Oral Vitamin C Supplementation Modulates Inflammatory Cytokines in Clonal Cytopenia of Undetermined Significance and Low-Risk Myeloid Malignancies: Results from EVI-2 Trial

Introduction
We have previously reported elevated levels of inflammatory cytokines, such as IL-6, IL-10, TNF-α, CXCL10, and decreased levels of CCR5/RANTES in peripheral blood (PB) plasma of patients with myeloid malignancies and precursor conditions compared to healthy elderly controls (Nielsen et al., 2022). Additionally, we showed that these levels correlate with poor clinical outcomes.
Preclinical studies have shown that malignant myeloid cells secrete excess IL-6 and that inflammation promote pre-leukemic development in mice. Vitamin C (vitC) is a known antioxidant that also possesses anti-inflammatory properties. In vitro studies have demonstrated that vitC significantly reduces pro-inflammatory cytokines, particularly IL-6 and TNF-α, in human cells. Thus, vitC supplementation may be an attractive preventive therapy for patients with early-stage myeloid malignancies and precursor conditions.

Aim
We aim to measure cytokine levels in plasma from PB of patients with low-risk myelodysplastic neoplasms (MDS; IPSS-R score ≤3), MDS/myeloproliferative neoplasms (MDS/MPN), and clonal cytopenia of undetermined significance (CCUS). We aim to investigate whether oral vitC supplementation can modulate cytokine levels and to correlate these levels with molecular and clinical characteristics.

Methods
In the completed randomized, placebo-controlled phase 2 study (NCT03682029), 109 patients with low-risk myeloid malignancies and CCUS received 1000 mg of oral vitC daily (n=55) or placebo (n=54) for 12 months. Concentrations of 19 cytokines and chemokines (IL-6, IL-12p70, IL-17, IL-1α, IL-1β, IL-4, IL-7, IL-8, IL-10, TNF-a, IFN-У, SDF-1α, TGF-β1, CXCL10, CCLR5/RANTES, G-CSF, GM-CSF, M-CSF, and VEGF) in PB plasma were measured at baseline and end-of-treatment using Multiplex assays.

Results
During the late-breaking oral session at the European Hematology Association (EHA) 2024 congress, we presented data from the secondary endpoint of the EVI-2 study, showing that overall survival in patients treated with vitC was significantly longer compared to those receiving placebo (hazard ratio 2.88; 95% CI: 1.41-5.89; P=0.002).
Now, we further extend our analysis with data from the secondary endpoint and describe unstratified cytokine data between the vitC and placebo groups. Stratification analysis of cytokine levels based on molecular factors (e.g., type of somatic mutation(s) and vitC concentration) and clinical disease characteristics are not yet available but will be presented at the conference.

After correction for multiple testing, the mean fold changes in plasma concentrations from baseline to end-of-treatment for IL-6 (p<0.001), IL-10 (p<0.001), CXCL10 (p=0.003), M-CSF (p<0.001), and G-CSF (p<0.001) were significantly lower in the vitC group compared to placebo, whereas CCL5/RANTES (p=0.023) was significantly higher in the vitC group. Differences in other cytokines were observed between the groups, but these did not reach statistical significance.
Notably, the mean fold changes of IL-6, IL-10, and CXCL10 demonstrated a decrease in cytokine concentrations in the vitC group from baseline to end-of-treatment, whereas the mean fold changes in the placebo group indicated an increase in their concentrations. Additionally, fold changes for CCL5/RANTES showed a mean increase in concentration in the vitC group and a mean decrease in the placebo group. Overall, vitC modifies concentrations of previously identified disease-related inflammatory cytokines in a manner that is associated with better clinical outcomes.

Conclusion
Oral vitC supplementation in patients with CCUS and low-risk myeloid malignancies modulates peripheral blood plasma concentrations of disease-related inflammatory cytokines, supporting its potential as a preventive therapy in patients with early-stage myeloid malignancies and precursor conditions.