Impact of Dietary Intervention on Tumor Immunity: the DigesT Trial

This trial aims to assess the immunological and metabolic changes induced by the Fasting Mimicking Diet (FMD) in the pre-operative and post-operative setting in breast cancer and melanoma patients. Three cohorts of patients will be enrolled: 1) Cohort A: patients with resectable breast cancer (cT1N0M0 stage or cT1cN1M0-cT2cN0M0 stages not requiring pre-operative systemic treatment at the judgment of the investigator) who are candidate to curative surgery; 2) Cohort B: patients with malignant melanoma patients candidate to dissection of the lymph node basin because of a positive sentinel lymph node (stage IIIA-IIIB-IIIC); 3) Cohort C: patients with resected malignant melanoma (including radicalization and, in case, lymph node dissection) who are not candidate to any adjuvant treatment, but only to clinical and radiological follow-up (stage IIB-IIC). Patients in cohorts A and B will undergo one 5-days FMD cycle about 13-15 days before surgical removal of primary tumor (breast) or lymph nodes (breast, melanoma). Patients in cohort C will undergo 4 consecutive FMD cycles every 28 days, starting one month after surgery.

Preclinical evidences suggest that reducing the concentration of blood metabolites and growth factors reduces the in vivo growth of several tumor models, while protecting normal tissues from the cytotoxic effects of chemotherapeutical treatments. In recent years, a plant-based, calorie-restricted, low-carbohydrate, low-protein diet, also known as Fasting Mimicking Diet (FMD), has been proposed as a potential anticancer dietary intervention. The FMD is safe when administered cyclically (every 21-28 days) to healthy volunteers, and is capable of significantly reducing the concentration of plasma glucose, serum insulin and IGF-1, while increasing levels of plasma IGFBPs and ketone bodies. The FMD has been shown to inhibit the in vivo growth of several tumor models, including breast cancer and melanoma mice models. The anticancer effects of the FMD are likely mediated by two concomitant mechanisms: 1) one direct anticancer effect that is mediated by the inhibition of energy production and anabolic pathways, such as protein and fatty acid synthesis, in cancer cells; 2) one indirect effect that is mediated by the activation of antitumor immunity, with the result of enhanced tumor infiltration by cytotoxic CD8+ T-lymphocytes and reduced infiltration by immunosuppressive populations. According to the currently accepted model, the anticancer and immunomodulatory effects of the FMD mostly derive from the reduction of circulating glucose, insulin and IGF-1 levels, and a parallel increase of ketone body and IGF-1 binding protein concentration. However, recent observations in healthy volunteers and cancer patients, suggest that FMD-mediated changes in many other metabolites, such as specific amino acids or fatty acids, could contribute to the cell-autonomous or immune-mediated anticancer effects of the FMD. While the study of the effects of the FMD in combination with standard treatments (e.g. chemotherapy, molecular targeted therapy) in advanced cancers represents the final objective of the ongoing studies, fully uncovering the metabolic and immunological effects of the FMD alone is essential to design future combination studies. From this perspective, the pre- and post-operative clinical settings in cancer patients who are not candidate to other medical treatments represent an ideal context to assess the effects of the FMD without other confounding factors. This trial primarily aims to assess the immunological and metabolic changes induced by the FMD in the pre-operative and post-operative setting in breast cancer and melanoma patients. Three cohorts of patients will be enrolled: 1) Cohort A: patients with resectable breast cancer (cT1N0M0 stage or cT1cN1M0-cT2cN0M0 stages not requiring pre-operative systemic treatment at the judgment of the investigator) who are candidate to curative surgery; 2) Cohort B: patients with malignant melanoma patients candidate to dissection of the lymph node basin because of a positive sentinel lymph node (stage IIIA-IIIB-IIIC); 3) Cohort C: patients with resected malignant melanoma (including radicalization and, in case, lymph node dissection) who are not candidate to any adjuvant treatment, but only to clinical and radiological follow-up (stage IIB-IIC). Patients in cohorts A and B will undergo one 5-days FMD cycle about 13-15 days before surgical removal of primary tumor (breast) or lymph nodes (breast, melanoma). Patients in cohort C will undergo 4 consecutive FMD cycles every 28 days, starting one month after surgery.

Eligibility Criteria:
Inclusion Criteria:

1. Age ≥ 18 and ≤ 75 years.
2. Evidence of a personally signed and dated informed consent document (ICD) indicating that the patient has been informed of all pertinent aspects of the study before enrollment and FMD prescription.
3. Willingness and ability to comply with the FMD protocol, the scheduled visits, treatment plans, laboratory tests and other procedures.
4. Histologically confirmed diagnosis of invasive breast cancer candidate to curative surgery (Cohort A), or resected malignant melanoma requiring dissection of the regional lymph node basin for sentinel lymph node involvement (Cohort B), or malignant melanoma treated with curative surgery (including, in case, lymph node removal and lymph node dissection) (Cohort C). For breast cancer patients, any biological subgroup (including estrogen receptor-positive, HER2-positive, triple-negative breast cancer) will be admitted; HER2-positive tumors will be defined on the basis of an IHC score of 3, or a score of 2 with ISH evaluation indicative of gene amplification.
5. Availability of archival FFPE tissue blocks of primary breast cancer (Cohort A) or melanoma (Cohort B, Cohort C).
6. Presence of an Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1.
7. Presence of adequate bone marrow and organ function as defined by the following laboratory values:

   * ANC ≥ 1.5 x 109/l
   * platelets ≥ 100 x 109/l
   * hemoglobin ≥ 9.0 g/dl
   * calcium (corrected for serum albumin) within normal limits or ≤ grade 1 according to NCI-CTCAE version 4.03 if not clinically significant
   * potassium within the normal limits, or corrected with supplements
   * creatinine \< 1.5 ULN
   * blood uric acid \< 10 mg/dl
   * ALT and AST ≤ 2.5 x ULN
   * total bilirubin \< ULN except for patients with Gilbert syndrome who may only be included in the total bilirubin is \< 3.0 x ULN or direct bilirubin \< 1.5 x ULN
   * Albumin \> 3 g/dL
8. Fasting glucose ≤ 200 mg/dl.
9. Total Cholesterol ≤ 300 mg/dl.
10. Triglycerides ≤ 300 mg/dl.
11. Female patients of childbearing potential must agree to sexual abstinence or to use two highly effective method of contraception throughout the study and for at least 30 days after the end of the FMD. Abstinence is only acceptable if it is in line with the preferred and usual lifestyle of the patient. Examples of contraceptive methods with a failure rate of \< 1% per year include tubal ligation, male sterilization, hormonal implants, established, proper use of combined oral or injected hormonal contraceptives, and certain intrauterine devices. Alternatively, two methods (e.g., two barrier methods such as a condom and a cervical cap) may be combined to achieve a failure rate of \< 1% per year. Barrier methods must always be supplemented with the use of a spermicide. A patient is of childbearing potential if, in the opinion of the Investigator, she is biologically capable of having children and is sexually active.

Female patients are not of childbearing potential if they meet at least one of the following criteria:

* Have undergone a documented hysterectomy and/or bilateral oophorectomy
* Have medically confirmed ovarian failure
* Achieved post-menopausal status, defined as: (≥ 12 months of non-therapy-induced amenorrhea) or surgically sterile (absence of ovaries) and have a serum FSH level within the laboratory's reference range for postmenopausal females.

Exclusion Criteria:

1. Prior systemic treatment for breast cancer or melanoma.
2. Diagnosis of a concurrent malignancy other than breast cancer or melanoma, or malignancy other than breast cancer or melanoma diagnosed within 5 years of treatment enrollment, with the exception of adequately treated, basal or squamous cell carcinoma, non-melanomatous skin cancer or curatively resected cervical cancer.
3. Body Mass Index (BMI) \< 20 Kg/m2.
4. Anamnesis of alcohol abuse.
5. Unintentional weight loss ≥ 5% in the last three months, unless the patient has a BMI \> 25 Kg/m2 at study enrollment. Intentional weight loss is permitted if \< 10% in the last three months and patient BMI is \> 22 kg/m2.
6. Severe heart, liver, pulmonary, kidney comorbidities.
7. Current status of pregnancy or lactation, where pregnancy is defined as the state of a female after conception and until the termination of gestation, confirmed by a positive hCG laboratory test (\> 5 mIU/mL).
8. Active HBV or HCV infection.
9. Severe infections within 4 weeks prior to FMD initiation, including, but not limited to, hospitalization for complications of infection, bacteremia, or severe pneumonia.
10. Active autoimmune diseases that require systemic treatment (i.e. with use of disease modifying agents, corticosteroids or immunosuppressive drugs).
11. History of recent diagnosis of hypothyroidism for which replacement therapy (eg., thyroxine) and blood endocrine profile are not stabilized yet.
12. Established diagnosis of diabetes mellitus type I or diabetes mellitus type II that requires pharmacological treatment (including, but not limited to, insulin, insulin secretagogues and metformin).
13. Severe impairment of the gastrointestinal (GI) function or GI disease that may alter the digestion and absorption of nutrients during the re-feeding phase (e.g. active ulcerative diseases of the stomach or intestine, uncontrolled nausea, vomiting, diarrhea, malabsorption syndrome, or small bowel resection).
14. Known history of Human Immunodeficiency Virus (HIV) infection.
15. Clinically significant heart disease and/or recent cardiac events including:

    * history of angina pectoris, coronary artery bypass graft (CABG), symptomatic pericarditis, or myocardial infarction within 12 months prior to the start of study treatment;
    * history of documented congestive heart failure (NYHA III-IV);
    * documented cardiomyopathy.
16. History of cardiac arythmias, (e.g. ventricular tachycardia, chronic atrial fibrillation), complete left bundle branch block, high grade AV block (e.g. bifascicular block, Mobitz type II and third degree AV block), supraventricular, nodal arrhythmias, or conduction abnormality in the previous 12 months.
17. Uncontrolled hypertension defined by a Systolic Blood Pressure (SBP) ≥ 160 mmHg and/or Diastolic Blood Pressure (DBP) ≥ 100 mmHg, with or without anti-hypertensive medication.
18. Known reduction of left-ventricular ejection fraction (LVEF) to less than 50%, as assessed by multigated radionuclide scintigraphic scan (MUGA) or echocardiography.
19. Previous episodes of symptomatic hypotension causing unconsciousness.
20. Baseline fasting plasma glucose ≤ 65 mg/dl.
21. Ongoing therapy with systemic corticosteroids, or systemic corticosteroid therapy ≤ 2 weeks before study enrollment, or who have not recovered from side effects of such treatment. The following uses of corticosteroids are permitted: topical applications (e.g. for rash), inhaled sprays (e.g. for obstructive airways diseases), eye drops.
22. Any serious medical or psychiatric illness that in the assessment of the investigator renders the patient not suitable for participation in this clinical study.