Vitamin C following allogeneic SCT
Patients were prospectively enrolled in the study between October 2018 – October 2021 and propensity scores matched with historical controls transplanted between 2015-2018. Follow up was updated as of February 2023. Patient characteristics are described in Table 1; 55 patients received IV vitamin C, including 10/10 HLA-MRD and MUD (n=48) and 9/10 HLA MUD recipients (n=7; 4 HLA-DQ, 2 HLA-B and 1 HLA-A mismatch). The number of fludarabine and melphalan recipients was higher in the vitamin C group as compared to historical controls. A safety lead-in cohort of 14 patients was initially enrolled. All patients enrolled were deficient in vitamin C at day 0, median level 0.3 mg/dL (range: 0.1-0.5); the median neutrophil recovery was by 12 days (range 9-15 days); similarly, platelets engrafted by day 12 (8-21 days) as well, with 100% donor myeloid engraftment at day 30. Safety endpoints of myeloid engraftment, grade 3 acute GVHD and NRM were evaluated in these patients, without triggering any of these endpoints allowing enrollment to continue.
Engraftment in vitamin C recipients
In the patients treated with Vitamin C (N=55), the median time to neutrophil and platelet engraftment were 11 and 12 days respectively (neutrophils: 9-15 days platelet 8-21 days). T cell chimerism at day 30, 60, and 90 following HSCT was > 95% in the Vitamin C group. Concomitantly measured median CD3+ cell count on days 30, 60, and 90 were 236 (2-2024), 485 (5-5580) and 390 (112-2565)/mL respectively and were not different compared historical controls. Median CD3+/4+ cell count on day 30 was 80/mL (range 30-416), CD3+/8+ was 198/mL (25-1924), CD19+ cell count was 8/mL (range 5-84) and CD3-/56+ cell count was 260/mL (range 25-656), indicating early T and NK cell recovery in these patients.
Clinical outcomes in vitamin C recipients
Non-relapse mortality was lower in the vitamin C treated group (11%) compared to the historical control (25%), but was not statistically significant in the multivariate model (HR = 0.4, 95% CI: 0.1, 1.0, p-value = 0.069) (Figure 1). Overall survival was improved in study participants ((HR = 0.5, 95% CI: 0.2, 1.0, p-value = 0.065) (Figure 2); AML patients experienced significantly worse outcomes in multivariate analysis. Overall relapse rate was similar between the two cohorts.
The vitamin C cohort had seven HLA mismatched unrelated donors and despite this there was no increase observed in the risk of acute GVHD in the control vs. study cohorts (grade II-IV, (33%) vs. (33%), p-value = 0.81 or III-IV, (24%) vs. (17%), p-value = 0.35). Organ distribution of acute GVHD grade 3-4 in vitamin C recipients was; skin only (2), GI (3), skin and liver (1), skin and GI (2) and steroid refractory GI (2). Moderate to severe chronic GVHD rates when accounting for competing risks of relapse and mortality were lower in the Vitamin C group (11%) compared to the historical controls (24%), (adj HR = 0.60, 95% CI: 0.20, 1.76, p-value = 0.352) (Supplementary Figure 2).
In the vitamin C vs. historical control cohorts (7 vs. 14%, P=0.03) received rabbit anti-thymocyte globulin (ATG) as a part of GVHD prophylaxis regimen. Of note the schedule on which ATG was administered was predominantly day -3 to day -1 in the historical control and day -9 thru -7 in the Vitamin C recipients, with similar rates of acute and chronic GVHD observed despite the earlier administration of ATG, and a less profound in vivo T cell depleting effect.
Clinical outcomes in myeloid malignancy
Because of the frequency of DNA hypermethylation in myeloid malignancies, the clinical outcomes in patients with AML, MDS and CML were examined comparing trial patients with their propensity-matched historical controls (Table 2). Overall survival was superior in these patients amongst the vitamin C recipients (p = 0.0167) (HR = 0.32, 95% CI: 0.12, 0.84, p = 0.02) (Figure 3). This was attributable to a lower risk of NRM in the vitamin C treated patients (10%) than historical controls (37%) (HR = 0.22, 95% CI: 0.07, 0.69, p-value = 0.009) (Figure 4). Relapse and acute GVHD were not different in this subgroup, however the rate of chronic GVHD tended to be lower in Vitamin C recipients (12 vs. 24%; adj HR = 0.56, 95% CI: 0.17, 1.82, p-value = 0.383) between the two groups.
Infections and toxicities in the Vitamin C recipients
CMV reactivation rate was higher in historical controls (36%) than intervention (24%) (p-value = 0.35), and a similar trend was observed in myeloid malignancy patients. EBV reactivation rate was similar in historical controls (33%) and in the Vitamin C recipients (35%) (p-value = 0.88). There were no grade 3 – 5 adverse events attributable to vitamin C therapy in this trial, particularly no nephrotoxicity or renal calculi were observed.
Cytokine levels in Vitamin C recipients
Vitamin C level (normal 0.4-2.0 mg/dL) was universally low prior to intervention and was restored to normal by day 14 (Figure 5), while CRP (normal 0-0.5 mg/dL) rose in the early aftermath of transplant following conditioning and engraftment to come down later. The peaks for both the parameters coincided, suggesting potential benefit of vitamin C may have come from its physiologic effect at the time of maximum inflammatory cytokine surge. The first 14 patients treated with vitamin C had pro-inflammatory cytokines IL-1b, IL-2, IL-6, IL-12, TNF-α, IFN-γ, and soluble thrombomodulin measured, which remained unchanged between day 0 and day 14 & day 30 after HSCT (P=NS for all direct comparisons). Heat maps demonstrating change of cytokine levels post-transplant showed that most of the patients showed either a reduction or stabilization of their inflammatory cytokine levels following SCT, with few patients demonstrating an increase at all the time points tested following HSCT (Supplementary Figure 3). However, in most instances the cytokine levels were back down in the lower ranges by day 30. Elevated cytokine levels on day 14 appear to correspond to higher magnitude of donor derived T cell recovery by day 30 following transplant (Supplementary Figure 4).