Genome-wide methylation analysis identifies genes specific to breast cancer hormone receptor status and risk of recurrence

Abstract

To better understand the biology of hormone receptor-positive and-negative breast cancer and to identify methylated gene markers of disease progression, we carried out a genome-wide methylation array analysis on 103 primary invasive breast cancers and 21 normal breast samples, using the Illumina Infinium HumanMethylation27 array that queried 27,578 CpG loci. Estrogen and/or progesterone receptor-positive tumors displayed more hypermethylated loci than estrogen receptor (ER)-negative tumors. However, the hypermethylated loci in ER-negative tumors were clustered closer to the transcriptional start site compared with ER-positive tumors. An ER-classifier set of CpG loci was identified, which independently partitioned primary tumors into ER subtypes. A total of 40 (32 novel and 8 previously known) CpG loci showed differential methylation specific to either ER-positive or ER-negative tumors. Each of the 40 ER subtype-specific loci was validated in silico, using an independent, publicly available methylome dataset from the Cancer Genome Atlas. In addition, we identified 100 methylated CpG loci that were significantly associated with disease progression; the majority of these loci were informative particularly in ER-negative breast cancer. Overall, the set was highly enriched in homeobox containing genes. This pilot study shows the robustness of the breast cancer methylome and illustrates its potential to stratify and reveal biological differences between ER subtypes of breast cancer. Furthermore, it defines candidate ER-specific markers and identifies potential markers predictive of outcome within ER subgroups.

Figure 1

Schema outlining study design for analysis of association of methylation with (A) ER-status and (B) disease outcome.

Figure 2

A. Unsupervised hierarchical cluster analysis of the most varied 5% of CpG loci probes among tumors (1378 loci; SD > 0.160). 2D-hierachial cluster analysis was performed using the Manhattan distance on 103 tumors, and 1378 loci showed two distinct clusters. Cluster 1 is enriched for ER+ tumors (pink bars); Regions of Cluster 2 appear enriched for ER-negative tumors (purple bars). Normal organoids cluster together (yellow bars). Examples of breast cancer related gene loci are indicated at left. B. Histogram plot showing the frequency of loci differentially methylated in ER-positive and ER-negative tumors. Plotted on the X-axis is the fold-difference in the mean locus methylation of ER-positive tumors/ER-negative tumors, among 8376 loci with (SD>0.1 across all tumors). The 200 (left and right shaded boxes) are CpG loci most differentially methylated between ER-positive and ER-negative tumors. C. Distance to the transcriptional start site (TSS) of the top CpG loci identified in ER-positive versus ER-negative tumors. Location of 100 ER-positive (pink line), 100-ER negative (purple line) and all 8376 (black line) CpG loci was plotted relative to the TSS indicated as 0 on the X axis. D. Validation of ER Subtype Markers. An ROC plot shows AUC 0.961 for detection of ER-subtype in 50 independent tumors within TCGA, using the ER-specific 40 loci marker set (data in Figure 3, Supp Table 2, Supp Fig 2).

Figure 3

CpG Methylation Biomarkers of ER-positive versus ER-negative subtypes in primary tumors. In the left panel (Discovery set, JH) CpG loci were evaluated using 103 primary tumors. The Y-axis: β-methylation, X-axis: Breast cancer samples, and N: normal breast organoids. The ER-status of the tumors is denoted on top of each box. Right panel (TCGA validation set), shows validation on an independent dataset (n=50) from TCGA. Shown are representative novel (A) and previously known (B) CpG loci specifically hypermethylated in ER+ and ER− breast cancers.

Figure 4

Methylated CpG Loci Associated with Disease Progression. Kaplan-Meier plots show CpG loci with strong associations between hypermethylation and high rate of disease progression among 82 tumors. Recurrence is shown for ER+ (dashed line), and ER-negative tumors (solid line). High (red line) and low (blue line) methylation was defined relative to the median methylation level for a given CpG loci within ER subtype. CpG loci associated with a high rate of recurrence in (A) ER+ and ER-negative breast tumors. (B) ER-negative breast tumors. Cox regression p-values for each of the 100 CpG loci set is presented in Supp Table 4A. (C) Verification of HumanMethylation27 array data (top panel, blue bars) by QM-MSP analysis (bottom panel) for AKR1B1. X-axis: Tumors; Y-axis: methylation levels (β-value for array, % methylation for QM-MSP). Kaplan-Meier curves using array and QM-MSP values. Figure 4A: CpG loci associated with high rate of recurrence in ER+ and ER− breast cancer Figure 4B: CpG loci associated with high rate of recurrence in ER-negative breast cancer Figure 4C: Translation from array to QM-MSP for AKR1B1

Figure 5

2-D Hierarchical cluster analysis of CpG loci in homeobox genes. Methylation levels of homeobox (A) 18 loci from the 100 recurrence markers; and (B) 60 additional unique loci in 82 primary invasive tumors. Kaplan-Meier plots of homeobox (C) 18 loci set, log rank p-value=0.033, (D) 60 loci set, p-value=0.025, and (E) unsupervised top 5% most varied array probes across tumor samples, p-value=0.438. Strong enrichment of homeobox genes was observed within the 100 recurrence loci set (table). The bars under the case dendrogram indicate recurrence (black, recurred < 60 mo; salmon, did not recur by 60 mo; gray, censored by 60 mo) and ER (pink, ER-positive; purple ER-negative) status. Analysis of methylation array homeobox gene loci in primary invasive tumors (n=82)