Eugenia dysenterica plants. For germination tests, the seeds were soaked in Al solution and evaluated for twenty days in growth chambers. In a second experiment, young plants were cultivated in hydroponic systems with various Al concentrations to evaluate the morphological, anatomical and physiological characteristics of E. dysenterica. Anatomical changes and low germinative vigor were observed in seeds germinated in 600 and 800 μmol Al3+ L−1. In the hydroponic system, 200 μmol Al3+ L−1 stimulated root growth in young plants. The activity of antioxidant enzymes and the accumulation of phenolic compounds were greatest at the highest Al doses, preventing changes in gas exchange and chlorophyll a fluorescence. Starch grain accumulation was noted in plant cells exposed to 200 and 400 μmol Al3+ L−1. Adult E. dysenterica trees also accumulated Al in leaves, bark and seeds. These data suggest that E. dysenterica is tolerant to Al.Native Cerrado plants are exposed to soils with low pH and high availability of Al. In this study, we measured the Al content in adult plants, and investigated the effects of various Al doses on germination and early development of  Where CiAlx: initial root length measured before exposure to the nutrient solution at an “x” level of Al; CfAlx: final root length measured after n days of exposure to the nutrient solution at an “x” level of Al; CiAl0: initial root length before exposure to the solution without any Al; and CfAl0: final root length measured after n days of exposure to the nutrient solution without Al.Root measurements were performed daily during the 20 days of Al plant exposure. At the end of the exposure period, measurements of the main root were taken and the total root growth rate (TRG) was calculated as (TRG = [(Cf/Ci)*100]-100, where Ci and Cf indicate the initial and final root lengths, respectively). Relative root elongation (RRE%) was calculated according to the equation proposed by Vasconcelos et al. :  coupled to an IRGA . Initially, the leaves were adapted to the dark for at least 30 min (when the photosystem II (PSII) reaction centers were open), followed by the application of the measurement light (~0.03 µmol m−2 s−1) and a saturation pulse (>3000 µmol m−2 s−1), used to obtain the minimum (F0) and maximum (Fm) fluorescence, respectively. The potential quantum yield of the PSII was determined as Fv/Fm = (Fm-F0)/Fm /Fm′), the photochemical extinction coefficient (qP = [Fm′-F]/[Fm′-Fo′]), the non-photochemical extinction coefficient (NPQ = [Fm-Fm′]/Fm′), the apparent rate of electron transport (ETR = Φ PSII x RFA x 0.5 × 0.84), and the maximum photochemical PSII efficiency in light-adapted leaves (Fv′/Fm′ = [(Fm′–Fo′)/Fm′]) [Chlorophyll m-F0)/Fm . After l′)/Fm′]) .a fluorescence data to determine photosynthetic rate , stomatal conductance , transpiration rate , ratio of internal to external CO2 (Ci/Ca), and the ratio of photosynthetic rate to internal CO2 concentration (A/Ci). Measurements were performed using an infrared gas analyzer . Assessments were performed between 9:00 AM and 11:00 AM under constant photosynthetically-active radiation  and CO2 concentration (~415 μmol mol−1), ambient temperature (~25.5 °C), and relative humidity (~74%).Gas exchange was assessed on the same leaf as the chlorophyll 2 leaflet E. dysenterica samples were collected from the root tip and from the last fully expanded leaf of all replicates (n = 4) from each treatment (n = 5) after 20 days of hydroponic cultivation in Al-containing solutions. The material was washed and processed as described in Item 2.3. The plant material was stained with toluidine blue to obtain epidermis images for morphoanatomical observations, i.e., the adaxial and abaxial surfaces, palisade and spongy parenchyma, other mesophyll tissues and the meristematic root region. Starch staining was performed using histochemistry with Lugol solution at 10 g L−1 [For the morphoanatomical analyses, 3–5-cm root and 3 cm10 g L−1 .E. dysenterica seeds after twenty days of exposure to various Al concentrations and in young leaves and roots after twenty days of growth in a nutrient solution containing various Al concentrations.Al content was determined in both adult trees from which fruits and seeds were collected and from experimental plants. Leaf and bark samples were collected from five adult plants in full production. Al content was also evaluated in −1.The collected material was previously washed in distilled water to remove adhered Al, dried in a circulation oven, heated at 70 °C for 78 h, and ground in a Wiley mill . Plant samples (500 mg) were added to tubes containing a nitroperchloric solution (2:1) and were digested in a digester block at 160 °C. Subsequently, the tube volume was brought to 25 mL with deionized water, as described by Malavolta et al. [g for 15 min at 4 °C and the supernatant (enzyme extract) was used for the enzymatic determinations.Superoxide dismutase , catalase  and peroxidase  activities were determined by preparing plant extracts using maceration of approximately 300 mg of roots in 2 mL of an extraction medium, consisting of 0.1 M potassium phosphate buffer, pH 6.8; 0.1 mM ethylenediaminetetraacetic acid (EDTA); 1 mM phenylmethylsulfonic fluoride (PMSF) and 1% (w/v) polyvinylpolypyrrolidone (PVPP). The maceration solution was subjected to centrifugation at 12,000 x −1 mg−1 protein. One unit of SOD was defined as the amount of enzyme required to inhibit NBT photoreduction by 50% [SOD activity was determined by adding 40 μL of the enzyme extract to 5 mL of the reaction medium, consisting of a 50 mM sodium phosphate buffer, pH 7.8, containing 13 mM methionine, p-nitro tetrazolium blue (NBT) 75 μM, 0.1 mM EDTA and 2 μM riboflavin. The reaction was conducted at 25 °C in a chamber under illumination of a 15 W fluorescent lamp for 10 min. Blue formazan absorbance produced by the NBT photoreduction was determined at 560 nm . The res2O2 [2O2 degradation was determined at 240 nm in the first minute of the reaction at 25 °C. Enzymatic activity was calculated using the molar extinction coefficient of 36 M−1 cm−1 [−1 mg−1 protein.Catalase activity was determined by adding 0.1 mL of the enzyme extract to 2.9 mL of the reaction medium, consisting of a 50 mM potassium phosphate buffer pH 7.0 and 12.5 mM H2O2 . The decM−1 cm−1  and was 2O2 [−1 cm−1 [−1 mg−1 protein.Peroxidase activity was determined by the addition of 0.1 mL of the enzyme extract to 4.9 mL of the reaction medium, consisting of a 25 mM potassium phosphate buffer pH 6.8, 20 mM pyrogallol and 20 mM H2O2 . Purpuro[−1 cm−1  and was The protein content in the enzymatic extracts was quantified according to the methodology proposed by Bradford  at 595 np < 0.05). All statistical analyses were performed using ASSISTAT v. 7.7 software.The quantitative data were first subjected to homogeneity analysis (Levene test) and error normality assessment (Shapiro-Wilk test). Because data normality was confirmed, ANOVA was performed, followed by Dunnett’s test, to determine significant differences between the Al treatments and the control (E. dysenterica decreased with increasing Al3+ concentrations (3+ dose led to a 70.25% decrease in the GRI and 30% decrease in germination percentage compared to the control (The germination rate index (GRI) and germination percentages in trations . The hig control .3+ doses caused endosperm region cell destruction in E. dysenterica , root growth rate (58%) and relative root length (30%) than the control seedlings  was altered, with a 16.07% increase observed for the 200 μmol L−1 Al3+ treatment in relation to the control (Regarding  control .A), stomatal conductance (gs), transpiration rate (E), internal to external CO2 concentration ratio (Ci/Ca) and photosynthetic rate to internal CO2 concentration (Ci) ratio (A/Ci) were not affected by Al in E. dysenterica plants , and also able to accumulate high Al content in leaves (≥552.64 mg kg−1). Seeds inherited Al in their tissues (111.07 mg kg−1) from their parent plants and increasing Al doses in the germination test led to higher Al content found in seeds, although this was significantly different only for 800 μmol L−1 Al3+ (E. dysenterica plants were shown to contain Al in roots (521.04 mg kg−1) and leaves (140.94 mg kg−1), even in the absence of Al in the growth solution. Roots abundantly accumulated Al in all treatments, reaching 2332.46 mg kg−1 in the 800 μmol L−1 Al treatment. Similar results were verified for leaves; however, the amount of accumulated Al was much lower compared to the values in roots.Adult L−1 Al3+ . In the E. dysenterica seeds altered the germination process, compromising embryo development through endosperm cell destruction and solute extrusion. Despite the fact that E. dysenterica matrix plants store Al in seeds, external contact with high Al concentrations may interfere with germination and may impair root growth and seedling establishment. The hypothesis we proposed was that seeds would not present Al cell detoxification mechanisms or complexation processes against absorption, greatly increasing the direct interference of Al on cell division, inhibiting germination. In this manner, direct environmental Al contact with E. dysenterica seeds would lead to impaired germination processes.The phytotoxic action of Al on E. dysenterica, affecting cell division and/or elongation and root protrusion. Toxic effects of Al have also been observed in Conyza seeds, with a 35% decrease in Conyza canadensis and 60% in Conyza bonariensis seed germination [Erythrina speciosa and Eugenia brasiliensis seeds. This suggests that Al compromises seed vigor, even in tolerant species, resulting in seedlings with less robust root systems, as observed in the present study.The low seed germination levels in the presence of Al suggests that the amount of Al accumulated in seeds is sufficient to inhibit embryo growth resumption and seedling formation . In thismination . Moreovemination  found thE. dysenterica plants grown at various Al concentrations did not display morphological, anatomical or physiological damage; rather, root growth was stimulated by the 200 μmol L−1 Al treatment. This root growth increment under low Al concentrations was previously reported for E. dysenterica by Rodrigues et al. [E. dysenterica roots in the hydroponic assay. Corroborating the findings of the present study, Rodrigues et al. [Hancornia speciosa grown in a nutrient solution, observed root growth stimulation at 300 μmol L−1 Al. These responses may be associated with Al detoxification via phenolic compounds and starch accumulation, increased root nutrient uptake and the formation of Al detoxification mechanisms.s et al. , who poss et al. . This sus et al. , when asE. dysenterica root system cells is a result of the inhibition of carbohydrate translocation, an energy source for plants that stimulates greater root growth. Påhlsson [Fagus sylvatica roots during 31 days of growth under Al treatment, with no root growth decreases. These data suggest that carbohydrate accumulation is associated with root growth stimulation caused by Al, attributable to the greater availability of energy sources such as starch and sucrose.The greater starch accumulation in Påhlsson  reportedE. dysenterica mesophyll cell and root system integrity suggests that Al does not affect anatomical characteristics, because Al stress in sensitive plants usually leads to changes in leaf and root structures [E. dysenterica absorbs Al by the root system; therefore, cell walls and vacuoles are the primary storage sites for accumulation, suggesting that the tolerance system is based on internal chelation and Al compartmentalization, instead of absorption restriction [E. dysenterica phenol accumulation suggests an Al detoxification strategy and subsequent complexation with these metabolites [3+ [ructures ,50,51, wructures  reportedtriction . Reinforabolites . Phenoliabolites , and plaites [3+ .E. dysenterica did not present changes to chlorophyll a fluorescence or gas exchange. Al-tolerant species may exhibit stimulated growth over a wide range of Al concentrations [In sensitive species, Al affects plant physiological characteristics such as net photosynthetic rate and stomatal conductance ,54. In ttrations  with higtrations . In additrations  was not E. dysenterica plants is related to the activation of a defense mechanism to protect plants against the formation of reactive oxygen species by the action of SOD, CAT and POX enzymes [E. dysenterica cultivated with Al, mainly at 200 and 400 μmol L−1 Al3+, when root growth was stimulated. Similar results were reported by Ghanati et al. [Camellia sinensis plants, where SOD and CAT activities in roots increased in the presence of Al, suggesting that these antioxidant enzymes are beneficial for tea plants and for stimulating root growth. SOD activity is associated with increased superoxide radical production, which is metabolized into hydrogen peroxide (H2O2) [2O2 levels in plants; however, CAT and POX have higher affinities for H2O2 removal [The increase of the antioxidant capacity of  enzymes . Increasi et al.  in Camele (H2O2) . Several removal .E. dysenterica can be characterized as an Al-tolerant species as demonstrated by root growth stimulation, anatomical integrity and maintenance of gas exchange and chlorophyll fluorescence parameters. Furthermore, Al promotes plant growth, primarily at lower concentrations.This species naturally accumulates Al in its tissues  when it is grown in soils containing Al. Metal accumulation, predominantly heavy metals, is a characteristic present in over 450 species of vascular plant families, orders and genera . Native E. dysenterica germination was affected by exposure to various Al concentrations. Nevertheless, young E. dysenterica plants showed Al tolerance. Anatomical and physiological traits were not changed by increasing Al doses. The accumulation of phenolic compounds and the activation of antioxidant enzymes system acted as Al-detoxification mechanisms in cells. Starch accumulation may be related with the highest root growth observed at 200 μmol L−1 Al3+.


Recently, evidence has emerged that palliative gastrectomy in patients with stage IV gastric cancer may offer some survival benefits. However, the decision whether to perform primary tumor surgery remains challenging for surgeons, and investigations into models that are predictive of prognosis are scarce. Current study aimed to develop and validate prognostic nomograms for patients with metastatic gastric adenocarcinoma treated with palliative gastrectomy.The development dataset comprised 1186 patients from the Surveillance, Epidemiology, and End Results Program who were diagnosed with metastatic gastric adenocarcinoma in 2004–2011, while the validation dataset included 407 patients diagnosed in 2012–2015. Variables were incorporated into a Cox proportional hazards model to identify independent risk factors for survival. Both pre- and postoperative nomograms for predicting 1- or 2-year survival probabilities were constructed using the development dataset. The concordance index (c-index) and calibration curves were plotted to determine the accuracy of the nomogram models. Finally, the cut-off value of the calculated total scores based on preoperative nomograms was set and validated by comparing survival with contemporary cases without primary tumor surgery.Age, tumor size, location, grade, T stage, N stage, metastatic site, scope of gastrectomy, number of examined lymph node(s), chemotherapy and radiotherapy were risk factors of survival and were included as variables in the postoperative nomogram; the c-indices of the development and validation datasets were 0.701  and 0.699 (95% CI: 0.682–0.716), respectively. The preoperative nomogram incorporated age, tumor size, location, grade, depth of invasion, regional lymph node(s) status, and metastatic site. The c-indices for the internal (bootstrap) and external validation sets were 0.629 (95% CI: 0.620–0.639) and 0.607 (95% CI: 0.588–0.626), respectively. Based on the preoperative nomogram, patients with preoperative total score > 28 showed no survival benefit with gastrectomy compared to no primary tumor surgery.Our survival nomograms for patients with metastatic gastric adenocarcinoma undergoing palliative gastrectomy can assist surgeons in treatment decision-making and prognostication. Gastric cancer is one of the leading causes of cancer-related mortality worldwide . AdenocaA randomized clinical trial REGATTA which aimed to evaluate whether the addition of gastrectomy to chemotherapy improves survival for advanced gastric cancer patients with a single non-curable factor was terminated ahead of time due to the negative results from the interim analysis . HoweverHowever, the decision regarding whether to perform primary tumor surgery remains challenging, and investigations into models that are predictive of prognosis are scarce. As such, there is a need to identify and appraise factors that predict the outcomes of palliative gastrectomy in patients with metastases at the time of initial diagnosis of gastric adenocarcinoma, either for predicting the individuals’ prognoses or for clinical decision-making. Therefore, we performed this study using the SEER database to construct nomograms that predict the survival of patients with metastatic gastric adenocarcinoma subsequent to palliative gastrectomy.The recently released SEER database submission  includes cancer patients diagnosed from 1973 to 2015. We accessed the database using the SEER*Stat software version 8.3.5  with permission from the SEER program office. There were 16,588 patients with metastatic gastric adenocarcinoma  in the database who were diagnosed between 2004 and 2015. Of these patients, 1971 were known to have been undergone a gastrectomy and 14,344 had not had surgery on the primary tumor. After excluding patients with missing essential data, 1593 patients with advanced gastric adenocarcinoma  who received palliative gastrectomy were analyzed; 1186 patients (approximately three-quarters of the dataset) who were diagnosed between 2004 and 2011 were used as the development cohort to construct predictive models, while the remaining 407 patients (who were diagnosed between 2012 and 2015) were used as the validation cohort. Figure Some variables in the original dataset were re-coded for purposes of statistical analysis. Tumors located in the fundus and body of the stomach, gastric antrum, pylorus, and lesser or greater curvature of stomach were re-classified as non-cardia tumors. Consequently, the primary lesion sites were categorized as “cardia tumors”, “non-cardia tumors”, “overlapping lesions”, and “stomach, not otherwise specified (NOS)”. Because relatively few patients had well-differentiated (grade [G]1) and undifferentiated (G4) tumors, they were combined with patients with moderately differentiated tumors (G2) and poorly differentiated tumors (G3), respectively, and the tumor grades were thus re-classified as G1/2 and G3/4. Because patient records spanned more than 10 years, staging according to both the American Joint Committee on Cancer (AJCC) sixth and seventh editions coexisted in the dataset; therefore, we translated the AJCC sixth edition T and N staging codes into their corresponding seventh edition codes to generate a uniform dataset.Three continuous variables, “age at diagnosis”, “tumor size”, and “number of regional lymph nodes examined” were transformed into categorical variables. The X-tile plotting software was used to determine the cut-off value for continuous variables in terms of their impact on survival . The anaP-values < 0.01 and excluding those with P-values > 0.10, was used in combination with multivariate Cox proportional hazards regression analysis to select prognosis predictive variables for the nomogram. The results were described as hazards ratios (HRs) and 95% confidence intervals (CIs). All P-values were 2-tailed, and values of less than 0.05 were considered statistically significant.The development dataset  was used to construct the nomograms. Parameters that were patient-related , tumor-related , and treatment-related  were included in the Cox regression analysis. Cox regression was performed using SPSS 22.0 statistical software . The backward stepwise method, including parameters with We hypothesized that survival can be predicted preoperatively in patients who underwent palliative gastrectomy; hence, a preoperative model was constructed. This model incorporated “age at diagnosis”, “sex”, “race”, “grade”, “tumor size”, “tumor location”, “pathological subtype”, “tumor invasion” (according to T stage), “regional LN(s) status”  and “metastatic sites” into Cox regression analyses. It was assumed that these parameters could be evaluable pre-surgically. Although “T stage” and “N stage” are generally derived from postoperative pathological findings, modern imaging techniques can produce accurate staging algorithms, at least for negative or positive regional lymph node (LN) status.http://www.r-project.org/).Two nomograms were devised based on the above Cox models by scoring the independent variables according to the regression coefficient. Nomograms were plotted using the “nomogram” function in ‘R’ version 3.5.0  using the ‘rms’ and ‘survival packages’ . Similar to the area under the receiver operating characteristic curve, the c-index estimates the probability of concordance between predicted and observed outcomes in rank order and ranges between 0.5 and 1.0; higher values indicate better discrimination. Calibration refers to the discrepancy between predictions and actual outcomes, and is usually measured by graphic calibration curves that represent the relationship between the observed outcome frequencies and predicted probabilities.The validation procedures were also performed using ‘R’ version 3.5.0. For internal validation of predictive models, 1000 bootstraps with sample sizes of 200 were generated from the original dataset. We calculated c-indices and plot calibration curves to compare the nomogram-predicted 1-year and 2-year survival probabilities with the actually-observed survival outcomes. Plots that approached a 45-degree line indicate a well-calibrated nomogram. The external validation dataset comprised patients diagnosed during a different period (2012–2015), and 1000 times bootstrapping (size 130) was performed to calculate and plot the calibration curves.The development dataset comprised 1186 patients diagnosed between 2004 and 2011, while the validation dataset included 407 patients diagnosed between 2012 and 2015. Table P < 0.05). The risk of death was also greater in patients with ≥N1 tumors than in those with N0 tumors; moreover, older age and poorer tumor differentiation were also associated with poorer survival. However, patients with only distant lymph node involvement had a significantly lower risk of death than patients with metastases at other sites; non-cardia tumors showed a lower risk of death than cardia tumors. Chemotherapy and radiotherapy significantly decreased the risk of death with HRs of 0.5 (95% CI: 0.4–0.6) and 0.8 (95% CI: 0.6–0.9). Sex, race, pathological subtype and surgery to other site(s) or node(s) did not have a significant impact on survival. Tumor size > 50 mm was associated with a marginally decreased risk of death compared to the “≤30 mm” group . Based on the backward stepwise method, “age”, “tumor size”, “tumor location”, “grade”, “T stage”, “N stage”, “metastatic site”, “scope of gastrectomy”, “number of examined lymph node(s)”, “chemotherapy” and “radiotherapy” were retained in the final regression equation.Fifteen parameters were initially incorporated the into Cox regression analyses, including three demographic variables, seven tumor-related variables, and five treatment-related variables (Postoperative Model); the analysis results are shown in Table In the preoperative Cox regression model, older age, poorer differentiation, deeper tumor invasion, and positive regional LN(s) were shown to increase the risk of death significantly. Pure distant LN(s) metastasis had a lower risk of death than metastases in other site(s). After performing the backward stepwise procedure, the above variables together with tumor location and tumor size were retained in the final regression equation. The results were also shown in Table After running the R program, we plotted a nomogram for the determination of postoperative prognosis Fig. . Eleven A similar procedure to the above was performed to generate a preoperative nomogram based on a preoperative model Fig. , which wP = 0.071). In fact, survival for patients with a total score ≤ 28 was shown to be superior to the “no primary tumor surgery group” ; survival curves are showed in Fig. P = 0.844). Those with a total score ≤ 28 group showed improved survival (mOS: 12.0 (10.1–13.8) vs. 5.0 (4.7–5.3) months, HR: 0.540 (95% CI: 0.473–0.616), P = 0.000); survival curves are showed in Fig. Cases in the development dataset were scored base on preoperative nomogram, and total scores were calculated. A cut-off value was attempted several times manually by comparing survival with that of contemporarily registered cases with no primary tumor surgery. A total score of 28 was found to be an optimal cut-off point. After setting the “no primary tumor surgery group” as reference in the Cox regression, there was no survival benefit for patients with a total score > 28  , 3-TeslaThere are some limitations associated with nomograms such as ours. First, the validation cohort had similar characteristics to the development cohort, which restricted the extrapolation to more diverse patient groups. Ideally, such nomograms should be validated in an external cohort or using data from other institutions. Second, our nomograms showed moderate predictive capabilities, especially for the preoperative nomogram with c-indices no greater than 0.7. One possible explanation was that the parameters used to construct the models were limited to those available in the database. The database cannot provide more details of the patients, for example, performance status of the patients, oliogo- or multi-metastasis of tumor, et al.; as to the information on treatment, only sequence of radiotherapy with surgery was displayed in database, other details such as residues of tumor resection , chemotherapy sequence and concurrent chemo-radiation were omitted in current cohort. All these factors were considered to be have impact on survival of patients who received palliative gastrectomy. Finally, constructing the preoperative nomogram depended in part on postoperative parameters such as T and N staging as surrogates; if more relevant preoperative parameters been available, they would likely have been more valuable.We constructed first-of-their-type nomograms that are predictive of survival in patients with metastatic gastric adenocarcinoma who are candidates for palliative gastrectomy. Despite some limitations, these nomograms may assist surgeons in their decision-making regarding treatment as well as in evaluating their patients’ prognoses.


With most countries in sub-Saharan Africa (SSA) lagging behind schedule to implement a comprehensive viral hepatitis elimination strategy, several barriers to accurate information and hepatitis B virus (HBV) services still exist, that are unique to different regions. In an obstetric population of a high HBV burden SSA setting without antenatal HBV services, we systematically evaluated perceptions and prevention behavioral intentions in relation to HBV and liver cancer.Eligible consenting pregnant women were recruited from public health care facilities in the central and northern regions of Uganda, between October 2016 and December 2017. Standardized procedures and instruments based on the health belief model and theory of planned behavior were used to collect data on socio-demographic characteristics, HBV perceptions and behavioral intentions. Descriptive analysis using Chi-square tests was done to obtain distribution of respondents by levels of perceived risk of HBV and liver cancer for themselves, their child under 5 years and their spouse. Modified Poisson regression analyses were used to evaluate relationships between perception variables and different behavioral outcomes .p = 0.055) was inversely associated with intention to screen for HBV. Conversely, perceived self-efficacy showed a consistent association with intention to screen for HBV (PRR = 1.18(1.10–1.23) p = 0.005), to vaccinate (PRR = 1.20(1.05–1.36) p = 0.006) and to seek treatment for HBV (PRR = 1.40(1.18–1.67) p < 0.001). Women from the north, compared to the central region (PRR = 1.76 (1.13–2.72) p = 0.012), and those who self-identified as Catholic (PRR = 1.85 (0.99–3.56) p = 0.056), and as Protestant, (PRR = 2.22 (1.22–4.04) p = 0.002), were more likely to have higher perceived self-efficacy, compared to Muslims. Age and education were not related to perceived self-efficacy.Perceived risk (PRR = 0.95(0.90–1.00), Women in both regions hold incorrect perceptions of HBV and liver cancer risk, with women from the central reporting higher perceived risk than those from the north. High perceived self-efficacy influenced intention to participate in HBV prevention. Programs and policies geared towards enhancing HBV prevention in this sub-population may consider socio-cultural factors observed to influence prevention behaviors. These findings may guide HBV interventions aimed at improving capacity to seek HBV prevention services, thereby promoting HBV micro-elimination in this sub-population. Globally, chronic infection with hepatitis B virus (HBV) is a public health challenge, affecting more than 350 million individuals –3. ChronWith Uganda’s high HBV national prevalence , pregnanSelf-perceived health is defined as “an individual’s evaluation of his or her own health” . Theoriea person’s perceived likelihood or “subjective probability that he or she will engage in a given behavior” [. The theory of planned behavior has been utilized in disease prevention studies including liver cancer prevention research [Intention to prevent HBV was hinged on the theory of planned behavior , which iehavior” , has beeehavior” –26. The research , 28. Altresearch –31 whichresearch . ApplyinIn this study, we aimed to measure pregnant women’s perceptions about risk and prevention of HBV and liver cancer; perceived disease severity, barriers, benefits and self-efficacy for hepatitis B and liver cancer, and also determined the relationship between perception variables, socio-demographic characteristics and intention to test, treat and vaccinate against hepatitis B, as proxy measures for actual behaviors.This was a cross-sectional study. Participants were recruited from antenatal clinics in public health care facilities. These were considered appropriate settings to access pregnant women across a range of cultural and socio-demographic profiles. Also, antenatal clinic settings would equally be efficient to initiate hepatitis B-specific health education and culturally-suitable prevention messages. Arua hospital was selected in North western region, because it is the main public health facility that offers antenatal care to a large population of the surrounding region and neighboring districts, while in the central region which has a much larger urbanized population, there are many public health facilities that receive high volumes of antenatal clients, therefore two health facilities  were randomly selected from the central region, as previously described  .thwoman waiting in the antenatal clinic line was approached about the study and provided with detailed information, and after completing informed consent procedures was enrolled into the study. This process was conducted until the total sample size was accrued. Women received information about the study on each clinic day from trained study personnel during general antenatal gatherings. Eligible participants had to be pregnant women, at least 18 years of age, residing in the region, able to provide written informed consent and to undergo study procedures. Following information provision, eligible women were approached for details about the study and for permission to participate.A sample size of 455 was estimated using Kish Leslie formula (1965) based on the following assumptions: a proportion who intend to screen for HBV to be 50%, a precision of 0.05, type 1 error  of 5%, a design effect of 1.2 and a non-response fraction of 10%. Enrollment of expectant women from antenatal clinics was performed each Monday, Tuesday and Thursday of the week in Kiswa and Kasangati health units, and each Monday and Thursday in Arua Hospital, days when the respective antenatal clinics were scheduled to work. Participants were sampled using a systematic sampling approach . Every 5This study sought and obtained ethical clearance. Approval was sought in relation to performing investigation among pregnant women, as a vulnerable group, from Makerere University School of Public health’s Higher Degrees, Research and Ethics Committee.A structured questionnaire based on the health belief model and the theory of planned behavior was administered to each eligible consenting woman by trained interviewers. To formulate the study instrument, a thorough review of existing literature and of health behavioral theories was conducted. The initial questionnaire was then developed and reviewed by a panel of experts who included 2 hepatologists, a behavioral scientist and a health promotion expert. Their review provided feedback on content and coverage which was used to get a revised version. This was piloted in a sample of 20 pregnant women in a region outside the study area. The pilot provided feedback on language appropriateness and cultural suitability of the questions and from this a final version was obtained and authorized by all experts. The questionnaire had a section on socio-demographic information: age (years) highest education level achieved , region of birth , current region of residence , marital status  and religion . The questionnaire had other sections on knowledge, perceptions and behavioral intentions in relation to HBV, and it has been provided as Additional file “What is the likelihood that you will get hepatitis B disease during your lifetime?” and “What is the likelihood that you will get liver cancer during your lifetime?”;ii) conditional risk  with likert-scale responses “very low”, “low”, “moderate”, “high and “very high”). The third approach assessed ‘comparative risk’  compared to another woman your age?”(b) “compared to your spouse?”, with responses of “much higher”, “higher”, “same”, “lower”, “much lower”). To evaluate perceived risk for their child and spouse, participants were asked these questions “What is the likelihood that your young child (aged 5 years or less) will get (i) hepatitis B (ii) liver cancer during their lifetime?” and “What is the likelihood that your spouse will get (i) hepatitis B (ii) liver cancer during their lifetime?”. Responses were as described for individual risk.Individual perceived risk was assessed using three approaches: i)Absolute lifetime risk (questions: If I had liver cancer i)my career would be endangered, ii)my marriage would be endangered, iii) my financial status would deteriorate, iv) I believe that hepatitis B is a serious disease, v)hepatitis B can be fatal and vi) if I got liver cancer, it would more serious than other diseases.”Perceived severity was assessed with these statements ; “If I hIf I vaccinate my child against HBV, I do not worry about the child getting liver cancer later in adulthood”, “Testing for HBV will help me find and treat HBV early, before it causes liver cancer”, “If I am tested and found to have HBV, the treatment may not be as bad as treatment for liver cancer” “Testing for HBV is the only way to find out if I have the disease”, “Testing , Immunizing against, and treating HBV is an easy way to prevent liver cancer”, “The HBV test will help you not to worry as much about liver cancer”, “Testing and treating HBV will decrease my chances of dying from liver cancer”.Perceived benefits were assessed with these statements; “Compared with your other health problems, having to test for HBV is not important”, “You are not aware that hepatitis B has a vaccine”, “Adults do not need to test for HBV”, “At your age, you do not vaccinate against HBV”. “You do not need a hepatitis B test or vaccine if you do not have liver symptoms.”, “You are afraid to have a hepatitis B test because it might show that you are infected”, “Having the HBV test is a lot of trouble for you”, “You are worried about having the HBV test because you don’t understand what will be done.”, “Having a hepatitis B test is painful for you.”, “Cost would keep you from having the HBV test.”, “Getting vaccinated for hepatitis B when pregnant will result in a miscarriage”.Perceived barriers were assessed by these statements; “I am certain that I can take my infant for all the recommended immunizations, even if the immunization center is far from where I live”, “I am certain that I can take myself for a hepatitis B test, even if I have to pay for the test”, “I am certain that I can take myself for a hepatitis B vaccination, even if I have to pay for the vaccination”, “If I am tested and found to have hepatitis B, I am certain that I can take myself for hepatitis B treatment, even if the treatment center is far from where I live”. Responses to perceived severity, benefits, barriers and self-efficacy were “strongly disagree”, “disagree”, “neutral”, “agree”, “strongly agree”, with participants requested to select which of these best represents their belief.Perceived self-efficacy was assessed with these statements; “How likely is it that you will take your child for vaccination against HBV, as part of the routine infant immunization schedule?”, “How likely is it that you will take a hepatitis B test over the next 12 months?”, “How likely is it that, if tested and found infected, you will seek treatment for hepatitis B over the next 12 months?”, “How likely is it that, if tested and found unimmunized, you will seek and obtain hepatitis B vaccination over the next 12 months?”. Responses for this section were on a 5-point likert scale, of “very unlikely”, “unlikely” “neutral”, “likely” and “very likely”, score = 1, if response was “likely” or “very likely”, otherwise score = 0.Behavioral intentions were assessed by four questions; “p-value of 0.10 were entered into the multivariable regression model. Interaction terms were also included in the model, and where they were significant, were retained, else they were dropped, for a more parsimonious model. Second, regression analyses were done to further examine the potential relationship between socio-demographic factors and perception variables that were predictive of behavioral intention outcomes. Statistical significance in the multivariable models were determined at the 5% cut-off. Estimated prevalence risk ratios and their corresponding 95% confidence intervals were reported.Descriptive univariate analysis of socio-demographic and perception variables was performed to obtain distribution for continuous variables and frequencies for categorical variables. The dataset used for this analysis is provided as Additional file The study involved a total of 455 pregnant women, 300 from the central region and 155 from the north region. Average age (sd) of participants was 24.9 (5.2) years. Just over three quarters (79%) were Christian, and about half (53%) had at least a secondary level education while 34% had primary or no education. Other participant characteristics are shown in Table p < 0.01), that of their children to be low , and that of their spouses to be low . Similarly, as shown in Fig. p < 0.001), that of their children to be high , and that of their spouses to be high .About half of all participants perceived their lifetime risk of acquiring HBV  and liver cancer  to be low. More than a third,, overall, reported low perceived risk of acquiring liver cancer, given HBV infection. There were regional differences in levels of perceived risk of acquiring HBV and liver cancer, by the three measures of risk . As shown in Fig. p = 0.447) perceived themselves to have high risk, while about two thirds  perceived risk to their spouse as high  believed that risk to their children getting liver cancer, if infected with HBV was high.Regarding perceived risk of acquiring liver cancer, if one were infected with HBV, about half of women from both regions  perceived their risk of acquiring liver cancer to be higher than that of another woman their age. Regarding women’s perceived risk of getting liver cancer compared to their spouses, a third of women from the central compared to a quarter from the north (31.3% versus 24.5% p = 0.003) reported high perceived risk.For perceived risk compared to others, approximately a quarter of women in both regions . In multivariable models, perceived self-efficacy showed a consistent association with intention to screen for HBV (PRR = 1.18(1.10–1.23) p = 0.005). In addition, there was an inverse association between high perceived HBV risk and intention to screen for HBV (PRR = 0.95(0.90–1.00), p = 0.055), after adjusting for other perception variables. See Table In bivariable regression analyses, perceived barriers and perceived self-efficacy showed an inverse and a direct association with intention to screen for hepatitis B, respectively. For each unit increase in level of perceived barriers to screening, there was a 3% lower prevalence of intention to screen for HBV (PRR = 0.97(0.95–0.99) 181.10–1.3 p = 0.0p < 0.001); perceived disease severity (PRR = 1.07(1.01–1.12) p = 0.013) and perceived benefits (PRR = 1.10 (1.04–1.17) p = 0.002). There was an inverse association between perceived barriers (PRR = 0.97(0.95–0.98) p < 0.001) and intent to seek HBV treatment. However, only perceived self-efficacy persisted in adjusted models for intention to seek HBV treatment ((PRR = 1.40(1.18–1.67) p < 0.001).In bivariate analysis, intention to seek HBV treatment was significantly associated with perceived self-efficacy (PRR = 1.38 (1.18–1.62) p = 0.001); associations with borderline significance were also observed with perceived disease severity PRR = 1.05 (1.00–1.10) p = 0.040; and perceived benefits 1.10 (1.02–1.13) p = 0.010) and perceived barriers (PRR = 0.98 (0.96–0.99) p < 0.018). In the multivariate analysis, only perceived self-efficacy (PRR = 1.20(1.05–1.36) p = 0.006) was statistically significantly associated with intent to get HBV vaccination.In bivariate analyses, intention to get HBV vaccination was significantly related to perceived self-efficacy (PRR = 1.24 (1.09–1.40) p = 0.012) compared to those from the central region. In addition, respondents who identified as catholic, (PRR = 1.85 (1.21–3.56) p = 0.056) or protestant, (PRR = 2.22 (1.22–4.04) p = 0.002) had higher prevalence of perceived self-efficacy compared to those who self-identified as Muslims. As displayed in Table We performed further analysis to determine which socio-demographic variables were related to having high perceived self-efficacy., In a multivariate regression model, respondents from the northern region had 76% higher prevalence of high perceived self-efficacy (PRR = 1.76 1.13–2.72) –2.72 p =HBV education triggers formulation of decisions to seek HBV care and prevention services, but is more effective if it is rooted in a clear understanding of existing population perceptions regarding disease risks and prevention. In this study, we have assessed perceptions and behavioral intentions related to HBV risk and prevention among pregnant women in two regions of Uganda, a country of moderate to high HBV prevalence with inadequate programs for preventing mother to child HBV transmission. We identified that high perceived self-efficacy was associated with intention to screen, vaccinate and seek treatment for hepatitis B. Further assessment also showed that individuals residing in the north, compared to the central region, plus those belonging to Christian religious following compared to Muslims, were more likely to have high perceived self-efficacy for participating in HBV prevention actions.Few pregnant women, all-inclusive, had high perceived risk of acquiring HBV and liver cancer during their lifetime, and a significant proportion still believed they were at low risk of liver cancer, even if they were to contract HBV. Our results also showed regional differences in risk perceptions, where a higher fraction of women from the central region tended to have high perceived risk of acquiring HBV and liver cancer, for themselves, their children and their spouses, compared to those from the northern region. This finding is similar to a study by Kue and colleagues among Chinese immigrants in the USA , where pPerceived self-efficacy in this study was independently related to hepatitis B prevention behavioral intentions, including intention to screen for hepatitis B, to seek treatment, and to receive a hepatitis B vaccination. In this, our data stand with the theory of planned behaviour, indicating that if individuals are self-assured in their ability to prevent HBV, they are more likely to participate in preventive actions. A recent study among Iranian patients with non-alcoholic fatty liver disease also found that both perceived duration of illness and self-efficacy were predictive of adopting healthy nutritional habits . SimilarAlthough perceived risk and severity have been reported in several studies to positively influence disease screening behaviors , 47–50, Links between HBV-related perception constructs and socio-demographic characteristics have been barely evaluated, particularly in SSA pregnant populations, or in developed countries among migrants from Africa. In our study, both region and religion influenced perceived self-efficacy, a socio-cognitive construct shown to positively influence uptake of HBV preventive behaviors. Pregnant women who resided in the central, compared to the northern region were more likely to have low perceived self-efficacy for taking up HBV prevention measures. In addition, individuals who self-identified as Muslims were also more likely to have low self-efficacy for participating in HBV prevention services, compared to those who self-identified as Christians, in adjusted models. These findings mirror those in a study among Moroccan immigrants in Europe , where aWe note that this study had important limitations. Assessment of behavioral intentions relied on participants’ self-reports and HBV-related perceptions were gauged in a setting without a national program for HBV testing or vaccination for pregnant women. This might have influenced how respondents perceived their risk and how they might have reported their intent to participate in HBV prevention services, which may limit comparability to other research. Moreover, we did not include assessment of cues to action. Nonetheless, our study is among very few investigations to evaluate hepatitis B related perceptions and preventive intentions among indigenous African pregnant women. As such, it contributes to filling an existing gap on available evidence to inform programs that aim to reinforce hepatitis B prevention behaviors among pregnant women within the SSA region. In addition, we performed a rigorous assessment of HBV-related perceptions and preventive behavioral intentions, followed by further analysis to specifically identify correlates of perceived self-efficacy in this pregnant population, given that perceived efficacy was directly related to positive HBV preventive behavioral intentions. This work, in line with the current global health sector strategy on viral hepatitis elimination by 2030 , has sigIn an obstetric population of a SSA setting without antenatal HBV services, we systematically evaluated perceptions of hepatitis B and liver cancer risk and attendant benefits, barriers and self-efficacy in relation to prevention behaviors. We found low perceptions of risk of both HBV and liver cancer. We identified that high perceived self-efficacy, of which region and religion were significant determinants, was associated with intention to screen, to vaccinate and to seek treatment for hepatitis B. In environments like this one, where specific evidence needed to refine HBV risk and prevention communication is insufficient, these findings may be relied on to lay a foundation for strengthening HBV and liver cancer risk communication and prevention programming, in order to maximize their impact on national HBV elimination strategies.Additional file 1. Knowledge, perceptions and behavioral intentions questionnaire. This is a questionnaire that was developed for this study and used to collect data on HBV knowledge, perceptions and preventive behavioral intentions.Additional file 2. Knowledge, perceptions and behavioral intentions dataset. A dataset provided as excel spread sheets. It contains data on socio-demographic, knowledge, perceptions and behavioral intentions variables that were used to perform analyses for the findings reported in this manuscript. Dataset contains variables pertaining to each respondent: identification number, age, gender, nationality, religion , primary tribe (respondent’s ethnic tribe), marital status , education level (highest education attained), variables c1-c6 describe participant’s personal risk of getting HBV or liver cancer, c7-c13 describe participant’s perceived risk of their child under five years getting HBV or liver cancer, c14-c18 describe participant’s perceived risk of their spouse getting HBV or liver cancer. c1ds-c8ds describe perceived HBV disease severity, pm1-pm7 describe perceived benefits, pb1-pb11 describe perceived barriers, pse12-pse15 describe perceived self-efficacy, bi01-bi04 describe behavioral intentions.


Background: This study evaluated the characteristics of subependymal giant cell astrocytoma (SEGA) in patients with tuberous sclerosis complex (TSC) entered into the TuberOus SClerosis registry to increase disease Awareness (TOSCA).Methods: The study was conducted at 170 sites across 31 countries. Data from patients of any age with a documented clinical visit for TSC in the 12 months preceding enrollment or those newly diagnosed with TSC were entered.Results: SEGA were reported in 554 of 2,216 patients (25%). Median age at diagnosis of SEGA was 8 years , with 18.1% diagnosed after age 18 years. SEGA growth occurred in 22.7% of patients aged ≤ 18 years and in 11.6% of patients aged > 18 years. SEGA were symptomatic in 42.1% of patients. Symptoms included increased seizure frequency (15.8%), behavioural disturbance (11.9%), and regression/loss of cognitive skills (9.9%), in addition to those typically associated with increased intracranial pressure. SEGA were significantly more frequent in patients with TSC2 compared to TSC1 variants . Main treatment modalities included surgery (59.6%) and mammalian target of rapamycin (mTOR) inhibitors (49%).Conclusions: Although SEGA diagnosis and growth typically occurs during childhood, SEGA can occur and grow in both infants and adults. Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder characterized by growth of hamartomas in several organs, including the brain, kidneys, lungs, heart, eyes, and skin . SubepenTSC2 mutations compared with those with TSC1 mutations  inhibitors are the recommended treatment options for SEGA associated with TSC. Surgical resection should be considered for acutely symptomatic SEGA, while either surgical resection or medical treatment with mTOR inhibitors may be considered for growing, but not acutely symptomatic SEGA . HoweverTuberOus SClerosis registry to increase disease Awareness) registry was designed with the aim of providing deeper insights into the manifestations of TSC and its management. The baseline core data of the TOSCA registry published previously provided understanding of the overall manifestations and natural history of TSC  and SEGA characteristics using Chi-square test or fisher exact test, and median test. Since baseline data were collected prior to the 2013 international consensus on SEGA definition, no specific inclusion criteria were defined. The TOSCA cohort therefore reflects worldwide clinical practice.Data collected specific to SEGA included tumour characteristics such as presence of single or multiple SEGA, clinical signs and symptoms associated with SEGA, and management. Characteristics of SEGA according to the age at consent were evaluated. The study also assessed the association between genotype  or as median (range), unless stated otherwise.TOSCA was designed and conducted according to the Guidelines for Good Clinical Practice and ethical principles outlined in the Declaration of Helsinki , 29. AftAs of September 30, 2015, 2,216 patients  with TSC were enrolled in the TOSCA registry from 170 sites across 31 countries. The demographic and clinical characteristics of the enrolled patients are shown in Overall, SEGA were reported in 554 patients (25%); 275 (49.6%) were males and 279 (50.4%) were females. Of these, SEGA were present at baseline in 463 patients (83.6%), resolved with treatment before baseline in 80 patients (14.4%), and were reported to have resolved spontaneously in 10 patients (1.8%), the latter possibly due to measurement errors in small lesions. Detailed information was lacking for one patient. The median age at SEGA diagnosis was 8 years . SEGA were diagnosed before 2 years of age in 26.6%, before 18 years in 81.9% of patients, and after 18 years in 18.1% patients . The oldOf the 463 patients with SEGA at baseline, 209 (45.1%) had multiple SEGA and in 208 patients (44.9%) SEGA were present bilaterally . Among pTSC1 and TSC2 are shown in TSC2 mutation compared to those with a TSC1 mutation . However, there was no significant difference with respect to SEGA diagnosis before 2 years of age (p = 0.3812), multiple (p = 0.8368), bilateral (p = 0.9550) or growing SEGA (p = 0.3302), and presence of SEGA-related symptoms (p > 0.05) in patients with mutations in TSC1 compared to TSC2  should trigger an earlier scan. Similarly, a growing SEGA should prompt a more frequent clinical and radiological follow-up. Parents and patients should be educated regarding relevant symptoms that should prompt referral to medical evaluation . The TOSTSC2 mutations are associated with a more severe clinical phenotype  and mTOR inhibitor (49%) were the most common treatment modalities at baseline. Current international recommendations propose the use of surgical resection for acutely symptomatic SEGAs. For growing but asymptomatic SEGA, both surgical resection and mTOR inhibitors are potential treatments. In determining the best option, discussion of the complication risks, adverse effects, cost, length of treatment, family preference, surgical expertise in SEGA, and potential impact on TSC-associated comorbidities should be included in the decision-making process , 33. mTOThe median time from SEGA diagnosis to treatment initiation was 319 days. This likely reflects a watch and wait approach to document growth and the need for intervention.The current study has the following limitations: firstly, the observational nature allowed collection of only those data that were already available from clinical practice and hence reflects “real world” data. Secondly, a major challenge for this registry was to ensure that data about all the disease manifestations for each patient were reported although the sites involved in the registry did not always follow patients for all disease manifestations in the same way. However, the low number of missing data for SEGA (4.7%) reflects good quality of data collection.In summary, the study highlights that the rates of SEGA in patients with TSC might be higher than previously reported. Increase in seizure frequency, behavioural disturbance, regression/loss of cognitive skills were identified as frequent symptoms associated with SEGA, over and above headaches, typically associated with raised intracranial pressure. SEGA may already be present and grow at a very young age. Although SEGA mostly occur in childhood, it is important to be vigilant in adults as well, since SEGA growth does occur also in these age groups.http://www.encepp.eu/ (EU PAS Register Number EUPAS3247).Novartis supports the publication of scientifically rigorous analysis that is relevant to patient care, regardless of a positive or negative outcome. Qualified external researchers can request access to anonymized patient-level data, respecting patient informed consent, contacting study sponsor authors. The protocol can be accessed through EnCePP portal The study protocol and all amendments were reviewed and approved (if applicable) by independent ethics committee/institutional review board for each centre: National Hospital Organization Central Ethics Committee; Gazi University Clinical Research Ethics Committee; Independent Multidisciplinary Committee on Ethical Review of Clinical Trials; Peking Union Medical College Hospital; Commissie Medische Ethiek UZ Brussel; CNIL , CCTIRS (Comité Consultatif sur le traitement de l'information en matière de recherche dans le domaine de la santé); Comité Etico Investigación Clínica de Euskadi (CEIC-E); Consejeria de Salud y Bienestar Social, Dirección General de Calidad, Investigación, Desarrollo e Innovación, Comité Coordinador de Ética de la Investigación Biomédica de Andalucía; Research Ethics Committee of the University of Tartu (UT REC); Ethikkommission der Medizinischen Universität Graz; North Wales REC—West; Regionala Etikprövningsnämnden i Göteborg; REK—Regionale komiteer for medisinsk og helsefaglig forskningsetikk; Komisja Bioetyczna przy Instytucie Pomnik Centrum Zdrowia Dziecka; Ethikkommission bei der Ludwig-Maximilians-Universitat München; Hokkaido University Hospital Independent clinical research Institutional Ethics Committee; Medical Juntendo University Institutional Ethics Committee; National Center for Chile Health and Deveropment of IRB; Osaka University Hospital of IRB; Ethics Committee at Moscow Institute of Pediatrics and Pediatric Surgery; Peking University First Hospital; Sanbo Brain Hospital Capital Medical University; Tianjin Children's Hospital; Childrens Hospital Of Fudan University; Zhongshan Hospital Fudan University; Fudan University Shanghai Cancer Center; The Second Affiliated Hospital of Guangzhou Medical University; The First Affiliated Hospital, Sun Yan-Sen University; The First Affiliated Hospital Of Guangzhou Medical University; Shenzhen Children's Hospital; West China Hospital, Sichuan University; Xijing Hospital; Children's Hospital of Chongqing Medical University; Wuhan Children's Hospital; The second affiliated hospital of Xi'an jiaotong university; Guangdong 999 brain hospital; Seoul National University Hospital Institutional Review Board; National Taiwan University Hospital (NTUH) Research Ethics Committee (REC); Institutional Review Board of the Taichung Veterans General Hospital; Institutional Review Board of Chung Shan Medical University Hospital; Institutional Review Board, Tungs' Taichung MetroHarbor Hospital; Institutional Review Board of National Cheng Kung University Hospital; Metro South Human Research Ethics Committee; Sydney Children's Hospital Network Human Research Ethics Committee; St. Vincents Hospital Human Research Ethics Committee; Royal Melbourne Hospital Human Research Ethics Committee; Siriraj Institutional Review Board; The Institutional Review board, Faculty of Medicine, Chulalongkorn University, Third Floor, Ananthamahidol Building, King Chulalongkorn Memorial Hospital; The committee on Human Rights Related to Research Involving Human Subjects; Institutional Review board, Royal Thai Army Medical Department IRB RTA, Fifth Floor, Phramongkutklaowejvitya Building, Phramongkutklao College of Medicine; Research Ethics Committee, Faculty of Medicine, Chiang Mai University; Research and Development, Queen Sirikit National Institute of Child Health; Human Research Ethics Committee, Faculty of Health Sciences, University of Cape Town; Shaare Zedek Meidcla center Helsinki comittee; Sheba Medical center Helsinki comittee; Tel Aviv Sourasly Medical center Helsinki comittee; General University Hospital of Patras Ethics Committee; Pendeli Children's Hospital Ethics Committee; General University Hospital of Athens 'G. Gennimatas Ethics Committee; Evaggelismos General Hospital Ethics Committee; General University Hospital of Thessaloniki AHEPA Ethics Committee; General University Hospital of Ionnina Ethics Committee; METC UMC Utrecht; Direcció General de Regulació, Planificació i Recursos Sanitaris; Comité Ético de Investigación Clínica del Hospital Universitario Vall d'Hebron de Barcelona, Generalitat de Catalunya.Departament de Salut; Comité Ético de Investigación Clínica Hospital Universitario La Paz; Dirección General de Ordenación e Inspección, Consejería de Sanidad Comunidad de Madrid, Servicios de Control Farmacéutico y Productos Sanitarios; Comité Etico Investigación Clínica del Hospital Universitario y Politécnico de La Fe; Dirección General de Farmàcia i Productes Sanitaris, Generalitat de Valencia; Comité de Ética de la Investigación de Centro de Granada; Instituto Aragonés de Ciencias de la Salud (IACS); Comité Etico Investigación Clínica Regional del Principado de Asturias; Comité Etico Investigación Clínica Hospital 12 de Octubre; Comité Etico Investigación Clínica Hospital Universitario Virgen de la Arrixaca; Sección de Ordenación e Inspección Farmacéutica Departamento de Salud; Comité Ético de Investigación Clínica del Hospital Universitario del Río Hortega de Valladolid; Comissão de Ética para a Saúde (CES), Centro Hospitalar de Lisboa Ocidental, EPE; Comissão de Ética para a Saúde (CES), Centro Hospitalar do Porto, E.P.E; Comissão de Ética para a Saúde (CES), Centro Hospitalar Lisboa Central, EPE; Comissão de Ética para a Saúde (CES), Hospital Garcia de Orta, EPE; Comissão de Ética para a Saúde (CES), Centro Hospitalar de São João, EPE; Comissão de Ética para a Saúde (CES), Hospital Professor Doutor Fernando Fonseca, EPE; Comissão de Ética para a Saúde (CES), Centro Hospitalar do Algarve, EPE (Unidade de Faro); LUHS Kaunas Regional Biomedical Research Ethics Committee; Paula Stradiņa klīniskās universitātes slimnīcas, Attīstības biedrības Klīniskās izpētes Ētikas komiteja, Ethics Committee for Clinical Research; Komisija Republike Slovenije za medicinsko etiko; Comitato Etico Indipendente Presso La Fondazione Ptv Policlinico Tor Vergata Di Roma; Comitato Etico Regione Calabria Sezione Centro c/o A.O.U. Mater Domini Di Catanzaro; Comitato Etico Azienda Ospedaliera Universitaria Di Cagliari; Comitato Etico Cardarelli-Santobono c/o Ao Cardarelli; Comitato Etico Per La Sperimentazione Clinica Delle Province Di Verona E Rovigo, Presso Aoui Verona; Eticka Komise Fn Brno; Eticka Komisia Dfnsp Bratislava; Eticka Komisia Pri Dfn Kosice; Eticka Komisia Bratislavskeho Samospravneho Kraja; Comisia Natională de Bioetică a Medicamentului şi a Dispozitivelor Medicale; Comitato Etico Milano area 1 c/o ASST FBF Sacco—P.O. L. Sacco; Comité de Ética de la Investigación de Centro Hospital Universitario Virgen del Rocío; Comité Ético de Investigación Clínica Fundació Sant Joan de Déu Generalitat de Catalunya. Departament de Salut; Comité Ético de Investigación Clínica Hospital Infantil Universitario Niño Jesús; Consejería de Sanidad Dirección General de Salus Pública Junta de Castilla León; Dirección General de Asistencia Sanitaria, Consejería de Sanidad Gobierno del Principado de Asturias; Dirección General de Planificación, Ordenación Sanitaria y Farmacéutica e Investigación, Consejeria de Sanidad y Política Social Región de Murcia; Ethics Committee at Moscow Institute of Pediatrics and Pediatric Surgery; Paula Stradiņa klīniskās universitātes slimnīcas, Attīstības biedrības Klīniskās izpētes Ētikas komiteja, Ethics Committee for Clinical Research; The First Affiliated Hospital of The Fourth Military Medical University; Zhongshan Hospital Fudan University.AJ, EB, MB, PC, MD, JF, MF, CH, SJ, JL, AM, RN, VS, MS, RT, BZ, and JK: designing the study, patient accrual, clinical care, data interpretation, drafting, revising, final review, and approval of the manuscript. TC, VC, GBdA, PdV, CF, FO, JQ, YT, and SY: designing the study, data interpretation, drafting, revising, final review, and approval of the manuscript. LD: designing the study, trial management, data collection, data analysis, data interpretation, drafting, revising, final review, and approval of the manuscript. RM: designing the study, data analysis, data interpretation, drafting, revising, final review, and approval of the manuscript. SS: designing the study, trial statistician, data analysis, data interpretation, drafting, revising, final review, and approval of the manuscript.AJ, EB, TC, VC, PC, GBdA, PdV, JK, JF, MF, CF, CH, SJ, RN, FO, JQ, MS, RT, MD, JL, AM, SY, MB, and BZ received honoraria and support for the travels from Novartis. VC received personal fees for consulting, lecture fees and travel from Actelion, Bayer, Biogen Idec, Boehringer Ingelheim, Gilead, GSK, MSD, Novartis, Pfizer, Roche, Sanofi; grants from Actelion, Boehringer Ingelheim, GSK, Pfizer, Roche; personal fees for developing educational material from Boehringer Ingelheim and Roche. PdV has been on the study steering group of the EXIST-1, 2, and 3 studies sponsored by Novartis, and co-PI on two investigator-initiated studies part-funded by Novartis. RN received grant support, paid to her institution, from Eisai and lectures fees from Nutricia, Eisai, Advicenne, and GW Pharma. YT received personal fee from Novartis for lecture and for copyright of referential figures from the journals, and received grant from Japanese government for intractable epilepsy research. SJ was partly financed by the EC Seventh Framework Programme , the Polish Ministerial funds for science (years 2013-2018) for the implementation of international cofinanced project and the grant EPIMARKER of the Polish National Center for Research and Development No STRATEGMED3/306306/4/2016. JK, PC, CH, JL, and JQ received research grant from Novartis. RM and SS are employees of Novartis. LD was Novartis employee at the time of manuscript concept approval. This study was funded by Novartis Pharma AG. All authors approved the final version of the manuscript prior to submission.


APOE ε4 influences the association between white matter hyperintensities (WMH) and cognitive impairment in Alzheimer disease (AD) and dementia with Lewy bodies (DLB).To determine if APOE ε4 genotyping. Total WMH volumes were quantified. Neuropsychological test scores were included in a confirmatory factor analysis to identify cognitive domains encompassing attention/executive functions, learning/memory, and language, and factor scores for each domain were calculated per participant. After testing interactions between WMH and APOE ε4 in the full sample, we tested associations of WMH with factor scores using linear regression models in APOE ε4 carriers (n = 167) and noncarriers (n = 122). We hypothesized that greater WMH volume would relate to worse cognition more strongly in APOE ε4 carriers. Findings were replicated in 198 patients with AD from the Alzheimer's Disease Neuroimaging Initiative (ADNI-I), and estimates from both samples were meta-analyzed.A total of 289 patients  underwent volumetric MRI, neuropsychological testing, and APOE ε4 for language, but not for memory or executive functions. Separate analyses in APOE ε4 carriers and noncarriers showed that greater WMH volume was associated with worse attention/executive functions, learning/memory, and language in APOE ε4 carriers only. In ADNI-I, greater WMH burden was associated with worse attention/executive functions and language in APOE ε4 carriers only. No significant associations were observed in noncarriers. Meta-analyses showed that greater WMH volume was associated with worse performance on all cognitive domains in APOE ε4 carriers only.A significant interaction was observed between WMH and APOE ε4 may influence the association between WMH and cognitive performance in AD and DLB. We tested associations with domain-specific cognitive impairment instead of global cognition because at different disease stages, impairment might be more apparent in certain domains and not others. We hypothesized that (1) higher WMH burden would be more strongly associated with worse cognition in APOE ε4 carriers than noncarriers and the association would be APOE ε4 allele dosage dependent, (2) this association would be irrespective of the clinical diagnosis, and (3) if indeed WMH burden is associated with worse cognition in APOE ε4 carriers, WMH in carriers might be a result of a more toxic vascular pathology, i.e., CAA.Therefore, we examined the role of APOE ε4 on the association of WMH volume and cognitive functions in patients with AD and patients with DLB.This is a cross-sectional study examining the effect of 15 The majority of participants in the SDS are Caucasian of European descent.This work was embedded within the Sunnybrook Dementia Study (SDS), a prospective observational study of patients with dementia.16 ADNI was launched in 2003 as a public–private partnership. For the most up to date information, see adni-info.org.For replication of study findings, data from the Alzheimer's Disease Neuroimaging Initiative Phase I (ADNI-I) (2004) were utilized.3) at recruitment and cognitive impairment is largely attributed to AD pathology with minimal confounding comorbid SVD. The SDS represents a heterogeneous real-world clinical case series followed longitudinally, and reflects a similar vascular risk factor and SVD burden profile to community and population-based studies.17ADNI-I is characterized by a low WMH burden  is approved by the local Research Ethics Board at Sunnybrook Health Sciences Centre and written informed consent was obtained from participants or their surrogate caregivers according to the Declaration of Helsinki.APOE ε4 genotype, MRI volumetrics, and neuropsychological battery, were available. This included 239 patients with AD and 50 patients with DLB with varying degrees of SVD. Of the 289 patients included, 36 had autopsy data available.Data from 289 MRI-confirmed stroke-free patients with dementia, including APOE ε4 genotype, MRI volumetric, and neuropsychological data available were included. We used data from the 24 month follow-up visit instead of baseline for better comparability to the SDS sample given the mild initial nature of participants included in ADNI, i.e., progression of the AD stage and that of WMH burden, and ensuring a sufficient number of participants to obtain valid estimates.A total of 198 patients with AD with 18 while DLB (SDS only) was diagnosed using the Third Report of DLB Consortium criteria.19 Diagnoses were confirmed on clinical follow-up.For both study samples, AD was diagnosed on recruitment, using the Neurologic and Communicative Disorders and Stroke and Alzheimer's Disease and Related Disorders Association criteria,Diagnostic consensus in the SDS was achieved through review by at least 2 physicians (M.M. and S.E.B.) with expertise in dementia diagnosis.APOE genotyping was performed using DNA extracted from leukocytes in both SDS20 and ADNI.21 Genotype frequencies in both samples did not deviate from that predicted by Hardy-Weinberg equilibrium.15MRI scans were acquired on a 1.5T Signa system . Three sets of structural MRI sequences were used: T1-weighted, T2-weighted, and proton density–weighted (PD). Details of MRI acquisition are provided elsewhere.22 WMHs were identified as lesions that appear as punctate or diffuse regions of hyperintense signal on T2/PD MRI. These images were used to quantify global, deep, and periventricular WMH volumes (cm3). For analyses, total WMH volumes adjusted for total intracranial volume (TIV) were used: TIV adjusted WMH volumes = (raw WMH volume/TIV) × 103.MRIs were processed using the Semi-Automated Brain Region Extraction and Lesion Explorer processing pipeline.23Methods for MRI data acquisition, processing, and WMH quantification are described in detail elsewhere.24 The following tests for global cognition and domain-specific functioning were administered: (1) Mini-Mental State Examination (MMSE); (2) Dementia Rating Scale; (3) California Verbal Learning Test (CVLT), total acquisition score through 5 trials, CVLT long delay–free recall, and CVLT long delay–cued recall; (4) Wechsler Memory Scale (WMS) visual recognition immediate and delayed recall; (5) forward digit span (FDS); (6) backward digit span (BDS); (7) Boston Naming Test (BNT); (8) Semantic Fluency (SF); (9) Wisconsin Card Sorting Test (WCST); (10) Controlled Oral Word Association task–Phonemic Fluency (PF-FAS); (11) Trail-Making Test A; and (12) Digit Symbol Substitution Task (DSST). The number of patients who completed each test differed; this variability was dependent on dementia severity. Ninety percent of patients had completed at least 8 neuropsychological tests.The neuropsychological battery was performed within 90 days of MRI acquisition. Trained psychometrists blinded to neuroimaging, dementia diagnosis, and genotype information administered all tests.25The cognitive test battery in ADNI-1 included (1) MMSE; (2) Rey Auditory Verbal Learning Test (RAVLT), total acquisition score through 5 trials and delayed recall; (3) logical memory immediate and delayed recall; (4) FDS; (5) BDS; (6) BNT; (7) category fluency ; (8) Trail-Making Test A; and (9) DSST. Details are described elsewhere.For all test scores, higher scores correspond to better cognition, except for WCST , and Trail-Making Test A (time taken to complete the task in seconds), for which a higher score corresponds to worse performance.Age, sex, years of education, diabetes mellitus type 2 (present vs absent), systolic and diastolic blood pressure (mm Hg), hypertension (present vs absent), smoking status , and dementia diagnosis (AD or DLB) were considered potential confounders.Available covariates in ADNI-I included age, sex, education, and systolic and diastolic blood pressure.For consistency across both study samples, we included systolic and diastolic blood pressure as covariates and not hypertension.15 This workup included a screen for CAA using immunohistochemistry for β-amyloid (Aβ)  in at least 2 brain sections . For 34 of these 36 cases, the original autopsy reports were reviewed by a neuropathologist (J.K.) to determine the presence or absence of CAA. For 2 of the 36 cases, the reports were not available. For 3 of the 34 cases with available reports, the presence or absence of amyloid angiopathy was not stated in the autopsy report; the slides from the original autopsy were retrieved, reviewed by J.K., and the presence or absence of CAA was determined. Given that only 2 anatomical areas of the brain had been screened for CAA, applying a formal CAA grading scheme was not feasible. Using these data (n = 34), we aimed to explore if there was a higher prevalence of CAA in APOE ε4 carriers.Thirty-six of the SDS cases had a postmortem neuropathologic examination to diagnose and stage neurodegenerative disease phenomena.z scores.TIV-adjusted WMH volumes were log-transformed to achieve a normal distribution and standardized by calculating APOE ε4 carriers and noncarriers using t tests for continuous and χ2 tests for categorical variables.We compared participant characteristics between 26 and calculated scores for each cognitive factor, i.e., attention/executive functions, learning/memory, and language for each participant. These cognitive factor scores were then used as outcomes in our analyses instead of individual test scores. CFA uses all available information for any model specified instead of a complete case analysis, and obtained factors are allowed to correlate. We present standardized measures in this article to facilitate interpretation. Adequacy of model fit to the data was assessed by comparative fit index (CFI) , root mean square error of approximation (RMSEA) , and the standardized root mean square residual (SRMR) .27In both samples, we aimed to reduce the number of tests by making comprehensive factor scores (latent constructs) for each cognitive domain, based on the specific tests and the domain that they are known to assess. Therefore, we conducted a Confirmatory Factor Analysis (CFA)APOE ε4 carrier status as a predictor, and also tested the interaction between WMH and APOE ε4 carrier status.Subsequently, in both study samples, we first tested associations between WMH volume and each of the 3 cognitive factor scores with all covariates including APOE ε4 carriers and noncarriers separately, based on our a priori hypothesis, i.e., higher WMH burden would be more strongly associated with worse cognition in APOE ε4 carriers than noncarriers, because of the known strong biological effects of the APOE ε4 allele.28Second, we investigated the associations between WMH volume and each cognitive factor score in Relationships between the following cognitive factors and observed test scores were hypothesized and tested using CFA: (1) attention/executive functions , (2) learning/memory , and (3) language . Scores for WCST and Trails A were inverse-coded for consistency with other test scores.APOE ε4 carrier status:We used the following multiple linear regression model in the SDS sample (n = 289) to test associations of WMH with executive functions, memory, and language, and an interaction between WMH and APOE ε4 carrier status + β3 * (WMH volume × APOE ε4 carrier status) + β4 * age + β 5 * sex + β6 * education + β7 * diabetes mellitus + β8 * systolic blood pressure + β9 * diastolic blood pressure + β10 * smoking + β11 * clinical dementia diagnosis.Cognitive factor score = β0 + β1 * WMH volume + β2 * APOE ε4 carriers and noncarriers separately using a similar model, but without APOE ε4 and its interaction term.Further, we tested associations of WMH with the cognitive domains in For each regression, 2 models were fitted. Model I was adjusted for age and sex; II was additionally adjusted for years of education, diabetes mellitus type 2, systolic and diastolic blood pressure, smoking status, and dementia diagnosis. We also repeated model II by replacing systolic and diastolic blood pressure by hypertension.The following variables had missing values and were dealt with by multiple imputation using chained equations in Stata: systolic and diastolic blood pressure and smoking , diabetes , and years of education . All available covariates were used as predictors for imputation.5 we repeated the analyses in APOE ɛ4 carriers and noncarriers excluding DLB cases.Since studies suggest that WMH are not associated with cognition in DLB, but in AD only,APOE ɛ4 carriers were dependent on APOE ε4 allele dosage. After comparing study characteristics and WMH volumes by APOE ɛ4 allele dosage  using analysis of variance (ANOVA) (Tukey post hoc) and χ2 tests for continuous and categorical variables, respectively, we repeated our analyses in APOE ɛ4 heterozygotes (n = 130) and APOE ɛ4 homozygotes (n = 37).In a post hoc analysis, we tested if associations between WMH and cognitive domains in APOE ɛ4 carrier status in our autopsy subsample (n = 34). This analysis was conditional on our primary results, i.e., to be performed if indeed WMH were associated with worse cognition more strongly in APOE ε4 carriers than noncarriers. In this case, we hypothesized that since APOE ε4 is a risk factor for CAA, the likely etiology of WMH in carriers is CAA, which might be more toxic than WMH caused by vascular compromise or ischemia due to cardiovascular risk factors alone. We compared the numbers of patients with CAA by APOE ε4 carrier status and by allele dosage using the Fisher exact test. Since studies suggest that CAA is more prevalent in APOE ε2 carriers,29 we also examined the number of persons with CAA across genotypes: ε2-ε3 (n = 2), ε3-ε3 (n = 12), ε3-ε4 (n = 13), and ε4-ε4 (n = 7); however, statistical comparisons could not be made due to small numbers within some cells.We explored the prevalence of CAA by Relationships between the following cognitive factors and observed test scores were hypothesized and tested: (1) attention/executive , (2) learning/memory , and (3) language .APOE ε4) was tested , and then analyses were repeated in APOE ε4 carriers and noncarriers separately. For regression, model I was adjusted for age and sex only; II was additionally adjusted for education and systolic and diastolic blood pressure. Analyses were also repeated in APOE ε4 heterozygotes (n = 91) and homozygotes (n = 40).As in the SDS, a full model with an interaction term  for all statistical tests, and all analyses were performed using Stata Software Version 14.1 .The authors have carefully documented all data, methods, and materials used to conduct the research in this article and agree to share anonymized data by request from any qualified investigator.APOE ε4 carriers and noncarriers. APOE ε4 carrier status.Characteristics of the study sample are presented in In the CFA, single confirmatory factor models for all 3 cognitive factors tested showed excellent fit to the data: attention/executive , learning/memory , and language .APOE ε4  was observed for language, but not for executive functions  or memory . With our a priori hypothesis that WMH relate to cognition differently in carriers and noncarriers, and a significant interaction observed between WMH and APOE ε4 for language, we performed analyses separately in APOE ε4 carriers and noncarriers for all cognitive domains.In the full model (n = 289), WMH volume was not associated with attention/executive functions, learning/memory, or language. An interaction between WMH and APOE ε4 carriers; no associations were observed in noncarriers , a similar pattern of results was obtained .APOE ε4 carriers were younger than noncarriers and heterozygous carriers . Homozygous APOE ε4 carriers also had lower WMH volume than noncarriers and heterozygous carriers . Heterozygous carriers had a greater burden of cardiovascular risk factors .Homozygous  factors .WMH were15 A total of 66.6% (n = 8/12) of the APOE ε4 noncarriers had CAA compared to 76% (n = 16/21) of APOE ε4 carriers. Sixty-four percent (n = 9/14) of heterozygous APOE ε4 carriers had CAA, whereas 100% (n = 7/7) of the homozygous APOE ε4 carriers had CAA. However, differences across these groups were not significant . Fifty percent (n = 6/12) of patients with ε3-ε3 genotype had CAA, 50% (n = 1/2) of the ε3-ε2 patients, 39% (n = 8/13) of ε3-ε4 patients, and 100% (n = 7/7) of the ε4-ε4 patients had CAA. There were no patients with ε2-ε2 genotype.In the autopsy subsample, 21 patients were neuropathologically diagnosed with AD and 15 with DLB. All AD cases were pathologically confirmed to have AD, including one case with coexisting Lewy bodies. All DLB cases were confirmed to have DLB, with varying degrees of neurofibrillary tangle pathology.APOE ε4 carriers and noncarriers except that carriers were significantly younger than noncarriers .Characteristics of the study sample are summarized in APOE ε4 homozygotes were younger than heterozygotes and noncarriers , learning/memory , and language .p value <0.001), but not with memory or language. No interaction was observed between WMH and APOE ε4 for executive functions , memory (0.97), or language (0.34).In the full model (n = 198), WMH volume was associated with attention/executive functions  but not in homozygotes . For language, however, effect estimates for both homozygotes and heterozygotes were nonsignificant.As in the SDS, WMH volume was associated with executive functions in p value 2.117 × 10−3), learning/memory , and language  in carriers, with no effects seen in noncarriers. No heterogeneity was observed between the 2 studies and variance in effect estimates attributable to heterogeneity for all domains was ∼0%.Meta-analyses of estimates from SDS and ADNI-I showed a strong association of WMH with attention/executive functions  as well. Therefore, studies with larger sample sizes are required. However, in an attempt to obtain more robust estimates, we conducted meta-analyses of estimates from SDS and ADNI, which resulted in stronger results. The SDS and ADNI-I used a different neuropsychological battery; however, there were similar tests available in both cohorts tapping into the major cognitive domains. This would not have affected our results as replication is more robust if performed using a different methodology to test the same research question. The number of patients who completed each cognitive test differed, which was related to dementia severity. Missing data from more severe cases might have resulted in an underestimation of the associations. Smoking and diabetes were not documented for most ADNI-I participants, hence were not included as covariates; these were not significant confounders in the SDS sample, so models in the 2 samples are fairly comparable. The numbers in the autopsy-based dataset were not sufficient to draw definitive conclusions; however, they provided important insights and can possibly direct future research.We examine the effect of APOE ε4 may influence the association of WMH with executive functions and language across the spectrum of AD and DLB. Our meta-analysis results showed significant associations of greater WMH volume with cognitive impairment across all 3 cognitive domains tested. Information about the APOE ε4 status of patients may be useful to understand the relative contributions of different pathologies to an individual's unique dementia syndrome, and to guide therapy as well. Future studies should aim to extend these findings to other dementia diagnoses and larger datasets. These findings emphasize the importance of WMH (as a marker of SVD) across the AD/DLB spectrum, and open avenues for further research to understand shared etiologies and risk factors across the dementias.