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. 2020 Jan-Dec:19:1533033819896331.
doi: 10.1177/1533033819896331.

Methylome Variation Predicts Exemestane Resistance in Advanced ER+ Breast Cancer

Xiao-Ran Liu  1   2 , Ru-Yan Zhang  1   2 , Hao Gong  3   2 , Hope S Rugo  4 , Ling-Bo Chen  3 , Yuan Fu  3 , Jian-Wei Che  3   5 , Jian Tie  1 , Bin Shao  1 , Feng-Ling Wan  1 , Wei-Yao Kong  1 , Guo-Hong Song  1 , Han-Fang Jiang  1 , Guo-Bing Xu  1 , Hui-Ping Li  1
Affiliations

Methylome Variation Predicts Exemestane Resistance in Advanced ER+ Breast Cancer

Xiao-Ran Liu et al. Technol Cancer Res Treat. 2020 Jan-Dec.

Abstract

Background: More than 30% of estrogen receptor-positive breast cancers are resistant to primary hormone therapy, and about 40% that initially respond to hormone therapy eventually acquire resistance. Although the mechanisms of hormone therapy resistance remain unclear, aberrant DNA methylation has been implicated in oncogenesis and drug resistance.

Purpose: We investigated the relationship between methylome variations in circulating tumor DNA and exemestane resistance, to track hormone therapy efficacy.

Methods: We prospectively recruited 16 patients who were receiving first-line therapy in our center. All patients received exemestane-based hormone therapy after enrollment. We collected blood samples at baseline, first follow-up (after 2 therapeutic cycles) and at detection of disease progression. Disease that progressed within 6 months under exemestane treatment was considered exemestane resistance but was considered relatively exemestane-sensitive otherwise. We obtained circulating tumor DNA-derived methylomes using the whole-genome bisulfide sequencing method. Methylation calling was done by BISMARK software; differentially methylated regions for exemestane resistance were calculated afterward.

Results: Median follow-up for the 16 patients was 19.0 months. We found 7 exemestane resistance-related differentially methylated regions, located in different chromosomes, with both significantly different methylation density and methylation ratio. Baseline methylation density and methylation ratio of chromosome 6 [32400000-32599999] were both high in exemestane resistance. High baseline methylation ratios of chromosome 3 [67800000-67999999] (P = .013), chromosome 3 [140200000-140399999] (P = .037), and chromosome 12 [101200000-101399999] (P = .026) could also predict exemestane resistance. During exemestane treatment, synchronized changes in methylation density and methylation ratio in chromosome 6 [32400000-32599999] could accurately stratify patients in terms of progression-free survival (P = .000033). Cutoff values of methylation density and methylation ratio for chromosome 6 [149600000-149799999] were 0.066 and 0.076, respectively.

Conclusion: Methylation change in chromosome 6 [149600000-149799999] is an ideal predictor of exemestane resistance with great clinical potential.

Keywords: advanced breast cancer; circulating tumor DNA; exemestane resistance; methylomes.

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Conflict of interest statement

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Methylome variations during exemestane (EXE) treatment. Four of the 16 patients were exemestane resistant (EXEr), and 12 were exemestane sensitive (EXEs). Differential variations in methylation regions between samples taken at different time points (baseline [upper], first follow-up [middle], and diagnosis of disease progression [lower]) are shown. Each value is calculated using the formula: 2 × ([methylation rate] − methylome variations during EXE treatment. Four of the 16 patients were EXEr and depicted in different colors (orange or green).
Figure 2.
Figure 2.
Comparison of baseline methylation status of 7 selected DMRs between EXEr and EXEs patients. Comparison of baseline MD of certain DMRs between EXEr and EXEs group (A, C, E, G); comparison of baseline MR of certain DMRs between EXEr and EXEs groups (B, D, F, H). Receiver operating characteristic curve analysis for 7 selected DMRs in terms of MD (I) and MR (J). Kaplan-Meier survival analysis for baseline MR of Chr3 [67800000-67999999] (K), Chr3 [140200000-140399999] (L), and Chr12 [101200000-101399999] (M). “Resistant” indicates EXEr patients, “sensitive” indicates EXEs patients. Chr indicates chromosome; DMRs, differential methylation regions; EXEr, exemestane resistance; EXEs, exemestane sensitive; MD, methylation density; MR, methylation ratio.
Figure 3.
Figure 3.
Methylation status changes in EXE-resistant (EXEr) and EXE-sensitive (EXEs) groups during EXE treatment. (A), Red: Hierarchical clustering analysis of methylation ratio (MR) variation in all 15 patients and EXEr patients. (B) and (C), Receiver operating characteristic curve analysis shows changes in methylation density (MD) and MR for 7 selected differentially methylated regions. (D-F) Kaplan-Meier survival analyses for MD variation (D) and MR variation (E) and synchronized MD and MR variation (F) of Chr6 [149600000-149799999]. Demethylation: synchronized decrease in MD and MR, Remethylation: synchronized increase in MD and MR. Chr indicates chromosome.
Figure 4.
Figure 4.
Genomic methylation status in MCF-7 human breast cancer cells and in exemestane-resistant MCF-7 (MCF-7/EXE) cells. (A) Viability assay of MCF-7 and MCF-7/EXE cells subjected to increasing concentrations of exemestane. (B) IC50 of MCF-7 and MCF-7/EXE were calculated using cell viability curve. Morphologies of (C) MCF-7 and (D) MCF-7/EXE (magnification: ×200). Comparisons between MCF-7 and MCF-7/EXE for (E) average genomic methylation ratio (MR) and (F) average genomic methylation density (MD). Comparisons between MCF-7 and MCF-7/EXE for (G) MR and (H) MD of chromosome 6 [32400000-32599999].
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