Epigenetic resensitization to platinum in ovarian cancer

Abstract

Preclinical studies have shown that hypomethylating agents reverse platinum resistance in ovarian cancer. In this phase II clinical trial, based upon the results of our phase I dose defining study, we tested the clinical and biologic activity of low-dose decitabine administered before carboplatin in platinum-resistant ovarian cancer patients. Among 17 patients with heavily pretreated and platinum-resistant ovarian cancer, the regimen induced a 35% objective response rate (RR) and progression-free survival (PFS) of 10.2 months, with nine patients (53%) free of progression at 6 months. Global and gene-specific DNA demethylation was achieved in peripheral blood mononuclear cells and tumors. The number of demethylated genes was greater (P < 0.05) in tumor biopsies from patients with PFS more than 6 versus less than 6 months (311 vs. 244 genes). Pathways enriched at baseline in tumors from patients with PFS more than 6 months included cytokine-cytokine receptor interactions, drug transporters, and mitogen-activated protein kinase, toll-like receptor and Jak-STAT signaling pathways, whereas those enriched in demethylated genes after decitabine treatment included pathways involved in cancer, Wnt signaling, and apoptosis (P < 0.01). Demethylation of MLH1, RASSF1A, HOXA10, and HOXA11 in tumors positively correlated with PFS (P < 0.05). Together, the results of this study suggest that low-dose decitabine altered DNA methylation of genes and cancer pathways, restoring sensitivity to carboplatin in patients with heavily pretreated ovarian cancer and resulting in a high RR and prolonged PFS.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

D. Matei and K.P. Nephew are consultants and on the advisory board of Supergen (now Astex Pharmaceuticals, Inc.).

©2012 AACR

Figures

Figure 1

Clinical activity. A, waterfall plot of RECIST defined responses (n = 16 patients with measurable disease). B, Kaplan–Meier curve for PFS. The solid line represents the estimation of the survival function and the dashed lines represent the 95% CIs.

Figure 2

Pharmacodynamic activity of decitabine on global DNA demethylation. A, LINE1methylation measured by pyrosequencing in PBMCs on days 1 and 8 (C1D1, C1D8) of cycles 1 and 2 (C2D1, C2D8), and at end of study. Significant demethylation was found on C1D8 and C2D8 during the treatment. There was no difference between days 1 of both cycles. Bars/error bars represent mean ± SD. *, P < 0.05. B, LINE1 in biopsy samples from patients on days 1 and 8 of cycle 1. Bars/error bars represent mean ± SD. C, Venn diagrams show the numbers of demethylated and remaining hypermethylated genes in PBMCs after decitabine treatment in patients PFS less than 6 months versus PFS more than 6 months. Dotted line divides the demethylated gene numbers and remained hypermethylated gene numbers after first cycle treatment (β value greater than 0.5 is considered as hypermethylated). D, Venn diagrams show the numbers of demethylated genes and remaining hypermethylated genes in biopsies after decitabine treatment in patients achieving PFS less than 6 months versus PFS more than 6 months.

Figure 3

Differentially hypermethylated genes at baseline for PFS > 6 months versus PFS n = 14). Methylation levels are color coded as red (high β value) to green (low β value). P values less than 0.01, using 2-sample t tests comparing responders to nonresponders, were used for selecting differentially methylated genes. B, a map of enriched functional clusters based on functional clustering, using the online webtool DAVID, for genes that were hypermethylated in biopsies from responders (PFS > 6 months) versus nonresponders (PFS < 6 months), respectively. The size of the bars (wheel spokes) and the circles represent the gene counts and enrichment scores, respectively. Hypermethylation was defined by β values greater than 0.5 for all patients in the same PFS group. See Supplementary Table S3 for lists of specific gene members of each cluster group and enrichment scores. C, the pathways enriched for unique hypermethylated genes in PFS more than 6 months versus PFS less than 6 months, using the online gene functional analysis tool Pathway Express.

Figure 4

Specific ovarian cancer gene methylation levels. A, MLH1, showing plasma DNA demethylation after cycle 2, day 8 (C2D8); B, RASSF1A, similarly demethylated after C2D8; C, AKT1S1, demethylated after cycles 1, day 8 (C1D8), and 2, days 1 (C2D1) and 8 (C2D8), as measured by pyrosequencing on those specific cycles/days, and at end of study. D, baseline (pretreatment) methylation levels of HOXA10 in tumors or ascites, correlates with extended PFS. E, treatment-induced reduction of HOXA10 methylation levels in tumor biopsies, as correlated with patient PFS more than 6 months, versus patient PFS less than 6 months (P < 0.05). In A, B, C, and E, bars/error bars represent mean ± SD. *, P < 0.05.