Mindfulness and its efficacy for psychological and biological responses in women with breast cancer

Abstract Many breast cancer survivors have to deal with a variety of psychological and physiological sequelae including impaired immune responses. The primary purpose of this randomized controlled trial was to determine the efficacy of a mindfulness‐based stress reduction (MBSR) intervention for mood disorders in women with breast cancer. Secondary outcomes were symptom experience, health status, coping capacity, mindfulness, posttraumatic growth, and immune status. This RTC assigned 166 women with breast cancer to one of three groups: MBSR (8 weekly group sessions of MBSR), active controls (self‐instructing MBSR) and non‐MBSR. The primary outcome measure was the Hospital Anxiety and Depression Scale. Secondary outcome measures were: Memorial Symptom Assessment Scale, SF‐36, Sense of Coherence, Five Facets of Mindfulness Questionnaire, and Posttraumatic Growth Index. Blood samples were analyzed using flow cytometry for NK‐cell activity (FANKIA) and lymphocyte phenotyping; concentrations of cytokines were determined in sera using commercial high sensitivity IL‐6 and IL‐8 ELISA (enzyme‐linked immunosorbent assay) kits. Results provide evidence for beneficial effects of MBSR on psychological and biological responses. Women in the MBSR group experienced significant improvements in depression scores, with a mean pre‐MBSR HAD‐score of 4.3 and post‐MBSR score of 3.3 (P = 0.001), and compared to non‐MBSR (P = 0.015). Significant improvements on scores for distress, symptom burden, and mental health were also observed. Furthermore, MBSR facilitated coping capacity as well as mindfulness and posttraumatic growth. Significant benefits in immune response within the MBSR group and between groups were observed. MBSR have potential for alleviating depression, symptom experience, and for enhancing coping capacity, mindfulness and posttraumatic growth, which may improve breast cancer survivorship. MBSR also led to beneficial effect on immune function; the clinical implications of this finding merit further research.


Introduction
Still, individuals report persistent coexistent physical and psychological symptoms that contribute to interference with daily life after breast cancer treatment [9].
An increasing body of research has established an association between distress and changes in immune function [10,11]. Distress seems to have a significant negative effect on immune function, such as lowered natural killer cells (NK cells) and T lymphocytes (T cells) [11,12]. T cells have been linked to breast cancer recurrence and survival [13][14][15]. Other important parameters are cytokines, such as interleukin-6 (IL-6) and interleukin-8 (IL-8), which are independently correlated with breast cancer disease stage and progression [16][17][18][19].
Thus, previous research indicates a significant need for interventions to improve well-being, alleviate distress and symptom burden, and to reinforce immunity in women during breast cancer diagnosis, treatment, and recovery.
Originating from ancient Buddhist and yoga traditions, mindfulness-based interventions have become increasingly popular in the Western world. Mindfulness is described as a "way of being" and defined as the capacity for awareness in each moment, by "paying attention in a particular way: on purpose, in the present moment, and nonjudgmentally" [20]. The use of mindfulness-based interventions in oncology has proliferated over the past decade and research in the field has rapidly expanded [21,22] While there appears to be evidence to support the use of mindfulness-based interventions with cancer patients, the overall quality of existing trials varies considerably [23].
Mindfulness-based stress reduction (MBSR) is an 8-week, standardized program combining mindfulness meditation, yoga and other techniques designed to reduce stress and improve well-being and quality of life in patients with a wide range of chronic pain and stress disorders [20,24,25]. MBSR and has also been shown to improve mood disorders [21,26]and reduce stress in cancer patients [27,28]. Furthermore, MBSR reduces fear of recurrence and improves physical functioning which in turn leads to reduced stress and anxiety in women with breast cancer [29]. Evidence from nonrandomized, uncontrolled studies suggests that MBSR improves quality of life and coping, decreases stress and alters cortisol and immune patterns [30][31][32][33].
These results raise important questions as to whether MBSR is related to positive outcomes in mood disorders, symptom burden and health status, as well as strengthened immune system functioning in breast cancer survivors. Despite growing evidence that MBSR influences immune function, there is a need for studies to determine how biomarkers relate to changes in mindfulness and psychosocial outcomes, including symptom reduction and wellbeing [34]. While noting the effectiveness of MBSR, authors of several reviews have pointed out the inherent methodological problems in the published studies [35,36]. There is also a need for randomized controlled studies with long-term follow-up [36].
The primary purpose of our study was to determine the efficacy of MBSR intervention for mood disorder symptom improvements in women with breast cancer. Secondary goals were to evaluate their symptom experience, distress, health status, coping capacity, mindfulness, posttraumatic growth, and immune status.

Study design
In this 3-month follow-up study, we present the first results of a 5-year longitudinal, randomized, controlled trial (RTC). Details of this trial have been described elsewhere [37].
The trial was designed in accord with Consort recommendation [38][39][40]. In an unblinded RTC, participants' expectations about the intervention may lead to a placebo effect in the intervention group and/or a negative response among controls. In order to minimize a potential placebo effect in the active intervention group and a "frustrebo response" [41] in controls, a three-armed design was chosen.
Patients diagnosed with breast cancer were consecutively recruited to participate after completion of adjuvant chemotherapy and/or radiation therapy, with or without endocrine therapy. Patients were excluded on the basis of having another advanced illness at diagnosis that might interfere with the ability to participate, ongoing major depression, ongoing Herceptin therapy, or who had previously, as well as during the intervention, used MBSR and other mind-body programs (including yoga). This trial was approved by the Regional Ethical Review Board, University of Gothenburg, and informed consent was obtained before enrolment.

Procedures
Eligible patients were contacted by research nurses at the first follow-up appointment for patients receiving hormonal therapy or at the last treatment for patients undergoing chemotherapy. After oral and written information, interested patients provided written consent to participate in the study. Participants were first invited to a baseline health check-up appointment, which included blood sample collection and questionnaire completion. Randomization was computerized and conducted in blocks of 9, 12, and 15, varied randomly. Assignment codes were kept in sequentially numbered, opaque, sealed envelopes, prepared by the research coordinator.

Intervention
Participants were randomized into one of three groups: MBSR (8 weeks self-instructing MBSR program + instructor and weekly group sessions), active controls (8 weeks self-instructing MBSR program) or non-MBSR (no intervention).
Participants in the MBSR group attended a standardized, group-based, 8-week course once a week for an average of 2 h each week with homework assignments consisting of 20 min sessions, 6 days/week. Participants were provided with information material, including a 20-page introduction to mindfulness training, a compacted disk (CD) with meditation exercises, the training program and a diary in order to report the time allotted to mindfulness training including patients′ reflections about the meditation exercises. Led by a certified MBSR instructor, these weekly group sessions focused on the participants' experiences of mindfulness, and including gentle meditation and yoga training. Active controls received information material, a CD, 8 weeks of selfinstructing training program and a diary. The only difference between MBSR group and active controls was the weekly group sessions. Participants in both MBSR group and active controls were provided with written and oral instructions how to use information material, CD and diary. All participants received standard care for follow-up for breast cancer according to the national and local guidelines [42].

Measures
Socio-demographic data were collected through chart review and interviews. Clinical characteristics, patient selfreported outcomes and biomarkers were collected at health checks both pre and postintervention. Follow-ups for MBSR group and active controls were conducted 1 month after the intervention, and at similar time points. The same procedures, at similar time points of 3 months were conducted for those in the non-MBSR group.

Mood disorder
Mood disorder was measured using the Hospital Anxiety and Depression scale (HAD), which is one of most widely used instruments to screen for anxiety and depression in cancer patients [43][44][45]. The HAD is a 14-item questionnaire consisting of two subscales: anxiety and depression. Subscale scores range from 0 to 21; scores for each subscale are defined as: 0-7 (normal), 8-10 (possible cases), and 11-21 (cases of psychological morbidity) [46]. The internal consistency of reliability for both subscales are satisfactory, with Cronbach's alpha 0.72-0.89, respectively, 0.78-0.93 [45] Secondary outcomes measures

Symptom experience
Symptom experience was evaluated using the Memorial Symptom Assessment Scale (MSAS), a questionnaire consisting of 32 symptoms and symptom frequency, severity, and distress [47]. The MSAS generates two subscales including physical and psychological symptoms, and two global indicators: Total Symptom Burden Scale (TMSAS) and the Global Symptom Distress Index (GDI). The MSAS is a reliable and valid multidimensional measure of symptom experience in cancer populations [47,48] including the Swedish version of the MSAS [49].

Health status
Health status was measured using the 36-item Short Form Health Survey (SF-36), which consists of eight scaled scores: vitality, physical functioning, bodily pain, general health perceptions, physical, emotional and social role functioning, and mental health. The maximum score is 100 points. Reliability measurements of the SF-36 are consistently good [50][51][52][53].

Mindfulness
Mindfulness was measured using the 29-item short form Five Facets of Mindfulness Questionnaire (FFMQ-Swedish version), consisting of five key facets of mindfulness: observing, describing, acting with awareness, nonjudging, and nonreactivity to inner experience [59,60].

Personal growth
Personal growth was evaluated using the Posttraumatic Growth Inventory (PTGI), which measures positive life changes after traumatic events. The PTGI yields a total score based on five dimensions: relating to others, new possibilities, personal strength, spiritual change, and appreciation Mindfulness in women with breast cancer E. Kenne Sarenmalm et al. of life [61]. The PTGI has shown good reliability in previous research with a total score Cronbach's α of 0.96 [62].

Lymphocyte distribution in peripheral blood
Lymphocyte distribution in peripheral blood was analyzed by flow cytometry using a FACSCanto II flow cytometer and the FACSDiva software. The absolute number of blood lymphocytes was determined with Trucount reference beads using the method recommended by the manufacturer. The following subpopulations were reported CD3 + , CD3 + 4 + and CD3 + 8 + T cells, CD19 + B cells, and CD3-16 + 56 + NK cells. The results for each subpopulation were expressed as the percentage of lymphocytes and as the number of cells × 10 9 /L. Antibodies to the antigens above. Trucount beads, the FACSCanto II flow cytometer and the FACSDiva software were all from BD Biosciences, Mountain View, CA.

NK-cell activity
NK-cell activity was measured using a Flow-cytometric Assay of Natural Killer cell Immune response in Activated whole blood (FANKIA) a modified version of a previously published method using flow cytometry and stained K562 cells as target cells [63]. Whole blood was mixed with a defined number of target cells transfected with the gene for green fluorescent protein (GFP) [64]. After incubation the same volume was collected from tubes with: blood and target cells, target cells and medium; and blood and medium and analyses were performed as described above. The lytic activity was defined as the reduction in the number of target cells after mixing with the blood, expressed in percentage of target cells.

Determination of cytokine concentrations
Determination of cytokine concentrations were determined in sera using commercial high sensitivity IL-6 and IL-8 ELISA kits (R&D Systems, Inc., Abingdon, UK) according to the instructions from the manufacturer.

Data analysis
Sample size calculation was based on the primary outcome: breast cancer patient's mood disorder symptoms. A oneunit change on the HAD-subscales from baseline to 3-month follow-up was regarded as clinically relevant. The detection of such a difference would require 50 participants per group (a total of 150 participants) to achieve a statistical power of 80%. Descriptive statistics were used to summarize sociodemographic and clinical characteristics. Spearman's correlation coefficients were calculated to determine the strength of relationships between selected variables. As most of the variables we explored were of ordinal data type and most of the continuous variables deal with skewed distributions deviating from normal-distribution, we used nonparametric tests (Wilcoxon's test for comparison within groups and Mann-Whitney's test for comparison between groups). P < 0.05 was considered as statistically significant result.

Results
A total of 177 women consented participation and were randomly assigned to one of three groups. There were 11 drop-outs after randomization, that is, two patients were excluded as they did not complete the intervention, two patients withdraw their participation due to rapid breast cancer disease progression, and seven patients did not visited first follow-up (MBSR = 4; active controls = 5; non-MBSR = 2). The final groups were MBSR (n = 62), active controls (n = 52) and non-MBSR (n = 52). Postintervention data were missing for one active control participant. A participation flowchart is depicted in Figure 1.
Participants' ranged from 34 to 80 years (mean = 57.2, SD = 10.2). No statistical differences were found between groups on demographic or clinical characteristics. Participant descriptions are listed in Table 1.

Psychological response
Study results revealed significant changes in psychological and biological responses to the MBSR intervention, summarized in Tables 2-8.
On the primary outcome measures, MBSR participants reported significant improvements on depression symptoms both within group (mean = 4.3; SD = 3.7 to mean = 3.3; SD = 3.3; P = 0.001) as well as compared to non-MBSR (P = 0.015), but not on anxiety symptoms ( Table 2).
The number of reduced cases of depression were 7 (11%) in MBSR participants compared to 4 (8%) reduced cases of depression in active controls and non-MBSR, respectively.
Pre and postintervention HAD scores for depression and anxiety are presented in Figure 2.
Within the MBSR group, changes in health status were seen in improved vitality (mean = 49.5; SD = 27.5 to  (Table 4).
There were also some significant changes pre and postintervention in active controls and in the non-MBSR group Mindfulness in women with breast cancer E. Kenne Sarenmalm et al. suggesting a decrease in the absolute number of NK cells (CD3-16-56 + NKx10e9/I) for active controls (P = 0.011) compared to non-MBSR. There were also significant changes for active controls regarding CD3-16 + 56 + NK% versus non-MBSR (P = 0.025) ( Table 8). There were no significant differences in serum concentrations of IL-6 or IL-8 between any of the study groups.

Discussion
Our trial provides evidence in support of the efficacy of MBSR for psychological and biological response among women with breast cancer. The primary purpose of this study was to determine the efficacy of MBSR intervention on mood disorder, that is, depression and anxiety. Our finding of improvements in depression is consistent with other RTCs that have evaluated MBSR and mood disorders in breast cancer patients [21,26]. Unlike those studies, our findings revealed no significant changes in anxiety. However, consistent with our trial, a meta-analysis of mindfulness-based interventions that included participants who met the diagnostic criteria for a current episode of anxiety or depressive disorder show that MBSR is effective for reducing symptoms of depression, but not anxiety [65].
MBSR participants reported significantly greater improvements in symptoms, especially psychological symptoms. In addition, their symptom burden and distress significantly decreased. MBSR participants also improved in functional status; in line with previous research showing significant intergroup improvements in mental health   Mindfulness in women with breast cancer E. Kenne Sarenmalm et al. [66], our findings indicate significant improved mental health between groups.
A common assumption is that mindfulness increases the individual's ability to cope, but few RCTs have examined the effect of MBSR on coping capacity. The MBSR intervention appears to improve coping effectiveness in breast cancer patients [32], and behavioral and cognitive coping [67]. Results from our trial show that women who participated in the MBSR experienced improved coping capacity, here measured as sense of coherence (SOC). Previous research has identified SOC as a significant predictor of distress, number and type of coping strategies in women with breast cancer [68], suggesting the lower the SOC, the higher the levels of symptom burden [3]. While Antonovsky [54] believed that SOC is a relatively stable personality state, our findings show evidence that MBSR may influence SOC (i.e., to improve patients' ability to manage, comprehend, and finding meaning living with breast cancer). Significant changes are marked in bold. 1 36-items short form health survey (SF-36). 2 Change over time (P-value) within groups (Wilcoxon signed test). 3 Change over time (P-value) between groups (comparison of MBSR-Active controls vs. Non-MBSR (ref.) (Mann-Whitney test). Enhanced elements of mindfulness were shown for nonreactivity and observing in the MBSR group. Future research is needed to explore the complexity and relations among the different dimensions of mindfulness, and to gain a deeper understanding about which factors facilitate the cultivation of mindfulness [69].
Our trial indicates that the benefits of MBSR may also extend to posttraumatic growth. Relatively little research has investigated the relationship between posttraumatic growth and immunity. A study of patients with hepatoma suggests that higher PTGI scores are associated with higher peripheral blood leukocytes and longer survival [70]. Further research addressing the interrelationship of MBSR with posttraumatic growth and immune response is warranted.
In terms of biological response, changes in NK-cell activity and numbers of both NK cells and B cells within the MBSR group as well as between groups were seen. Of note was the finding that there were no changes in numbers of IL-6 or IL-8. The clinical relevance of these discrete findings is difficult to estimate and more research is needed to fully explain the clinical meaning of these biological parameters. However, consistent with our findings, there is intriguing evidence suggesting that finding meaning and personal growth is associated with T-cell levels [71] and NK-cell activity [72]. Furthermore, there have been prior reports that distress is associated with immune downregulation, including reduced NK-cell activity [12,32]. In addition, participation in mindfulness   training leads to a shift from proinflammatory response in cancer patients [33] and a pilot study has suggested that improvements in well-being following MBSR was associated with increased NK activity and decreased CRP levels [73].
Given the beneficial efficacy of MBSR on both psychological and biological outcomes, future longitudinal studies may be needed to investigate the effect of these outcomes on disease progression and survival. There were several tendencies in favor for MBSR group, although not statistically significant, and for some outcome variables statistically significant changes could be detected only within MBSR group but not between the groups. A larger sample size might have resulted in more significant differences between groups. Several improvements were also seen in active controls (i.e., physical and social role functioning, and observing) as well as between groups (i.e., global distress, mental health, and posttraumatic growth). Future research is needed to explore who benefits from participating in an MBSR intervention with weekly group sessions versus using a self-instructing training program. This study is characterized by several strengths, including use of active and non-MBSR controls, random assignment, inclusion of patient-reported outcomes and immune response among a homogenous group of women diagnosed with breast cancer. To our knowledge, this is the first MBSR intervention study to include a comparison between standardized MBSR and both active controls using a self-instructing program and passive controls. The notable study limitation was that all women who fulfilled the inclusion criteria were invited without undergoing screened for mood disorder before study invitation. Furthermore, despite randomization, there were differences in distribution regarding disease stage (tumor size and type of breast cancer) which might have affected study results.
In conclusion, results from this RCT suggest that MBSR is beneficial and leads to psychological and biological improvements. MBSR may hold potential for alleviating depression, distress and symptom experience, and to strengthen coping capacity, which may improve breast cancer survivorship. Since there were also positive changes in the active control group, it is important to provide the self-training program to patients who prefer to practice themselves, without weekly group exercises. Finally, longitudinal studies are required to investigate whether these positive psychological and biological responses remain constant, increases or decreases over time.

Mindfulness in women with breast cancer
Enhanced psychosocial well-being following participation in a mindfulness-based stress reduction program is associated with increased natural killer cell activity. J. Altern. Complement. Med. 16:531-538.

Supporting Information
Additional supporting information may be found in the online version of this article: Figure S1. X. Typical result with dot plots showing CD3+, CD3+4+, CD3+8+, 19-,and CD3-16+56+ cells. Absolute numbers of cells were calculated using Trucount reference beads. Lymphocytes and Trucount beads were defined in a CD45 versus side scatter area (SSC-A) and a CD19 versus SSC-A plot, respectively. CD3+ and CD3-cells were then defined in a CD3 versus SSC-A plot. Then CD3+4+ and CD3+8+ were defined in a plot of CD8 versus CD4. Finally CD19 and CD3-16+56+ cells were defined in a plot of CD16+56+ versus CD19. Figure S2. Y Typical result with dot plots showing regulatory T cells. Lymphocytes were defined in a forward scatter area (FSC-A) versus side scatter area (SSC-A) plot. CD3+4+ cells were then defined in a CD3 versus CD4 plot. Then CD3+4+25+ were defined in a plot of CD4 versus CD25. Finally CD3+4+25+FOXP3++ (regulatory T cells) cells were defined in a plot of CD25 versus FOXP3.