Effects of Acute and Chronic Exercise on Myeloid-Derived Suppressor Cells in Melanoma Patients (Agility)

This study is designed to investigate the effects of ketogenic milieu induced by acute exercise as well as the effects of recurrent exercise bouts on functional status and the accumulation in peripheral blood of MDSCs and the consequent balance on antitumor immunity in melanoma patients.

The screening period starts once a patient has provided written informed consent to participate in the study and ends on the day of exercise initiation. Screening assessments have to be done within 28 days prior to exercise initiation. The informed consent will adhere to IRB/ERC requirements, applicable laws and regulations. After the sign of an informed consensus, patients will be assigned to a specific exercise program. The first series of patients will be assigned to the Acute Exercise Program (AEP group), the second series to the Chronic Exercise Program (CEP group). The protocol will be implemented in the time between diagnosis and surgical intervention without any delay in the therapeutic pathway. Basal evaluation: patient's life style will be investigated using the short version of Physical Activity Questionnaire (Italian version) and MeDiet questionnaire. Age, weight and eight will be registered for all patients. The Lean Body Mass will be measured by Dual Energy X-ray Absorptiometry (DEXA). A cardiorespiratory test will be performed to calculate an appropriate exercise intensity and subsequently the Six Minutes WalkingTest and the Comfortable Walking Speed test will be run before exercise protocol. In the CEP group the DEXA and the walking tests will be repeated after the end of exercise program. Exercise prescription: the physical exercise consists in walking at a brisk pace. In order to produce a constant metabolic stress in patients with different aerobic power, walking intensity will be prescribed in accordance with the method of the Heart Rate Reserve (HRR), ie difference between resting heart rate (HR-resting) and the heart rate corresponding to muscle's maximal oxygen uptake (HR-max). This prescribing method is based on the linear relationship between heart rate reserve and muscle workload calculated as a percentage of the difference between resting and maximum muscle oxygen consumption (VO2R%). The training intensity will be set to the 30% HRR, the lowest intensity that produces a training effect. To minimize the impact of the research protocol on the patient's therapeutic path, the investigators refrain from the execution of a Cardio Pulmonary Exercise Test and therefore opted for calculating the HR-max using the ACSM formula. Given the entry criteria of this study the investigators believe this choice will minimally affect the exercise-dose prescription and study results. Acute Exercise Protocol (AEP): The acute program consists in a single walking session on a treadmill lasting 80 minutes. The exercise session will begin with a 10-minute warm-up at the patient's self selected comfortable speed. On the ending of warm up, treadmill speed will be increased until reaching target HRR zone. Then the treadmill speed will be maintained stable throughout the exercise session. Cardiac workload will be monitored by a Sport-watch (Garmin Forerunner 35) and the walking biomechanics by a foot sensor (Garmin Running Dynamic Pod Sensore). Chronic Exercise Protocol (CEP): The chronic program consists in a walking session on a treadmill lasting 80 minutes (depending on the AEP results), repeated three time a week for three weeks. The walking intensity will be defined during a treadmill test performed before the beginning of the exercise program. Each exercise session will begin with a 10-minute warm-up at the patient's self selected comfortable speed. On the ending of warm up, patients will increase treadmill speed until reaching target HRR zone. Then the treadmill speed will be maintained stable throughout the exercise session. The first training session will be organized in hospital with supervision of research staff. Cardiac workload will be monitored by a Sport-watch(Garmin Forerunner 35) and walking biomechanics by a foot sensor (Garmin Running Dynamic Pod Sensore). Diet intervention: Modifications occurring during AEP strictly depend on the baseline metabolic conditions. For this reason, patients must remain fasting for 4 hours before the workout and avoid glucose rich foodies for 120 minutes after the end of exercise session in CEP group. During the exercise session patients will have free access to water only. All patients will complete a food diary during the study period.

Pre/post intervention, two-arms, sequential study. Patients with newly diagnose of melanoma are eligible.The study will enroll a total of 20 patients (10 in the AEP and 10 in the CEP), who will be enrolled approximately in 12-13 months.The first series of patients will be assigned to the Acute Exercise Program (AEP group), the second series to the Chronic Exercise Program (CEP group). The final endpoint is to verify whether short-term physical exercise can represent a feasible and safe tool for re-setting antitumor immunity in early melanoma patients. Investigators will analyze how the acute physical exercise modifies systemic immunity and biochemical/metabolic parameters. Specifically, Investigators will verify whether the exercise will modify:1 the immunological profiling of blood including frequency and function of MDSC, together with the concomitant status of antitumor immunity; 2 blood concentration of biochemical and metabolic parameters related to physical exercise and immunity

First series of patients (10 pts) will be assigned to the Acute Exercise Protocol Group (AEP Group). The acute program consists in a single walking session on a treadmill lasting 80 minutes. The exercise session will begin with a 10-minute warm-up at the patient's self selected comfortable speed. On the ending of warm up, treadmill speed will be increased until reaching target HRR zone. Then the treadmill speed will be maintained stable throughout the exercise session. Cardiac workload will be monitored by a Sport-watch (Garmin Forerunner) and the walking biomechanics by a foot sensor (Garmin Running Dynamic Pod Sensore).

Second series of patients (10 pts) will be assigned to the Chronic Exercise Protocol Group (CEP Group). The chronic program consists in a walking session on a treadmill lasting 80 minutes (depending on the AEP results), repeated three time a week for three weeks. The walking intensity will be defined during a treadmill test performedbefore the beginning of the exercise program. Each exercise session will beginwith a 10-minute warm-up at the patient's self selected comfortable speed. On the ending of warm up, patients will increase treadmill speed until reaching target HRR zone.Then the treadmill speed will be maintained stable throughout the exercise session. The first training session will be organized in hospital with supervision of research staff. Cardiac workload will be monitored by a Sport-watch (Garmin Forerunner) and walking biomechanics by a foot sensor (Garmin Running Dynamic Pod Sensore).

The chronic program consists in a walking session on a treadmill lasting 80 minutes (depending on the AEP results), repeated three time a week for three weeks.

Quantification of the immune cell subsets (to determine if occur the re-setting antitumor immunity by short-term physical exercise)

Immune cells will be analyzed for frequency and function by 10-color flow cytometry to test the reciprocal balance of protumor vs antitumor immune subsets. The analysis, based in the application of multiple panels of different antibodies, will be performed on peripheral blood mononuclear cells (PBMC) isolated from peripheral blood.

Quantification of the immune cell subsets (to determine if occur the re-setting antitumor immunity by short-term physical exercise)

Immune cells will be analyzed for frequency and function by 10-color flow cytometry to test the reciprocal balance of protumor vs antitumor immune subsets. The analysis, based in the application of multiple panels of different antibodies, will be performed on peripheral blood mononuclear cells (PBMC) isolated from peripheral blood.

Quantification of the immune cell subsets (to determine if occur the re-setting antitumor immunity by short-term physical exercise)

Immune cells will be analyzed for frequency and function by 10-color flow cytometry to test the reciprocal balance of protumor vs antitumor immune subsets. The analysis, based in the application of multiple panels of different antibodies, will be performed on peripheral blood mononuclear cells (PBMC) isolated from peripheral blood.

Quantification of the immune cell subsets (to determine if occur the re-setting antitumor immunity by short-term physical exercise)

Immune cells will be analyzed for frequency and function by 10-color flow cytometry to test the reciprocal balance of protumor vs antitumor immune subsets. The analysis, based in the application of multiple panels of different antibodies, will be performed on peripheral blood mononuclear cells (PBMC) isolated from peripheral blood.

Quantification of the immune cell subsets (to determine if occur the re-setting antitumor immunity by short-term physical exercise)

Immune cells will be analyzed for frequency and function by 10-color flow cytometry to test the reciprocal balance of protumor vs antitumor immune subsets. The analysis, based in the application of multiple panels of different antibodies, will be performed on peripheral blood mononuclear cells (PBMC) isolated from peripheral blood.

Quantification of the immune cell subsets (to determine if occur the re-setting antitumor immunity by short-term physical exercise)

Immune cells will be analyzed for frequency and function by 10-color flow cytometry to test the reciprocal balance of protumor vs antitumor immune subsets. The analysis, based in the application of multiple panels of different antibodies, will be performed on peripheral blood mononuclear cells (PBMC) isolated from peripheral blood.

Quantification of the immune cell subsets (to determine if occur the re-setting antitumor immunity by short-term physical exercise)

Immune cells will be analyzed for frequency and function by 10-color flow cytometry to test the reciprocal balance of protumor vs antitumor immune subsets. The analysis, based in the application of multiple panels of different antibodies, will be performed on peripheral blood mononuclear cells (PBMC) isolated from peripheral blood.

Quantification of the immune cell subsets (to determine if occur the re-setting antitumor immunity by short-term physical exercise)

Immune cells will be analyzed for frequency and function by 10-color flow cytometry to test the reciprocal balance of protumor vs antitumor immune subsets. The analysis, based in the application of multiple panels of different antibodies, will be performed on peripheral blood mononuclear cells (PBMC) isolated from peripheral blood.

Transcriptional Immune cell profiling (to determine if occur the re-setting antitumor immunity by short-term physical exercise)

CD14+, CD15+ and total immune cells will be immunosorted directly from whole blood (3 ml) and frozen for the targeted expression profiling of signaling pathways and functional patterns (Nanostring or customized PCR cards).

Transcriptional Immune cell profiling (to determine if occur the re-setting antitumor immunity by short-term physical exercise)

CD14+, CD15+ and total immune cells will be immunosorted directly from whole blood (3 ml) and frozen for the targeted expression profiling of signaling pathways and functional patterns (Nanostring or customized PCR cards).

Transcriptional Immune cell profiling (to determine if occur the re-setting antitumor immunity by short-term physical exercise)

CD14+, CD15+ and total immune cells will be immunosorted directly from whole blood (3 ml) and frozen for the targeted expression profiling of signaling pathways and functional patterns (Nanostring or customized PCR cards).

Transcriptional Immune cell profiling (to determine if occur the re-setting antitumor immunity by short-term physical exercise)

CD14+, CD15+ and total immune cells will be immunosorted directly from whole blood (3 ml) and frozen for the targeted expression profiling of signaling pathways and functional patterns (Nanostring or customized PCR cards).

Transcriptional Immune cell profiling (to determine if occur the re-setting antitumor immunity by short-term physical exercise)

CD14+, CD15+ and total immune cells will be immunosorted directly from whole blood (3 ml) and frozen for the targeted expression profiling of signaling pathways and functional patterns (Nanostring or customized PCR cards).

Transcriptional Immune cell profiling (to determine if occur the re-setting antitumor immunity by short-term physical exercise)

CD14+, CD15+ and total immune cells will be immunosorted directly from whole blood (3 ml) and frozen for the targeted expression profiling of signaling pathways and functional patterns (Nanostring or customized PCR cards).

Transcriptional Immune cell profiling (to determine if occur the re-setting antitumor immunity by short-term physical exercise)

CD14+, CD15+ and total immune cells will be immunosorted directly from whole blood (3 ml) and frozen for the targeted expression profiling of signaling pathways and functional patterns (Nanostring or customized PCR cards).

Transcriptional Immune cell profiling (to determine if occur the re-setting antitumor immunity by short-term physical exercise)

CD14+, CD15+ and total immune cells will be immunosorted directly from whole blood (3 ml) and frozen for the targeted expression profiling of signaling pathways and functional patterns (Nanostring or customized PCR cards).

In plasma we will quantify pH, Lactate, Ketone bodies, Free Fatty Acids, Glucose and Insulin serum levels that will be combined to report the metabolic parameters

In serum we will quantify ACTH, cortisol, epinephrine and norepinephrine concentrations that will be combined to report the hormonal parameters

In blood we will quantify IL-6, CRP, inflammatory cito/chemokines such as IL8, CCL2, IFN, IL10, IL1b, TNFa, TGFb that will be combined to report the inflammatory parameters

We will analyze the Lipidomics and amino acid profiles by laboratory test that will be combined to report the composition of cell membranes

Whole blood cell counts will be also performed; this parameter is characterized by the absolute number as well as relative ratio.

In plasma we will quantify pH, Lactate, Ketone bodies, Free Fatty Acids, Glucose and Insulin serum levels that will be combined to report the metabolic parameters

In serum we will quantify ACTH, cortisol, epinephrine and norepinephrine concentrations that will be combined to report the hormonal parameters

In blood we will quantify IL-6, CRP, inflammatory cito/chemokines such as IL8, CCL2, IFN, IL10, IL1b, TNFa, TGFb that will be combined to report the inflammatory parameters

We will analyze the Lipidomics and amino acid profiles by laboratory test that will be combined to report the composition of cell membranes

Whole blood cell counts will be also performed; this parameter is characterized by the absolute number as well as relative ratio.

In plasma we will quantify pH, Lactate, Ketone bodies, Free Fatty Acids, Glucose and Insulin serum levels that will be combined to report the metabolic parameters

In serum we will quantify ACTH, cortisol, epinephrine and norepinephrine concentrations that will be combined to report the hormonal parameters

In blood we will quantify IL-6, CRP, inflammatory cito/chemokines such as IL8, CCL2, IFN, IL10, IL1b, TNFa, TGFb that will be combined to report the inflammatory parameters

We will analyze the Lipidomics and amino acid profiles by laboratory test that will be combined to report the composition of cell membranes

Whole blood cell counts will be also performed; this parameter is characterized by the absolute number as well as relative ratio.

In plasma we will quantify pH, Lactate, Ketone bodies, Free Fatty Acids, Glucose and Insulin serum levels that will be combined to report the metabolic parameters

In serum we will quantify ACTH, cortisol, epinephrine and norepinephrine concentrations that will be combined to report the hormonal parameters

In blood we will quantify IL-6, CRP, inflammatory cito/chemokines such as IL8, CCL2, IFN, IL10, IL1b, TNFa, TGFb that will be combined to report the inflammatory parameters

We will analyze the Lipidomics and amino acid profiles by laboratory test that will be combined to report the composition of cell membranes

Whole blood cell counts will be also performed; this parameter is characterized by the absolute number as well as relative ratio.

In plasma we will quantify pH, Lactate, Ketone bodies, Free Fatty Acids, Glucose and Insulin serum levels that will be combined to report the metabolic parameters

In serum we will quantify ACTH, cortisol, epinephrine and norepinephrine concentrations that will be combined to report the hormonal parameters

In blood we will quantify IL-6, CRP, inflammatory cito/chemokines such as IL8, CCL2, IFN, IL10, IL1b, TNFa, TGFb that will be combined to report the inflammatory parameters

We will analyze the Lipidomics and amino acid profiles by laboratory test that will be combined to report the composition of cell membranes

Whole blood cell counts will be also performed; this parameter is characterized by the absolute number as well as relative ratio.

In plasma we will quantify pH, Lactate, Ketone bodies, Free Fatty Acids, Glucose and Insulin serum levels that will be combined to report the metabolic parameters

In serum we will quantify ACTH, cortisol, epinephrine and norepinephrine concentrations that will be combined to report the hormonal parameters

In blood we will quantify IL-6, CRP, inflammatory cito/chemokines such as IL8, CCL2, IFN, IL10, IL1b, TNFa, TGFb that will be combined to report the inflammatory parameters

We will analyze the Lipidomics and amino acid profiles by laboratory test that will be combined to report the composition of cell membranes

Whole blood cell counts will be also performed; this parameter is characterized by the absolute number as well as relative ratio.

In plasma we will quantify pH, Lactate, Ketone bodies, Free Fatty Acids, Glucose and Insulin serum levels that will be combined to report the metabolic parameters

In serum we will quantify ACTH, cortisol, epinephrine and norepinephrine concentrations that will be combined to report the hormonal parameters

In blood we will quantify IL-6, CRP, inflammatory cito/chemokines such as IL8, CCL2, IFN, IL10, IL1b, TNFa, TGFb that will be combined to report the inflammatory parameters

We will analyze the Lipidomics and amino acid profiles by laboratory test that will be combined to report the composition of cell membranes

Whole blood cell counts will be also performed; this parameter is characterized by the absolute number as well as relative ratio.

In plasma we will quantify pH, Lactate, Ketone bodies, Free Fatty Acids, Glucose and Insulin serum levels that will be combined to report the metabolic parameters

In serum we will quantify ACTH, cortisol, epinephrine and norepinephrine concentrations that will be combined to report the hormonal parameters

In blood we will quantify IL-6, CRP, inflammatory cito/chemokines such as IL8, CCL2, IFN, IL10, IL1b, TNFa, TGFb that will be combined to report the inflammatory parameters

We will analyze the Lipidomics and amino acid profiles by laboratory test that will be combined to report the composition of cell membranes

Whole blood cell counts will be also performed; this parameter is characterized by the absolute number as well as relative ratio.

Capillary sampling (prick sampling) to analysis metabolic parameters at systemic blood level will be performed during the first, in-hospital, training session and further will be continued at home, self-managed by the patient who will have to measure glycaemia and ketones every morning (finger prick sampling) and after each training sessions (earlobe prick sampling).

Capillary sampling (prick sampling) to analysis metabolic parameters at systemic blood level will be performed during the first, in-hospital, training session and further will be continued at home, self-managed by the patient who will have to measure glycaemia and ketones every morning (finger prick sampling) and after each training sessions (earlobe prick sampling).

Capillary sampling (prick sampling) to analysis metabolic parameters at systemic blood level will be performed during the first, in-hospital, training session and further will be continued at home, self-managed by the patient who will have to measure glycaemia and ketones every morning (finger prick sampling) and after each training sessions (earlobe prick sampling).

Capillary sampling (prick sampling) to analysis metabolic parameters at systemic blood level will be performed during the first, in-hospital, training session and further will be continued at home, self-managed by the patient who will have to measure glycaemia and ketones every morning (finger prick sampling) and after each training sessions (earlobe prick sampling).

Capillary sampling (prick sampling) to analysis metabolic parameters at systemic blood level will be performed during the first, in-hospital, training session and further will be continued at home, self-managed by the patient who will have to measure glycaemia and ketones every morning (finger prick sampling) and after each training sessions (earlobe prick sampling).

Capillary sampling (prick sampling) to analysis metabolic parameters at systemic blood level will be performed during the first, in-hospital, training session and further will be continued at home, self-managed by the patient who will have to measure glycaemia and ketones every morning (finger prick sampling) and after each training sessions (earlobe prick sampling).

Capillary sampling (prick sampling) to analysis metabolic parameters at systemic blood level will be performed during the first, in-hospital, training session and further will be continued at home, self-managed by the patient who will have to measure glycaemia and ketones every morning (finger prick sampling) and after each training sessions (earlobe prick sampling).

Capillary sampling (prick sampling) to analysis metabolic parameters at systemic blood level will be performed during the first, in-hospital, training session and further will be continued at home, self-managed by the patient who will have to measure glycaemia and ketones every morning (finger prick sampling) and after each training sessions (earlobe prick sampling).

Inclusion Criteria: Age from 18 to 65 years at the time of recruitment Histologically confirmed diagnosis of primary melanoma Patients who are candidate to curative surgery for primary melanoma, skin radicalization or sentinel node biopsy. American Society of Anaesthesiologists (ASA) status I Willingness and ability to comply with the protocol, the scheduled visits, treatment plans, laboratory tests and other procedures. Understanding and signature of the informed consent Female patients of childbearing potential must agree to sexual abstinence or to use highly effective method of contraception throughout the study (for CEP patients) Exclusion Criteria: American Society of Anaesthesiologists (ASA) status > I Pregnancy or lactation Known infection from HIV virus Regular physical exercise (more than 1 exercise workout of moderate to intense exercise activity per week) Any physical or mental conditions interfering with the ability to perform exercise or to complete the testing procedures.

Since the study data could be compared with those obtained from other clinical studies, focused on the immune system, this part is not yet defined. Anyhow it will depend on the results obtained.

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