Monitoring of Serum DNA Methylation as an Early Independent Marker of Response and Survival in Metastatic Breast Cancer: TBCRC 005 Prospective Biomarker Study

Abstract

Purpose Epigenetic alterations measured in blood may help guide breast cancer treatment. The multisite prospective study TBCRC 005 was conducted to examine the ability of a novel panel of cell-free DNA methylation markers to predict survival outcomes in metastatic breast cancer (MBC) using a new quantitative multiplex assay (cMethDNA). Patients and Methods Ten genes were tested in duplicate serum samples from 141 women at baseline, at week 4, and at first restaging. A cumulative methylation index (CMI) was generated on the basis of six of the 10 genes tested. Methylation cut points were selected to maximize the log-rank statistic, and cross-validation was used to obtain unbiased point estimates. Logistic regression or Cox proportional hazard models were used to test associations between the CMI and progression-free survival (PFS), overall survival (OS), and disease status at first restaging. The added value of the CMI in predicting survival outcomes was evaluated and compared with circulating tumor cells (CellSearch). Results Median PFS and OS were significantly shorter in women with a high CMI (PFS, 2.1 months; OS, 12.3 months) versus a low CMI (PFS, 5.8 months; OS, 21.7 months). In multivariable models, among women with MBC, a high versus low CMI at week 4 was independently associated with worse PFS (hazard ratio, 1.79; 95% CI, 1.23 to 2.60; P = .002) and OS (hazard ratio, 1.75; 95% CI, 1.21 to 2.54; P = .003). An increase in the CMI from baseline to week 4 was associated with worse PFS ( P < .001) and progressive disease at first restaging ( P < .001). Week 4 CMI was a strong predictor of PFS, even in the presence of circulating tumor cells ( P = .004). Conclusion Methylation of this gene panel is a strong predictor of survival outcomes in MBC and may have clinical usefulness in risk stratification and disease monitoring.

Fig 1.

Kaplan-Meier curves of (A) progression-free survival and (B) overall survival by CMI at week 4 for women with metastatic breast cancer. Landmark analysis at week 4 excluded patients encountering events before blood draw at week 4. A horizontal line indicates median survival times. Gehan tests were stratified by subset resulting from random split of the data with the two-fold cross-validation. CMI, cumulative methylation index; HR, hazard ratio.

Fig 2.

Association of disease status at first restaging with (A) CMI and (B) CTCs across three time points. The length of the box is the interquartile range and represents the middle 50% of the data. The horizontal line inside the box shows the median. The lower and upper edges of the box represent the 25th and 75th percentiles, respectively. The vertical dashed lines extend from the box to the upper and lower 1.5 interquartile values from the upper and lower edges. Jonckheere-Terpstra test for trend in change from baseline among ordered disease status at first restaging. *Significant difference (P < .001) at the given time point compared with baseline using Wilcoxon signed rank test. Natural log transformed CMI and CTC data were graphed and the y-axes were formatted with back-transformation values. CMI, cumulative methylation index; CTCs, circulating tumor cells; PD, progressive disease; PR/CR, partial response/complete response; SD, stable disease.

Fig A1.

TBCRC-005 study schema. For this study, we had duplicate serum samples from 141 women at baseline. In addition, for 129 of the 141 women, we had duplicate measures at a second time point, and for 112 of the 129 women, duplicate measures at a third time point.

Fig A2.

Flow chart describing study population. Patients were enrolled from the following seven cancer centers: Johns Hopkins Sidney Kimmel Cancer Center, Baltimore, MD; University of Alabama at Birmingham, Birmingham, AL; Indiana University, Bloomington, IN; University of Chicago, Chicago, IL; Dana-Farber Cancer Institute, Boston, MA; University of North Carolina, Chapel Hill, NC; and Mayo Clinic, Rochester, MN. QCs, quality control samples.

Fig A3.

Scatter plot summarizing the MI for each of the 10 genes evaluated. MI = [(No. methylated copies)/(No. methylated + gene standard copies)] × 100. MI for each sample was averaged across duplicates. MI, methylation index.

Fig A4.

Forest plot of the association of CMI and CTCs as a continuous marker with both PFS and OS. Baseline and week 4 CTCs and CMI modeled as a continuous marker and both PFS and OS. All markers were treated as continuous variables and log transformed. The bars represent 95% CIs. The size of the box is indicative of the precision of the point estimate. CMI, cumulative methylation index; CTCs, circulating tumor cells; HR, hazard ratio; LCL, lower confidence limit; OS, overall survival; PFS, progression-free survival; UCL, upper confidence limit.

Fig A5.

Baseline and week 4 distributions of CTCs. CTCs, circulating tumor cells.

Fig A6.

Forest plot of the association of CMI and CTCs as a continuous marker with disease status at first restaging. All markers were treated as continuous variables and were log transformed. The bars represent 95% CIs. The size of the box is indicative of precision of the point estimate. CMI, cumulative methylation index; CTCs, circulating tumor cells; LCL, lower confidence limit; OR, odds ratio; UCL, upper confidence limit.