Tumor and serum DNA methylation in women receiving preoperative chemotherapy with or without vorinostat in TBCRC008

Abstract

Background: Methylated gene markers have shown promise in predicting breast cancer outcomes and treatment response. We evaluated whether baseline and changes in tissue and serum methylation levels would predict pathological complete response (pCR) in patients with HER2-negative early breast cancer undergoing preoperative chemotherapy.

Methods: The TBCRC008 trial investigated pCR following 12 weeks of preoperative carboplatin and albumin-bound paclitaxel + vorinostat/placebo (n = 62). We measured methylation of a 10-gene panel by quantitative multiplex methylation-specific polymerase chain reaction and expressed results as cumulative methylation index (CMI). We evaluated association between CMI level [baseline, day 15 (D15), and change] and pCR using univariate and multivariable logistic regression models controlling for treatment and hormone receptor (HR) status, and performed exploratory subgroup analyses.

Results: In univariate analysis, one log unit increase in tissue CMI levels at D15 was associated with 40% lower chance of obtaining pCR (odds ratio, OR 0.60, 95% CI 0.37-0.97; p = 0.037). Subgroup analyses suggested a significant association between tissue D15 CMI levels and pCR in vorinostat-treated [OR 0.44 (0.20, 0.93), p = 0.03], but not placebo-treated patients.

Conclusion: In this study investigating the predictive roles of tissue and serum CMI levels in patients with early breast cancer for the first time, we demonstrate that high D15 tissue CMI levels may predict poor response. Larger studies and improved analytical procedures to detect methylated gene markers in early stage breast cancer are needed. TBCRC008 is registered on ClinicalTrials.gov (NCT00616967).

Keywords: Biomarkers; Breast cancer; Methylation; Preoperative chemotherapy; cMethDNA.

Conflict of interest statement

Conflicts of interest: VS has received research grants from Merck, Celgene Corporation, Abbvie, Pfizer, Novartis, Medimmune, and Puma Biotechnology. RC has received research grants from Novartis, Puma Biotechnology, Genentech, Merrimack, Clovis, Merck. AMS has received consulting fees from Eli Lilly and Co., and Pfizer. SS has received research grants from AVON Foundation, grants, licensing/royalty fees and consulting fees from Cepheid for QM-MSP and cMethDNA assays. MJF has received licensing/royalty fees and consulting fees from Cepheid. No other authors have disclosed any conflicts of interest.

Figures

Figure 1. TBCRC008 Study Schema

Figure 2. Tissue and Serum DNA Methylation

QM-MSP and cMethDNA analyses were performed on tissue (n=58 baseline and 50 D15) and serum (n=59 baseline and 61 D15) samples, respectively. Scatter plots show CMI methylation levels as the sum of methylation for each marker in 10-gene panel. Median methylation is indicated (bar) and only samples with methylation values for all 10 genes were evaluated.

Figure 3. Tumor Content and DNA Methylation of Tumors in Vorinostat vs Placebo Arms

Tumor content in paired samples of the same individual. Change between baseline and D15 tumor content (% tumor in biopsy section) and methylation was quantified using Wilcoxon matched pairs test. Significant reductions in both tumor content and methylation were observed at D15 within the study population (“All patients”), as well as vorinostat and placebo groups. Only samples having methylation values for all 10 genes were evaluated.

Figure 4. Tissue CMI at D15 by pCR Status and Treatment Arm

CMI of the 10-gene panel is indicated by scatter plots for each treatment group (median shown as bar). For each individual sample (x-axis) the bar graphs indicate the level of CMI (height of the bar, y-axis) and the relative methylation of each gene (shown by colored segment). A. D15 methylation levels of tissue samples in pCR versus no pCR groups. B. D15 methylation levels of tissue and serum samples in vorinostat (V) versus placebo (P) groups. C. D15 methylation levels of tissue samples grouped pCR versus no pCR, ± vorinostat. Only samples having methylation values for all 10 genes were evaluated (nonparametric Mann-Whitney U test).