Social determinants such as education, income, marital status, and social networks play a crucial role in cancer outcomes, including breast cancer. One potential influencing factor is diet, and the Mediterranean diet has been shown to benefit both the health and the microbiome.

This raises the question: Does adherence to the Mediterranean diet impact the microbiome, quality of life, overall well-being, and outcomes in breast cancer patients undergoing (neo)adjuvant treatment, particularly among those with unfavorable socioeconomic determinants?

The Mediterranean diet consists of plant foods such as vegetables, fruits, nuts, seeds, berries, legumes, herbs, and spices. It also features animal proteins like fish, meat, and cheese, along with fat from olive oil. Thus, adherence to the Mediterranean diet is adherence to the official Danish diet recommendations.

This phase II clinical trial is an interdisciplinary study combining nutrition, sociology, and health research. Breast cancer patients candidates for (neo)adjuvant treatment at four oncology departments in Region South Denmark will be randomized 2:1 to the Mediterranean diet (with individual dietary guidance from a nutrition therapist aiming at daily consumption of a minimum of 30 grams of dietary fiber and weekly consumption of a minimum of 30 different plant foods; minimizing the amount of ultra-processed food) versus the patient's regular diet, in conjuction with the (neo)adjuvant chemotherapy.

The trial's primary endpoint will be changes in gut microbiota composition in feces.

The study evaluates how the Mediterranean diet affects the microbiota (evaluated using Oxford Nanopore Technology 16S sequencing on fecal samples); short-chain fatty acids in stool and plasma (assessed by mass spectrometry); immune system (using flow cytometry for immune cell determination in blood and immunohistochemical determination of immune cells in the tumor tissue); and patients' social quality of life, overall well-being, sense of empowerment, and personal feeling of agency (using the WHOQOL-100 questionnaire); taking into account the impact of the distance from the patient residence to the hospital (tracked from GEOTEAM at Statistics Denmark), and socioeconomic factors (using the DREAM database).

The evaluation of biological parameters is based on the suggested mechanism of action. A diet rich in various plant foods and dietary fiber will alter the gut microbiota, promoting bacteria producing high amounts of short-chain fatty acids. These fatty acids will activate immune cells, aiding in the destruction of cancer cells.

The study will also assess whether the Mediterranean diet will positively impact the patients' social quality of life, overall well-being, sense of empowerment, and personal feeling of agency. The Mediterranean diet may not only lead to improved dietary habits but also serve as a crucial tool for breast cancer patients, particularly among those with unfavorable socioeconomic determinants. Enhanced empowerment and improved coping tools could be an important step in combating cancer-related inequalities.

STUDY PURPOSE The project investigates whether adherence to the Mediterranean diet can influence the microbiome, improve quality of life, overall well-being, sense of empowerment, personal feeling of agency, immune activity, levels of short-chain fatty acids and patient outcomes in breast cancer patients undergoing chemotherapy. Additionally, it examines if these effects are more pronounced in individuals with challenging socioeconomic determinants.

BREAST CANCER Among women, breast cancer is the most diagnosed cancer, and is the leading cause of cancer deaths globally in most countries, with 2.3 million new cases recorded in 2022 and approximately 665,000 deaths. In Denmark, there are 5000 new cases, and 1.050 deaths annually, of those 250 in the Region of Southern Denmark. In men, breast cancer is rare with an average of 42 new cases annually in Denmark.

ONCOLOGICAL TREATMENT The oncological treatment of early breast cancer encompasses neoadjuvant (pre-surgery) and adjuvant (post-surgery) treatments.Chemotherapy comprising anthracyclines and taxanes is a cornerstone in both neoadjuvant and adjuvant settings for breast cancer treatment. These drugs have been shown to significantly improve patient outcomes as these regimens are particularly effective in shrinking tumors before surgery, reducing the risk of recurrence and improving overall survival. HER2-targeted therapies, such as trastuzumab, are specifically used for HER2-positive breast cancers in conjunction with chemotherapy and have significantly improved outcomes. Recently, checkpoint immunotherapy, pembrolizumab, has been approved in Denmark for triple-negative early breast cancer together with chemotherapy due to increased pathological complete response (pCR).

SOCIOECONIC INEQUALITY At the national level, disparities in breast cancer outcomes are commonly observed along socioeconomic and demographic lines in both low-, middle- and, high-income countries. These disparities are evident across all stages of the breast cancer continuum, from exposure to modifiable risk factors to access to palliative care and mortality rates. Consequently, women with lower socioeconomic status are more likely to present with late-stage cancer and experience poorer breast cancer prognoses compared to those with higher socioeconomic status. Cancer inequalities at the individual level are predominantly shaped by systemic social determinants of health.

In Denmark an estimated 21% of breast cancer deaths five years after the diagnosis of breast cancer could have been avoided, had patients in all income groups had the same survival rate as the high-income group. Thus, social inequality in cancer survival is a critical problem, and patients with little education, low income, or living alone have poorer survival than better educated cancer patients, with higher income, and living in a relationship. This applies despite women with higher socioeconomic status having significantly higher breast cancer incidence.

GUT MICROBIOTA AND MEDITERRANEAN DIET The human gut hosts approximately 38 trillion cells called the microbiota; the human gut microbiome consists of the genes these cells harbor.

In the 2022 update of Hallmarks of Cancer, the microbiome was introduced as a new important hallmark of cancer. Importantly, the microbiome modulates four other hallmarks: tumor growth, tumor-promoting inflammation, immune evasions, and genomic instability; additionally, the microbiome modulates therapy resistance. The implication is, therefore, that the microbiome is a critical link in cancer development, progression, and treatment response.

The Mediterranean diet is defined as the consumption of fresh fruits, vegetables, nuts, berries, legumes, and non-refined cereals. Olive oil is the principal source of lipids. Moderate intake of alcohol, preferably red wine, with meals; moderate consumption of fish, poultry, eggs, low-fat dairy products, and sweets; monthly consumption of red meat; and regular physical activity.

As the Mediterranean diet includes a wide variety of plant foods and proteins from animal sources, it is a flexible option for many patients. It is not restrictive and allows for many different types of food, with the main limitation being processed foods. This flexibility was a key reason why Harvey et al. chose this diet for a randomized clinical trial involving cancer patients. The study aimed to measure fatigue during chemotherapy and included a sub-study to qualitatively assess how patients felt about making dietary changes during their treatment, identifying facilitators and barriers. Importantly, patients reported that the diet gave them a sense of control and empowerment. They enjoyed learning about nutrition, trying new foods, feeling in control, setting goals, doing something constructive to aid their treatment, and having a positive focus. Barriers included chemotherapy side effects (mouth sores, taste changes, and lack of energy) and food preferences.

Higher adherence to the Mediterranean diet was associated with a 23% lower risk of all-cause mortality. The Mediterranean diet was feasible and safe for adults with cancer. Mediterranean diet has been associated with a reduced risk of breast cancer development. Its impact on survival outcomes after breast cancer diagnosis remains an area of active investigation. Changes in the microbiome composition were observed already after four weeks of the Mediterranean diet.

The Mediterranean diet has several effects, including lipid-lowering and anti-inflammatory actions, reducing chronic inflammation. Moreover, bacteria in the gut ferment dietary fiber to short-chain fatty acids; factors that upregulate immune functions. Short-chain fatty acids are important as an energy source for intestinal epithelial cells, but some enter the bloodstream and act as signaling molecules. They target G-protein-coupled receptors (GPCR) GPCR41, GPCR43, and GPCR109A.

Several studies in patients with melanoma, and cancers of the kidney, lung, and bladder have found that bacterial diversity at baseline can predict response to immunotherapy treatment; some types of bacteria are often reported to be found in larger quantities at baseline in responders than in non-responders, especially Akkermansia mucinifila, Ruminococcaceae, Faecalibacterium, and Lachnospiraceae.

In patients with primary HER2-positive breast cancer undergoing trastuzumab treatment, a recent study revealed the direct involvement of the gut microbiota in trastuzumab efficacy. To the extend of our knowledge, the association with response to checkpoint immunotherapy in breast cancer patients has not been reported.

MICROBIOME AND VITAMIN D Vitamin D has recently been associated with changes in the microbiome and cancer outcomes. Vitamin D is the only vitamin not normally consumed sufficiently even in a well-balanced diet. The main source of vitamin D is solar exposure to the skin. Denmark is situated between the 55th and 57th northern latitudes and therefore UV exposure is insufficient for vitamin D production for large parts of the year. Measurements of vitamin D will be performed at baseline and at Day 1 of cycle 6, and daily supplementation with 50 micrograms of vitamin D is encouraged.

POTENTIAL BENEFITS AND RISKS The potential benefits are that the Mediterranean diet will result in greater microbial diversity during (neo)adjuvant therapy, increase the amounts of short-chain fatty acids in serum and stool, activate the immune system, and improve patient outcomes.

Moreover, the Mediterranean diet may positively impact the patient's social quality of life, overall well-being, sense of empowerment, and personal feeling of agency.

Additionally, these effects may be more pronounced in individuals with challenging socioeconomic determinants.

The potential risk of harm is considered minimal since the intervention is food, and the investigated Mediterranean Diet is comparable to the diet recommended by the Danish Health Authorities (foedevarestyrelsen.dk) The study will be carried out according to the principles outlined in the Declaration of Helsinki. With food as the intervention, the study does not need monitoring from the GCP unit, according to Danish rules.

INTERVENTION The intervention is the Mediterranean Diet.

STUDY POPULATION The study population consists of patients with newly diagnosed early breast cancer, candidates for (neo)adjuvant chemotherapy-based treatment at one of the participating sites in the Region of Southern Denmark.

PATIENT INVOLVEMENT A patient panel, comprising four breast cancer survivors and one representative from a cancer patient association, is actively involved in the design and execution of the MEDITERRACARE study helping developping patient information material, review and provide feedback on questionnaires, and offer practical insights for conducting the trial.

RELEVANCE OF THE TRIAL Changes in bacterial composition can occur within a few weeks of switching to a diet rich in dietary fiber and various plant foods. A positive correlation between gut microbiota and favorable outcomes to checkpoint immunotherapy has been demonstrated in cancer patients. Gut microbiota is important for HER2+ therapy efficacy.

Socioeconomic inequalities are critical for breast cancer patients. The study will evaluate whether adherence to the Mediterranean diet can influence the microbiome, and improve quality of life, overall well-being, sense of empowerment, personal feeling of agency, immune activity, amounts of short-chain fatty acids, and patient outcomes in breast cancer patients undergoing chemotherapy. Additionally, it examines if these effects are more pronounced in individuals with challenging socioeconomic determinants.

This study addresses a knowledge gap. No study worldwide has assessed the Mediterranean diet in early breast cancer patients during chemotherapy with special emphasis on socioeconomic inequality.

HYPOTHESIS The hypothesis is that the Mediterranean diet results in greater microbial diversity during (neo)adjuvant therapy, increases the amounts of short-chain fatty acids in serum and stool, increases the numbers of T- and Natural Killer cells in blood and tumor tissue, reduces inflammation in blood and tumor tissue, and improves patient outcomes.

Moreover, the Mediterranean diet will positively impact the patient's social quality of life, overall well-being, sense of empowerment, and personal feeling of agency.

Additionally, these effects will be more pronounced in individuals with challenging socioeconomic determinants.

STUDY DESIGN The MEDITERRACARE study is designed as a phase II randomized, controlled trial with two arms in a 2:1 allocation ratio evaluating the Mediterranean diet versus regular diet, and a primary endpoint of changes in gut microbiota composition. The study protocol is reported following the CONSORT guidelines.

STUDY SETTING AND RECRUITEMENT Enrolment takes place at four Danish oncology outpatient clinics in the Region of Southern Denmark (Odense, Vejle, Sønderborg, Esbjerg).

Randomization will be performed as block randomization with 2:1 allocation using a computer-generated table of block sizes of 6 or 9, created and supervised by a data manager at OPEN, Open Patient Data Explorative Network, Odense University Hospital, Region of Southern Denmark.

Blinding of patients and health care professionals involved is not intended.

DETAILS ON INTERVENTION The patients randomized to the intervention arm, and if possible a partner or other family member, will receive nutritional guidance during the weekly outpatient chemotherapy visits or via online calls or home visits. Initially 1x weekly during the first month and every other week onwards. In addition, patients will get a booklet on how to implement the Mediterranean Diet in daily life, and access to a password-protected website with teaching videos and Mediterranean diet recipes. To ensure compliance regarding sampling of feces and reporting of food intake patients in the control arm will also be called on during visits to the department.

Group sessions will be arranged for patients randomized to the Mediterranean diet and a hot-line is open during working hours.

DATA COLLECTION Study data will be collected and managed using the REDCap electronic data capture tool (Vanderbilt University, v.12.0.33) hosted at OPEN (Open Patient Data Explorative Network, Odense University Hospital).

At the first consultation at the Department of Oncology, a blood test will be drawn for baseline values and kits for stool sampling at home will be provided to the patient. The stool sample should be obtained at home before the chemotherapy starts.

TIMING DATA COLLECTION The WHOQOL-100 questionnaire and baseline dietary and lifestyle questionnaire will be provided to the patient's E-boks. Data will be captured in REDCap.

Stool samples (1-2 grams per collection) will be collected by patients at home and immediately frozen at -18°C in their home freezer. Patients will bring their samples to the hospital in a cooling bag at the next planned visit and the samples will immediately be transferred to -20°C until DNA extraction. Stool samples will be obtained at baseline, and at Day 1 of cycle 2, 4, 6, or in case of early discontinuation of chemotherapy and stored in a research biobank until completion of primary analyses. Potential left-over stool or Bacterial DNA after the analyses for this study are stored in the OPEN BIOBANK for future, not yet specified, research. Participants will be contacted and asked for consent in case of future research.

Microbiome evaluation will use Oxford Nanopore Technology 16S sequencing of fecal samples.

Metabolome assessment will detect short-chain fatty acids (SCFAs) in patient stool and plasma samples using ultrahigh-performance liquid chromatography-tandem mass spectrometry.

Adhesion to the diet will be measured via food diaries (www.myfood24.org). The software provides feedback to the user about reaching the daily/weekly goals, thereby nudging the participant to reach the target. Patients will also fill out a paper record of different plant foods eaten every week.

Quantification of CD4+ T-cells, CD8+ T-cells, CD19+ B cells, and 56+ Natural Killer cells will be performed by flow cytometry.

Hematology (hemoglobin, white blood cell count, absolute neutrophils, monocytes, lymphocytes, and platelet count) CRP, Vitamin D status will be measured at initiation of the study and at day 1 of chemotherapy cycle 6.

GlycA and lipoproteins will be examined using Nuclear Magnetic Resonance (NMR)-technique at Nightingale, Finland, (https://nightingalehealth.com) as an expression of metabolic improvements.

Blood samples drawn after a minimum of 6 hours fasting will be treated and stored according to current research ethical practice in Denmark in a research biobank assigned for this trial. Blood samples of a total of 40 ml/time are drawn at four time points: at baseline, and Day 1 of cycle 2, 4, and 6 chemotherapy, or in case of early discontinuation of chemotherapy.

Samples are stored as plasma, serum, buffy coat, and whole blood at minus 80 degrees Celsius at the Departments of Biochemistry of participating hospitals and final storage at the OPEN BIOBANK and stored in a research biobank until completion of primary analyses. Potential left-over blood after the analyses for this study are stored in the OPEN BIOBANK for future, not yet specified, research. Participants will be contacted and asked for consent in case of future research.

Archived tumor tissue from the primary breast tumor will be sampled, including the diagnostic biopsy and surgery specimens in neoadjuvant-treated patients, and the surgery specimens in adjuvant-treated patients, which are then stored in a research biobank. Primary tumor tissue will be analyzed for tumor-infiltrating immune cells.

Pathological samples are stored according to clinical routines at the Departments of Pathology affiliated with the trial sites. Immunohistochemical staining will be performed at the Department of Pathology, Esbjerg Hospital.

The following immune cells will be identified within the tumor tissue: CD8+ T cells, CD4+ T cells, FoxP3 regulatory T Cells, CD20+ B Cells, CD57+ NK Cells, CD66b+ Neutrophils granulocytes, CD68+ Macrophages, Granzyme B (GZMB)+ Cells, PD-L1. These immune cells will be quantified using digital image analysis combined with artificial intelligence at the Department of Clinical Medicine, Aarhus University. The median cell density of immune cells per mm2 tumor tissue will be calculated.

DATA REGISTRATION Data will be collected in a secure and central REDCap database. Groups and subgroups Participating patients should have newly diagnosed early breast cancer and be considered eligible for initiating either adjuvant or neoadjuvant chemotherapy-based treatment.

DURATION OF PARTICIPATION In MEDITERRACARE, patients will be followed from randomization before chemotherapy commencement until the start of treatment cycle 6. If a patient stops chemotherapy before cycle 6, blood and fecal samples are collected and participation in the trial ends. Recruitment is planned to begin uniformly across centers from April 2025. The recruitment period is expected to last 18 months.

Data collection is planned to cease latest April 2027, with primary study reporting completed by April, 2028. Follow-up for clinical events, i.e. recurrence, will be conducted through Danish Breast Cancer Cooperative Group(DBCG) database until December 2036. Follow up for for vital status will be followed up via the Central Person Register (CPR) registry until December 2036.

STOPPING THE TRIAL The sponsor can stop the trial in case of practical issues such as insufficient inclusion of patients.

RANDOMIZATION CODE The randomization code will be stored in the REDCap database. Code breaking is not relevant in the MEDITERRACARE study.

CASE REPORT FILES (CRFs) Data to be extracted from the hospital records will be directly typed into the central REDCap database.

Questionnaires filled out by the patient electronically at home will directly be stored in the central REDCap database. Questionnaires filled out by the patient on paper (plants consumed per week) will be collected and typed into the REDCap database.

Patients can stop participating in the trial at any time point without explanation.

EXCLUSION OF GENDER OR AGE GROUPS There are no restrictions on age and gender except for restricting the participation to adults. Breast cancer is extremely rare below the age of 18.

As breast cancer is rare in males, we expect only a few males to be recruited. As the oncology department typically handles breast cancer in males, we do not expect males to be underrepresented.

SOCIOECONOMIC EQUALITY AND PATIENT QUALITY OF LIFE The driving question in this part of the study is whether the Mediterranean diet will positively impact the patients' social quality of life, overall well-being, sense of empowerment, and personal feeling of agency, using the WHOQOL-100 questionnaire.

We will explore both geographic and social inequality, based on our previous extensive research in this area encompassing both geographic and social inequality in health.

The distance to the healthcare system is defined as the geographical distance between the patient's place of residence and the hospital. The distance calculation is carried out by GEOTEAM at Statistics Denmark.

Co-variables include socio-demographic and socio-economic characteristics, such as age, gender, cohabitation status, employment, income, and education, in addition to disease stage and co-morbidity. These will be obtained from relevant registers in the DREAM database, such as income, education, and employment.

In the qualitative study, the focus is on the experience of the intervention, and the benefits or barriers associated with it. The emphasis is on patients' everyday experiences, how the intervention affects their practical lives, and whether they encounter challenges and, if so, what those challenges are. The qualitative interview study is based on semi-structured interviews with 20 patients, of whom 15 have received the Mediterranean diet intervention and 5 have not.

First, the register analysis will be performed to establish a foundational understanding of the participants' socio-economic and socio-demographic conditions. Second, responses from utilizing the WHOQOL-100 questionnaire will analyzed. Third, individual interviews will be conducted with the patient group, focusing on uncovering their experiences, practices, and perspectives regarding their life situations and overall well-being. Moreover, data on family/relatives support on dietary changes and support in general for women under critical risk will be extracted.

ADHERENCE Adherence to the protocol will be monitored via food registration in the MyFood24 system. Patients are required to report their daily food intake at least 3 days per week. Every week a separate report on weekly intake of different plants should be filled out.

Patients are required to fill out the forms, and will be encouraged to fulfill the goals of consuming 30 grams of dietary fiber daily and 30 different plants per week in the intervention group and follow their normal diet in the control group.

COMPLIANCE AND MONITORING Compliance will be monitored via MyFood24 and by collection of plant registration forms.

SUBSEQUENT TREATMENT If a patient leaves the trial, they will continue their normal medical treatment without the diet intervention.

SAFETY EVALUATION Since the MEDITERRACARE study is a clinical trial investigating a diet similar to that recommended by the Danish Health Authorities, there will be no safety issues to assess.

STATISTICS Data will be stored in a secure server in OPEN Analysis. Descriptive analyses will be performed to explore exchangeability between the two study arms and presented in a table describing the study population on key characteristics.

The WHOQOL-100 questionnaire will be analyzed. A DREAM database analysis will be performed to establish an understanding of participant's socioeconomic and socio-demographic levels. Individual interviews will be conducted to uncover experiences, practices, and perspectives regarding their life situation and general well-being.

The analysis will separate participants into three socio-economic groups (low-risk vs. median-risk vs. high-risk). This stratification will be used as the key to interpreting data for gut bacteria composition and immune cell activity For comparison between groups two-sample Student's t-test will be applied for normally distributed numerical data and the Wilcoxon-Mann-Whitney test will be applied for non-normally distributed numerical data while categorical data will be compared by chi-squared test.

Correlation analyses of median values (measured at the start of cycle 6) of the six domains in the WHO-100 questionnaire, and median estimates (measured at the start of cycle 6) of blood CRP, monocytes, neutrophils, CD4/CD8/CD19/CD56 cells as well as median values of daily dietary fiber and median weekly plant food intake; concentrations of fecal metabolites including SCFA, and alpha diversity, beta diversity as well as microbial differential abundance will be performed with relevant stats package within the statistical programming language R (v.3.5.1).

A recurrence will be defined as any invasive breast cancer recurrence irrespective of localization.

Information on overall survival will be retrieved by linkage to the national CPR registry.

Invasive disease-free survival among groups will be analyzed in crude analysis using the Kaplan-Meier and distant recurrence-free survival will be analyzed using competing risk analysis with cumulative incidences. Cox regression hazards analysis will be performed allowing for controlling for confounders in multivariable analysis.

A 2-sided p-value of ≤ 0.05 will be used to determine significance. All statistical data analysis will be carried out using R.

SAMPLE SIZE CONSIDERATIONS Assuming a larger heterogeneity in the intervention group of a factor of sqrt(2) (due to compliance and the dietary flexibility for the intervention) and with a distribution of 2:1 between intervention and standard diet, the relative effect size detectable on the change from baseline to the end of intervention should be 0.69 with n = 33 in the standard diet group and n = 66 in the intervention group to have a study power of 0.8 with a significance level of 0.05. Thus, assuming a 5% drop-off, the study needs 105 patients to be included. There will be stratification for neoadjuvant chemotherapy versus adjuvant chemotherapy.

INTERIM ANALYSIS IS NOT PLANNED Missing data will be handled according to the scoring instructions of the instruments or imputations.

Deviations from the statistical analysis plan will be reported in the final publications.

All analyses will be performed as intention-to-treat analyses.

Inclusion Criteria:

1. Signed written informed consent approved by the Ethical Review Board.

2. Age ≥ 18

3. Histologically confirmed unilateral adenocarcinoma of the breast, stage I-III, candidates for neoadjuvant chemotherapy followed by curative surgery, or adjuvant chemotherapy after curative surgery, according to the Danish Breast Cancer Group guidelines 48. The addition of trastuzumab and pertuzumab in HER2-positive tumors, or pembrolizumab in triple-negative tumors, per Danish Breast Cancer Group guidelines 48, is allowed as an adjunct to chemotherapy.

4. Good performance status (WHO performance status 0 or 1).

5. Willingness by the patient to undergo treatment and study-related procedures according to the protocol.

6. Have a smartphone or computer to which it is possible to receive emails (for food diary; www.myfood24.org)

7. Must be willing to restrict from probiotic (living microorganisms) supplements during the study. Fermented foods are allowed.

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Exclusion Criteria:

1. Clinical or radiological signs of metastatic disease.
2. History of other malignancy within the last 5 years, except for carcinoma in situ of the cervix or non-melanoma skin cancer.
3. Previous chemotherapy for cancer or other malignant disease.
4. Major altered digestive system disorders (e.g. gastric by-pass or Crohn's disease or ulcerative colitis)
5. Unable to communicate effectively in Danish or English.

When the clinical trial ends, the anonymized data will be made available to other researchers through public databases such as the Zenodo open data repository (CERN) or other equivalent databases.

when the clinical trial ends and data are mature, anticipated 1 January 2031

Zenodo open data repository (CERN) or other equivalent databases.

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