DNA methylation patterns in luminal breast cancers differ from non-luminal subtypes and can identify relapse risk independent of other clinical variables

Abstract

The diversity of breast cancers reflects variations in underlying biology and affects the clinical implications for patients. Gene expression studies have identified five major subtypes- Luminal A, Luminal B, basal-like, ErbB2+ and Normal-Like. We set out to determine the role of DNA methylation in subtypes by performing genome-wide scans of CpG methylation in breast cancer samples with known expression-based subtypes. Unsupervised hierarchical clustering using a set of most varying loci clustered the tumors into a Luminal A majority (82%) cluster, Basal-like/ErbB2+ majority (86%) cluster and a non-specific cluster with samples that were also inconclusive in their expression-based subtype correlations. Contributing methylation loci were both gene associated loci (30%) and non-gene associated (70%), suggesting subtype dependant genome-wide alterations in the methylation landscape. The methylation patterns of significant differentially methylated genes in luminal A tumors are similar to those identified in CD24 + luminal epithelial cells and the patterns in basal-like tumors similar to CD44 + breast progenitor cells. CpG islands in the HOXA cluster and other homeobox (IRX2, DLX2, NKX2-2) genes were significantly more methylated in Luminal A tumors. A significant number of genes (2853, p < 0.05) exhibited expression-methylation correlation, implying possible functional effects of methylation on gene expression. Furthermore, analysis of these tumors by using follow-up survival data identified differential methylation of islands proximal to genes involved in Cell Cycle and Proliferation (Ki-67, UBE2C, KIF2C, HDAC4), angiogenesis (VEGF, BTG1, KLF5), cell fate commitment (SPRY1, OLIG2, LHX2 and LHX5) as having prognostic value independent of subtypes and other clinical factors.

Figure 1

(a) Differentially methylated loci between tumors and normal tissue of 3 loci proximal to genes GPR10, DRD5 and CDKN1C. The samples are grouped by expression subtype (b) Bisulfite sequencing of selected loci in independent sample set validates significant methylation in tumors compared to normal tissue.

Figure 2

(a) Unsupervised clustering of breast tumors using methylation data groups samples similarly to those obtained by expression based analysis. Luminal A (Blue) subtypes (Cluster 1) form a distinctly separate group from the basal‐like(red) and ErbB2+(purple) subtypes. ER status, TP53 mutational status and Her status by FISH are plotted under the dendrogram (Black = positive, Gray = Negative, white = NA). Correlations to Luminal, Basal and ErbB2+ Centroid derived from expression data are plotted under the heatmap. Clusters I and II have unambiguous assignment of expression subtype, while cluster III contains samples with samples having weak correlations to multiple centroids. (b) fragment subset clusters luminal and basal groups. (c) Addition of 25 validation samples to the clustering retains the separation of luminal subtypes from basal/ErbB2 enriched subtypes.

Figure 2

(Continued)

Figure 3

(a) Deregulation of methylation at HOX A genes in Luminal cancer subtypes (b) Heatmap of average methylation values of 100 selected genes groups by subtype. Transcription factors belonging to Homeobox family (HOXA, IRX, LHX1), forkhead family (FOXC1, FOXD4, FOXP2), cell adhesion molecules (KRT7, COL7A1, COL14A1, COL16A1) and protocadherins (PCDHAC2, PCDHGA10) have increased methylation levels in luminal tumors when compared to basal and ErbB2 subtypes.

Figure 4

Loci whose methylation status predicts likelihood of relapse.

Figure 5

(a) Methylation status of TP53BP2 is associated relapse risk, (b) risk associated with TP53 mutation status (c) BP2 methylation status is informative of relapse risk only in patients with wild type TP53 but not (d) TP53.

Figure 6

Correlation of expression levels to methylation states of the associated fragments. Heatmap of the mean of the expression values for a selected list of 50 genes with significant anti‐correlation to methylation states is plotted. White colors indicate no samples were present in that methylation state for the gene. The number of individual samples in each state varies by gene and thus is not plotted.