IntroductionDuctal carcinoma in situ (DCIS) is a non-invasive lesion of the breast that is frequently detected by mammography and subsequently removed by surgery. However, it is estimated that about half of the detected lesions would never have progressed into invasive cancer. Identifying DCIS and invasive cancer specific epigenetic lesions and understanding how these epigenetic changes are involved in triggering tumour progression is important for a better understanding of which lesions are at risk of becoming invasive.MethodsQuantitative DNA methylation analysis of ABCB1, CDKN2A/p16INK4a, ESR1, FOXC1, GSTP1, IGF2, MGMT, MLH1, PPP2R2B, PTEN and RASSF1A was performed by pyrosequencing in a series of 27 pure DCIS, 28 small invasive ductal carcinomas (IDCs), 34 IDCs with a DCIS component and 5 normal breast tissue samples. FOXC1, ABCB1, PPP2R2B and PTEN were analyzed in 23 additional normal breast tissue samples. Real-Time PCR expression analysis was performed for FOXC1.ResultsAberrant DNA methylation was observed in all three diagnosis groups for the following genes: ABCB1, FOXC1, GSTP1, MGMT, MLH1, PPP2R2B, PTEN and RASSF1A. For most of these genes, methylation was already present at the DCIS level with the same frequency as within IDCs. For FOXC1 significant differences in methylation levels were observed between normal breast tissue and invasive tumours (P < 0.001). The average DNA methylation levels were significantly higher in the pure IDCs and IDCs with DCIS compared to pure DCIS (P = 0.007 and P = 0.001, respectively). Real-time PCR analysis of FOXC1 expression from 25 DCIS, 23 IDCs and 28 normal tissue samples showed lower gene expression levels of FOXC1 in both methylated and unmethylated tumours compared to normal tissue (P < 0.001). DNA methylation levels of FOXC1, GSTP1, ABCB1 and RASSF1A were higher in oestrogen receptor (ER) positive vs. ER negative tumours; whereas methylation levels of FOXC1, ABCB1, PPP2R2B and PTEN were lower in tumours with a TP53 mutation.ConclusionsQuantitative methylation analysis identified ABCB1, FOXC1, PPP2R2B and PTEN as novel genes to be methylated in DCIS. In particular, FOXC1 showed a significant increase in the methylation frequency in invasive tumours. Low FOXC1 gene expression in both methylated and unmethylated DCIS and IDCs indicates that the loss of its expression is an early event during breast cancer progression.

A B S T R A C TBreast cancer is a heterogeneous disease that can be divided in subtypes based on histol-

The majority of human breast cancers exhibit luminal epithelial differentiation. However, most aggressive behavior, including invasion and purported cancer stem cell activity, are considered characteristics of basal-like cells. We asked the following questions: Must luminal-like breast cancer cells become basal-like to initiate tumors or to invade? Could luminally differentiated cells within a basally initiated hierarchy also be tumorigenic? To answer these questions, we used rare and mutually exclusive lineage markers to isolate subsets of luminal-like and basal-like cells from human breast tumors. We enriched for populations with or without prominent basal-like traits from individual tumors or single cell cloning from cell lines and recovered cells with a luminal-like phenotype. Tumor cells with basal-like traits mimicked phenotypic and functional behavior associated with stem cells assessed by gene expression, mammosphere formation and lineage markers. Luminallike cells without basal-like traits, surprisingly, were fully capable of initiating invasive tumors in NOD SCID gamma (NSG) mice. In fact, these phenotypically pure luminal-like cells generated larger and more invasive tumors than their basal-like counterparts. The tumorigenicity and invasive potential of the luminal-like cancer cells relied strongly on the expression of the gene GCNT1, which encodes a key glycosyltransferase controlling O-glycan branching. These findings demonstrate that basal-like cells, as defined currently, are not a requirement for breast tumor aggressiveness, and that within a single tumor there are multiple "stem-like" cells with tumorigenic potential casting some doubt on the hypothesis of hierarchical or differentiative loss of tumorigenicity.clonal isolation | prospective | signatures