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Statistical analysis | dysplasia | ESOPHAGEAL CANCER, DYSPLASIA | Summary statistics for continuous variables (means and standard deviations) and categorical variables (counts and proportions) were presented for both the intervention and control groups. An aggregate of the compliance for all 12 weeks for each individual was obtained by dividing 100/12 for example, 100% compliance if patients have full compliance for all 12 weeks, 92% if they have missed 1 week, 83% if missed 2 weeks, etc, and presented as mean.All available data were included in the statistical analysis referring to the intention‐to‐treat approach with respect to the original study group assignment and regardless of the patient's participation rate in the exercise intervention. Complete case analysis was used and no attempt to impute missing data was made. Changes in muscle strength and muscle mass were assessed as continuous outcomes. Mean differences (MD) with 95% confidence intervals (CI) were calculated to compare changes between baseline and post‐intervention assessments for the two groups. Owing to limited sample size, randomization might not be sufficient to control for confounding. Therefore, three models were used. In model 1, no confounders were adjusted for, and independent Baseline characteristics of study participants in a Swedish nationwide randomized controlled trial of a home‐based physical activity program in patients treated with surgery for esophageal cancer
Abbreviations: CDS, Clavien Dindo Score; HGD, high‐grade dysplasia.Results are mean ± SD.Missing values <4%. | PMC9939163 |
RESULTS | PMC9939163 | |||
Study participants | esophageal cancer, D) Muscle mass | OESOPHAGEAL CANCER | Patient enrolment was consecutive and lasted from January 2016 to August 2020. A total of 243 survivors from the OSCAR study were assessed for eligibility. Of them, 233 met the inclusion criteria, but 72 declined to participate. The remaining 161 participants (69.1% of those eligible) were randomly assigned to either the intervention group (Patient characteristics are presented in Table The compliance rate in the intervention group was 91%. The changes in the means between baseline and post‐intervention, comparing the intervention with control group for all the primary outcome measures are presented as a panel graph in Figure Panel graph showing changes in means of the primary outcomes. (A) Maximal hand grip strength; (B) Average hand grip strength; (C) Lower extremity strength; (D) Muscle mass assessed between 1 year after surgery for esophageal cancer (baseline) and after approximately 12 weeks of physical activity‐based intervention (follow‐up) | PMC9939163 |
Hand grip strength | esophageal cancer | OESOPHAGEAL CANCER | Among the 70 patients with completed measures in the control group, two patients had missing values for hand grip strength and hence were not included in the final analysis (Figure Differences in primary outcomes between baseline and post‐intervention for the two allocation groups of the Swedish nationwide randomized controlled trial of a home‐based physical activity program in patients treated with surgery for esophageal cancer presented as per the three statistical models
Abbreviations: CI, Confidence intervals; MD, Mean differences.Significant at Significant at | PMC9939163 |
Lower extremity strength | From those who had completed measures in the control group ( | PMC9939163 | ||
Muscle mass | Among the participants with complete end of study measures in the intervention group ( | PMC9939163 | ||
DISCUSSION | esophageal cancer, lower extremity muscle strength, muscle mass | ESOPHAGEAL CANCER, RECRUITMENT, OESOPHAGEAL CANCER | This trial demonstrated that a specially designed 12‐week home‐based PA program improved lower extremity muscle strength among patients having undergone surgery for esophageal cancer 1‐year earlier, while no influence on hand grip strength or muscle mass was identified.Some methodological strengths and weaknesses of the trial should be considered. A major strength was the nationwide multicentered design that ensured that all eligible patients were identified yielding a high enrolment rate. Adherence to a rigorous protocol enabled homogeneity in the recruitment process also uniformity and completeness of the data collected. Moreover, a comprehensive variable list with patient and clinical data allowed for adjustment for predefined confounders and variables observed to vary between intervention and control groups at baseline. Another notable strength is the unique home‐visit design for data collection by the research nurse at baseline and research dietician at follow‐up, which aided increased completeness and reduced missing values at both time points. One potential limitation may have been the exclusion of patients not willing to start a PA intervention may present a potential for selection bias. In addition, a slightly higher attrition rate in the intervention group than the control group also may have contributed to some extent of selection bias. Another limitation is that although preoperative BMI was adjusted for in the analysis, unknown confounders such as pre‐operative muscle mass and distribution of body fat among patients may have still differed between the groups but unaccounted for in the analysis but the RCT design may help reduce these imbalances. In addition, the higher drop‐out rate (17% of those randomized) than assumed in the original sample size calculated is another limitation in the present study.To our knowledge, this is the first RCT to examine the effectiveness of a home‐based PA intervention in long‐term survivors of esophageal cancer. The results cannot be directly compared to other exercise trials among post‐operative esophageal cancer survivors since they were not conducted at the same time point after surgery. In other studies testing exercise interventions after esophageal cancer surgery, the intervention was either multidimensional including both PA and nutritional components or conducted immediately after surgery.A distinctive strength of our trial was the home‐based exercise intervention, that was designed to personalize the intervention according to patient specific considerations such as their average age, invasive treatment history and a nationwide recruitment. The intervention was designed to be simple, safe, easy to perform, monitored from distance, and inexpensive. Home‐based exercise programs show promising potential for effective improvement of symptoms, exercise capacity, and quality of life in patients with lung,The long and incomplete recovery with substantial limitations in long‐term recovery in postoperative survivors of esophageal cancer is a concern for patients, healthcare, and society. Most patients need many follow‐up appointments with healthcare during several years, and the limited availability of interventions that can help these patients is a great concern. Thus, the focus on the role of a physical training program after surgery for esophageal cancer in improving muscle strength, which is a major determinant of physical recovery, is imperative. The results of this trial, testing the intervention of a tailored 12‐week home‐based PA program contribute to the development in the post‐operative management of these patients. The results show promising potential for encouraging patients to be physically active after surgery and in turn enhance the rehabilitation and provide a novel and robust tool for better recovery. An improved recovery would decrease the burden on healthcare by increasing patient self‐care and reducing the need for further or extra appointments. A research partnership group consisting of patients operated on for esophageal cancer were presented with the results of the present study and inputs from their interpretation of the results are included in the discussion above.In conclusion, this nationwide multicenter RCT demonstrated that a tailored home‐based PA intervention induced improvement in lower extremity muscle strength among esophageal cancer patients who underwent surgery for esophageal cancer 1‐year earlier. Although further investigations are warranted to determine the optimum intensity of the intervention and potential measurement bias, the results should be enough for encouraging patients to be physically active after surgery in the longer term to improve the recovery. | PMC9939163 |
FUNDING INFORMATION | ALF, Cancer | CANCER | This work is supported by the Swedish Cancer Society 180685, the Swedish Research Council 2017–01744, the Swedish Research Council for Health, Workinglife and Welfare 2017–00812, The Cancer Research Funds of Radiumhemmet 171103 and Region Stockholm (ALF) LS 2018–1157. Pernilla Lagergren is supported by the NIHR Imperial Biomedical Research Centre (BRC) for her position at Imperial College London, London, United Kingdom. | PMC9939163 |
CONFLICT OF INTEREST | The authors declare no conflict of interest. | PMC9939163 | ||
ETHICAL APPROVAL STATEMENT | Ethical approval was granted by the Regional Ethical Review Board in Stockholm, Sweden (Dnrs: 2015/2142–32; 2016/1696–32/1; 2018/1447–32). | PMC9939163 | ||
CONSENT TO PARTICIPATE | Written and oral information was given, and informed consent was collected from all participants by the research nurse before the randomization envelope was opened. | PMC9939163 | ||
TRIAL REGISTRATION | PMC9939163 | |||
ACKNOWLEDGMENTS | We acknowledge patients of the PA study for their valuable participation and our Surgical Care Science patient research partnership group for valuable inputs on the study. | PMC9939163 | ||
DATA AVAILABILITY STATEMENT | The data sets generated and/or analyzed in the current study will not be publicly available due to the ethical review act but will be available from the principal investigators of the study on reasonable request. | PMC9939163 | ||
REFERENCES | PMC9939163 | |||
2. Materials and Methods | PMC10346321 | |||
2.1. Study Design and Participants | chronic non-communicable diseases, amenorrhea | VASODILATION, CARDIOVASCULAR DISEASES | A randomized trial conducted on 48 individuals compared the effects of resveratrol supplementation (500 mg/day) and energy restriction (1000 kcal/day) for 30 days on SIRT1 and vascular reactivity parameters. The study participants were 24 postmenopausal women (01 years of natural amenorrhea) and 24 men aged 55 to 65, all without previous cardiovascular diseases. Participants were healthy volunteers without chronic non-communicable diseases, normal physical examination, and normal resting electrocardiogram. After a washout period of 15 days without using any medications or supplements, the participants were randomly assigned to either caloric restriction or resveratrol groups in a 1:1 ratio according to sex. Subsequently, participants underwent a standardized interview, blood sample collection, anthropometric assessment, blood pressure and heart rate measurement, and vascular reactivity test. Such procedures were repeated at the end of the study.Exclusion criteria were: BMI ≥ 30 kg/mThe analyzed clinical variables were age, weight, body mass index (BMI), waist circumference, blood pressure, and heart rate. Biochemical parameters included serum concentrations of triglycerides, total cholesterol, high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), apolipoprotein A–I (apoA–I), apolipoprotein B (apoB), lipoprotein (a) (Lp(a)), non-esterified fatty acids (NEFA), glucose, insulin, high-sensitivity C-reactive protein (hsCRP), noradrenaline (NA), and serum levels and gene expression of SIRT1. Vascular reactivity parameters included endothelium-dependent and endothelium-independent vasodilation.The energy restriction group consisted of a low-calorie diet (1000 kcal/day), corresponding to an approximate 50% energy intake reduction. Food diaries were used to analyze the adherence to the proposed interventions. Subjects were instructed to write down all the food intake daily and not exceed 1000 kcal/day. Participants included in the resveratrol group were instructed to take resveratrol capsules (250 mg of resveratrol each) twice a day (The study was approved by the Ethics Committee of the University of São Paulo Medical School Hospital (CAAE:00788012.8.0000.0068), and all participants signed informed consent. Trial registration: | PMC10346321 |
2.2. Biochemical Analysis | Laboratory tests were performed with biological samples collected after a 12-h fast. Serum samples were obtained after collecting venous blood in tubes without an anticoagulant and centrifuged for 20 min at 1800G (Eppendorf, Hamburg, Germany). Citrated blood samples were centrifuged for 10 min at 200× | PMC10346321 | ||
2.3. Sirtuin 1 Assessment | SIRT1 serum concentration was determined using an ELISA kit (Uscn Life Science, Wuhan, Hubei, China). Serum samples, before and after interventions, were analyzed in duplicate and in the same ELISA plate using the Multiscan FC plate reader (Thermo Scientific, Waltham, MA, USA), with a coefficient of variation of 12%, according to the manufacturer’s instructions. All tests were performed according to the manufacturer’s instructions.Gene expression of SIRT1 was evaluated at pre- and post-inclusion in the protocol by using the specific assay Hs01009005_m1 (Applied Biosystems, Life Technologies, 151, Waltham, MA, USA). Total RNA was obtained from peripheral leukocytes using the TRIzol reagent (Life Technologies). cDNA synthesis was performed using a Superscript II kit (Life Technologies) with 1 μg from total RNA in a final volume of 20 μL, according to the manufacturer’s instructions. Expression of the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), used as the normalizing gene, was evaluated by using the specific assay # Hs02758991_g1 (Applied Biosystems). The reaction mix was prepared using 5 μL of Universal Master Mix (Life Technologies), 0.5 μL of primers and probes mix (×20), and 2.5 μL of cDNA diluted samples (1:5).PCR was performed according to the following protocol: enzymatic activation for 2 min at 50 °C, initial denaturation for 10 min at 95 °C, followed by 40 cycles of denaturation for 15 s at 95 °C and annealing for 20 s at 60 °C. Reactions were run in triplicate, and the number of copies of the gene transcript was determined using Ct (“threshold cycle”) values. For calculations, Ct values of SIRT1 were subtracted from Ct values for the GAPDH gene. The results are expressed in arbitrary units (AU). | PMC10346321 | ||
2.4. Resveratrol Purity and Formulation Analysis | The resveratrol administered to participants was obtained from a compounding pharmacy (Buenos Aires Pharmacy, São Paulo, Brazil). The purity of the product supplied was analyzed by capillary electrophoresis using a Proteome Lab PA800 (Beckman Coulter, Fullerton, CA, USA) at the Laboratory of Capillary Chromatography and Electrophoresis at the Chemistry Institute of the University of São Paulo. Samples of the manipulated capsules and the standards of resveratrol were performed in triplicate, and areas under the peak were compared. The purity of resveratrol was 87 ± 1.1% on average (coefficient of variation: 1.2%). | PMC10346321 | ||
2.5. Vascular Reactivity Assessment | Endothelium-dependent flow-mediated vasodilation | VASODILATION | Endothelium-dependent flow-mediated vasodilation (FMD) and endothelium-independent vasodilation (NMD) were assessed according to a previous guideline [ | PMC10346321 |
2.6. Statistical Analyses | REGRESSIONS | The sample size of 48 patients, with 24 subjects per treatment arm, was determined to yield a power of 80% with a 5% significance level to detect a 30% difference in Sirt1 serum concentrations. Participants were randomly assigned in a 1:1 ratio using computer-generated numbers to include participants in the resveratrol or energy restriction groups.Pre- and post-intervention variables were described as the median and interquartile range (IQR). Wilcoxon’s test was used for pre- and post-treatment analysis. Mann-Whitney’s U test was used for intergroup comparisons of baseline variables and changes. Partial correlations controlled by intervention group (resveratrol and energy restriction) and sex (men and women) were used to evaluate the relationship between changes (Δ) of SIRT1 and vascular reactivity. Multiple linear regressions with a backward method were used to assess this association further, using changes (Δ) of triglycerides, BMI, total cholesterol, and noradrenaline as adjustment variables and changes (Δ) of FMD and NMD as dependent variables. Predictive variables with weak association (The level of significance was set at | PMC10346321 | |
3. Results | REGRESSIONS | Baseline and post-intervention data are described in At the end of the study, individuals in the energy restriction group had a significant reduction in weight (When comparing groups, there was a statistically significant difference between the changes (∆) in weight (Both interventions increased serum SIRT1 (Correlations adjusted by group and sex are described in Multiple linear regressions are shown in Regarding the impact of SIRT1 on FMD ( | PMC10346321 | |
4. Discussion | cerebral ischemic stress | VASODILATION, CARDIOVASCULAR DISEASES, INFLAMMATION, OXIDATIVE STRESS, ENDOTHELIAL DYSFUNCTION | The main results of our study showed that 30 days of energy restriction, i.e., a 1000 kcal diet, or 500 mg daily resveratrol supplementation similarly increased circulating SIRT1 and decreased plasma noradrenaline. Plasma noradrenaline reduction was correlated with higher baseline circulating SIRT1. After the interventions, circulating SIRT1 was positively correlated with NMD independently of sex and interventions. Furthermore, circulating SIRT1 was independently associated with NMD in the energy restriction group. Regarding SIRT1 gene expression, we did not find any statistically significant differences at the end of the study, but we found a statistically significant association with FMD in men.Dietary energy restriction is the only physiological intervention that increases life expectancy in mammals [Resveratrol increases cell cAMP content, which in turn activates monophosphate-activated protein kinase (AMPK), a protein that acts as an upstream molecule to regulate expression of SIRT1 [SIRT1 has gained attention due to its metabolic regulation effect with concomitant activation of AMPK (adenosine5′-monophosphate (AMP)-activated protein kinase), a key molecule in cellular metabolism. Both proteins induce mitochondrial biogenesis, fatty acids β-oxidation, and gluconeogenesis through the activation of peroxisome proliferator-activated receptor- γ coactivator 1α (PGC1α) [Inflammation and oxidative stress are key factors for nitric oxide (NO) degradation and, subsequently, endothelial dysfunction, which is a critical factor for the development and progression of cardiovascular diseases [Our results showed that baseline SIRT1 gene expression positively correlated with changes in FMD, suggesting that individuals with higher SIRT1 gene expression presented higher FMD. Furthermore, we showed that SIRT1 expression was positively associated with FMD in men. This may be explained by the lower levels of circulating estrogen in men than in women, resulting in lower basal activation of SIRT1 in this group. Laboratory studies showed that, in female rats, estrogen activated the SIRT1/AMPK signaling pathway, increasing protection against cerebral ischemic stress [Regarding endothelium-independent vasodilation, our results showed that circulating SIRT1 was independently associated with NMD in the energy restriction group. Vascular reactivity measured by NMD provides information about the response of vascular smooth muscle cells (VSMCs) to a NO donor, i.e., endothelium-independent vasodilation [Both interventions reduced plasma noradrenaline, indicating a sympatholytic effect of resveratrol and dietary energy restriction. We also found that higher baseline levels of circulating SIRT1 were inversely correlated with plasma noradrenaline, suggesting that SIRT1 regulates plasma noradrenaline. Vascular reactivity is regulated by sympathetic nervous system (SNS) activation, which can be assessed by plasma noradrenaline [Our study has limitations. Firstly, we analyzed a small number of subjects. Our sample size was calculated using circulating SIRT1. Therefore, variations in parameters such as gene expression of SIRT1 and vascular reactivity could be undetected. On the other hand, we used multivariate analysis and correlations to detect change tendencies. Another limitation was that all subjects were at low cardiovascular risk, which could mask alterations in vascular reactivity parameters after interventions. In addition, the applicability of vascular reactivity tests on low cardiovascular risk populations is still debated [As far as we know, this is the first study that associated SIRT1 with well-established markers of vascular function in humans. Our results corroborate a previous laboratory study that showed that SIRT1 improves endothelial function [ | PMC10346321 |
Author Contributions | Conceptualization, A.d.P.M. and G.H.F.G.; Methodology, A.d.P.M.; Validation, A.d.P.M.; Data collection, A.R., M.F.d.S.G., S.D.A., C.M.C.S. and A.d.P.M.; Data analysis, A.d.P.M. and G.H.F.G.; Writing—Original draft preparation, A.d.P.M., G.H.F.G., K.L.K. and N.F.d.O.F.; Writing—review and editing, A.d.P.M. and G.H.F.G.; Project administration, A.d.P.M.; Funding acquisition: A.d.P.M. All authors have read and agreed to the published version of the manuscript. | PMC10346321 | ||
Institutional Review Board Statement | HEART | The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of Heart Institute of University of São Paulo Medical School (protocol code CAAE:00788012.8.0000.0068). | PMC10346321 | |
Informed Consent Statement | Informed consent was obtained from all subjects involved in the study. | PMC10346321 | ||
Data Availability Statement | Not applicable. | PMC10346321 | ||
Conflicts of Interest | The authors declare no conflict of interest. | PMC10346321 | ||
References | AD | VASODILATION | Research protocol design.Clinical and biochemical characteristics of the participants at baseline and post-treatment.Data are presented as median (interquartile range—IQR). *: Comparison of intra-groups post–pre-tests. **: Comparison between groups at baseline. Significant values are bold.Comparison of the median differences (post-test—pre-test; ∆) between groups.Data are presented as median (interquartile range—IQR). Significant values are bold.Partial correlations between vascular reactivity parameters, noradrenaline, and Sirtuin 1 adjusted by treatment group and sex.Values are detailed as correlation coefficient (r). AD: artery diameter; FMD: flow-mediated vasodilation; NA: noradrenaline; NMD: nitrate-mediated vasodilation; *: Effects of Sirtuin 1 on flow-mediated vasodilation stratified by sex and intervention.The first model included all variables (i.e., triglycerides, BMI, total cholesterol, noradrenaline, circulating, and expression of Sirtuin 1). Excluded variables in the final model are indicated by “n/a”. Significant values are bold.Effects of Sirtuin 1 on nitrate-mediated vasodilation stratified by sex and intervention.The first model included all variables (i.e., triglycerides, BMI, total cholesterol, noradrenaline, circulating, and expression of Sirtuin 1). Excluded variables in the final model are indicated by “n/a”. Significant values are bold. | PMC10346321 |
1. Introduction | Obesity, obesity, death, breast cancer, Breast cancer, Smartphone-Based Cancer, deaths, Cancer | OBESITY, OBESITY, BREAST CANCER, BREAST CANCER, SECONDARY, CANCER | Breast cancer prevalence has increased globally, with 12.2% of breast cancer cases identified in China. Obesity and unhealthy lifestyles are major risk factors for breast cancer. We conducted a randomized control trial to assess the feasibility and evaluate the preliminary effect of the Smartphone-Based Cancer and Obesity Prevention Education (SCOPE) program among adult biological women with a waist circumference greater than 80 cm. The SCOPE program includes tailored and culturally appropriate educational information for obesity and breast cancer prevention delivered by the research team via WeChat. The control group received non-tailored general health information via WeChat. A total of 102 women (52 intervention, 50 control) participated, and 87 (85%) completed 6-month follow-up assessments. For the primary study outcome at 6 months, women using SCOPE significantly reduced waist circumference (Cohen’s Cancer is China’s leading cause of death (about 7500 deaths per day). Cancer not only adversely affects the health of individuals but also imposes an economic burden on families and healthcare systems [In China, smartphone ownership is about 71%; thus, a smartphone-based intervention could offer an effective and feasible tool to reduce the risk of obesity and breast cancer [Thus, we piloted a smartphone-based intervention designed to meet the needs and lifestyles of Chinese mothers using the popular communication platform WeChat. This study aimed to assess the feasibility and estimate the preliminary efficacy of a Smartphone-Based Cancer and Obesity Prevention Education Program for Chinese Women (SCOPE) using a randomized control trial (RCT) design. To prevent the risk for breast cancer, this study focused on obesity as the primary outcome and healthy lifestyle and knowledge and attitude regarding breast cancer prevention as secondary outcomes. Two major aims of this study include:Aim 1: To assess the feasibility of SCOPE, a tailored smartphone-based lifestyle intervention.Aim 2: To estimate the preliminary efficacy of SCOPE intervention on the primary study outcome (waist circumference) and secondary outcomes (body mass index, fruit and vegetable consumption, physical activity, self-efficacy in diet and physical activity, social support in diet and physical activity, and knowledge and attitudes toward breast cancer) in the SCOPE intervention compared to the control groups at 3 months and 6 months. | PMC10218157 |
2. Materials and Methods | DISEASE, RECRUITMENT | This pilot study used a randomized control trial design. The social cognitive theory (SCT) [Women who met the inclusion criteria from two community health centers in Changsha, Hunan Province, China, were invited to this study and randomized to either the SCOPE or control programs. Inclusion criteria included (1) being biologically female, (2) being at least 18 years old, (3) having a waist circumference greater than 80 cm, (4) owning a smartphone, (5) being able to read Chinese and speak Mandarin, (6) being premenopausal, and (7) having a child between the ages of 1 and 17 years old. Exclusion criteria included (1) being pregnant, (2) giving birth less than 12 months before the enrollment date, or (3) having an acute or life-threatening disease (e.g., renal or heart failure).Trained research assistants worked with the study sites to recruit eligible women. An invitation letter that explains the study’s purpose and WeChat contact information was sent to all women with dependent children. They were asked to message the study’s WeChat within two weeks of receiving the invitation to indicate their interest in participating. Those who were interested were invited to an in-person screening session during health fairs hosted at the recruitment sites. Several in-person screening sessions were offered at each location. During these sessions, eligibility verification, informed consent, administration of a baseline survey, and measurements were obtained. The committee on human research at the University of California San Francisco and Central South University approved the study (IRB 18-27025).The SCOPE intervention consisted of three components: (1) the use of the Fitbit activity tracker, (2) 12-weekly education modules delivered via WeChat, and (3) bi-weekly tailored messages. The 12-week active intervention included the Fitbit tracker and 12 weekly, culturally appropriate, evidence-based educational modules delivered via WeChat, a popular communication app in China. After the 12 weekly modules, the maintenance phase included six bi-weekly tailored tips and messages based on Fitbit data and personal goals sent via WeChat. The control group received general health information without tailoring via WeChat at the same frequency as the SCOPE program. Data on outcomes were collected at baseline, 3 months, and 6 months. | PMC10218157 | |
2.1. Intervention Contents | Obesity, WeChat-based, Cancer | OBESITY, BREAST CANCER, DISEASE, CANCER | Fitbit tracker. All participants received a Fitbit Alta HRTwelve modules. Modules were sent via WeChat. Participants received 12 weekly, culturally appropriate, evidence-based educational modules along with tailored tips and messages via WeChat. Each module includes an educational session/information that lasts less than 20 min. The educational modules include (1) Assess Your Risk, (2) Prevention Through Cancer Screening and Vaccination, (3) Eat Well to Reduce Cancer Risk for You and Your Family, (4) Smart Shopping and Cooking, (5) Abs Exercise, (6) Active Lifestyle with Your Child to Boost Your Energy, (7) Fitness for You and Your Child, (8) Healthy Lifestyle for Healthy You, (9) Happy Motherhood and Stress Management, (10) Effective Coping and Getting Support, (11) Stay Motivated to Prevent Cancer and Obesity, and (12) Healthy Weight and Flat Abs for Long-Term Health. The contents of the modules were based on relevant evidence from the literature as well as professional organizations in China, such as the Chinese Center for Disease Controls (Biweekly WeChat messages. Biweekly WeChat messages are based on individual characteristics, behavior patterns, and preferences. Individual characteristics include information related to their health and family environment, such as the number of children at home and family history of breast cancer. Preferences include information collected at baseline on when they would like to receive the WeChat message, and behavior patterns include physical activity and diet data from Fitbit. Text messaging is a convenient way to facilitate realistic goal setting, self-monitoring, and information exchange. Interventions using WeChat-based text messages have been found to promote behavioral change and reduce weight in adults [ | PMC10218157 |
2.2. Control Group | The control group received a Fitbit Alta HR | PMC10218157 | ||
2.3. Data Collection and Measures | obesity, Diabetes | OBESITY, BREAST CANCER, CREST, DIABETES | Data were collected at baseline, 3 months, and 6 months. The research assistants and a registered nurse conducted all measurements. The trained research assistant and registered nurse measured WC, weight, and height. Study participants completed all questionnaires in a separate room where a research assistant could answer questions. All measurements and survey items have been used in China and demonstrated adequate reliability and validity.Feasibility. The number of eligible participants, percentage of completed assessments, and module access were assessed.Usability and acceptance. The frequency of accessing the 12 modules and satisfaction of reviewing the modules were collected to measure the usability and acceptance of these modules.Demographic survey. The socio-demographic questionnaire assesses participants’ age, education level, parents’ occupation, residence, and household income.Waist circumference (WC). This study followed the waist circumference measurement protocol set by the National Institute of Health. Waist circumference was measured midway between the lowest rib and the superior border of the iliac crest. The circumferences were given as the mean of the 2 measurements to the nearest 0.1 cm. Central obesity is a waist circumference ≥ 80.0 cm for Chinese women [Body mass index BMI. BMI is determined by measuring weight and height (weight [kg]/height [m]Fruit/vegetable intake and physical activity. Two items measured vegetable and fruit intake (>5 servings/per day) and regular physical activity (a total of 150 min per week) were used in this study. These two items were from the Canadian Diabetes Risk Questionnaire for the Chinese population survey used in China (CHINARISK). Adequate reliability (test-retested reliability = 0.98) and validity (sensitivity = 0.73 and specificity = 0.63) have been reported [Physical activity/average daily steps. The average daily steps on the week we collected data were used. Previous studies indicated that the Fitbit step count outputs are stable at different speeds and substantial with observer counts (0.97–1.0) [Self-efficacy for diet and physical activity. The 8-item survey was used in the study. Participants were asked about their confidence level in engaging in activities related to a healthy diet and regular physical activities. Scoring was based on five points (0- not confident to 5- very confident), and a higher score indicates a higher level of self-efficacy. Reliabilities have been documented in the exercise domain (Cronbach’s alpha 0.87 to 0.97) and the diet domain (Cronbach’s alphas 0.93 to 0.95) [Social support for diet and physical activity. The social support scale consists of six items measuring social support for diet and physical activity from family members and friends. The survey includes three items to evaluate the support of diet and exercise from family members and three from friends. The diet social support scale is based on a 4-point scale: 0 (never), 1 (sometimes), 2 (frequently), or 3 (always), while the physical activity social support scale is rated on a 5-point scale: 0 (never) to 4 (always). Higher scores indicate more social support for the participant. The survey has adequate reliability, with Cronbach’s alpha ranging from 0.80 to 0.87 for the diet domain and 0.84 to 0.91 for the physical activity domain [Perceived stress scale (PSS). This 14-items survey measures the degree of perceived stress the mothers perceived in the past month. The survey asked participants to respond to 14 questions on how often they have felt (such as upset, nervous, angry, or having difficulties) over the past month. Participants report the frequency of these questions on a five-point Likert scale (0 = never to 4 = very often), with the higher the score reflecting the higher level of the perceived stress. This survey has been used in China and has good psychometric properties in the Chinese population [Breast cancer risk knowledge, attitude, and barriers questionnaire (CBCSBQ). A 12-item self-report questionnaire measures knowledge (4 items), attitude (4 items), and barriers to mammographic screening (4 items) [ | PMC10218157 |
2.4. Data Analysis | Because this is a pilot study aimed at testing the feasibility, acceptance, and preliminary efficacy, a power analysis was not conducted. Since this pilot RCT has a limited sample size, analyses were intended to be exploratory, and we set α = 0.05 (two-sided). Descriptive statistics (means and standard deviations, or medians and interquartile ranges if skewed) were reported for quantitative demographic and outcome variables. Frequencies and percentages were reported for categorical variables. T, chi-square, or analysis of variance (ANOVA) tests were used to compare differences between the intervention and control group at baseline. We used mixed effects models to estimate the effect of intervention vs. control from before/after treatment (linear for continuous outcomes; logistic for binary, e.g., yes to 5 servings of fruit and vegetables). Because the sample size was small, we used the nonparametric bootstrap with 5000 draws to obtain bias-corrected confidence intervals and evaluated significance [ | PMC10218157 | ||
3. Results | PMC10218157 | |||
3.1. Sample Characteristics | A total of 102 women (52 in the SCOPE intervention and 50 in the control group) participated in this study (see | PMC10218157 | ||
3.2. Effect of the Intervention | Results from the mixed-model analysis found that at the 3-month follow-up, compared to baseline, participants in the SCOPE program compared to control significantly reduced WC (At 6 months, compared to baseline, women in the SCOPE group compared to control significantly reduced WC ( | PMC10218157 | ||
3.3. Feasibility and Satisfaction of the SCOPE Modules | A total of 85% of participants completed 6 months of follow-up. The main reasons for missing the follow-up assessment include lack of time and inability to come to the site for assessment. The average number of modules viewed was 8.5 out of 12 modules. Most participants in the SCOPE intervention program (93%) indicated they learned a great deal or a lot from these modules. | PMC10218157 | ||
4. Discussion | obesity, abdominal obesity, breast cancer | OBESITY, SDH, BREAST CANCER | As breast cancer is a significant health issue, and obesity increases the risk for breast cancer, especially for women with children, we developed a smartphone-based intervention using a popular communication app tailored to Chinese mothers. Our intervention aimed at reducing obesity, increasing knowledge and attitude, and reducing barriers to breast cancer. In our pilot study, less than 50% of participants who have abdominal obesity reported eating five servings of fruit and vegetables and exercising daily at baseline. We observed a significant reduction in WC and BMI in the intervention group compared to the control group and small to large effect sizes of the intervention on improving PA steps, breast cancer knowledge, and breast cancer attitude at three months follow-up. Smaller effects on behavior and psychosocial outcomes were found at the six-month follow-up. Our pilot study revealed good feasibility and high satisfaction in accessing the modules.The lower level of engagement in healthy eating and active lifestyle found in women with abdominal obesity in China is problematic and even worse during the COVID pandemic. The results of our study are consistent with other studies. Previous work examining the effects of the COVID pandemic in China found that more than half of people gained weight, with 16.3% of women gaining more than 2.5 kg [The results of our study revealed great potential in reducing obesity and the risk of breast cancer. Although studies focusing on Chinese women are limited, current evidence demonstrates that premenopausal Asian women with abdominal obesity have a higher risk of breast cancer compared to non-Hispanic white women [Our study also revealed small to large effects on the behaviors and psychosocial factors, such as increasing fruit/vegetable intake, PA steps, and social support regarding diet and PA. As studies have supported the impact of a poorer health lifestyle, including a sedentary lifestyle and inadequate fruit and vegetable intake, on an increased risk for abdominal obesity, it is important to build a healthy lifestyle and a supportive environment for maintaining a healthy lifestyle to ensure the sustainability of health behavior changes [In addition, this intervention also improved women’s knowledge and attitudes regarding breast cancer. Our study is consistent with other studies suggesting that adequate educational programs can increase women’s knowledge and attitudes about breast cancer [Our pilot study revealed good feasibility and high satisfaction in accessing the modules. Traditional in-person weight management and breast cancer prevention programs are time-consuming and costly, especially during a pandemic. The promising effect of our intervention can contribute to the use of communication apps and theoretical-based intervention. Our intervention integrates the SCT and SDH, the use of everyday technology (WeChat), and an adaptation of an evidence-based program tailored to the behavior patterns, preferences, and cultures of women in China. WeChat is a popular multifunctional social media mobile app, and connectivity is widely available without restrictions [There are several limitations in this pilot study. The study’s results need to be interpreted with caution. Because this pilot study was conducted with a small sample size and a homogeneous population, we have limited power to detect small differences. The results may also only be generalized to similar populations. Moreover, this study did not include other potential risk factors such as alcohol drinking and smoking. Other limitations included convenience sampling, self-reported measures, and only 6 months of follow-up. Future studies would also need to investigate whether specific subgroups of women can benefit more from this program and include other potential risk factors (such as alcohol consumption and smoking) in the study. | PMC10218157 |
5. Conclusions | obesity, breast cancer | OBESITY, BREAST CANCER | This pilot study demonstrates a promising way to prevent obesity and breast cancer. The SCOPE intervention delivered via a communication app is feasible and may positively reduce women’s risk for obesity and improve breast cancer knowledge and attitude. This intervention has great potential to promote the health and wellness of women and the community. | PMC10218157 |
Author Contributions | Conceptualization, J.-L.C. and J.G.; methodology, J.-L.C.; software, T.J.H. and C.-X.L.; validation, J.-L.C., J.G. and T.J.H.; formal analysis, T.J.H., J.-L.C. and C.-X.L.; investigation, Q.Z., Y.J., P.M., H.Z. and Q.H.; resources, P.M., H.Z. and Q.H.; data curation, C.-X.L., Q.Z., Y.J. and Q.H.; writing—original draft preparation, J.-L.C. and C.-X.L.; writing—review and editing, J.-L.C. and J.G.; visualization, C.-X.L.; supervision, J.-L.C.; project administration, Q.Z., Y.J., P.M., H.Z. and Q.H.; funding acquisition, J.-L.C. All authors have read and agreed to the published version of the manuscript. | PMC10218157 | ||
Institutional Review Board Statement | The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Ethics Committee on Human Research of the University of California San Francisco (IRB 18-27025). | PMC10218157 | ||
Informed Consent Statement | Informed consent was obtained from all subjects involved in the study. | PMC10218157 | ||
Data Availability Statement | The data presented in this study are available on request from the corresponding author. The data are not publicly available due to them containing personal information. | PMC10218157 | ||
Conflicts of Interest | The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. | PMC10218157 | ||
References | RECRUITMENT | Sample session in WeChat. Patient recruitment flow chart.Demographics.Baseline data.Comparisons between control and SCOPE experimental groups.Mixed model and effect size of the intervention. | PMC10218157 | |
Visual Abstract | breast cancer | METASTATIC DISEASE, BREAST CANCER | Published online Jul. 13, 2023.
Imaging using the human epidermal growth factor receptor 2 (HER2)–binding tracer Up to 20% of breast cancer cases have human epidermal growth factor receptor 2 (HER2) overexpression with or without HER2 oncogene amplification (Currently, treatment failure is relatively common, and cure remains rare in metastatic disease (ABY-025 was labeled with positron-emitting Trastuzumab resistance has been documented both as primary and as acquired, prominently in previously treated patients (The primary endpoint was to correlate [ | PMC10478820 |
MATERIALS AND METHODS | PMC10478820 | |||
Patient Population | tumor, PBC, neutropenia | TUMOR, HER2-POSITIVE BREAST CANCER, PBC, NEUTROPENIA, MALIGNANCIES, CONGESTIVE HEART FAILURE, CONCOMITANT DISEASE, HER2-NEGATIVE BREAST CANCER | The current study was a planned interim analysis of a phase II study embedded within a larger academically driven prospective open-label phase II and phase III diagnostic trial, approved by the Swedish Medical Products Agency (EudraCT 2017-002115-34; diarienummer, 5.1-2018-30296; Women with newly diagnosed stage II or stage III PBC and planned for neoadjuvant therapy or women with confirmed progression in MBC and planned for HER2-targeted therapy concomitant with chemotherapy were considered as candidates for this study. The inclusion criteria included women with biopsy-confirmed HER2-positive or borderline HER2-positive breast cancer, at least 1 tumor lesion of at least 1.0 cm, at least 1 tumor available for biopsy, a negative pregnancy test and active contraceptive measures for women of child-bearing age, at least 18 y of age, a predicted survival of more than 12 wk, and a World Health Organization performance status of 2 or lower.The exclusion criteria included women with biopsy-confirmed HER2-negative breast cancer before enrollment in this study; other coexisting malignancies; uncontrolled serious concomitant disease including congestive heart failure, inadequate organ function such as neutropenia, or abnormally high liver or kidney function tests (absolute neutrophil count < 1,500 cells/mmPatients fulfilling the above-mentioned criteria underwent [ | PMC10478820 |
Production of [ | The ABY-025 peptide was provided by Affibody AB. Production of [ | PMC10478820 | ||
[ | All scans were performed using a Discovery MI PET/CT scanner (GE Healthcare), and scans included a section from the skull apex to mid thigh. [The first 10 patients were closely monitored by electrocardiograms and clinical examinations during their stay at the PET center. All patients received a phone call after 24 h from the time of [ | PMC10478820 | ||
[ | [ | PMC10478820 | ||
PET Measurements | Images obtained from both [ | PMC10478820 | ||
Biopsies | Lesions were selected for study biopsies on the basis of uptake on both PET scans and accessibility at a multidisciplinary trial conference the day after the last baseline PET scan. The biopsied lesions were carefully located and manually defined on [ | PMC10478820 | ||
HER2-Targeted Treatment | multiple recurrent disease | RECURRENT DISEASE | Patients planned for neoadjuvant treatment and patients with first-time recurrent disease received trastuzumab, pertuzumab, and chemotherapy according to guidelines, whereas patients with multiple recurrent disease received trastuzumab emtansine. | PMC10478820 |
Statistical Analysis | PET metrics were reported as mean ± SD unless otherwise stated. Receiver operating characteristic curve analysis was used to investigate the predictive value of the variables and to define a cutoff for [ | PMC10478820 | ||
RESULTS | PMC10478820 | |||
Patient Characteristics | RECRUITMENT, BREAST CANCER | Patient characteristics and descriptive data are shown in Patient Characteristics and Descriptive DataBased on immunohistochemistry and ISH results.Qualitative data are number and percentage; continuous data are median and range.Anatomic Distribution and [Number of biopsied lesions is in parentheses, 1 per patient. SUVDiagram of recruitment and diagnostic classification of study subjects, according to the Standards for Reporting Diagnostic Accuracy. BC = breast cancer; ABY = [Color-coded clinical response in patients with breast cancer. | PMC10478820 | |
Tumor Lesions | We measured tracer uptake in up to 5 of the largest lesions per patient, including the biopsied lesion. Anatomic distribution of measured lesions is shown in [ | PMC10478820 | ||
[ | In total, 12 patients showed a mismatch between PET and HER2 status, using SUV | PMC10478820 | ||
Clinical and Metabolic Response | therapy-naïve, PBC, breast cancer | DISEASE, PBC, BREAST CANCER | Thirty-two patients had either partial or complete metabolic response after receiving 2 cycles of treatment. Clinically, 22 patients had complete response, 11 had partial response, 2 had stable disease, and 5 had progressive disease. Global Δ-TLG was significantly associated with the clinical response ([Receiver operating characteristic curve analysis of positive metabolic response after 2 cycles of HER2-targeted treatment in breast cancer patients according to [A previously proposed SUVA multivariate model for patient-level prediction of global Δ-TLG using the number of previous treatments, global TLG at baseline, [Number of previous treatments and their effect on response rate measured as Δ-TLG after 2 cycles of HER2-targeted treatment. One-way ANOVA showed significantly different response rates among 3 groups (In contrast to MBC, all PBC patients achieved metabolic response regardless of [Δ-TLG was significantly associated with the number of previous treatments. All therapy-naïve patients achieved a metabolic response regardless of the [ | PMC10478820 |
DISCUSSION | coronavirus disease 2019 | CORONAVIRUS DISEASE 2019 | The potential of [The results showed that immunohistochemistry staining did not always reflect the biologic availability of the receptors; a HER2-positive biopsy sample with low [We found an inverse association between the number of previous treatments and the metabolic response to current treatment. The more treatments previously received, the higher the [Predicting positive metabolic response to HER2-targeted therapy in lymph nodes and soft-tissue lesions was possible using a prespecified cutoff SUVThe ability of [This study was initially intended as a multicentric phase II and phase III study, and the current data are the results from a prespecified interim analysis after inclusion of 40 patients. Unfortunately, the coronavirus disease 2019 pandemic coincided with the study, delaying our inclusion rate and prohibiting wider inclusion. In effect, intersite reproducibility remains to be studied. As we could not show a significant association between biopsy results and PET, which was the main endpoint, the planned phase III trial was aborted. Instead, further trials should focus on changes in clinical management and outcome using HER2 imaging. | PMC10478820 |
CONCLUSION | BREAST CANCER | Immunohistochemistry staining and ISH are currently the gold standards to determine HER2 status in breast cancer. However, limitations in the metastatic setting still hinder accurate biopsy-based evaluation of heterogeneous HER2 expression. Hence, the advantage of noninvasive techniques such as [ | PMC10478820 | |
DISCLOSURE | Breast Cancer, Cancer | BREAST CANCER, CANCER | This work was partially supported by grants from the Swedish Breast Cancer Association, Swedish Cancer Foundation (19 0507 Pj), Roche AB Sweden, and the Percy Falk Foundation. Fredrik Frejd and Joachim Feldwisch are employees and own shares in Affibody AB. Jens Sörensen received clinical advisor remunerations from Affibody AB. Johan Hartman obtained speaker’s honoraria or advisory board remunerations from Roche, Novartis, Pfizer, Eli Lilly, MSD, Veracyte, and ExactSciences and received institutional research support from Cepheid, Roche, and Novartis. No other potential conflict of interest relevant to this article was reported. | PMC10478820 |
REFERENCES | PMC10478820 | |||
Subject terms | AML, cancers, myelofibrosis | CANCERS, ACUTE MYELOID LEUKEMIA, AML, MYELOFIBROSIS, MUTANT, MYELODYSPLASTIC SYNDROMES | Telomerase enables replicative immortality in most cancers including acute myeloid leukemia (AML). Imetelstat is a first-in-class telomerase inhibitor with clinical efficacy in myelofibrosis and myelodysplastic syndromes. Here, we develop an AML patient-derived xenograft resource and perform integrated genomics, transcriptomics and lipidomics analyses combined with functional genetics to identify key mediators of imetelstat efficacy. In a randomized phase II-like preclinical trial in patient-derived xenografts, imetelstat effectively diminishes AML burden and preferentially targets subgroups containing mutant Bruedigam et al. explore the mechanism of action and therapeutic benefit of telomerase inhibitor imetelstat in a collection of PDXs from patients with AML and demonstrate that, in combination with chemotherapy, it re-sensitizes treatment-resistant AML cells. | PMC10824665 |
Main | blood cancer, AML | BLOOD CANCER, DISEASE, AML, LEUKEMIA STEM CELL | AML is an aggressive and lethal blood cancer with a 5-year overall survival rate of less than 45% for patients younger than 60 years of age and less than 10% for older patients, predominantly due to disease relapse after chemotherapy or targeted treatments. AML has been extensively classified based on biological features and advances in sequencing technologies have led to a comprehensive genetic classification strategy (European LeukemiaNet, ELN2017)Telomerase is an attractive target as it is highly expressed and reactivated in the majority of AML and absent in most cell types including healthy hematopoietic cells. We have previously shown that genetic depletion of telomerase eradicates leukemia stem cells, particularly upon enforced replicationIn addition to its canonical role as critical regulator of telomere length maintenance, telomerase fulfills important non-canonical roles contributing to stress elimination, regulation of Wnt/β-catenin, NF-κB and p65 signaling, as well as resistance to ionizing radiationPreclinical trials in patient-derived xenografts (PDXs) provide genetically diverse, tractable models to define the efficacy of drugs and to identify biomarkers of response and resistance in AMLIn this study, we aimed to assess the preclinical efficacy of imetelstat in a large AML PDX resource that reflects the diversity of genetic abnormalities found in large patient cohorts. We utilized this AML PDX resource to identify biomarkers of resistance and response to imetelstat therapy and to test potentially synergistic combination therapies. To elucidate the mechanism of action of imetelstat in an unbiased manner, we performed genome-wide CRISPR/Cas9 editing allowing the identification of gene knockouts that confer resistance to imetelstat therapy. This study reveals that imetelstat is a potent inducer of ferroptosis that effectively diminishes AML burden and delays relapse following oxidative stress-inducing therapy. | PMC10824665 |
Results | PMC10824665 | |||
Generation of a comprehensive AML PDX resource | AML, thrombocytopenia, anemia, splenomegaly, AML PDX | DISEASE, THROMBOCYTOPENIA, AML, ANEMIA | To generate a representative AML PDX inventory, primary bone marrow or blood samples from 50 patients were tested for engraftment and development of AML in NOD/SCID/IL2gR−/−/hIL3,CSF2,KITLG (NSGS). The overall success rate for primary engraftment in NSGS was 70%, defined by bone marrow, spleen or peripheral blood donor chimerism of at least 20%, splenomegaly (spleen weight >70 mg), anemia (HCT < 35%) or thrombocytopenia (PLT < 400 × 10From the individual samples from patients with AML that successfully engrafted in NSGS, 30 were randomly selected and characterized based on clinical parameters, including patient age, sex, ELN2017 risk, World Health Organization (WHO) disease classification and molecular profiles obtained by transcriptional and mutational sequencing (Fig. | PMC10824665 |
A phase II-like preclinical trial of imetelstat in AML PDX | AML | AML | To test the preclinical efficacy of imetelstat in AML, the characterized 30 individual samples from patients with AML were each transplanted into 12 NSGS recipients ( | PMC10824665 |
A CRISPR/Cas9 screen to identify key effectors of imetelstat | AML | To investigate the mechanism of action of imetelstat in AML in an unbiased manner, we applied the Brunello guide RNA (gRNA) library | PMC10824665 | |
Lipophagy precedes imetelstat-induced ferroptosis | AML | By integrating transcriptomics and functional genetics, we aimed to investigate the mechanism by which imetelstat induces ferroptosis. We performed an overlay of the in vivo AML PDX RNA-seq datasets from imetelstat and vehicle-treated mice with the Brunello library CRISPR/Cas9 knockout screen data (cutoff criteria of RNA-seq adjusted | PMC10824665 | |
Oxidative stress signatures distinguish sustained responders | AML, hematological malignancies | DISEASE, AML, HEMATOLOGICAL MALIGNANCIES | We next aimed to identify biomarkers of imetelstat response and resistance. Improved survival in imetelstat-treated AML PDXs correlated with significantly reduced engraftment and disease burden; however, there were clear differences in the magnitude and duration of individual responses (Extended Data Fig. We next aimed to identify genetic biomarkers of response and resistance to imetelstat therapy by analyzing the data from individual samples from patients with AML at baseline that were generated by genomic sequencing using a comprehensive panel of 585 genes frequently mutated in hematological malignancies | PMC10824665 |
Oxidative stress induction sensitizes AML PDX to imetelstat | OXIDATIVE STRESS | The finding that responses to imetelstat are associated with baseline molecular signatures annotated as oxidative stress and that the mechanism of action of imetelstat features ROS-mediated ferroptosis led to the hypothesis that oxidative stress induction can sensitize to imetelstat therapy.Standard induction chemotherapy composed of cytarabine and an anthracycline is a potent inducer of ROS | PMC10824665 | |
Discussion | AML, death, hematological myeloid malignancies, myelofibrosis | ESSENTIAL THROMBOCYTHEMIA, OXIDATIVE STRESS, AML, MYELOFIBROSIS | Imetelstat is a first-in class telomerase inhibitor with clinical efficacies in hematological myeloid malignancies, including essential thrombocythemia, myelofibrosis and lower-risk myelodysplastic syndromesFerroptosis is a recently discovered type of non-apoptotic regulatory cell death that relies on the balance of the production of ROS during lipid peroxidation and the antioxidant system and it is generally characterized by three hallmarks: (1) loss of peroxide repair capacity through GPX4; (2) availability of redox-active iron; and (3) oxidation of polyunsaturated fatty acid-containing phospholipidsOur functional genetic experiments using Brunello library CRISPR/Cas9 editing have provided further evidence that imetelstat restricts leukemic progression via ferroptosis, revealing a closely related functional network of seven genes. One of the identified targets, ACSL4, has previously been identified as a key regulator of ferroptosis sensitivity through the shaping of the cellular lipid compositionG-quadruplexes are recognized by and regulate the activity of many proteins involved in telomere maintenance, replication, transcription, translation, mutagenesis and DNA recombinationUsing a newly established, comprehensive AML patient-derived xenograft resource that reflects the overall genetic abnormalities found in large clinical cohorts, we demonstrated a proof of concept for the sequential administration of standard induction chemotherapy followed by imetelstat consolidation to induce oxidative stress and sensitize AML patient samples to imetelstat treatment. This approach was able to cause significant delay or prevention of AML relapse. The efficacy of sequential therapy suggests that imetelstat may be particularly useful in preventing relapse after chemotherapy, for example, as a maintenance therapy. Recently, maintenance therapy with oral CC486 has shown a survival benefit in AML; however, there is no survival plateau and therefore, most patients still relapse and die of their diseaseIn conclusion, imetelstat is a potent inducer of ferroptosis that effectively diminishes AML burden and delays relapse following oxidative stress-inducing chemotherapy.Clinical trials will address the efficacy of imetelstat in AML and may focus on this compound as a consolidation strategy for preventing relapse or potentially together with targeted therapies to improve outcomes in patients with AML. | PMC10824665 |
Methods | Our research complies with all relevant ethical regulations, including QIMR Berghofer human research ethics committee protocol P1382 (HREC/14/QRBW/278) and QIMR Berghofer animal research ethics committee protocol A11605M. Animals were monitored daily and immediately killed based on the scoring criteria detailed below. | PMC10824665 | ||
Mouse monitoring | weight loss, hunching, paralysis | Animals were monitored daily and always immediately killed as soon as a cumulative clinical score of 3 or above was reached, based on weight loss (score 1, >10–20%; and score 2, >20% or >15% if maintained for >72 h), posture (score 1, hunching noted only at rest; and score 2, severe hunching), activity (score 1, mild to moderately decreased; and score 2, stationary unless stimulated, hind limb paralysis) and white cell count (score 1, 10–60 × 10 | PMC10824665 | |
Mouse models | All mouse experiments were approved by the institutional (QIMR Berghofer) ethics committee protocol A11605M. NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjz/SzJ), NSGS (NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg[CMV-IL3,CSF2,KITLG]1Eav/MloySzJ) and NRGS (NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl Tg[CMV-IL3,CSF2,KITLG]1Eav/J) were imported from Jackson Laboratories. All mice were kept pathogen-free in the animal facility of QIMR Berghofer. Mice received autoclaved Baytril-treated (100 mg l | PMC10824665 | ||
Xenograft transplantation experiments | AML | AML | AML samples were obtained from patients, after informed consent in accordance with the Declaration of Helsinki. Ficoll density gradient was used to recover viable mononuclear cells. Viably frozen AML cells were thawed and CD3-depleted with biotinylated anti-human CD3 (SK7) and biotin-binder Dynabeads (Invitrogen) and subsequently injected via the lateral tail vein into 2.8 Gy irradiated (24 h before transplant) female NSGS or NRGS recipients (6–8 weeks old). For normal hematopoiesis studies, viable mononuclear cells were isolated from cord blood samples (provided by the Wesley-St Andrew’s Research Institute Tissue Bank with appropriate ethics approval) by Ficoll density gradient, CD3-depleted as above and subsequently enriched for CD34 | PMC10824665 |
Oligonucleotide sequences of imetelstat and mismatch controls | Imetelstat (GRN163L): 5′ R-TAGGGTTAGACAA-NH2 3′.Mismatch 1 (GRN140833): 5′ R-TAGGTGTAAGCAA-NH2 3′.Mismatch 2 (GRN142865): 5′ R-TAGGGATTCAGAA-NH2 3′. | PMC10824665 | ||
Drug treatment studies | NSG, NSGS or NRGS mice were treated with 15 mg kg | PMC10824665 | ||
Blood analysis | BLOOD | Blood was collected into EDTA-coated tubes and analyzed on a Hemavet 950 (Drew Scientific). PB smears were stained with Wright-Giemsa according to the manufacturer’s protocol (BioScientific). | PMC10824665 | |
Histology | Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin and stained with hematoxylin and eosin. Images of histological slides were obtained on a ScanScope FL (Aperio). | PMC10824665 | ||
Flow cytometry analysis of AML PDX and cord blood transplants | AML | LYSIS, AML | For monitoring AML engraftment, 25–50 μl of PB were stained after red cell lysis (BD Pharm Lyse, BD Biosciences) with anti-human CD45-AF647 (H130) and anti-mouse CD45.1-PE (A20). For AML phenotyping, cell populations were purified from bone marrow (both femurs and tibiae) or SPL and after red blood cell lysis stained with anti-human CD45-FITC (H130), anti-mouse Cd45.1-PerCP/Cy5.5 (A20), anti-human CD34-PE (581), anti-human CD33-APC (WM53), anti-human CD38-APC/Cy7 (HIT2) and anti-human GPR56-PE/Cy7 (CG4). Flow cytometry analysis of lipid peroxidation was performed using C11-BODIPY 581/591 (Sapphire Bioscience) according to a previously published protocol | PMC10824665 |
Terminal restriction fragment analysis | TRFs were obtained from genomic DNA by complete digestion with the restriction enzymes HinfI and RsaI. TRFs were separated by pulsed-field gel electrophoresis. Gels were dried, denatured and subjected to in-gel hybridization with a γ-[32P]-ATP-labeled (CCCTAA)4 oligonucleotide probe. Gels were washed and the telomeric signal visualized by phosphorimage analysis. TRFs were processed by ImageJ 1.52a analysis software to quantitate mean telomere length. | PMC10824665 | ||
Telomere length qPCR | BLOOD | Samples were purified using the DNeasy Blood and Tissue kit (QIAGEN). DNA isolation was performed as described previously | PMC10824665 | |
Cell lines | The | PMC10824665 | ||
Cell culture and in vitro cell growth analysis | AML | AML | AML cell lines were cultured in RPMI with 10% fetal calf serum, 2 mM glutamine and 200 U ml | PMC10824665 |
Imaging flow cytometry | Lipophagy was detected by assessing colocalization of C12-BODIPY and LAMP1 using a previously published method with modifications | PMC10824665 | ||
Flow cytometry analysis of AML cell lines | Before staining, 2 × 10Flow cytometry analysis of lipid peroxidation was performed using C11-BODIPY 581/591 (Sapphire Bioscience) according to a previously published protocol | PMC10824665 | ||
Western blotting | LYSED, SECONDARY, LYSIS | Cells were lysed on ice using m-PER lysis buffer (Thermo Fisher Scientific) supplemented with a protease and phosphatase inhibitor (Cell Signaling, 5872) and protein was quantified using Pierce BCA protein assay kit (Thermo Fisher Scientific). In total, 20–50 μg of protein extract was electrophoresed on a 4–15% SDS gradient gel (Mini-PROTEAN TGC Gel, Bio-Rad) and transferred to an activated PVDF membrane for 1 h at 4 °C. Unspecific binding sites were blocked in 5% BSA in Tris-buffered saline with 1% Tween-20 (TBS-T) for 1 h at 4 °C. Primary antibodies were incubated overnight at 4 °C in constant motion in 5% BSA–TBS-T. The mouse anti-Cas9 (S. pyogenes) antibody (Cell Signaling; cat. 14697, clone 7A9-3A3) and rabbit anti-ACSL4 antibody (Abcam; cat. ab155282; clone EPR8640) were used at 1:1,000 dilution in 5% BSA–TBS-T. The mouse anti-actin Ab-5 antibody (BD Biosciences; cat. 612656; clone C4/actin (RUO)) was used at 1:3,000 dilution in 5% BSA–TBS-T. The membrane was washed with TBS-T three times for 5 min before incubation with the secondary antibody for 1 h at room temperature. Polyclonal goat anti-rabbit immunoglobulins/HRP (Dako; cat. P0448) and polyclonal rabbit anti-mouse immunoglobulins/HRP (Dako; cat. P0260) were used at 1:4,000 dilution. After subsequent membrane washing, protein was detected using Immobilon chemiluminescent HRP substrate (WBKLS0500, Millipore) and imaged with the iBright CL1500 imaging system. | PMC10824665 | |
Confocal microscopy | FLUOR, COLD | Cytospins were fixed and permeabilized with methanol:acetone (prechilled) at a ratio of 1:1 for 10 min at room temperature, then washed twice with cold PBS and once with room temperature PBS (5 min each). Cytospins were then incubated with 1% BSA–PBS at room temperature for 1 h, washed three times in PBS for 5 min each and then incubated in primary antibody (anti-human Vimentin XP rabbit monoclonal antibody Alexa Fluor 488 conjugate, Cell Signaling; cat. 9854; clone D21H3) or isotype control (rabbit monoclonal antibody IgG XP Isotype Control Alexa Fluor 488 conjugate, Cell Signaling; cat. 2975; clone DA1E) at a dilution of 1:400 in 1% BSA–PBS for 1 h at room temperature or O/N at 4 °C. Cytospins were then washed three times in PBS for 5 min each. Coverslips were mounted in pro-long DAPI Gold. Images were acquired on a Zeiss 780-NLO confocal microscope. | PMC10824665 | |
CRISPR/Cas9 editing | The Brunello genome-wide gRNA library contains 76,441 gRNAs targeting 19,114 genes and was obtained from Addgene (cat. 73178) | PMC10824665 | ||
Mutational sequencing | cancer | CANCER, HEMATOLOGICAL MALIGNANCIES | Genomic alterations were profiled using the HemePACT assay (integrated mutation profiling of actionable cancer targets related to hematological malignancies) | PMC10824665 |
RNA sequencing | RNA was isolated from a maximum of 0.5 × 10 | PMC10824665 | ||
Lipidomics | Targeted lipidomics was performed on a 1290 Infinity II UHPLC coupled to a 6470 QQQ mass spectrometer via AJS ESI source (Agilent) in positive ionization mode, using a scheduled multiple reaction monitoring (MRM) method | PMC10824665 | ||
Statistical analyses | Unless otherwise stated, statistical analyses were carried out using GraphPad Prism v.9.4.0. Microsoft Excel for Mac v.16.75.2 was used to re-calculate those | PMC10824665 | ||
Statistics and reproducibility | Study design was based on sample sizes that proved to be adequate in previous experiments using similar approaches and thus no statistical methods were used to predetermine sample sizes for this study | PMC10824665 |
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