Participants

IRB approval for this study was obtained from both Indiana Wesleyan University and Lutheran Hospital (Fort Wayne, IN) and informed written consent was obtained from all women prior to study participation. All patients were recently diagnosed with Stage 1 to 3a breast cancer (using the Tumor, Node, Metastasis (TNM) Stage Grouping System) at the Lutheran Hospital Breast Center and Womens Health clinic in Fort Wayne, Indiana (see Table1). They were contacted over a two-month period, and 20 of them (66%) agreed to participate. Following initial surgery (lumpectomy and/or mastectomy) all participating women (active treatment group) in the present study underwent radiation therapy (N=20). Depending on the staging and recommended treatment guidelines, most active treatment women then had chemotherapy (Cyclophosphamide, Methotrexate, Fluorouracil or CMF) or Taxotere (Docetaxel) (Table 1). These were all women in the active treatment group with a positive lymph node biopsy during surgery (N=9) as per the hospital oncology department standard of care for breast cancer at that time. Unfortunately, the molecular subtype of breast cancer (e.g., triple negative, HER2+, HR+) was not available for the active patient or breast cancer survivor (remission) groups in the present study. Though data on molecular subtype of breast cancer is not available, some patients in both groups received tamoxifen (Table 1), suggesting a hormone-dependent (luminal) disease. Those not receiving the drug most likely belonged to HER2-positive or triple-negative subtype.

Women were excluded if their medical chart review revealed significant neuropsychological risk from a history of central nervous system disease or infection (e.g., meningitis, HIV, stroke), seizures, prior cancer diagnoses, or traumatic brain injury or accident.

Through a breast cancer survivors support group meeting monthly at the Lutheran Hospital Breast Center, a comparison group was enrolled. These were all women who had completed treatment for breast cancer at least a year previously (N=26), including chemotherapy (N=18). All present women in the active treatment group given chemotherapy completed the full course of treatment in accordance with the American Cancer Association guidelines for early stage breast cancer, either CMF or Taxotere. The cancer survivor comparison group members were also excluded if a medical history revealed any of the exclusion neuropsychological risk factors as noted above for the active treatment group. Although the breast cancer survivor comparison group could report their treatment history, we did not have access to their original disease stage at diagnosis. The two groups were equivalent on age and education, all were Caucasian and the majority were married, employed, and with at least some college education (Table 1).

Zung Self-Rating Depression Scale [19, 20]. The Zung Self-Rating Depression Scale (Zung SDS) is a self-administered 20-item questionnaire that includes a variety of statements associated with depressed moods and is a helpful tool used to assess depression in individuals. The inventory looks at various symptoms of depression such as insomnia, fatigue, suicidal thoughts, and anxiety. The 20 items are based on a Likert scale and the four possible responses range from None or little of the time to Most or all of the time. The higher the score, the more depressed the respondent.

The State-Trait Anxiety Inventory [21,22,23]. The State-Trait Anxiety Inventory (STAI) is a measure that looks specifically at anxiety. It measures a persons current anxiety (state) and characteristic anxiety (trait). The full STAI is a 40-item self-report anxiety inventory, but a shortened version of the STAI consisting of five items was used in this study [21, 22]. The higher the score, the more anxious the respondent.

Hope Quality of Life Scale Breast Cancer Patient Version [24,25,26,27]. This version of the Quality of Life Scale (Ferrell, Dow, & Grant, 1995) is based on previous versions created by the City of Hope National Medical Center. The assessment is a 46-item questionnaire that looks at the four domains of quality of life: physical well-being, psychological well-being, social well-being, and spiritual well-being. This instrument has been validated and reliability has been insured over multiple uses in hospital studies [26,27,28]. The higher the score, the better the quality of life.

Pittsburgh Sleep Quality Index [29, 30]. A modified version of the Pittsburgh Sleep Quality Index (PSQI) was used to evaluate sleep quality in the patients. The PSQI is broken down into seven parts for scoring. They are subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbances, use of medication, and daytime dysfunction. The higher the score on these seven components the more likely the person is to have sleep disturbances. We also included the Sleep Hygiene Likert Scale optional items with this questionnaire.

Functional Assessment of Cancer Therapy - General (FACT-G) [31]. This assessment is a comprehensive compilation of questions which measures health-related quality of life (QOL) in patients with cancer and other chronic illnesses. This study used the FACT-G in conjunction with the subscale for assessing specific problems related to anemia in cancer patients (FACT-An), a subscale which is represented by questions addressing the cardinal symptom of anemia, which is fatigue. This measure also helped gauge the impact of cancer treatment, such as chemotherapy, on other measures of emotional, psychological, and spiritual well-being. For two of the scales (Physical, Functional) higher scores indicate poorer functionality. For the other two scales (Social/Family Well-being, Emotional Well-being), higher scores indicate higher well-being.

Fatigue Symptom Inventory (FSI) and the Multidimensional Fatigue Symptom Inventory (MFSI) [32,33,34]. The MFSI (Hann, et al., 1998) is a 14-item self-report measure designed to measure the daily patterns of fatigue including intensity, frequency, and impact on overall quality of life. The MFSI (Stein, Martin, Hann, & Jacobsen, 1998) is an 83-item self-report measure that evaluates the principal manifestations of fatigue; and includes the subscales of global, somatic, affective, cognitive, and behavioral manifestations of fatigue. These instruments have been validated in a variety of clinical contexts with cancer patients, and provide for a sensitive overview of the impact of the disease on patients overall quality of life as well as activities of daily living (ADL) [32, 34]. Lower scores indicate less intense symptoms of fatigue, so the lower the score the better.

Spiritual Beliefs Inventories (SBI) [35, 36]. In a landmark publication which gained credibility for the inclusion of spiritual well-being assessment within the broader evaluation of quality of life (QOL) issues in psycho-oncology research, Holland and her co-workers (Holland et al., 1998) developed and published the Spiritual Beliefs Inventory. Unlike previous spiritual well-being inventories (e.g., the Spiritual Well-Being Scale in Paloutzian and Ellison, 1991) used in health-related research (e.g., Carson & Green, 1992; Kaczorowski, 1989), the SBI does not rely exclusively on spiritual and religious beliefs, but also includes a social support measure derived from involvement with church or religious groups. The result of their efforts is a well-validated 15-item questionnaire that is a brief, yet robust, measure of the more universal aspects of religious, spiritual, and religious community social support coping with a life-threatening illness as well as the subsequent QOL impact. Also included was the Paloutzian and Ellison 20-item Spiritual Well-Being scale (SWB) that was developed to look at just spiritual and religious beliefs [37]. For both instruments, a higher score indicates a stronger sense of spiritual wellbeing.

Bottomley Social Support Breast Cancer Scale [38,39,40]. This is a cancer-specific questionnaire that is well validated in the clinical cancer-care context and also quick and easy to use in a busy clinical environment. The Bottomley Social Support Scale has good construct validity with the Hospital Anxiety and Depression Scale. It is reliable, allowing medical and support staff to assess the levels of social support and implement any appropriate social support interventions and sensitive to the duress of breast cancer treatment and care [41]. The higher the score, the less the perceived social support on the part of the respondent.

Automated Neuropsychological Assessment Metric (ANAM) [42]. This is a computerized neuropsychological assessment battery developed by researchers at the Walter Reed Medical Center for use in human performance factor studies (e.g., neuropsychological effects of fatigue, chronic stress, sleeplessness, toxic exposure). It takes about 45 to 60min to complete for the average adult. The components of the assessment are derived from well-validated brain/behavior tests such as those encompassed in the Halstead-Reitan Neuropsychological Assessment Battery and the Wechsler Adult scales for both intelligence and memory. The principal measures that we were taken from this assessment pertain to executive functioning and problem solving (Tower of Hanoi Task, Symbolic Logical Reasoning Test), encoding and memory, and a continuous performance task measure of attentional capacity and response time. Furthermore, the ANAM battery has been validated in the hospital or clinic setting [42,43,44]. For all of the ANAM tests, the higher the score the better the performance with the exception of simple reaction time.

Twenty-five ml blood samples were drawn (210ml Na-heparin, 15ml clot) immediately before the computerized questionnaire and ANAM assessment at each of our four study assessment points. Blood was placed in heparinized tubes and was evaluated for hematocrit (percent of red blood cells) and total plasma protein (g/dl). Standard Wrights staining procedure was used to obtain complete blood counts (CBC). The heparinized samples were then separated into the leukocyte and erythrocyte fractions by Histopaque-1077 gradient centrifugation. FITC-conjugated murine monoclonal antibodies were used to label and measure total T cell count (CD3), helper T cell (CD4+), cytotoxic T cell (CD8+), B cell (CD19+), and NK cell (CD16+) using flow cytometry. Each of these assays was conducted at the Indiana Wesleyan University (IWU) research laboratory facility under the direction of Burton Webb. To accomplish this, blood specimens at room temperature were transported from the Lutheran Hospital Fort Wayne study site (about a 1-h drive) within 24h of the blood draw by an IWU student lab assistant trained in the safe handling of biohazard material, employed by Dr. Webb in his lab on a work-study program. Upon receipt at IWU, the blood specimens were immediately processed and placed in deep freeze frozen storage (70C) for later analysis. Dr. Webb, who at the time taught immunology to first-year medical students at Indiana University-Purdue University Indianapolis (IUPUI) Medical School-Muncie, supervised all immunology specimen processing and assay procedures directly in his laboratory at Indiana Wesleyan University, Marion.

Neuman and colleagues documented a relationship between immunological response and mental health [45]. A protocol similar to theirs for assessing the integrity of immunological response as it relates to psychological, emotional, neuropsychological, and spiritual well-being in the breast cancer patients was used. Blood samples were drawn immediately before the ANAM (computerized neuropsychological assessment) battery. Blood placed in heparinized tubes were evaluated for microhematocrit % (percent of red blood cells) using a centrifuge; and plasma protein values (g/dl) using a standard refractometer were determined from these samples. A radioimmunoassay kit was used to measure plasma cortisol (a stress hormone emitted by the adrenal cortex) and Toxicity-preventing activity (TxPA) (a serum albumin factor correlated with cardiovascular risk), along with lipid density measures. Standard staining procedures were used to obtain total white blood cells (WBC) as well as specific types of WBC important in combating disease and infections (mononuclear cells or lymphocytes, neutrophils, and monocytes). Fluorescein-labeled murine monoclonal antibodies were used to label and measure CD3, CD4, CD8, CD19, and CD16/CD56 lymphocytes using fluorescence-activated cell sorting (FACS) scan. NK (natural killer) white blood cells were also measured, since these provide protection against cancer cells and are related to blood vessel constriction following a stressor condition (Neumann & Chi, 1999). These assays were completed at the Indiana University Medical School Muncie Center for Medical Education immunology laboratory.

For the newly diagnosed breast cancer group, the first assessment point was at enrollment, within the first few weeks of diagnosis, usually following lumpectomy surgery and just before radiation treatment. The second assessment point was generally after surgery and during or just after radiation therapy, usually about one to 2 months after initial diagnosis for the active treatment group and enrollment for the remission breast cancer survivor group (Fig.1). The third assessment point was at 2 months after treatment had started (after the conclusion of radiation therapy and in the initial stages of chemotherapy if recommended). The fourth assessment point occurred a month following completion of all chemotherapy if recommended. This was usually at the six- to eight-month point for the active treatment group and about 6 months following enrollment for the remission group. The second, third, and fourth assessments occurred 23days after treatment (radiation and/or chemotherapy).

legend. The approximate timing of the 4 principal assessment domains (Spiritual Well-being, Quality of Life, Computerized Neurocognitive Performance, Immunological Biomarkers) is depicted for both the active treatment and remission (breast cancer survivor) groups in the present study. Timing of the assessments coincided with the approximate course of treatment from diagnosis/enrollment (Assessment 1), initial surgery and radiation treatment (Assessment 2), continuation/completion of radiation and initiation of chemotherapy (if needed) (Assessment 3), and completion of radiation and/or chemotherapy if needed (Assessment 4)

For the breast cancer survivor comparison group, the assessments occurred at enrollment, 1 month, 2 months, and at 6 months. The completed surveys and computerized ANAM neuropsychological assessment were completed in a quiet counseling room at the Womens Cancer Center at Lutheran Hospital. Each blood drawn was completed by a registered nurse immediately following the assessments. Specimens then were processed at Muncie Center for Biomedical Education, where immunological biomarkers were assayed using flow cytometry.

Descriptive statistics at enrollment for active treatment breast cancer group and the breast cancer post-treatment (remission) comparison group not on active treatment, were compared using item averages for all questions (reverse coded where necessary) for each of the questionnaire measures (psychosocial, emotional wellbeing, quality of life, spiritual wellbeing, physical symptoms of cancer treatment). Statistically significant differences between the two groups were compared for these measures at p<0.05 using an independent samples Tukey t test (nonparametric). The ANAM neuropsychological performance measures were computed automatically as age- and gender-based standardized scores using the ANAM norms built into the software package for American adults. Adjusting for age and years of formal education at enrollment (diagnosis), partial correlation coefficients were computed for the emotional wellbeing, quality of life, physical and health symptoms, and spiritual wellbeing measures when correlated with the immunology T cell biomarkers (CD4, CD8, CD16) for the active treatment and for the cancer survivor comparison groups separately. The most significant correlations were visually depicted in a scatterplot in the study figures.

Active treatment women were also compared across principal outcomes based on type of treatment prescribed (chemotherapy and radiation versus radiation only). For the active treatment subgroup comparisons (chemotherapy and radiation versus radiation only), an ANOVA repeated-measures analysis was used to evaluate whether a significant group by time interaction effect was apparent, indicating a greater degree of change over time for one treatment subgroup compared to the other. The most significant questionnaire-based (psychosocial support, quality of life, emotional wellbeing, spirituality, physical symptoms) time by treatment subgroup interaction effects was then depicted in the form of visual plot. The same was done with the most significant time by treatment subgroup interaction effect for the immunology biomarkers (CD4, CD8, CD16). IBM SPSS statistical software version 21 was used for all analyses, graphs, and plots [46].

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Preliminary study on the effects of treatment for breast cancer: immunological markers as they relate to quality of life and neuropsychological...