Background The efficacy of intravenous thrombolysis (IVT) is time-dependent.Objective To compare the door-to-needle (DTN) time of stroke neurologists (SNs)versusnon-stroke neurologists (NSNs) and emergency room physicians (EPs). Additionally, we aimed to determine elements associated with DTN ≤ 20 minutes.Methods Prospective study of patients with IVT treated at Clínica Alemana between June 2016 and September 2021.Results A total of 301 patients underwent treatment for IVT. The mean DTN time was 43.3 ± 23.6 minutes. One hundred seventy-three (57.4%) patients were evaluated by SNs, 122 (40.5%) by NSNs, and 6 (2.1%) by EPs. The mean DTN times were 40.8 ± 23, 46 ± 24.7, and 58 ± 22.5 minutes, respectively. Door-to-needle time ≤ 20 minutes occurred more frequently when patients were treated by SNs compared to NSNs and EPs: 15%, 4%, and 0%, respectively . In univariate analysis DTN time ≤ 20 minutes was associated with treatment by a SN (p = 0.002), coronavirus disease 2019 pandemic period (p = 0.21), time to emergency room (ER) (p = 0.21), presence of diabetes (p = 0.142), hypercholesterolemia (p = 0.007), atrial fibrillation (p < 0.09), score on the National Institutes of Health Stroke Scale (NIHSS) (p = 0.001), lower systolic (p = 0.143) and diastolic (p = 0.21) blood pressures, the Alberta Stroke Program Early CT Score , vessel occlusion (p = 0.05), use of tenecteplase (p = 0.18), thrombectomy (p = 0.13), and years of experience of the physician (p < 0.001). After multivariate analysis, being treated by a SN , NIHSS  and lower systolic blood pressure  remained significant.Conclusion Treatment by a SN resulted in a higher probability of treating the patient in a DTN time within 20 minutes. Intravenous thrombolysis (IVT) for patients with acute ischemic stroke (AIS) who meet eligibility criteria is efficacious and increases the probability of patient independence at 3 months from the onset of symptoms.The aim of this study was to compare DTN time in patients with AIS when treated on arrival by SNs and NSNs and emergency room physicians (EPs). Furthermore, we analyzed which elements were associated with very short DTN time (≤ 20 minutes); this was carried out in a single medical center in Santiago, Chile.In this prospective cohort study, patients with suspected AIS who consulted the ER of Clínica Alemana of Santiago (CAS) from June 2016 to September 2021 were evaluated by the neurologist on call.The ER is staffed by six SNs, defined as neurologists with 1 or more years of training in Stroke Units in Chile, Europe, and/or the USA, who regularly take care of stroke patients in the Stroke and/or Intensive Care Units. Additionally, the ER is staffed by five NSNs, who underwent training as neurologist's specialists in areas such as extrapyramidal disorders, and who are knowledgeable of our stroke protocols but who do not treat stroke patients in their practice. In the absence of any of the ER staff neurologists, two SNs or three NSNs who are not ER staff could treat the case..The time from stroke symptomatology onset  to arrival to the ER was registered, as was the assessment of stroke severity according to the National Institute of Health Stroke Scale (NIHSS). Systolic and diastolic blood pressures were measured prior to the administration of the thrombolytic bolus.In patients with AIS, prestroke modified Rankin Scale (mRS) score, sex, age, and cardiovascular risk factors were recordedPatients were then studied with a neuroimaging protocol, which has been previously describedPatients eligible for IVT were treated within a 4.5-hour time window. The alteplase bolus was usually administered after the NCCT and prior to the results of blood parameters, CTA, and DWI. Patients with a wake-up stroke or an unknown time of evolution (UTE) Underwent and additional fluid-attenuated inversion recovery (Flair imaging) and were treated with IVT if the AIS was not visible on this additional imaging or if a CT perfusion (CTP) study demonstrated a good profile. The imaging protocol used for UTE patients was left at the discretion of the attending physician. At our center, there is no established upper NIHSS limit for IVT; patients with low NIHSS may be treated if they had suffered a relevant intracranial large vessel occlusion (LVO), defined as symptomatic disease involving the terminal internal carotid artery or segments M1 or M2 of the middle cerebral artery, the A1 segment of the anterior cerebral artery, the P1 segment of the posterior cerebral artery or the basilar and vertebral arteries, or a neurological deficit that could cause significant disability.The information obtained from the imaging protocol included NCCT Alberta Stroke Program Early CT Score (ASPECTS), the presence of a hyperdense artery sign and the existence of LVO.During April of 2020, and due to the coronavirus 2019 (COVID-19) pandemic, our center authorized the use of tenecteplase for AIS treatment in order to evacuate these patients from the ER as soon as possible.Patients with relevant intracranial large vessel disease are usually treated with bridging therapy with thrombectomy.Clínica Alemana has two peripheral divisions in Santiago: La Dehesa, located 13.8 kilometers from CAS, and Chicureo, located 24.8 kilometers from the main installation. Both divisions have an ER and could perform our stroke imaging protocol 24/7. They are covered by EPs who frequently work with our stroke team in CAS and who received training in stroke treatment. In the case of an AIS patient, they could discuss the case by telemedicine with the neurologist on call in CAS, and if IVT is provided, the patient is immediately transferred by ambulance to CAS.For all patients treated with IVT, DTN time was registered according to the type of attending physician , staff, or replacement doctors. Finally, it was registered whether the treatment was carried out during regular hours, defined as from 8:00 AM to 7:59 PM or during the night shift, from 8 PM to 7:59 AM, and if it was performed on a weekday or weekend/national holiday.After the initial treatment, patients were admitted to the Stroke Unit, where additional evaluations could be performed.Symptomatic intracranial hemorrhage (ICH) was defined as the presence of intracranial hemorrhage on NCCT with an increase of the NIHSS of four or more in the first 36 hours of admission. Favorable outcome was defined as stroke patients with a mRS score ≤ 2 points at the 90-day follow-up. To simulate real-world stroke-treating scenarios, stroke mimics (SM) were not excluded from the analysis.The Ethics Committee of Clínica Alemana de Santiago/Universidad del Desarrollo approved the registry, and patients, or their relatives, provided written informed consent.Door-to-needle times between SNs and non-stroke physicians (NSN and EPs) were compared; the percentage of DTN time > 60 minutes and ≤ 20 minutes for these groups were also calculated.After excluding those cases treated by EPs , we performed a univariate analysis with Fisher exact test of independence to evaluate the associations of DTN time ≤ 20 minutes with basal mRS, sex, age, cardiovascular risk factors, time to the ER, admission NIHSS, and pretreatment systolic and diastolic blood pressures. Other factors considered were leucoaroisis on NCCT, ASPECT score, the presence of the hyperdense artery sign, bridging endovascular treatment, SN versus NSN, years of experience of the physician, being treated during the day versus the night shift, weekdays or weekends/national holidays, COVID-19 pandemic period versus no pandemic, the use of alteplase versus tenecteplase, and a final diagnosis of SM at discharge were also analyzed.p < 0.25, Hosmer Lemeshow criteria for fishing variables) in the univariate analysis to select the final multivariate model.A logistic regression analysis was performed for those variables that were associated .Between June 2016 and September 2021, 386 consecutive patients with suspected AIS and who were possible candidates for IVT were admitted to our center. Of these, 85 cases did not receive IVT, 31 due to use of an anticoagulant whose effect could not be reversed, 13 because of very mild deficits, 7 suffered from conditions that could increase the risk of ICH, 12 due to non-favorable RAPID (software results) or DWI/FLAIR images, 5 patients because of unfavorable basal medical conditions, and, finally, 17 for a variety of other reasons.p = 0.04) and performed more IVT during weekends (p = 0.03) and during the COVID-19 pandemic (p = 0.000). Stroke neurologists had more years of experience (p = 0.001) and used alteplase more frequently (p = 0.000).The remaining 301 patients were treated with IVT. Their mean age was 67 ± 18,5 years and 49.2% of these were male. The mean DTN time was 43.3 ± 23.6 minutes, with a median of 37.1 minutes. Only 58 patients (19.2%) had a DTN time > 60 minutes. One hundred seventy-three patients (57.4%) were evaluated by SNs, 122 (40.5%) by NSNs, and 6 (2.1%) by EPs. The baseline characteristics of the patients are shown inp = 0.004). The median DTN time was 35, 39, and 62.5 minutes for these groups. There was no difference between the DTN time prior to and throughout the COVID-19 pandemic period, 43.8 ± 23 versus 41.5 ± 23 minutes.The mean DTN time was 40.8 ± 23, 46 ± 24.7 and 58 ± 22.5 minutes for SNs, NSNs, and EPs, respectively (p = 0.004). Stroke neurologists had more patients with a DTN time ≤ 60 minutes: 82.6%, 79.1%, and 50%, respectively , but these were not statistically significant.Door-to-needle time ≤ 20 minutes was more frequently observed in patients treated by SNs when compared to NSNs and EPs: 15%, 4%, and 0%, respectively , treatment during the COVID-19 pandemic (p = 0.21), time to ER (p = 0.21), presence of diabetes (p = 0.142), hypercholesterolemia (p = 0.007), atrial fibrillation (p = 0.09), NIHSS (p = 0.001), lower systolic (p = 0.143) and diastolic (p = 0.21) blood pressures; ASPECTS on CT (p = 0.09), the presence of a vessel occlusion on CTA (p = 0.05), the use of tenecteplase (p = 0.18), bridging thrombectomy (p = 0.13), and years of experience of the physician who performed the IVT treatment (p = 0.001).The variables associated with DTN time ≤ 20 minutes are shown inp = 0.007), NIHSS , and lower systolic blood pressure on admission  remained statistically significant. An addition of 1 point in NIHSS increases by 7% the chance of being treated with a DTN time ≤ 20 minutes; on the other hand, every increase of 10 mmHg of the systolic blood pressure increased the DTN time by 1 minute.In the multivariate analysis , being p = 0.63).An ICH was present in 12 (4.4%) of the 271 stroke patients treated (none of the SM suffered an ICH); 6 of these patients had been treated by a SN (3.8%), 6 (5.4%) by a NSN, and none had been treated by EPs. These differences were not statistically significant (p = 0.69). After adjustment for age, presence of diabetes mellitus, NIHSS, systolic blood pressure on admission, and the presence of leukoaraiosis and LVO, the results remained statistically nonsignificant.Favorable outcome (mRS score ≤ 2) was not statistically different between those treated by a SN: 112 out of 154 patients (72.2%), and NSN 77 (70%) of 110 (p = 0.69), even for those subjects who had suffered a LVO (p = 0.89).On day 90, there was no difference in the distribution of mRS between patients treated by SNs and NSNs  and fewer cases. Currently, to the best of our knowledge, there have been no direct comparisons of DTN times between SN and neurologists not dedicated to stroke care, with both being neuro-hospitalists.Two previous studies, by Lee et al.In our experience in CAS, EPs were the physicians that showed the longest DTN time. In all cases, telemedicine was activated, and final DTN was 58 ± 22.5 minutes. This result is very similar to that of the TeleStroke Unit (TeleACV) of the Southern Metropolitan Area, Ministry of Health, Chile, in which 115 cases treated with IVT had a median DTN time of 56.5 minutes.One out of every 10 patients treated in our institution had a DTN time ≤ 20 minutes; this number is low when compared to highly specialized centers,The main factor associated with a DTN time ≤ 20 minute at our institution is being treated by a SN, who is the only physician who is involved in the management of AIS in the ER and the Stroke and Critical Care units, and who are the most experienced and trained in stroke management.Higher NIHSS scores also correlated with DTN time ≤ 20. This is not surprising as more severe clinical manifestations correlate with more expeditious detection of AIS in the ER.In our experience, patients treated by a SN show a similar incidence of ICH and functional outcome at 3 months as those treated by NSN. A previous study suggested that when stroke patients are treated by a SN, they have better outcomes.The presence of a SN in the ER is desirable in view of his expertise in this field. In our experience, this is associated with shorter DTN time; however, SN specialists constitute a limited resource in many countries. Other strategies could also be used in order to reduce the DTN time, such as prenotification of arrival to the Emergency Medical Services, single-call activation of the treatment team, moving the patient directly to NCCT, and administration of alteplase in the scanner.This study has limitations: it represents the experience of a single center and a rather small sample. Additionally, we did not evaluate the reasons why SNs demonstrated shorter DTN time and DTN time ≤ 20 in particular. There are 10% of SM patients, and there was no prehospital stroke notification, which would clearly decrease DTN time. Finally, a stroke fellow is sent to the ER every time a stroke patient arrives, and this could influence the DTN time of patients being treated by a NSN. On the other hand, our research represents a real-world scenario in which the ER is populated by neurologists with different types of training.In conclusion, SNs demonstrated shorter DTN time and higher probabilities of treating patients in ≤ 20 minutes after arriving when compared to NSNs.


Optimal diabetes care and risk factor management are important to delay micro‐ and macrovascular complications in individuals with type 1 diabetes (T1D). Ongoing improvement of management strategies requires the evaluation of target achievement and identification of risk factors in individuals who do (or do not) achieve these targets.Cross‐sectional data were collected from adults with T1D visiting six diabetes centers in the Netherlands in 2018. Targets were defined as glycated hemoglobin (HbA1c) <53 mmol/mol, low‐density lipoprotein‐cholesterol (LDL‐c) <2.6 mmoL/L (no cardiovascular disease [CVD] present) or <1.8 mmoL/L (CVD present), or blood pressure (BP) <140/90 mm Hg. Target achievement was compared for individuals with and without CVD.Data from 1737 individuals were included. Mean HbA1c was 63 mmol/mol (7.9%), LDL‐c was 2.67 mmoL/L, and BP 131/76 mm Hg. In individuals with CVD, 24%, 33%, and 46% achieved HbA1c, LDL‐c, and BP targets respectively. In individuals without CVD these percentages were 29%, 54%, and 77%, respectively. Individuals with CVD did not have any significant risk factors for HbA1c, LDL‐c, and BP target achievement. In comparison, individuals without CVD were more likely to achieve glycemic targets if they were men and insulin pump users. Smoking, microvascular complications, and the prescription of lipid‐lowering and antihypertensive medication were negatively associated with glycemic target achievement. No characteristics were associated with LDL‐c target achievement. Microvascular complications and antihypertensive medication prescription were negatively associated with BP target attainment.Opportunities for improvement of diabetes management exist for the achievement of glycemic, lipid, and BP targets but may differ between individuals with and without CVD. HighlightsAchievement of glycemic, lipid, and blood pressure targets are suboptimal.Individuals with type 1 diabetes and cardiovascular disease (CVD) have more difficulty reaching treatment targets than individuals with diabetes without CVD.More consideration may be required for individuals with a previous cardiovascular event and type 1 diabetes. Despite the large burden of CVD in individuals with T1D, reports on the achievement of LDL‐c and blood pressure targets are limited. In addition, percentages of individuals with T1D reaching treatment targetsIn this study, we assess the percentage of adults with T1D who achieve HbA1c, LDL‐c, and blood pressure targets, with and without known CVD across six diabetes centers in the Netherlands. Furthermore, differences between those who achieve or do not achieve targets are described to find potential subgroups that require more, or different, attention for cardiovascular risk management.22.1This is a cross‐sectional registry‐based study. Electronic patient data were collected from two centers: Diabeter, a specialized T1D treatment and research center with five locations throughout the Netherlands, and the University Medical Center Groningen (UMCG), the Netherlands. Individuals visiting these clinics between 1 January 2018 and 31 December 2018 were included if they were over the age of 18, were diagnosed with T1D, and had used insulin for at least 1 year. T1D diagnosis was determined by the presence of American Diabetes Association (ADA) criteria for diabetes mellitus and a clinical presentation typical for T1D or the presence of autoantibodies.The Medical Ethical Review Board of the UMCG, Groningen, the Netherlands, declared that this study was not subject to the Dutch Medical Research Involving Human Subjects Act (WMO) and a waiver was granted. The institutional review board approved the study protocol (202000883).Data extraction for this study is described in detail elsewhere.2.2Demographic data included age and diabetes duration, sex, and ethnicity or parental country of birth. Ethnicity was classified as either western European or non‐western European, as no meaningful subgroups could be formed in the latter. When ethnicity was not available, parental country of birth was used to determine ethnicity. If at least one parent was born outside of western Europe, the individual was considered non‐western European.CVD was defined as the presence of a positive medical history for coronary artery disease, cerebrovascular disease or transient ischemic attack, peripheral arterial disease, or the prescription of platelet aggregation inhibitors. An individual was considered to have microvascular complications if they had a positive medical history for retinopathy, neuropathy, or nephropathy.Anthropometric data on blood pressure, height, and weight were also extracted. Body mass index (BMI) was calculated by dividing weight in kilograms by the height in meters squared.Laboratory measurements included HbA1c, serum creatinine, and LDL‐c. Estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration formula.Medication prescribed was extracted from the EMRs and relevant medication was coded based on their mechanism of action: antihypertensive, lipid lowering, and platelet aggregation inhibitors. For antihypertensive medication, the number of antihypertensive medications prescribed was calculated and classified as either none, one, two, or three or more.2.3The achievement of targets was assessed for the outcomes glycemic control, LDL‐c, and blood pressure. The ADA guidelines recommend the HbA1c target to be <53 mmol/mol (7.0%), unless stricter targets can be achieved without risk of hypoglycemia (HbA1c <48 mmol/mol [6.5%]) or when an individual's life expectancy is limited (HbA1c <64 mmol/mol [8.0%]), or the harms outweigh the benefits.LDL‐c targets were <2.6 mmoL/L for those without CVD and <1.8 mmoL/L for those with CVD.Blood pressure targets were achieved if the measured blood pressure in the outpatient clinic setting was <140/90 mm Hg.2.4All statistical analyses were conducted using R Statistical software,t tests, Wilcoxon rank‐sum, chi‐square, or Fisher exact tests where appropriate.Differences in the characteristics between those with and without CVD were evaluated with unpaired The achievement of targets was further analyzed for those without CVD and those with CVD. Percentages of individuals achieving the target were calculated for each target separately for those with and without CVD.For the CVD positive (CVD+) and CVD negative (CVD−) group, age‐adjusted odds ratios (aORs) were calculated using multiple logistic regression analysis to determine which risk factors may have an impact on target achievement. Adjustment was made for age as age is a confounder for the achievement of HbA1c, LDL‐c, and blood pressure.3A total of 2293 individuals visited the six diabetes centers for annual diabetes screening. After excluding individuals with missing data for HbA1c, LDL‐c, and blood pressure, 1737 were included in this study. Table Participants with CVD were significantly older, had a longer diabetes duration, a greater BMI and higher systolic blood pressure, and were more often smokers or former smokers in comparison to the CVD‐ group. The percentage of individuals with microvascular complications, and with any medication prescription ‐‐ for all working mechanisms ‐‐ was significantly greater in individuals with CVD, whereas CSII use was significantly lower among those with CVD.3.1p = .17). The LDL‐c target was achieved by about half of the study population. Despite lower overall LDL‐c in the CVD+ group, the achievement of this target was significantly lower in the CVD+ group in comparison to the CVD‐ group . Blood pressure targets were achieved by about three quarters of the study population and were achieved more often by individuals in the CVD‐ group .The achievement of HbA1c, LDL‐c, and blood pressure targets is shown in Figure 3.2Characteristics of individuals with CVD achieving glycemic, LDL‐c, and blood pressure targets were heterogenous, and only a few characteristics differed significantly between those who did or did not achieve targets Table . IndividFigures 3.3In contrast, in the CVD− group several factors were found to differ significantly between those who did or did not achieve targets Table . In indip = .013) and CSII users  , with microvascular complications , and prescribed LLM  and AHM  were significantly less likely to achieve glycemic targets  Figure . Those ss Figure . Other cAssociations between characteristics and LDL‐c achievement are illustrated in Figure p < .001), individuals with microvascular complications , and those prescribed AHM  were significantly less likely to achieve blood pressure targets , with brand‐agnostic prescription under EU/Dutch health care laws. The research presented here was independently performed and there are no conflicts of interest.Table S1. Differences in characteristics between individuals with cardiovascular disease (CVD) achieving glycated hemoglobin (HbA1c), LDL‐cholesterol (LDL‐c), and blood pressure (BP) targets.Table S2. Differences in characteristics between individuals without cardiovascular disease (CVD) achieving glycated hemoglobin (HbA1c), LDL‐cholesterol (LDL‐c), and BP targets.Click here for additional data file.


Lactobacillus paracasei, highly sensitive probiotic species to gastric acid, was encapsulated with polyacrylate resin. C57BL/6 male mice were equally divided into three groups; control group was fed with basal diet without any additives, the un‐encapsulated group was fed with 0.1% of a mixture of encapsulating material and L. paracasei, and encapsulated group was fed with 0.1% encapsulated L. paracasei (microcapsule) for 4 weeks. The result showed elevated fecal moisture percentage in the encapsulated group, but not in the un‐encapsulated group. Further study showed that the ratio of villus height to crypt depth in the small intestine was significantly higher compared to un‐encapsulated and the control group. Microencapsulated probiotics also remarkably increased intestinal mucin and secretory immunoglobulin A (sIgA) concentration, intestinal MUC‐2, and tight junction protein mRNA expression levels improving the intestinal barrier function of mice. In addition, microcapsules also reduced proinflammatory factor mRNA expression, while considerably increasing anti‐inflammatory factor mRNA expression. Microbiota metabolites, fecal LPS (Lipopolysaccharide) were downregulated, and acetate and lactate were upraised compared to control. Furthermore, glutathione peroxidase (GSH‐Px) and TAOC levels were increased and Malondialdehyde (MDA) was decreased improving antioxidant capacity. Microflora and bioinformatic predictive analysis of feces showed that encapsulated probiotics remarkably increased Lactobacillus proportions. Mice's intestinal health can thus be improved by using microencapsulated probiotics.The beneficial effect of probiotics on host health is impaired due to the substantial loss of survivability during gastric transit caused by small intestinal enzymes and bile acids. Encapsulation helps to preserve the probiotics species from severe environmental factors.  We employed a new protective material to create microcapsules that safeguard probiotic strains against harsh environmental conditions. Our findings demonstrate that the microcapsules we developed have a positive effect on the gut health of mice. The diversity of the microbiome is closely associated with intestinal health  was sprayed onto the pellets using a spray bottle. The purpose of this step was to ensure that the bacteria are evenly distributed on the surface of the starch pellets.First, starch pellets are used as a carrier and added to the coating pot which was preheated to 37°C. A probiotic bacterium known as Once the bacterial liquid was applied, the enteric coating material was then sprayed onto the pellets. This coating material is pH‐dependent, it will remain intact as it passes through the stomach and will only dissolve in the intestines where the pH is higher. Polyacrylic resin is commonly used as an enteric coating material due to its pH sensitivity and ability to protect the bacteria until it reaches the intestines.By encapsulating the bacteria in starch pellets and then applying an enteric coating, the survivability of the probiotics is increased. This process protects the bacteria from the harsh acidic environment of the stomach and ensures that they can reach the intestines intact, where they can provide their beneficial effects.2.2The artificial gastric juice  and small intestine fluid  were prepared according to the manufacturer's instructions. The coated and uncoated bacteria were inoculated into separate Petri dishes of the artificial gastric juice and then incubated at a temperature of 37°C for 1.5 h. Following this, the bacteria were moved from the artificial gastric juice to separate Petri dishes of the artificial small intestine fluid and incubated again at 37°C for another 1.5 h. After spending 1.5 h in the artificial small intestine fluid, the bacterial solution was diluted by a factor of 10,000. This diluted bacterial solution was then plated on a suitable agar medium and incubated under appropriate conditions to promote the formation of colonies. Finally, the number of monoclonal colonies that formed was counted, and a comparison between the number of colonies that formed from the coated and uncoated bacteria was performed to determine whether the coating provided any protection against the simulated digestive fluids.2.3n = 10) according to their weight: one group was blank control (Control) which received a basal diet without any additives, the second group was un‐encapsulated L. paracasei (0.1% MELP), which received 1 g/Kg of a mixture of L. paracasei GDMCC 1.649 (0.25 g/Kg) and coating material, polyacrylate resin (0.75 g/Kg), and the last group was 0.1% encapsulated L. paracasei (0.1% ECLP) which received 1 g/Kg of encapsulated L. paracasei. Here, encapsulated Lactobacillus was prepared by encapsulating L. paracasei GDMCC 1.649 with polyacrylate resin by Hefei Ansheng Pharmaceutical Technology Co., Ltd, Hefei, China. Feed was prepared with water by maintaining a pH of 3.5 to protect encapsulating material from disintegration in the presence of water. The animals were individually housed in cages. Each animal's body weight and feed intake were recorded weekly. The Nuclear magnetic resonance system  was used to measure the body composition.A total of 30 C57BL/6 male mice (5 weeks) were obtained from the Animal Experiment Center of Guangdong Province  and were caged individually, in a sterile and controlled environment with a temperature of a 24°C ± 2°C, relative humidity of 65% ± 10% and 12 h light 12 h/dark cycle. For 2 weeks, mice were provided with mouse feed and water ad libitum. After 2 weeks of adaptation, mice were assigned into three groups /weight of feces before drying × 100%. Similarly, all the feces were also collected regularly from the litter on 10 mL tubes to measure total dry fecal weight and use the feces for further studies. Feces collected in sealed tubes (10 mL) were dried for 48 h at 37°C to protect them from the loss of volatile nutrients that can be evaporated and lost at a higher temperature. After drying, feces were weighed to obtain dry fecal weight and stored at −80°C for later use.2.5After feeding for 28 days, all mice were sacrificed. Blood was harvested from an eyeball. Blood samples were collected in a 1.5 mL centrifuge tube, placed for 1 h at room temperature for clotting, and centrifuged . The serum of each sample was separated and stored at −20°C for subsequent detection and analysis. From the dissected mice, tissues and internal organs were observed and weighed. Intestinal tissues and their contents were stored at −80°C for future use. The parts of separated specimens of the intestinal section  were fixed in a 4% paraformaldehyde solution for morphological study.2.6The specimens placed in paraformaldehyde were embedded in paraffin wax, and slices were sectioned at 5 μm. The obtained sections were stained with hematoxylin and eosin (HE) by mounting in a glass slide. Afterward, the slides were observed under the microscope  for villi length (V) and crypt depth(C). The measurement of V and C was done using Image . Finally, the ratio of V and C (V/C) was determined.2.7The protein was extracted from intestinal tissues using RIPA lysis buffer , and its concentration was determined using a BCA protein assay kit . The protein concentration was then adjusted to 20 μg/20 μL and denatured with protein loading buffer  by boiling it in water for 10 min. The western blot (WB) procedures followed a previous study  were computed using commercial kits  as per the instructions of the company. Furthermore, total antioxidant capacity (TAOC) in blood serum was detected by a commercial product of Solrabio Life Science Beijing, China. For fecal/intestinal digesta contents, the feces and intestinal digesta were diluted with double‐distilled water at the ratio of 1:5 (weight/volume), homogenized, and centrifuged . The obtained supernatant was used to detect different contents by following the kit instructions. Digesta pH was measured by inserting the sterile glass electrode of a pH meter (Thermoscientific™ Eutech Elite pH Spear) to a tube containing digesta, and the values were noted down.The level of inflammatory factor  in duodenum tissue, serum malondialdehyde (MDA), glutathione peroxidase (GSH‐PX), fecal mucin, secretory immunoglobulin A (sIgA), albumin, lipopolysaccharide (LPS), and antrimethylamine N‐oxide (TMAO) were carried out by enzyme‐linked immunosorbent assay (ELISA) kits from Shanghai Ruifan Biotechnology Co., Ltd., China, by following the company's instructions. Similarly, blood and fecal urea nitrogen (BUN), ammonia, and hydrogen peroxide . For RNA extraction, intestinal tissues stored at −80°C were taken. Total RNA was extracted from intestinal sections  using RNA extraction kit  as per the manufacturer's instructions, and then determined using a Nanodrop spectrophotometer. Complementary DNA (cDNA) was synthesized by using 2 μg of total RNA by treating with DNase I  to obtain a final volume of 20 μL by using Random Primer 9  and M‐MLV Reverse Transcriptase  by following the protocol of the company. Then, total cDNA was mixed with antisense primers, SYBR green Real‐Time PCR master Mix and Nucleic acid‐free water. The qPCR reaction had a final volume of 20 μL. The qPCR was carried out with the Applied Biosystems QuantStudio 3 Real‐Time PCR System . GAPDH was used as control, and the relative gene expression of mRNA was calculated by 22.10g at 4°C. Around 400 μL of the supernatant was extracted via pipette after centrifugation. By the use of a disposable syringe, the supernatant was filtered via a 0.22 μm filter, placed in a glass sample bottle, and injected into a glass column of 4.6 × 250 mm dimension. As per the protocol of the company and the parameters of the machine, the test was carried out.The determination of SCFA and lactic acid concentrations were done via HPLC (high‐performance liquid chromatography). In brief, the fecal and jejunal contents were taken out from a refrigerator, thawed at 4°C, and mixed thoroughly. Around 0.2 g of the contents from each sample was diluted with double‐distilled water at the ratio of 1:5 (w/v) and mixed using a vortex for around 20 min to break all the feces. The mixture was centrifuged for 15 min, 12,000× 2.11After 21 days of feeding, fresh fecal samples from four mice of control and encapsulated groups were collected on a well‐sterilized 1.5 mL tube twice a day for 5 days, placed on liquid nitrogen, and stored at −80°C until DNA extraction. 16S rRNA sequencing was performed at Beijing Novogene Co., Ltd. The steps and procedure of the experiment are described in the previous article    and effective tags were obtained by the removal of chimera sequences . Sequences with ≥97% similarity were assigned to the same OTUs  database  in a body weight gain or feed intake throughout the experiment  compared to the control  in encapsulated group compared to control. Additionally, the weight of white adipose tissue (WAT), that is, inguinal (iWAT) and gonadal (gWAT), as well as the weight of brown adipose tissue (BAT) showed no significant differences among the groups. To investigate the morphological change and structure, initially the intestinal and colon length were measured. The length was found to be numerically higher in ECLP group but had no significant difference (p > .05) . In addition, the dietary treatment of microcapsule increased the villus height significantly (p < .05) in the duodenum and jejunum with respect to control. Meanwhile, compared to the un‐encapsulated group, encapsulated group raised villus height in duodenum. Furthermore, the ileal villus height was increased numerically (p = .08), and crypt depth was decreased significantly (p < .05) in the encapsulated group. But, no notable improvement in intestinal morphology was observed in the un‐encapsulated group, as there was no remarkable change in villus height, crypt depth, or their ratio compared to control. Furthermore, when we studied the effects of microcapsules by WB on the intestinal barrier in the duodenum by which is crucial for tissue morphology and absorption function, we noted an elevation in Claudin‐1 in the encapsulated group  Figure . Using H) Figure . All thep Figure .The above results clearly show that the un‐encapsulated group had no differences in fecal output, body mass, tissue index, intestinal morphology, and intestinal barrier when compared to the control group, but the encapsulated group had significant changes in fecal output and improved intestinal morphology and barrier function when compared to both control and un‐encapsulated groups. One possible explanation for this could be the use of a lower amount of probiotics, which may have resulted in reduced survivability during passage through the gastrointestinal tract.L. paracasei improves intestinal histomorphometry and fecal quality. Thus, we used only control and encapsulated group mice samples for further investigation, if the microcapsule has other beneficial impacts on gut health. Initially, we observed an increase in the mRNA expression levels of tight junction proteins including claudin‐1, occludin, and zonula occludens‐1 (ZO‐1) in the duodenum, jejunum, and ileum sections in the encapsulated group compared to the control, as shown in Figure Ultimately, it concludes that supplementing with encapsulated 3.4Lactobacillus raised the quantity of SIgA (p < .05) and lowered the level of albumin (p = .054) , and ileum (p = .06). In addition, the expression of IL‐6 was reduced in the duodenum (p = .07), jejunum (p = .05), and ileum (p < .05). We also found that IL‐8 expression was downregulated in the duodenum, jejunum (p = .05), and ileum (p = .07). A significant decrease (p < .05) in TNF‐α mRNA expression was observed in the ileum section, but no significant difference was found in the duodenum and jejunum (p = .05). Additionally, the consumption of encapsulated Lactobacillus increased the level of IL‐10 compared to those in the control group, suggesting the improvement of inflammation. Similarly, an ELISA test was conducted to confirm the presence of inflammatory cytokines in the duodenal tissue. The results of the test indicated a significant decrease in IL‐1β (p < .05) and IL‐6 (p = .05), as well as an increase in IL‐10 (p < .05). As shown in Figure Lactobacillus elevated the level of total antioxidant capacity (TAOC) and glutathione peroxidase (GSH‐Px) in blood serum. In addition, we observed the reduction of malondialdehyde (MDA) level (p < .05) in the encapsulated group  and albumin contents were quantified as an indicator of the intestinal barrier. Encapsulated ) Figure . Mucin l) Figure . An incrn Figure . To examn Figure . IL‐1β lp Figure .3.5Lactobacillus group compared to control with a significant difference. In addition, the pH of digesta was reduced in the jejunum, colon (p = .08), and cecum (p < .05)  in its level  and trimethylamine‐N‐oxide concentration (TMAO) were decreased Figure  in encap) Figure . The vall Figure . Similarl Figure . The effd Figure . To show3.6Lactobacillus on fecal microbiota, feces were analyzed by 16S rRNA sequencing. We investigated that the consumption of encapsulated Lactobacillus decreased (p < .05) the observed species  in the encapsulated group  enrichment analysis was used to express the enrichment of the microbiome on a different level  and reduces the Bacteroidota   Figure . In addip Figure .Lactobacillus and Bacteroides were dominant bacteria at the genus level. The relative abundance of Lactobacillus, Turicibacter, and Lachnoclostridium was elevated in the treatment group, and Bacteroides was slightly reduced. There was a statistically significant reduction of Romboutsia in the treatment group , and other diseases on the host  and L. murinus species (0.3194 vs. 0.1314), which could be the reason for the decrease in richness and diversity as found in our study. Moreover, we observed a clear difference between the control and encapsulated probiotic‐treated group, that is, beta diversity, which illustrates L. paracasei affects the microbiota in mice.Probiotics have a vital role in maintaining the gut microbiota by increasing the population of beneficial bacteria and reducing the pathogens by competing with them  after supplementing the encapsulated probiotic, which is believed to be the marker for obese animals. However, we did not find any difference in weight gain in mice between those groups. A recent study has presented that this biomarker is still difficult to associate with the weight and health of an individual , which exert a positive effect on the host's health by maintaining immune homeostasis, improving gastrointestinal barrier function, and suppressing proinflammatory cytokines  ; data curation ; investigation ; methodology (lead); writing – original draft (lead); writing – review and editing (Lead). Canjun Zhu: Data curation ; investigation ; supervision ; writing – review and editing . Guilian Zhou: Funding acquisition ; project administration ; resources . Guli Xu: Methodology (supporting). Yuxian Zeng: Methodology (supporting). Jincheng Li: Methodology (supporting). Jingjing Zhou: Methodology (supporting). Gang Shu: Conceptualization ; validation ; writing – review and editing . Qingyan Jiang: Conceptualization ; funding acquisition ; investigation ; project administration . Genghui Li: Methodology (supporting). Yujun Wang: Methodology (supporting).The project was supported by grant of the Guangdong key research and development program (2019B020218001) and the National Natural Science Foundation of China (32102626), the Local innovative and research teams project of Guangdong province (2019BT02N630), Quality Control for Feed and Products of Livestock and Poultry Key Laboratory of Sichuan Province (NH2021202206).The authors confirm that they have no conflict of interest.Animal care and procedures were performed as per the guidelines and were approved by the Animal Subjects Committee of South China Agricultural University and Department of Science and Technology of Guangdong Province (permission number: SYXK [Yue] 2014‐0136).


No patient died within 100 days after auto‐PBSCT, and no unexpected serious adverse events were observed. Although 1‐year event‐free survival was 100%, hematological relapse was observed in three patients at a median of 801 days  after auto‐PBSCT. Molecular progressive disease was observed in the other two patients, although they maintained their first hematological remission at the last visit. Auto‐PBSCT can be safely performed for Ph+ALL with TKIs. A limitation of auto‐PBSCT was suggested, despite the increase in the intensity of a single treatment. The development of long‐term therapeutic strategies by including new molecular targeted drugs is warranted to maintain long‐term molecular remission.Autologous hematopoietic stem cell transplantation (SCT) is not a standard treatment option for Philadelphia chromosome‐positive acute lymphoblastic leukemia (Ph+ALL); however, its position has been reassessed since the introduction of tyrosine kinase inhibitors (TKIs). We prospectively analyzed the efficacy and safety of autologous peripheral blood SCT (auto‐PBSCT) for Ph+ALL patients aged between 55 and 70 years who had achieved complete molecular remission. Melphalan, cyclophosphamide, etoposide, and dexamethasone were used for conditioning. A total of 12 courses of maintenance therapy, including dasatinib, were performed. The required number of CD34 AdvaAllogeneic SCT  is a standard treatment option for Philadelphia chromosome‐positive acute lymphoblastic leukemia (Ph+ALL) , despiteBecause autologous SCT has not been recommended in Japan since the introduction of TKIs, there is no data on autologous SCT for Japanese Ph+ALL patients. In this study, we planned to perform autologous SCT for Ph+ALL patients (aged 55 years or older) with complete molecular remission (CMR) who had transplant toxicity concerns for allo‐SCT, with the expectation of a more potent antileukemic effect than conventional chemotherapy.22.1This was a multicenter prospective exploratory study with a target sample size of 5. The primary end point was the proportion of deaths within 100 days of autologous peripheral blood SCT (auto‐PBSCT). Secondary end points examining treatment efficacy included the proportion of molecular or hematological relapse, overall survival (OS), event‐free survival (EFS), molecular or hematological relapse rate, and non‐relapse mortality (NRM) at 100 days, 1 year, and 3 years after auto‐PBSCT. Regarding safety, the proportion of therapy‐related deaths, adverse events, engraftment failures, BCR‐ABL detections in harvested peripheral blood stem cells and BCR‐ABL mutation at relapse, the success rate of peripheral blood stem cell harvesting, and the ratio of the actual to the planned dose of dasatinib during maintenance were measured. De novo Ph+ALL patients aged between 55 and 70 years who achieved CMR within three chemotherapy regimens were registered in this study. The eligibility criteria have been reported elsewhere . Surviva2.2BCR‐ABL1 chimeric gene was planned to be analyzed at designated time points: before conditioning for auto‐PBSCT, 30 days after auto‐PBSCT, before maintenance courses #4, #8, and #12, and 3 years after auto‐PBSCT.The study protocol has been described in detail elsewhere . Briefly2.3We assessed the numerical and phenotypic features of peripheral T cells at the same time as MRD analyses using multiparametric flow cytometry (FCM).Peripheral blood mononuclear cells were isolated by density gradient with Ficoll‐Paque  and were stored in liquid nitrogen until analysis. Cells were washed with phosphate‐buffered saline containing 2% fetal calf serum  and subjected to staining with surface antibodies, shown in Table 2.4BCR‐ABL1 signal was detected after CMR was achieved. Molecular progressive disease was defined as an increase of 1‐log or more in the number of copies of BCR‐ABL1 for a second consecutive time with an interval of at least 1 week. Hematological relapse was defined as (1) the appearance of leukemic blasts in peripheral blood, (2) 5% or more bone marrow blasts, or (3) the appearance of extramedullary lesions, after the hematological remission was achieved. Neutrophil engraftment was defined by an absolute neutrophil count of at least 0.5 × 109/L for three consecutive days, and platelet engraftment was defined by a count of at least 20 × 109/L without transfusion support.CMR was defined by the absence of detectable MRD with a sensitivity of at least 0.01%. Molecular relapse was defined when the 2.5The Kaplan–Meier method was used to estimate OS and EFS . OS was 33.1Between September 2017 and February 2019, five patients were enrolled in this study. The patient flowchart is shown in Figure 3.2+ cells (2 × 106/kg) or more was harvested during the first PBSCH regimen in three patients, a median of 2 days of the procedure . The median number of CD34+ cells was 1.91 × 106/kg  in all five patients. In the second PBSCH regimen, a sufficient number of CD34+ cells were harvested in both patients in 1 day of procedure using plerixafor. Most non‐hematological adverse events were grades 1 or 2, and no specific adverse events were observed in more than half of the patients . Neutrophil and platelet engraftments were achieved in all patients at a median of 10 days  and 13 days , respectively. No patient died up to 100 days after auto‐PBSCT. Febrile neutropenia was the most common severe adverse event (grades 3–4) during the auto‐PBSCT period . All but one patient were able to start dasatinib at 50 mg/day as planned and gradually increased to 100 mg/day. Only 1 patient was able to receive dasatinib at 100 mg/day throughout the 12 courses; the other patients needed a dose reduction . The secondary end points are summarized in Table Although molecular relapse was observed at the checkup 30 days after auto‐PBSCT in one patient, MRD became negative again with the initiation of maintenance therapy. Molecular progressive disease was observed at a median of 731 days after auto‐PBSCT  Figure . In 4 pa3.6+ and CD8+ cells among T cells were almost constant after auto‐PBSCT, and the CR4/CD8 ratio remained low throughout the observation period  cells tended to decrease once at pre‐M4  and was particularly high in AP‐03, who received a lower dose of dasatinib Figure . In addi3 Figure .4+ cells was collected in all cases. All patients survived for at least 100 days after auto‐PBSCT with no unexpected serious adverse events, indicating that there were no safety concerns. However, all patients eventually experienced molecular progressive disease or hematological relapse. If the goal was to develop a cure, the long‐term results were not satisfactory.This study prospectively evaluated the safety and efficacy of auto‐PBSCT for Ph+ALL. For PBSCH, the required number of CD34As NRM in allo‐SCT becomes higher as patient age increases, especially for patients aged 55 years or older, the ageThe 3‐year OS after allo‐SCT with reduced intensity conditioning was reported to be 64.2% in a study that analyzed Japanese registry data . As for This study was planned with the concept of aiming for a cure by eliminating leukemia with high‐dose chemotherapy followed by auto‐PBSCT. However, the antileukemic effect achieved by donor immunity could not be obtained, unlike allo‐SCT. Relapse after a long period since auto‐PBSCT suggested a limit to increasing the intensity of a single‐chemotherapy treatment. Recently, TKI plus blinatumomab showed excellent outcomes for de novo Ph+ALL patients , 18. TheIt is interesting that the patient with the earliest relapse (AP‐02) had higher eTreg cells and a lower CD8/eTreg ratio about 4 months before relapse. Increased eTreg cells may have led to early relapse by suppressing the antileukemic immune response. However, a careful interpretation of this outcome is required due to the small number of cases in this analysis. An increase in eTreg cells after autologous SCT is reportedly associated with early relapse in multiple myeloma  and atte+ T cells was reported to be inhibited by dasatinib in a dose‐dependent manner [+ T cells indicated that the selective depletion of eTreg cells was established by low‐dose dasatinib therapy in patient AP‐03 (Figure A high CD8/Treg ratio in solid tumor tissue correlates with a favorable prognosis . In the t manner , which st manner . The rel3 Figure .In conclusion, auto‐PBSCT can be safely performed for patients with Ph+ALL. In all cases, molecular remission was sustained for more than a year after auto‐PBSCT, but disease progression was eventually observed. Long‐term treatment strategies, such as continued maintenance therapy incorporating new molecular targeted drugs, could be promising.Satoshi Nishiwaki, Isamu Sugiura, and Hitoshi Kiyoi designed the study, analyzed the data, and wrote the manuscript. Takahiko Sato, Daisuke Sugiyama, Sachiko Ito, and Hiroyoshi Nishikawa performed immunological analyses. Miki Kobayashi, Masahide Osaki, Masashi Sawa, Yoshitaka Adachi, Motohito Okabe, Shigeki Saito, Takanobu Morishita, Akio Kohno, Takahiro Nishiyama, Hiroatsu Iida, and Shingo Kurahashi registered patients and performed the protocol treatment. Yachiyo Kuwatsuka contributed to the data management and analysis of the data. All authors critically reviewed the manuscript and checked the final manuscript.M.S. received honoraria from Kyowa Kirin Co., Ltd., Chugai Pharmaceutical Co., Pfizer Inc., Astellas Pharma Inc., Nippon Shinyaku, Ono Pharmaceutical, MSD, Bristol‐Myers Squibb, Asahi Kasei, Novartis Pharma K.K., Eisai Co., Ltd., Otsuka Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Sanofi S.A., Takeda Pharmaceutical Co., Ltd., Celgene, Mochida Pharmaceutical Co., Ltd., Shire plc, Mundipharma K.K., AbbVie Inc., CSL Behring, SymBio, Janssen Pharmaceutical K.K., AstraZeneca plc, Daiichi Sankyo Co., and GlaxoSmithKline plc outside of this study. H.I. received research funding from Chugai Pharmaceutical Co. and honoraria from Astellas Pharma Inc., Novartis Pharma K.K., AbbVie Inc., and Janssen outside of this study. H.N. received research funding and honoraria from Ono Pharmaceutical, MSD, Bristol‐Myers Squibb, and Chugai Pharmaceutical, and research funding from Taiho Pharmaceutical, Daiichi Sankyo, Kyowa Kirin Co., Ltd., Zenyaku Kogyo, BioPharma Inc., Debiopharm, Asahi Kasei, Sysmex, FUJIFILM Corporation, SRL, Astellas Pharma Inc., Sumitomo Dainippon Pharma Co., and BD Japan outside of this study. H.K. received research funding from FUJIFILM Corporation, Kyowa Hakko Kirin Co., Ltd., Bristol‐Myers Squibb, Otsuka Pharmaceutical Co., Ltd., Perseus Proteomics Inc., Daiichi Sankyo Co., Ltd., AbbVie Inc., CURED, Inc., Astellas Pharma Inc., Chugai Pharmaceutical Co., Ltd., Zenyaku Kogyo Co., Ltd., Nippon Shinyaku Co., Ltd., Eisai Co., Ltd., Takeda Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Novartis Pharma K.K., and Sanofi K.K., and honoraria from Astellas Pharma Inc., AbbVie Inc., Chugai Pharmaceutical Co., Ltd., and Novartis Pharma K.K. outside of this study. The remaining authors declare no competing financial interests.This study was approved by the institutional review board of each participating hospital.All participants gave their informed written consent to participate in this study.UMIN000026445.Supporting InformationClick here for additional data file.


Diagnosing and treating synchronous multiple primary lung cancers (sMPLC) are complex and challenging. This study aimed to report real‐world data on the comprehensive diagnosis and treatment of patients with early‐stage sMPLC.A single‐center cohort study was carried out and a large number of patients with early‐stage sMPLC were included. A single‐ or two‐stage surgery was performed to remove the primary and co‐existing lesions. The “X” strategies, including ablation, SBRT, and EGFR‐TKIs treatment, were applied to treat the high‐risk residual lesions. Wide panel‐genomic sequencing was performed to assess the genetic heterogeneity of the co‐existing lesions.p = 0.014) and the solid presence of a primary lesion (p < 0.001) were significantly related to tumor recurrence. And a T2‐stage primary tumor had a significantly higher rate of developing new lesions after the initial surgery (p < 0.001).A total of 465 early‐stage sMPLC patients with 1198 resected lesions were included. Despite most patients being histologically different or harboring different genetic alternations, about 7.5% of the patients had the same histological type and driver gene mutation changes, comprehensive re‐evaluation is thus needed. The “Surgery + X” strategy showed remarkable efficacy and safety in treating multiple lesions. Follow‐up data revealed that the T2 stage (In real‐world practice, histopathological and radiological evaluation combined with genetic analyses could be a robust diagnostic approach for sMPLC. The “Surgery + X” treatment strategy showed remarkable efficacy, superiority, and safety in the clinical treatment of early‐stage sMPLC. A major challenge in managing these cases is determining whether co‐existing nodules should be diagnosed and treated as separate primary cancers or intrapulmonary metastases, as the treatment strategies and outcomes differ significantly between the two forms of the disease.Surgical resection remains the most employed strategy for treating early‐stage sMPLC.The present study summarized our center's real‐world data on the clinical diagnosis and treatment of patients with sMPLC. Based on multidisciplinary diagnostic analyses of sMPLC, a comprehensive treatment strategy entitled “Surgery + X” was provided to patients with sMPLC. The clinicopathological, diagnostic, therapeutic, genomic status. and prognostic factors were analyzed.22.1Between April 2015 and December 2020, patients who underwent curative‐intent surgical resection for lung cancer were retrospectively reviewed, and a total of 582 patients were diagnosed with multiple primary lung cancer (MPLC) according to the Martini‐Melamed criteria.2.2Surgical procedures for each patient with sMPLC were determined based on age, cardiopulmonary function, tumor size, CT features, lesions distribution, and comorbidities severity.In the cases with bilateral lesions, a two‐stage procedure was recommended, first on the side of the lesion with an advanced stage based on preoperative assessment. The time of the second operation was suggested based on the patient's condition . Systemic and sampling lymph node dissection was performed in cases with a lobectomy and sublobar segmental and wedge resections, respectively. The largest T and highest N stages of all lesions in each case were treated as the final comprehensive T and N stages. The highest stage of all lesions determined the comprehensive TNM stage according to the eighth TNM classification guidelines.Despite curative intent resection, there were still some high‐risk residual lesions in some of the sMPLC patients, which were not fit for additional resection due to impaired pulmonary function, improper location, or other medical conditions. Our multidisciplinary team assesses the properties of these residual lesions based on previous study strategies, considering changes in radiologic and biological behavior. GGO or mGGO residual nodules larger than 0.8 cm in diameter, or those that gradually increase in size or density during follow‐up, were considered high‐risk or progressed residual lesions that need to be treated.The “X” strategies, including ablation, SBRT, and EGFR‐TKIs treatment, were applied to treat the high‐risk residual lesions in the following situation: (a) a co‐existing lesion that had a high risk of malignancy but was improper for additional surgical resection due to impaired pulmonary function or other medical conditions after initial surgery; (b) a progressed residual lesion; (c) a newly emerged lesion, existed persistently and evaluated by the multidisciplinary team to be a high risk of malignancy. The selection of the “X” strategy should take into account the location, anatomical variations, CT features, and patients' willingness. For GGO lesions located in the outer 1/3–1/2 of the lung, CT‐guided percutaneous puncture microwave ablation is usually recommended. For lesions located near the bronchi and closer to the center of the lung, electromagnetic navigation bronchoscopy‐guided microwave ablation (ENB‐MWA) is a common choice. For lesions with a high risk of complications from ablation, SBRT is an alternative option. For patients with a primary EGFRm lesion that reached stage IB and with high‐risk factors for tumor recurrence, adjuvant EGFR‐TKI treatment was recommended.2.3Fresh frozen tissues were collected from each resected nodule. Genomic DNA was extracted using the QIAamp DNA Tissue Kit (Qiagen). The samples were subjected to wide panel‐genomic sequencing  at the coverage depth of 1800×. Germline DNA from peripheral blood mononuclear cells of the same patients was used as a reference. The detection and data analysis were performed as described previously.2.4t‐test was used for continuous variables. The Kaplan–Meier analysis calculated cumulative survival rates, and survival differences were compared using the log‐rank test. All analysis was performed with SPSS software for Windows (Version 22.0), R (version 4.0.5), and PRISM software . A p‐value <0.05 were considered statistically significant.Continuous variables were presented as mean ± standard deviation, while categorical variables were presented as frequencies and numbers (percentages). The Fisher's exact test or chi‐square test was used to compare nominal categorical variables, and the 33.1n = 9), large‐cell neuroendocrine carcinoma (n = 3), adenosquamous carcinoma (n = 3), and large‐cell undifferentiated carcinoma (n = 2)). Sixty‐seven lesions diagnosed as atypical adenomatous hyperplasia (AAH) were resected as concomitant lesions of the primary lesions. Based on CT findings and clinicopathological results, all 465 patients were diagnosed as T1‐2N0M0 (stage Ia‐Ib) sMPLC patients. Regarding the time of surgery, the number of patients with early‐stage sMPLC has increased dramatically since 2018 . Among them, exon 19 deletions , L858R missense mutation , and EGFR amplification  occurred most frequently. TP53 mutations were found in 76 lesions (15.4%), and high mutation frequency of RBM10 (13.2%), ERBB2 (12.3%), BRAF (9.3%), MED12 (9.1%), and KRAS (7.3%) were also detected. Only 10 lesions (2.0%) harbored EML4‐ALK fusions. As described previously, lesions from sMPLC showed significant genetic heterogeneity.Despite most patients with sMPLC being histologically different or harboring different genetic alternations, about 7.5% (35/465) of the patients had the same histological type and driver gene mutation changes. For these patients, radiological and histopathological analyses were re‐evaluated by the multidisciplinary team of thoracic oncology to confirm the sMPLC diagnosis. Lesions presenting as GGO or mGGO or with an element of carcinoma in situ (AIS) on pathological examination were diagnosed as primary lung cancers.3.4p = 0.343). The gender, smoking history, CT features, and surgical procedures were unrelated to patients' survival . There were 11 patients (1.1%) who experienced tumor recurrence or distant metastases, and 6 (1.3%) died at the end of the follow‐up course. There was no significant difference in recurrence‐free survival (RFS) between patients with T1 and T2 stage tumors , and 3 patients with three lesions underwent electromagnetic navigation bronchoscopy‐guided microwave ablation (ENB‐MWA). Among the 43 lesions, 32 were GGO lesions, and 11 were mGGO lesions. The median size of the lesions was 1.23 cm (range from 0.6 cm to 2.0 cm). No serious adverse events occurred during the ablation. Two patients suffered mild pneumothorax without symptoms. Lesion responses were evaluated 3 months after ablation. According to RECIST 1.1 criteria,3.5.2Seven patients underwent a standard SBRT procedure to treat the high‐risk residual lesions. Six of them had one lesion treated, and one patient had two lesions treated. The SBRT was safe and effective. No severe complications occurred during the treatment.3.5.3Thirty‐four patients with 53 residual lesions were treated with postoperative EGFR‐TKI. All these patients were pathologically diagnosed as stage Ib after initial surgery and harbored EGFR mutations in the resected primary lesions. The EGFR‐TKIs treatment was provided according to the latest study results.4The diagnosis and treatment of sMPLC are challenges in clinical practice. Previous studies have demonstrated the safety and efficacy of surgical resection for the primary lesions of sMPLC. However, the alternative strategies for treating high‐risk residual lesions remain controversial. The present study demonstrated real‐world data and outcomes on the clinical diagnosis and comprehensive treatment of early‐stage sMPLC. Several essential findings were present.First, the safety and efficacy of anatomical resection combined with sublobar resection under a minimally invasive approach were confirmed. Previous studies reported that lobectomy is the optimal choice for the second primary lung cancer, and sublobar resection will not compromise the survival of tumors smaller than 2 cm. However, aggressive surgical treatment, especially pneumonectomy, will bring more comorbidities and worsen the prognosis.Second, for early‐stage sMPLC, most residual lesions were small GGO or mGGO lesions. According to the evaluation of the multidisciplinary team, a close follow‐up strategy was adopted for most of these residual lesions. The “X” strategies were only provided when a progressed residual lesion or a persistent lesion with a high possibility of invasive adenocarcinoma was found. Recent case‐based studies have reported favorite outcomes of the clinical treatment of residual lesions using ablation and SBRT.1‐2N0M0 sMPLC, patients with T2 stage primary tumor or solid primary lesions had a higher rate of tumor recurrence, and a T2 stage primary tumor also significantly related to developing new lesions after the initial surgery, indicating closer follow‐up policy or a more aggressive treatment strategy should be adopted for these patients.Third, the previous study has demonstrated that lymph node metastasis was an independent unfavorable prognostic factor of sMPLC.Fourth, it is noteworthy that despite the majority of patients being histologically different or harboring different genetic alterations, about 7.5% of patients had the same histological type and driver gene mutation changes. In this situation, it is rather challenging to differentiate a second primary cancer from a metastatic lesion. The multidisciplinary team re‐evaluated CT features and histopathological analyses to confirm the diagnosis according to previous reportsThere are some limitations of this study. First, this study used a single‐institution Chinese cohort and included surgically resected cases since 2015, an external validation study is needed. Second, for the residual lesions, it is impossible to perform biopsies on every lesion in real‐world practice. We selected the lesions presenting as GGO or mGGO appearance suspected to be malignancy based on the evaluations of the multidisciplinary team of thoracic oncology. Third, the follow‐up period is relatively short for early‐stage sMPLC, especially for the residual lesions after treatment. Long‐term follow‐up will provide more information on the efficacy of the comprehensive treatment. In addition, a small number of patients were lost to follow‐up during the study, which might bias the prognosis results.In conclusion, our study demonstrated the comprehensive diagnosis and treatment of patients with early‐stage sMPLC in real‐world practice. Histopathological and radiological evaluation combined with genetic analyses could be the robust diagnostic approach for differentiating primary cancer from a metastatic lesion. The “Surgery + X” treatment strategy showed remarkable efficacy, superiority, and safety in the clinical treatment of early‐stage sMPLC.Danting Zhou: Data curation ; investigation ; writing – original draft . Tianyu Yao: Data curation ; writing – original draft . Xiaojie Huang: Data curation (supporting); formal analysis ; investigation (lead). Fang Wu: Project administration ; supervision . Yi Jiang: Methodology ; validation ; visualization . Muyun Peng: Investigation ; project administration ; resources ; software . Banglun Qian: Formal analysis ; resources ; software . Wenliang Liu: Methodology ; software ; validation . Fenglei Yu: Data curation ; funding acquisition ; resources ; supervision ; validation ; writing – review and editing (supporting). Chen Chen: Conceptualization (lead); data curation ; funding acquisition ; investigation ; methodology (lead); Supervision ; Writing – review and editing (lead).This study was funded by the Hunan Provincial Natural Science Foundation , Hunan Provincial Key Area R&D Program NO. 2019SK2253, Hunan Clinical Medical Technology Innovation Guidance Project of China Hunan Provincial Science & Technology Department, No. 2020SK53427, CSCO Cancer Research Foundation (CSCO‐Y‐young2019‐034 and CSCO‐2019Roche‐073), and the Changsha Municipal Natural Science Foundation NO. kq2014246.The authors declare that there are no financial/commercial conflicts of interest involved in this study.The research protocol of this study was approved by the Institutional Review Board of the Second Xiangya Hospital. Written informed consent was obtained from all subjects.Not Applicable.Figure S1:Figure S2:Figure S3:Figure S4:Click here for additional data file.


Leisure sedentary behavior (LSB) is associated with the risk of cancer, but the causal relationship between them has not been clarified. The aim of this study was to assess the potential causal association between LSB and risk of 15 site‐specific cancers.The causal association between LSB and cancer were assessed with univariate Mendelian randomization (UVMR) and multivariate Mendelian randomization (MVMR). 194 SNPs associated with LSB  were adopted as the instrument variables. Sensitivity analyses were performed to ensure the robustness of the results.p = 0.04) ，breast cancer  . Although causal association was not found between television watching and ovarian cancer, it was seen in low grade and low malignant potential serous ovarian cancer . However, significant results were not obtained in the UVMR analysis between driving, computer use and the 15 types of cancer. Further MVMR analysis indicated that the above results are independent from most metabolic factors and dietary habits, but mediated by educational attainment.UVMR analysis revealed that television watching significantly increased the risk of endometrial cancer (OR = 1.29, 95% CI = 1.02–1.64, LSB in form of television watching has independent causal association with the risk of endometrial cancer, breast cancer, and ovarian cancer. The detailed information of these studies is shown in Table 2.3F‐statistic, equal to (n–2)R2/(1–R2), can be used to evaluate the effect of weak instrumental variables.R2 was the degree of sedentary behaviors explained by the SNP, and was calculated using the formula R2 = 2 × (1 – EAF) × EAF×β2/(se2 × n) . Weak instrument bias is considered when F > 10. In addition, variation between individual genetic variant (I2GX) was calculated to assess potential weak instrument bias in the MR‐Egger regression analysis. Low risk of measurement bias was considered at I2GX > 95%.Weak instrument bias refers to bias produced by genetic variants with low power to explain exposure owing to insufficient sample size. According to the literature, the 2.4β‐value   was also calculated, and heterogeneity was considered as significant if I2 > 25%.p‐value for outcomes should be excluded, or random‐effects models and weight‐median are more appropriate for estimating MR effect , weighted‐median, MR‐Egger, MR‐PRESSO, and weighted‐mode, were performed to account for presence of horizontal pleiotropy, heterogeneity, potential violations of the MR assumptions, and invalid instrument‐exposure associations Figure .30, 3Horizontal pleiotropy, outliers, and heterogeneity were visually analyzed by using scatterplots, leave‐one‐out plots, and funnel plots.p‐value threshold, we added genetic variants for sedentary behavior with higher p‐value  and repeated the MR analysis.To determine if the results were robust to the 2.5GWAS studies of LSB have revealed some degree of genetic association between sedentary behaviors and other traits including triglycerides, total cholesterol, HDL, LDL, BMI, T2D, and educational attainment.To investigate whether the effect of LSB on cancers may be mitigated by its effect on other traits and prevent potential MR assumption violations, MVMR was used to estimate the direct effect of multiple exposures on different outcomes Figure .40 The2.6p < 0.05. Correction for p‐value was made using the Bonferroni method in the secondary analysis of LSB on metabolic factors (p < 0.05/9 = 0.006). Associations were considered suggestive at 0.006 < p < 0.05. Causal associations were estimated using the odds ratio (OR). All analyses were finished using the R package TwoSampleMR (version 0.5.6). Data analyses were conducted between January 2021 and June 2022.Primary univariate and multivariate analyses of the relationship between LSB and cancers were conducted with a two‐sided significance threshold of 33.1F‐value of IVs related to television watching, computer use, and driving ranged from 23.94 to 144.19, 24.07 to 79.56, and 24.00 to 45.01, respectively. None of them were considered to have weak instrument bias (F > 10)  Table . As calc3.2Complete results of UVMR of the association between 3 sedentary behaviors and 15 site‐specific cancers are summarized in Figure p = 0.010). It occurred in endometrioid subtype  rather than non‐endometrioid subtype  (Table p‐intercept >0.05). However, there was significant heterogeneity except for non‐endometrioid subtype  increase in watching television increased endometrial cancer risk by nearly 30%  Table . There wpe Table . After cp = 0.007) in the IVW fixed‐effects analyses. The association appeared to be stronger for the estrogen receptor negative (ER–) subtype  than for estrogen receptor positive (ER+) subtype   Table . In the 5) Table , S6. Giv5) Table . In addip = 0.018) without significant pleiotropy and heterogeneity   Tables . This asAside from the above results, television watching made no significant difference to the other 11 cancers. There was no evidence of horizontal pleiotropy or heterogeneity except for prostate cancer. But even when we used control analyses to correct the heterogeneity, no significant results were obtained Tables .3.3p = 0.019) and cervical cancer . Both of the latter findings, however, were supported by only one MR method, whereas all other methods consistently showed null results for computer use and endometrial and cervical cancer , TC , LDL , and T2D , while negatively associated with HDL  and years of schooling . For dietary habits, TV viewing was a risk factor of most unhealthy dietary habits , but was negatively associated with other dietary habits . Computer use was showed to be positively associated with years of schooling  and intake of cereal . However, there were no significant associations between computer use and all metabolic factors as well as all unhealthy dietary habits. As for driving, it was only showed to be associated with year of schooling   Tables . InteresFor endometrial cancer, adjustment of BMI, T2D, and educational attainment made the previously causal association non‐significant, indicating that BMI, T2D, and educational attainment were mediators in this association. Adjustment of BMI also indicated it as the mediators in the association between television watching and low‐grade serous ovarian cancer. Besides, only educational attainment changed the association between television watching and breast cancer, indicating it as a mediator Figure .4Our study explored the causal relationship between LSBs and 15 site‐specific cancers, using MR analyses. After complete sensitivity analyses, the associations were converged on three common female cancers – endometrial, breast, and ovarian cancer. Furthermore, the associations varied among subtypes of the three female cancers. However, computer use and driving were not seen as risk factors for the selected cancers. Further MVMR analyses indicated the direct effect of television watching on the three female cancers. Given the high stability of the positive results, we considered television watching as a risk factor for endometrial cancer, breast cancer, and ovarian cancer. Whether computer use and driving were risk factors was not conclusive.We found that television watching increased the risk of endometrial cancer, especially in endometroid subtype. This is in accordance with the previous large meta‐analyses that found a consistent association between television watching and incidence of endometrial cancer.We indicated television watching as a risk factor for breast cancer. Contrary to our findings, no significant causal association was shown in two other meta‐analyses.We also demonstrated causal associations between LSB and ovarian cancer in line with a large meta‐analysis.Another key point to note is that causal effects of driving and computer use on the selected cancers were either non‐significant or unestablished in several sensitivity analyses. For driving trait, inadequate SNPs and low statistical power might be the reasons. For computer use, the follow‐up pre‐UVMR and MVMR analysis gave the possible explanation. Pre‐UVMR analysis revealed high genetic correlations between sedentary behaviors and educational attainment, which were negative for TV viewing and driving, and positive for computer use. In addition, causal associations were found between TV viewing and cardio‐metabolic factors as well as unhealthy dietary habits, which were insignificant for computer use. It seemed that people spending more time in using computer had higher educational attainment and healthier dietary habits. And education had been presumed to causally influence health because it generally confers greater access to salubrious resources such as economic security, healthy lifestyles, social ties, fulfilling jobs, a sense of personal control, and learned effectiveness.The multivariable MR analyses showed an effect of television watching on female cancer independent of most cardio‐metabolic factors and dietary habits. It also indicated vertical pleiotropy of BMI, T2D, intake of fizzy drink and biscuits, as the direct effects of TV viewing on cancers were attenuated compared with the total effects. This provides genetic insights in how sedentary behaviors are associated with cancers. LSB could cause increased snack intake and decreased energy expenditure, accompanied by weight gain and obesity, which could increase risk of cancer. Obesity facilitates carcinogenesis through a number of pathways, including insulin resistance, perturbations in the insulin‐like growth factor axis, and low‐grade systemic inflammation.The multivariable MR in which we corrected for education indicated pleiotropy due to education. Pre‐UVMR analysis in our study revealed correlations between sedentary behaviors and educational attainment in line with previous study. Besides, we have also verified the inverse association between educational attainment and sedentary behaviors in line with a previous study. This study also found that higher educational attainment levels were positively with vigorous physical activity levels and alcohol consumption.The strengths of our study are, initially, that it is the first to explore and find a causal association between sedentary behavior and three common female cancers using MR and that different associations were found within different subtypes with reliable evidence. The results suggest a potential role of LSB in cancer prevention. We may need to promote lifestyle changes that reduce sedentary time for the general public. Second, the instrumental variables used in this study were derived from a large GWAS with significant statistical power. In addition, this study carried out sensitivity analyses using several different methods to test the heterogeneity and pleiotropy of IVs and correct the directionality of causal associations. The test results confirmed one another, making the results more reliable. Finally, MVMR was performed on the positive results obtained from UVMR, which revealed the direct effect of LSB on the corresponding cancers.The first limitation of this study is that the population included in this study was European, and it is not clear whether the conclusions of the study can be extended to other ethnic groups. Second, SNPs obtained by statistical methods rather than biological methods inevitably have pleiotropy, for which we used multiple sensitivity analyses to minimize the impact of pleiotropy. We also performed MVMR to correct the effects of confounders. However, this does not rule out the effects of other unknown, potentially confounding variables. In addition, the acquisition of leisure sedentary time was based on subjective measurements rather than with measurement tools; although this avoided measurement bias, it was pointed out that subjective and objective measurement standards are non‐uniform.Jinwei Chen: Conceptualization (lead); data curation (lead); formal analysis (lead); visualization (lead); writing – original draft (lead). Kai‐Bin Yang: Conceptualization (lead); formal analysis (lead); visualization (lead); writing – original draft (lead). Youyu Qiu: Conceptualization (supporting); formal analysis (lead); visualization ; writing – original draft (lead); writing – review and editing . Weijie Lai: Data curation (lead); visualization . Sifan Qi: Data curation (lead); visualization (supporting). Gaoyuan Wang: Data curation ; visualization (supporting). Lin Chen: Data curation ; writing – original draft . Kunpeng Li: Data curation (supporting); writing – original draft . Dan Zhou: Data curation (supporting); writing – original draft (supporting). Qing Liu: Formal analysis ; visualization (supporting); writing – original draft (lead). Ling‐Long Tang: Formal analysis (supporting); visualization (supporting); writing – original draft . Xu Liu: Formal analysis (supporting); visualization (supporting); writing – review and editing . Xiao‐Jing Du: Conceptualization (lead); project administration (lead); writing – review and editing . Rui Guo: Conceptualization (supporting); supervision (lead); writing – review and editing . Jun Ma: Conceptualization (lead); funding acquisition (lead); project administration (lead); supervision (lead); writing – review and editing (lead).This study was supported by grants from the National Natural Science Foundation of China , the Key‐Area Research and Development Program of Guangdong Province (2019B020230002), and the Overseas Expertise Introduction Project for Discipline Innovation .No potential conflict of interest was reported by the authors.Not applicable.The study strictly followed the Declaration of Helsinki. The data were anonymized and publicly available, thus institutional ethics committee approval and consent from the participants were not required.Figures S1–S99.Click here for additional data file.Tables S1–S9.Click here for additional data file.


Previous observational studies have showed that certain psychiatric disorders may be linked to breast cancer risk, there is, however, little understanding of relationships between mental disorders and a variety of breast diseases. This study aims to investigate if mental disorders influence the risks of overall breast cancer, the two subtypes of breast cancer (ER+ and ER-), breast benign tumors and breast inflammatory diseases.During our research, genome-wide association study (GWAS) data for seven psychiatric disorders  from the Psychiatric Genomics Consortium (PGC) and the UK Biobank were selected, and single-nucleotide polymorphisms (SNPs) significantly linked to these mental disorders were identified as instrumental variables. GWAS data for breast diseases came from the Breast Cancer Association Consortium (BCAC) as well as the FinnGen consortium. We performed two-sample Mendelian randomization (MR) analyses and multivariable MR analyses to assess these SNPs’ effects on various breast diseases. Both heterogeneity and pleiotropy were evaluated by sensitivity analyses.p = 3.84 × 10−6; multivariable MR: OR 1.06, 95%CI [1.04-1.09], p = 2.34 × 10−6), ER+  and ER-  breast cancer. Nevertheless, major depressive disorder only showed significant positive association with overall breast cancer  according to the two-sample MR analysis, but not in the multivariable MR analysis. In regards to the remainder of the mental illnesses and breast diseases, there were no significant correlations. While as for the data from the UK Biobank, schizophrenia did not significantly increase the risk of breast cancer.When the GWAS data of psychiatric disorders were derived from the PGC, our research found that schizophrenia significantly increased the risks of overall breast cancer (two-sample MR: OR 1.05, 95%CI [1.03-1.07], The correlation between schizophrenia and breast cancer found in this study may be false positive results caused by underlying horizontal pleiotropy, rather than a true cause-and-effect relationship. More prospective studies are still needed to be carried out to determine the definitive links between mental illnesses and breast diseases. Among all breast malignancies, breast cancer has the greatest impact on human beings, the incidence of which has been the highest amongst lady cancers, and it is additionally the leading cause of death among women with cancers , 2. BreaAmong the potential elements that may additionally influence risks of breast diseases, mental factors are receiving growing attention . PreviouMendelian randomization (MR) is a promising causal reasoning device developed in current years , 14, whiTwo-sample MR and multivariable MR analyses were conducted in this study using genome-wide association study (GWAS) statistics to investigate causal relationships between seven psychiatric disorders and the risks of breast cancer, breast benign tumors, and breast inflammatory diseases. This work can be used to provide insights to further screening or prevention of breast diseases in clinical settings.2.2.1.https://www.med.unc.edu/pgc/, in order to identify genetic variations connected with these psychiatric disorders among the population of European descent. The number of single nucleotide polymorphisms (SNPs) differed significantly across the GWAS data for each psychiatric disorder, consequently, to make the analyses as accurate as possible, we extracted effect sizes of genetic variations conforming to the genome-wide significance levels (p < 5 × 10−8) and not falling into linkage disequilibrium  when exploring schizophrenia, major depressive disorder and bipolar disorder. Nevertheless, the conditions for selecting instrumental variables were set to p < 5 × 10−6 and LD r2 < 0.001 as for the remaining four mental disorders. Several previous analyses also adopted the settings described above (2: R2 = [2 × EAF × (1-EAF) ×(β)2]/[(2 × EAF × (1-EAF) × (β)2)+(2 × EAF × (1-EAF) × N ×se (β)2)], where EAF was the effector allele frequency, β was the effector size, N was the sample size and se (β) was the standard error of the genetic effect. And then we calculated the F statistic: F = [R2 × (N-k-1)]/[(1-R2) × k], assessing the strength of the statistics  in ed above , 27. Furatistics , 28, 29,atistics . SNPs wiiscarded . Detaile2.2.Data on overall breast cancer and two kinds of molecular subtypes (ER+ and ER-) were obtained from the Breast Cancer Association Consortium (BCAC), which were based on GWAS studies of 228,951 women of European descent  . Additio2.3.p < 0.05/n (n was the number of exposures) was statistically significant, p > 0.05 meaned no statistical significances, and 0.05/n < p < 0.05 represented suggestive statistical significances. While for the FDR method, we obtained adjusted p values corrected by the multiple corrections, and p < 0.05 was statistically significant.The SNPs of mental disorders were extracted from outcome data, excluding those that were strongly associated. Further, we aligned the alleles of SNPs from exposures and outcomes, and then discarded SNPs with incompatible alleles or palindromic SNPs with intermediate allelic frequencies. We also excluded SNPs associated with confounding factors of the exposures and outcomes. Finally, exposures with more than three SNPs were retained for MR analyses. The two-sample MR analyses mainly used inverse variance weighted(IVW), Weighted median and MR Egger to examine the causal relationships between each mental disease and risks of the overall breast cancer, the two subtypes (ER+ and ER−), breast benign tumors and inflammatory diseases. In order to make the conclusions more reliable, we used two methods of multiple tests which called the Bonferroni method and the false discovery rate (FDR) method. The Bonferroni method was more stringent than the FDR method. In the Bonferroni method, we defined that p < 0.05 was recognized as existing heterogeneity  and schizophrenia  , but not for major depressive disorder (Ppleiotropy > 0.05). Based on the leave-one-out analyses, none of the instrumental variables significantly altered the extent of causation between the two psychiatric disorders and overall breast cancer risk . The results of the LDSC regression analyses between mental disorders and overall breast cancer were presented in When GWAS data of exposures were derived from the PGC, the two-sample MR analyses using IVW method revealed a significant increase in overall breast cancer risk for individuals with major depressive disorder  . The resvariable . The estvariable . For schdisorder . Neverthcer risk . On the cer risk . Howevercer risk , no sign3.2.p = 5.94 × 10−5) and ER−  breast cancer based on the IVW analyses. The results of Bonferroni and FDR multiple correction methods were consistent (p = 0.030), but there was no statistical significance for ER+ breast cancer , whereas major depressive disorder did not play a significant role , breast benign tumors and breast inflammatory diseases using Mendelian randomization analyses.Our study found some associations between schizophrenia and breast cancer, but it might be false positive results due to underlying horizontal pleiotropy, and there were no evidences of causal relationships between psychiatric disorders and these breast diseases.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Only publicly available GWAS data were used in this study, and the ethics approval could be available in the original GWAS studies.XinW and XiaW designed the study. SZ, CY, KF, JL, XK, and RZ were responsible for data extraction and collation. QS was responsible for statistical analyses. FR wrote the manuscript. All authors contributed to the article and approved the submitted version.This research was supported by the National Key Research and Development Program of China (Grant No. 2019YFE0110000), National Natural Science Foundation of China (Grant No. 82072097), CAMS Innovation Fund for Medical Science (CIFMS) , Beijing Hope Run Special Fund of Cancer Foundation of China (Grant No. LC2020A18).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.