The Effects of a Remote-based Weight Loss Program on Adipocytokines, Metabolic Markers, and Telomere Length in Breast Cancer Survivors: the POWER-Remote Trial

Excess body weight is a major risk factor for many malignancies, including breast cancer (1, 2). Once diagnosed with breast cancer, women who are overweight or obese experience inferior survival outcomes (3, 4). Furthermore, most women gain weight following a diagnosis of breast cancer, which is associated with a 1.5-fold increased risk of breast cancer recurrence and death (4, 5).

Preliminary studies suggest that weight loss in breast cancer survivors leads to improvements in cancer outcomes; however, data have been inconsistent. In the Women's Intervention Nutrition Study (WINS), women with early breast cancer who received a dietary intervention experienced modest weight loss and had reduced risk of breast cancer recurrence (6). Conversely, in the Women's Healthy Eating and Living (WHEL) study, a different dietary intervention did not lead to significant weight loss and did not reduce the risk of breast cancer recurrence (7). Results from WINS and WHEL suggest that weight loss may be required to demonstrate a reduction in breast cancer recurrence (8). Furthermore, obesity is associated with quality-of-life factors, including neuropathy, cardiotoxicity, chronic fatigue, and lymphedema (9). Studies have demonstrated that weight loss in breast cancer survivors is feasible; however, most interventions have not been validated in other cohorts or used in-person counseling, which limit scalability into clinical practice (10–13). There is an urgent need to develop scalable weight loss interventions that can be readily integrated into clinical practice.

The Practice-based Opportunities for Weight Reduction (POWER) study, a randomized controlled comparative effectiveness trial, demonstrated that among a general medicine cohort of obese adults with at least one cardiovascular risk factor, 38% of participants randomized to a remote-support intervention achieved and sustained a ≥5% weight loss over 24 months, with a maximum weight loss observed at 6 months (14). In this study, we adapted the remote-support arm of the POWER study, designated POWER-remote, for breast cancer survivors.

Obesity is associated with specific biological changes in various metabolic and inflammatory factors, as well as factors related to cancer risk. Obesity is also associated with high concentrations of inflammatory cytokines, which drive pro-proliferative pathways (15). Recent data demonstrate that both breast cancer and energy excess states are associated with shorter telomere length, and a healthy lifestyle may favorably modulate telomerase and telomere length (16–18), but several methodologic concerns have been raised about the measurement tools used in these studies (19, 20).

The primary objective of this study was to compare the proportion of breast cancer survivors who could achieve at least 5% weight loss between those randomized to the adapted POWER-remote program versus a control arm of self-directed weight loss. Translational objectives were to characterize alterations in circulating concentrations of adipocytokines, and a clinically validated measurement of telomere length at 6 months.

We designed a randomized clinical trial to assess the effects of POWER-remote versus self-directed weight loss in overweight/obese women who completed local and systemic chemotherapy for early-stage breast cancer. Eligible participants had stage 0–III breast cancer and completed recommended primary breast surgery, radiation, and/or chemotherapy prior to enrolling into the trial. Endocrine therapy was allowed if started at least 3 months prior to randomization and if expected to continue for the duration of the study. Concurrent anti-HER2 therapy was permitted. Women had to have a body mass index (BMI) ≥25 kg/m², weigh ≤400 lbs, and be willing to lose at least 5% of their body weight. The study protocol conducted in accordance with the Declaration of Helsinki was approved by the Institutional Review Board, and enrolled subjects provided signed written informed consent.

The infrastructure of the adapted POWER-remote intervention was similar to that of the original POWER trial; however, educational materials included oncology-relevant information such as lymph edema prevention exercises and general information about breast cancer. In addition, instead of engaging the primary care provider as in the original POWER study, each patient's treating oncologist was involved and received information regarding the patient's weight loss. Participants randomized to POWER-remote received a 12-month behavioral weight loss intervention based on the original POWER study including telephone-based behavioral weight loss coaching and use of a web-based self-monitoring and learning platform developed by Healthways Inc (14). Participants could record dietary intake, exercise, and weight on a web-based platform (Innergy). Dietary recommendations included a reduced calorie, high vegetable and fruit diet based on the Dietary Approaches to Stop Hypertension (DASH) diet (21). Weight goals and behavioral and self-monitoring recommendations are described in Supplementary Table S1. Coaches trained in both behavioral weight loss principles and motivational interviewing reviewed self-monitoring data through the Innergy website and provided behavioral weight loss counseling during telephonic coaching sessions. The website and an accompanying smartphone application allowed participants to track their weight, food and beverage intake, and exercise; the website provided access to the weight loss educational materials for review during coaching calls. Additional platform features included a message center to communicate with the study health coach and a group wall for weight loss support from other participants in the study. Participants were offered 21 phone calls over the 1-year study period (weekly for 3 months, monthly for an additional 9 months). The approximately 20-minute calls were with a designated coach and included review of self-monitoring, problem solving, and identification of barriers and strategies for overcoming these barriers and goal setting. The theoretical framework for the active intervention draws upon behavior change theory related to weight loss, specifically social cognitive theory and utilized a Motivational Interviewing approach. The health coach had a background in delivering weight loss interventions (including the original POWER trial) and was trained by experienced coinvestigators.

This arm served as the comparison group. It reflected standard medical care, where oncologists encourage participants to achieve and maintain ideal BMI (9). The same coach as in the POWER-remote arm delivered the one-time coaching session to self-directed participants. The content of this call included the importance of gradual weight loss, promoted lifestyle change related to diet and exercise, and encouraged self -monitoring. Participants in this arm were provided the National Heart, Lung, and Blood Institute (NHLBI) publication “Aim for A Healthy Weight,” and met with a weight-loss coach one time during the baseline visit (22).

Weight and height were collected at baseline, 6, and 12 months by trained and certified staff using a high-quality, calibrated digital scale. Outcome assessment staff were masked to randomization assignment. Each weight measurement was collected twice; if the difference between the two measurements was more than 0.1 kg, the measurements were repeated until the difference between two measurements was less than 0.1 kg. BMI was calculated as the Quetelet index (kg/m²).

Waist circumference was measured in centimeters and collected by trained, certified staff using a measuring tape at a horizontal plane one cm above the navel. Measurements were repeated once; if the difference between the two measurements was more than 0.5 cm, measurements were repeated until the difference between two measurements was less than 0.5 cm.

Serum samples from study participants were collected at baseline and 6 months. Samples were examined using HADCYMAG-61K | MILLIPLEX MAP Human Adipocyte Magnetic Bead Panel - Endocrine Multiplex Assay (EMD Millipore) following manufacturer's instructions. The analytes included in this multiplex assay were adiponectin, leptin, resistin, HGF, NGF, PAI1, TNFα, MCP1, IL1β, IL6, and IL8.

Fasting levels of insulin, glucose, hs-CRP, total cholesterol, high-density lipoprotein cholesterol (HDL), low density lipoprotein (LDL), and triglycerides were analyzed by standardized enzymatic methodology at Clinical Laboratory Improvement Amendments (CLIA)-certified laboratories. Total cholesterol ratio was calculated by the total cholesterol number divided by HDL.

Telomere length was measured on lymphocytes and granulocytes, which were cryopreserved from Ficoll-separated blood samples, using a CLIA-approved flow cytometry and FISH assay as described previously (23, 24). Cells were preserved in freezing media at the time of isolation and preserved in liquid nitrogen until thawing at the time of analysis. There is high interlab concordance for measurement of leukocyte telomere length by flowFISH (23). The data for each subject and timepoint were plotted relative to a validated nomogram derived from (192 controls) from across the age spectrum (23, 25). The deviation from the age-adjusted range was calculated as the difference in telomere length from the median value for age (delta telomere length, deltaTL; ref. 23). The effect of the intervention was tested by comparing the deltaTL differences in lymphocytes at 6 months versus baseline. The age at the time of draw was used for the median control value.

The original POWER study demonstrated that 53% participants in the remote-support intervention group and 14.2% participants in the self-directed group had at least a 5% weight loss at 6 months, and 38% participants in the intervention group and 19% in the self-directed group maintained this weight loss at 24 months (14). Although our primary endpoint was the proportion of participants achieving at least 5% of weight loss at 6 months, given that individuals with breast cancer may undergo treatments that affect weight and because we enrolled persons who were both overweight and obese compared with obese only individuals in the original POWER trial, we powered our study to detect the smaller difference of 38.2% versus 18.8%. We estimated that a sample size of 80 participants was required to yield approximately 88% power to detect a differential weight loss response of 19% in the self-directed arm and 38% in the POWER-remote arm with a one-sided type I error of 10%. Women were randomized 1:1, stratifying by use of hormonal therapy and menopausal status.

Analyses were by intention to treat. The proportion of women who achieved at least 5% weight loss at 6 months was estimated with an exact 95% confidence interval (CI). The primary analysis would conclude a significant benefit for the POWER-remote versus the self-directed arm if the one-sided Fisher exact test P value was <0.10 for the difference in the proportion of women who lost 5% or more weight at 6 months. Rejection of the null hypothesis in this phase II randomized study would suggest that POWER-remote is an effective intervention in overweight/obese breast cancer survivors, and justify further investigation in future trials. The OR for the association between 5% or more weight loss and study arm was estimated using logistic regression that included the randomization stratification variables as covariates. This analysis was also performed for response as measured at 12 months, to describe differences in weight loss maintenance between arms. Changes in weight at 6 and 12 months were measured continuously. Differential changes in weight between groups across time points were assessed with a mixed effects regression model, with a random intercept for each patient and terms for time point, treatment group, and their interaction. Changes in waist circumference were analyzed similarly. Exploratory analyses were performed to evaluate weight loss in specific subgroups using interaction analysis.

We summarized biomarkers at baseline and 6 months with descriptive statistics. We calculated within-patient changes in biomarkers from baseline to 6 months and compared the differences according to treatment arm with Wilcoxon rank-sum tests. We explored the differences in biomarkers according to weight loss. These analyses separating weight loss from treatment arm were of interest because the likely mechanism to biomarker changes is weight loss rather than the randomized group. The telomere length comparisons were made using the ΔTL values to adjust for age-related changes and the data and the statistics were plotted and calculated using GraphPad Prism. Changes in TL according to exposure to chemotherapy versus no chemotherapy were assessed by the Mann–Whitney test. Two-sided P values <0.05 were considered statistically significant for all analyses except for the primary analysis, which used a one-sided P < 0.10. Because of the exploratory nature of the secondary and correlative analyses, we did not adjust for multiple comparisons in reporting the results of the correlatives. Analyses were completed using R version 3.4.2.

From July 2013 to December 2015, 458 women were assessed for eligibility, and a total of 96 signed a written informed consent and were randomized; 87 participants were eligible for the primary analysis (Fig. 1). Of the 362 who were excluded, 305 did not meet eligibility criteria, only 12 declined, and 45 had other reasons. The most common reasons for not meeting eligibility criteria were (i) no correspondence after first contact (n = 105), (ii) medical exclusion (i.e., second malignancy, excluded medication, uncontrolled comorbidity; n = 59), (iii) BMI less than 25 kg/m² (n = 35); the most common “other” reasons were (i) travel (n = 18), (ii) no breast cancer (n = 4), (iii) limited access to computer/internet (n = 3), and (iv) on another trial (n = 3). Characteristics were balanced between study arms; 74% of participants were postmenopausal, with an average BMI of 32 kg/m², more than half had received chemotherapy (52% POWER-remote, 59% self-directed), and the majority of women had estrogen receptor–positive disease and were on endocrine therapy (Table 1). At 6 months, 45 participants (90%) had measured weights in POWER-remote arm, and 42 (91%) in the self-directed; at 12 months, 41 (82%) had measured weights in POWER-remote, and 36 (78%) in the self-directed arms.

Those randomized to the POWER-remote had high participation rates. Median call completion was 14 of 15 calls in the first 6 months and seven of seven calls from months 7–12. There was a median of 24 weekly logins during the first 6 months and 22.5 weekly logins during months 7–12.

At 6 months, 51% of women in the POWER-remote and 12% in the self-directed arm had lost at least 5% of their baseline body weight (one-sided Fisher P < 0.0001, adjusted OR, 7.9; 95% CI, 2.6–23.9; P = 0.0003). Significant differences between groups were observed at 12 months, with 51% of women in the POWER-remote arm exhibiting at least 5% loss of their baseline body weight compared with 17% in the self-directed arm (adjusted OR, 5.2; 95% CI, 1.8–14.2; P = 0.003). Significant differences between study arms were also noted in the proportion of women losing at least 10% of their baseline body weight at 6 (22% vs 0, P < 0.001) and 12 months (32% vs 5.6%, P = 0.004). Patients enrolled in POWER-remote were more likely to achieve weight loss than those in the self-directed arm (Fig. 2A). In the POWER-remote group, mean weight loss at 6 months was 4.6 kg (SD = 4.8 kg), which was sustained at 12 months (mean weight loss 4.7, SD = 6.3), compared with mean weight loss of 0.5 kg (SD = 3.3) at 6 months, and 0.4 kg (SD = 4.7) at 12 months in the self-directed arm (Fig. 2B). In subgroup analyses, weight loss did not differ by age, race, prior chemotherapy, endocrine therapy, or baseline BMI category (each P_interaction > 0.05; Supplementary Fig. S1).

At 6 months, patients in both study arms had reductions in waist circumference [POWER-remote arm: -5.4 cm (−10.3 cm, −0.55 cm), self-directed: −1.7 cm (−3.8 cm, 0.37 cm)], but the decreases were not different between groups (P_interaction = 0.26). By 12 months, however, the change in the POWER-remote arm was -6.6 cm (−11.5 cm, −1.7 cm), which was significantly lower compared with the self-directed group [increase by 0.3 cm (−2.0 cm, 2.1 cm), P_interaction = 0.003; Supplementary Fig. 2].

Compared to the self-directed arm, we observed reduction in leptin concentrations at 6 months in women randomized to the POWER-remote arm. We did not observe a significant effect on resistin, adiponectin, HGF, NGF, IL1β, IL8, IL6, MCP1, PAI1, and TNFα (Table 2). Leptin concentration in the POWER-remote arm decreased by 918.8 pg/mL (SD = 2,127) compared with an increase by 395.1 pg/mL (SD = 1,596) in the self-directed arm (P < 0.01). Participants who achieved 5% or greater weight loss, versus not, had a significant decrease in leptin by 1,615 pg/mL (SD = 2,388, P < 0.01). Those achieving 5% weight loss, compared with those who did not, had favorable improvements in inflammatory cytokines including lower HGF, IL1β, and hs-CRP, and a smaller increase in MCP1 (Table 2).

Those who achieved at least 5% weight loss had lower triglycerides and total cholesterol ratio at 6 months (Table 2). When percent weight change was assessed as a continuous variable, percent weight loss was again associated with decreases in leptin and triglyceride concentrations, and, decreases in insulin were also observed (Fig. 3). Differences between study arm and insulin, glucose, and lipid panels were not observed.

A total of 84 participants with available samples underwent telomere length testing by flowFISH (41 self-directed, and 43 POWER-remote; Fig. 4A). The following analyses are listed for lymphocytes but similar patterns were seen for granulocyte telomere length. While there was no difference between the two groups in telomere length at baseline, the overall telomere length in this cohort and in each of the groups was slightly but significantly shorter than the historic controls from a validated nomogram based on healthy age-matched controls [mean ΔTL -0.8 (95% CI, −1.1 to −0.5) vs -0.8 kb (−1.0 to −0.5), respectively; ref. 23]. This effect was not related to a history of chemotherapy exposure as we detected no difference in telomere length between treated and untreated cases (P = 0.21, Mann–Whitney test; Fig. 4B). When we examined differences in telomere length at 6 months, we found no difference compared with baseline (mean +0.1 vs +0.1, P = 0.7, Mann–Whitney test). These changes are also within the assay variability of the flowFISH telomere length measurement (23). Indeed, there was no change in lymphocyte or granulocyte telomere length detected when we compared those who lost 5% or 10% of baseline body weight versus with those who did not (P = 0.67, Wilcoxon rank-sum test), or when assessed continuously by percentage weight loss (P = 0.09, Pearson correlation test; Table 2; Fig. 4C).

In this prospective study, we demonstrate that breast cancer survivors randomized to a remotely supported scalable weight loss intervention can experience clinically significant weight loss known to improve cardiovascular risk factors, irrespective of chemotherapy or use of endocrine therapies (26, 27). Participants in this study who were randomized to the POWER-remote arm had near perfect adherence as demonstrated by the high call completion and login rates, and only one patient randomized to POWER-remote who participated was lost to follow up.

POWER-remote is inherently scalability, cost-effective relative to in-person interventions and thus may be implemented in clinical practice (28). This is particularly relevant as more definitive studies that assess the benefits of weight loss are ongoing, in particular the Breast Cancer Weight Loss (BWEL) study (NCT02750826; ref. 29). Indeed, in a recent interim analysis of the SUCCESS-C trial, patients who completed a 2-year lifestyle intervention program had better disease-free survival (DFS) than noncompleters (HR = 0.35; 95% CI, 0.27–0.45, P < 0.001); however, only 48.2% of patients originally randomized to the lifestyle intervention group completed the entire program (30). The American Society of Clinical Oncology (ASCO) recommends counselling breast cancer survivors to achieve and maintain an ideal weight, limit consumption of high-calorie foods and beverages, and increase physical activity; however, there is no specific intervention or program to help patients accomplish this goal (31).

Weight loss in patients with breast cancer is feasible, as demonstrated by several studies. The Lifestyle Intervention in Adjuvant Treatment of Early Breast Cancer (LISA) Study, which evaluated a mail-based general health information with or without a telephone-based lifestyle intervention on postmenopausal breast cancer survivors on letrozole found that participants lost a mean 5.3% versus 0.7% of their baseline body weight at 6 months (32). The Lifestyle, Exercise, And Nutrition (LEAN) Study evaluated an in-person, versus remote, versus usual care intervention, and found an average of 6.4%, 5.4%, and 2.0% weight loss, respectively, at 6 months (33). The Enhance Recovery and Good Health for You (ENERGY) Trial, which compared a group-based versus a less intensive intervention in breast cancer survivors found that at 12 months, mean weight loss favored the group intervention (6.0% vs 1.5%; ref. 34). Another intervention, which evaluated the addition of metformin to a weight loss intervention found similar percent weight loss at 6 months in the weight loss group (5.5% vs 2.7%; ref. 35). While these mean changes in weight loss are comparable with our study (Fig. 2B), the proportion of patients who achieved at least 5% weight loss in these other studies is not reported, a metric that identifies the percentage of the population that may benefit from the intervention. Our intervention included premenopausal women, did not have an in-person component, nor included pharmacotherapy; thus, builds upon existing literature. Furthermore, we demonstrate that by the end of the intervention, there was a significant decrease in waist circumference in those who underwent the POWER-remote intervention. Waist circumference is associated with increase breast cancer risk in both pre-and postmenopausal women, and may be a better marker than BMI in determining risk of metabolic syndrome (36, 37).

We consistently observed that weight loss and study arm correlated with decreases in leptin. This is particularly noteworthy as leptin has been demonstrated to increase breast cancer cell proliferation, invasion, and migration by activation of prosurvival pathways (38–41). Weight loss was associated with relatively lower concentrations of MCP1, HGF, and IL1β, which are inflammatory markers that may have prooncogenic properties (39). While in this small study, the variability in these markers was high, the general trend suggests a favorable effect from weight loss.

We observed a clinically significant decrease in triglycerides, and small decreases in total cholesterol ratio in patients who met the primary endpoint of 5% weight loss. High triglycerides and total cholesterol ratio are associated with increased cardiovascular risk (42, 43). This is particularly relevant because cancer treatment in patients with obesity can further increase risk for adverse cardiac effects, especially in those treated with anthracyclines or trastuzumab (44, 45).

We did not demonstrate differences in leukocyte telomere length by randomized group or by weight loss. Our data are in contrast to studies in prostate cancer suggesting that a dietary intervention was associated with an increase in telomerase activity in peripheral blood mononuclear cells, and results from the LEAN study which found an association with randomization to a weight loss intervention with telomere lengthening after 6 months by measuring relative telomere length by qPCR done on buffy coat–extracted genomic DNA (16, 17, 46). We chose to evaluate telomere length because it is a primary mediator of cellular and clinical phenotypes, whereas telomerase activity is only detectable in cycline lymphoctyes, and assays are semiquantitative. Furthermore, we utilized a more robust and clinically validated telomere length measurement technique (flowFISH; refs. 19, 20). Similar to our findings, a recent retrospective study found that assignment to a weight loss intervention did not result in a significant change in telomere length compared to the control group (3% vs 5% shortening, respectively, P = 0.12; ref. 46). Thus, there is insufficient evidence to suggest short-term weight loss is associated with changes in telomere length.

The major strength of POWER-remote, compared with other weight loss interventions in breast cancer populations, is that it is based on an intervention demonstrated to be effective in a general medicine population, where a remote-supported intervention was equal in efficacy to an in-person intervention (14). Given its scalability and exportability the POWER-remote arm serves as a control arm for next-generation studies evaluating relationships of weight loss and sleep interventions in patients with insomnia (NCT03542604), biomarker modulation (NCT02431676), and pharmacologic weight loss management. Our randomized design used a standard-of-care approach as a control arm, further validating our findings. Our comprehensive biomarker analysis, although exploratory due to small sample size, further validates biological changes observed with weight loss. Limitations of this study include a short follow up period of 12 months. Notably, the original POWER study had 24-month follow up and weight loss was sustained (14).

With increasing data demonstrating the benefits of weight loss in breast cancer survivors, practical integration of weight loss programs that offer guidance to patients will be of paramount importance. The POWER-remote intervention is a scalable and effective weight loss program that can significantly reduce weight in breast cancer survivors.

A. Dalcin and G.J. Jerome are employees/paid consultants for Healthways Inc. G.I. Cohen reports receiving speakers bureau honoraria from Amgen. K.L. Smith reports receiving other commercial research support from Pfizer, and holds ownership interest (including patents) in Abbvie and Abbott Lab. C. Snyder reports receiving commercial research grants from Genentech. V. Stearns reports receiving commercial research grants from Abbvie, Novartis, Pfizer, Biocept, and Puma, and other remuneration from Immunomedics. C.A. Santa-Maria has research funding from Pfizer, Astrazeneca, Tesaro, Novartis; has served on advisory boards for Bristol Meyers Squibb, Genomic Health, Athenex, Polyphor, and Halozyme. No potential conflicts of interest were disclosed by the other authors.

Conception and design: C.A. Santa-Maria, J.W. Coughlin, D. Sharma, G.I. Cohen, R.M. Connolly, L.J. Appel, V. Stearns

Development of methodology: C.A. Santa-Maria, J.W. Coughlin, D. Sharma, G.J. Jerome, V. Stearns

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C.A. Santa-Maria, D. Sharma, M. Armanios, C. Schreyer, A. Dalcin, A. Carpenter, G.J. Jerome, D.K. Armstrong, G.I. Cohen, R.M. Connolly, J. Fetting, R.S. Miller, K.L. Smith, A. Wolfe, A.C. Wolff, L.J. Appel, V. Stearns, M. Chaudhry

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C.A. Santa-Maria, D. Sharma, M. Armanios, A.L. Blackford, A. Dalcin, G.J. Jerome, D.K. Armstrong, G.I. Cohen, R.S. Miller, K.L. Smith, C. Snyder, C.-Y. Huang, V. Stearns

Writing, review, and/or revision of the manuscript: C.A. Santa-Maria, J.W. Coughlin, D. Sharma, A.L. Blackford, C. Schreyer, A. Dalcin, A. Carpenter, G.J. Jerome, D.K. Armstrong, G.I. Cohen, R.M. Connolly, J. Fetting, R.S. Miller, K.L. Smith, C. Snyder, A.C. Wolff, C.-Y. Huang, L.J. Appel, V. Stearns, M. Chaudhry

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C.A. Santa-Maria, A.L. Blackford, C. Schreyer, A. Carpenter, G.J. Jerome, L.J. Appel, V. Stearns

Study supervision: C.A. Santa-Maria, J.W. Coughlin, A. Dalcin, L.J. Appel, V. Stearns

This work was supported by Breast Cancer Research Foundation, Cigarette Restitution Fund, National Institutes of Health [P30 CA006973], and Commonwealth Foundation Johns Hopkins Precision Medicine Initiative (to M. Armanios).

We thank Dr. Robert Donegan and Dr. Ben Park for patient referral, Brandon Luber for assistance in study design, and David Lim for assistance with data analysis.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.