follow-up interval, patients treated with nivolumab-ipilimumab spent 34.4% of their time (12.4 of 36 months) not receiving systemic anticancer treatments compared with 30.8% (11.1 of 36 months) and 24.7% (8.9 of 36 months) of the time for the pembrolizumab and nivolumab treatment groups, respectively.A total of 316 patients with advanced melanoma receiving first-line nivolumab , pembrolizumab , or combination nivolumab-ipilimumab  were included. Treatment groups were similar with regard to sex, primary tumor location, and presence of metastasis, although patients receiving combination nivolumab-ipilimumab had a lower Eastern Cooperative Oncology Group status, were younger, and were more likely to be This cohort study of patients with advanced melanoma receiving first-line ICI therapy suggests that TFS represents a patient-centric, informative end point. Patients treated with combination nivolumab-ipilimumab spent more time alive and free from systemic anticancer therapy than those treated with anti–PD-1 monotherapy alone. This cohort study evaluates treatment-free survival outcomes for patients with advanced melanoma receiving first-line immune checkpoint inhibitor therapy outside of a clinical trial setting. Patients who discontinue ICI therapy due to toxic effects or prolonged duration of disease control are able to receive the ongoing benefit from these agents as they are able to remain free of subsequent systemic anticancer (SSAC) therapy. A better understanding of this dimension of care is crucial because multiple studies have found an associaton between increased time spent free of SSAC and improvements in patient quality of life.8 Treatment-free survival (TFS) quantifies time spent free of SSAC therapy by measuring the period between cessation of first-line treatment and initiation of subsequent lines of therapy. Treatment-free survival has been examined among patients with advanced melanoma receiving first-line ICI therapy in the clinical trial setting10; however, an examination of TFS in a routine care setting for this patient population has yet to be reported, to our knowledge. A more thorough understanding of the TFS outcomes experienced by patients with advanced melanoma treated with first-line ICI therapy may help inform therapeutic decision-making, given the increasing number of treatment choices available to clinicians and patients in this setting.Analyses of the effectiveness of these treatment regimens have largely relied on conventional time-to-event outcomes, such as overall survival.STROBE) reporting guideline.Patients were selected from the Alberta Immunotherapy Database (AID) study, which is a multicenter observational cohort study being conducted in Alberta, Canada. The AID study retrospectively captured demographic, clinical, pathologic, and outcome data for patients having received ICI therapy between August 1, 2013, and May 31, 2020. Of the patients included in the AID cohort, those selected for this cohort study were required to have a histologically confirmed, locally advanced form of metastatic melanoma (stage III unresectable or stage IV metastatic melanoma), agnostic of primary site of origin, as well as to have received at least 1 cycle of first-line ICI therapy with either single-agent nivolumab, single-agent pembrolizumab, or combination nivolumab-ipilimumab. This study was reviewed and approved by the health research ethics board of the Alberta–Cancer Committee, which waived patient consent given the low-risk, deidentified, retrospective nature of this work. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology . All statistical tests were 2-sided, and P < .05 was considered to indicate statistical significance. No formal statistical testing was undertaken due to the observational nature of this work, and all results are presented in a descriptive manner alone.Statistical analysis was conducted in August 2022. Treatment-free survival was calculated for each ICI therapy subgroup of patients with advanced melanoma and defined as the area under the curve between 2 time-to-event end points: time to ICI therapy discontinuation and time to subsequent SSAC therapy initiation. Time to ICI therapy discontinuation was defined as the number of months between ICI therapy initiation and the first of therapy discontinuation for any reason, death, or censoring at last known time alive. Time to SSAC therapy initiation was defined as the number of months from ICI therapy initiation to the first of SSAC therapy initiation, death, or censoring at last known time alive. The difference between these 2 time-to-event outcomes represents the time that patients were able to spend free of SSAC therapy and was determined using a restricted mean survival time approach at 36 months. This time point was selected given that the median follow-up of included patients in this study was 36.1 months , as assessed by the reverse Kaplan-Meier method.BRAF (OMIM 164757) V600E variant status . ECOG status represents an individual’s level of function with regard to their ability to care for themselves and perform daily activities rated on a scale from 0, which indicates no restrictions, to 5, which is death.A total of 316 patients were included in this analysis, including patients receiving single-agent pembrolizumab , single-agent nivolumab , or combination ipilimumab-nivolumab . Baseline participant characteristics at the time of ICI therapy initiation as stratified by therapy received are shown in the To provide a descriptive analysis for the patterns of TFS seen among patients with advanced melanoma receiving first-line ICI therapy, swimmer plots for a representative selection of 50 patients from each ICI therapy subgroup are shown in BRAF V600E–positive patients in the combined nivolumab-ipilimumab group, as this subgroup experienced a numerically longer period receiving subsequent SSAC therapy  compared with those without this variant .Patients who received single-agent nivolumab appeared to show a trend toward a longer period receiving ICI therapy  compared with those receiving single-agent pembrolizumab  or nivolumab-ipilimumab . Patients receiving nivolumab-ipilimumab spent a numerically longer period of the 36-month follow-up time alive and not receiving ICI therapy  compared with those receiving nivolumab  and pembrolizumab  . This co10In this study, we present the first observational analysis of TFS outcomes for patients with advanced melanoma receiving first-line ICI therapy outside the clinical trial setting, to our knowledge. Our analysis found that patients receiving combination nivolumab-ipilimumab experienced improved overall survival, as well as a longer period free of SSAC therapy and death compared with patients receiving single-agent anti–PD-1 agents. These findings are in line with previous studies that have examined TFS in the advanced melanoma clinical trial setting.13 However, the subset of patients included in the combination nivolumab-ipilimumab cohort also had a lower ECOG status, were younger, and were more likely to be BRAF V600E variant positive, all of which may have been associated with the trend toward improved TFS in this group.The longer TFS seen for patients receiving combination nivolumab-ipilimumab therapy may be associated with a more durable initial period of disease control, permitting SSAC therapy withdrawal, or the development of treatment-limiting adverse events followed by a treatment-free interval. These immune-related adverse events have been shown to be an established treatment biomarker of the likely benefit from ICI therapy.BRAF V600E–positive patients in this group, as this subgroup of patients has effective second-line therapy options. Moreover, a subset of patients receiving anti–PD-1 monotherapy may have been treated with second-line ipilimumab, which has a comparatively shorter duration of therapy, thus limiting their duration receiving SSAC therapy.In addition, differences in baseline patient characteristics, as well as choice of second-line therapies, were likely associated with differences in the duration receiving SSAC therapy seen between groups. Specifically, the numerically longer time receiving SSAC therapies seen for the combination nivolumab-ipilimumab group may have been associated with the larger proportion of Taken in context, these findings suggest that combination nivolumab-ipilimumab may provide an additional benefit to patients with advanced melanoma by lengthening the time they spend not receiving SSAC therapy, which has been associated with improvements in quality-of-life outcomes. Moreover, this work emphasizes the importance of examining outcomes such as TFS to understand how time is spent by patients receiving different therapy regimens. Future studies investigating this outcome among patients with advanced melanoma may also use different study methods, such as a target trial approach, to further enhance the conclusions drawn.BRAF V600E–positive patients, which were not accounted for in this analysis. Furthermore, previous analyses10 had further stratified TFS based on the presence of immune-related adverse events into TFS with toxic effects and TFS without toxic effects, which could not be replicated in this cohort. Therefore, it is possible that, although patients receiving combination nivolumab-ipilimumab experienced longer TFS, a greater portion of this time was spent with additional toxic effects, as was seen in previous clinical trial analyses.13 Another limitation is the lack of information regarding choice of SSAC therapy for patients included in this analysis, which may have provided additional context for interpreting differences in duration receiving SSAC therapy seen in this study.This study has some limitations. It was retrospective and was restricted to patients who received therapy at centers in Alberta, Canada, which may limit the generalizability of the findings. In addition, given the observational nature of this study, differences in TFS between groups may also be partially associated with underlying differences in key clinical variables, such as ECOG status and patient age, as well as differences in tumor characteristics, such as the proportion of In this cohort study of patients with advanced melanoma, those receiving first-line combined nivolumab-ipilimumab experienced a numerically longer TFS period compared with those receiving single-agent anti–PD-1 therapy. Additionally, TFS represented a patient-centric and informative end point.


This cross-sectional study compares COVID-19 pandemic-era excess deaths associated with Alzheimer disease or related dementias between year 1 and year 2 overall and by age, sex, race and ethnicity, and place of death. How did mortality with Alzheimer disease and related dementias (ADRD) as an underlying or contributing cause change during the COVID-19 pandemic?This cross-sectional study found a large increase in ADRD-related mortality during pandemic year 1 (March 2020 to February 2021) compared with prepandemic mortality rates but substantial declines from year 1 to year 2 (March 2021 to February 2022). The largest declines occurred in nursing home/long-term care settings, but excess mortality occurring at home and medical facilities remained high in year 2.Major reductions in pandemic-era ADRD-related excess mortality were achieved in pandemic year 2 for nursing home/long-term care settings residents. Adults with Alzheimer disease and related dementias (ADRD) are particularly vulnerable to the direct and indirect effects of the COVID-19 pandemic. Deaths associated with ADRD increased substantially in pandemic year 1. It is unclear whether mortality associated with ADRD declined when better prevention strategies, testing, and vaccines became widely available in year 2.To compare pandemic-era excess deaths associated with ADRD between year 1 and year 2 overall and by age, sex, race and ethnicity, and place of death.This time series analysis used all death certificates of US decedents 65 years and older with ADRD as an underlying or contributing cause of death from January 2014 through February 2022.COVID-19 pandemic era.Pandemic-era excess deaths associated with ADRD were defined as the difference between deaths with ADRD as an underlying or contributing cause observed from March 2020 to February 2021 (year 1) and March 2021 to February 2022 (year 2) compared with expected deaths during this period. Expected deaths were estimated using data from January 2014 to February 2020 fitted with autoregressive integrated moving average models.Overall, 2 334 101 death certificates were analyzed. A total of 94 688  pandemic-era excess deaths with ADRD were estimated in year 1 and 21 586  in year 2. Declines in ADRD-related deaths in year 2 were substantial for every age, sex, and racial and ethnic group evaluated. Pandemic-era ADRD-related excess deaths declined among nursing home/long-term care residents , but excess deaths at home remained high .This study found that large increases in mortality with ADRD as an underlying or contributing cause of death occurred in COVID-19 pandemic year 1 but were largely mitigated in pandemic year 2. The most pronounced declines were observed for deaths in nursing home/long-term care settings. Conversely, excess deaths at home and in medical facilities remained high in year 2. Common comorbidities among older adults with ADRD increase the risk of death if they are infected with SARS-CoV-2.3 Social isolation resulting from COVID-19 lockdowns may exacerbate depression and loneliness among individuals with ADRD, increasing their risk of hospitalization and mortality.5 Disruptions in care and services may have disproportionately affected individuals with ADRD.6 In the early pandemic period, excess mortality disproportionately affected older adults with ADRD in long-term care facilities, likely due to the high prevalence of comorbidities among residents, staffing shortages, isolation from family members and/or other essential caregivers, and challenges in implementing and enforcing infection prevention and control measures.1 Sex and racial and ethnic disparities in excess mortality from ADRD were also noted, with more excess deaths in females  and non-Hispanic Black and Hispanic (vs non-Hispanic White) older adults in the early pandemic.7Older adults with Alzheimer disease and related dementias (ADRD) are particularly vulnerable to the direct and indirect impacts of the COVID-19 pandemic.1 Pharmaceutical and nonpharmaceutical preventive measures became widely available in pandemic year 2, but vaccine distribution and uptake of other measures were inconsistent.9 Tracking excess mortality associated with ADRD over the pandemic is critical to inform policy and research priorities, as changes in mortality provide insights into whether current preventive measures are effective at protecting older adults with ADRD. This study assessed pandemic-era changes in mortality with ADRD as an underlying or contributing cause comparing the COVID-19 pandemic year 1 (March 2020 to February 2021) to year 2 (March 2021 to February 2022), in the overall population and by age, sex, race and ethnicity, and place of death.While research has documented substantial excess mortality among individuals with ADRD in the early pandemic, it is unclear how its magnitude changed as the pandemic evolved.11 We considered deaths with any mention of ADRD on the death certificate, including as the underlying cause or any of up to 19 listed contributing conditions as deaths with ADRD. ADRD was classified by International Statistical Classification of Diseases and Related Health Problems, Tenth Revision codes and included unspecified dementia, Alzheimer disease, vascular dementia, and other degenerative diseases of the nervous system  reporting guideline.Final death certificate data from January 2014 to December 2021 and provisional death certificate data from January 2022 to February 2022 were extracted from the National Center for Health Statistics mortality surveillance system.The National Center for Health Statistics provided bridged-race death data from 2014 to 2020 and single-race death data from 2018 to 2022. We combined 2014-2017 bridged-race data and 2018-2022 single-race data to estimate race-specific excess mortality. To evaluate the bias associated with combining single- and bridged-race data, we compared both numerators (death counts) and denominators (population size) using single- vs bridged-race categories for the period during which both data sources were available, ie, 2018-2020 , Alpha wave , Delta wave , and Omicron wave .Additional stratification variables included age , sex , race and ethnicity , and place of death .13 We selected the model with the lowest Akaike information criterion and calculated monthly excess deaths as observed minus expected deaths. We calculated total excess deaths by summing all monthly excess deaths and calculated corresponding 95% prediction intervals (PIs) by simulating the expected death model 10 000 times, selecting the 2.5 and 97.5 percentiles, and subtracting them from the number of observed deaths. We calculated risk ratios as the observed number of deaths divided by the expected number of deaths. We calculated excess mortality rates per capita, as the number of excess deaths divided by the corresponding population size and multiplied by 100 000.We defined pandemic-era excess ADRD-related deaths as the difference between observed and expected deaths over the same period. To estimate expected deaths, we fitted auto-regressive integrated moving-average models (ARIMA) to monthly ADRD-related death count data from January 2014 to February 2020. We used this prepandemic prediction model to forecast the number of ADRD-related deaths that would have been expected in the absence of the pandemic, accounting for historical mortality trends and seasonality.We performed stratified analyses by age, sex, race and ethnicity, and place of death. To facilitate comparisons across groups, excess deaths were age standardized in 10-year categories to the 2000 population when applicable.To validate the performance of the ARIMA model, we performed sensitivity analyses using data from 2014 to 2018 to forecast monthly death rates from January to December 2019. All observed deaths fell within the 95% PI of the expected deaths, suggesting accurate predictions . Third, we used weekly COVID-19 vaccination data from the US Centers for Disease Control and Prevention COVID Data Tracker to understand how vaccination may have affected excess mortality. Specifically, we assessed how changes in annualized excess ADRD-related deaths differed by vaccine coverage across states .We conducted exploratory analyses to investigate potential reasons for notable declines in excess deaths between years 1 and 2. First, we evaluated how changes in the number of NH/LTC residents during the pandemic might have affected the estimation of excess deaths in NH/LTC settings. To this end, we obtained the monthly number of NH/LTC residents from the Centers for Medicare & Medicaid Services (CMS) for 2019 to 2022. Specifically, we adjusted the observed number of deaths, accounting for the relative change in the population size of NH/LTC residents by comparing each month between March 2020 to February 2022 to the corresponding month in 2019  ADRD-related deaths were expected in year 1, implying a year-1 excess of 94 688 deaths . In year 2, ADRD-related deaths declined to 435 156, implying an excess of 21 586 deaths  . The panPandemic-era excess ADRD-related death rates declined substantially between the first and second year of the pandemic for every age, sex, and racial and ethnic group evaluated .Year 2 of the pandemic saw major declines in ADRD-related deaths occurring in NH/LTCs  for Asian adults, 64  for Black adults, 33  for Hispanic adults, and 30  for White adults . After tLong-term care facilities were hard hit in the early pandemic . After vr = −0.54 ) and velocity   in year 1 and an estimated −2914  in year 2. Furthermore, the overall trends in the adjusted and unadjusted monthly death counts were similar , in contrast with a pronounced increase during the Delta wave . These divergent patterns across waves may reflect not only changes in public health policies and interventions but also differences in transmissibility, infectivity, and mortality rates among COVID-19 variants.People living with ADRD were uniquely vulnerable to the pandemic. Our findings are consistent with prior research showing large adverse effects of the early pandemic on older adults with ADRD.17 In year 2, several initiatives to reduce SARS-CoV-2 transmission implemented in NH/LTC facilities may have further contributed to declines in excess mortality in these settings, including limiting the use of shared spaces and improved surveillance testing of staff members.18ADRD-related deaths fell in pandemic year 2, primarily due to reductions in deaths in NH/LTC facilities. This finding cannot be explained by declines in the number of NH/LTC residents as the overall trend remained similar after we accounted for changes in population size using the CMS data. The frailty selection hypothesis alone is not sufficient to explain the results. If the frailty selection hypothesis is supported, we would expect to see reductions in all leading causes of death. However, cancer deaths in nursing homes remained similar between years 1 and 2. Our finding that faster vaccine rollout and greater coverage were associated with larger reductions in ADRD-related deaths in year 2 suggests that access to vaccines, both for persons living with ADRD and their care professionals, may play a key role in reducing excess deaths. Vaccination among staff and residents may lower excess deaths in nursing homes directly through preventing viral transmission and infection and indirectly through reducing social isolation.19 They have also been found to have higher mortality following a COVID-19 diagnosis than their counterparts without dementia, and the disparities persisted in 2021 despite the availability of vaccines.20 Increased policy efforts are imperative for reducing excess deaths among community-dwelling older adults.Relatedly, the persistently high levels of ADRD-related deaths occurring at home suggest that community-dwelling older adults with ADRD may not have benefited similarly from COVID-19 preventive measures. Community-dwelling older adults with dementia have been found to be more likely to put off care during the pandemic than those who live in nursing homes, potentially contributing to high-excess home deaths.25 While we could not directly assess racial and ethnic disparities in ADRD-related mortality without population data on the number of individuals living with ADRD by race and ethnicity, our analysis of mortality differences by race and ethnicity highlights concerning patterns in excess ADRD-related deaths during the pandemic. Our findings for year 1 suggest that Black older adults with ADRD experienced substantially higher excess deaths in the early pandemic. Due to the racial segregation of nursing home facilities, Black individuals with ADRD may be more likely to cluster in nursing homes that have worse infection rates.28 Discrimination in medical settings and differential prevalence of comorbidities may also have contributed to this excess mortality. Racial and ethnic disparities in excess ADRD-related deaths persisted despite declines in ADRD-related deaths across all racial and ethnic groups in year 2. These findings highlight the need to monitor inequalities and attend to how structural racism can exacerbate vulnerability to ADRD-related and COVID-19 mortality.Research conducted prior to the pandemic has yielded inconsistent results on racial and ethnic disparities in ADRD mortality.29 However, the patterns of temporal changes in ADRD-related excess deaths that our study revealed are corroborated by Medicare data, which showed a decline in excess deaths among individuals with ADRD in 2021.30 Our findings may underestimate the number of ADRD-related deaths from racial and ethnic minority groups in whom ADRD underdiagnosis is common.31 Finally, the 2022 death certificate data are provisional. Although analyzing finalized death certificates would be ideal, using currently available data is critical to inform timely policy responses.Our data may not fully capture deaths among all individuals with ADRDs, if ADRD was undiagnosed, or if the diagnosis was not considered to contribute to death.16 Our inclusion of both the underlying and contributing causes of death likely lessens the impact of misclassification of both ADRD and COVID-19.33 Our time series models starting in 2014 accounted for prepandemic temporal trends in deaths, for example due to population aging or increased ADRD diagnoses.34This study has several strengths. The inclusion of 2020, 2021, and early 2022 data allowed us to capture the dynamics of the pandemic’s impact on individuals who lived with ADRD. Most prior research relied on underlying cause of death classification.COVID-19 pandemic-era mortality with ADRD as an underlying or contributing cause has been extremely high. These deaths are often preventable, and ADRD-related mortality fell markedly in NH/LTC settings later in the pandemic. Vaccinations were likely critical to these improvements. Our findings underscore the urgent need to mitigate the pandemic’s impacts on community-dwelling older adults with ADRD.