Phase I study of epigenetic modulation with 5-azacytidine and valproic acid in patients with advanced cancers

Abstract

Purpose: 5-Azacytidine (5-AZA) is a DNA-hypomethylating agent. Valproic acid is a histone deacetylase inhibitor. Combining hypomethylating agents and histone deacetylase inhibitors produces synergistic anticancer activity in vitro and in vivo. On the basis of this evidence, we conducted a phase I study of the combination of 5-AZA and valproic acid in patients with advanced cancers.

Experimental design: 5-AZA was administered s.c. daily for 10 days. Valproic acid was given orally daily with a goal to titrate to plasma levels of 75 to 100 mug/mL (therapeutic for seizures). Cycles were 28 days long. 5-AZA was started at 20 mg/m(2) and escalated using an adaptive algorithm based on the toxicity profile in the prior cohort (6 + 6 design). Peripheral blood mononuclear cell global DNA methylation and histone H3 acetylation were estimated with the long interspersed nucleotide elements pyrosequencing assay and Western blots, respectively, on days 1 and 10 of each cycle when patients agreed to provide them.

Results: Fifty-five patients were enrolled. Median age was 60 years (range, 12-77 years). The maximum tolerated dose was 75 mg/m(2) of 5-AZA in combination with valproic acid. Dose-limiting toxicities were neutropenic fever and thrombocytopenia, which occurred at a dose of 94 mg/m(2) of 5-AZA. Stable disease lasting 4 to 12 months (median, 6 months) was observed in 14 patients (25%). A significant decrease in global DNA methylation and induction of histone acetylation were observed.

Conclusion: The combination of 5-AZA and valproic acid is safe at doses up to 75 mg/m(2) for 5-AZA in patients with advanced malignancies.

Figure 1

Global DNA methylation was observed at different 5-AZA doses (mg/m²). C1D1, cycle 1 day 1; C1D10, cycle 1 day 10; C2D1 cycle 2 day 1. In red is represented the median percentage of methylated CpG sites on day 0 [63.4% (range 59%-70%)], day 10 [57.4% (range 53%-63%)] before returning close to baseline by day 1 of the next cycle [60.6% (range 46%-68%)] (p <0.001 by Wilcoxon sign-rank test). Abbreviations: C1D1 = course 1, day 1; C1D10 = course 1, day 10; C2D1 = course 2, day 1 (which is day 29 from the start of therapy).

Figure 2A

Increased acetylation of histone H3 was observed in 20 of 33 evaluable patients (61%). Panel B: Three representative Western blot of histone H3 acetylation analyzed using conventional Western blot techniques on days 1 and 10 of treatment. Histone acetylation was measured in positive control (HL-60 cells treated with valproic acid), and in patients on day 1 (pre-therapy) and day 10. Histone acetylation was considered if at least a twofold increase in histone acetylation was detected comparing day 10 with day 1. AC-H3, acetylated histone H3.