Short summary

The MEDITERRACARE study investigates whether adherence to the Mediterranean diet can influence the microbiota and 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.

Rationale

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 deaths1. In Denmark, there are 5000 new cases, and 1.050 deaths annually. In men, breast cancer is rare with an average of 42 new cases annually in Denmark2 Oncological treatment: The oncological treatment of early breast cancer encompasses neoadjuvant (pre-surgery) and adjuvant (post-surgery) treatments. Adjuvant therapy is given to the patients to minimize the risk of recurrence. Neoadjuvant treatment is recommended for patients with large tumors or nodal spread or who are technically inoperable and can change the surgical options to less invasive surgery. The oncology therapy is based on the patient's tumor characteristics (Estrogen receptor status, human epidermal growth factor receptor 2 (HER-2) status, and mRNA expression of 50 genes (PAM50) molecular subtype) (NK) and menopausal status, and is thus tailored to the individual patient. 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 3. HER2-targeted therapies, such as trastuzumab, are specifically used for HER2-positive breast cancers in conjunction with chemotherapy and have significantly improved outcomes 4. 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) 5. However, chemotherapy-based treatments are faced with treatment-related side effects of fatigue and cognitive impairment that reduce quality of life6. Socioeconomic disparities: 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, patients 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 7. Cancer inequalities at the individual level are predominantly shaped by systemic social determinants of health 8. 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 9. Thus, it is important to note that social inequality in cancer survival is a critical problem, and patients with little education, low income, or living alone have poorer survival than more highly educated cancer patients, with higher income, and living in a relationship 9-11. This applies also to breast cancer patients, despite women with higher socioeconomic status having significantly higher breast cancer incidence 8, 12. The gut microbiome: The human gut hosts approximately 38 trillion cells called the microbiota; the human gut microbiome consists of the genes these cells harbor 13. In the 2022 update of the Hallmarks of Cancer, the microbiome was introduced as a new important hallmark of cancer 14. Importantly, the microbiome modulates four other hallmarks: tumor growth, tumor-promoting inflammation, immune evasions, and genomic instability; additionally, the microbiome modulates therapy resistance 14. The implication is, therefore, that the microbiome is a critical link in cancer development, progression, and treatment response. In a recently published review, our research group evaluated the data from thirty-one clinical trials measuring data on the microbiome and clinical outcomes in various types of cancer patients on treatment with single-agent immunotherapy or immunotherapy combination treatments15. The studies showed that specific types of gut bacteria present in the gut at baseline, including Akkermansia mucinifila and Faecalibacterium prausnitzii, but also bacterial diversity was linked to clinical outcomes such as response rate, progression-free survival, or overall survival15. Similarly, in two studies, patient reported intake of dietary fiber and plant foods at baseline showed a link between high intake of dietary fiber and many plant foods and cancer outcomes15. However, there are currently no data from intervention studies with high fiber diet and plant foods, studying the microbiome and cancer outcomes. 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 16. 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 17. 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 (dislike of the food by the patients or their families, especially children)17. The Mediterranean diet has several effects, including lipid-lowering 18 and anti-inflammatory actions, reducing chronic inflammation 17,19, 20. Higher adherence to the Mediterranean diet was associated with a 23% lower risk of all-cause mortality 21. The Mediterranean diet was feasible and safe for adults with cancer 22. The Mediterranean diet has been associated with a reduced risk of breast cancer development 23. Its impact on survival outcomes after breast cancer diagnosis remains an area of active investigation 24, 25. Changes in the microbiome composition were observed already after four weeks of the Mediterranean diet 26. Moreover, bacteria in the gut ferment dietary fiber to short-chain fatty acids (e.g., acetate, propionate, and butyrate), factors that upregulate immune functions 27, 28. 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.29 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 (NK) 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. Vitamin D: has recently been associated with changes in the microbiome and cancer outcomes30-32. 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, ultraviolet (UV) exposure is insufficient for vitamin D production for large parts of the year. Measurements of vitamin D will be performed at baseline and cycle 6, and daily supplementation with 50 micrograms of vitamin D is encouraged.

Description of the cohort

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 population will consist 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. Inclusion Criteria: • Signed written informed consent approved by the Ethical Review Board. • Age ≥ 18 • 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 34. The addition of trastuzumab and pertuzumab in HER2-positive tumors, or pembrolizumab in triple-negative tumors, per Danish Breast Cancer Group guidelines 34, is allowed as an adjunct to chemotherapy. • Good performance status (WHO performance status 0 or 1). • Willingness by the patient to undergo treatment and study-related procedures according to the protocol. • Have a smartphone or computer to which it is possible to receive emails (for food diary; www.myfood24.org) • Must be willing to restrict from probiotic (living microorganisms) supplements during the study. Fermented foods are allowed. Exclusion Criteria: • Clinical or radiological signs of metastatic disease. • History of other malignancy within the last 5 years, except for carcinoma in situ of the cervix or non-melanoma skin cancer. • Previous chemotherapy for cancer or other malignant disease. • Major altered digestive system disorders (e.g. gastric by-pass, Crohn's disease, or ulcerative colitis) • Unable to communicate effectively in Danish or English.

Data and biological material

1 The WHOQOL-100 and baseline dietary and lifestyle questionnaires will be provided electronically to patients. 2 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, before cycles 2, 4, 6, or in case of early discontinuation. 3 Microbiome evaluation will use Oxford Nanopore Technology 16S sequencing on fecal samples, including quality control, sequence alignment, and taxonomic classification 4 Metabolome assessment will detect short-chain fatty acids (SCFAs) in patient stool and plasma samples using ultrahigh-performance liquid chromatography-tandem mass spectrometry 35. 5 Adhesion to the diet will be measured via food diaries (www.myfood24.org). The software provides feedback to the user about reaching the daily fiber intake goals, thereby nudging the participant to reach the target. Patients will also fill out a paper record of different plant foods eaten every week. 6 Quantification of Cluster of Differentiation (CD)4+ T-cells, CD8+ T-cells, CD19+ B cells, and 56+ Natural Killer cells will be performed by flow cytometry. 7 Hematology (hemoglobin, white blood cell count, absolute neutrophils, monocytes, lymphocytes, and platelet count) C-reactive protein (CRP)Programmed, Vitamin D status will be measured at initiation of the study and cycle 6 8 GlycA and lipoproteins will be examined using Nuclear Magnetic Resonance (NMR)-technique as an expression of metabolic improvements 9 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, cycle 2, 4, and 6 of 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. 10 Archived tumor tissues 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. Primary tumor tissue will be analyzed for tumor-infiltrating immune cells.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. The median cell density of immune cells per mm 2 tumor tissue will be calculated.

Collaborating researchers and departments

Department of Oncology, University Hospital of Southern Denmark, Vejle

• Lone Marie Volmer, MD

Department of Oncology, Hospital of Southern Jutland, Sønderborg

• Erik Hugger jakobsen, MD

Department of Oncology, Odense University Hospital

• Søren Cold, MD

Department of Public Health, University of Southern Denmark

• Professor Pernille Tanggaard Andersen

Department of Chemistry and Bioscience, University of Aalborg, Esbjerg

• Ass. ProfessorJens Laurids Sørensen, PhD

Department of Pathology, Aarhus University Hospital

• Professor Torben Steiniche, MD

Department of Food Science, University of Copenhagen

• Professor Morten Arendt Rasmussen

Department of Oncology, Copenhagen University Hospital

• Professor Bent Ejlertsen, MD

Department of Clinical Immunology and Biochemistry, Vejle Hospital

• Line Nederby, PhD

Publications associated with the project

Gamrath L, Pedersen TB, Møller MV, Volmer LM, Holst-Christensen L, Vestermark LW, Donskov F. Role of the Microbiome and Diet for Response to Cancer Checkpoint Immunotherapy: A Narrative Review of Clinical Trials. Curr Oncol Rep 2025 27:45-58