Elderly patients (aged ≥ 65 years) represent an increasing proportion of patients with psoriasis and 15% of these have moderate to severe disease. Biologics are being used frequently in this group of patients even though safety and efficacy data are limited. In addition, owing to anti‐interleukin (IL)‐23 therapies being a relatively recent option, no data have been reported about their use in elderly patients with psoriasis.To evaluate and compare the safety and efficacy of guselkumab, risankizumab and tildrakizumab in real‐world practice in elderly patients.This was a single‐centre retrospective study that enrolled patients aged ≥ 65 years with moderate to severe plaque psoriasis, treated with guselkumab, risankizumab or tildrakizumab. The length of the study for each group depended on the drug .n = 20 on guselkumab; n = 8 on risankizumab; n = 6 on tildrakizumab). At Week 4, 29.4% reached 90% improvement in Psoriasis Area and Severity Index (PASI90) and 8.8% reached 100% improvement in PASI (PASI100); at Week 28, PASI90 and PASI100 was reached by 58.8% and 29.4%, respectively. At the final follow‐up , data were available only for the risankizumab (Week 40) and guselkumab (Week 44) and groups, and showed that 71.4% of patients had reached PASI90 and 53.5% had reached PASI100. Four patients (11.7%) discontinued treatment. No significant differences were found between the three groups. The limitations of the study included its retrospective nature of the study, small sample size, and different numbers of patients and follow‐up duration for the different groups .In total, 34 patients were enrolled (The three anti‐IL‐23 therapies assessed are promising, safe and effective options in elderly patients, and there was no significant difference between them. However, more data are needed to confirm our results and to understand their role in the management of this group of patients. Psoriasis is a chronic inflammatory disease affecting 1%–3% of global population.Conventional systemic treatments including acitretin, methotrexate and ciclosporin, are often avoided due to the risk of adverse events (AEs) and frequent contraindications. Hence, elderly patients with moderate to severe psoriasis may be undertreated, resulting in severe psychological and physical impacts.The study was approved by the ethics committee of the University of Naples Federico II , and performed in accordance with the principles of the Declaration of Helsinki. All patients signed an informed consent before starting the study, and gave consent to publication of case details and images, where applicable.This was a single‐centre retrospective study performed in Italy, which enrolled patients with moderate to severe psoriasis who attended the Psoriasis Care Center, University of Naples Federico II, Naples, Italy during the period October 2018 to March 2021.Inclusion criteria were: (i) moderate to severe plaque‐psoriasis diagnosis since at least 1 year; (ii) patients aged ≥ 65 years; (iii) patients starting treatment with guselkumab, risankizumab or tildrakizumab; and (iv) treatment period of at least 44 weeks for risankisumab, 40 weeks for guselkumab and 28 weeks for tildrakizumab .At baseline, the following items were registered for each patient: (i) personal and demographic data; (ii) duration of psoriasis and any eventual psoriatic arthritis (PsA); (iii) comorbidities; (iv) previous systemic treatments for psoriasis; and (v) psoriasis severity as measured using the Psoriasis Area and Severity Index (PASI) and body surface area (BSA) tools.At every follow‐up, psoriasis severity (PASI and BSA), routine blood tests  and AEs were evaluated. Safety was assessed by treatment‐emergent AEs, physical examination and laboratory monitoring.t‐test, with P < 0.05 considered statistically significant. All statistical analyses were performed using GraphPad Prism software .Data are presented as mean ± SD (for continuous variables) or as number and percentage of patients . The significance of differences in mean values obtained at the different time points of treatment was assessed by the unpaired Student In total, 34 patients were enrolled . Psoriasis duration was 22.6 ± 14.1 years. Half of the patients (52.9%) also had PsA. The most frequent comorbidity was hypertension (70.5%), followed by dyslipidaemia (47.1%), diabetes (32.3%) and depression (26.4%). All patients had been treated with at least one conventional systemic drug, with methotrexate (67.6%) and ciclosporin (35.3%) being the most common.Only 8.8% of the patient group had never received biologics, while 91.2% had been treated previously with at least one biologic, of whom 85.2% had failed to respond to an anti‐TNF, 35.3% to an anti‐IL‐12/23 and 67.6% to an anti‐IL‐17 drug. Mean anti‐IL‐23 treatment duration was 43.7 ± 8.2 weeks.The mean clinical outcomes were 90% and 100% improvement in PASI (PASI90 and PASI100 respectively). In the total patient group, 29.4% reached PASI90 and 8.8% PASI100 at Week 4, while at Week 28, PASI90 and PASI 100 were observed in 58.8% and 29.4%, respectively, and at the end of the study , 71.4% had reached PASI90 and 53.5% PASI100. There were no statistically significant differences in efficacy between the three drugs, except for PASI100 at Week 4, which was lower for the tildrakizumab group compared with the other two groups  discontinued the treatment: three patients stopped because of secondary inefficacy; one patient stopped because of a diagnosis of bladder cancer 18 weeks after guselkumab initiation. Mild and nonclinically relevant blood test alterations were experienced by 23.5% of patients.Candida infection, malignancy or cardiovascular events (CVEs) were reported. Detailed data on overall study population and on the three groups separately are displayed in Table Potential registered AEs were found in 29.4% of patients, including pharyngitis 12.5%), headache (12.5%), influenza‐like illness (8.3%) and diarrhoea (2.9%), none of which required treatment discontinuation. Again, no significant differences in discontinuation rate or safety were observed between the three investigated drugs. No other cases of serious AEs, injection site reaction, 2.5%, heaWith ageing populations worldwide, there is a higher proportion of elderly patients with psoriasis.post hoc analysis of two phase III RCTs of etanercept found no significant difference in terms of efficacy between elderly and younger patients, but older patients did have a higher risk of AEs.post hoc analysis of adalimumab, the REVEAL trial, found that the drug had lower effectiveness in elderly than in younger patients.To date, most of the reported relevant clinical studies on biologics in elderly patients have focused on the efficacy and safety of anti‐TNF‐α drugs, owing to their longer market availability.post hoc analysis of three phase III secukinumab trials  showed comparable efficacy profiles between elderly and younger patients; however, the rates of serious AEs and discontinuation were higher in older participants.Regarding anti‐IL‐17 inhibitors, a recent study including 114 elderly patients showed that these drugs were an effective and safe therapeutic option for patients with psoriasis aged ≥ 65 years, with low rates of only mild AEs; however, the discontinuation rate was 28.9%, mostly related to psoriasis relapses.Owing to their recent availability, data on anti‐IL‐23 therapies for elderly patients in real‐world clinical practice are still scant. Phase III clinical trials showed that anti‐IL‐23 agents have an acceptable safety profile in elderly patients.The limitations of our study included its retrospective nature, relatively small sample size, unequal follow‐up duration among the three groups, the lower number of tildrakizumab‐treated patients and higher components of guselkumab .The three anti‐IL‐23 therapies assessed are promising, safe and effective options in elderly patients, and there was no significant difference between them. However, more data, both from dedicated trials and real‐life reports, are needed to confirm our results in order to better understand the role that anti‐IL‐23 therapies could play in the management of this group of patients.


Recent studies have highlighted the increased risk of low bone mineral density (BMD) in adults with cardiovascular disease. However, little is known about BMD in adults with congenital heart disease (CHD), particularly in developing countries. We hypothesized that factors related to BMD would lead to a high prevalence of low BMD in adults with CHD. This study aimed to determine the prevalence of low BMD and its related factors in Vietnamese adults with CHD.Z‐scores and T‐scores at the posteroanterior lumbar spine and left proximal femur. Logistic regression analyses were performed to evaluate factors related to low BMD.We conducted a cross‐sectional study of 73 adults diagnosed with CHD in Vietnam. Low BMD was classified based on their site‐specific p < 0.001). Moreover, the prevalence of low BMD was significantly higher in adults with CHD than in those without polycythemia and vitamin D deficiency . A stratified multivariate logistic regression analysis revealed that low BMD was associated with polycythemia .Low BMD was confirmed in one‐third of the adults with CHD. There were trends of more bone loss in certain parts of the body than in others, with the prevalence of low BMD at the sites of the lumbar vertebrae (L1‒L4) and left proximal femur  of 43.9%, 31.8%, 28.8%, 33.3%, 8.8%, 1.5%, and 6.1%, respectively. The prevalence of low BMD in the lumbar spine was significantly higher than that in the left proximal femur (34.3% vs. 2.9%, Low BMD is common among adults with CHD in Vietnam and related to polycythemia. As longevity and healthcare quality improve, many patients with CHD now survive until adulthood, markedly changing the disease pattern and requiring management in adulthood.Osteoporosis involves a gradual decrease in bone mineral density (BMD) and microarchitectural deterioration of bone tissue, leading to bone fragility. The gold standard for osteoporosis diagnosis is BMD, which is measured by dual‐energy X‐ray absorptiometry scans (DXA). Osteoporosis has a complex pathophysiology that is determined by factors such as sex, age, hormones, nutrition, and lifestyle.It is hypothesized that CHD shares many biological risk factors with low bone quality. Recent evidence suggests that low bone quality, including osteoporosis, is common among patients with CHD.22.1From May 2019 to September 2020, we conducted a cross‐sectional study of adults with CHD who were followed up at the Vietnam National Heart Institute, a national referral cardiovascular center specializing in CHD.2.2We selected patients who were confirmed to have CHD on cardiac imaging. All patients underwent transthoracic echocardiography performed by a cardiologist with cardiac sonography certification at least once. If diagnosis was unclear, patients underwent another form of imaging, such as transesophageal echocardiography, chest computed tomography, cardiac magnetic resonance imaging, and right heart catheterization. The final diagnosis was confirmed after consultation with a CHD specialist. Patients were included if their BMD was determined by DXA, and if they were aged ≥18 years.2); and those with a history or current use of drugs associated with osteoporosis or long‐term bone effects, such as corticosteroids, aromatase inhibitors, gonadotropin‐releasing hormone agonists, androgen‐deprivation therapy, injection contraception, medroxyprogesterone acetate, selective serotonin reuptake inhibitors, antiepileptic drugs, calcineurin inhibitors, and chemotherapy agents. We also excluded those who were pregnant or breastfeeding.We excluded patients with any of the following characteristics: peri‐ and postmenopause; acute medical status for the past 3 months, and known disorder(s) affecting bone metabolism d2, where N is the sample size, Z is the statistic corresponding to the confidence level, P is the expected prevalence, and d is the precision. We selected P from a recent study that found an osteoporosis prevalence of 43% among Vietnamese women aged ≥50 years.We calculated the sample size for this cross‐sectional study using the following formula: 2.42) was measured using a Life Scope BSM3500 (Nihon Kohden). Cyanosis was defined as a SpO2 ≤92% in room air. Pulmonary artery hypertension (PAH) was defined as a systolic pulmonary artery pressure ≥50 mmHg as estimated from tricuspid valve regurgitation on echocardiography. Anemia was defined as a hemoglobin level <12.0 g/dL in women and <13.0 g/dL in men. Polycythemia was defined as a hemoglobin level ≥185 g/L in men or ≥165 g/L in women with a hematocrit value ≥51% in men and ≥48% in women.Demographic and clinical information were obtained at consultation. We used the International Classification of Disease, 10th revision, for CHD diagnosis. Young adults were defined to be aged between 18 and 35 years.2. Liver dysfunction was diagnosed if plasma total bilirubin and aspartate transaminase and/or alanine transaminase levels were elevated more than the reference ranges for laboratory values . Plasma total calcium levels were measured by Cobas 8100 (Roche), while the levels of plasma 25‐hydroxyvitamin D (25[OH]D) and parathyroid hormone (PTH) were measured using Architect i2000SR (Abbott). We defined cutoff points of vitamin D deficiency as plasma 25(OH)D levels <20 ng/mL, severe vitamin D deficiency as plasma 25(OH)D levels <12 ng/mL, hypocalcemia as plasma total calcium levels <2.15 mmol/L, and hyperparathyroidism as plasma PTH levels >7.2 pmol/L.Biochemical testing was performed at the Department of Biochemistry, Bach Mai Hospital, according to the ISO 15189:2007 standards. Renal dysfunction was defined as an eGFR ≤90 ml/min/1.73 mZ‐scores were calculated as the number of standard deviations (SDs) away from the average value of the reference group, with the following formula: Z‐score = (BMD‐expected BMD)/SD. Site‐specific T‐scores were calculated for participants aged >50 years. The T‐score was the number of SDs of the patient's measured BMD from a reference peak BMD (mean BMD of sex‐matched young adults) calculated using the following formula: T‐score = (patient's BMD‐peak BMD)/SD. In premenopausal women and men aged <50 years, low BMD was defined as a site‐specific Z‐score ≤−2.0, as described by the recommendations of the International Society for Clinical Densitometry. In men aged >50 years, low BMD was defined as a site‐specific T‐score ≤−2.5 (osteoporosis) according to the World Health Organization criteria. Notably, a site‐specific T‐score between −1 and −2.5 was considered to indicate osteopenia.BMD was evaluated at the posteroanterior lumbar spine  and left proximal femur  using the Discovery DXA system software version 13.5.4 (Hologic) for baseline scans. Site‐specific 2.5T‐score, and Z‐score) and low BMD prevalence at the evaluated site in adults with CHD overall and in subgroups. The secondary outcomes were low BMD‐related factors in these patients.The primary outcome measures were bone characteristics . In all analyses, statistical significance was defined as a two‐tailed p < 0.05.Data were analyzed using SPSS version 22.0 (IBM). Continuous variables are described as mean  for normally distributed data or median (interquartile range) for non‐normally distributed data. Numbers and percentages are used to describe nominal variables. Among subgroups, normally distributed continuous variables were compared using Student's 33.1The characteristics of 73 adult patients with CHD are summarized in Table 3.2p < 0.001, respectively).The prevalence of low BMD is shown in Figure T‐ and Z‐scores were low at any site of the lumbar spine (<−1.0) but in the normal range (>−1.0) at the left proximal femur. The overall mean BMD, T‐score, and Z‐score of the lumbar spine and left proximal femur were similar, except for the mean BMD at the trochanteric femur in women, which was significantly lower than that in men.Table 3.3p = 0.001) and in patients with vitamin D deficiency than in those without . The relationship between BMD and age in patients with CHD is described in Supporting Information: Table R = 0.505, p = 0.004 and R = 0.503, p = 0.004, respectively). Meanwhile, at other sites in both sexes, age‐related BMD was low. The results of the stratified logistic regression for predicting low BMD are summarized in Table p = 0.004).Figure 4To the best of our knowledge, this study provided previously unreported evidence on the significant prevalence of low BMD in adults with CHD in Vietnam. Low BMD was more prevalent in patients with polycythemia than in those without, and vitamin D deficiency was prevalent in patients with the aforementioned health conditions. Moreover, polycythemia was an independent risk factor for low BMD in adults with CHD.4.1Patients were predominantly female with simple cardiac malformations, similar to a previous report.4.2Z‐scores <−2.0.We found reduced BMD levels in the lumbar spine as measured by DXA in adults with CHD, with no sex‐related differences in mean BMD at any site, except the trochanteric femur. It was previously reported that bone health is linked to low BMD and cardiovascular disease.2, and women, 0.853 vs. 0.87 g/m2). The same trend was also noted in the mean T‐score and Z‐score at the lumbar spine.Furthermore, our study demonstrated that, at the lumbar spine, the mean BMD in adults with CHD seemed to be lower than the Vietnamese population reference ranges  have been explained.The protocol of the current study was approved by the Science Board and Ethics Committee of the Bach Mai Hospital . The study complied with the principles of the Declaration of Helsinki. All participants provided written informed consent.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.


Malus domestica) core‐collection.Tree architecture shows large genotypic variability, but how this affects water‐deficit responses is poorly understood. To assess the possibility of reaching ideotypes with adequate combinations of architectural and functional traits in the face of climate change, we combined high‐throughput field phenotyping and genome‐wide association studies (GWAS) on an apple tree  scanning and airborne multispectral and thermal imagery to monitor tree architecture, canopy shape, light interception, vegetation indices and transpiration on 241 apple cultivars submitted to progressive field soil drying. GWAS was performed with single nucleotide polymorphism (SNP)‐by‐SNP and multi‐SNP methods.Large phenotypic and genetic variability was observed for all traits examined within the collection, especially canopy surface temperature in both well‐watered and water deficit conditions, suggesting control of water loss was largely genotype‐dependent. Robust genomic associations revealed independent genetic control for the architectural and functional traits. Screening associated genomic regions revealed candidate genes involved in relevant pathways for each trait.We show that multiple allelic combinations exist for all studied traits within this collection. This opens promising avenues to jointly optimize tree architecture, light interception and water use in breeding strategies. Genotypes carrying favourable alleles depending on environmental scenarios and production objectives could thus be targeted. Throughout the experiment, ψsoil remained constant for WW trees (c. −8 kPa) while it gradually decreased for WD trees and reached on average −72 kPa on 27 July ; 12 July, light WD ; 17 July, moderate WD ; 27 July, severe WD . Predawn leaf water potential  and midday leaf  and stem  water potentials were also measured on 45 trees across the orchard, with two Schölander pressure chambers previously cross‐calibrated using a distributed, pressurized nitrogen source .At the flight dates, soil dryness increased on WD rows Fig. : 5 July,et al., et al., et al., penAlea  assuming a cylinder trunk shape. TCSA is reported to be a good indicator of tree size  on the variables related to tree architecture using individual tree values. Tree coordinates on the two first PCA dimensions (respectively representative of vegetative development and light interception capacity) were further used as additional traits.All data analyses were performed using R  with a random effect of the genotype (G), fixed effects of the watering scenario (S), spatial design (row), date of measurements (D), and flight within each date (F). For T‐LiDAR variables, a fixed‐effect of the tree position relative to the LiDAR (R) was included. The best model was selected (Table H2) as:G the genotypic variance, varRes the residual variance, and n the number of replicates per genotype.Different mixed‐effect models were tested  with the function BootMer (R/lme4).A 95% confidence interval (CI) for Best linear unbiased predictors (BLUPs) of genotypic values were extracted for each trait. To quantify the genotypic response to soil water deficit, we calculated at each date another variable named resp.TSTA computed as the difference between BLUPs of TSTA under WD condition (TSTA.WD) and BLUPs of TSTA under WW condition (TSTA.WW). The ratios resp.TSTA_12.07/resp.TSTA_27.07 (named contrib_12.07) and resp.TSTA_17.07/resp.TSTA_27.07 (contrib_17.07) were further calculated to quantify the proportion of variation in resp.TSTA observed at severe soil dryness intensity (27 July) that was achieved at light (12 July) and moderate (17 July) intensities of soil dryness.Multivariate analyses of correlations between all traits were performed on BLUPs.et al., et al., et al., GWAS was performed to look for associations between BLUPs and genomic markers. We used the Axiom®Apple 480 K array that resulted in 275 223 robust SNPs for GWAS upon stringent filters  matrix estimated based on SNPs with MAF > 0.05, using VanRaden kinship estimator  were computed as the ratio of variance explained by the polygenic effects (G) to the total BLUP variance, in the null GWAS model, i.e. without considering the effect of individual SNPs (β). This model, accounting only for relatedness, was found  to be the best to control confounding factors as compared to models accounting for structure  from the Wald test statistic was smaller than the Bonferroni threshold, controlling the family‐wise error rate at 5% to account for multiple testing. This threshold was calculated on a subset of nearly independent SNPs using a LD pruning method implemented in Plink/indep  = 5.63.Two types of GWAS models were used. A SNP‐by‐SNP model implemented in et al.  proposed by Segura et al. . The modgemma and MLMM were compared. SNPs selected by both methods were considered ‘highly reliable’ and further used for analysing interaction effects and candidate genes. R/rutilstimflutre . For the particular situation of chr13 where significant SNPs were detected for several traits, a haplotype‐based analysis was attempted to decipher the genetic control of these traits . Annotations for gene biochemical functions were enriched by the biological functions inferred from the putative orthologues identified in Arabidopsis thaliana retrieved from (www.rosaceae.org). We searched UniProt (www.uniprot.org/) and TAIR (www.arabidopsis.org/) databases to get a complete description of the gene functions based on gene ontology and annotation.In order to investigate candidate genes in the associated regions, QTLs were defined as intervals of ±100 kb around the most significant SNPs (in line with Urrestarazu r > 0.7) were observed between dates. We thus estimated a single BLUP for subsequent analyses for each of these three indices, integrating the four dates and both scenarios. Corresponding H2 values were higher than 0.9  and light interception (e.g. STAR varied from 0.55 to 1.06). High values of H² were observed .Canopy development, tree architecture and light interception related traits were computed at a single date, in either summer or winter. The models selected sometimes accounted for the watering scenario but its effect remained low Table , revealir = 0.85 and 0.95, respectively) while the correlation between GNDVI and MCARI2 was lower (r = 0.67). BLUPs of these three variables were significantly, positively correlated with the ones related to canopy development, particularly a_volume and Dim.1 . To estimate BLUPs of TSTA.WW independent of atmospheric variations, we used data collected on the 4 d with a mixed‐effect model including a fixed effect of the measurement date. These TSTA.WW BLUPs displayed a substantial range of variability among the cultivars (0.4–2.3°C) with H2 = 0.67 (CI 0.65–0.68). This suggests a genotype‐dependent control of water loss in WW conditions.TSTA, the difference between canopy surface and air temperature computed from thermal imaging, was first evaluated for the WW trees as a proxy for water loss in nonlimiting watering conditions (TSTA.WW). Despite the stability of tes Fig. , mean vates Fig. , likely tes Fig. . HoweverH² values were similar between dates . TSTA.WD displayed a marked and progressive increase along with intensification of soil drying , and contrib_12.07 and contrib_17.07 did not correlate with the absolute value of maximal resp.TSTA observed on 27 July  and canopy development, suggesting a tendency for more vigorously growing cultivars to maintain stronger canopy temperature regulation than small cultivars. However, the weakness of these correlations (r < 0.43) indicated that multiple combinations of these traits exist depending on the cultivar. Regarding the relationship between canopy surface temperature (either TSTA.WW or resp.TSTA_27.07) and light interception efficiency, correlations appeared even weaker (0.04 < r < 0.26), although significantly different from zero.We further investigated correlations between BLUPs for traits related to canopy development or light interception on the one hand, and canopy surface temperature under WW (TSTA.WW) or in response to severe WD conditions (resp.TSTA_27.07) on the other hand Figs , S6. Corgemma  = 5.73), had a −log10 of 4.68 for STAR and of 3.84 for ci  = 5.77), had a −log10 of 4.35 for Dim.2 and 3.12 for STAR  7 (Dim.2) and one at the top of chr15 (ci). The results obtained on the corresponding SNPs for the other traits related to light interception confirmed their substantial, although nonsignificant, effects. For instance, the SNP on chr7 found significant for Dim.2  up to 7.7  ABNORMAL SHOOT 5, that was shown to have a crucial role for leaf development and morphogenesis  distinct from those associated with vegetative development, highlighting the complementary nature of the information they carry. These associations were consistent with the chlorophyll‐related characteristics often attributed to vegetation indices. For instance, on chr13, the gene homologous to STN7, a protein kinase involved in photosynthesis regulation was found in the region specifically associated to MCARI2 and NDVI. This kinase plays a well‐described role in the redistribution of light excitation energy between photosystem II and photosystem I  considered as a proxy of canopy transpiration  increased with its severity, and a strong and increasing genotypic variability was observed along the month of measurements. It should be noted that in such an orchard experiment, the evolution of the soil WD was only partly controlled, resulting in variability for oil Fig.  as well wn Table  at any dA major outcome of this study is that phenotypic and genetic analyses indicate independent controls of traits respectively related to (1) vegetative architecture; (2) light interception; and (3) water loss estimated by canopy surface temperature. This represents an important step in our understanding of the physiological and genetic basis of these three groups of traits, which are all relevant breeding targets in the context of climate change. As a consequence, multiple allelic combinations exist within the apple tree collection for the 16 highly reliable SNPs Table . This alAC‐L, BP, MD, J‐LR, and EC designed research. AC‐L, BP, MD, and J‐LR performed the experiments. MD performed the MS and TIR images post‐processing. BP performed the T‐LiDAR scans post‐processing. AC‐L and BP analysed data and carried out GWAS analyses with contribution from VS and BG. HM and C‐ED generated the genotyping data. AC‐L and BP wrote the article with the contribution of EC, J‐LR, C‐ED, HM, VS and MD. All authors approved the manuscript.Fig. S1 Evolution of soil water potential during the experiment.Fig. S2 Tree architecture and light‐interception related traits retrieved from T‐LiDAR measurements.Fig. S3 Vegetation indices  are stable and correlated across dates and watering scenarios.Fig. S4 Canopy surface temperature (TSTA) under the well‐watered scenario across dates.Fig. S5 Correlations between the proportion of maximal canopy temperature increase observed at the light and moderate intensities of soil dryness, and the maximal canopy temperature increase.Fig. S6 Weak correlations between tree canopy temperature and traits related to vegetative architecture and light interception.Fig. S7 Two regions associated with traits related to light interception capacity on chromosomes 7 and 15.Fig. S8 Three regions associated with traits related to tree architecture on chromosomes 2, 4, and 6.Fig. S9 Boxplots of allelic effects for a large region associated with several architecture‐related traits on chromosome 13.Fig. S10 Heatmap of linkage disequilibrium in a large region associated with several architecture‐related traits on chromosome 13.Fig. S11 A large genomic region controls architecture‐related traits on chromosome 13.Fig. S12 Haplotype analysis of a genomic region controlling architecture‐related traits on chromosome 13.Fig. S13 Different regions controlling respectively the canopy temperature in well‐watered conditions, or its response to water deficit.Fig. S14 Heatmap of allelic effects for the 16 highly reliable associations and the 241 cultivars of the apple tree core‐collection.Methods S1 Haplotype analysis of a genomic region controlling architecture‐related traits on chromosome 13.Table S1 Summary of the meteorological variables during the drones’ flights at the four dates of measurements in July 2017.Table S2 Leaf and stem water potentials measured on 45 trees among the apple tree core‐collection.Click here for additional data file.Table S3 Complete set of associations detected for the traits related to vigour, light interception and canopy temperature response to water deficit computed from T‐LiDAR, multispectral and thermal imaging on a collection of 241 apple tree varieties.Table S4 Summary of the GWAS results per trait.Table S5 List of genes underlying the most highly reliable SNPs.Table S6 Co‐localizations between associated SNPs on the apple core collection and SSR associated on bi‐parental populations for similar or related traits.Table S7 List of allelic effects for the 16 highly reliable associations and the 241 cultivars of the apple tree core‐collection.New Phytologist Central Office.Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries  should be directed to the Click here for additional data file.


Type 2 diabetes mellitus (T2DM) is a prevalent public health problem worldwide, and the economic burden of the disease poses one of the main challenges for health systems in low‐ and middle‐income countries. This study aimed to estimate the economic burden of T2DM in Iran, in 2018.This was a cost‐of‐illness study. Three hundred and seventy‐five patients with T2DM who were referred to Imam Reza and Sina's educational and therapeutic centers and Asad Abadi clinic in Tabriz, Iran, in 2018 were included. A researcher‐constructed checklist was used for data collection. Data were analyzed using EXCEL and SPSS software version 22.Total economic burden of diabetes was estimated at 152,443,862,480.3  . The mean total direct and indirect costs were 11,278.68 (PPP)  and 6808.88  , respectively. The mean total direct medical cost and the direct nonmedical cost were 10,819.43   and 459.24   per patient, respectively. Besides, the mean direct medical cost was 6.18 times the total per capita expenditure on health, and the total direct medical cost was 8.9% times the total expenditure on health.Diabetes imposes a substantial economic burden on patients, health systems, and the whole economy. Besides, since the cost of the disease in patients treated with insulin and those with diabetes complications is significantly higher, the reinforcement of self‐care measures and focusing on modifying lifestyle  in patients with T2DM can significantly reduce the costs of the disease. According to the IDF report in 2021, diabetes caused at least USD 966 billion in health expenditure ,Iran, as one of the countries of the Middle East and North Africa (MENA) region, has been affected by the diabetes epidemic.The global prevalence of diabetes and the economic and social burden of diabetes is expected to rise due to changing demographics and lifestyle, especially in middle‐ and low‐income countries.Given the increasing prevalence of T2DM in Iran and the effects of type 2 diabetes costs on patients, third‐party payer organizations, and the healthcare system, it is crucial for health policymakers to know how much costs are imposed on the health system because of diabetes. Knowing the economic burden of diabetes can help policymakers make better decisions in prioritizing cost‐effective measures. Thus, the principal objective of this study was to estimate the economic burden of type 2 diabetes in Iran, in 2018.22.1This prevalence‐based cost of illness study was conducted from the societal perspective using bottom‐up approach costing.2.2The statistical population included all patients with type 2 diabetes in East Azerbaijan province referring to Imam Reza and Sina's educational and therapeutic centers and Asad Abadi clinic in Tabriz, Iran, in 2018. The population of East Azerbaijan province is estimated at 4 million, and the rate of diabetes incidence is 10%. So, the population of patients with diabetes is estimated at 40,000, and based on the Morgan table, 375 patients with T2DM were selected. Hence, based on the calculated population size and incidence rate, the estimated population is estimated at 40,000. Due to the inaccessibility to all of the statistical population in our study, the convenience sampling method was used consecutively.n = 7), interview with researchers who had conducted at least one cost of illness study (n = 3), interview with professors in Health Economics (n = 2), interview with T2DM patients (n = 15), and the review of records of inpatients and outpatients of T2DM patients. The checklist consists of demographic variables , duration of the disease, treatment type, and questions about diabetes‐related costs. The questionnaires were completed during a 20‐min face‐to‐face interview by one of the research team.A researcher‐made checklist was used for data collection. The checklist design process was as follows: interview with endocrinologists , lost productivity due to missed workdays, and premature death due to diabetes. First, to estimate the cost of missed workdays per patient, we calculated the annual average number of missed workdays of the patients and their families due to diabetes and then multiplied it by the GDP per capita; in this way, we estimated the cost of missed workdays per patient. To calculate the cost of premature death due to disease, the average duration of premature death was multiplied by GDP per capita (Formulas (1) and (2)):Formula (1): Lost productivity due to missed workdays of patients and their families = (the number of missed workdays of patients and their families/the number of workdays of the year) × GDP per capita .Formula (2): (retirement age in Iran − the median age of death) × GDP per capita .https://data.worldbank.org/indicator/NY.GDP.PCAP.PP.CD). A detailed description of unit costs is presented in Table All costs in this study were adjusted to current international dollars based on the World Bank's PPP conversion factors  were used for cost estimates. Spearman correlation coefficient (CC) was used to examine the association between cost items and age, duration of the disease, the number of hospitalization, and Mann–Whitney for the association between mean costs and gender, informal payment, basic insurance coverage, supplemental insurance coverage, and habitation status. Kruskal–Wallis for the association between mean costs and age, Insurance type, treatment type, and cause of hospitalization. The tests were carried out at a 5% significance level, and a 2.6This study was approved by the Ethics Committee of Tabriz University of Medical Sciences (Reference No: IR.TBZMED.REC.1395.672). All participants were informed about the purpose of the study and assured of confidentiality and anonymity. Moreover, written informed consent was taken from each of them before the total procedure. All methods were carried out in accordance with the relevant guidelines and regulations.3p = 0.01, CC = 0.13), disease duration , hospitalization number , and total medical direct costs. With an increase in age, disease duration, and hospitalization number, the total medical direct costs would increase  were male. The mean (SD) age of patients, disease duration, and the number of hospitalization were 60.73 ± 14.15, 11.32 ± 8/03 year, and 1.75 ± 0.87, respectively. 98.1% of patients were covered by basic insurance, and only 13.1% of them were covered by supplemental insurance (Table According to the results of Table Table The lost productivity due to missed workdays of patients and their families was estimated via formula (1):Formula (1): Lost productivity due to missed workdays of patients and their families = (the number of missed workdays of patients and their families/the number of workdays of the year) × GDP per capita.Lost productivity due to missed workdays of patients and their families = 0.3711 + 0.1341 = 0.5052 × 14,535.9 = 7343.53 PPP, Current International $.Lost productivity due to missed workdays of patients and their families (patients residing in urban areas) = 0.3292 + 0.1327 = 0.4619 × 14,535.9 = 6714.13 PPP, Current International $.Lost productivity due to missed workdays of patients and their families  = 0.4311 + 0.1376 = 0.5687 × 14,535.9 = 8266.56 PPP, Current International $.Estimating total costsTotal patients with T2DM in Iran = 8,428,105Hospitalization rate = 30.2%1.Total medical direct costs of diabetes in Iran =  + ((inpatient rate) × the number of patients with T2DM in Iran × mean total inpatient costs) =  + (0.302) × 8,428,105 × 3517.97) = 82,233,104,766.05 + 8,954,245,805.414 = 91,187,350,571.19 .2.Total nonmedical direct costs of diabetes in Iran =  +  =  +  = 2,175,521,122.21 + 1,695,034,350.47 = 3,870,555,472.68  95,057,906,043.87.3.Total indirect costs of diabetes in Iran = total mortality costs + total morbidity costs = 1,135,953,666.66 + 56,250,002,769.77 = 57,385,956,436.43 .3.1Total mortality costs = total number of death × mean lost years of life (premature years) × GDP per capita PPP, Current International $ = 12,660 × 10 × 14,535.9 = 1,840,244,940Outpatient rate = 0.698i) NPV = FV/ × mean lost productivity costs of inpatients) + (the number of patients with T2DM in Iran × (0.698) × mean lost productivity costs of outpatients) =  +  = 17,703,901,384.16 + 38,546,101,385.61 = 56,250,002,769.77.4.Total economic burden of diabetes in Iran = total medical direct costs + total nonmedical direct costs + total indirect costs = 91,187,350,571.19 + 3,870,555,472.68 + 57,385,956,436.43 = 152,443,862,480.3 .Cost components as a percentage of total costsTotal medical direct costs as percentage of total costs = total medical direct costs/total costs = 91,187,350,571.19/152,443,862,480.3 = 0.59.Total nonmedical direct costs as percentage of total costs = Total nonmedical direct costs/total costs = 3,870,555,472.68/152,443,862,480.3 = 0.02.Total indirect costs as percentage of total costs = total indirect costs/total costs = 57,385,956,436.43/152,443,862,480.3 = 0.37.Medical direct costs as a percentage of health expenditurePPP, Current International $) (https://data.worldbank.org/indicator/SH.XPD.CHEX.PP.CD).Total per capita expenditure on health in Iran (2017) = 1748.40 .Total Health Expenditure in Iran (2017) = 1,015,528,323,000  of Iran (2017) = 1,172,665,500,000.https://data.worldbank.org/indicator/NY.GDP.MKTP.PP.CDGDP per capita, PPP   = 14,535.9 .https://data.worldbank.org/indicator/NY.GDP.PCAP.PP.CD?end=2018&locations=MW.&start=1960Mean direct Medical costs of inpatients/Total per capita expenditure on health = 13,274.98/1748.40 = 7.59.Mean direct Medical costs of outpatients/Total per capita expenditure on health = 9757.01/1748.40 = 5.58.Mean medical direct costs  = outpatient costs + ((inpatient rate) × inpatient costs = 9757.01 + ((0.302) × 3517.97 = 10,819.43.Mean direct medical costs /Total per capita expenditure on health = 10,819.43/1748.40 = 6.18.Total medical direct costs/total health expenditure = 91,187,350,571.19/1,015,528,323,000 = 0.089 = 8.9%.Total costs /total GDP  =  152,443,862,480.3/1,172,665,500,000 = 7.69%.4In this study, a total of 375 patients with type 2 diabetes were studied. The total economic burden was 18,087.56 PPP, Current International $ ($US 6877.39) per patient, representing 1.24 times GDP per capita  (2017) = 14,535.9 . Our study showed that direct medical costs were the major component of the total economic burden of patients with T2DM. The mean total direct medical cost was estimated at 10,819.43  per patient, representing 74.43% of GDP per capita. Diabetes is a chronic disease that requires consistent healthcare, and people with T2DM may spend years with the disease and its complications. Therefore, in contrast to acute and fatal diseases, most of the economic burden of this disease is related to direct medical costs.Although the results of some previous studies are almost similar to the results of the present study in some cases, the estimated economic burden of the disease differs according to the design and methods of the studies. The reason for these differences can be attributed to methodological differences and a difference in the quantity and quality of services provided to patients with T2DM in some areas, differences in currencies, and the timing of studies. So, it is difficult to compare results across studies because they were assessed with different currencies.Nguyen Tu et al., in Vietnam, conducted a cross‐sectional study and analyzed 392 patients with T2DM. The authors showed that the annual cost was $24,610 per patient, equivalent to 12% of GDP per capita. The highest amount of cost was direct medical cost at $12,730, and pharmaceuticals constituted 27.5% and 53.2% of the total cost and total medical direct cost, respectively.Of the direct medical costs, the proportion of outpatient costs to the total economic burden of diabetes was 2.77 times higher than inpatient costs (9757.01 vs. 3517.97). Outpatient costs occur most frequently in any of the patients with T2DM, while hospitalization costs occur only in some patients with T2DM. Hence, much of the direct medical costs were due to the costs of outpatient.In this study, the mean outpatient cost was estimated at 9757.01 , accounting for 53.94% of the mean total cost. Insulin and wound healing costs were the most costly contributor to outpatient costs, with 3087.35   and 1794.46  , respectively. But, in a study by Barcelo et al., in Latin America and the Caribbean, insulin was the second‐highest estimated cost after treatment of complications (between $6 and $11 billion).In this study, different variables such as the history of informal payment, the incidence of complications, insurance coverage, type of insurance, type of treatment received, and region of residence affected the direct costs of diabetes. The direct medical costs among those undergoing insulin treatment and oral medications were, respectively, 3.64 and 2.85 times higher than those who did not take any medicine and just relied on modifying their lifestyle  to control their disease. Hence, it appears that adequate focus on self‐care measures and modifying lifestyle in T2DM patients can lead to a significant decrease in disease costs, in addition to improving clinical outcomes.Direct medical costs in patients with a foot ulcer, neuropathy, nephropathy, and retinopathy were 2.13, 1.25, 1.06, and 1.04 times higher, respectively than in other patients. Patients with foot ulcers and neuropathy  incurred the highest costs. It seems that the incidence of diabetes complications increases the disease costs by increasing the need for more complicated treatment care and admission to the hospital, in addition to a reduction of quality of life and increased mortality and disability in T2DM patients. So, to manage the costs of patients with type 2 diabetes, health policymakers should focus more on preventive and self‐care measures and improvement of service quality in the first and second levels of the referral system. Also, integrated personalized diabetes management can be a solution to reduce complications and improve treatment outcomes.According to the results, 18.1% of patients had informal payment, and direct medical costs among patients who incurred informal payment were 1.46 times higher than those without informal payment. These payments are made by patients to better access healthcare services. Informal payments are most of the time regarded as a sign, indicating a wide range of underlying problems in the healthcare system. It is representing of inefficiencies of the health system in providing equitable access to health services and imposes an additional economic burden on patients. Hence, appropriate control measures should be taken to prevent receiving informal payments by physicians.Direct medical costs among patients under health insurance coverage were 7.47 times as much as those who were not under the coverage of health insurance. It seems that those who were not under the coverage of health insurance had poor financial access to healthcare services and were more likely to be deprived of receiving health services. Although equitable access to health services is considered a fundamental human right regardless of ability to pay in Iran, 1.9% of patients with T2DM are not covered by health insurance, and these patients are deprived of receiving essential services due to their inability to pay for medical costs. Therefore, it is necessary to identify these patients and take the necessary supportive measures to improve their access to essential healthcare services and financial protection of these patients against the catastrophic costs incurred by the disease by the government, health insurance organizations, charities, and other related institutions.The cost of the disease was more likely to be higher among those under Iranian health insurance coverage than those under social security insurance. This difference may be due to two reasons: first, the Social Security Administration may have been more successful in managing the costs of diabetes, and second, Iranian health insurance may have provided more services to these patients. However, without sufficient information about the quantity and quality of services provided to patients covered by these two insurance organizations, as well as information about the quality of life‐related to healthcare and clinical outcomes of these two groups, a definite judgment is not possible, and needs further investigation.Direct medical costs among patients residing in rural areas were 23% higher when compared with those residing in urban areas. Most patients living in rural areas who were referred to urban healthcare centers had a more complicated situation, and they need more advanced and expensive treatment care, which may imply that patients residing in rural areas were more likely to delay or not receive needed medical care due to the long distance from medical centers and limited access to the tertiary level of health services and have more complex and expensive services need.The mean direct nonmedical cost in our study was 459.24  per patient . Commuting costs accounted for the highest direct nonmedical costs. In 2016, in Vietnam, the estimated nonmedical direct cost was $34.40.The mean indirect cost in this study was 6808.88  per patient, accounting for 37.64% of the mean total cost . In Vietnam, in 2016, the indirect cost of T2DM accounted for 34.3% of the total cost.Direct nonmedical costs especially, commuting costs, were higher for those living in rural areas. Also, the mean cost of lost productivity was higher for patients living in rural areas  than those living in urban areas . It has meant that they spent more time and had a higher number of missed workdays. It seems that despite all the efforts made by the Ministry of Health of Iran to achieve equitable access to health services, financial, physical, and temporal access to health services is still largely unfair. This disparity is remarkable; hence, policymakers should adopt supportive measures for those residing in rural areas or away from advanced treatment centers.In our study, mean direct medical costs  were 6.18 times the total per capita expenditure on health. This suggests that the health expenditure of each patient with T2DM is more than six times as high as the average health expenditure of other people. The total medical direct costs of diabetes were equivalent to 8.9% of total health expenditure. This has meant that diabetes requires prevention measures and some interventions should be taken into consideration in the health system program.The contribution of basic insurance, patient, subsidy, supplementary insurance, and hospital discount in the reimbursement of hospitalization costs was 82.54%, 7.25%, 6.95%, 2%, and 1.22%, respectively. Moreover, the patient contribution to the total outpatient cost was estimated at 44.44%. The patient contribution of total medical direct costs was 0.36. Except for hospitalization costs, health insurance does not provide adequate support for people with T2DM, and these patients incurred a significant share of outpatient costs. Although these people pay these costs gradually over time, the share of people with T2DM in reimbursing outpatient costs puts significant economic pressure on them, especially in low‐income and vulnerable groups. Therefore, it is necessary for the government to revise the services coverage and financial coverage for these patients and provide more support for them. Besides, strengthening the role of supplementary insurance can also be considered as an adjuvant solution. Currently, private insurance in Iran plays a minor role in reimbursing costs. A systematic review showed that health insurance coverage could have protective effects against out‐of‐pocket expenditures, but mainly for those with higher incomes, while the poor often lacked coverage.5This study has some limitations. The cost of decreased productivity at work due to disability and its complications and loss of income due to job changes caused by diabetes and its complications have not been estimated.5.1Diabetes imposes a substantial economic burden on patients, health systems, and the whole economy. Besides, since the cost of the disease in patients treated with insulin and those with T2DM complications is significantly higher, the reinforcement of self‐care measures and focusing on modifying lifestyle  in patients with T2DM can significantly reduce the costs of the disease. Since the cost of the disease for those living in rural areas and those who were forced to refer to the tertiary level treatment centers in large cities was higher than for those living in urban areas, more financial and practical support should be provided for these people by the government. Also, due to the high rate of patients’ copayment, especially in the reimbursement of outpatient costs, it is necessary to strengthen the role of health insurance in reimbursing the cost of illness and reducing the economic pressure on patients. More accurate monitoring of service providers to prevent informal payments can reduce direct medical costs, especially patient payments.6Diabetes imposes a substantial economic burden on patients, health systems, and the whole economy. Besides, since the cost of the disease in patients treated with insulin and those with diabetes complications is significantly higher, the reinforcement of self‐care measures and focusing on modifying lifestyle  in patients with T2DM can significantly reduce the costs of the disease.Habib Jalilian: conceptualization; data curation; formal analysis; methodology; project administration; software; validation; visualization; writing–original draft; writing–review & editing. Somayeh Heydari: writing–original draft; writing–review & editing. Ali Imani: supervision. Mozhgan Salimi: data curation. Nazanin Mir: data curation. Farzad Najafipour: project administration; supervision. All authors have read and approved the final version of the manuscript. Farzad Najafipour had full access to all of the data in this study and take complete responsibility for the integrity of the data and the accuracy of the data analysis.The authors declare no conflicts of interest.The lead author Farzad Najafipour affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned  have been explained.


For patients with high‐risk stage II or stage III colorectal cancer (CRC), adjuvant chemotherapy (AC) improves survival, yet use varies substantially across medical oncology settings.Utilization of guideline concordant CRC AC was assessed at a Veterans Health Administration (VHA) facility to determine quality improvement initiatives.p < .01) in the NCCN eligible cohort (N = 180), and  in an analysis of patients who completed appointments and had AC recommended by providers (N = 146).The study was a retrospective review of CRC surgeries from January 1, 2000 to December 31, 2015 at a South Regional VHA. Inclusion criteria consisted of pathologic high‐risk stage II or stage III CRC, with exclusion for age ≥80, age ≥75 hospitalized with major co‐morbidity in the prior year, and death or discharge to hospice within 30 days of the index surgery. The primary predictor was year‐group; partitioned 2000–2005, 2006–2010, 2011–2015 to account for changes in NCCN high risk stage II definitions. Primary outcome was AC receipt. Secondary outcome was reason for chemotherapy omission. Among 180 eligible surgeries , patients were mostly male (96%), white (79%) and with median age 64 years. Overall, 117 (65%) received AC. Compared to 2000–2005, patients undergoing surgery between 2011 and 2015 were less likely to receive AC , due to more patients declining AC (27% vs. 6%, Survival benefitting AC decreased over time among a nonelderly Veteran cohort eligible for AC. Evaluating care decisions and trends within other VHA facilities and outside the VHA are warranted. For example, from 2003 to 2006, 77.5% of Veterans treated at a Veterans Health Administration (VHA) facility were referred to medical oncology and received timely AC.22.1The study was a retrospective secondary data review cohort of Veterans undergoing colorectal resections at a south regional VHA between January 1, 2000 and December 31, 2015, identified using two sources: (1) Veterans Information Systems and Technology Architecture (VistA) and (2) the VHA gastroenterology surveillance initiative database. The study site was a 238‐bed tertiary care Veterans Affairs hospital. In FY2018 the site had 9313 hospital admissions and cared for 103 207 unique Veterans. Most surgical oncology care is performed at the primary campus, which hosts a weekly interdisciplinary Tumor Board. Veterans receive CRC care from multiple services including primary care, oncology, surgery, gastroenterology, radiology, and pathology. From November 2015 through November 2019, the outpatient medical oncology clinic consulted on ~1600 newly diagnosed oncology patients, averaging 4400 infusion visits per year. During the study time‐period, total neoadjuvant therapy for rectal cancer had not been adopted into practice.The VHA Tennessee Valley Healthcare System Institutional Review Board (IRB) and Research and Development Committee approved this study, with a waiver for subject consent under secondary data review.2.2Patients were identified using Current Procedural Terminology (CPT) codes for colorectal surgeries: 44140–44 147, 44 150–44 153, 44 155–44 158, 44 160, 44 204–44 208, 44 210–44 212, 45 110–45 119, 45 126, 45 160–45 170, 45 395–45 397Due to equivocal evidence of benefit in elderly patients and older patients with significant co‐morbidities,2.3The primary predictor was year of the index surgery, partitioned  to account for changes in NCCN guidelines.2.4The primary outcome was receipt of any chemotherapy (yes/no). Secondary outcomes included the type of AC regimen received . Exploratory outcomes regarding patient and provider reasons for omission of AC were abstracted from chart‐level information contained within documented notes.2.5Study data were collected and managed using REDCap electronic data capture tools hosted through the VHA.Two researchers  abstracted data on patient metrics, process measures, and patient outcomes from the VA Computerized Patient Record System (CPRS), VistA, and Joint Legacy Viewer (J.L.V.). To ensure data accuracy, 8% of charts were independently abstracted by both reviewers, and two randomly chosen variables  were assessed for inter‐reviewer agreement, yielding very‐good agreement (kappa 0.79 and 0.92).2.6Patient characteristics at time of index surgery included age, sex, race , marital status, zip code, co‐morbidities , and >15 pounds of weight loss prior to the index surgery. Cardiovascular disease was defined as a history of systolic heart failure, coronary artery disease, myocardial infarction, stroke, transient ischemic event, peripheral artery disease, or peripheral vascular disease, or documentation of ejection fraction <50%. Obstructive pulmonary disease was defined as history of chronic obstructive pulmonary disease with or without supplemental oxygen use or a pulmonary function test with FEV1/FVC ratio of <0.80. Type 2 diabetes was defined as history of type 2 diabetes (regardless of any macro or microvascular complications) or an active hypoglycemic medication on the patient's medication list. Chronic kidney disease (CKD) was defined as history of CKD 3–5 in history or laboratory evidence of >3 months of glomerular filtration rate consistently <60 ml/min. From patient zip codes, median annual incomes were calculated using the American Community Survey Census Data 2007–20112.7STATA IC v.16.0 was used for all statistical analyses. Descriptive statistics were calculated on the baseline characteristics of patients by year‐group . The primary analysis considered the proportion of patients receiving AC (numerator) among all those eligible for AC by NCCN guidelines (denominator).t‐tests evaluated whether patient and provider reasons for omitting AC changed over time. The first analysis considered all patients eligible for AC by NCCN guidelines. The second analysis restricted the denominator to only those patients who received an oncology evaluation and had a provider recommendation for AC.Multivariable logistic regression tested associations between year‐group and receipt of AC. Covariates were selected based on prior observed associations with receipt of AC or having suspected or known temporal changes during 2000–2015. Final multivariable model covariates were age, race, tumor stage, tumor type, travel distance, cardiovascular disease, length of stay, and 30‐day readmission. A subgroup analysis by cancer type  was performed to evaluate for practice differences in treatment. As these subgroup models were over‐fitted for their sample size, they should be viewed as exploratory. Two post hoc exploratory analyses using chi square and student 33.1N = 121 colon and N = 59 rectal cancers , 2006–2010 (N = 64), and 2011–2015 (N = 56).Of 1107 unique colorectal resections, 623 were excluded for no evidence of cancer, 212 for stage I or low‐risk stage II disease, 47 for metastatic disease, and 45 for age, active co‐morbidity, death, or hospice, yielding a final cohort of Patients were predominantly male (96%) and white (79%) with a median age of 64 years  Table . Approxi3.2Median LOS for the index surgery hospitalization was 7 days (IQR 5–9 days) with no difference observed across year‐groups. Use of pre‐operative tumor board discussions improved over time; 48% between 2000 and 2005, 73% between 2006 and 2010, and 78% between 2011 and 2015. Improvements were also seen in the metric of ≥12 lymph nodes evaluated at time of surgery; 50% versus 69% versus 86% in the 3‐year groups.3.3Of the 180 patients eligible to receive AC, 117 (65%) received chemotherapy Table . Receipt3.4N = 13), oncology providers recommending against AC (N = 18), and patients declining AC (N = 24), with (N = 5) patients not receiving AC for unknown reasons, and (N = 1) having loss to follow up . No patterns of change were observed in the proportion of providers not recommending AC.The most common reasons for not receiving AC were lack of medical oncology referrals , 4 of 60 patients (6%) declined AC in 2000–2005, 5 of 60 (8%) declined in 2006–2010, and 15 of 56 (27%) declined during 2011–2015 (p = .002). Evaluating patient refusal using the denominator restricted to those patients who received an oncology visit and had a provider recommendation for AC (N = 146), demonstrated that 4 of 48 patients (8%) declined AC between 2000 and 2005, 5 of 51 (10%) declined between 2006 and 2010, and 15 of 47 (32%) declined AC between 2011 and 2015 (p < .001).Patient AC refusal increased over time. Evaluating for the full cohort eligible for AC  were statistically associated with patients declining AC.Regarding regimen choice, in the 2000–2005 year‐group, 5FU/LV was the predominant regimen (58%) followed by oxaliplatin doublets (13%) and capecitabine (0%). In 2006–2010, oxaliplatin doublets (45%), and capecitabine (1%) increased, while 5FU/LV decreased (23%). By 2011–2015, oxaliplatin doublets plateaued (37.5%) with minimal further increase in capecitabine (7%), but a continued decrease in 5FU/LV (9%)  and 2006–2010 (69%) are similar to those of the Surveillance, Epidemiology, and End Results (SEER) program estimates (72% and 66%)Our findings add to several studies on AC quality for CRC. Using a SEER‐based US cohort, Murphy et al observed an initial increase in CRC AC utilization from 1995 to 2005 followed by a possible decrease in 2010 compared to 2005.Walter et al observed decreased AC utilization among German patients treated in 2009–2012 compared to 2005–2008, driven primarily by patient refusal and age, especially with respect to oxaliplatin.Our study has limitations that should be noted. First, our study is retrospective and observational. As a cohort of predominantly white men, our VHA sample was underpowered to test for associations with sex and race and has limits on generalizability. The higher rates of co‐morbidities and rurality in our population likely biased our findings toward lower overall receipt of AC, however, these factors remained stable across the study period, and are unlikely to explain the temporal reduction in AC.It is possible that unmeasured confounders impacted our findings. For example, our study did not account for co‐insurance. Prior to the 2008 recession, approximately 77% of Veterans were co‐insured with non‐VHA insurance, however, the number of Veterans enrolling or utilizing VA services increased during 2009–2013 (particularly in non‐Medicaid expanding states).It is possible that 5FU and LV drug shortages contributed to the reduction in AC. 5FU and LV are two of the more common chemotherapies with shortages, and oncology providers often respond to drug shortages by substituting, delaying, or even omitting therapy.Our findings raise the possibility that declining rates of CRC AC may not simply be a phenomenon of frail and elderly patients, but a more general trend occurring across ages driven primarily by factors influencing patient attitudes toward AC. While these specific factors remain unknown, they do not appear to be related to worsening Veteran health status nor do they impact other CRC post‐operative care such as surgical follow up or colonoscopies.In summary, our study provides several novel insights on how quality care patterns can change over time and how reliance on national‐level data can lead to underestimation of important local barriers and facilitators that are critical to improving high quality cancer care. Future work should investigate underlying reasons why patients chose not to receive AC despite meeting NCCN eligibility and whether our observed reduction in AC is specific to our institution and/or CRC or reflects a broader trend of patients declining potentially curative chemotherapy.Gretchen Edwards: Conceptualization; data curation; formal analysis; investigation; methodology; writing‐review & editing. Lauren Samuels: Conceptualization; formal analysis; methodology; software; visualization; writing‐review & editing. Cathy Eng: Formal analysis; supervision; validation; writing‐review & editing. Christianne Roumie: Conceptualization; methodology; project administration; resources; supervision; writing‐review & editing.The authors have no conflicts of interest to report.The VHA Institutional Review Board (IRB) and Research and Development Committee approved this study, with a waiver for subject consent under secondary data review.


Curcumin is well known for its anticancer properties. Its cytotoxic activity has been documented in several cancer cell lines, including breast cancer. The pleiotropic activity of curcumin as an antioxidant, an antiangiogenic, antiproliferative, and pro‐apoptotic, is due to its diverse targets, such as signaling pathways, protein/enzyme, or noncoding gene.This study aimed to identify key miRNAs and mRNAs induced by curcumin in breast cancer cells MCF7, T47D (hormone positive), versus MDA‐MB231 (hormone negative) using comparative analysis of global gene expression profiles.RNA was isolated and subjected to mRNA and miRNA library sequencing to study the global gene expression profile of curcumin‐treated breast cancer cells. The differential expression of gene and miRNA was performed using the DESeq R package. The enriched pathways were studied using cluster profileR, and integrated miRNA–mRNA analysis was carried out using miRtarvis and miRmapper tools.Curcumin treatment led to upregulation of 59% TSGs in MCF7, 21% in MDA‐MB‐231 cells, and 36% TSGs in T47D, and downregulation of 57% oncogenes in MCF7, 76% in MDA‐MB‐231, and 91% in T47D. Similarly, curcumin treatment led to upregulation of 32% TSmiRs in MCF7, 37.5% in MDA‐MB231, and 62.5% in T47D, and downregulation of 77% oncomiRs in MCF7, 50% in MDA‐MB231 and 28.6% in T47D. Integrated analysis of miRNA–mRNA led to the identification of a common NFKB pathway altered by curcumin in all three cell lines. Analysis of uniquely enriched pathway revealed non‐integrin membrane–ECM interactions and laminin interactions in MCF7; extracellular matrix organization and degradation in MDA‐MB‐231 and cell cycle arrest and G2/M transition in T47D.Curcumin regulates miRNA and mRNA in a cell type‐specific manner. The integrative analysis led to the detection of miRNAs and mRNAs pairs, which can be used as biomarkers associated with carcinogenesis, diagnostic, and treatment response in breast cancer. MiRNAs are ~22 nucleotides noncoding RNAs that regulate gene expression by RNA interference post‐transcriptionally.Integrated RNA‐seq and miRNA‐seq analysis provide insight into drug‐induced global alterations in the transcriptome. It also helps in deciphering the molecular mechanism of the disease via gene expression and gene regulation.RNA‐seq has been employed to study the changes in tumorigenesis in the presence of different drugs such as curcumin,22.12. 100 mM curcumin stocks were prepared in DMSO, and the treatment was given so that all treatments had equal concentrations of dimethyl sulfoxide (DMSO) between 0.1% and 0.2%. The molecular signatures of each cell line are tabulated in Table MDA‐MB‐231, MCF7, and T47D cells were purchased from the National Centre of Cell Culture (NCCS), Pune, Maharashtra, India. All the cell lines were authenticated at NCCS by short tandem repeat (STR) analysis. Dulbecco's Modified Eagles Medium  was used for MDA‐MB‐231 cells, Eagle's Minimum Essential Medium  was used for MCF7 cells, and Roswell Park Memorial Institute‐1640  media was used for T47D cells. All three media were supplemented with heat‐inactivated 10% fetal bovine serum (Gibco), 100 IU mg/ml penicillin/streptomycin (Gibco) at 37°C in a humidified atmosphere containing 5% CO2.22.2.15 cells were seeded in each well of a 6‐well plate. After 24 h, MCF7, T47D, and MDA‐MB‐231 cells were treated with 10 μM curcumin. After 48 h of treatment, cells were trypsinized, and cells from three wells having the same treatment were pooled together. After two PBS washes, RNA extraction was done using Trizol Reagent (Ambion) following the manufacturer's recommendations. RNA concentration and purity were checked using Qubit , and its integrity was examined by capillary electrophoresis  to ensure RNA integrity number >9, for a good RNA library preparation. Paired‐end RNA‐seq libraries were prepared using Illumina TruSeq RNA Library Prep Kit v2.Drug treatment and RNA isolation: 0.75 × 10mRNA library preparation: In brief, from the total RNA, mRNAs were separated using oligo‐dT beads and fragmented to 200–250 bp. After cDNA was synthesized, the ends were repaired for blunt ends, and the 3' ends were adenylated. To the adenylated sites, adapters were linked, and subsequently, PCR amplification of the library was done. After constructing the libraries, their concentrations and insert sizes were detected using Qubit and Agilent Tapestation, respectively. High throughput sequencing was performed using Illumina HiSeq2500 to obtain 100‐bp paired‐end reads.2.2.2RNA isolation was done as mentioned above, and RNA sample was given for library preparation. miRNA‐library preparation was outsourced to SciGenom Labs, India. In brief, after checking the quality, RIN of RNA, 3′ and 5′ adapters were ligated to the short mature miRNA sequences. After adapter ligation, reverse transcription was done to obtain single‐stranded cDNAs. The cDNA was then PCR amplified, and the amplicons were run on 8% native PAGE. The ~150 bp library was gel purified and the quality of libraries was checked using Tapestation 2200, Agilent. The libraries were pooled and sequenced using Hiseq 2500, Illumina.2.32.3.1Data analysis was carried out, beginning with filtering raw reads output from Illumina Hiseq2500 platform. The sequencing depth for each sample was >40 million reads. The quality of the reads was checked using the FastQC tool.2.3.2Q) >30. Trimming was done using trim_galoreData analysis was carried out in the following steps: filtering was done on raw reads output from Illumina Hiseq2500 platform. The sequencing depth for each sample was >10 million reads. The quality of the reads was checked using the FastQC tool,2.4p‐value.For pathway analysis of the differentially expressed genes, we have used clusterprofileR.2.5miRtarvis+2.6cDNA was synthesized from 1–2 μg RNA using cDNA synthesis kit from Takara Bio according to the manufacturer's instructions. The PCR reaction was performed using StepOnePlus™ real‐time PCR system from Applied Biosystems using iTaq™ Universal SYBR® Green supermix from Bio‐Rad with PCR primers for genes  and miRNAs . The sequences for the primers are provided in 2.75 MCF7 cells/ml were seeded and treated with curcumin  for 48 h, and whole cell lysate was prepared as described in.To perform this assay, 0.8 × 1033.1The dose–response study of curcumin in breast cancer cells  was performed,The PCA plots of differentially expressed genes among three breast cancer cell lines MCF7, MDA‐MB231, and T47D, treated with curcumin . A transcriptome summary shows the percentage of up and down‐regulated genes among the total DE genes   PCR. The qRT‐PCR results showed a similar differential expression pattern, validated the expression of genes, that is upregulation/ downregulation, as obtained from the RNA‐Seq results. However, the logFurther, DE genes were subjected to pathway analysis in all three cell lines to identify altered pathways upon curcumin treatment. Common pathways altered in MCF7, MDA‐MB‐231 by curcumin were ER‐Phagosome pathway, Antigen processing‐Cross presentation Interferon gamma signaling, Endosomal/Vacuolar pathway, Antigen Presentation: Folding, assembly and peptide loading of class I MHC pathways. Extracellular matrix (ECM) related interactions with non‐integrin membrane and laminin pathways were regulated by curcumin uniquely in MCF7Figure  cells, wCancer is driven by an imbalance of oncogene and tumor suppressor gene (TSG) expression. Thus, we analyzed the significant DE genes for TSGs and oncogenes. For TSGs, we used the TSG database3.2The chromosomal distribution of the altered transcripts (significant DE genes) across all cell lines was plotted using Circos.3.3The MCF7, MDA‐MB‐231, and T47D cells were treated with curcumin as mentioned above, and miRNA‐seq was performed. To prepare the miRNA library, three biological replicates were pooled. Sequencing was performed with 2 sets of biological replicates. More than 20 million reads were obtained for each sample. 82%–97% alignment was achieved with processed reads using the reference genome (hg38) , and dif819 miRNAs were commonly regulated between all three breast cancer cells Figure . ~4% of 3.4p‐value <0.05 were given as input for miRTarVisThe integrated analysis of mRNA–miRNA of breast cancer cell lines namely, MCF7, MDA‐MB‐231, T47D upon curcumin treatment was performed and all the DE miRNAs with log fold change >0.5 and mRNA DE genes with log fold change >1.5, 3.5We built a miRNA–mRNA network for pathways regulated by curcumin in each cell line. Genes enriched in non‐integrin membrane‐ECM interactions and laminin interactions were selected for MCF7; genes enriched in Extracellular matrix organization and degradation were selected for MDA‐MB‐231, and genes enriched in cell cycle arrest and G2/M transition were selected for T47D. Using miRtarvis+, we built an interaction network of miRNA–mRNA Figure . We got The genes enriched in each network were subjected to GEPIA analysis.Network analysis showed miR‐363, curcumin commonly regulated miR‐363‐3p in MCF7, MDA‐MB‐231, and T47D. miR‐363 targeted BMP7 in MDA‐MB‐231 cells and regulated the ECM pathway. miR‐363 targeted integrins like ITGAV, ITGA6 in MCF7, and AURKA in T47D cells to regulate the cell cycle G2/M phase. Dolati et al. have reported regulation of miR‐363 by nano curcumin treatment in multiple sclerosis,4In this study, the whole transcriptome (mRNA and miRNA) post curcumin treatment in mesenchymal MDA‐MB‐231 (TNBC), epithelial hormone‐responsive MCF7 , and T47D  breast cancer cells have been explored. To evaluate the transcriptomic changes, IC50 values of curcumin on all three cell lines were obtained.Identification of DE genes and DE miRNAs can be good predictors of response to chemotherapy, biomarker selection, early diagnosis of disease subtypes, and the development of targeted therapeutics.The study showed that 22 DE genes and 819 miRNAs were common in all cell lines after curcumin treatment. More than 48% of genes were uniquely regulated after curcumin treatment. Similarly, RNA‐seq post Shikonin treatment showed more than ~60% DE genes uniquely regulated in MCF7, MDA‐MB‐231, and SK‐BR‐3.Curcumin regulated Antigen processing and presentation, Interferon alpha/beta/gamma signaling in MCF7, MDA‐MB‐231, whereas pathways related to cell cycle regulation in T47D. In a previous study, Ruihua Li et al. reported that curcumin induced ferroptosis in MCF7, MDA‐MB‐231 cells by upregulating HO‐1 and downregulating GPX4.The overall tilt in the balance between oncogenic to tumor suppressor after all treatments were evident. Breast cancer progression involves genetic events like an amplification of oncogenes and loss of function of TSGs, leading to malignancy.miR‐21, miR‐27b, miR‐10b‐3p, and miR‐200a‐5p have been reported oncomiRs in breast cancer whereas miR‐26a/b, miR‐628, miR‐205, and miR‐124 have been reported TS‐miRs in breast cancer.A practical and unique approach was used for mRNA‐miRNA data integration using two tools, miRTarVismiRmapper analysis showed that in MCF7 miR‐590‐3p, MDA‐MB‐231 miR‐106a‐3p, and T47D, let‐7c‐3p regulates the maximum number of DE genes upon curcumin treatment. All these miRs regulate the NFKB pathway indicating the pivotal role of curcumin in the pathway. Thus, we mined for NFKB targets in the DE gene list and their respective regulating DE miRNAs. We obtained 526 DE NFKB targets in MCF7 cells, 89 in MDA‐MB‐231, and 56 in T47D cells. CCL21 was commonly DE in all the cells after curcumin treatment, indicating its role in immunomodulating breast cancer cells. Curcumin is known to interact with various immunomodulators like dendritic cells, macrophages, both B and T lymphocytes, cytokines, and various transcription factors with their downstream signaling pathways.miRNA−mRNA networks were generated for Non‐integrin membrane‐ECM interactions and Laminin interactions in MCF7, Extracellular matrix organization and its degradation pathways in MDA‐MB‐231 and Cell Cycle Arrest and G2/M transition pathways in T47D. We got intricate networks for these pathways in curcumin‐treated cells. In addition, GEPIA analysis of these genes in breast cancer helped classify them as TSG or oncogene and further interpret the effect of curcumin on breast cancer cells. SDC1 regulates ECM fiber organization in breast cancer stromal fibroblasts and thus is involved in cell motility.Downregulation of MMP11, LOXL2, and BMP7 by curcumin in aggressive, mesenchymal MDA‐MB‐231 cells indicates a positive outcome. miR‐125b is known to regulate MMP11 expression in breast cancer.CCNA2 and CCNB1 are prognostic markers for ER +ve breast cancer and are closely associated with hormone therapy resistance.miR‐363‐3p functions as TS miRNA in lung cancer,In conclusion, curcumin regulates miRNA and mRNA in a cell type specific manner. Curcumin altered different pathways in breast cancer cell lines such as cell cycle, migration, invasion, and so forth. The integrative analysis led to the detection of miRNAs and mRNAs pairs, which can be used as biomarkers, associated with carcinogenesis, diagnosis and treatment response in breast cancer.The authors have stated explicitly that there are no conflicts of interest in connection with this article.Conceptualization, S.N. and B.C; Methodology, S.N., S.D., B.C.; Investigation, S.N. and B.C.; Formal Analysis, S.N.; Resources, B.C.; Writing ‐ Original Draft, S.N.; Writing ‐ Review & Editing, S.N. and B.C; Visualization, S.N. and B.C.; Supervision, B.C.; Funding Acquisition, B.C. SN and BC conceived the idea, designed the experiments, analyzed the data and wrote the manuscript. SN performed experiments on breast cancer cells. SN and SD performed the bioinformatics analysis. All authors reviewed the manuscript. All authors read and approved the final manuscript.All authors had full access to the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. The study was given institutional approval.Supplementary Table 1 Inhouse Sequencing mRNA Data Summary for total reads and overall alignment with Hg38 Reference genomeSupplementary Table 2: Inhouse Sequencing miRNA Data Summary for total reads and overall alignment with Hg38 Reference genomeSupplementary Table 3: List of TS miRs and oncomiRs regulated in breast cancerSupplementary Table 4: Sequences for RT‐PCR primers used un the studySupplementary Figure 1: PCA plots for Curcumin treated MCF7,MDA‐MB‐231 and T47D mRNA(A) and miRNA(B) samples.Supplementary Figure 2: Transcriptome Summary of % Upregulation and % downregulation of DE significant mRNAs(A) and miRNAs(B) in Curcumin treatment (.(Abbreviations‐ Curcumin:Cur)p value < 0.05, **: p value < 0.005)Supplementary Figure 3: miRNA‐mRNA network for NFKB targets regulated by Curcumin in MCF7(A), MDA‐MB‐231(B) and T47D(C) cells. (D) Assessment of NFkB in MCF7 breast cancer cells treated with Curcumin: Western blot analysis NFkB protein was done on Curcumin treated MCF7 cell lysates. The experiment was done in duplicates and representative image is shown. Quantification was done (B) and is represented as bar graph of mean +/− SEM. One sample t test and one way ANOVA test was performed and the p value was calculated between control and Curcumin treated groups (*: Click here for additional data file.


Our findings demonstrated that patients with objective responses had favorable survival outcomes and suggested that pembrolizumab could be discontinued safely in this population. This study should drive further efforts to optimize the treatment duration for pembrolizumab responders.Pembrolizumab, an anti‐programmed death 1 monoclonal antibody, has revolutionized the treatment of metastatic urothelial carcinoma. However, the optimal treatment duration for treatment responders has not been established. To address this, we retrospectively assess the treatment outcomes and duration of pembrolizumab for patients whose best response was complete response (CR) or partial response (PR) in a Japanese nationwide cohort of platinum‐refractory metastatic urothelial carcinoma. Of 203 patients whose best response was CR or PR, 83 patients discontinued pembrolizumab before progression. The median pembrolizumab treatment duration was 6.9 months. The 2‐year relapse‐free survival (RFS), treatment‐free survival, and OS rates after discontinuation were 49.0%, 57.4%, and 74.5%, respectively. CR, higher hemoglobin levels, and a better Eastern Cooperative Oncology Group performance status at the time of discontinuation were associated with significantly better RFS. Pembrolizumab was re‐administered to 12 patients. Pembrolizumab re‐challenge resulted in CR, PR, stable disease, and progressive disease in six, three, two, and one patient, respectively. Propensity score‐matched landmark analysis revealed no significant OS difference between patients who continued or discontinued pembrolizumab at 6, 12, and 18 months ( Favorable relapse‐free survival was observed after pembrolizumab discontinuation in patients with urothelial carcinoma, especially in those with complete responses, higher hemoglobin levels, and better ECOG‐PS at the time of discontinuation. Re‐administration upon progression was effective in 90.9% of the patients, and no difference in overall survival was observed between the patients who continued or discontinued treatment after complete or partial responses.  Median overall survival (OS) in mUC ranges 10–12 months, and the 5‐year OS rate is less than 30%.22.1n = 4) and those who discontinued pembrolizumab because of death from other causes (n = 4) were also excluded. Because the survival data were collected for the initial cohort at the end of 2019, we assessed the survival outcomes of all censored patients up to December 2021.The clinical data were collected from the Japan Urological Oncology Group nationwide cohort of patients with platinum‐refractory mUC. The cohort characteristics and initial outcome data were reported elsewhere.The study was approved by the institutional review board at Kyoto University  and review board of all institutions. This study conformed to the Declaration of Helsinki.The primary outcome was relapse‐free survival (RFS), and the secondary outcomes were treatment‐free survival (TFS) and OS. RFS was defined as the time from treatment discontinuation to cancer progression based on RECIST, pembrolizumab retreatment, subsequent chemotherapy, or death from any cause. TFS was defined as the time from pembrolizumab discontinuation to subsequent treatment or death from any cause. The parameters evaluated in this study included age, sex, primary cancer site, histological subtype, prior surgery, and the interval since prior chemotherapy. The Eastern Cooperative Oncology Group performance status (ECOG‐PS), hemoglobin level at pembrolizumab initiation, and site of metastasis were collected at pembrolizumab initiation; after 6, 12, and 18 months of treatment; and at the time of treatment cessation.2.2p < 0.05 indicated statistical significance.All statistical analyses were performed using R . Fisher's exact test and the Mann–Whitney U test were used to analyze categorical and continuous variables, respectively. The Kaplan–Meier method with the log‐rank test was used to assess RFS, TFS, and OS. We adopted landmark analysis to address immortal bias (lead‐time bias).33.1Among the 203 patients whose initial response was PR or CR, 83 (40.9%) patients discontinued pembrolizumab treatment before disease progression Figure . The bas3.2p = 0.02). The 2‐year RFS rates were 63.9% (95% CI = 48.5–84.3) and 37.0% (95% CI = 24.2–56.6) in patients with CR and PR, respectively. The Bellmunt risk factors  at the start of pembrolizumab were not associated with RFS  and hemoglobin levels (p = 0.04), and the number of Bellmunt risk factors (p < 0.01) at pembrolizumab discontinuation were significantly associated with RFS . To further validate this finding, we conducted time‐dependent Cox proportional hazard regression analysis (Table p = 0.07). The hemoglobin level , ECOG‐PS , achievement of complete disease remission  at each time point , and prior chemotherapy within <90 days  were significantly associated with OS.Because our data suggested that the patient condition at treatment discontinuation, not the initial stage, have significant effect on outcomes, we conducted propensity score‐matched landmark analysis at different time points. Patients who discontinued pembrolizumab after <6, 6–12, and 12–18 months were matched with those who continued pembrolizumab at 6, 12, and 18 months, respectively. The baseline covariates were well‐balanced between the two groups Table . The OS is Table . The dis4To the best of our knowledge, this was the first real‐world study of the outcomes of patients with mUC who discontinued pembrolizumab before disease progression. We observed favorable disease control and survival even after discontinuation. CR, higher hemoglobin levels, and better ECOG‐PS at the time of discontinuation were the significant factors for prolonged response. Furthermore, patients responded to pembrolizumab re‐challenge even after disease relapse. Comparison to a matched drug continuation group indicated that treatment can be safely discontinued among responders to pembrolizumab. These observations provided new insights into the appropriate treatment duration of immune checkpoint inhibitor therapy in patients with mUC.In this study, approximately 10% of all patients and 40% of responders (CR or PR) discontinued pembrolizumab before progression. There are no other real‐world reports of pembrolizumab interruption in UC, and it is difficult to assess whether these rates of treatment discontinuation were high. The KEYNOTE‐045 phase III trial of second‐line pembrolizumab in patients with mUC was designed to end treatment after 2 years.We observed favorable survival outcomes after drug discontinuation, especially in patients with CR. Although the follow‐up data were immature, half of all patients and two‐thirds of patients with CR were free from treatment and progression for more than 2 years. A long‐lasting response is a hallmark of immunotherapy,When considering pembrolizumab interruption, it is important that the efficacy of treatment is maintained. In our study, tumors that relapsed after discontinuation remained sensitive to pembrolizumab re‐challenge in most patients. Little is known about immunotherapy re‐challenge in patients with mUC.Our observation has given us an opportunity to reconsider the appropriate treatment duration for immune checkpoint inhibitors. The current standard of care is continuous treatment for all patients until progression or up to 2 years based on FDA approval. However, our data indicated that a shorter duration of treatment might provide substantial benefits in some patients. Moreover, a long duration of treatment might increase the risk of immune‐related adverse events (irAEs) and financial difficulties. Although whether early cessation of therapy can reduce the risk of irAEs is undetermined, serious irAEs after discontinuation of immunotherapy are rare.This study had several limitations. First, because of the retrospective nature of this cohort, patients who discontinued pembrolizumab were carefully selected by physicians. This cohort lacks precise clinical data and information about tumor size at the time of drug cessation. For patients with PR, those who discontinued may have smaller tumors than those who continued. Moreover, some patients received multimodal evaluations such as fluorodeoxyglucose positron emission tomography to confirm CR, whereas other patients had limited access to these examinations. Consequently, the characteristics of patients at pembrolizumab discontinuation were heterogeneous. Since our data are not enough to conclude early cessation in responders is safe or not, the appropriate criteria for discontinuing pembrolizumab should be prospectively examined. Our work will support the planning of trials to determine the criteria for safely stopping treatment. Second, the 2‐year follow‐up period and cohort size were not sufficient to conclude that early discontinuation is safe in patients with mUC. Because patients in this cohort remain under observation, we will provide longer‐term follow‐up results in the future. Third, although landmark analysis with propensity score matching effectively minimized selection and immortal bias, it does not achieve complete randomization. In addition, landmark analysis eliminated some patients who did not reach the different time points. Nevertheless, these cases of early progression were not the focus of this study.In conclusion, this nationwide cohort study revealed that the survival outcomes after pembrolizumab discontinuation before progression were favorable, especially for patients with CR, higher hemoglobin levels, and better ECOG‐PS at the time of discontinuation. Tumors that relapsed after discontinuation remained sensitive to pembrolizumab in most patients. No difference in OS was observed between patients who continued or discontinued pembrolizumab after PR or CR. This study should spur further prospective trials to optimize the treatment duration for pembrolizumab responders.Katsuhiro Ito: Conceptualization, data curation, formal analysis, writing—original draft and writing—review and editing. Yuki Kita: conceptualization, data curation, writing—reviewing and editing. Akira Yokomizo, Jun Miki, Yuko Yoshio, Hiroaki Matsumoto, Takehiko Segawa, Takashi Karashima, Naotaka Nishiyama, Kazuto Imai, Shigetaka Suekane, Seiji Nagasawa, and Shin Higashi: data curation, writing—review and editing. Hiroyuki Nishiyama: Project administration, supervision. Hiroshi Kitamura: supervision. Takashi Kobayashi: supervision, conceptualization, data curation, writing—review and editing.The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.All authors declare there is no conflict of interest.The study was approved by the institutional review board at Kyoto University  and review board of all institutions. This study conformed to the Declaration of Helsinki. Informed consent was obtained from all individual participants included in the study.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.