Clinical Trials /

Epigenetics, Vitamin C, and Abnormal Blood Cell Formation - Vitamin C in Patients With Low-Risk Myeloid Malignancies

NCT03682029

Description:

The primary purpose of this multi-centre, randomized, placebo-controlled, double-blind phase II study is to investigate if oral vitamin C may change the biology of low-risk myeloid malignancies; i.e., clonal cytopenia of undetermined significance (CCUS), low-risk myelodysplastic syndrome (MDS), and chronic myelomonocytic leukemia (CMML)-0/1 by reversing some of the epigenetic changes characteristic of these disease entities. The epigenetic regulator TET2 is the gene most often affected in CCUS. Preclinical studies have shown that active demethylation by the TET enzymes is dependent on vitamin C, and the investigators have shown that plasma vitamin C levels are exceedingly low in hematological cancer patients but are easily corrected by oral vitamin C. This study is part of an array of EVITA studies aimed at clarifying whether the standard of care of patients with myeloid malignancies should be changed and oral vitamin C supplement added to the treatment recommendations.

Related Conditions:
  • Chronic Myelomonocytic Leukemia-0
  • Chronic Myelomonocytic Leukemia-1
  • Myelodysplastic Syndromes
Recruiting Status:

Recruiting

Phase:

N/A

Trial Eligibility

Document

Title

  • Brief Title: Epigenetics, Vitamin C, and Abnormal Blood Cell Formation - Vitamin C in Patients With Low-Risk Myeloid Malignancies
  • Official Title: Epigenetics, Vitamin C, and Abnormal Hematopoiesis - Role of Vitamin C in Epigenetic Regulation in Hematopoiesis Sub-Study on CCUS, Low-Risk MDS, and CMML-0/1

Clinical Trial IDs

  • ORG STUDY ID: H-16022249 low-risk cohort
  • NCT ID: NCT03682029

Conditions

  • Myelodysplastic Syndromes
  • Chronic Myelomonocytic Leukemia-1
  • Cytopenia

Purpose

The primary purpose of this multi-centre, randomized, placebo-controlled, double-blind phase II study is to investigate if oral vitamin C may change the biology of low-risk myeloid malignancies; i.e., clonal cytopenia of undetermined significance (CCUS), low-risk myelodysplastic syndrome (MDS), and chronic myelomonocytic leukemia (CMML)-0/1 by reversing some of the epigenetic changes characteristic of these disease entities. The epigenetic regulator TET2 is the gene most often affected in CCUS. Preclinical studies have shown that active demethylation by the TET enzymes is dependent on vitamin C, and the investigators have shown that plasma vitamin C levels are exceedingly low in hematological cancer patients but are easily corrected by oral vitamin C. This study is part of an array of EVITA studies aimed at clarifying whether the standard of care of patients with myeloid malignancies should be changed and oral vitamin C supplement added to the treatment recommendations.

Detailed Description

      BACKGROUND Recent investigations have shown that mutations in epigenetic regulators are
      common, both in the apparently normal hematopoiesis of the elderly and in patients (pts) with
      myeloid cancers. It was long anticipated that DNA methylation was a permanent silencing mark,
      but with the discovery of the ten eleven translocation (TET) enzymes it became clear that
      active demethylation occurs. The initial steps in this process are catalyzed by TET enzymes,
      which are, however, frequently mutated and methylated in hematological cancers. The Jumonji
      enzymes, which catalyze histone demethylation, are also aberrantly regulated in hematological
      cancers.

      Vitamin C (VitC) was identified in the 1930'ies as the necessary micronutrient in the
      prevention of scurvy. Unlike plants and most animals, humans are unable to synthesize vitC
      from glucose due to lack of the required enzyme, L-gulonolactone oxidase. Therefore, vitC
      must be provided through the diet. Recent studies recognize vitC as an important cofactor for
      the Fe(II)- and 2-oxoglutarate dioxygenase family. These include the TET enzymes, which are
      involved in the conversion of 5-methylcytosine (5-mC) to its oxidized derivatives
      5-hydroxymethylcytosine (5-hmC), 5-carboxyl cytosine (5-caC), and 5-formylcytosine (5-fC),
      and the Jumonji enzymes that are involved in histone demethylation. Accordingly, vitC may
      potentially play an important role in the regulation of DNA and histone demethylation.
      However, > 80 percentage of hematological cancer pts were found to be severely vitC
      deficient. Interestingly, analyses of 20 participants included in the investigators' recently
      conducted randomized, placebo-controlled pilot study (NCT02877277) show that the level of
      vitC in MDS and CMML pts undergoing treatment with azacitidine, is easily elevated to the
      normal range by oral vitC supplement (unpublished data). When pts that were already taking
      vitC supplements were switched to placebo, the vitC levels quickly dropped below the normal
      range.

      It has also been shown that the formation of 5-hmC and its derivatives may be compromised in
      healthy individuals and pts with TET mutations. However, since many of these mutations are
      heterozygous, and since the three TET enzymes (TET1, TET2, and TET3) may have some
      redundancy, restoration of vitC to physiological levels might have an impact on the level of
      5-hmC/5-mC in individuals with TET mutant clonal hematopoiesis or hematological cancer.

      Analyses of 5-hmC/5-mC levels in peripheral blood (PB) mononuclear cells (MNCs) from the
      participants in the pilot study also showed a clear trend toward increased 5-hmC in the vitC
      arm, however, after designing the trial the investigators realized that 5-hmC/5-mC levels are
      better measured in hematopoietic stem cells in the bone marrow (BM) where the levels are
      10-20 fold higher. Thus, the pilot study will be followed-up with a randomized
      placebo-controlled trial of oral vitC in individuals with low-risk myeloid malignancies;
      i.e., CCUS or low-risk MDS/CMML, to investigate if oral vitC can change the biology of these
      disease entities and ultimately prevent progression.

      Hypotheses:

        1. The investigators have previously shown that cancer pts are vitC deficient, and
           individuals with CCUS, which represents pre-MDS, might also be vitC deficient. The
           hypothesis is that this may lead to reduced levels of 5-hmC/5-mC in vivo in both cancer
           pts and individuals with CCUS

        2. Elevating serum vitC levels to the normal range in CCUS and low-risk MDS/CMML pts by
           oral supplementation with vitC may

             -  increase the 5-hmC/5-mC ratio

             -  change the plasma cytokine profile towards a less inflammatory, less tumorigenic
                profile

             -  reduce the malignant clone

             -  change gene expression

      Aims:

      To determine if restoring vitC to the normal range in CCUS and low-risk MDS/CMML pts will:

        1. Increase the 5-hmC/5-mC ratio in CCUS and low-risk MDS/CMML pts

        2. Reduce accumulation of 5-mC at promoters/enhancers/long terminal repeats (LTRs), or at
           other regulatory genomic regions of tumor suppressors/methylated driver genes/genes
           involved in hematopoietic development

        3. Upregulate the expression of these genes

        4. Reduce the malignant clone

        5. Entail any safety risks

      RESEARCH PLAN A total of 70 participants is planned for enrolment. Individuals with CCUS,
      low-risk MDS, or CMML-0 or -1 will be included from Rigshospitalet and Herlev Hospital, both
      newly diagnosed individuals and individuals already being followed at the hematological
      department. Members of the Van Andel Research Institute consortium in US will also be
      participating in enrolling participants at University of Southern California.

      The participants will enter block randomization with a ratio of 1:1; vitC 1000 mg/day p.o.
      versus placebo for one year.

      MATERIAL AND MEASUREMENTS PB: PB samples (45 mL) will be taken at study entry and every 3
      months (or more if required according to physician's choice) during the first year. After one
      year PB will be taken with a frequency determined by the treating physician.

      Measurements include blood counts including differential count, plasma vitC levels, levels of
      folic acid, vitamin B12, interleukin (IL)-6, vitamin D, iron, ferritin, transferrin, and
      markers of inflammation, immune response, and oxidative stress/damage. Mutations in genes
      frequently involved in myeloid cancer/clonal hematopoiesis, including epigenetic regulators,
      and variant allele frequencies (VAF) will be investigated in PB at study entry, at 12 months,
      and at progression. A cytokine panel (ELISA) will be used to assess the levels of an array of
      cytokines.

      BM: BM samples (18 mL) will be taken at study entry, after 3 months, and after one year.
      Hereafter, BM samples will be taken with a frequency determined by the treating physician.

      Levels of vitC, gene expression, and total 5-hmC/5-mC will be measured in sorted mesenchymal
      cells, stem cells, and progenitor cells, and compared to the corresponding levels in PB MNCs
      and granulocytes.

      If progression to MDS/acute myeloid leukemia occurs: Locus specific measurements of
      5-hmC/5-mC will be performed in regulatory genomic regions before and after progression and
      compared to RNA expression of disease-related genes in selected cases.

      RESEARCH BIOBANK A research biobank will be established at The Epi-/Genome Laboratory,
      Rigshospitalet / Biotech Research and Innovation Centre to store the biological samples from
      the participants. The research biobank is approved by the Regional Science Ethics Committee
      and the Danish Data Protection Agency in accordance with the Act on Processing of Personal
      Data (license no. H-16022249 and 04864/RH-2016-259, respectively). Cryopreserved separated
      MNCs from BM and PB will be stored in addition to granulocyte pellets and plasma. The date
      for closing the research biobank is 31-12-2030.

      For correlative studies, biological samples will be sent to Van Andel Research Institute,
      Grand Rapids, US, and Imperial College, London, UK (external collaborators), for RNA
      sequencing and analyses of DNA methylation and hydroxymethylation, respectively. Biological
      samples will also be sent to Life Science Faculty, University of Copenhagen, and The National
      Veterinary Institute, Technical University of Denmark (external collaborators) for
      measurement of serum and intracellular vitC concentrations and analyses of immunological
      response, respectively.

      METHODS VitC measurement: Ascorbate and total vitC, i.e., ascorbate + dehydroascorbic acid
      (the oxidized form of vitC; DHA), are quantified by high-performance liquid chromatography
      (HPLC) with coulometric detection; DHA is assessed by subtraction of ascorbate from total
      vitC. Uric acid is used as endogenous internal standard.

      Cell sorting: Fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting
      (MACS, using a RoboSep device).

      Total 5-hmC/5-mC measurement: Dot blot analysis of 5-hmC. 5-hmC/5-mC measurement by Mass
      Spectrometry.

      Locus specific 5-hmC/5-mC measurement: "EPIC" 850 K BeadChips. 5-hmC/5-mC at selected sites
      will be measured by pyrosequencing.

      Gene expression: Total RNA sequencing and reverse transcriptase-quantitative polymerase chain
      reaction.

      Mutation detection: Targeted next generation sequencing of a panel of genes recurrently
      mutated in myeloid cancer as described.

      STATISTICAL CONSIDERATIONS AND POWER CALCULATION This study is the first study to examine the
      effects of vitC as monotherapy on 5-hmC/5-mC levels in hematopoietic stem cells in humans in
      vivo. Therefore, it is not possible to perform a power calculation or a sample size
      calculation. The number of participants (n=70) is set as an estimate of the number needed to
      observe a significant difference between the groups (vitC vs. placebo) in the primary
      endpoint; 5-hmC/5-mC level. The final number of participants will be determined by power and
      sample size calculations based on preliminary findings in the present cohort.

      Efficacy analyses are by intention-to-treat. Safety analyses include all participants who
      receive at least one dose of protocol therapy.

      The Wilcoxon matched-pairs signed rank test is used for comparing paired quantitative
      variables, and Fisher's exact test for contingency analysis of response groups.

      ETHICAL CONSIDERATIONS The study has been approved by the Regional Science Ethics Committee
      (H-16022249) and the Danish Data Protection Agency (04864/RH-2016-259).

      All participants included in the project will be informed orally and in writing.
      Participation will only be accepted after written consent. Participants will be informed that
      they can at any time for any reason withdraw from the study without it affecting their
      treatment in the health care system.

      Using the targeted DNA sequencing approach described, there is a small risk of detecting a
      germline mutation in the participants related to myeloid malignancy. The participants will be
      asked to state in the informed consent if they do not want to receive any further relevant
      health-related information that may appear during the project analyses. Unless the
      participant explicitly states that he or she does not want to be informed of any potential
      health-related random findings in the study, the participant will be informed and offered
      further investigations and genetic counseling at the local hospital.

      Patient disadvantages, side effects, risks, and complications Blood sampling is associated
      with brief discomfort and/or pain. No significant risks are associated with the blood
      sampling. Local bleeding can occur, which in rare cases can cause discomfort and
      discoloration for a few days. Rarely, a blood sampling can cause vasovagal reaction leading
      to a brief loss of consciousness.

      BM aspiration is associated with brief pain while the local anesthesia is given. Furthermore,
      many individuals experience an uncomfortable feeling in the nates and leg while the BM is
      aspirated. This lasts approximately 30 seconds. Finally, some tenderness can occur for a few
      days after the procedure. Possible complications to undergoing a BM investigation include
      bleeding and infection. However, the incidence of these complications is extremely low.

      According to the Nordic Nutrition Recommendations there is no evidence that intake of vitC
      above 1000 mg/day are either carcinogenic or teratogenic. However, high intakes (> 1000-2000
      mg/day) may cause diarrhea and other gastrointestinal disturbances and susceptible
      individuals may experience kidney stone formation from increased oxalate formation. Since
      vitC is only given at physiological doses, it is anticipated that the study is safe and will
      provide no additional risk for the participants; an assumption that is supported by the
      experience from the pilot study.
    

Trial Arms

NameTypeDescriptionInterventions
Vitamin CExperimentalVitamin C (ascorbic acid) 500 mg/capsule. Ingestion of 2 capsules (1000 mg) daily for 12 months.
    PlaceboPlacebo ComparatorPlacebo capsule. Ingestion of 2 capsules daily for 12 months. Placebo will be prepared as capsules that look and taste identical to the vitamin C supplement capsules. The content of the placebo is lactose, potato starch, gelatin, magnesium stearate, and talc.

      Eligibility Criteria

              Inclusion Criteria:
      
              A diagnosis of CCUS:
      
                -  Persistent cytopenia for > 6 months defined as hgb < 11.3 g/dL (7 mmol/L) in women and
                   hgb < 12.9 g/dL (8 mmol/L) in men, thrombocyte count < 150 x 10^9/L or neutrophil
                   count < 1.8 x 10^9/L
      
                -  Normal cytogenetics (with the exception of deletion of the Y chromosome which can be
                   accepted)
      
                -  A bone marrow morphology that is not diagnostic of MDS or any other malignancy
      
                -  Other common causes of cytopenia (vitamin or other deficiencies, virus infection,
                   etc.) have been ruled out
      
                -  Hematolytic conditions have been ruled out
      
                -  The presence of a detectable mutation in genes recurrently affected in myeloid
                   malignancy representing a clonal marker (excluding germline mutations)
      
              OR
      
              A diagnosis of MDS as according to World Health Organization (WHO) 2016 diagnostic criteria
      
              • Revised international prognostic scoring system (IPSS-R) risk score ≤ 3 AND bone marrow
              blast percentage < 5 defining low-risk
      
              OR
      
              A diagnosis of CMML-0 or -1 as according to WHO 2016 diagnostic criteria
      
              AND
      
              (All diagnostic categories) The presence of a detectable mutation in genes recurrently
              affected in myeloid malignancy representing a clonal marker (excluding germline mutations)
      
              Exclusion Criteria:
      
                -  Unwillingness to discontinue any and all use of vitamin C medication/supplementation
                   including multivitamin at least 24 hours prior to Baseline investigations and sampling
      
                -  Lack of ability to understand the information given, or lack of willingness to sign a
                   written informed consent document
      
                -  Treatment with chemotherapy within the past 6 months
      
                -  Patients receiving active treatment for their myeloid malignancy, including
                   investigational agents, with the exception of granulocyte colony-stimulating factor
                   (G-CSF) and erythropoietin
      
                -  History of allergic reactions to ascorbic acid
      
                -  Unwillingness to comply with all aspects of the protocol
            
      Maximum Eligible Age:N/A
      Minimum Eligible Age:18 Years
      Eligible Gender:All
      Healthy Volunteers:No

      Primary Outcome Measures

      Measure:Mean Change from Baseline in 5-hmC/5-mC Level at 12 Months
      Time Frame:At baseline and at 12 months
      Safety Issue:
      Description:Mean change from baseline to 12 months in global 5-hmC/5-mC measured in hematopoietic stem cells is compared between the vitamin C group and the placebo group. In addition, the mean change from baseline in 5-hmC/5-mC at 12 months is compared between participants in the vitamin C arm with baseline plasma vitamin C level in the normal range vs. participants in the vitamin C arm with baseline plasma vitamin C level below the normal range.

      Secondary Outcome Measures

      Measure:Mean Change from Baseline in 5-hmC/5-mC Level at 3 Months
      Time Frame:At baseline and at 3 months
      Safety Issue:
      Description:Mean change from baseline to 3 months in global 5-hmC/5-mC measured in hematopoietic stem cells is compared between the vitamin C group and the placebo group. In addition, the mean change from baseline in 5-hmC/5-mC at 12 months is compared between participants in the vitamin C arm with baseline plasma vitamin C level in the normal range vs. participants in the vitamin C arm with baseline plasma vitamin C level below the normal range.
      Measure:Mean Change from Baseline in 5-mC at Selected Sites at 12 Months
      Time Frame:At baseline and at 12 months
      Safety Issue:
      Description:Mean change from baseline to 12 months in 5-mC at promoters/enhancers/long terminal repeats, and other regulatory genomic regions of tumor suppressors/methylated driver genes/genes involved in hematopoietic development.
      Measure:Mean Change from Baseline in H3K9 Methylation at Selected Sites at 12 Months
      Time Frame:At baseline and at 12 months
      Safety Issue:
      Description:Mean change from baseline to 12 months in methylated histone H3 lysine at position 9 at promoters/enhancers/long terminal repeats, and other regulatory regions of tumor suppressors/methylated driver genes/genes involved in hematopoietic development.
      Measure:Mean Change from Baseline in Variant Allele Frequency at 12 Months
      Time Frame:At baseline and at 12 months
      Safety Issue:
      Description:Mean change from baseline to 12 months in mutant allele burden as measured by the variant allele frequency (VAF).
      Measure:Mean Change from Baseline in Plasma Cytokine Levels at 12 Months
      Time Frame:At baseline and at 12 months
      Safety Issue:
      Description:Mean change from baseline to 12 months in plasma cytokine levels (e.g., IL-6, S100A9, etc.).
      Measure:Mean Change from Baseline in mRNA Levels of Selected Genes at 12 Months
      Time Frame:At baseline and at 12 months
      Safety Issue:
      Description:Mean change from baseline to 12 months in mRNA levels of tumor suppressor genes, oncogenes, epigenetic regulators, cytokines, and genes involved in hematopoietic development.
      Measure:Mean Change from Baseline in mRNA Levels of Selected Genes at 3 Months
      Time Frame:At baseline and at 3 months
      Safety Issue:
      Description:Mean change from baseline to 3 months in mRNA levels of tumor suppressor genes, oncogenes, epigenetic regulators, cytokines, and genes involved in hematopoietic development.
      Measure:Number of Participants with Serious Adverse Events and Treatment-Related Adverse Events as Assessed by CTCAE v5.0
      Time Frame:Through study completion, 12 months
      Safety Issue:
      Description:Number of serious adverse events and treatment-related adverse events as assessed by CTCAE v5.0. Adverse events, that are not considered serious or related to vitamin C intake, will not be recorded.
      Measure:Percentage of Participants with Vitamin C Deficiency
      Time Frame:Day 1
      Safety Issue:
      Description:Percentage of participants with baseline plasma vitamin C level below the normal physiological range.

      Details

      Phase:N/A
      Primary Purpose:Interventional
      Overall Status:Recruiting
      Lead Sponsor:Rigshospitalet, Denmark

      Trial Keywords

      • Clonal Cytopenia of Undetermined Significance
      • Myelodysplastic Syndromes
      • Chronic Myelomonocytic Leukemia
      • Low-Risk Myeloid Malignancies
      • Vitamin C
      • Epigenetics
      • TET2
      • Randomized, Placebo-Controlled Trial
      • Clinical Study

      Last Updated

      September 27, 2018