Patients (≥12 years) with ≥2 asthma diagnoses, newly initiated on medium-/high-dose ICS/LABA (Japanese asthma guidelines), from 01 April 2009 to 31 March 2015 were included, using Japan Medical Data Center Claims Database. Primary objective: proportion of patients with uncontrolled asthma in the year following ICS/LABA initiation. Secondary objectives: predictors of uncontrolled asthma and healthcare resource utilization. In medium-dose  and high-dose (N = 8661) ICS/LABA cohorts, 23% and 21% patients, respectively, were uncontrolled. Treatment step up and exacerbation were most common indicators of uncontrolled asthma. Predictors of uncontrolled asthma, analyzed by multivariable Cox model, included systemic corticosteroid use, exacerbation history, comorbidities, and being female. In both cohorts, healthcare resource utilization was higher in patients with uncontrolled asthma. Over 20% patients with persistent asthma who initiated medium- or high-dose ICS/LABA were uncontrolled, highlighting unmet need for novel therapies in these patients.Many asthma patients remain uncontrolled despite guideline-based therapies. We examined real-life asthma control in Japanese patients prescribed with inhaled corticosteroid/long-acting β In Japan, approximately 3 million people suffer from asthma, making it the leading chronic respiratory disease nationally2.Asthma is a major chronic respiratory disease affecting >350 million people worldwide3 report and regional Japanese asthma guidelines (JGL)4. Inhaled corticosteroid (ICS) is the cornerstone in asthma management and the first choice for patients requiring regular maintenance treatment3. For patients who experience persistent symptoms and exacerbations, guidelines recommend use of ICS with a long-acting β2-agonist (ICS/LABA) or a higher dose of ICS. However, a considerable number of asthma patients receiving guideline-based therapies continue to have poor disease control, diminished quality of life, reduced work productivity, and emergency or hospital-based medical care6.Recommendations for asthma care are established by well-known clinical entities such as the Global Initiative for Asthma (GINA)7; however, there is limited evidence on the prevalence, characteristics, and burden of poor asthma control in patients with ICS/LABA prescription8. Particularly in Asia, there is a lack of data on the level of disease control following prescription with ICS/LABA, and frequency of treatment escalation when asthma remains poorly controlled, and healthcare resource utilization.This subpopulation of patients with uncontrolled asthma accounts for considerable healthcare expendituresIn order to understand the clinically meaningful response to ICS/LABA therapy in the Japanese population, we conducted a retrospective cohort study using the Japan Medical Data Centre (JMDC) Claims Database to identify the proportion of patients with uncontrolled asthma in the 1-year period following newly initiated treatment with medium- or high-dose ICS/LABA. Additionally, we investigated the demographics and clinical characteristics associated with uncontrolled asthma in patients treated with ICS/LABA, treatment pathways following the first sign of uncontrolled asthma, and healthcare resource utilization burden in these patients.4. Patient disposition according to JGL-defined and GINA-defined9 medium- and high-dose ICS/LABA cohorts is shown in Fig. A total of 24,937 patients met the inclusion criteria for the medium-dose ICS/LABA cohort and 8661 patients for the high-dose cohort based on the definition of JGL9 medium- and high-dose ICS/LABA cohorts are detailed in Supplementary Table The mean ± standard deviation (SD) age at index date was 38.9 ± 13.31 years in the medium-dose ICS/LABA cohort and 40.8 ± 11.94 years in the high-dose ICS/LABA cohort. Gender distribution was comparable between the cohorts as was body mass index (BMI). A total of 2260 patients (9.1%) in the medium-dose cohort and 840 patients (9.7%) in the high-dose cohort were habitual smokers. In the 1-year prior to the index date, 10.8% of patients in the medium-dose cohort and 15.7% of patients in the high-dose cohort experienced ≥1 exacerbation. Rhinitis/rhinosinusitis was the most common comorbidity reported in both cohorts , with allergic rhinitis/rhinosinusitis being the most common subtype (Supplementary Table n = 5707) in the medium-dose ICS/LABA cohort and 20.7% patients (n = 1793) in the high-dose ICS/LABA cohort (both cohorts were defined based on JGL4) were found to have uncontrolled asthma , theophylline, and a maintenance systemic corticosteroid in the 1-year pre-index compared with controlled patients in both the cohorts. Patients with uncontrolled asthma also tended to have more comorbidities than controlled patients  with uncontrolled asthma did not receive treatment step up following the first event indicating loss of asthma control. Of the patients who stepped up, the prescription trend for first step up was: addition of LTRA, 43.0%; addition of theophylline, 12.1%; increased ICS dose, 6.6%; addition of more than one asthma controller including LTRA, 4.4%; maintenance corticosteroids, 1.1%; addition of more than one asthma controller excluding LTRA, 0.4%; and addition of long-acting muscarinic antagonist (LAMA), 0.2%. Second and third lines of step up medications were also evaluated. The large majority of patients received no further treatment step up (between 76% and 100%) at the second line; however, of those who did, addition of LTRA was generally the most common. Treatment pathways to LTRA after the first episode of uncontrolled asthma in the medium-dose ICS/LABA cohort are described in Fig. A total of 176 patients (40.8%) with uncontrolled asthma and an additional 1 year of follow-up had no treatment step up following the first episode of uncontrolled asthma. Of the patients who stepped up, the prescription trend for first step up was: addition of LTRA, 36.4%; addition of theophylline, 15.6%; addition of more than one asthma controller including LTRA, 3.9%; maintenance corticosteroids, 2.8%; and addition of LAMA, 0.5%. Second and third lines of step-up medications were also examined, again with the large majority of patients having no further step up. Figure Overall, the rates of asthma- and non-asthma specific healthcare resource utilization events tended to be lower in the medium-dose ICS/LABA cohort, compared with the high-dose ICS/LABA cohort. In the medium-dose cohort, patients with uncontrolled asthma had significantly higher incidence rates for all asthma- and non-asthma-specific healthcare resource utilization items compared with patients with controlled asthma  in the medium-dose and 20.7% (n = 1782) in the high-dose cohort , 55.8% had not well-controlled asthma, which was defined as the asthma control test (ACT) score <208; this proportion was higher compared with that observed in the current study. Similarly, in a cross-sectional analysis of patients with asthma treated with ICS/LABA in the UK, approximately two-third of the population (n/N = 452/701) were not well controlled (ACT < 20)6. In a retrospective cohort study using the UK-Clinical Practice Research Datalink database11, in which uncontrolled asthma was defined similarly to this study , 35.1% of patients in the medium-dose ICS/LABA cohort  and 45.7% of patients in high-dose cohort  remained uncontrolled during 1 year after the initiation of treatment with medium- or high-dose ICS/LABA. These results suggest that Japanese asthma patients newly initiated on medium- or high-dose ICS/LABA may tend to be better controlled compared with those in other countries like Germany and the UK. However, caution needs to be exercised in comparing disease control between studies as the way in which asthma control is measured tend to vary between studies.In a study evaluating disease control in German patients with self-reported asthma  were associated with uncontrolled asthma13. These findings suggest that patients with uncontrolled asthma represent a more medically “at-risk” group, being older, having more comorbidities, and a higher rate of exacerbations. Therefore, uncontrolled asthma is associated with susceptibility for negative health consequences.In general, these findings concur with the evidence from the previous reports on patient-related risk factors for uncontrolled asthma14.In terms of indicators for uncontrolled asthma in these two cohorts, a treatment step up with LTRA or presentation with moderate exacerbation were the two most frequent reasons identified in both cohorts. It has been suggested that the popularity of LTRA as an add-on treatment for asthma, over other add-on treatment options, may be due to the Japanese patient preference for oral medications over inhaled agents15, with uncontrolled asthma more likely in those patients with more frequent exacerbations16. Exacerbation rate is known to increase with disease severity16 and a history of exacerbations is predictive of an increased risk of subsequent exacerbations17. Being able to identify and subsequently target patients at risk of exacerbations is likely to lead to more effective asthma management. Index year was also shown to significantly impact patients’ risk of uncontrolled asthma, suggesting that asthma patients in earlier years  were more likely to be uncontrolled. This may reflect the introduction, in Japan, of different ICS/LABAs  during the timeframe of 2010 to 2015 , and therefore asthma patients in subsequent years may have been better controlled as a result. In the present study, female sex was found to be a significant predictor of uncontrolled asthma in both ICS/LABA cohorts. This is in line with previous studies which have shown that female sex was associated with increased risk of time-to-first exacerbations19. Factors such as greater susceptibility to allergen triggers and increased prevalence of comorbidities have been suggested to contribute to poor symptom control in female patients with asthma compared to male patients20.For both ICS/LABA cohorts, the predictors suggesting the greatest risk of uncontrolled asthma were prescription of maintenance systemic corticosteroids and evidence of asthma exacerbation in the pre-index period. Evidence of these two factors in the pre-index period may be suggestive of poor asthma control or severe asthma. The association between asthma exacerbation and poor asthma control is well documented21.Treatment pathways in patients who were found to be uncontrolled revealed that 32% of the medium-dose and 41% of the high-dose ICS/LABA cohort had no treatment step up or add-on despite their asthma being uncontrolled. The study was not designed to capture a wider range of intervention possibilities, such as non-prescription therapies or anti-infectives, and this observation likely represents an incomplete picture of actual clinical practices, including, but not limited to, difference in perception of disease control between patients and physicians, lack of proper communication between patient and physician, etc. Moreover, in patients who received treatment step up, LTRA was the most frequent prescribed add-on medication, which might be due to the prevalent use of LTRA as add-on asthma medication in Japan22.Rates of healthcare resource utilization events and associated healthcare costs were higher in the high-dose ICS/LABA cohort compared with medium-dose cohort and for uncontrolled patients versus controlled patients. These findings were in line with previous findings with healthcare resource utilization being high in asthma patients with higher severity and poorer control24 while acknowledging the purpose of health insurance claims are not for clinical research and may omit relevant clinical parameters. Several limitations of the claims database include: frequently missing data for height, weight, and tobacco smoking history; lack of primary diagnosis specification for patient encounters reporting multiple diagnoses codes; under-representation of senior aged and unemployed adults; and missing data for laboratory, spirometry information, and Single Inhaler Maintenance and Reliever Therapy use as rescue therapy. Moreover, the JMDC database includes Japanese company employees and their family members and therefore representation over 65 years old is very limited (nearly 3%)25. In addition, patient’s prescription compliance and persistence and inhaler technique were not available for analysis as predictors of control. According to the definition of adherence based on time over which a patient continues treatment or continues to refill the prescription, from starting to stopping therapy26, every patient in this study was considered adherent until the first episode of treatment discontinuation (a gap of >90 days between subsequent medium- or high-dose ICS/LABA prescription) during the post-index date. Also, data pertaining to patient-reported outcomes and objective asthma control score  and lung function parameters were not available in the JMDC database. Therefore, the level of poor asthma control may be underestimated in this study. On the other hand, it is possible to overestimate medication use, since in most pharmaco-epidemiological studies conducted using routinely collected health data such as this study, drug prescription is used as a proxy for medication use. The performance of the model was assessed using Harrell’s C-statistic, which does not test for proportionality. An article by Stensrud and Hernan highlighted some of the issues of testing proportionality in medical studies, such as this. The authors explain that, due to the nature of the data in medical studies, it is likely that they may contradict the proportional hazard assumption27. The large sample size included in the analysis could have led to statistically significant difference between the evaluated parameters even with a difference in mean values, which may not be necessarily clinically meaningful. The study results should be interpreted with clinical perspectives.The use of secondary databases such as JMDC for research has demonstrated utility in assessing drug utilization in various disease areasReal-world evidence on the disease control status of patients with asthma in Japan is expected to improve the understanding of a challenging yet urgently important topic among physicians and health authorities and thus support the adoption of new modalities and policies in clinical practice to achieve better clinical outcomes.Despite universal health access in Japan, disease control appears uncontrolled in >20% patients with asthma who initiated treatment with medium- or high-dose ICS/LABA, highlighting unmet medical needs and opportunity for novel therapies in these patients.29. The data are patient level, anonymized, and longitudinal. The database includes information on age, gender, period of healthcare coverage, healthcare claims, disease diagnoses (standard disease codes), and costs associated with healthcare claims29. The study period was between 01 April 2008 and 31 March 2016. Patients with ≥2 asthma diagnoses who were newly initiated on ICS/LABA during the identification period between 01 April 2009 and 31 March 2015 and fulfilled the inclusion and exclusion criteria (section below) were enrolled, and divided into two cohorts—medium- or high-dose ICS/LABA treatment cohort, according to JGL , representative of approximately 4.4% of the total Japanese populationPatients were followed for up to 1 year post-index date. The first event indicating uncontrolled asthma was evaluated during this period, classifying patients as either controlled or uncontrolled in the medium- and high-dose ICS/LABA cohorts. Study design for patients initiated on medium- or high-dose ICS/LABA are presented in Fig. All study data were accessed using techniques compliant with the Health Insurance Portability and Accountability Act of 1996; therefore, there was no extraction of identifiable protected health information during the course of the study, and this study did not meet requirements for informed consent from patients. This study was approved by Institutional Review Board of the Clinical Research Review Network of Japan, 1-4-9, Itachibori, Nishi-ku, Osaka, 550-0012, Japan.Eligible patients were aged ≥12 years at the index date with the following: evidence of ≥2 claims of asthma diagnoses as defined by the International Classification of Diseases (ICD-10) of J45 or J46 occurring between April 2009 and March 2015, newly initiated on maintenance treatment with medium- or high-dose ICS/LABA between 01 April 2009 and 31 March 2015, and at least 1 year of enrollment (continuous data available in the database) prior to the index date.Patients with a diagnosis of COPD (ICD-10 J44) were excluded. Patients were excluded from the medium-dose ICS/LABA cohort if they had a prescription for medium- or high-dose ICS/LABA in the pre-index period or a prescription for low- or high-dose ICS/LABA on the index date. Patients were excluded from the high-dose ICS/LABA cohort if they had a prescription for high-dose ICS/LABA in the pre-index period or a prescription for low- or medium-dose ICS/LABA on the index date.9.The primary objective of the study was to identify the proportion of patients with uncontrolled asthma in the 1-year period following newly initiated treatment with medium- or high-dose ICS/LABA, based on JGL. Secondary objectives were to describe and compare demographic and clinical characteristics of patients with controlled and uncontrolled asthma on medium- or high-dose ICS/LABA treatment in the 1-year period after the index date, to identify predictors of uncontrolled asthma in both study cohorts, and to describe treatment pathways following the first sign of uncontrolled asthma in both the cohorts. Other secondary endpoints included healthcare resource utilization in controlled and uncontrolled patient groups in the 1-year follow-up period after the index date and percentage of patients with uncontrolled asthma in the 1-year period after initiating treatment with a medium- or high-dose ICS/LABA fixed-dose combination, based on the GINA report , addition of LAMA, addition of LTRA, addition of maintenance systemic corticosteroids, addition of more than one asthma controller in the same day, including LTRA, or addition of more than one asthma controller in the same day, excluding LTRA.2. All healthcare resource utilization variables were assessed for both asthma specific and all cause (excluding asthma specific). Healthcare costs were calculated based on ER visit cost, inpatient cost, outpatient cost, and outpatient prescription drug cost.Healthcare resource utilization was evaluated in the follow-up period based on the number of outpatient visit, hospitalizations, outpatient emergency room (ER), length of stay in hospitalization, and prescriptionsn) and proportion (%) of total patients having the given characteristics; continuous variables are summarized with the mean, standard deviation (SD), median, 25th percentile, 75th percentile, interquartile range, minimum, and maximum. Categorical variables were compared using chi-square tests, while continuous variables were compared using Wilcoxon rank test. No imputation methods were used in this study and complete case analysis was used in the Cox regression analysis.Categorical variables are reported as frequency , using univariate analysis. All healthcare resource utilization-related rates were divided by the patient-specific days of follow-up. Rate and 95% CI were calculated with SAS procedure GENMOD, using Poisson distribution, with log (follow-up) as offset, and Wald test was used to compare rates between controlled versus uncontrolled asthma. Annualized healthcare costs were reported using the following calculation: costs during follow-up period/(duration of follow-up period/365), and Wilcoxon rank test was used to compare costs between controlled versus uncontrolled asthma. All analyses were performed using SAS version 9.4.Further information on research design is available in the Supplementary InformationReporting Summary


To the Editor—The coronavirus disease 2019 (COVID-19) outbreak is a global public health problem. After its occurrence in the Republic of China in December, 2019, the disease spread worldwide, occurring in >160 countries.1 The virus is highly contagious, and close contact in crowded places is an important contributing factor to SARS-CoV-2 transmission. Medical personal are an important at-risk population that can get COVID-19 due to close contact with patients in daily practice.3 Sporadic case reports about COVID-19 from many countries have been published.5 Hospital-acquired infection is possible, as are hospital outbreaks. Here, we present data from Thailand, the second country where the disease occurred in early January 2020.6The setting is a rural district hospital in Yala province, the southernmost province of Thailand that shares an international border with Malaysia, another country where COVID-19 outbreaks occur. This small, 30-bed hospital serves local people in that rural district. The outbreak occurred on March 22, 2020, when 3 medical personnel (2 nurses and 1 physician) developed fever and COVID-19 was confirmed after they had provided regular care to local people with a history of COVID-19 contact. All 3 medical personnel are presently under respiratory isolation, but none has had lung complications. All of the other 21 medical personnel of the hospital, including 7 physicians, are under quarantine. The hospital had to be closed and no longer provides any service. According to our best knowledge, this is the first report of COVID-19 outbreak in a hospital that resulted in total disruption of hospital function.The COVID-19 outbreak in hospitals is serious because it can result in abrupt cessation of local medical care and especially management of COVID-19 during the outbreak. Hospitals usually have protective systems in place, but a high-volume load might result in unsuccessful disease control. A good hospital infection control program during COVID-19 should be simple but strict, with aggressive procedures that might differ from routine clinical practice. Strategic planning to reduce unnecessary physical examinations, to implement universal drug distribution, or to postpone unnecessary procedures during the crisis period, is necessary.


COVID-19 is an infectious disease caused by the SARS COV-2 virus. Patients with COVID-19 are susceptible to thrombosis due to excessive inflammation, platelet activation, endothelial dysfunction, and circulatory stasis, resulting in an increased risk of death due to associated coagulopathies. In addition, many patients receiving antithrombotic therapy for pre-existing thrombotic diseases can develop COVID-19, which can further complicate dose adjustment, choice and laboratory monitoring of antithrombotic treatment. This review summarizes the laboratory findings, the prohemostatic state, incidence of thromboembolic events and some potential therapeutic interventions of COVID-19 associated coagulopathy. We explore the roles of biomarkers of thrombosis and inflammation according to the severity of COVID-19. While therapeutic anticoagulation has been used empirically in some patients with severe COVID-19 but without thrombosis, it may be preferable to provide supportive care based on evidence-based randomized clinical trials. The likely lifting of travel restrictions will accelerate the spread of COVID-19, increasing morbidity and mortality across nations. Many individuals will continue to receive anticoagulation therapy regardless of their location, requiring on-going treatment with low-molecular weight heparin, vitamin K antagonist or direct-acting anticoagulants. Travel has greatly accelerated the global spread of COVID-19 and has so far affected over 107 million people with more than 2.3 million deaths (as of 10 February 2021) . The virThere is a complex interplay between pro-inflammatory cytokine/chemokine release, increased endothelial dysfunction/damage and potential sepsis-induced coagulopathy during the acute phase of the disease, which in severe cases can increase the risk of thrombosis . IncreasPost-mortem histological similarities suggest that Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV), the cause of a previous endemic between 2002 and 2003, also causes ARDS with visible localized pulmonary hemorrhage, pulmonary oedema, desquamation with hyaline membrane formation and interstitial mononuclear inflammatory infiltrates. Localized pulmonary arteriolar thrombosis observed with SARS has not yet been described in autopsy reports of patients with COVID-19 . PulmonaThrombocytopenia and increased D-dimer levels are consistently associated with an increased need for mechanical ventilation, admission to the intensive care unit (ICU) or death ,13. The Thrombotic microangiopathy (TMA) can occur in many different clinical conditions, including pathogenic complement activation. The complement system mediates the innate immune response that promotes inflammation, defends against bacterial infections, and often neutralizes infectious viruses . Two murExcessive complement activation occurs in humans in a number of pathological settings, leading to diffuse TMA and end-organ dysfunction, e.g., atypical hemolytic-uremic syndrome (aHUS), a rare disorder of uncontrolled complement activation characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure. TMA in aHUS results in renal dysfunction and, in rare cases, cardiac dysfunction. Importantly, aHUS is treatable with eculizumab, a C5 complement inhibitor. Early treatment with eculizumab can reverse both renal and cardiac dysfunction . AlthougThere are several potential control points in the pathophysiological cascade COVID-19, starting from the initial infection to later development of ARDS where targeted therapeutic interventions could reduce the severity of disease. There is a role for dexamethasone in the treatment of ARDS in moderate to severe COVID-19 infection; dexamethasone reduces mortality and has become the standard of care in addition to using anti-viral and immunomodulatory therapies. Excessive systemic inflammation in patients with severe COVID-19 is likely to deplete levels of Vit C, Vit D and Zn in many individuals. Several human and animal studies highlight the potential efficacy of supplementation with a combination of Zn, intravenous Vit C and oral Vit D. Inhibition of IL6 by tocilizumab shows beneficial effects in several clinical trials and could reduce microthrombosis. Moreover, when used at appropriate doses, these treatments generally have an exceptionally good safety record. Aspirin , the macrolide antibiotic azithromycin, oral or intravenous administration of NAC (N-acetylcysteine) has a role in inhibiting NF-κB and reducing the activation of the coagulation cascade in severe cases of COVID-19 .p = 0.0033) in patients with COVID-19 .,26.25,26COVID-19 . Levels COVID-19 . Nearly COVID-19 ,14. Incrp < 0.0001), which was higher compared to other biomarkers such as D-dimers and lymphocyte counts [p < 0.001) or non-invasive  ventilation, develop complications such as ARDS  or acute kidney injury . Clinicians should remain alert that increased levels of hs-TnI can also be related to non-ischemic causes of myocardial injury and thereby avoid inappropriate use of other resources [Increased troponin levels in patients with COVID-19 are associated with poor outcomes , but thee counts . Anothere counts . These pesources .Venous thromboembolism (VTE) is common in patients with COVID-19, although the prevalence remains unknown. A recent scoping review reported the incidence of VTE to be 20%, with a risk of stroke of 3%; both VTE and risk of stroke are increased in severely ill patients . ARDS inTherapeutic anticoagulation is the mainstay of VTE management in patients either with or without COVID-19 ,41,42. PPatients with mild symptoms of COVID-19 should stay at home and the routine use of thromboprophylaxis is not recommended; they should be assessed for potential risks of VTE or bleeding and should continue anticoagulant treatment for other indications. Such patients should be counselled on the transition to DOAC after considering the risks of bleeding, potential drug interactions, affordability and availability of drugs, and recent INR status. There may be limitations to monitoring INR at home or at nearby laboratories due to the risk of exposure to SARS-CoV-2. Patients not suitable for treatment with DOAC should use LMWH as a reasonable alternative. Patients with a stable INR and who did not require changes in dosage within the last six months can safely continue warfarin therapy .Hospitalized patients with moderate to severe COVID-19 should be assessed for risks of VTE and DIC. Routine screening for VTE  in hospitalized patients with COVID-19 with elevated D-dimer levels (>1500 ng/mL) is not currently recommended. Signs of active bleeding should be monitored if DIC is suspected or confirmed. Every patient with moderate to severe symptoms should be offered thromboprophylaxis if not strictly contraindicated . The choice of drugs, the dose and duration of treatment should follow national guidelines. Laboratory data monitoring, especially of D-dimers, should be checked every 2−3 days. Intermediate or therapeutic doses of thromboprophylaxis should be used at the discretion of the treating physician based on the risk of bleeding. A study of 92 ICU patients indicates a 21% overall rate of hemorrhagic events, of which nearly half (48%) received anticoagulation treatment . ParenteProphylactic anticoagulation should be administered to patients with moderate or severe COVID-19 diagnosed with DIC but without significant bleeding. There are currently insufficient data to consider routine therapeutic or intermediate-dose parenteral anticoagulation with UFH or LMWH in hospitalized patients with COVID-19 with suspected or confirmed DIC but with no overt bleeding. It is reasonable to consider the indications for anticoagulation therapy during dose adjustment or discontinuation in patients with moderate or severe COVID-19 already receiving chronic anticoagulation treatment and who develop suspected or confirmed DIC without overt bleeding. A common recommendation in such conditions is to reduce the dose of anticoagulant if the thrombotic risk is not excessive ,46. PatiRisk assessment of VTE is reasonable when using pharmacological prophylaxis for up to 45 days post discharge. Pharmacological prophylaxis should be considered if there is an elevated risk for thrombotic events without a high bleeding risk. Patients should be counselled on the importance of ambulation and physical activity at home [Decisions regarding percutaneous coronary intervention or fibrinolytic therapy should be taken after assessing the severity of ST-elevation myocardial infarction (STEMI) and potential COVID-19 in patients and transmission risk to clinicians and healthcare providers .Post discharge extended thromboprophylaxis is recommended with LMWH or DOACs; even though these therapies reduce the risk of VTE, there remains the risk of bleeding events, including major bleeding ,52,53,54In view of the hemostatic derangements discussed above and from observations of other viral illnesses, some clinicians prefer the use of intermediate- or full-dose parenteral anticoagulation (rather than prophylactic dosing) for routine care of patients with COVID-19 based on the hypothesis that it could prevent microvascular thrombosis ,57. HoweHospital-associated venous thromboembolism (HA-VTE) includes VTE presentation while hospitalized, and for up to 90 days post-discharge. Patients infected with COVID-19 are at increased risk of HA-VTE, especially if they become immobilized during critical care. It is unclear if hospitalized patients with COVID-19 are at increased risk for VTE compared to other patients with chest infections and elevated D-dimer values. Elevated D-dimer levels can also be used in a scoring system to identify those at increased risk of VTE ,59. PatiThe management of PE requires a multidisciplinary team ,61,62,63A lack of data makes it difficult to recommend transfusion thresholds in patients with COVID-19 that differ from those recommended for other critically ill patients. The prophylactic transfusion of platelets, use of fresh frozen plasma, fibrinogen, and prothrombin complex concentrate may be considered if invasive procedures are planned . Lastly,9/L in DIC patients with active bleeding or >20 × 109/L in those with a high risk of bleeding or requiring invasive procedures, (b) fresh frozen plasma (15 to 25 mL/kg) in patients with active bleeding with either prolonged PT or aPTT ratios  or decreased fibrinogen (<1.5 g/L), (c) fibrinogen concentrate, or cryoprecipitate in patients with persisting severe hypofibrinogenemia (<1.5 g/L), and (d) prothrombin complex concentrate if fresh frozen plasma transfusion is not possible. Tranexamic acid is not recommended for routine use in COVID-19-associated DIC.Clinically overt bleeding is uncommon in patients with COVID-19. Bleeding in COVID-19-associated DIC requires support with blood products and should be managed as per local guidelines . The guiThe main management goals for patients with pre-existing or new onset thrombotic disease but without COVID-19 is to provide adequate antithrombotic protection, while minimizing physical contact between patients and healthcare workers. Outpatient management or early discharge for acute VTE is recommended whenever possible , and it In general, pharmacotherapy in patients without COVID-19 but with thrombotic disease should be provided according to usual management plans. There is little evidence that antiplatelet agents or anticoagulants increase vulnerability to infection with COVID-19, or of developing severe COVID-19. Patients education on (a) self-monitoring of symptoms is important, and (b) visits to the emergency department for minor bleeding is discouraged.Patients receiving vitamin K antagonist who need frequent INR checks face logistical challenges due to the lockdowns, with an increased risk of exposure to SARS-CoV-2 in public places. It is useful to consider alternatives such as extended INR testing intervals if previous INR values were stable . Other aWhenever switching of anticoagulant is planned, care should be taken to ensure the patients of affordability and accessibility of the agent. DOACs should be used with caution in the elderly  and those with acute kidney injury/renal impairment. Contraindications to treatment with DOACs include patients with mechanical heart valves, valvular atrial fibrillation (AF), antiphospholipid syndrome (APLS), concomitant use of drugs that inhibit cytochrome P450-family 3-subfamily A, and P-glycoprotein and patients who are pregnant or breastfeeding. Patient education on appropriate dietary habits while receiving vitamin K antagonists is also important. The use of LMWHs should be considered in cases where DOACs are not available or not approved by insurance providers.Multiple studies reported that environmental and physiological changes occur during routine commercial flights that could lead to mild hypoxia and gas expansion, and which can exacerbate chronic medical conditions or even induce acute in-flight medical events. Long-haul flights increase the risk of VET several fold. COVID-19 has at least two effects relevant to air travel. Unlike normal pneumonia, in which patients experience cough, chest discomfort and significant breathing difficulties, patients with COVID-19 pneumonia initially may not always experience such symptoms, causing a condition termed “silent” or “happy” hypoxia. This may be aggravated by the hypobaric cruising cabin altitude pressure. Silent hypoxia occurs because the virus only causes the air sacs to collapse to reduce oxygen levels without affecting the removal of carbon dioxide. It is important to detect hypoxia in these patients before they begin to experience dyspnea so that an early intervention can prevent the lungs from deteriorating further. Secondly, COVID-19 is associated with coagulopathy and endothelial damage resulting in VTE. As many economies return to normal, commercial aircrafts will resume operations whilst implementing preventative strategies. Pre- and on-board pulse oximetry screening can be used for early detection of silent hypoxia in unwell passengers boarding aircrafts. This is a simple procedure and can avoid on-flight emergencies. Identified risk factors may be accentuated by the procoagulant effects of undiagnosed COVID-19 that can increase the risk of VTE’s. The “new normal” pre- and post-travel consultation will need to take all these considerations into account. Travelers with known risk factors  should consider appropriate thromboprophylaxis .Many patients become sedentary given the recommendations to remain at home, and are thus at increased risk for VTE ,72,73. TAs daily routines continue to be disrupted, many will experience dietary changes  that can affect treatment with vitamin K antagonists. As the quarantine measures become even more restrictive, changes in physical activity, diet and vitamin K intake are likely to impact INR values further.The COVID-19 pandemic has devastated the economy of many countries, with the United Nations estimating that COVID-19 could cost the world economy more than $1 trillion in 2020 ,76. ThisGovernments have enacted mandatory home quarantine for all non-essential personnel in areas most affected by COVID-19. There are several issues to consider related to thrombotic disease at a community level:More information and data are needed to better define thromboembolic disease due to de novo COVID-19 and differentiate it from pre-existing thrombotic disease to guide optimal management strategies. A large international registry is currently accruing data from COVID-19 patients with VTE ,67 and a


The world-wide Coronavirus Disease 2019 (COVID-19) pandemic was triggered by the widespread of a new strain of coronavirus named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Multiple studies on the pathogenesis of SARS-CoV-2 have been conducted immediately after the spread of the disease. However, the molecular pathogenesis of the virus and related diseases has still not been fully revealed. In this study, we attempted to identify new transcriptomic signatures as candidate diagnostic models for clinical testing or as therapeutic targets for vaccine design. Using the recently reported transcriptomics data of upper airway tissue with acute respiratory illnesses, we integrated multiple machine learning methods to identify effective qualitative biomarkers and quantitative rules for the distinction of SARS-CoV-2 infection from other infectious diseases. The transcriptomics data was first analyzed by Boruta so that important features were selected, which were further evaluated by the minimum redundancy maximum relevance method. A feature list was produced. This list was fed into the incremental feature selection, incorporating some classification algorithms, to extract qualitative biomarker genes and construct quantitative rules. Also, an efficient classifier was built to identify patients infected with SARS-COV-2. The findings reported in this study may help in revealing the potential pathogenic mechanisms of COVID-19 and finding new targets for vaccine design. In late 2019, the Coronavirus Disease 2019 (COVID-19) pandemic was triggered by the spread of a new strain of coronavirus named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). With the first confirmed case reported, the pandemic has rapidly spread all over the world, affecting 227 countries and territories. Based on the reported public health statistics from World Health Organization and Johns Hopkins University , more thvia respiratory droplets and indirect contact via contaminated surfaces, especially for raw processed foods. Therefore, lockdown of epidemic areas  from Gene Expression Omnibus database at1 . A totalBoruta feature filtering  is usual2, which was implemented by python. Default parameters were used for convenience.This study adopted the program of Boruta downloaded from a public websitex and y can be computed byIrrelevant features (genes) were excluded by Boruta method. The remaining features were further analyzed by the mRMR method . This mep(x) is the marginal probabilistic density of x and p is the joint probabilistic density of x and y. Evidently, the higher the mutual information is, the stronger associations of the two variables are. The importance of a feature evaluated by mRMR is reflected by its rank in a feature list. To construct such list, mRMR method performs a loop procedure. Initially, an empty list is constructed. Features are added to such list one by one in a way that each loop determines an added feature. In each loop, for each remaining feature, calculate its relevance to class labels and mean redundancy to features already in the list. A feature with the maximum difference of relevance and mean redundancy is picked up and appended to the list. When all features are in the list, the loop stops. The obtained feature list was called the mRMR feature list in this study.where 3. Similar to the program of Boruta, default parameters were used.This study adopted the mRMR program downloaded from another public websiteBy integrating a supervised classification algorithm, IFS can be used to determine the optimal number of features used to build a classifier with best performance . Based ox. Compute its distance to other samples in the same category. Some samples with the smallest distances are picked up. From these samples, randomly select one sample, say y, and the linear combination of x and y is deemed as the new sample. Such new sample is added to the minor category. Above procedures execute several times until the predefined number of new samples have been generated. For the used dataset, each category contained 100 samples after SMOTE was applied on it. This study used the tool “SMOTE” from Weka  approach  was adoprom Weka , which iIn this study, the SMOTE approach was only used when evaluating the performance of classifiers in the IFS method. It was not used in the procedure for evaluating features.As mentioned in Section 2.4, IFS method needs a classification algorithm. This research tried four classification algorithms: (1) RF , (2) supRandom forest is widely adopted in the investigation of biological and biomedical data , and it Support vector machine is another classic classification algorithm and also has wide applications in bioinformatics and computational biology ,b. Such s, determination of its class consists of the following steps: (1) calculate the distances  between s and all samples in the training dataset; (2) find out k training samples with the smallest distances; (3) determine the class of s according to the distribution of classes of these k training samples, i.e., the class with the most frequency is assigned to s. In this study, the kNN implemented in the Scikit-learn package was adopted.K-nearest neighbor  is a simDecision tree  attemptsThe MCC  was adopX has binary values indicating the predicted sample class, matrix Y also has binary values representing the true classes of all samples, and cov is the covariance of two matrices. MCC has a value ranging between −1 and +1. When the classifier achieves the best performance, MCC equals +1.where matrix Besides, the accuracy on each category and overall accuracy (ACC) were also computed, which can give full evaluation on the performance of different classifiers.Regarding the recently reported transcriptomic data on 234 ARI patients, which included 93 patients infected with SARS-COV-2, 100 patients with other viruses, and 41 patients with no viral infection, we employed several advanced machine learning algorithms on such data. The aim was to extract essential biomarker genes and rules of SARS-COV-2. The whole procedures are illustrated in 15,979 gene features were observed and collected in the transcriptomic data. Evidently, not all of them are related to ARI patients with SARS-COV-2 or other viruses. The Boruta approach was first applied on the transcriptomic data. 179 features were selected, which are provided in 179 features selected by Boruta were further analyzed by the RMR method. An mRMR feature list was obtained, which is also available in Based on the mRMR feature list, IFS was carried out with the interval set to 1. 179 feature subsets were constructed. Given a feature subset and a classification algorithm, a classifier was built on samples represented by features in the subset. tenfold cross-validation was employed to evaluate the performance of each classifier. When evaluating the performance of different classifiers, SMOTE was applied to produce balance data to improve the efficiency of each classifier. The performance of different classifiers is provided in The optimum DT classifier used the top 67 features and yielded the MCC of 0.696. Although its performance is much lower than other three optimum classifiers, especially the optimum RF classifier, such classifier can induce several IF-THEH format rules, which can provide much biological insights to uncover the SARS-CoV-2 infection and its difference from other viral infection. Thus, we constructed a DT on all samples represented by top 67 features. Then, 24 rules were obtained, which are provided in As described and summarized above, using the transcriptomics data from the upper respiratory tissues, we identified a group of potential biomarkers that can reveal the differences between SARS-CoV-2 infection and other diseases caused by viral infections, confirming the potentials of such biomarkers to contribute to the clinical diagnosis of COVID-19 and the development of new drugs/vaccines against such virus. Although current studies on COVID-19 are still limited, and many potential pathogeneses of such infectious diseases have not been revealed, all the identified biomarkers, together with related quantitative rules, are related to COVID-19-associated pathogenesis, according to recent publications.Based on our newly presented computational methods, we identified a group of significant genes that contribute to the discrimination of different infection statuses . According to recent publications, some top genes participate in the discriminative biological processes of upper respiratory tissue cells under either physical or pathological conditions. Here, we analyzed the top five genes, which are listed in PSMB8 (ENSG00000204264). PSPM8 participates in the regulation of influenza virus replication in infection-associated cells such as respiratory epithelial cells , which participates in the tumorigenesis of colorectal cancer . According to the data source (Eran’s publication)  , this geal cells , implyinLGALS3BP (ENSG00000108679) and IRF9 (ENSG00000213928). Both have been confirmed to be expressed in upper airway tissues  are shown to be down-regulated in this rule, contributing to the prediction of patients with other virus infection. According to recent publications, in 2009, researchers from Duke University confirmed that IFI6 was shown to be down-regulated during the pathogenesis of influenza and other symptomatic respiratory viral infections (RPS6KA5 (ENSG00000100784), as the second parameter gene, has been shown to be down regulated during SARS-CoV-2 pathogenesis comparing to other virus infection. In 2017, researchers from Brazil reported that patients with Zika virus infection has specific expression level on such gene (DNAJB1 (ENSG00000132002), CXCL9 (ENSG00000138755), and CD8A (ENSG00000153563) are well-known immune response associated proteins. As reported, DNAJB1 has been shown to be down-regulated during the infection of influenza A virus  during SARS-CoV-2 infection was reported (BTN3A3 (ENSG00000111801), PRMT7 (ENSG00000132600) and COLCA2 (ENSG00000214290) are all proliferation associated genes, which may participate in the repair procedures after lung tissue damage caused by viral infection. The low expression level of such three genes have been reported to be associated with infection of different virus subtypes involving different tissues but not SARS-CoV-2 (The first rule (Rule 0) involved in eight parameters, contributing to the identification of patients with virus infection but not with the SARS-CoV-2 infection. The first parameter fections . Howeveruch gene , partial A virus , but notnfection . Thereforeported , helpingRS-CoV-2 , helpingIFI6 (ENSG0000 709). As we have discussed above, no direct reports confirmed that such gene has specific expression level during SARS-CoV-2 infection. However, with reverse direction of expression level, this parameter can help us to exclude patients with other common infections. As for the second and third parameter, TIMP1 (ENSG00000102265) and LGR6 (ENSG00000133067) has been reported to be associated with various kind of virus, including SARS-CoV-2 (HMOX1 (ENSG00000100292), researchers from University of Queensland have confirmed that the low expression level of such gene are associated with SARS-CoV-2 infection (As for the second rule (Rule 1) involving four parameters, such rule helps us to identify patients with SARS-CoV-2 infection. Although up to now, there are still few publications presenting the host-virus relationships specifically for SARS-CoV-2 infection, we still found strong supports for this rule. The first parameter is RS-CoV-2 . Therefonfection , correspIFI6 (ENSG00000126709). We have discussed above that it is down-regulated during other virus infection but not SARS-CoV-2 infection, which is correspondence with the expression tendency in this rule. The next gene is TIMP1 (ENSG00000102265) which has been shown to be related to SARS-CoV-2 infection with lower expression level during pathogenesis (SPATS2L (ENSG00000196141), it has been reported that such gene are up-regulated during the proliferation of B cells (CXCL9 (ENSG00000138755), which is tightly correlated with SARS-CoV-2 infection as we discussed above. The direction of such parameter is the same as its regulatory directions during SARS-CoV-2, while the absolute value of its expression level is significant lower in normal controls comparing to SARS-CoV-2 patients.For the third rule (Rule 2), which help us identify patients without virus infection, four parameters were involved. The first parameter is also gene ogenesis . In this B cells , which iAll in all, as we have discussed above, the top quantitative rules have been supported by recent publications, validating the reliability of the obtained rules.TRO (ENSG00000067445), TIMP1 (ENSG00000102265), IFI6 (ENSG00000126709), LGR6 (ENSG00000133067), WDR74 (ENSG00000133316), IFI44L (ENSG00000137959), and FAM83A (ENSG00000147689) were reported by both of two studies. As we have discussed above, genes FAM83A, IFI6, TIMP1, and LGR6 have already been discussed above, confirming their significant roles for distinguishing SARS-CoV-2 and other diseases. As for WDR74 (ENSG00000133316), IFI44L (ENSG00000137959), and TRO (ENSG00000067445), recent publications have also confirmed their correlations with SARS-CoV-2. Recently, a single-cell sequencing based analyses on the peripheral mononuclear cells identified IFI44L as one of the potential biomarkers to monitoring immune responses of SARS-CoV-2. As for WDR74 and TRO, although no direct evidence confirmed their correlations with SARS-CoV-2, both of them have already been shown to be correlated with coronavirus infection (The COVID-19 pandemic now has turned into a world-wide pandemic. Large number of researchers from all over the world have been working on the biological and epidemic characteristic of such virus. In May, 2020, researchers from University of California, San Francisco has identified some altered gene expression patterns in the upper airway during the pathogenesis of SARS-CoV-2 , which cnfection , implyinPSMB8, COLCA2, LGALS3BP and IRF9 as discussed in Section 4.1. Besides biomarker genes, our study extracted several rules, listed in Furthermore, this study also reported some exclusive biomarker genes compared with the previous study, such as As discussed above, all the identified top-ranked qualitative biomarkers and quantitative rules are correlated with the identified COVID-19-associated pathogenesis and contribute to distinguishing COVID-19-infected cases from other respiratory patients with or without virus infection, validating the efficacy and accuracy of our prediction. Therefore, the application of machine learning model may efficiently assist in the identification of potential diagnostic biomarkers and candidate drug targets and help establish a standard workflow for related analyses in such field.https://www.ncbi.nlm.nih.gov/geo/.Publicly available datasets were analyzed in this study. This data can be found here: TH and Y-DC designed the study. Y-HZ, HL, and ZL performed the experiments. Y-HZ, HL, TZ, and LC analyzed the results. Y-HZ and HL wrote the manuscript. All authors contributed to the research and reviewed the manuscript.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.


Diffusion MRI allows non-invasive assessment of white matter connectivity in typical development and of changes due to brain injury or pathology. Probabilistic white matter atlases allow diffusion metrics to be measured in specific white matter pathways, and are a critical component in spatial normalisation for group analysis. However, given the known developmental changes in white matter it may be sub-optimal to use an adult template when assessing data acquired from children.By averaging subject-specific fibre bundles from 28 children aged from 6 to 8 years, we created an age-specific probabilistic white matter atlas for 12 major white matter tracts. Using both the newly developed and Johns Hopkins adult atlases, we compared the atlas to subject-specific fibre bundles in two independent validation cohorts, assessing accuracy in terms of volumetric overlap and measured diffusion metrics.Our age-specific atlas gave better overall performance than the adult atlas, achieving higher volumetric overlap with subject-specific fibre tracking and higher correlation of FA measurements with those measured from subject-specific fibre bundles. Specifically, estimates of FA values for cortico-spinal tract, uncinate fasciculus, forceps minor, cingulate gyrus part of the cingulum and anterior thalamic radiation were all significantly more accurate when estimated with an age-specific atlas.The age-specific atlas allows delineation of white matter tracts in children aged 6-8 years, without the need for tractography, more accurately than when normalising to an adult atlas. To our knowledge, this is the first publicly available probabilistic atlas of white matter tracts for this age group. Using a white matter atlas eliminates the need for computationally intensive methods of delineating tracts by segmenting streamlines generated by tractography . This isThe widely used Johns Hopkins University (JHU) white matter tract atlas  is constIn this study, we used robust tract reconstruction protocols  to develAs a demonstration, we then investigated tract-level differences in children treated with therapeutic hypothermia (TH) for neonatal encephalopathy (NE) at birth, compared with healthy controls, and compared results obtained using the age-specific atlas to those from the JHU atlas. The children who had TH, do not have CP and are in mainstream education still exhibit significantly reduced performance on cognitive tests  and have22.1Ethics approval was obtained from the North Bristol Research Ethics Committee and the Health Research Authority (REC ID: 15/SW/0148). Informed and written consent was obtained from the parents of participants before collecting data. The cohort was made up of 36 healthy children aged 6-8 years with no evidence of neurological disease, originally recruited as controls for a study of the long-term effects of TH (“CoolMRI”) on behavioural and imaging outcomes. The 36 controls were split randomly into 28 atlas and 8 validation subjects such that the group were matched for age, sex, socio-economic status (SES) and full-scale intelligence quotient (FSIQ). A further 15 validation subjects were obtained from an open data source see . For the2.2a, b; -2 in 60 diffusion directions, equally distributed according to an electrostatic repulsion model, as well as 8 interspersed b = 0 images, with one data set acquired with positive phase encoding steps, then repeated with negative steps , giving a total of 136 images.DWI data were acquired with a Siemens 3 tesla Magnetom Skyra MRI scanner at the Clinical Research and Imaging Centre (CRiCBristol), Bristol, UK. Subjects were placed supine within the 32-channel receive only head-coil by an experienced radiographer, and head movement minimised by means of memory-foam padding. Children wore earplugs and were able to watch a film. A multiband echo-planar imaging sequence was used with the following parameters: TE = 70 ms; TR = 3150 ms; field of view 192 × 192 mm; 60 slices; 2.0 mm isotropic voxels; phase encoding in the anterior-posterior direction, in-plane acceleration factor = 2 , throughSetsompop et al., 20122.3The quality of the diffusion data was assessed using the EddyQC tool  from FSL2.4DWI data were corrected for eddy current induced distortions and subject movements using EDDY  and TOPUThe process of generating probability maps from the whole-brain tractograms is summarised in To delineate the fornix, streamlines were included which pass through the body of the fornix and either of the posterior limbs which project to the hippocampus . These wFor spatial normalisation, the average diffusion weighted image (aDWI), created for each subject by averaging all DWI images, was registered to the JHU aDWI template by 12-degree of freedom affine registration using FSL’s FLIRT  (note thhttps://neurovault.org/collections/LWTAAAST/).This atlas is available at Neurovault  and each subject-specific tract by measuring the Dice score  over a r2.5.2i is the FA in voxel i and Pi is the probability in voxel i. We then calculated the correlation between the probabilistic FA and individual FA . In subject-specific tracts, average FA was calculated by taking the mean FA in all masked voxels. In the probabilistic atlases (age-specific or JHU), the FA was weighted by the probability in each voxel using the following equation: l FA see .2.5.3Whole-tract average FA was calculated in each subject, using both probabilistic and subject-specific tracts. Average FA was calculated in probabilistic tracts using equation  and in s2.5.4http://fcon_1000.projects.nitrc.org/indi/cmi_healthy_brain_network/)  , a data-volution  and tracvolution . This alvolution , in addi-2). For each subject, FSL’s eddy_correct was used to register all volumes to the b = 0 image (but not correct for eddy current induced distortions). Tractography was then performed using the tensor-based algorithm described above.In order to assess whether the modern scanning protocols and preprocessing tools (such as the multi-shell acquisition and susceptibility-induced distortion correction with TOPUP) favoured the age-specific atlas over the JHU adult atlas (which was produced before these tools were available) we also assessed performance using a minimally processed version of the HBN validation cohort. To produce this dataset, we used single-shell data from each subject . We then tested for group differences in whole-tract FA. Bilateral tracts were tested separately. For comparison, we repeated the analysis using the JHU adult atlas. In the absence of ground truth, only a qualitative comparison of results obtained with the two atlases was performed.2.7To assess whether the age-specific atlas gave better volumetric agreement with subject-specific tracts than the JHU adult atlas, we performed a two-tailed, paired t-test comparing the peak Dice scores.In the slice-wise FA analysis and whole-tract FA analysis, we measured the correlation between atlas measurements and individual measurements using a repeated measures correlation coefficient , which uFor each validation method, we compared the correlation coefficient given by the age-specific atlas with that given by the JHU adult atlas, by applying Fisher’s z-transform to each correlation coefficient and estimating the 95% confidence intervals of the difference between these z-transformed correlation coefficients. The confidence intervals were estimated with a percentile bootstrap method . In the In the CoolMRI demonstration, Mann-Whitney U tests were applied to test for differences in the median FA between cases and controls in each tract, with Bonferroni correction applied to correct for family-wise error. Significant results have corrected p < 0.05.33.1The CoolMRI study recruited 51 children, without CP, treated with TH for NE at birth and 43 control children matched for age, sex and SES . Of the 3.23.33.3.1The Dice score at a range of thresholds is plotted for each tract for the same-site validation data in 3.3.2The correlation between slice-wise FA measured by the age-specific atlas and that measured from subject-specific tracts is shown for the same-site validation data in 3.3.33.4Numerous tracts in children treated with TH for NE had reduced FA compared to controls see . After B4This study introduces an age-specific probabilistic white matter atlas constructed from children aged 6-8 years, providing a method of delineating white matter tracts without tractography. We have shown that this atlas accurately delineates tracts in children from a same-site cohort, and a cohort from a different site, imaged with different scanner and acquisition protocol. The strong correlation between FA sampled by the atlas and that measured in each individual (i.e. the “gold standard”), at both a whole-tract level and slice-wise level, shows that the atlas provides an accurate estimate for the underlying white matter diffusion properties. Additionally, the Dice scores between tracts in the atlas and those delineated by subject-specific tracts were higher with the age-specific atlas than with the JHU adult atlas, demonstrating improved anatomical accuracy of the age-specific atlas. In these validation methods, the age-specific atlas performed better than simply registering to an existing adult white matter tract atlas, as is routinely done in the literature. As a demonstration, we applied the age-specific atlas to the CoolMRI study, revealing significantly reduced FA in several major white matter tracts in children treated with TH for NE at birth compared to healthy controls.The correlation of whole-tract FA measured by the atlas with that measured in subject-specific tracts offers quantification of the performance of the atlas as a whole. In the same-site validation data, the HBN data with FOD-based tractography, the HBN data with tensor-based tractography and the HBN data with minimal processing and tensor-based tractography, the age-specific atlas exhibited stronger correlation with the individual measurements than for the JHU atlas , Table 2Those tracts which exhibit low correlation between atlas and individual slice-wise FA measurements  have very little spread in FA values, resulting in tightly grouped measurements with a low correlation coefficient . For theLong, thin tracts, such as the CST, IFOF and ILF, are particularly susceptible to partial volume effects when measuring volumetric overlap; a small radial translation will result in a large change to the Dice score. In these tracts, the high correlation in sampled FA values shows that the age-specific atlas gives accurate measurement of the tract diffusion properties.Multi-site validation alleviates bias associated with using the same scanner for validation data and atlas construction, thus validation with the HBN data demonstrates that the age-specific atlas generalises to data from a different site, acquired with a different scanning protocol. In this dataset, the age-specific atlas gave better correlation of whole-tract FA measurements , Table 2Further bias may be introduced by the use of the same tractography algorithm for both atlas generation and in estimating diffusion metrics for the validation data. Therefore, we also included a validation dataset in which subject-specific fibre bundles were delineated using a tensor-based tractography algorithm. Whereas the FOD-based tractography algorithm used to construct the age-specific atlas uses spherical deconvolution to find the peak FOD in the closest orientation to the propagating streamline, the tensor-based algorithm propagates the streamline along the principal eigenvector of the diffusion tensor at each step. This is comparable to the tensor-based tractography algorithm used in the construction of the JHU adult atlas, thus providing a conservative test case for validation. Despite this bias towards the JHU atlas, the age-specific atlas still gave stronger correlation of whole-tract FA measurements. In the tests of volumetric overlap  and slicThis introduces the question of how to provide the “gold-standard” of fibre tracking; the tensor-based algorithm was used in one of the HBN datasets in order to eliminate bias towards the age-specific atlas (by introducing bias towards the JHU adult atlas). However, this category of fibre tracking algorithm is widely acknowledged to give poor characterisation of diffusion in brain white matter due to its inability to resolve crossing fibres . Thus, tIn future, as well as providing coverage of other age ranges, atlases could offer more extensive labelling of additional tracts and regions of white matter throughout development. A comprehensive database of fibre bundles across a range of ages, potentially constructed by applying automated tractography-based white matter tract segmentation protocols  to data Applying the age-specific atlas to the CoolMRI study to investigate group differences in tract-level FA revealed selective reduction in FA, that was significantly reduced in the left CG, left CH, left SLF and the fornix . For comPrevious studies of neonates treated with TH for NE have investigated the relationship between white matter diffusion properties, measured in the first weeks following birth, and neurodevelopmental outcome at 2 years of age. These studies found a significant reduction in FA in infants with adverse outcomes, compared to those with favourable outcomes, in widespread areas of white matter including, but not limited to the corpus callosum, anterior and posterior limbs of the internal capsule, external capsule, fornix, cingulum, and ILF . Many of5The age-specific atlas provided by this study has been shown to robustly delineate white matter tracts in children aged 6-8 years. FA measurements sampled by the atlas correlate strongly with those measured by individual fibre tracking, allowing reliable investigation of white matter diffusion properties in cohorts. The closer agreement between FA measured in individually identified tracts and that estimated when registering to an age-specific atlas, suggests that such an approach would increase sensitivity to group differences, and is recommended for all studies performed in children.Supplementary Materials


No significant association was observed in postmenopausal women. In addition to a luminal phenotype and axillary lymph node involvement, low levels of n-3 LC-PUFA in breast adipose tissue may constitute a risk factor that contributes to breast cancer bone metastases formation in premenopausal women.In the present study, we investigated various biochemical, clinical, and histological factors associated with bone metastases in a large cohort of pre- and postmenopausal women with breast cancer. Two hundred and sixty-one consecutive women with breast cancer were included in this study. Breast adipose tissue specimens were collected during surgery. After having established the fatty acid profile of breast adipose tissue by gas chromatography, we determined whether there were differences associated with the occurrence of bone metastases in these patients. Regarding the clinical and histological criteria, a majority of the patients with bone metastases (around 70%) had tumors with a luminal phenotype and 59% of them showed axillary lymph node involvement. Moreover, we found a negative association between the levels of  Breast cancer is the most commonly diagnosed cancer in women and is the leading cause of cancer death with 2,088,849 new cases and 626,679 specific mortality worldwide in 2018 [Several clinical studies have been conducted to identify the demographic, clinical, pathological, and genetic factors influencing BM development in breast cancer patients. They have reported a number of risk factors for BM, and they include axillary lymph node involvement, a large tumor size, and expression of the estrogen receptor by the tumor. Tumor phenotype is, therefore, widely accepted as a major risk factor since patients with luminal subtype tumors are at an increased risk of developing BM compared to patients with other breast cancer phenotype . In a reBisphosphonate remains the drug of choice to limit bone resorption, pain, and skeletal-related events, in addition to delay bone metastasis recurrence. Nonetheless, a collaborative meta-analyses from the early Breast Cancer Trialists’ Collaborative Group has recently observed reduced bisphosphonate or other drug efficacy in the treatment of bone lesions in premenopausal patients compared to postmenopausal patients . This obn-specific metastases, several studies have proposed alternative or complementary hypotheses to explain the existence of preferred metastasis sites for each cancer type or determine how metastasis growth can be supported by the host tissue microenvironment [The molecular pathways that regulate bone metastasis formation involve several steps, which include breast cancer cell detachment from the primary site, homing to the bone site with attachment and extravasation through the vascular barrier, colonization, and proliferation of breast cancer cells in the bone with a complex crosstalk between breast cancer cells and the bone microenvironment ,8,9. In ironment . Similarironment .n-6 and n-3 polyunsaturated fatty acids  content may be associated with a modulation of breast cancer risk [n-3 polyunsaturated fatty acids (n-3 PUFA) has been shown to have beneficial effects in various types of cancer including breast cancer [n-6 and n-3 PUFA) can also be evaluated by food frequency questionnaire. Nevertheless, numerous studies have shown that the fatty acid composition of adipose tissue can directly be used as a surrogate for the evaluation of past lipid intake [n-3 long-chain polyunsaturated fatty acids (LC-PUFA) capsules [The lipid composition of the diet in terms of saturated, monounsaturated, and cer risk . In addit cancer ,15. The d intake . In thatd intake ,18, the d intake ,19. Thiscapsules .n-3 LC-PUFA-enriched diets can reduce breast tumor and bone metastasis formation [n-3 LC-PUFA and bone metastasis. In the present study, we selected more than 250 breast cancer patients and examined if the levels of fatty acids  stored in breast adipose tissue are associated with the onset of bone metastasis formation according to the menopausal status. For this purpose, clinical and histological parameters were collected and the fatty acid profile of the mammary adipose tissue was determined by biochemical analysis (via gas chromatography) of cryopreserved adipose tissue samples.Several preclinical studies have reported that ormation ,22, but n = 40 HER2), all patients of this group with tissues harvested and stored in the cryobank between January 2009 and June 2012 were selected using the cryobank database. We also selected all the adipose tissues associated with triple negative tumors (n = 67 triple negative) from the same period. As expected, during this period, the majority of breast tumor phenotypes were of the luminal phenotype, and a selection had to be made. Patients with luminal A or B tumor phenotypes  were also selected throughout the same period according to sample availability and alphabetical order, and they were chosen to match the age distribution of the HER2 and triple negative tumor-carrying patient groups. As much as it was possible, samples of the luminal phenotype groups were matched with HER2 and triple negative patients of the same year. To prevent a potential selection bias, staff involved in patient selection (L. Ouldamer and C. Goupille) was only aware of the patient first letters’ name and their date of birth as well as the corresponding tumor phenotype. A total of 286 samples were selected at the beginning for the study. However, 16 samples were not found in the cryobank, and 9 were not large enough to perform a complete lipid analysis. Therefore, we used 261 tissues samples for which we were able to obtain a complete fatty acid profile. The main outcome was fatty acid composition of mammary adipose tissue according to the occurrence or not of bone metastasis in women managed for invasive breast cancer. The secondary outcome fatty acid composition of mammary adipose tissue according to the occurrence or not of bone metastasis stratified on patient’s menopausal status.In this retrospective study, we analyzed data from consecutive women treated for invasive breast cancer between January 2009 and June 2012 at our Tertiary Breast Care center in Tours (France). The population of patients was from Central France with mostly Caucasian origins. The present study was exploratory in nature, and we initially planned to include approximately 250 consecutive patients with tumors representative of all breast cancer phenotypes and for whom adipose tissue biopsies had previously been harvested for fatty acid analysis of triglyceride. The inclusion criterion was women treated between January 2009 and June 2012 for invasive breast cancer for which the associated breast adipose tissue samples had been excised during surgery and stored in our University Hospital cryobank. To have a substantial number of patients with the less frequent HER2 phenotype  analyses were performed by an experienced pathologist (F. Arbion). This pathologist reviewed histology reports, biopsy slides, surgical excision specimens, and determined grade, and tumor phenotype using the estrogen/progesterone receptors and HER2 expression profile .The adipose tissue collection cryobank was authorized and declared to the French Ministry of higher Education and Research (reference number DC-2008-308), and the study was performed with approval of the Tours University Teaching Hospital Review Board.Clinical information was obtained using medical records held at our institution in a computerized database. Post-treatment imaging consisted of breast/chest ultrasound/mammograms once a year. Follow-up visits were conducted every 4 months for the first 2 years, every 6 months for the following 3 years, and once a year thereafter. Disease recurrence was diagnosed by biopsy or imaging techniques adapted to clinical symptoms . Time-to-event analyses were calculated from the date of primary surgery as the starting point and women who were alive and without recurrence were censored at the date of last follow-up. During follow-up, the occurrence of BM was recorded and presented according to the menopausal status.Breast adipose tissue samples and lipid analysis were obtained from 261 patients. Samples were excised during surgery from the external (tumor-free) region of the lumpectomy or mastectomy, and they were stored in liquid nitrogen to avoid degradation until lipid analysis.n-Fallavier, France) and analyzed by capillary gas chromatography  using a polar column  and an on-column injector. Chromatography analysis was supervised and validated by two experienced analytical biochemists (C. Goupille and M. Pinault) blinded to the clinical data. Fatty acid levels were expressed as percentages of the total integrated area. In this study, long-chain fatty acids included fatty acids containing between 20 and 24 carbons.Determination of the adipose tissue fatty acid content was performed as previously described ,20. Briet-test or analysis of variance (ANOVA) for continuous variables. The main outcome measure in the present study was the occurrence of BM. A p-value of less than 0.05 was considered statistically significant. Data were managed with an Excel database  and analyzed with the R 3.0.2 software available online .Characteristics of women, tumor, and adipose tissue composition were analyzed using Chi-square statistics or Fisher’s exact test for categorical variables and p = 0.01) and the proportion of patients with positive axillary lymph node was significantly increased (54.7%) compared to patients without bone metastasis (39.5%) (p = 0.01).Characteristics of the population are described in Among the included women, 159 of them were postmenopausal, among which 32 (20.1%) presented bone metastasis, and 99 presented a premenopausal status, among which 21 (21.2%) presented BM. Three patients (1%) had an unknown menopausal status.The main outcome was to explore fatty acid composition of mammary adipose tissue according to the occurrence or not of bone metastasis in women managed for invasive breast cancer. No significant differences were observed in terms of fatty composition between patients with BM and patients without BM (data not shown).n-3 LC-PUFA levels were significantly decreased in the adipose environment of women with BM (p = 0.02). When examining fatty acids individually, docosapentaenoic acid (DPA) levels were significantly decreased (p = 0.03), and the decrease in docosahexaenoic acid (DHA) levels was marginally significant (p = 0.06).The main fatty acids observed in breast adipose tissue of premenopausal women are presented in In postmenopausal women, levels of the main breast adipose tissue fatty acids are presented in Finally, we compared the characteristics of patients with BM . The pron-3 LC-PUFA in breast adipose tissue and the development of BM in premenopausal women.The aim of this study was to evaluate if alterations in breast adipose tissue lipid composition may be associated with the development of BM in women with breast cancer. A luminal phenotype and lymph node invasion are factors associated with BM, and we found a significant association between low levels of Among the various clinical and histological factors examined, only lymph node involvement appears to contribute to BM, and a relationships between menopausal status or age and BM risk has not been clearly identified . On the n-3 LC-PUFA in breast adipose tissue are associated with the development of BM in premenopausal women. To explain the reduced levels of n-3 LC-PUFA in these patients, several hypotheses can be made. The first one is that PUFA depletion may be related to the presence of a tumor and its metabolism. To supply precursors for the synthesis of membrane phospholipids, fatty acids may be synthetized from nutrients  or directly obtained from the microenvironment. In the past few years, reprogramming of fatty acid metabolism has been the focus of new studies in cancer cells, and targeting lipid metabolism in this context may become a new therapeutic approach for cancer treatment [n-6 PUFAs and n-3 PUFAs in colonic cancer cells compared to normal colonocytes. Low levels of PUFA in adipose tissue may also be observed when specific types of PUFAs are released by adipose tissue during lipolysis [n-6 PUFA and n-3 PUFA) were reduced [n-3 LC-PUFAs by breast tumors suggests other hypotheses, i.e., the specific consumption of n-3 LC-PUFAs associated with the expression of specific fatty acid transporters . To datddressed , but theddressed . If the pathways . For exaferation . In addiferation ,34. Theyferation . In thatn-3 LC-PUFA in adipose tissue of premenopausal women may be reduced n-3 LC-PUFA intake. In fact, the best suppliers of n-3 LC-PUFA  are fish oil, marine fatty fishes, and seafood. n-3 LC-PUFA are essentially obtained via food intake since their endogenous synthesis (by elongation and desaturation) from the essential fatty acid alpha linolenic acid  is extremely reduced in human (less than 0.2% of ALA is converted into DPA or DHA) [The second hypothesis to explain the low levels of  or DHA) .n-3 LC-PUFA-enriched diets can reduce breast tumor bone metastasis formation [n-3 LC-PUFA of the mammary tumor microenvironment [n-3 LC-PUFA can attenuate breast cancer bone metastasis formation and the associated osteolysis by inhibiting the migration of breast cancer cells to the bone as well as by inhibiting osteoclastic bone resorption [n-3 LC-PUFA may associated with bone metastasis. They include action on primary tumor cells and modification of their metastatic capabilities and regulation of tumor cell homing to the bone via CXCR4 [n-3 LC-PUFA deficiencies may lead to foci of bone degradation, which provides a hospitable environment for metastasis formation.Several studies have identified tumor cell profiles associated with bone metastasis formation ,38,39, bormation ,22. Amonironment , a reducironment , a decreironment  have beesorption . Severalia CXCR4 . n-3 LC-n-3 LC-PUFA for bone health are well illustrated in the literature with experimental models. The skeletal metabolism is sensitive to n-3 LC-PUFA-deficient or -enriched diets [n-3 LC-PUFA supplementation can potentiate mesenchymal stem cell differentiation and increased osteogenesis [n-3 LC-PUFA diets are associated with benefits for bone health, and these data have been essentially reported in postmenopausal women in an osteoporosis context [n-like diet with an almost total absence of n-3 fatty acids, a decrease in bone mineral density was observed [n-3 LC-PUFA. Insufficient intake of n-3 PUFA may create a bone imbalanced microenvironment favorable to metastases implantation and/or development.Beneficial effects of ed diets ,49. Seveed diets . More reogenesis , suppresogenesis ,53, inhiogenesis , or inhiogenesis . In huma context . Importaobserved . Taken tn-3 LC-PUFA and bone metastases only observable in premenopausal women. Estrogen is known to promote breast cancer cell proliferation. Importantly, fatty acid metabolism can interfere with estrogen activity and vice versa. A study examining mRNA levels in tumors has shown that tumors with luminal phenotypes favor endogenous fatty acid synthesis, whereas tumors that do not express hormonal receptors are more likely to import fatty acids [n-3 PUFA can alter estrogen-signaling pathways in the model luminal A MCF-7 cell line. In that case, it was shown that n-3 LC-PUFA (EPA and DHA) could inhibit estrogen-mediated proliferative signaling pathways and promote a proapoptotic pathway [n-3 LC-PUFA effects may be more observable in an estrogenic context in patients with hormone-sensitive tumors. Furthermore, taking into account diet consumption, studies have shown that adipose tissues (and blood) from postmenopausal women contain more n-3 LC-PUFA than those from premenopausal women [n-3LC-PUFA levels and bone metastasis formation during the postmenopause. In premenopausal women, double beneficial effects of n-3 LC PUFA on bone and cancer cells may explain, at least in part, the observed protective effects of n-3 PUFAs. The effects of DPA are less described in the literature, but its activities are consistent with those described for EPA and DHA [The final interrogation is why is the association between low levels of ty acids . Accordity acids . It is a pathway . Taken tal women . The reaal women . We cann and DHA .n-3 LC PUFA levels and bone metastasis formation in premenopausal breast cancer patients. Although an efficient activity of bisphosphonates may be likely observed in postmenopausal women [n-3 LC-PUFA supplementation may be an interesting therapeutic approach to promote a beneficial remodeling of the bone microenvironment in premenopausal women. Two distinct bone microenvironments observed in pre- versus postmenopausal women have been reported, and different mechanisms in the development of bone metastasis according to the menopausal status have been described [n-3 LC-PUFA on the bone microenvironment will have to be further explored in premenopausal women.Our study described for the first time in humans an association between al women , n-3 LC-escribed ,63. Howen-3 LC-PUFA dietary intake remains likely, but additional investigations will be required to support this hypothesis. Fourth, despite our precautions, we cannot exclude that the patient selection method, particularly that of patients with a luminal phenotype, may be biased. At the starting point, the objective of this project was to explore the potential relationships between breast cancer aggressiveness and fatty acid composition of breast adipose tissue. We had previously identified an association between inflammatory breast cancer and low levels of n-3 LC-PUFA [n-3 LC-PUFA. Adipose tissue may serve as a biomarker of past lipid intake, but it appears that this statement is particularly more accurate for all PUFAs than SFA and MUFA, as previously and recently shown [n-3 LC PUFA levels and eliminate a potential bias associated with the bone metastasis group size, which remains small in our study.Some limitations deserve to be mentioned. First, we cannot exclude an inherent bias related to the retrospective and exploratory nature of the study, especially concerning the recurrence diagnosis, which represents a concern. However, all the included women were treated at our regional referral center applying the current French/European guidelines after systematic multidisciplinary committee approval. Second, we may not have recorded in full the data regarding subsequent sites of distant metastases after initial distant failure; although such an information may be more readily available in a retrospective institution review. We acknowledge that most events for luminal tumors may occur after many years of follow-up. Third, we did not have access to data regarding the eating habits of our patients. The reduced or lack of  LC-PUFA . In the ly shown ,65. Addin-3 LC-PUFAs in cancer, international organizations recommend that current n-3 LC-PUFA intake be between 200 and 500 mg/day. Our retrospective and exploratory study shows that low levels of n-3 LC-PUFA in the adipose tissue of premenopausal women with breast cancer are associated with bone metastasis formation. Additional studies in human will be required to determine the origin of these low levels of n-3 LC-PUFA before proposing an n-3 LC-PUFA supplementation to prevent or contain bone metastasis in premenopausal women with breast cancer.Alternative therapies are needed for premenopausal women targeting bone tumor cells and to maintain them in a dormant state or modify the bone microenvironment to make it less hospitable. While some experimental and epidemiological studies have suggested a beneficial effect of