Each patient will be asked to document her daily sporting activities in a standardized self-assessment study diary. The patients will be instructed by study personnel how to use the diary during recruitment and during each follow-up visit. The study personnel check the completeness and accuracy of the diary and discuss potential improvements with the patients on each follow-up visit. The diary will be used to document the following points:

As a standardized measure of physical activity the metabolic equivalent task (MET) will be used to describe the metabolic turnover of the patients even when performing different sportive activities (46, 47).

During each of the five study visits the patients perform a spiroergometry on a treadmill (XRCISE RUNNER MED™ by Cardiowise, ERGO-FIT™, Pirmasens, Germany) for the assessment of cardiopulmonary fitness (48). After a technical introduction for the patient, the calibration of the aeroman™ and a baseline spirometry in seated position, the patients start walking at an individually determined speed, typically 4 kmph. Subsequently, the patient is challenged according to a standardized, validated protocol that ensures a linear increase in Oxygen uptake response (49). For inter- and intraindividual comparison, the time course of work rate in watts (WR_(t)) is calculated using the formula published by Porszasz et al. (49) WT(t) = m * g * ν(t) * sin (α), where m is body mass in kg, g is the gravitational acceleration (9.81 m*s^-2), ν(t) is the time course of velocity in meters per second, and α is the angle of inclination. The speed or gradient of the treadmill is increased by 0.5 kmph or 1 percent every 2 minutes to intensify the level of exertion. After each interval, the patient breathes through a tightly fitting spirometer mouthpiece (aeroman™ professional, ACEOS GmbH, Fürth, Germany) according to a validated protocol (50). The measurement will be terminated once the patient reaches a maximum heart rate defined as 220 – age (bpm) or a respiratory quotient >1. Furthermore, the subjective perception of exertion according to Borg >17 (51), dizziness, dyspnea, nausea or pain will lead to termination of the spiroergometry (52). The patient can interrupt the measurement due to subjective exhaustion at any time. This will reveal intraindividual longitudinal changes during the first year after the diagnosis of breast cancer. The ventilatory threshold (VT) will be used as a submaximal indicator of general cardiopulmonary fitness (53). VT represents the excessive increase of carbon dioxide output compared to oxygen uptake. In addition, heart rate (HR), oxygen uptake (VO₂), carbon dioxide release (VCO₂), respiratory quotient (RQ), breathing frequency (BF) and respiratory minute volume (VE) will be assessed to allow the analysis of endurance performance. The measurements obtained with the spiroergometry are given in Table 2 .

Physical activity is assessed daily by supplying each patient uses a commercial fitness tracker (Fitbit charge 3™ (Fitbit Inc., San Francisco) that will be linked to her smartphone (54). Study personnel assists the patients and – if required – their associate to install the smartphone app and to setup the measurements. The patients are requested to transcribe the measurements of their fitness tracker into their study diary weekly. Those values are shown in Table 3 .

The body composition will be determined based on bioelectrical impedance analysis (BIA) (TANITA scale™, Tanita Europe BV, Stuttgart). The test person stands barefoot on a body scale and holds sensors in both hands. Using the impedance between the four measuring points, numerous data can be collected that provide information on the muscle, fat, bone and water content of the whole body and individual compartments. Bioimpedance analysis is routinely used in the context of nutritional advice and sports medicine, but it can also shed light into disease processes, such as hemodialysis patients (55) ( Table 4 ). When accessible, the body composition will also be estimated by using routine CT scans of the study patients as previously published (56).

Plicometry allows a standardized estimate of the body fat status which can change under influences such as sport, chemotherapy or cancer (57–60). In the BEGYN study, fat distribution is assessed using the skinfold measurement using the 3 point-method (triceps, suprailiac skinfold, and thigh) according to Jackson and Pollock (61). ( Figure 2 ). The total body subcutaneous fat tissue (in kg) was estimated from the plicometry measurements using the formula (61):

S is the sum of the skinfold thicknesses measured at the triceps, the suprailiac skinfold and the thigh.

For disease monitoring and adjustment of the antineoplastic therapy, multiple biomarkers are routinely assessed during the quarterly study visits ( Table 5 ). In addition, the BEGYN study will include vitamin D and selenium concentrations since they are often discussed as potentially relevant to cancer biology (69–72).

Peripheral blood mononuclear cells (PBMCs) will be obtained from 9,6 ml anticoagulated blood samples by Ficoll density gradient centrifugation and resuspended in 90% fetal calf serum + 10% dimethylsulphoxide. Plasma supernatant will be collected and all biosamples will be cryopreserved at -80°C until further processing (73).

The underlying disease, the antineoplastic therapies and physical activity can have a profound impact on the immune system, respectively (26, 74–77). The number of 36 circulating leukocyte subsets will be assessed by flow cytometry using a panel of validated antibody stainings ( Table 6 ), based on previous studies (73, 78). Cells will be analyzed with FACSCelesta™ (BD Biosciences, Franklin Lakes, New Jersey, USA) (79).

Plasma cytokine profiles will be measured by Luminex^® xMAP™ technology (Austin Texas, USA) since they reflect the activity of various immune cells ( Table 7 ). Luminex^® xMAP™ technology performed on MAGPIX™ instruments, enables the simultaneous quantification of up to 50 target proteins or nucleic acids (80) and have been validated for numerous immunological studies, including breast cancer (20, 62, 81) and infections (82). Superparamagnetic microsphere beads are conjugated with a distinct monoclonal antibody. The beads themselves are dyed with varying amounts of red and infrared fluorophores to allow clear assignment to a specific bead region. MAGPIX™ fluorescence imager-based instruments use a LED illumination/CCD camera detection system for both bead region identification and reporter fluorophore-based analyte quantification (83).

The expression of selected mRNA transcripts related to breast cancer and inflammation will be measured by a quantitative real-time PCR (qPCR). The method used is based on a previously published technique (73). Total RNA will be isolated from PMBCs using the High Pure RNA Isolation Kit (Roche, Basel, Suisse). Reverse transcription will be performed using SuperScript™ VILO IV cDNA Synthesis Kit (Invitrogen, Thermo Fisher Scientific) followed by the cDNA purification using QIAquick™ PCR Purification Kit (Qiagen, Hilden). Concentration and purity determination of the isolated RNA will be performed with NanoDrop™ (Thermo Fisher Scientific, Waltham, Massachusetts, USA). TaqMan™ Gene Expression Assays (Applied Biosystems, Thermo Fisher Scientific, Waltham, Massachusetts, USA) will be used to perform qRT-PCR of immunological key transcription factors TBX21, RORC, GATA3, FOXP3 (84–88). The Δct-values will be calculated using ACTB, EEF1A1, and 18S as a standard. Values will be normalized to the data from the samples taken at the baseline visit.

The overall quality of life is assessed using the Quality of Life Questionnaire (QLQ-C30 version 3.0) which has been developed and validated by the European Organisation for Research and Treatment of Cancer (EORTC) to explore quality of life among cancer patients (90). The supplementary questionnaire QLQ-BR23 will be used to record symptoms specific for breast cancer (95). In sum, a score from 0 – 100 can be calculated allowing the quantification of the impairments regarding the patient’s health and quality of life.

The questionnaire “Mental health scales” (MHS) (92) was used to assess the psychological integrity of the study participants, in order to evaluate the development of the patients’ personality throughout the study duration. The questionnaire consists out of seven scales (autonomy (17 items), willpower (14 items), acceptance of live (8 items), self-reflection (12 items), finding of a meaning (7 items), naturalness (10 items) and social integration (8 items)), which yields a total of 76 items. The questionnaire is answered with the help of a five-point Likert-scale (1 = I fully agree; 5 = I fully disagree) and an example item is “Generally I am confident”.

Symptoms of chronic fatigue syndrome (CFS) (93) that affects more than 50% of breast cancer patients and the extent of psychological stress will be assessed using the Distress Thermometer (DT) (96).

The self reported anxiety and distress will be assessed using the HADS (Hospital Anxiety and Depression Scale) questionnaire (89, 97) and the German adaptation of the Distress Thermometer (DT) (94, 96–98).

To get an overview of the mental state of the patients, the German questionnaire “Multidimensional Well-being Questionnaire” (MDBQ) (91) has been used in its short version with twelve items (short version A). This questionnaire captures three bipolar dimensions of the current mental state (good mood – bad mood, alertness – fatigue, tranquility – inquietude) of the breast cancer patients. The questionnaire presents twelve adjectives (e.g., satisfied, flabby, good etc.) and with the help of a five-point Likert-scale (1 = not at all; 5 = very much) the patients rate their instant feeling. Cronbachs’α is between 0.86 and 0.94, indicating a good consistency of the scale.

To our knowledge, the BEGYN study is one of the largest studies with a very broad approach in breast cancer patients that will allow to identify associations between physical, psychological and laboratory variables, thus linking aspects that are often studied separately in breast cancer patients. Regarding the assessment of physical activity, it will be of particular interest to discuss the results of the BEGYN study in the light of other ongoing highly innovative studies such as the PROTECT study which also includes patients with colorectal cancer and lung cancer (6). One of the major strengths is the continuous recording of the patient’s physical activity and well-being by diary and by use of a fitness tracker since physical activity encompasses not only explicit sports activities, which may last a few hours per week, but also activity during everyday activities. Various forms of physical activity have been proposed, such as yoga, Tai Chi Chuan, Nordic walking, jogging, weight training, cycling, dancing and many others (20, 36, 37, 47). However, it has been claimed that individualized recommendations are needed to meet the needs of the patient (6, 105). By using the concept of metabolic equivalents (MET), the BEGYN study will allow comparing effects of physical activities of similar intensities independent of the type of exercise. Since nutrition, food supplements and lifestyle may significantly influence the effects of exercise and antineoplastic therapies, these variables are assessed by using questionnaires and the self-assessment diary.

The BEGYN study will yield an overview on a relatively long period of 12 months after diagnosis, starting before any specific antineoplastic therapy. Thus, the patient will typically be observed beyond the initial steps of therapy, which are also often associated with psychoemotional distress. This may give insight into predictors of a more favorable long-term management towards a new physical and psychological balance. Moreover, the 12 months study period will reduce potential artifacts due to seasonal effects which can, in example, influence outdoor activities and Vitamin D concentrations. The role of Vitamin D in carcinogenesis and cancer therapy is still under debate. Importantly, serial measurements of 2,5 OH Vitamin D concentrations in peripheral blood and assessment of therapeutic Vitamin D intake may give insight into the role of Vitamin D metabolism and may also provide data on the interaction between antineoplastic therapy and Vitamin D. In addition, the BEGYN study will yield serial selenium concentrations and insight on the consumption of selenium by the patients, which often is provided as a self-medication, causing significant financial burden for some patients.

The BEGYN study will allow to compare patients with various antineoplastic therapies, i.e., endocrine therapy, chemotherapy, surgery, and radiotherapy. Due to the strict adherence to the national guideline, therapy concepts are to be expected representative for the German national standard.

The BEGYN study will yield a deep insight into the innate and adaptive immune system during the first year after diagnosis of non-metastatic breast cancer (62). Inflammatory processes are a crucial part of the physiological response to cancer cells. Simultaneously, the BEGYN study will shed light on the effects of endocrine therapy and of chemotherapy on the immune system. Moreover, it is well recognized that moderate physical activity is associated with an improved immune status whereas excessive physical activity leads to an increase susceptibility towards (viral) infections (22). Interestingly, natural killer (NK) cells play a key role in sports physiology and in restricting tumor growth (64). Thus, the BEGYN study will be among the first studies to quantify four distinct subsets of NK cells in relation not only to breast cancer and antineoplastic therapy, but also in relation to the extent and type of physical activity.

The BEGYN study also has some limitations, such as the single center approach. However, the data will serve as a basis for designing future multicenter studies. In example, the results of the BEGYN study might help focusing the highly detailed immunophenotyping regarding the characterization of leukocyte subsets, mRNA transcripts and cytokines to the most promising variables in future study protocols. One further limitation of the study is that the quality of self-assessments may underlie intra-individual and inter-individual variations when filling out the self-assessment diary (e.g. daily documentation of physical activities, weekly read outs of the measurements of the fitness tracker etc.). Due to the need of serial measurements of the body composition, ethical concerns on X-ray exposure were the reason to use bioimpedance analysis and plicometry rather than Dual-energy X-ray absorptiometry, which is regarded as the gold standard (106). With regard to the known limitations of bioimpedance analysis and plicometry, we will compare the intra-individual relative changes over time instead of absolute values (107). In addition, we will further extend the data by estimating the body composition from the routine CT scans of the study patients (56). The BEGYN study is an observational study, thus potential causal relationships must be interpreted carefully. Ethical concerns would not allow randomizing patients into a group that would not be encouraged to exercise since this would conflict with the high evidence guidelines. However, the BEGYN study might help defining valid methods of continuous registration of physical activity for future studies to compare the role of various types of exercise, e.g., training of strength versus endurance.