A proof-of-principle study of epigenetic therapy added to neoadjuvant doxorubicin cyclophosphamide for locally advanced breast cancer

Claudia Arce, Carlos Pérez-Plasencia, Aurora González-Fierro, Erick de la Cruz-Hernández, Alma Revilla-Vázquez, Alma Chávez-Blanco, Catalina Trejo-Becerril, Enrique Pérez-Cárdenas, Lucia Taja-Chayeb, Enrique Bargallo, Patricia Villarreal, Teresa Ramírez, Teresa Vela, Myrna Candelaria, Maria F Camargo, Elizabeth Robles, Alfonso Dueñas-González, Claudia Arce, Carlos Pérez-Plasencia, Aurora González-Fierro, Erick de la Cruz-Hernández, Alma Revilla-Vázquez, Alma Chávez-Blanco, Catalina Trejo-Becerril, Enrique Pérez-Cárdenas, Lucia Taja-Chayeb, Enrique Bargallo, Patricia Villarreal, Teresa Ramírez, Teresa Vela, Myrna Candelaria, Maria F Camargo, Elizabeth Robles, Alfonso Dueñas-González

Abstract

Background: Aberrant DNA methylation and histone deacetylation participate in cancer development and progression; hence, their reversal by inhibitors of DNA methylation and histone deacetylases (HDACs) is at present undergoing clinical testing in cancer therapy. As epigenetic alterations are common to breast cancer, in this proof-of-concept study demethylating hydralazine, plus the HDAC inhibitor magnesium valproate, were added to neoadjuvant doxorubicin and cyclophosphamide in locally advanced breast cancer to assess their safety and biological efficacy.

Methodology: This was a single-arm interventional trial on breast cancer patients (ClinicalTrials.gov Identifier: NCT00395655). After signing informed consent, patients were typed for acetylator phenotype and then treated with hydralazine at 182 mg for rapid-, or 83 mg for slow-acetylators, and magnesium valproate at 30 mg/kg, starting from day -7 until chemotherapy ended, the latter consisting of four cycles of doxorubicin 60 mg/m2 and cyclophosphamide 600 mg/m2 every 21 days. Core-needle biopsies were taken from primary breast tumors at diagnosis and at day 8 of treatment with hydralazine and valproate.

Main findings: 16 patients were included and received treatment as planned. All were evaluated for clinical response and toxicity and 15 for pathological response. Treatment was well-tolerated. The most common toxicity was drowsiness grades 1-2. Five (31%) patients had clinical CR and eight (50%) PR for an ORR of 81%. No patient progressed. One of 15 operated patients (6.6%) had pathological CR and 70% had residual disease <3 cm. There was a statistically significant decrease in global 5mC content and HDAC activity. Hydralazine and magnesium valproate up- and down-regulated at least 3-fold, 1,091 and 89 genes, respectively.

Conclusions: Hydralazine and magnesium valproate produce DNA demethylation, HDAC inhibition, and gene reactivation in primary tumors. Doxorubicin and cyclophosphamide treatment is safe, well-tolerated, and appears to increase the efficacy of chemotherapy. A randomized phase III study is ongoing to support the efficacy of so-called epigenetic or transcriptional cancer therapy.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Flow diagram of the trial.
Figure 1. Flow diagram of the trial.
Figure 2. Global DNA methylation.
Figure 2. Global DNA methylation.
The five patients had a reduction in 5mC content varying from 0.7−2.59% decrease. The mean 5mC in the five pre-treatment samples was 5.71% (standard deviation [SD], 1.43) which decreased to 4.2% (SD, 0.95). This difference was statistically significant (p = 0.043).
Figure 3. Histone deacetylase activity.
Figure 3. Histone deacetylase activity.
The five patients assayed showed a reduction in enzymatic activity. In Y axis are the absolute values in optical density (OD) units. Higher and lower decreases were 0.3845 and 0.0175, for a mean decrease of 0.1624. This difference was statistically significant (p = 0.042).
Figure 4. Hierarchical Cluster Analysis.
Figure 4. Hierarchical Cluster Analysis.
The cluster shown represents 3,117 genes. Each row represents a gene, whereas each column corresponds to a tissue sample. The relative abundance of the gene in the tissue correlates with color intensity (red, induced; green, repressed; black, no change). On the dendogram, post-treated clinical samples clustered together.
Figure 5. Principal Component Analysis.
Figure 5. Principal Component Analysis.
Principal Component Analysis, showing in three-dimensional space relationship between samples.
Figure 6. Gene expression by RT-PCR.
Figure 6. Gene expression by RT-PCR.
RT-PCR of NDUFA13 and DAPPER genes in biopsies from the primary tumor showing that these genes were reactivated after treatment. This corresponds to one of the three patients whose biopsy (only post-treatment) was analyzed by microarray and showed over-expression of these genes.

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