Characterization of NK Cells Under First Line Advanced Therapy Either as Curative Therapy for Metastatic Melanoma or as Adjuvant Therapy for High-risk of Recurrence

Study Overview
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Brief Summary
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Cutaneous melanoma is a tumor with a serious evolution if its initial diagnosis is late. Since 2011, the treatment of advanced forms involves two therapeutic approaches : targeted therapies (BRAF and MEK inhibitors) if the tumor carries a BRAF mutation or immunotherapies (anti-PD1, anti-CTLA-4) regardless of tumor BRAF mutation status. Current data support the hypothesis that combinations of agents targeting the tumor and its environment will be required for durable responses in the majority of patients. Investigators will study the role of NK lymphocytes in tumor immunosurveillance in patients undergoing first-line innovative therapy with metastatic melanoma or at high-risk of recurrence.

Detailed Description
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Natural Killer (NK) lymphocytes are cytotoxic effectors of the innate immune response that are involved in different phases of tumor immunosurveillance. These lymphocytes are activated upon contact with tumor cells and exert direct cytotoxic activity without prior immunization. Activated NK lymphocytes produce cytokines, including IFN-γ and TNF-α, which induce and maintain an activation of the adaptive immune response by CD8+ lymphocytes. Numerous studies in various experimental tumor models highlight the role of NK cells in the control of metastasis. Our previous work has shown that NK cells infiltrate primary melanomas, that metastatic patients have altered blood NKs, and furthermore, that chemotherapy modulates their functional status. Investigators also observed a particular distribution of gene polymorphisms encoding NK activating receptors of stage IV melanoma patients compared to healthy donors. Finally, the investigator described a novel population of NK lymphocytes in metastatic lymph nodes draining melanoma NK cell activation is regulated by a balance between activating receptors (NKG2D, NCR) and inhibitory receptors that bind to modulatory classical and non-classical HLA-I molecules (HLA-E and G). More recently it has been shown that Lc NKs also express checkpoint receptors including CD96/TIGIT, NKG2A, TIM3 and PD-1 (the latter 2 are present on LcTs) that negatively modulate NK activation. The possibility of activating the lytic and secretory function of Lc NKs by interfering with these receptors may represent an alternative yet to be explored in immunotherapy treatments. More recently, th investigator have developed a program to understand the interactions between tumor mutational profile, treatment with targeted therapies and NK immunogenicity. the investigator have a panel of melanoma lines with and without the BRAF V600E mutation and vemurafenib resistant variants have been obtained from some mutated lines. the investigator evaluated the impact of treatment and resistance to BRAF inhibition on Lc NK recognition and lysis. yhe investigator showed that a BRAF inhibitor, vemurafenib, decreased membrane and soluble expression of MICA/B and ULBP2, ligands for NKG2D, an activating receptor present on Lc NKs and certain populations of Lc T cells, in all BRAF mutated lines treated. For 6 of the 7 mutated lines, modulations of NK ligands expression by vemurafenib correlated with a slight decrease in NK cytotoxic functions. Vemurafenib-resistant (R) variants were generated and their characteristics were compared to those of sensitive (S) lines. The acquisition of resistance to vemurafenib induces a significant increase in NK functions (IFNg secretion and target lysis). The responsible mechanisms involve both the expression of NK receptor ligands and the modulation of death domain receptors (Fas, TRAILRII). Transcriptome analyses reveal different targets of interest in the three pairs of S/R melanoma lines carrying the BRAF V600E mutation and distinct additional mutations (submitted manuscript). Based on our recent results our hypothesize that Lc NKs are important players in tumor immunosurveillance during current treatments of melanoma patients. Immunotherapy approaches targeting these effectors may be of interest in combinations with targeted therapies or immunotherapies. The frequency of intratumoral Lc NKs has been shown to correlate with the presence of stimulatory dendritic cells and this environment is required for a response to anti-PD1 immunotherapy. Innovative antibodies are being developed to activate the antitumor functions of NK cells

Official Title
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Characterization of NK Cells Under First Line Advanced Therapy Either as Curative Therapy for Metastatic Melanoma or as Adjuvant Therapy for High-risk of Recurrence

Conditions
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Melanoma

Intervention / Treatment
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* Other: Blood samples
* Other: Skin biopsy


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Patient ≥ 18 years Histologically confirmed melanoma patient Patient for whom treatment by targeted therapy or immunotherapy is prescribed as an adjuvant or curative treatment In the case of adjuvant treatment, the tumor must be completely removed Patient included in the Ric-Mel cohort Patient informed of the objectives and modalities of the study and having received the information form and having given his/her written consent to participate in the research Patient affiliated to a social security system Exclusion Criteria: Patient already treated medically for melanoma Palliative care patient management Pregnant or breastfeeding women Patient under guardianship or curatorship refusal of the patient to participate in the study, or refusal of the patient to allow a portion of his/her previously collected skin sample to be used in the present research

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Other
Allocation: Non-Randomized
Intervention Model: Parallel Assignment
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Other: Group 1: BRAF-mutated metastatic patients treated with 1st line targeted therapy<br>BRAF-mutated metastatic patients treated with 1st line targeted therapy | Other: Blood samples<br>* Blood samples at each protocol visit, T and NK cell analysis<br>Other: Skin biopsy<br>* Biopsy of the skin lesion (optional)<br>|
| Other: Group 2: Metastatic patients treated with 1st line immunotherapy<br>Metastatic patients treated with 1st line immunotherapy | Other: Blood samples<br>* Blood samples at each protocol visit, T and NK cell analysis<br>Other: Skin biopsy<br>* Biopsy of the skin lesion (optional)<br>|
| Other: Group 3: BRAF-mutated patients treated with adjuvant targeted therapy<br>BRAF-mutated patients treated with adjuvant targeted therapy | Other: Blood samples<br>* Blood samples at each protocol visit, T and NK cell analysis<br>Other: Skin biopsy<br>* Biopsy of the skin lesion (optional)<br>|
| Other: Group 4: Patients treated with adjuvant immunotherapy<br>Patients treated with adjuvant immunotherapy | Other: Blood samples<br>* Blood samples at each protocol visit, T and NK cell analysis<br>Other: Skin biopsy<br>* Biopsy of the skin lesion (optional)<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| characterize lymphoid cells, in particular NK lymphocytes, LcTs before and during a first line of curative or adjuvant treatment by targeted therapy or immunotherapy in order to identify early markers of response/resistance to treatment. | NK cells are recovered from blood samples taken before the start of treatment and after 3-4 weeks | Before the start of treatment and after 3-4 weeks | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Characterize lymphoid cells before and during a first line of curative or adjuvant treatment with targeted therapy or immunotherapy to identify late markers of response/resistance to treatment. | NK cells are recovered from blood samples taken at 2-3 months to 6 of treatment Late changes from Month2-Month 3 to Month 6 in the expression of membrane or soluble markers of NK or LcT cells during treatment and according to treatment groups (targeted therapy or immunotherapy used in a curative or an adjuvant setting). | From Mont 2-Month 3 to Mmonth 6 | 
| Evaluate the association between soluble or membrane markers modified during treatment and clinical evolution | analysis of soluble or membrane markers altered during treatment as a function of responders and non-responders according to RECIST 1.1 criteria. | During treatment up to 6 month | 
| Immunohistochemical analysis of markers of interest on primary tumors and/or metastases at baseline and during treatment if possible and according to the response to treatment | Tumor biopsy analysis to Identification of tumor markers or combination of tumor markers whose expression is associated with response or resistance to therapy according to RECIST 1.1. If sequential biopsies are obtained in patients with skin metastases, investigation of an association between tumor phenotype in the microenvironment at S3-S4 of treatment and response to treatment | Before the start of treatment and during treatment up to 6 month | 
| Assess the predictive capacity of soluble tumor markers in blood cells before the beginning of the treatment on the clinical evolution | Blood analysis evaluat soluble and membrane markers at baseline and during treatment as a function of treatment response | Before the start of treatment | 
| Comparison of soluble or membrane markers modified according to the treatment | Cellular analysis to see modification of the expression of membrane markers (+/- 30% of expression of the marker or Mean Fluorescence) or soluble markers (+/- 20% of expression of the marker) of NK or LcT cells during the treatment and according to the treatment received: targeted therapy or immunotherapy | During treatment up to 6 months | 
| Comparison of soluble or membrane markers modified according to the presence or not of metastasis | Cellular analysis to study modification of the expression of membrane markers (+/- 30% of expression of the marker or of the Mean Fluorescence) or soluble markers (+/- 20% of expression of the marker) of NK or LcT cells during the treatment and according to the presence or not of metastasis at the inclusion | During treatment up to 3 months | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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melanoma, NK cells, targeted therapies, immunotherapies