New Frontiers in Breast Pathology
Moderator: Dr. Sunil Lakhani
Section 2 -
Epigenetics of Breast Cancer
During the last years, it has been established the relevance of aberrant epigenetic patterns in human
tumours. For DNA methylation, it is known that two apparently contrasting phenomena coexist in the
cancer cell: a profound loss of global 5-methylcytosine genomic content with discrete areas of dense
hypermethylation. Overall hypomethylation takes place predominantly in DNA repetitive and endoparasitic
sequences and has been linked to the generation of chromosomal instability. On the other hand,
hypermethylation occurs in the CpG islands located in the promoters of certain tumour-suppressor genes,
leading to gene silencing (Esteller 2006).
It is well known that these two kinds of changes in the DNA methylation pattern occur in breast
cancer. This paradoxical coexistence of a global decrease in methylation with regional hypermethylation
implies that independent and different processes are responsible for hypomethylation and
hypermethylation. It has been suggested that global hypomethylation is caused by a global increase in
demethylase activity whereas regional hypermethylation results from local changes in chromatin structure,
which prevent access to the abundant demethylase(s) (Szyf et al, 2004).
The phenomenon of regional DNA hypermethylation and silencing of tumor suppressor genes in cancer has
been the focus of attention in the last decade. A significant amount of data has established a list of
genes hypermethylated in cancer and recently whole genome approaches have identified methylation
signatures of breast cancer cells (Yan et al, 2000). These methylation signatures, which are the unique
combination of methylated CpG islands in a cancer cell were correlated with breast cancer stage and have
been proposed to be a diagnostic marker of breast cancer cells. In addition to their diagnostic value in
breast cancer it is clear from the repertoire of methylated genes that silencing of these genes by DNA
methylation plays a role in the transformation process. Amongst the methylated genes are tumor
suppressor genes such as p16 whose methylation is proposed to silence this gene and override cell growth
regulatory signals (Silva et al, 2003). p16 methylation in DNA prepared from plasma of breast cancer
patients was associated with nodal metastasis. Another group of methylated genes in
breast cancer is composed of damage response genes such as BRCA1 (Esteller et al, 2000), which is also
mutated in familial breast cancer. Disruption of repair genes might increase sporadic mutations
frequency, a hallmark of cancer cells. Steroid receptor genes family members such as the estrogen
receptor (Ottaviano et al, 2000; Widschwendter et al, 2004)) and retinoic acid beta 2 (RARβ 2) receptor
are methylated and silenced in a fraction of breast cancers (Widschwendter et al, 2000). Interaction of
receptor with retinoic acid might have an antiproliferative effect and its silencing confers a selective
advantage on advanced breast cancer cells. Cell adhesion and cell surface molecules such as E-cadherin
(Nass et al, 2000) and inhibitors of proteases such as TIMP-3, whose silencing might promote metastasis
are also found to be methylated in breast cancer. In addition, E-cadherin hyemethylation occurs in
around 40% of lobular carcinomas and is implicated in the absence of protein expression that is
characteristic of this histotype of breast cancer (Sarriˇ et al, 2003).
The methylation status of several genes has been analyzed on samples obtained from nipple aspiration,
ductal lavage, and duct endoscopy, to evaluate their diagnostic potential. A seminal study
conducted by Evron et al examined fluid derived from cancer-bearing ducts, identified by
concomitant ductoscopy. Exfoliated ductal cells were tested by methylation-specific
PCR. Methylated alleles of cyclin D2, RAR▀, and TWISTgenes frequently were detected in cells isolated from
cancer-bearing ducts (17 of 20), but rarely in cells from healthy ducts (five of
45). In a study conducted by Krassenstein et al, the methylation status of a panel
of six tumor suppressor and other cancer genes (GSTP1, RAR▀, P16inka, p14ARF, RASSFIA, and DAPK) known to be hypermethylated in breast neoplasia was
analyzed in 22 NAF DNA samples and compared with their respective tumor samples.
These investigators found that each of the 22 tumors had hypermethylation of at least one gene
from the panel, thereby providing a target that was successfully detected in 18 (82%)
of the matched aspirate fluids with 100% specificity.
Recently, promoter methylation in the non-neoplastic cells of breast cancer stroma has been reported.
This alteration in tumor microenvironment may advance our understanding of breast cancer development and
progression and lead to new diagnostic and prognostic markers and therapeutic targets.
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