This study aims to evaluate the possible association between cancer cachexia and Gut Microbiota (GM) in patients with Breast Cancer (BC) cared at home.

Cancer cachexia is a multifactorial condition causing weight loss, muscle loss, and reduced physical strength in people with cancer. Moreover, the study will also analyze whether different stages of cachexia are linked to changes in the GM.

The study will also explore whether a supplementation with a probiotic mixture for twelve weeks is associated with changes in the GM and participants' nutritional and clinical condition.

The effect of the probiotic mixture (experimental group) will be compared with a placebo (control group) to evaluate whether changes in the GM and in cachexia-related measures differ between the two groups.

Participants will:

• Provide blood, stool, and urine samples to study the GM and biological markers.
• Answer questionnaires about symptoms and quality of life.
• Undergo assessments of nutritional status and body composition.

Participants eligible for the intervention will take either a probiotic mixture or a placebo for twelve weeks.

All study procedures will take place during home visits as part of the participants' regular home care program.

Cancer cachexia is a multifactorial syndrome defined by an ongoing loss of skeletal muscle mass that cannot be fully reversed by conventional nutritional support and leads to a progressive functional impairment. Moreover, cachexia is characterized by involuntary weight loss, reduced food intake, systemic inflammation, and metabolic alteration. Its pathophysiology is characterized by a negative protein and energy balance driven by a variable combination of reduced food intake and abnormal metabolism. It is common in people with advanced cancer and is associated with lower quality of life, poorer functional status, and shorter survival. Cachexia syndrome can develop progressively through various stages - precachexia, cachexia, and refractory cachexia and its severity can be classified according to degree of depletion of energy stores and body protein in combination with degree of ongoing weight loss. Refractory cachexia, which is considered no longer reversible, represents a very advanced stage associated with progressive worsening of quality of life and severe hypercatabolism, with an expected survival of less than three months.

The Gut Microbiota (GM) is increasingly recognized as a possible factor involved in the development and progression of cancer cachexia. Alterations in the GM, often referred to as dysbiosis, may contribute to systemic inflammation, intestinal barrier dysfunction, altered nutrient metabolism, and loss of muscle mass. Previous studies have shown that people with cancer cachexia may have specific alterations in GM, including differences in the abundance of certain bacterial groups and lower production of anti-inflammatory metabolites such as short-chain fatty acids.

The association between alterations in the GM and the development of cancer cachexia suggests that modifying the GM to correct cachexia-related dysbiosis may lead to new integrative approaches for the management of this highly burdensome condition.

Recent studies suggest that Breast Cancer (BC) is associated with GM dysbiosis and that changes in GM composition and function may play a crucial role in tumor development and progression. In these studies, patients with BC frequently show a different GM composition compared with healthy individuals, including specific alterations in certain bacterial taxa and a reduction in microbial alpha diversity. However, limited data are available on the relationship between GM and cachexia in people with BC, especially among those receiving home-based supportive and palliative care.

Considering that BC is one of the most common cancers among women assisted by the home supportive and palliative care program, it is particularly important to identify strategies that may improve the quality of life of these patients. A simple and feasible option to support the nutritional and functional status of these particularly vulnerable patients could be probiotic supplementation. At present, to our knowledge, no studies have evaluated the effect of probiotic supplementation on cachexia status in patients with BC.

This study was developed to better understand the role of the GM in the nutritional and functional condition of people with BC receiving home care and to explore whether modulation of the GM through a probiotic mixture may be associated with changes in cachexia-related measures.

The primary objective of the study is to evaluate whether GM composition, diversity, and function are associated with the stage of cachexia in people with BC receiving home care.

Secondary objectives include:

• Evaluate changes in GM composition, diversity, and function after 12 weeks in participants who received either a probiotic mixture or placebo.
• Evaluate the association between changes in GM composition and function and changes in cachexia status after 12 weeks in participants who received either a probiotic mixture or placebo.
• Evaluate inflammatory biomarkers and intestinal permeability biomarkers at baseline and after 12 weeks in relation to cachexia status in participants who received either a probiotic mixture or placebo.

All study procedures will be performed during home visits as part of the participants' regular home care program.

The study consists of two consecutive phases: an observational phase and an interventional phase.

I) Observational phase During the observational phase, all eligible participants (expected number: 200 patients) entering the home care program will undergo a baseline assessment. This phase is designed to evaluate the association between GM composition, diversity, and function and the stage of cachexia at baseline.

Participants will be classified into one of the following cachexia stages:

• No cachexia
• Precachexia
• Cachexia
• Refractory cachexia Cachexia stage will be defined using body weight loss, Body Mass Index, body composition, sarcopenia, oral intake, systemic inflammation, and Karnofsky Performance Status.

The observational phase aims to determine whether specific GM profiles are associated with different stages of cachexia at the entry and whether these profiles are related to inflammatory markers, intestinal permeability, symptoms, and quality of life.

Participants who do not have refractory cachexia and who have a life expectancy greater than six weeks will be eligible for the interventional phase.

II) Interventional phase The interventional phase is a randomized, double-blind, placebo-controlled study with two parallel groups. Approximately 140 participants are expected to enter the randomized interventional phase, with about 70 participants in each study group.

Participants will be randomly assigned in a 1:1 ratio to one of the following groups:

• Experimental group
• Control group Participants in the experimental group will take a probiotic mixture containing different strains of lactic acid bacteria and bifidobacteria orally twice daily for twelve weeks. Participants in the control group will receive a placebo in identical sachets for the same duration.

Neither the participants nor the study team performing the assessments will know the assigned intervention arm. Randomization will be centrally managed and stratified according to age and Karnofsky Performance Status to ensure balance between the groups.

Participants will undergo study assessments at three time points:

• Baseline
• Six weeks
• Twelve weeks At each assessment, clinical information, biological samples, and questionnaire data will be collected.

Clinical data collected during the study will include:

• Tumor stage and metastatic disease
• Previous and ongoing cancer treatments
• Pain treatment and other medications
• Karnofsky Performance Status
• Nutritional status
• Weight loss during the previous six months
• Body Mass Index
• Dietary intake
• Body composition and sarcopenia assessment
• Perceived symptom burden
• Quality of life Body composition and sarcopenia will be evaluated using bioimpedance analysis and handgrip strength measurement.

Participants will also complete questionnaires on symptoms and quality of life, including:

• Edmonton Symptom Assessment Scale
• EuroQol 5-Dimension questionnaire (EQ-5D) Blood, stool, and urine samples will be collected at baseline, six weeks, and twelve weeks.

Blood samples will be used to measure:

• Inflammatory markers
• Cytokines
• Markers of intestinal permeability

Stool samples will be used to evaluate:

• GM composition
• GM diversity
• Fecal calprotectin
• Functional and metabolomic profiles of the GM Urine samples will be collected for metabolomic analyses.

GM analysis will include:

• 16S Ribosomal RNA (16S rRNA) sequencing
• Shotgun metagenomic sequencing
• Alpha diversity indices
• Beta diversity analyses
• Relative abundance of major microbial groups
• Functional and metabolomic profiling The study will also evaluate biomarkers of systemic inflammation and intestinal permeability, including inflammatory cytokines, lipopolysaccharide-related markers, zonulin, intestinal fatty acid-binding protein, and other serum and fecal biomarkers.

Participants may continue to receive their prescribed oncologic treatments, including chemotherapy, radiotherapy, and hormonal therapy, provided that these treatments are documented and monitored during the trial.

Participants may also continue or start supportive treatments, including antiemetics, pain medications, and other drugs used to manage symptoms related to cancer. Participants who require antibiotic treatment or the initiation of total artificial nutrition during the 12-week trial period will discontinue the intervention phase of the study.

Inclusion Criteria

• Female patients with Breast Cancer (BC)
• Age ≥ 18 years
• Able to understand the study objectives and provide written informed consent
• Able to speak and understand Italian

Exclusion Criteria

• Antibiotic treatment within the previous 2 weeks
• Use of probiotic supplements within the previous month
• Artificial nutrition (enteral or parenteral)
• Severe dysphagia, intestinal obstruction, or impaired gastrointestinal function
• Diagnosis of dementia or cognitive impairment preventing understanding of the study information and/or provision of informed consent
• Severe organ failure
• Refusal to participate For the interventional phase: refractory cachexia and/or life expectancy of less than 6 weeks

Individual participant data will not be shared due to privacy and confidentiality considerations related to sensitive clinical and biological data.