Combining Active and Passive DNA Hypomethylation

EVI-3 is a phase 2 international, multicentre, randomized, parallel-group, placebo-controlled, double-blind study of the efficacy and safety of oral vitamin C supplement in combination with azacitidine (AZA) in patients with higher-risk myeloid malignancies with or without mutations in genes recurrently affected in myeloid malignancies. Treatment allocation is in 1:1 ratio (vitamin C vs. placebo) by block randomization stratified by clinical site. Study entry is staggered. Patients are randomized to either oral vitamin C 1000 mg daily or placebo from start of AZA treatment until end of study (EOS) or until AZA treatment is discontinued at the discretion of the treating physician, whichever occurs earlier. The accrual time is estimated to 48 months and 6 months follow-up, thus, maximum treatment duration will be approximately 54 months. A total of 196 patients is planned for enrollment.

Study visits are scheduled at baseline, after 1st AZA treatment cycle, after 6 AZA treatment cycles, and, if AZA treatment is continued, at EOS or end of AZA treatment. Evaluations at study visits include bone marrow investigation, peripheral blood tests, patient-reported outcome measures, adverse events and compliance. Bone marrow aspirate and peripheral blood will be collected for biobank at each study visit.

All patients will undergo follow-up once yearly from EOS. Follow-up will include information on duration of AZA therapy, survival and disease progression from myelodysplastic syndrome (MDS) or chronic myelomonocytic leukemia (CMML) to acute myeloid leukemia (AML), if diagnosed following a clinical indication for a bone marrow test.

• Vitamin C (Dietary supplement), also known as Ascorbic acid: Oral vitamin C (ascorbic acid) 1000 mg daily will be administered from day 1 in the 1st AZA cycle (D1/C1) and continued until discontinuation of AZA or EOS as combination treatment.
• Placebo (Dietary supplement): Placebo capsules (two capsules once daily) will be administered from day 1 in the 1st AZA cycle (D1/C1) and continued until discontinuation of AZA or EOS.

Primary outcomes

• Event-free survival [0-54 months]: Event-free survival in months in the group of patients receiving oral vitamin C + AZA (arm A) vs. the group of patients receiving placebo + AZA (arm B) calculated from the time of randomization to EOS. Event is defined as death, relapse, progression or lack of a response at 6 AZA cycles as defined by IWG 2006 (MDS and CMML) and ELN 2017 (AML) response criteria

Secondary outcomes

• Adverse events and serious adverse events [0-54 months]: Number and ratio of patients with adverse events in arm A vs. arm B assessed from the time of administration of intervention (day 1, AZA cycle 1 = D1/C1) to EOS. Total number of adverse events and serious adverse events and the number per year from D1/C1 to EOS in arm A vs. arm B. Number of patients discontinuing the intervention and discontinuation rate in arm A vs. arm B from D1/C1 to EOS
• Overall survival [0-54 months]: Overall survival in months in arm A vs. arm B calculated from the time of randomization to EOS
• Overall response rate [0-54 months]: Rate of overall response and rates of individual responses (as according to international consensus criteria), including best response, in arm A vs. arm B after 6 AZA cycles and at EOS
• Patient-reported outcome (PRO) measures [0-54 months]: Change in PRO measures including health-related quality of life scores (EORTC QLQ-C30 and Hematological Malignancy (HM)-PRO) from baseline to end of 1st AZA cycle, after 6 AZA cycles and EOS, if AZA treatment ongoing, respectively, in arm A vs. arm B. Numerical PRO scores after the 1st AZA cycle and after 6 AZA cycles (and EOS), respectively, in arm A vs. arm B
• Variant allele frequency (VAF) of mutated clones [0-54 months]: Change in VAF of mutated clones (in percentage points and in percentage) in bone marrow mononuclear cells from baseline to end of 6th AZA cycle and to end of treatment (if occurring before EOS) in arm A vs. arm B. Number and ratio of patients with appearance of new mutations between baseline and end of 6th AZA cycle (and end of treatment) in arm A vs. arm B. Total number of new mutations in arm A vs. arm B from baseline to end of 6th AZA cycle (and end of treatment)
• Global 5-hydroxymethylcytosine (5-hmC)/5-methylcytosine (5-mC) [0-54 months]: Change in global 5-hmC/5-mC in bone marrow CD34+ cells from baseline to end of 1st AZA cycle, end of 6th AZA cycle and end of treatment (if occurring before EOS), respectively, in arm A vs. arm B. Global 5-hmC/5-mC in bone marrow CD34+ cells at end of 1st AZA cycle and end of 6th AZA cycle (and end of treatment), respectively, in arm A vs. arm B
• Site specific 5-hmC/5-mC [0-54 months]: Change in 5-hmC/5-mC at specific loci at promoters/enhancers/long terminal repeats (LTRs) or at other regulatory genomic regions of tumor suppressors, oncogenes, genes involved in hematopoietic development or human endogenous retrovirus (HERV) in bone marrow CD34+ cells from baseline to end of 1st AZA cycle, end of 6th AZA cycle and end of treatment (if occurring before EOS), respectively, in arm A vs. arm B. Site specific 5-hmC/5-mC in bone marrow CD34+ cells at end of 1st AZA cycle and end of 6th AZA cycle (and end of treatment), respectively, in arm A vs. arm B
• Gene expression [0-54 months]: Change in expression of genes involved in viral defense pathways, cell differentiation and tumor suppression and oncogenes in bone marrow CD34+ cells from baseline end of 1st AZA cycle, end of 6th AZA cycle and end of treatment (if occurring before EOS), respectively, in arm A vs. arm B. Expression of genes involved in viral defense pathways, cell differentiation and tumor suppression in bone marrow CD34+ cells at end of 1st AZA cycle and end of 6th AZA cycle (and end of treatment), respectively, in arm A vs. arm B
• mRNA expression of HERV and HERV specific T-cell responses [0-54 months]: Change in levels of mRNA expression of HERV in bone marrow CD34+ cells and HERV specific T-cell responses from baseline to end of 1st AZA cycle, end of 6th AZA cycle and end of treatment (if occurring before EOS), respectively, in arm A vs. arm B. Levels of HERV mRNA in bone marrow CD34+ cells and HERV specific T-cell responses at end of 1st AZA cycle and end of 6th AZA cycle (and end of treatment), respectively, in arm A vs. arm B.
• Duration of azacitidine (AZA) therapy [0-54 months]: Duration of AZA therapy in patients randomized to AZA + oral vitamin C (arm A) compared to patients randomized to AZA + placebo (arm B) assessed at end of study (EOS)

Sex: All, ages: 18 Years to any

Inclusion Criteria:

• Patients eligible for treatment with azacitidine with one of the following diagnoses according to World Health Organization 2016:

* MDS Higher-risk MDS according to the IPSS-R, i.e., intermediate- to very high-risk (IPSS-R score > 3)
* CMML CMML with 10-29 percent marrow blasts without myeloproliferative disorder
* AML AML with 20-30 percent blasts (low-blast count AML)

Note: Patients with therapy-related MDS are eligible if they have not received radiation or chemotherapy for six months.

Exclusion Criteria:

* Patient eligible for allogeneic stem cell transplantation
* Prior therapy with hypomethylating agents
* Any matter constituting an exclusion criterion for treatment with azacitidine
* Patient receiving other active cancer treatment, including investigational agents, with the exception of hydroxyurea for white blood cell (WBC) control, G-CSF, and low permanent doses of steroid (≤ 25 mg oral prednisolone per day) for inflammatory disorders
* Therapeutic radiation or chemotherapy within the past 6 months
* History of allergic reactions to ascorbic acid
* History of kidney or urinary tract stones requiring intervention within the past year
* Lack of ability to understand the information given, or lack of willingness to sign a written informed consent document
* Unwillingness to comply with the protocol
* Unwillingness to discontinue any and all use of vitamin C medication/supplementation including multivitamin at least 3 days (but preferably longer) prior to inclusion and baseline sampling
* Planned azacitidine treatment after allogeneic stem cell transplantation
* Eastern Cooperative Oncology Group (ECOG) performance status ≥3
* Uncontrolled comorbidity including impaired hepatic function (total serum bilirubin >1.5 × upper limit of the normal range (ULN), serum alanine transaminase >3 × ULN, chronic hepatitis with decompensated cirrhosis), disabling psychiatric disease, severe neurologic disease, severe metabolic disease, or severe cardiac disease (NYHA class 3-4)

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• Kirsten Grønbæk, Prof., MD, Study director, Copenhagen University Hospital, Rigshospitalet
• Stine Ulrik Mikkelsen, MD, PhD, Principal investigator, Copenhagen University Hospital, Rigshospitalet
• Ali Al-Mousawi, MD, Principal investigator, Copenhagen University Hospital, Rigshospitalet