Co-stimulatory Markers and Vitamin D Status in Anti-PD1 Treated Melanoma Patients
Condition: Malignant Melanoma
MeSH terms: Melanoma
Study type: Observational

Summary: 
      An observational single center study designed to identify response-related biomarkers of
      anti-programmed death 1 (PD-1) therapy to advanced melanoma patients and to investigate if
      vitamin D levels are related to treatment response. 40 patients diagnosed with advanced
      melanoma will be included. Patients are included at the Department of Oncology, Aarhus
      University Hospital (AUH). All patients will be treated with Pembrolizumab as a standard
      procedure at the Department of Oncology. The protocol comprises blood samples at baseline, 3
      and 6 weeks after treatment initiation with anti-PD1 therapy and three years of observational
      follow-up. A total amount of 217 ml blood will be drawn during the study period. The study
      period is 6 weeks followed by 3 years of follow-up. Medical history, symptoms, response to
      treatment regarding the RESIST criteria and side affects will be recorded at each visit in
      both the study period and in follow-up. Biochemical markers will be obtained according to
      normal procedure during study and follow-up visits.

      20 Healthy volunteers (HV) are included, matched by age and gender. Collected blood samples
      (serum, plasma, peripheral blood mononuclear cells) will be analyzed after the last patient
      has ended the week 6 visit.
    

Detailed description: 
      Background Advanced melanoma still is associated with severe disease and early death. New
      immunotherapies including the checkpoint inhibitors anti-programmed death 1(PD-1) and
      anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) improve survival compared to
      chemotherapy. It still remains to be elucidated why the treatment only benefits some
      patients. The tumour cells expression of programmed death ligand 1 (PD-L1) and other
      co-stimulatory receptors, as well as the presence of soluble isoforms are speculated to
      influence treatment success. Possible modulators of the PD-1 pathway like vitamin D might
      also influence the treatment outcome. Investigating these relations could provide useful
      markers to predict response to anti-PD-1 therapy in advanced melanoma patients.

      PD-1, a transmembranous molecule present on activated T, B and natural killer (NK) cells is
      essential in maintaining self tolerance. PD-1 has two widely expressed ligands, PD-L1 and
      programmed death ligand 2 (PD-L2). Both PD-1 and its ligands are present in soluble (s)
      forms. In autoimmune disease like rheumatoid arthritis sPD-1 concentrations are associated
      with disease markers and scores.

      PD-L1 can be highly expressed by cancers and thereby silence the T cell immune attack. Using
      anti-PD-1 antibodies, the immune cells are not inhibited by the PD-1 pathway, and
      proliferation, activation and T cell survival increases, improving tumour cells targeting.
      Soluble PD-1, sPD-L1 and sPD-L2 may be potential markers in melanoma patients who would
      benefit from the anti- PD1 treatment.

      In autoimmune diseases as rheumatoid arthritis, Crohn's disease and in multiple sclerosis
      vitamin D deficiency is associated with increased disease activity. Active
      1.25-dihydroxyvitamin D3 (1.25-vitD) binds to the vitamin D receptor (VDR) and the
      1.25-vitD-VDR complex functions as a transcription factor. Regarding melanoma, VDR variants
      are associated with melanoma risk. In mice, vitamin D stimulation has been shown to increase
      the PD-L2 expression in dendritic cells (DCs). In humans vitamin D stimulation increased the
      DC PD-L1 expression and interleukin 10 (IL-10) production and PD-L1 blockage resulted in
      increased interferon gamma and decreased IL-10 production.

      Our preliminary pilot studies have demonstrated that vitamin D initially decreases PD-1
      expression in Crohn's disease T cells but after 26 weeks of vitamin D treatment PD-1
      expression increases in CD4+CD25+ T cells (unpublished data). These results might indicate
      that vitamin D treatment initially increase T cell activation replaced by a decreased T cell
      activation after long-time vitamin D treatment in inflamed patients. This might influence the
      anti-PD-1 treatment response in advanced melanoma. Improved knowledge of the interaction
      between anti-PD-1treatment, vitamin-D and sPD-1, PD-L1 and PD-L2 could provide new beneficial
      modifications to the treatment regimens currently available -for instance as a new predictor
      of treatment response.

      Methods and participants Advanced melanoma patients 40 patients diagnosed with advanced
      melanoma will be included in the present observational study designed to identify
      response-related biomarkers of anti-PD-1 therapy. Patients are included at the Department of
      Oncology, Aarhus University Hospital (AUH). The protocol is approved by the Central Denmark
      Regional Committee for Health Research Ethics and the Danish Data Protection Agency. The
      protocol comprises blood samples at baseline, 3 and 6 weeks after treatment initiation with
      anti-PD1 therapy. The study period is 6 weeks followed by 3 years of follow-up. Medical
      history and symptoms and serious immune side affects will be recorded at each visit in both
      the study period and in follow-up. Standard biochemical markers will be obtained according to
      guidelines during follow-up and will be independent of the project. Patients are follow-up
      every third week during the anti-PD-1 treatment. The treatment is stopped because of disease
      progression or server adverse events. After ended anti-PD-1 treatment patients are
      followed-up every 3rd month. The follow-up visits are used to detect time to treatment
      response and time to treatment failure.

      The included persons do not receive economic compensation for participating in the project.
      Aim one to three will be investigated when all patients have ended the study period of 6
      weeks. Aim five and six will be investigated when all patients have ended the follow-up
      period

      Healthy volunteers 20 Healthy volunteers (HV) are included as controls, matched by age and
      gender. Matched HV are obtained from buffy coats, an established collaboration with the Blood
      Bank, Aarhus University Hospital. It is not possible to trace the identity of a donor to a
      specific blood sample.

      Peripheral Blood Mononuclear Cells From advanced melanoma patients 36 ml of blood are
      collected at baseline and after 6 weeks of therapy (a total of 72 ml). From HV, blood from
      the buffy coat is used. Peripheral blood mononuclear cells (PBMCs) are isolated with ficoll,
      an established protocol. PBMCs are cryopreserved at -140C and stored in the INART bio bank of
      Biomedicine, Aarhus University. The PBMCs are used to investigate expression of
      co-stimulatory molecules; among these PD-1, PD-L1, PD-L2, CTLA-4, activation markers, cell
      proliferation, cell viability, cytokine production as well as vitamin D related cell markers
      together with mRNA expressions of vitamin D and PD-1 related genes.

      Plasma and Serum At baseline and after 3 and 6 weeks of therapy 18 ml of blood are obtained
      to isolate serum and plasma (a total of 54 ml blood). Plasma and serum from HV are obtained
      from the buffy coat. Plasma and serum is isolated, cryopreserved at -80C and stored in the
      INART bio bank of Biomedicine, Aarhus University. In plasma, sPD-1, sPD-L1, sPD-L2 and CTLA-4
      concentrations with more are determined.

      Study-related biochemical markers At baseline and after 6 weeks of initial treatment a total
      amount of 55 ml of blood are used for the following blood samples: 25-hydroxyvitamin D2+D3,
      1.25-dihydroxyvitamin D3, Parathyroid hormone, free calcium-ion and phosphate. Vitamin D
      binding protein concentration is measured at baseline. All the listed blood samples despite
      vitamin D binding protein concentration are measured as a standard operation procedure by the
      Clinical Biochemistry department at Aarhus University Hospital. Vitamin D binding protein
      concentrations are measured at Statens Serum Institut a public enterprise under the Danish
      Ministry of Health.

      Melanoma cell line The patient data will be supported by in vitro experiments using a
      melanoma cell line. PD-1 and vitamin D related surface markers, cytokine production and mRNA
      expressions will be associated to anti-PD-1 therapy, sPD-1, sPD-L1, sPD-L1 and vitamin D.

      Statistics ELISA-made results (sPD-1, sPD-L1, sPD-L2, sCLTA-4) are presented as median with
      total range and differences are calculated using Wilcoxon non-parametric statistics. Other
      results are examined for normal distribution. Normal distributed data are compared with
      student's t-test and non-normal distributed data are compared with Wilcoxon non-parametric
      statistics. Association between two variables will be estimated using Spearman's rank
      correlation coefficient (Spearman's rho). A p-value below 0.05 will be considered
      statistically significant.
    

Intervention name(s): Pembrolizumab
Intervention type(s): Drug
Intervention description(s): Pembrolizumab is already approved for treatment of malignant melanoma in advanced stages. We will investigate if sPD-1 or vitamin D status is changed in patients receiving pembrolizumab.
Intervention MeSH terms: Pembrolizumab
Observational model: Cohort
Time perspective: Retrospective
Sampling method: Non-Probability Sample
Study population: 
        40 patients with metastatic melanoma are included. Patients are admitted to Department of
        Oncology, Aarhus University Hospital (AUH). The patients are recruited to the study from
        the outpatient clinic of the Department of Oncology when they are about to begin treatment
        with pembrolizumab. Patients are informed that participation is voluntary and they can
        withdraw the informed consent at any time without a reason and without consequences.
      
Biospecimen description: 
      Peripheral blood cells.
    
Keywords: Vitamin D; Soluble PD-1